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O. thesis entitled ’ CELL-MEDIATED IMMUNITY _I_N vIvo AND _I_1~1 VITRO BY CHICKENS WITH BCG 0R MAREK'S DISEASE INFECTION AND THE EFFECTS OF BURSECTOMY AND THYMECTOMY ON THE EXPRESSION OF CELL-MEDIATED IMMUNITY presented by Inguna Silavs Fauser has been accepted towards fulfillment of the requirements for Ph.D. deg“, in Poultry Science . l E. E I n , - ‘ LIBRARY BINDEF'S ‘ nun-nu uIcIInI , a n v < " V - . h . u ‘t A i- -_- \-. - . , u ‘ ~- . ' .‘ ‘|. -' . . ‘ o . ." fl. , v . - E .- - I. ‘ Y ‘ . 1": ‘ 'l' \ ' 1.. , I ‘ .1? '- '5‘"! ‘ 7 "‘fi-"“ ' -t‘ifiwl. W!) Van c3141 1 . .1 “bush: (urn, at, .1: _ . LPN-W”: fr“ yeti: nu. ' a.“ E ' “a 3.01119 wiser.- th W). ' EM“: no run; NEH...” ‘ a t plastic ”‘3, mum (9-21! or beak-«may E " v.2 . ‘W 9"“ «mm tW'E-qu‘, , a. 1"“ in ilk m mm m Jr- : , i; ,v «3; ta ‘1ch at W ‘ ”a. EE-wax t (cm “to!!!“ 015W ABSTRACT CELL-MEDIATED IMMUNITY IN VIVO AND IN VITRO BY CHICKENS WITH BCG OR MAREK'S’DISEASE_TNFECTION AND THE EFFECTS OF BURSECTOMY AND THYMECTOMY ON THE EXPRESSION OF CELL-MEDIATED IMMUNITY BY Inguna Silavs Fauser A method was developed to detect the migration inhibi— tion faCtor (MIF), an [in 11252 correlate of delayed sensitivity, from peripheral leucocytes of chickens inocu- lated with Bacille Calmette-Guerin (ECG) or Marek's disease virus (MDV). There was no radial migration of the blood leucocytes from spots on a plastic petri dish in the presence of a tuberculo-protein (B-24) or A-antigen of Marek's disease (MD). Leucocytes from chickens sensitized with ECG or MDV were inhibited in in zitrg migration by B-24 and A-antigen respectively. MIF was not detected by B—24 in complete Freund's adjuvant (CFA) inoculated chickens. 3-24 elicited more delayed skin reactions in BCG than CFA sensitized groups: eight of eight as compared to five of eight. Skin reactions to old tuberculin (CT) were posi- ! tive in six of eight CFA inoculated chickens. Both OT and l 3-24 elicited delayed skin reactions in five of eight CFA Qf6£6\ Inguna Silavs Fauser (4 inoculated chickens. All eight CFA and seven of eight BCG inoculated chickens had granulomatous lesions and/or acid fast organisms at the site of inoculation and all inoculated chickens had precipitating antibody with puri- fied protein derivative (PPD). A-antigen elicited delayed skin reactions in all adult chickens infected with MDV. None of the five chickens inoculated with MDV in CFA had delayed skin reactions with A-antigen but all did with OT. MDV infected chickens had fluorescent antibody to MDV infected fibroblasts but no gross or microscopic MD lesions were detected. Three groups of chickens were used to determine if neonatal thymectomy would eliminate MIF production. Intact, bursectomized, and thymectomized chickens were negative on two tests three weeks apart for MIF production to B-24 prior to inoculation with BCG. They were also negative for pre- cipitating antibody to PPD and delayed skin reactions to 8-24. The tests were repeated after sufficient time for immune responses to have developed. Intact chickens had detectable antibody, MIF, and delayed skin reactions. All thymectomized chickens had antibody, three of seven had delayed skin reactions with no Arthus responses to B-24, and only one of seven had detectable MIF. Only the bur- sectomized chickens had true delayed skin reactions to OT. Inguna Silavs Fauser Three of seven intact and six of seven thymectomized chick— ens had Arthus reactions with OT. in 3239 graft-versus-host (GVH) reactions by blood leucocytes of chickens were greater by leucocytes from thymectomized donors than from the intact or bursectomized chickens. Detection of MIF in avian blood leucocytes is repro- ducible and correlates with delayed skin reactions. The results support evidence of the thymic role in the capacity to develop delayed sensitivity and MIF production, and indicate that the GVH reactivity by blood leucocytes may not be by the same population of thymic dependent cells as those capable of recruitment for MIF production. CELL-MEDIATED IMMUNITY IN_VIVO AND IN VITRO BY CHICKENS WITH BCG OR MAREK'S DISEASE INFECTION AND THE EFFECTS OF BURSECTOMY AND THYMECTOMY ON THE EXPRESSION OF CELL-MEDIATED IMMUNITY BY Inguna Silavs Fauser A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1974 DEDICATION Tb Dr. Anne vandeerude Miller, Instructor in the Life Science Department and Dr. Marinas swets, Chairman cf the English Department, Grand Rapids Junior College, Grand Rapids, Michigan, in appreciation of their talents as teachers. ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. Timothy S. Chang for his enthusiastic support, and Dr. Virginia H. Mallmann for her continued guidance and assistance. I am indebted to my other committee members, Dr. Theo Coleman, Dr. William Cooper and Dr. Howard Zindel for their individual academic involvement throughout and in their assistance in the preparation of this manuscript. I acknowledge the professional assistance of Dr. H. Graham Purchase in performing necropsies and pathologic diagnosis of chickens in experiments with Marek's disease and Dr. George 0. Winegar, for necropsy and pathologic diag- nosis of chickens sensitized with Mycobacteria. I thank Mrs. Amelia Carpenter, Mrs. Betty Leiby, Mrs. Jane Walsh and Mrs. Cecyl Fisher for processing tissues for histopathology and bacteriology. My thanks to Mrs. Marion Pontz for prep- aration of tissue culture medium and Dr. William Okazaki for his technical and professional assistance throughout. I thank Mr. Howard Stone for supplying the chickens used in my studies. My thanks to Mr. Sulo Hulkonen for preparation of pro— jection slides of results. iii I am grateful for the attentive care given by Mr. Lewis Moritz, Mr. Hugo Fox and Mr. Robert Lowe to all experimental chickens. My thanks to Mrs. Audré Gudanowski and Ms. Shrylene Z. Gaines for their technical assistance. I thank Ms. Cheryl Hunt for her technical assistance, help in the preparation of data for submission for computer analyses, and continued personal interest. I thank Dr. John Gill for assistance in the choice and development of statistical models and Dr. Larry Miller for their execution. I am grateful to Dr. Phil A. Long and Dr. Leland Velicer for sharing A-antigen with me. My thanks to all the members of the tuberculosis project for the preparation of Band 24.. I am grateful for the typing services of Mrs. Patty Jo Campbell, Mrs. Martha Eller, Miss Maureen Howe, Mrs. Evelyn Jones, Miss Sandy Leszynski, Mrs. Kathleen Long and Mrs. Virginia Ross. I thank all my family members for their continued financial and moral support. I wish to acknowledge the financial support in part by USDA, APHIS Cooperative Agreement #12-14-100-10,366(91) and the Department of Poultry Science, Michigan State University. Part of the research reported herein utilized the facilities of the USDA-RPL at East Lansing, Michigan. iv TABLE OF CONTENTS INTRODUCTION I I I I I I I I I I I I I I I I I I I I LITERATURE REVIEW I I I I I I I I I I I I I I I I I Dependence of Immunologic Development of the Chicken on the Thymus and Bursa of Fabricius. Structure and Development of the Thymus. . . . . Relationship of the Thymus to Other Lymphoid Organs. . . . . . . Structure and Development of the Bursa of. Fabricius . . Relationship of the Bursa to Other Lymphoid Organs. . . . . . . . . . . . Methods and Immunologic Effects of Thymectomy. . Methods and Immunologic Effects of Bursectomy. . In Vivo Tests of Cellular Immunity . . . . . . . In V1tro Tests and Correlates of Cellular Immunity. . . . . . . Cell-mediated Immunity and Its Clinical Manifes- tations . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . Chickens and Chick Embryos . . Collection of Blood for Tissue Serums. . . . . . . . . Experimental Sensitization . In Vivo Techniques . . . . . A. Thymectomy . . . . . . B. Bursectomy . . . . . C. Graft-versus- host Assay. D. Skin Testing . . . . . . In Vitro Techniques. . . . . . A. Leucocytes for Migration B. Antigens . . . . . . . . C. Antibodies . . . D. Bacteriological Techniques Postmortem Examinations. . . . . Statistical Analyses . . . . . . ulture and ('1' I I HI I I I I I I . OI IIIIIffIIIIIIIImI 'bition T :3 IIIIID‘III-ooo [.1 TABLE OF CONTENTS--Continued Page Experimental Designs . . . . . . . . . . . . . . 59 A. Experiment I . . . . . . . . . . . . . . . 59 BI Experiment II I I I I I I I I I I I I I I I 61 C. Experiment III . . . . . . . . . . . . . . 62 RESULTS I I I I I I I I I I I I I I I I I I I I I I 65 AI Experiment I I I I I I I I I I I I I I I I I I 65 B. Experiment II . . . . . . . . . . . . . . . . 76 C. Experiment III. . . . . . . . . . . . . . . . 85 DISCUSSIoNI I I I I I I I I I I I I I I I I I I I I 102 Factors Influencing In_Vitro Leucocyte Migration 102 CONCLUSIONS I I I I I I I I I I I I I I I I I I I I 118 APPENDICES 1. Nominal stainless steel hypodermic tubing specifications. . . . . . . . . . . . . . . . 121 2. Formulation of Medium 199 (M199). . . . . . . 122 3. Formulation of Ham's F-10 Medium (F-lO) . . . 123 4. Suggested operation of Jamesway incubator . . 124 LITEMTURE CITED. I I I I I I I I I I I I I I I I I 125 LIST OF TABLES TABLE Page 1. Migration in Vitro of leucocytes from control chickens With and without antigen in the cul- ture medium. . . . . . . . . . . . . . . . . . 66 2. Analysis of variance of data in Table l. . . . 67 w o Leucocyte migration by three groups of chick- ens with antigens. Migration summarized as least square means . . . . . . . . . . . . . . 69 4. Analysis of variance for data summarized in Table 3 and analyzed as a split plot random model I I I I I I I I I I I I I I I I I I I I I 7 o 5. Comparison of leucocyte migration means of control, CFA sensitized and ECG sensitized chickens with antigen. Supplementary testing for data analyzed in Table 4 . . . . . . . . . 72 6. Skin reactions, antibody responses and patho- logic reactions of 3 groups of chickens in Experilnent I I I I I I I I I I I I I I I I I I 7 4 7. Analysis of variance of migration of leuco- cytes with and without the antigens crude GA and CT from control chickens . . . . . . . . . 77 8. Mean migration in micrometer units by all leu— cocyte spots from 9 control chickens with no antigen, OT, and crude GA. . . . . . . . . . . 79 9. Analysis of variance of in Vitro migration of leucocytes from control and MDV sensitized chickens analyzed with the least square rou- tine as a split plot random model. . . . . . . 80 10. Antigen by sensitization interaction of data on Table 9 represented by least square means migration with and without antigen . . . . . . 81 LIST OF TABLES—-Continued TABLE 11. 12. 13. 14. 15. 16. 17. l8. 19. 20. 21. Arithmetic mean migration of leucocytes from control and MDV chickens with no antigen and A-antigen I I I I I I I I I I I I I I I I I I I Skin reactions and antibody responses of chick- ens in Experiment II . . . . . . . . . . . . . Least square mean spleen weights in grams of 19-day old embryos injected at 14 days embryo- nation with leucocytes from intact, bursecto- mized and thymectomized donors and mean spleen weights of control spleens. . . . . . . Analysis of variance for data summarized in Table 1 3 I I I I I I I I I I I I I I I I I I I Least square means ofspleen weightin grams by embryos of two sublines injected with leuco- cytes from intact and thymectomized donors . . Mean spleen weights of controls for data on Table 15 I I I I I I I I I I I I I I I I I I I Analysis of variance for data summarized in Table 15 I I I I I I I I I I I I I I I I I I I Analysis of variance of surgery, sensitization, and antigen in leucocyte migration in vitro. . Migration means for antigen by time inter- action Table 18. . . . . . . . . . . . . . . . Migration means in micrometer units for anti- gen by time by surgery interaction . . . . . . Skin reactions, antibody response, and lesions of adult chickens in Experiment III. . viii Page 83 84 86 88 89 91 92 93 95 97 100 INTRODUCTION The adaptive immunity of vertebrates includes the ability to produce immunoglobulins and to develop cellular immunity in response to antigenic stimulation. Cellular immunity which includes delayed sensitivity is mediated by specifically sensitized lymphocytes. It is responsible for homograft rejection, graft-versus-host reactivity and immun- ity to various microorganisms. It can not be transferred with serum, only by lymphoid cells or their extracts. The tests for varied functions of the sensitive lympho- cyte in git£g_were recently developed and are referred to as in zitrg correlates of delayed sensitivity. Because both immune systems, immunoglobulins and sensitive lymphocytes, can now be assayed in zitrg, there has been an intensive search for the mechanism(s) which control(s) the development 'and function of adaptive immunity. Thereby many untreatable diseases including neoplasms and autoimmunity may be treated by the appropriate "immunologic engineering", i.e., replace- ment of inadequate or defective immunologic function or sup- pression of immunologic responses. The chicken as a representative of the class Aves is a unique experimental laboratory model of adaptive immunity because two anatomically separated central lymphoid organs control the development of immunologic competence. The bursa of Fabricius, a hindgut lymphoid organ, is responsible for the system of immunoglobulin production which is com- prised of lymphocytes of the germinal centers and plasma cells of the peripheral lymphoid tissue. The other central lymphoid organ, the thymus, is responsible for the develop- ment of the thymus-dependent small lymphocytes of delayed sensitivity. The anatomical discreteness of the two central lymphoid organs in the chicken facilitates the selective study of either the immunoglobulin or cell-mediated immuno- logic system. Removal of either the bursa or thymus early in life compromises or eliminates the development of the respective immunologic capacity. This is a report of the results of research to investi- gate the following questions: (1) Is Migration Inhibition Factor (MIF), an in yitgg correlate of delayed sensitivity, one of the biological effector molecules elaborated when sensitive lymphocytes from chickens are incubated in yitgg with antigen? (2) Does Marek's disease, a lymphoprolifera— tive disease in chickens, evoke delayed sensitivity to a soluble antigen of the Marek's disease virus (MDV)? (3) Does MIF production correlate with in 2339 expressions of cellular hypersensitivity? (4) Does neonatal thymectomy results in compromised in yigg delayed sensitivity and in Vitro MIF production as predicted? LITERATURE REVIEW Dependence of Immunologic Development of the Chicken on the Thymus and Bursa of Fabric1us The development of adaptive immunity in the chicken is dependent upon the two anatomically separated central lymph- oid organs, the thymus comprised of 5-7 lobes on each side of the cervical vertebrae, and the bursa of Fabricius, a single hindgut lymphoid organ. A functional dissociation of the immune response in the chicken was first postulated by Szenberg and Warner (1962) and Warner 35 El' (1962), and confirmed by Aspinall gt El. (1963), Jankovié and Isvaneski (1963), Cooper 25 El. (1965), and Cain gt El- (1968). The thymus in chickens and mammals, and thymus- dependent small lymphocytes (T cells, T lymphocytes), are responsible for the development of delayed type hypersensi- tivity or cellular immunity (Miller, 1961; Lawrence and Landy, 1969). This includes immunologic surveillance as exemplified by homograft rejection and graft-versus-host reactivity, reactions induced by intracellular microbial chronic diseases with persistent delayed hypersensitivity skin reactions, and development of the untoward reactions of autoimmune diseases. It may also be involved in antigen recognition or other "helper" function in the humoral or antibody mediated system. The bursa of Fabricius is largely responsible for the capacity to develop humoral immunity (Chang gt gt., 1955; Glick gt gt., 1956). Humoral immunity is mediated by anti- bodies, the immunoglobulins which are complex protein globulins synthesized by plasma cells. The appendix of the rabbit is thought to function as a central lymphoid organ analogous to the avian bursa (Konda and Harris, 1966). The equivalent structure in other mammals has not been resolved, but it is generally considered to be some gut-associated tissue such as Peyer's patches, tonsils, and/or the appendix (McKneally and Good, 1971; Perey and Guttman, 1972). Van Alten and Menwissen in 1972 reported antibody pro- duction by bursal lymphocytes after sheep erythrocytes were injected into the bursal lumen. There is little evidence to indicate that the lymphoid cells within the bursa itself routinely produce specific antibody directed against a wide variety of experimentally injected antigenic substances (Dent and Good, 1965; Abramoff and Brien, 1968a; Choi and Good, 1973). Abramoff and Brien (1968a) reported marked cell differentiation occurred in the bursa after intravenous immunization with sheep erythrocytes. Bursal lymphocytes (B cells, B lymphocytes) do produce immunoglobulins but ' ‘have not always been characterized as reacting in some detectable way with the sensitizing antigen (Marinkovich and Baluda, 1966; Glick and Whatley, 1967; Thorbecke gt gt., 1968; Kincade and Cooper, 1971; Bankhurst gt gt., 1972). Nonetheless, higher levels of immunoglobulin bearing cells occur in the bursa than any other lymphoid organ in the chicken (McArthur gt gt., 1971; Rabellino gt gt., 1971). or in any mammalian lymphoid organ (Takahashi gt gt., 1971). Restoration of the capacity for immunoglobulin production follows transplantation of B cells into agammaglobulinemic recipients (Cooper gt gt., 1966b; Gilmour gt gt., 1970). It has been suggested that the pool size of B lymphocytes detected by surface immunoglobulins in bursa-dependent lymphoid areas is relatively fixed and dependent on the number seeded initially from the bursa and this is dependent upon the time allowed for the bursa to function before its removal (Kincade and Cooper, 1971; Kincade gt gt., 1973). Immunoglobulins are released and distributed into many fluids and secretions including serum (Leslie and Clem, 1969; Lebacq-Verheyden gt gt., 1972; Bienenstock gt gt., 1972), bile (Bienenstock gt §;., 1972; Lebacq-Verheyden gt gt., 1972; Leslie and Martin, 1973), bronchiorespiratory secretions (Leslie and Martin, 1973), saliva and oviduct washings (Orlans and Rose, 1972), extracts of the chicken caeca (Orlans and Rose, 1972) and the egg (Kramer and Cho, 1970). To date, three classes of immunoglobulins (19) have been identified in the chicken, IgM (Leslie and Clem, 1969) IgY or IgG (Leslie and Clem, 1969), and IgA (Bienenstock gt gt., 1972; Lebacq-Verheyden gt gt., 1972; Orlans and Rose, 1972). The small B lymphocytes proliferate and differentiate into plasma cells. Plasma cells can be identified £2 gtttg by immunofluorescence (Rabellino gt gt., 1971). Using this method, IgM production has been detected in the bursa as early as 14 days embryonation, and IgG production around the time of hatching (Kincade and Cooper, 1971; Cooper gt gt., 1972). It has not been determined whether these immuno- globulins are formed in response to antigenic stimuli. They are also present in embryonating chicks obtained from dams raised gnotobiotically (Thorbecke gt gt., 1968) and 8 day-old chicks reared under a germ-free environment had IgM on the bursa and spleen cells similar to controls. IgA is not detectable in the serum until chickens are several months old (Martin and Leslie, 1973). IgA production is thought to require "helper" function from thymus-dependent lymphocytes. IgA is found in high concentrations in most exocrine secretions such as bile (Orlans and Rose, 1972). Structure ggd Development of the Thygus In the adult chicken, the thymus consists of a variable number of paired lobes which extend from the anterior cervical to the anterior thoracic regions adjacent to the jugular veins (Jankovié and Isakovié, 1964; Warner, 1964; Lucas and Stettenheim, 1965; Druet and Janigan, 1966). The posterior lobes are intimately associated with the thyroid, parathyroid, and ultimobranchial bodies; sometimes, the capsule surrounding the posterior lobe is absent and lymphoid nodules penetrate the thyroid and parathyroid tissue (Payne and Breneman, 1952; Fauser, 1969; Panigrahi, 1970; Panigrahi gt gt., 1971; Payne, 1971). The thymus is reported to be the first lymphoid organ to begin development in the chick embryo. This commences around 5 days embryona- tion with outgrowths of the ventral and lateral walls of the third and fourth pharyngeal pouches with possible contribu- tion from branchial ectoderm (Venzke, 1952; Hammond, 1954). The origin of thymic lymphocytes (thymocytes) in all vertebrates reported is controversial. Originally two theories were proposed, neither mutually exclusive: The first proposed that epithelial cells within the developing mouse thymus transformed into thymocytes (Auerbach, 1961 as summarized in 1964) and the second theory stated that thymo- cytes were derived from extrinsic mesenchymal cells (stem cells) which migrated into the epithelial anlage, and then differentiated into thymocytes (Kingsbury, 1940). The second theory, on the basis of parabiotic studies of chick embryos of the opposite sex, is gaining acceptance (Moore and Owen, 1967). Large basophilic cells, derived from the embryo of the opposite sex, were found in the mesenchyme and blood vessels around the thymus at 8 and 9 days of embryonation. Owen and Ritter reported in 1969 that stem cells first enter the thymic anlage between 6 and 7 days incubation, and give rise to the basophilic cells, which are the pre- cursors of thymic lymphocytes, between 8 and 9 days of incubation. These cells are presumably derived from the yolk sac, as determined by grafting diffusion chambers con- taining embryonic thymus on the chorioallantois of 9 day chick embryos, and appear to differentiate into thymocytes under the inductive influence of thymic epithelium (Owen and Ritter, 1969). Lucas and Jamroz (1961) reported that lymphocytes are found in the thymus around 11 days embryona- tion, and by 13 days embryonation cortical and medullary zones are apparent in the thymus. Papermaster and Good (1962) reported large lymphoid cells at 14 days embryona- tion. The medulla of the thymus contains thymocytes and epithelial cells forming Hassalls' corpuscles, and the cortex is comprised of densely packed small thymocytes in a supportive network of reticular cells (Peterson and Good, 1965; Payne, 1971). The bone marrow is thought to serve as the source of thymic stem cells in the adult mouse (Moore and Owen, 1966; McGregor gt gt., 1971) and probably in other mammals as well. Relatiogghip of thg Thymus to Othgt Lygphoid Organs Thymus-dependent lymphocytes in mammals and chickens are established peripherally by a mechanism unresolved. A humoral factor of thymic origin_may affect cells in the peripheral lymphoid tissues (Osoba and Miller, 1963), or thymocytes may migrate to peripheral areas (Nossal and Goorie, 1964; Murray and Woods, 1964; woods and Linna, 1965; Larsson, 1966; Toro and Oléh, 1967; Owen and Ritter, 1969; Sainte-Marie and Peng, 1971). In the chicken, thymus- dependent small lymphocytes are located in the blood (Warner gt gt., 1962; Jankovié and Isakovié, 1964; Isakovié and Jankovié, 1964; Jaffe, 1966; Fauser, 1969; Fauser gt gt., 1969; Longenecker and Breitenbach, 1969; Fauser gt gt., 1973b), in the periarteriolar zone comprising the white pulp of the spleen Uankovié and Isakovié, 1964; Isakovié and Jankovié, 1964; Cooper gt gt., 1966a), caecal tonsils (Cooper gt gt., 1966a; Panigrahi, 1970), medullary lymphoid follicles of the bursa (Jankovié and Isakovié, 1964; Warner, 1967), and intestinal epithelium (Cooper gt gt., 1967; Panigrahi, 1970). Lymphoid tissue associated with the paraocular and paranasal organs of the chicken has been identified by Bang and Bang (1968). These include lymphoid accumulations in the mucosa around lacrimal ducts, Harderian glands and villi 10 of their ducts, as well as beneath the epithelium of the lateral nasal gland ducts. Small lymphocytes contained within these structures may be thymus-dependent, but it has not yet been established. Surgical removal of the chicken thymus within 24 hours of hatching results in a depletion of lymphocytes from the peripheral lymphoid tissue which is anatomically and func- tionally thymus-dependent (Cooper gt gt., 1966a). Thymectomy later in life or natural regression of the thy— mus does not grossly deplete thymus-dependent areas of small lymphocytes. Natural regression occurs at the onset of sexual maturity (Wolfe gt gt., 1962). Structure and Development of the Bursalof Fabricius) ‘- The mature bursa contains lymphoid follicles consisting of an outer cortex of densely packed lymphocytes supported by a reticular network and separated from the diffuse lymph- oid tissue and reticuloepithelial cells of the medulla by an epithelial zone and basement membrane (Payne, 1971). Epithelial and blast cells may pass from the medulla into the cortex (Ackerman and Knouff, 1959, as summarized in 1964). The bursa originates as a small sac dorsal to the cloaca with proposed derivation from embryonic endoderm and/or ectoderm (Romanoff, 1960; Ruth gt gt., 1964). On the twelfth 11 day of embryonation, epithelial buds form in the undifferen- tiated lining of the inner surface of the bursa and vascué larization commences (Ackerman and Knouff, 1963, as summar- ized in 1964). The epithelial buds give rise to lymphoid follicles and lymphocytopoiesis begins at 14 or 15 days embryonation (Ackerman and Knouff, 1959, as summarized in 1964; Papermaster and Good, 1962). The origin of bursal lymphocytes is not resolved, but there is evidence that yolk sac stem cells and possibly thymocytes "home" to and colonize the bursa (Moore and Owen, 1966; Linna gt gt., 1972; Potworowski, 1972; Toivanen gt gt., 1972a). The postem- bryonic stem cell originates from the bone marrow (Toivanen gt gt., 1972b). Presumably under the inductive influence of bursal epithelium, the stem cells differentiate into lymphoblasts which subsequently give rise to large, medium and small lymphocytes. Removal of the bursa after one month of age and its normal regression around the onset of sexual maturity do not deplete bursa-dependent lymphoid tissue or alter the ability to produce antibody (Chang gt gt., 1957; Wolfe gt gt., 1957; Graetzer gt gt., 1963a; Cooper gt gl., 1969). The normal regression or involution of the bursa occurs at the onset of sexual maturity (Wolfe gt gt., 1962). 12 Relationship of the Bursa to Other LymphOid Organs The bursa is essential for the development of germinal centers and small lymphocytes (B cells) which proliferate and become plasma cells with the ability to produce immuno— globulins and antibodies (Chang gt gt., 1955; Van Alten gt gt., 1965; Takahashi, 1967; Seto, 1970b). It is not resolved whether the bursa only seeds lymphocytes peri— pherally (Woods and Linna, 1965) or also elaborates a hor- mone (Glick, 1960; Jankovié and Leskowitz, 1965; St. Pierre and Ackerman, 1965) which regulates the differention of non-bursa-derived lymphocytes into immunoglobulin producing cells. The peripheral bursa-dependent system consists of small lymphocytes (B cells), plasma cells, and germinal centers (Warner, 1964; Cooper gt gt., 1966a). These are located in the red pulp of the spleen (Jankovié and Isakovié, 1964; Cooper gt gt., 1966a; Potworowski, 1972), caecal tonsils (Cooper gt gt., 1966a; Warner gt gt., 1969; Panigrahi, 1970), and gastrointestinal tract associated lymphoid accumulations (Warner, 1965; Payne, 1971), as well as Peyer's patches (Panigrahi, 1970). Lymphoid accumulations of germinal centers and/or plas- ma cells have been demonstrated also around the lacrimal ducts, Harderian glands and lateral nasal gland ducts (Bang 13 and Bang, 1968). Plasma cells within the paraocular and paranasal organs are thought to be responsible for immuno- globulin A (IgA) synthesis (Lebacq-Verheyden gt gt., 1972; Leslie and Martin, 1973). Relatively few large lymphocytes are found in the peripheral circulation of mature chickens (Lucas and Jamroz, 1961). Although the chicken does not possess lymph nodes analogous to those found in mammals, there are small collec- tions of lymphoid tissues (mural nodules) in the walls of lymphatic vessels. The mural nodules, which appear only after 27 days of age and develop germinal centers some 20 days later (Kondo, 1937, as cited by Payne in 1971; Biggs, 1957) contain supportive connective tissue and are vascu- larized by a plexus of small blood vessels. They lack a capsule and lymph sinuses, although germinal centers and plasma cells develop after antigenic stimulation (Good and Finstad, 1967, as reported by Payne in 1971). Small foci of lymphoid tissue occur in other organs and tissues, including connective tissue, bone marrow, skin, liver, lung, kidney, pancreas, endocrine glands, peripheral nerves, larynx and trachea (as reviewed by Payne, 1971). The lymphoid elements may consist of small, medium, and large lymphocytes and occasional germinal centers. Whether they are normal lymphopoietic structures, a response to lymphomatosis virus (Oakberg and Lucas, 1949; Lucas and 14 Oakberg, 1950; Oakberg, 1950), or occlusion of blood ves- sels (Lucas, 1949; Thorbecke gt gt., 1957, as reviewed by Payne in 1971), or some combination of these remains unresolved. Methods and Immunologic Effects of Thymectomy Thymectomy in the chicken can be performed surgically shortly after hatching but because of the anatomical loca— tion of the thymus, complete thymectomy by blunt dissection is difficult, if not impossible (Warner and Szenberg, 1964a, b; Metcalf, 1964; Panigrahi, 1970; Panigrahi gt gt., 1971). Panigrahi gt gt. (1971), reported that thymic remnants do not undergo hypertrophy or hyperplasia in response to demand for increased function. Functionally, thymectomized chickens have severely altered ability to reject homografts (Warner and Szenberg, 1962; Aspinall gt gt., 1963), have a reduced amount of experimental allergic encephalomyelitis (Jankovié and Isvaneski, 1963), and have reduced delayed sensitivity to diphtheria toxoid and tuberculin (Cooper gt gt., 1966a; Panigrahi, 1970; Fauser gt gt., 1973c). Compromised graft- versus-host reactivity by blood lymphocytes of thymectomized chickens has been reported (Cooper gt gt., 1966a). In the chickens that have thymic damage as a result of hormonal bursectomy, homograft rejection is abolished 15 whereas graft-versus-host (GVH) reactivity is retained (Warner and Szenberg, 1962). Therefore, Warner and Szenberg (1962) postulated that GVH competence may not be entirely thymus-dependent. It may have originated from a third cell source or from thymus-dependent lymphocytes which migrate peripherally from the thymus early in embryonation. Sheridan and his associates in 1969 reported that thymectomy of donors in the GVH reaction increases mild splenomegaly caused by non-B-locus histoincompatability as compared with thymectomy plus irradiation which decreases gross splenomeg- aly resulting from B-locus histoincompatability. The characteristic lesions of experimental allergic encephalomyelitis do not develop in thymectomized chickens (Blaw gt gt., 1967) and caseation necrosis of the spleen in experimental tuberculosis is decreased (Panigrahi gt gt., 1972). In thymectomized chickens, the thymus-dependent lym- phoid tissue is depleted of small lymphocytes. Depletions occur in the white pulp of the spleen, caecal tonsils, bur- sal follicles and intestinal epithelium. A reduction in the percentage of vascular lymphocytes in thymectomized chickens has been reported (Isakovié and Jankovié, 1964; Jankovié and Isakovié, 1964; Warner and Szenberg, 1964a; Jaffe, 1966). Cooper gt gt. (1966a), reported a decrease in the number of small lymphocytes in x-irradiated thymec- tomized chickens and no reduction in non-x-irradiated 16 thymectomized chickens. Fauser (1969) and Fauser‘gt;gt, (1969 and 1973b) reported a decrease in total lymphocytes in x-irradiated and non—x—irradiated thymectomized chickens. Thymectomy of day old chicks resulted in a prolonged deple— tion of vascular lymphocytes (up to 5 months of age). Coupling x-irradiation with thymectomy did not result in further depletion of vascular lymphocytes. The bursa-dependent functions remain in thymectomized chickens. Thymectomized chickens can produce antibody (Warner and Szenberg, 1962; Graetzer gt gt., 1963b; Isakovié gt gt., 1963; Cooper gt gt., 1966a) and natural haemagglutinins (Graetzer gt gt., 1963b). Some exceptions have been reported. Graetzer gt gt. (1963b), reported a diminished antibody response by thymectomized chickens, and Cooper gt gt. (1966a) found thymectomized.Xhirradiated chickens produced quantitatively less antibody after experimental injection with antigen. The thymusvdependent system may be necessary for the recognition of certain sub« stances as foreign which precedes the initiation of an immunologically specific response by either the thymus or bursa-dependent systems (Peterson gt gt., 1965). The co— operation of T cells in eliciting a B cell response has been demonstrated in chickens (McArthur gt gt., 1973; weinbaum gt gt., 1973) and other animals. The thymus may also be necessary to regulate or terminate the degree of bursa— dependent antibody response. Wick gt gt. (1970b) reported 17 higher antibody titers in thymectomized chickens. Recently, Rouse and Warner (1972a, b) reported func- tional thymectomy with antithymocyte sera, detected by loss of GVH reactivity. Lymphocytes from a human infant with congenital absence of the thymus failed to react as T cells tg ztttg and tg vivo (Lischner gt gt., 1967). Mgthods and Imgunologic Effects of Bursectomy Several methods for removal of the bursa are employed. Methods of bursectomy are evaluated as to effectiveness by observing depletion of bursa-dependent lymphoid tissue and absence of immunologic function. Reported methods of bur- sectomy include surgical bursectomy with or without x-ir- radiation thereafter, x-irradiation of the bursa itself, hormonal bursectomy, immunologically induced bursectomy in combination with another of the above methods, and cyclo- phosphamide treatment. The various methods of bursectomy vary in ease of performance as well as the degree of compro- mised bursa and bursa—dependent function. The early studies of bursa function utilized surgical bursectomy (Chang gt gt., 1955; Glick gt gt., 1956). Surgical bursectomy within a few days after hatching re- sulted in reduction or absence of the primary antibody response (Mueller gt gt., 1962; Jankovié and Isakovié, 18 1966; Cooper gt gt., 1966a), and a partially active anamnes- tic production of IgM antibodies but no IgG antibodies (Claflin gt gt., 1966; Arnason and Jankovié, 1967). The development of natural haemagglutinins may be suppressed (Graetzer gt _t., 1963a, b; Mueller gt gt., 1964). Surgical bursectomy does not result in total agammaglobulinemia (Cooper gt gt., 1966a). Immunoglobulin levels in serum may be normal or show deficiency in IgG with increased IgM levels (Ortega and Der, 1964; Cooper gt gt., 1966a). This IgG deficiency may be apparent within the first few weeks after hatching (Meter gt gt., 1969), or may not become apparent until several months of age (Arnason and Jankovié, 1967). Since the discovery of chicken IgA, it has also been shown that IgA is deficient in serum of bursectomized chickens (Martin and Leslie, 1973). IgA production may be somewhat thymus-dependent in other animal species (Clough gt gt., 1971). Bursectomized chickens have a higher mor- tality due to diseases in which antibodies confer protection. These include diseases resulting from infections with Salmonella typhimurium (Chang gt gt., 1959), Leptospira icterohaemorrhagiae (Kemmes and Pethes, 1963, as summarized by Payne, 1971), Eimeria tenella (Challey, 1962) and Treggnema angerinum (Soumrov gt gt., 1967 as summarized by Payne, 1971). Bursectomized chickens have a higher suscepti- bility to virus-induced myeloblastosis (Baluda, 1967). 19 There is a reduction of massive tumor formation of avian lymphoid leukosis by bursectomy as late as 4 months of age (Peterson gt gt., 1964; Peterson gt gt., 1966; Cooper gt gt., 1968). It is thought that the target cells of neoplastic transformation by the virus are the bursal lymphocytes (Peterson gt gt., 1964; Peterson gt gt., 1966; Cooper gt gt., 1968; Purchase gt gt., 1968). Hereditary autoimmune thyroiditis characteristic of the obese strain of White Leghorns is decreased by bursec- tomy (Wick gt gt., 1970a). It is not resolved whether the thyroid damage is a result of antibodies being produced which are specific for thyroid cells and destroy them and/or whether the B cell infiltration of thyroid tissue which occurs, mechanically causes thyroid damage. Bursa—dependent organs of bursectomized chickens have varying degrees of lymphocytic depletion (Isakovié and Jankovié, 1964; Claflin gt gt., 1966; Cooper gt gt., 1966a; Arnason and Jankovié, 1967; Cooper gt gt., 1969). The thymus-dependent functions reported to remain in- tact in bursectomized chickens are homograft rejection (Warner gt gt., 1962; Papermaster and Good, 1962; Aspinall 'gt gt., 1963; Isakovié gt gt., 1963; Jankovié gt gt., 1963), delayed skin sensitivity reactions (warner and Szenberg, 1962; Jankovié and I§vaneski, 1963; Jankovié gt gt., 1963; Cooper gt gt., 1966a), GVH reactions (Mueller gt gt., 1964; 20 Warner, 1965; Cooper gt gt., 1966a), and resistance to cer- tain diseases requiring cellular immunity to confer pro— tection (Pierce and Long, 1965; Longenecker gt gt., 1966; Long and Rose, 1970). Because some bursa-dependent function persists follow- ing surgical bursectomy after hatching, x-irradiation to destroy bursal lymphocytes seeded peripherally prior to bursectomy has been used (Cooper gt gt., 1966a). Because the recommended dosage of irradiation is high enough to be lethal for 50 percent of irradiated chicks, alternative methods of achieving functional bursectomy have been de- veloped. Weber and Weidanz reported in 1969 successful function- al bursectomy resulted from point irradiation of the bursa soon after hatching. Bursa-dependent areas of the spleen and caecal tonsils were depleted of germinal follicles and plasma cells. Surgical bursectomy tg ggg at 17 days of incubation induces total agammaglobulinemia (Van Alten gt gt., 1965; Cooper gt gt., 1969) but it is more difficult than post-hatching bursectomy. Hormonal bursectomy (Meyer gt gt., 1959; Mueller gt gt., 1960; Aspinall gt gt., 1961) is the prevention of embryonic bursal development by the use of certain hormones. The embryonating eggs may be dipped in a solution containing nortestosterone or embryos may be inoculated. Survival of chicks may be low if large doses of hormone or use early in 21 embryonation cause abnormal cloacal development resulting in fecal impaction after hatching. Hormonal bursectomized chicks may have some degree of thymic atrophy (Warner gt gt., 1962). Even if hormonal bursectomy does not result in agammaglobulinemia (Carey and Warner, 1964; Warner gt gt., 1969), the antibody response is severely reduced (Warner gt gt., 1969). Hormonally bursectomized chickens were unable to reject homografts of spleen cell suspensions (Papermaster gt gt., 1962). They also reported poorly developed thymus glands in hormonally bursectomized chicks. Sherman and Auerbach (1966) reported that doses of l9—nortestosterone sufficient to completely inhibit bursa development also retarded thymic morphogenesis. Functional bursectomy can be induced by immunological destruction of immunoglobulin producing cells. Kincade gt gt. (1970) reported that injection of IgM anti-mu-chain serum tg ggg, and surgical bursectomy at hatching, resulted in long term reduction of serum IgM and IgG and absence of germinal centers in bursa-dependent peripheral lymphoid tissue. Injection of anti IgM serum at hatching with sur- gical bursectomy resulted in lower serum IgM. Surgical bursectomy at hatching, followed by several injections of anti-mu—chain serum resulted in complete suppression of detectable serum IgM, IgG and IgA (Kincade gt gt., 1973). Cyclophosphamide is an alkylating agent toxic to rapidly dividing cells and has been used as an anti-tumor 22 drug in certain human malignancies. Cyclophosphamide in- jected repeatedly prior to and immediately after hatching (Purchase, 1973) or used in combination with irradiation (McArthur gt gt., 1973) renders chickens totally agamma- globulinemic. Lymphoid development in the bursa is ar- rested and bursa weights and sizes are significantly smaller than normal (Seto gt gt., 1971; Toivanen gt gt., 1972a, b). Toivanen gt gt. (1972a, b) reported depletion of lymphocytes in thymus- and bursa-dependent areas in spleens of cyclophosphamide treated chickens. In Vivo Tests of Cellular Immunity tg gtgg, cellular immunity can be manifested and de- tected by several methods such as the tuberculin type de- layed skin reaction, GVH reactivity, and homograft rejection. Injection of an antigen intradermally into an animal sensitive to the antigen can cause a marked local reaction that is visible grossly as erythema and induration. The delayed type skin reaction must be differentiated from the antibody mediated Arthus reaction. High levels of circu— lating precipitating antibody are necessary for the develop- ment of an Arthus reaction. The formation of antigen- antibody-complement complexes is thought to initiate the local inflammatory reaction (Dvorak gt gt., 1970; Straus, 1972). In 1969 Eisen reported that antigen localized in 23 the polymorphonuclear leucocytes present at the reaction site. The Arthus type response starts about 2 hours after antigen injection, reaches a maximum by 5 hours and generally diminishes by 24 hours. A severe reaction may, however, persist for 24 hours or more, making it dificult to distinguish from a delayed skin reaction. Histological- 1y the Arthus reaction is characterized by an acute inflam- matory response with edema and leucocytic infiltration. Vasculitis, hemorrhage, and necrosis can occur if the reac- tion is severe (Jawetz gt gt., 1970). The tuberculin reaction is the classic example and best studied of the delayed type skin reactions. The skin reaction is first visible after a few and up to 10 or 12 hours, reaching a maximum at 24 to 48 hours; it is a delayed reaction. An inflammatory response is critical to the expression of the skin reaction (Koster gt gt., 1971). Histologically, the inflammatory response and concommitant perivascular leucocytic infiltration of a delayed type skin reaction is biphasic. The first and minor phase at 3 to 4 hours is characterized by predominantly polymorphonuclear cells. At 5 to 6 hours polymorphonuclear leucocytes emi— grate from the perivascular spaces. The second and major peak commencing at 8 hours is characterized by massive granulocytic cell and mononuclear cell emigration. By 24 hours, the polymorphonuclear leucocytes have emigrated and 24 the mononuclear cells remain immobilized around the blood vessels (Spector, 1967). The emigrating lymphocytes of the guinea pig appear to belong to a subpopulation with a characteristic for surface binding of Thorotrast particles (Wiener gt gt., 1971). Grossly the reaction may persist for some days. According to Eisen (1969) the antigen be- comes localized on the cytoplasmic membrane of macrophages when cytophilic antibody is present. The expression of the delayed skin reaction depends upon the presence of sensitive lymphocytes (Waksman gt gt., 1961; Hill, 1969) discussed below. Zakarian and Billingham (1972) reported that in leucopenic guinea pigs the ability to develop delayed skin reactions was impaired. The skin reaction occurs even in the absence of antibody (Holtzer and Winkler, 1967) and is immunologically specific. In the case of contact sensitivity to haptens, specificity of sensitivity was to the carrier protein and not the hapten (Gell and Benecerraf, 1961). Furthermore, both carrier specificity and strong delayed skin reactions resulted equally in guinea pigs sensitized with lightly or heavily substituted conjugates (Benacerraf and Levine, 1962). Delayed skin sensitivity can be passively transferred from a sensitive to a nonsensitive individual only by liv- ing (Salvin and Nishio, 1972) sensitive lymphoid cells or some of their extracts or products such as transfer factor, 25 not by serum (Landsteiner and Chase, 1942; Chase, 1945; Lawrence and Pappenheimer, 1956; Najarian and Feldman, 1961; Lawrence gt gt., 1963; Salvin and Garvin, 1964; Lawrence and Valentine, 1970b). In the guinea pig, passive transfer of delayed sensitivity as measured by the delayed skin reaction was successful with lymphoid cells of the spleen, lymph nodes and peritoneal exudate but not with thymus or bone marrow cells of the same donors (Salvin gt gt., 1970). The duration of delayed sensitivity trans- ferred in this manner varies among different animal species (Lawrence and Pappenheimer, 1956). In passively sensitized animals, a large proportion cf mononuclear lymphoid cells at the reaction site are of host origin (Najarian and Feld- man, 1961; Turk, 1962; Oort and Turk, 1963; McCluskey gt gt., 1963) and probably originate in the bone marrow (Lubaroff and Waksman, 1967, 1968). Kay and Rieke (1963) reported numerous donor lymphoid cells were present at the reaction site only if skin testing was done immediately following cell transfer. The mechanism whereby a few sensitive lymphocytes can impart cellular sensitivity, or immunity, as demonstrated by a delayed skin reaction is not resolved. Sensitive lym— phocytes may impart an informational molecule to host lymphocytes, similar to the mechanism, via RNA transfer, for passive transfer of antibody production (Abramoff and 26 Brien, 1968b). These substances may stimulate or instruct host lymphocytes to divide and give rise to a clone of sensitive host lymphocytes. The passive transfer of cellu- lar immunity by leucocyte extracts and transfer factor (Lawrence, 1955; Lawrence and Pappenheimer, 1956; Jureziz gt gt., 1970; Paque and Dray, 1970; Rosenfeld gt gt., 1972) could support either hypothesis. Indeed, the mono- nuclear cells at the reaction site in passively sensitized guinea pigs have undergone recent proliferation (Spector, 1967). Alternatively, antigen may react with a few sensi- tive lymphocytes which in turn synthesize and release bio— logical effector molecules which initiate a wide spectrum of nonspecific effects. These then may nonspecifically cause the subsequent cellular emigration, infiltration and mononuclear cell stasis at the site of the skin test. Injection of antigen intraperitoneally leads to a disap- pearance of macrophages in sensitive guinea pigs and may be analogous to the tg_ztttg effect of migration inhibition factor (MIF) upon macrophages (Sonozaki and Cohen, 1971). MIF, or a skin reactive factor which may be associated with MIF produced tg gtttg, can cause skin reactions when injected intradermally (Bennett and Bloom, 1968; Pick gt gt., 1971). A shorter time is required for the skin reaction. Histologically it is identified as being the delayed type. Another alternative for the mechanism of passive transfer of 27 sensitivity is that sensitive lymphocytes may induce SYn- thesis of specific antigen receptor sites on nonsensitive T cells, converting them to sensitive T cells (Levin gt gt., 1973). Intradermal testing of chickens is difficult because the site generally used is the lateral aspect of the wattle (Karlson, 1972). As an alternative site to the wattle, the dermis of the metatarsal foot pad can be used. The dermis of the wattle of a young male chicken is approximately 0.45 mm thick (Lucas and Stettenheim, 1972). The outside di- ameter of a 26 gauge % inch needle, recommended for avian tuberculin testing, is 0.45 mm (Becton-Dickinson, 1964, Appendix 1). There are no published reports of the dermal thickness of the foot pad (Lucas, 1973), but intradermal injection of the foot is easier than of the wattle. Besides the Arthus type reaction, another type of skin reaction should be differentiated from the tuberculin type delayed skin reaction. The Jones-Mote reaction, or more recently termed cutaneous basophilic hypersensitivity (Bast gt gt., 1971), can be passively transferred with sensitive lymph node cells, not serum, requires protein or protein- antibody conjugates for induction of sensitivity, and skin reactions are delayed in onset without a persistent indura- tion. Any induration is transient and can be elicited for only a few weeks after initial sensitization. The polymor- phonuclear infiltrate at the site of the skin test consists 28 mainly of basophils. There is no detectable MIF produced by sensitive lymphocytes from guinea pigs with cutaneous basophilic hypersensitivity. Bast and his associates (1971) stress that a true delayed hypersensitivity can be induced experimentally only with complete Freund's adjuvant (CFA) or the equivalent. The wax D portion of the high lipid content of the Mycobacterium genus contained in CFA reportedly induces inflammation required for development of delayed sensitivity (Hiu and Amiel, 1971). Immunologically competent cells responsible for trans- plantation rejection are operationally defined as those cells which are capable of initiating a GVH response. Immunologically mediated homograft rejection is primarily cell-mediated (Eddteston gt gt., 1969; Falk gt gt., 1970; Ferraresi gt gt., 1970; Starzl gt gt., 1970; Dormant gt gt., 1972; Hellstrdm and Hellstrdm, 1972). Histologically in— compatible donor cells with this capability react immuno- logically to the recipient's tissues when inoculated into an immunologically incompetent recipient, and initiate a wide variety of pathological damage to the recipient. The severity of the reaction is determined by the size of the inoculum, the age of the donor, the cell type within the inoculum, the animal species test system, the age of the recipient and degree of incompatability between donor and recipient. The reaction of competent donor cells upon time recipient may cause death, splenomegaly, hepatomegaly, 29 anemia, pock formation on the chorioallantoic membrane of the embryonating chick, runting, or a combination of these (Simonsen, 1962). In contrast, if immunologically mature cells are inoculated into an immunologically competent non- isogenic recipient, the transplanted cells may in time be rejected. The rejection is by the recipient's immunologic mechanisms (Solomon, 1963). The route of the administration of the cells which initiate GVH reactions is not critical. The cells appear to "home" to organs that normally contain leucocytes (Simonsen, 1962). An intravenous injection results in more generalized and rapid pathologic damage. It has generally been assumed that the destruction of host tissue is de- pendent upon proliferation of donor cells (Simonsen, 1962; Warner, 1964; Longenecker gt gt., 1970), and a proliferative response by lymphocytes appears necessary for the expression of GVH (Seto, 1968a). On the basis of characteristic GVH membrane lesions, Delanny and Ebert (1962) challenged this theory. Recent evidence shows that the recipient actively contributes to the ensuing pathologic lesions (Walker gt gt., 1972; Hartmann and Fisher, 1973). Killby and her associ- ates (1972) proposed that, in the embryonating chick, the grafted immunocompetent lymphocytes provide the stimu- 1us, presumably via RNA transfer, to cause the ensuing [proliferative response by recipient hemopoietic stem cells. 111w proliferation of recipient, rather than or in addition 30 to donor cells, contributes to the pathologic damage of GVH disease (Walker gt §;., 1972). Induction of GVH reactions in 6-8 week old Fl’hybrid mice by injection of either of the parental strain's whole blood stimulated the recipients' spleen cells to become cytotoxic (Singh gg gl., 1972). They postulated that the cytotoxicity was both specifically directed by activated donor cells in the process of sensi- tization against host target cells and nonspecifically directed by host cells at damaging other host cells. Identification of the cell type which is immunologically competent in initiating GVH disease is of practical im— portance both in homotransplantation and in immunologic re- constitution of host deficiencies in immunocompetent cell populations. It has been established that where thoracic duct lymphocytes are used, a good correlation exists between the number of lymphocytes, particularly small lymphocytes, and the severity of GVH reactions in rodents. The cells collected from the thoracic duct are generally lymphocytes, 95% of which are the small long-lived recirculating type (Simonsen, 1962). The chicken has not been shown to have highly function- al lymph channels analogous to those of mammals and conse- quently whether long and short-lived lymphocytes exist is not known. Splenomegaly in embryonating chicks can be in- duced with as few as 30 blood lymphocytes (Terasaki, 1959) 31 but not by monocytes or red blood cells. The lymphocytes active in GVH reactions cannot be identified on the basis of morphology or bouyant density (Szenberg and Shortman, 1966). The immunologic basis for GVH disease is the cell— mediated mechanism (Potworowski gE_gl,, 1971), but a humoral response can occur and contribute to rejection. Porter (as summarized by Simonsen, 1962) reported that grafted immuno- logically competent cells in rabbits produce humoral anti— bodies against the recipients blood group factors and postulated that this accounted for the resulting anemia often associated with GVH disease. Antibody production by donor cells, restimulated with antigen at the time of in— jection into a recipient, has been demonstrated in the chick embryo (Seto, 1970a). T cells are believed to be the type of lymphocyte that initiates the GVH reaction (Cooper gg_gl., 1966a). The GVH reaction can be nullified or retarded by injecting the recipient with antibody to the donor tissues or by adding isologous (recipient type) adult spleen cells (Simonsen, 1962) to the inoculum to cause homograft destruc- tion. The genetic contribution to GVH reactivity is deter' mined by transplantation or histocompatability antigens on clones of immunocompetent lymphocytes (Burnet, 1960, 1961, 1962 as reviewed by Simonsen, 1962; Simonsen, 1962). When transplanted to a non-isogenic recipient the genetically 32 determined antigenic differences of the recipient are recog- nized. This recognition must precede the ensuing prolifera- tive response and subsequent destruction of the host or its organs. The GVH reaction is therefore a primary immunologic response requiring no known prior contact of competent cells with the foreign histocompatability antigens. Prior contact of the donor cells ig_yiyg_or $2.!iEEQ with the recipient's transplantation antigens can accelerate the GVH reaction; however, the increase in severity is inversely related to the strength or dominance of the transplantation antigens, i.e., the degree of antigenic dissimilarity of donor and recipient cells (Nielsen, 1972). The significance of the transplantation antigens in the GVH reaction was established in the mouse system and the major ones reside at the H-2 locus (reviewed by McDevitt, 1971). In chickens, the B blood group locus appears to be a major locus determining histocompatability (Longenecker ggflgl., 1973). It is possible that thymic-dependent functions which include homograft rejection (Miller, 1962; Eddteston g2_gl,, 1969; Falk g£_gl., 1970; Ferraresi 23.31., 1970; Starzl gg'gl., 1970; Dormont gg‘gl., 1972; Hellstrom and Hellstrom, 1972) and the GVH response are mediated by functionally dif- ferent T cell subpopulations. There are subpopulations of T cells in mice with differing sensitivities and/or func- tionally restricted potential. The lymphocyte population 33 which mediates cytotoxicity as measured i3 ZEEEQ is not the same one responsible for GVH reactivity (Mage and McHugh, 1973). Cytotoxicity increased following immunization if there was a strong histoincompatability. This was shown by selective ig.yit£g_binding and cytotoxicity by nonadherent small lymphocytes to allogeneic target cells grown in mono- layers. The nonbinding lymphocytes retained GVH reactivity but had no further cytotoxicity for additional target cells. It is not clear whether this indicates a difference between functionally differentiated (Sprent and Miller, 1970) and nondifferentiated T cells or in different functional capa- bilities of different T cell subpopulations (Bach and Brashler, 1970; Anderson ggwgl., 1972; Stites gg_gl., 1972). The capability to initiate the GVH reaction as well as homograft rejection in the chicken is thymus-dependent (Cooper ggjgl., 1966a). The manner in which the thymus exerts its influence is not known. The blood lymphocytes increase in GVH immunocompetence with age and reach a ‘ plateau by 4 weeks of age (Seto, l968k». Thymic lymphocytes generally initiate GVH reactions less effectively than lym- phocytes from peripheral blood. Seto (1966) and Droege and his associates (1973) reported a higher proportion of thymus cells were GVH competent in older chickens than in young chicks. This GVH reactive population was characterized by high electrophoretic mobility recovered from low speed 34 centrifugation fractions of thymus cell suspensions (Droege ggygl., 1973). The most sensitive means of assaying GVH reactions in the chicken is by measuring spenomegaly (Seto, 1966). Furthermore, intravenous injection resulted in greater splenomegaly than grafting techniques and the age of great- est sensitivity of the recipient was l3-l4 days embryonation (Seto, 1966). No reports were found in which cell populations from thymectomized or bursectomized chickens were tested for GVH reactivity as well as for immunologic function by one of the i3 yiE£g_correlates of delayed sensitivity. Tests and Correlates of Cellular Immunity Several ig_!i5£g_correlates of delayed sensitivity have been reported. They will not be individually reviewed, but only enumerated to aid in reference. Incubation of sensi— tive lymphocytes with the sensitizing antigen can yield in addition to MIF other so-called biological effector mole- cules or lymphocyte factors which affect the behavior of macrophages, polymorphonuclear leucocytes, or in the case of interferon, other cells susceptible to infection by the same virus. These factors include a leucotactic factor for mono- nuclear cells (Ward and David, 1969; Ward gg_gl., 1970). If antigen-antibody complexes are added to supernatants, a 35 chemotactic factor for eosinophils may be produced (Cohen and Ward, 1971; McGarry 22.2l'r 1971). Other factors pro“ duced ig|zi3£g_are macrophage aggregation factor (MAF) (Lolekha gg.gl., 1970), macrophage activating factor (Mooney and Waksman, 1970), cytotoxic factors (Granger and Williams, 1968; David and David, 1972), cloning inhibitory factor (Lawrence and Landy, 1969), skin reactive factor (Bennett and Bloom, 1968), mitogenic factor (Valentine and Lawrence, 1969), interferon (Green gg'gl., 1969; Milstone and Waksman, 1970), antibody (Tsuchimoto ggygl., 1972; Meyers gg‘gl., 1972), and transfer factor (Lawrence and Valentine, 1970a). The characteristic spreading of macrophages 12_ZEEE2 on glass and plastic surfaces may also be inhibited (Fauve and Dekaris, 1968), but a name for a factor has not been given. The species in which some or all of the above have been demonstrated are humans, guinea pigs, mice, chickens, rats and rabbits. The biologic effector molecules listed above (with the exception of MAP, Nathan g3_gl., 1971) may or may not all be produced simultaneously, or under identical cul- ture conditions, and appear to differ functionally and physiochemically from one another. An additional ig_yi§£g_assay, the mixed lymphocyte re- action (MLR) differs from those listed above. It is not a test which ascertains if sensitization has occurred, even though during the MLR effector molecules such as mitogenic 36 factor (Gordon and MacLean, 1965) and MIF (Bartfeld and Atoynatan, 1970) may be produced. The MLR tests for a primary reaction between genetic- ally distinct cell populations (Maclaurin, 1972; Wagner gt 31,, 1972). In the chicken, thymic medullary lymphocytes and splenic lymphocytes are equally active in the MLR (Weber, 1970). When incubated ig’yiggg_with antigen for several days lymphocytes from animals with cellular immunity undergo a cell transformation or proliferation detected by observa- tion of morphologic changes (Mills, 1966) or by tritiated thymidine uptake. The blastogenic or proliferative response requires the presence of macrOphages (Hersh and Harris, 1968). A proliferative response may also occur by B lympho- cytes stimulated lg yiggg with antigen. Whether the pro- liferative lymphocyte response is that of B or T cells is antigen dependent (Jacobs ggygl., 1972). A proliferative response not necessarily immunologically specific can be induced experimentally with substances such as phytohaemag- glutinin (PHA) and keyhole limpet hemocyanin (KLH). PHA has mitogenic erythroagglutinating and immunogenic properties (Markley 2E.2l°r 1972). Tissue explants from.sensitive chickens and/or guinea pigs (Aronson, 1931 and 1933; Rich and Lewis, 1932; Fabrizio, 1952) were early ig_vitro models of inhibition of cell 37 migration in the presence of the sensitizing antigen. Subse- quently, oil-induced peritoneal exudate cells (PEC), con- sisting of macrOphages and lymphocytes were incubated in capillary tubes with antigen added to the culture medium (George and Vaughan, 1962). Migration of PEC out from capillary tubes is inhibited if lymphocytes are from guinea pigs sensitive to the antigen, not from controls. Antigens unrelated to the sensitizing antigen or ones that induce only antibody production cause no release of MIF from lym- phycytes as detected by inhibition of migration of PEC (David gg_gl,, 1964a, c; David and Schflossman, 1968). Soluble as well as particulate antigens have been used‘ig. yg££g_(Carpenter, 1963; Al-Askari gg_gl,, 1965; David and Paterson, 1965; Malloy gg_gl., 1972). In the presence of the appropriate antigen, lymphocytes from sensitive guinea pigs release a factor, MIF, which in- hibits the migration of monocytes ig_yi§£g_(Bloom.and. Bennett, 1966, 1968; Bloom and Jimenez, 1970). This factor inhibits the migration of macrophages from sensitive (direct MIF test) and nonsensitive (indirect MIF test) guinea pigs. The MIF produced by human lymphocytes inhibits guinea pig macrophage migration (Rajapakse and Glynn, 1970), an indie rect test for MIF. In addition to inhibition of macrophage migration, lymphocyte migration in the presence of high amounts of serum.may also be inhibited in migration in 38 guinea pigs (Halpern gt gl., 1967). But this has been chal— lenged by Salvin EE.E$' (1971). Only a few lymphocytes from a sensitive animal are necessary to detect the production of MIF (David g£.gl., 1964b). The synthesis of MIF can be blocked i2 yiggg by chemical blockers of protein synthesis (David, 1965). It does not exist preformed in the lympho— cytes. Whereas antigen is reportedly necessary to stimulate ig_yiggg_MIF synthesis by sensitive lymphocytes, the action of MIF on macrophages from a nonsensitive individual (indirect assay) does not require additional antigen (Yoshida g§_gl,, 1972). Sensitive lymphocytes in the MIF test require a given amount of antigen to continue elaborate ing MIF. Escape from migration inhibition by macrophages can occur after prolonged iggyi3£g_culture but inhibition can be restored by addition of more antigen (David'gg;gl., 1964b; Nathan gg.gl., 1971). The action of MIF on macrophages may require cytophilic binding of MIF to macrophages. Bartfeld and Atoynatan (1971) were able to block migration inhibition of MIF by treating macrophages with N—acetylcysteine which inactivates the bind— ing site for MIF on the heavy chain of guinea pig macro- .phage-associated cytophilic antibody. In guinea pigs, delayed skin reactions can be elicited sooner after'sentie tization than can i5 yiE£g_MIF production be detected (Ferraresi gg_gl., 1969). 39 There is no method yet to distinguish single lympho- cytes of a given sensitivity (Gowens and McGregor, 1965). However, sensitive T cells support RNA virus replication (Bloom gg_gl., 1970; Bloom, 1971). Currently, the use of the term sensitive lymphocytes indicates lymphocytes from an animal with delayed sensitivity, without inference that all the lymphocytes present are sensitive to a given anti- gen (David and David, 1972). T lymphocytes, in general, can be identified in humans, rabbits, guinea pigs, rats and mice by their characteristic light refringence (Pompidou and Schramn, 1971) and in certain species by the presence of a characteristic T antigen (Potworowski and Nairn, 1967; Owen and Raff, 1970; Malchow SE.El-v 1972; Jacobs ggpgl., 1972). T cells have not been shown to bind antigen by means of cell-associated antibody. This property is used to identify B cells (Wigzell and Andersson, 1969). However, immuno- absorption at 37 C on mouse fibroblasts by rat lymphocytes via their receptor to strain specific antigens on the mouse fibroblasts has been reported to be a property of T cells in rats (Wekerle g3.gl., 1972). These specifically immune T cells have a short life span and a rapid turnover in mice (McGregor g2 g1,, 1971). Sensitive lymphocytes from PEC, lymph nodes, blood, and spleen of many mammals elaborate MIF (Salvin gg.gl., 1970; Winkelstein, 1972), whereas thymus and bone marrow cells do 40 not (Winkelstein, 1972). Spleen cells from sensitive chick- ens elaborate MIF (Morita and Soekawa, 1971; Zwilling g3_gl., 1972). Peripheral blood has been used as the source of sensitive lymphocytes from man (Clausen and S¢borg, 1969; Kaltrieder gg_gl., 1969; Mookerjee gg_gl., 1969; Bendixen and S¢borg, 1970; Tarnvik, 1970; Clausen, 1971) and the chicken (Fauser ggIgl., 1973a; Fauser gg_gl., 1973c). lg yiE£g_culture of blood leucocytes is more difficult than culture of leucocytes from other sources. Serum used in culture is reported to be one critical factor. Although serum free medium is available for ig.ziggg culture of chicken leucocytes (weber, 1970), it cannot be used success- fully if sensitive cells are concommittantly stimulated with antigen (Kirchner and Oppenheim, 1972). Peritoneal exudates, or chicken abdominal exudates, are difficult to obtain and peripheral blood is not. Another advantage of using blood experimentally as compared to spleen cells (Morita and Soekawa, 1971, 1972) is that the same animal can be used repeatedly, as his own control, before and after the induction of delayed sensitivity. Techniques other than explants and capillary tube migrations have been reported (Salvin and Nishio, 1969; Smyth and weiss, 1970; Mallmann gt_gl., 1971; Houck and Chang, 1973; Harrington and Stastny, 1973). These methods utilize, in one form or another, a monolayer or droplet of lymphocytes and macrophages or both to which various test 41 antigens and culture medium are added. Serum, but not com- plement, is required in most of these assays (George and Vaughan, 1962; Bennett and Bloom, 1968; David and David, 1972). Lymphocyte function(s) in delayed sensitivity is(are) not completely understood. The sensitivities and conditions necessary for elicitation of a response i2.!i££2 as compared to ig_yi!g vary (Thomas gg_gl,, 1971). If the various ig' giggg correlates of cellular immunity are duplicated iglyiyg, several ig_gigg_phenomena may possibly be explained. These phenomena include the following: (1) the reticuloendothelial systems activation and mitosis of macrophages (North, 1970) following infections which lead to delayed type sensitivity (Mackaness, 1971; Youmans, 1971), (2) the presence of re- cipient cells not donor cells, at the site of a skin test in passively transferred delayed sensitivity, and (3) the accumulation of mononuclear cells which give rise to granu- lomas and/or tissue destruction (Ruddle and Waksman, 1967) in certain diseases caused by intracellular parasitism. Very few specifically sensitive lymphocytes are requir- ed to affect and involve, by a mechanism or means not yet fully understood, many nonsensitive cells to participate in and cause many nonspecific reactions which are nonetheless immunologically specific both in induction and memory (McGregor gg.gl., 1971). This amplification of immunologic— ally activated responses, known collectively as cellular 42 immunity, and independent retention of immunologic memory is an obvious advantage to biological specialization and conservation. Cell-mediated Immunity and Its Clinical Manifestations Cell-mediated immunity which is mediated by and resides in the sensitive lymphocyte is involved directly and in- directly in many disease processes. It is manifested by both protective and untoward reactions. The clinical mani- festations of cell-mediated immunity are so varied that only recently, with the advent of ig.yi3£g tests, have the breadth and complexity of T cell-mediated reactions become more apparent. It has been recognized since the time of Koch (Mack- aness 1964, 1969, 1971), that delayed hypersensitivity, exemplified by the tuberculin type skin reaction, results from intracellular infections with many fungal, bacterial and protozoan agents. The various i3 yiggg correlates of cellular immunity have begun to enumerate the physiologic repertoire of reactions associated with cellular immunity. Functional and/or structural defects in the T cell in immunologic deficiency diseases may be associated clinical- ly and be responsible for increased susceptibility to recurrent or chronic infection with certain group of microorganisms(Chilgren gt 11., 1969; Mendes and Raphael, 43 1971). Many so-called autoimmune diseases, both naturally occurring and experimentally induced, are cell-mediated (Jankovié and Mitrovic, 1963; David and Paterson, 1965; Brostoff gg’gl., 1969; Behan gg‘gl., 1970; Werdelin and McCluskey, 1971). It has been proposed that in man heavy irradiation of the thymus gland during childhood predisposes to the develop- ment of neoplasms (Janower and Miettinen, 1971). Cellular immunity to tumor Specific antigens has been shown in natur- ally occurring and experimentally induced tumors (Kronman g£_gl., 1969; Sojogren and Borum, 1971; WOlberg, 1971; Churchill g£|gl., 1972) and regression of certain human neoplasms by a cell-mediated process has been reported (Stjernsward and Levin, 1971; DiSaia gg.gl., 1972). Intradermal injection in guinea pigs of MIF with hepatoma tumor cells suppressed local tumor formation (Bernstein g2 g1., 1971). Mice infected with leukemia virus and sensitized with CFA had no detectable MIF ig’zi3£g_(Friedman. and Ceglowski, 1971). Similarly, Hodgkin's disease patients have decreased MIF production ig_yiE£g_(Churchill g2_gl., 1971). Antibody may block the cytotoxic effect of immune T lymphocytes by a process referred to as immunologic enhance- Jnent, and may mask a functionally capable cell—mediated immunity (Hellstrom and Hellstrom, 1972). The antibody 44 coating the target (tumor) cells prevents the cell-mediated destruction by T lymphocytes (Prehn, 1971). The immune system may be functionally depressed secon- darily by infections (Notkins gg_gl., 1970; Purchase gg‘gl., 1968) or the cells of the immune system may be infected. Indications are that the lymphoid leukosis virus of chickens replicates within the cells of the bursa prior to its spread systemically. Surgical bursectomy prevents the cycle of the infection (Peterson g3 g1., 1964). The etiologic agent of Marek's disease of chickens is a group B virus which is a cell-associated herpes virus. The disease is lymphoproliferative (Calnek and Witter, 1972). Whether the disease is primarily neoplastic or in- flammatory in nature has been widely debated (Purchase, 1972). Marek's disease (MD) infected chickens have increas- ed quantities of circulating globulin (Purchase, 1972) and maternal antibodies appear to confer protection against naturally occurring spread to young chicks (Calnek and Witter, 1972). General immunologic function is depressed as determined by susceptibility to coccidia, and decreased antibody titers (Burg g3 g1., 1971) with concommitant ele- vated gammaglobulin levels (Calnek and Witter, 1972). Neither thymectomy or bursectomy prevents the disease. Chickens with MD are reported to have fewer thymus-dependent lymphoid areas in the Spleen (Evans gg gl., 1971). 45 Gonadal tumors in MD infected chickens consisted of T cells and there is a B cell lymphocytosis in the blood (Hudson and Payne, 1972). The presence of T lymphocytes in MD tumors has been confirmed and extended to include tumors of the spleen, ovary, and nerves as well as blood (Rouse g£.gl., 1973). MD viral antigens induce delayed hypersensitivity skin reactions and MIF production £2.2iEEQ (Dawe gg.gl., 1971; Fauser gg'gl., 1973a; Fauser gg_gl., 1973c). It is not re- solved if the pathogenesis of MD is partially autoimmunity (Rouse gg,gl., 1973), if cellular immunity has a role in the natural age regression of MD lesions (Calnek and Witter, 1972), or if the T cell response has any significant role in MD. The induction of cell-mediated immunity is one possible means for prevention or treatment of diseases in which anti- bodies are not protective. It can be by vaccination and/or passive transfer of immunity by sensitive lymphocytes, their extracts, or products (Lawrence, 1955; Bernstein gg'gl,, 1971; DiSaia gg‘gl., 1972). Vaccination with Bacille Calmette-Guerin (BCG), for tuberculosis of man, a disease in which antibodies do not confer protection, is done routinely in many countries. The passive transfer of cell-mediated immunity is still experimental (Levin gg'gl., 1973). Whether the partial protection given by BCG is specific is not resolved. One possibility is that the sensitive 46 lymphocytes, established by BCG vaccination, when stimu- lated i3 xiyg_with antigen, recruit effector cells such as macrophages to the foci of infection (Mackaness, 1971). The macrophages in turn can sequester and kill intracellular parasites. Another suggestion has been that the inflamma- tory reaction, which is initiated and maintained by BCG infection, may augment resistance by nonspecifically stimu- lating macrophages to a hyper-reactive state (Dumonde, 1967; Melnick, 1971). This in turn may enable macrophages to destroy tubercle baccilli, nonrelated organisms, and, perhaps even neoplastic cells. It is not known how vaccination of chickens with herpes- virus of turkeys (HVT) protects against Marek's disease. Protection may result by a nonimmunologically mediated mechanism(s) or by immunologically mediated humoral and/or cellular immunity. The host parasite relationship between the virus and infected cells of MD can serve as a model for the study of other infections of a chronic nature as well as neoplastic diseases of mankind. It is worthwhile to determine whether cell-mediated immunity results from in- fection with MDV. The chicken model is suitable for studying cell- mediated immunity and humoral immunity singly. Of specific interest is the perfection of a technique, suitable for use with chickens, to study cell-mediated immunity ig_vitro. 47 Whether MIF production is thymus-dependent can be tested. There are reports of MIF production by chicken leucocytes (Morita and Soekawa, 1971; Zwilling gg.gl., 1972). Suffi- cient numbers of leucocytes for ig.ziggg assays have been obtained from spleens. In the absence of abundant numbers of lymph nodes, blood is a more feasible source of leuco- cytes for repeated MIF assays. There are a number of ad- vantages to adapting the MIF procedure for use with blood leucocytes. Each chicken can be used as its own control, reducing the size of experimental error due to individual chicken differences. Leucocytes can be tested for MIF production prior and subsequent to sensitization. Addi- tional assays for T cell function, namely skin reactions and GVH reactions, can be used as ig_yizg tests for sensi- tive T cells in the chicken. Because BCG as a vaccine stimulates cell-mediated im- munity to tuberculosis of man and it is known that it in- duces as least delayed sensitiVity in chickens, it can be used as a positive control for cell-mediated sensitivity in the chicken as well. BCG is not pathogenic for chickens, but can cause a severe inflammation and delayed sensitivity. The MDV is less pathogenic for chickens after they are 8 weeks of age (Witter g5 g1., 1973). MDV infection can be induced in the adult chicken without high mortality. The discovery that the bursa of Fabricius in the chicken is the central lymphoid organ for the immunoglobulin 48 producing system has stimulated the subsequent search for the mechanisms underlying the establishment of and modus operandi of the thymus-dependent cell-mediated immune system. MATERIALS AND METHODS ChickenS‘and Chick Embryos The White Leghorn chickens used in all experiments were either Fl hybrids of Regional Poultry Research Laboratory Line 15x7, offspring of line 6x7 dg‘crossed with line 6 $2, or embryos of line 15, subline l and 4 (Stone, 1974). Collection of Blgod for Tissue Culture. and Test Serums Serum used in tissue culture was separated from blood of specific pathogen free (SPF) chickens of varying genetic background. Chickens had been maintained separately for genetic studies and blood was obtained after their SPF status was confirmed (Stone, 1974). Chickens were exsanguinated by cardiac puncture. Blood was conveyed by needle, plastic tubing, and a vacuum pump from the heart to glass bottles which were laid on the side until the blood clotted. Bottles were shaken to dis— lodge the clot, and stored at room temperature overnight. The following day, fluid was decanted, pooled, and centriv fuged at 1,000xg for 15 minutes. The serum was decanted from the sterile centrifuge tubes and filtered through a 49 50 0.45 p Millipore filter (Millipore Filter Corp., Bedford, Mass.). The serum was tested for sterility by incubating 0.5 ml serum in 4.5 ml each tissue culture medium (a 3:5 ratio of F10, M-l99) (Appendices 2 and 3) and tryptose phos- phate broth for one week. Only sterile sera were used in tissue culture. Serum was dispensed in either 20, 30 or 45 ml samples in sterile plastic screw cap tubes and stored at —20 C un- til used in tissue culture medium. Throughout each experi- ment serum from the same pooled lot was used. Blood, used as a source of leucocytes and test serum, was taken from the brachial wing vein with syringe and 22 gauge 1 inch needle. Test serum samples were separated from 2 1/2 to 5 cc of blood. Viable leucocytes were separated from 10 ml brachial wing blood collected into 0.05 cc heparin (sodium heparin, USP 1,000 units/ml, Fellows Medical Manufacturing Incorpor- ated, Oak Park, Mich.) with a 10 cc syringe and 22 gauge 1 inch needle. Experimental Sensitization Chickens were sensitized by injection with viable BCG in CFA, CFA, and/or HVT. Those sensitized with BCG were injected subcutaneously with a total of 1 ml containing 5 mg wet weight viable BCG in an equal amount with incomplete 51 Freund's adjuvant (IFA) (#063760, Difco LabOratories, Detroit, Mich.). CFA (Control 516160 Difco Laboratories),a total of 1 ml in an equal amount with sterile saline, was injected subcutaneously. Cloned GA strain of MDV (Purchase‘gtigl., 1971) was injected subcutaneously at a dose of 7x103 plaque 5 forming units (PFU). HVT, 1.4x10 PFU, was injected sub— cutaneously. lg Vng'Techniques A. Thymectomy Thymectomy was performed within 24 hours after‘hatche ing. The chicken was anesthetized with 0.05«0.08'cc Com— buthal (Diamond Laboratories Inc., Des Moines, Iowa) administered intra—abdominally. An incision approximately 4 cm long was made on the dorsal surface of the neck and each lobe of the thymus, with surrounding connective tissue and fat deposits removed by blunt dissection. The inciv sions were closed with Michel wound clips (Propper Manu— facturing Co., Inc., Long Island City, New York) which.were removed 3 weeks later. Aseptic techniques were used. Antibiotics were not administered postoperatively. B. Bursectomy Bursectomy was performed within 24 hours after hatch“ ing under anesthesia described above. An incision 52 approximately 2 cm long was made immediately dorsal to the cloaca and ventral to the tail. The bursa of Fabricius was dissected from the surrounding connective tissue and removed at the stalk. The incision was not closed and antibiotics were not administered postoperatively. Aseptic techniques were used. C. Graft-versus-host Assay Leucocytes were purified by the method described below in Leucocytes for Migration Inhibition Test except that they were kept in sterile plastic tubes at 4 C, not attached to petri dishes. On the basis of the total mononuclear leucocyte counts using Trypan Blue dye (Grand Island Bio- logical Company, Grand Island, New York) in phosphate buf- fered saline (PBS) diluent, the final volume was adjusted to contain 106 mononuclear leucocytes/0.05 cc. Embryos of fourteen day embryonating eggs (line 15 subline l and sub- line 4) were used as recipients. The chorioallantoic vein was located with the aid of a candler and its location marked on the shell. The shell, but not the shell membrane, was cut with a small electric disc saw and removed from the shell membrane with a sterile 26 gauge 1/2 inch needle. The eggs were stored, cut surface facing upward, for at least 20 minutes to allow the embryos to become relatively quiescent. Thereafter, the exposed shell membrane was covered with sterile mineral oil to increase transparency. 53 One million viable mononuclear leucocytes were injected in a 0.05 cc volume of complete tissue culture medium into the chorioallantoic vein. Thirty gauge l/2 inch needles were used to inject donor leucocytes, with the direction of the blood flow. A momentary clearing of the contents of blood from the vein aided in visually ascertaining the success of intravenous injection. Leucocytes from each donor were injected into a minimum of 10 embryos. The embryonating eggs were incubated in a single stage Jamesway incubator at standard operating conditions regarding temper- ature, humidity, air flow and rotation (Jamesway Incubators, Appendix 4). Nineteen day embryonating eggs to be assayed for GVH splenomegaly were removed from the incubator and stored overnight at 4 C. The intact spleens were removed from embryos. Each spleen was blotted with dry filter paper and stored individually in plastic preweighed weighing boats. Five weighing boats were placed in covered plastic petri dishes containing moisture-saturated filter paper. The weighing boats containing spleens were weighed individually within minutes of harvesting on a Mettler balance. weights were recorded to four decimal places. D. Skin Testing Skin tests were by intradermal (ID) injection in one wattle of 0.05 ml solution of antigen in sterile PBS. 54 In one experiment a skin test with sterile PBS was performed one week prior to skin testing with Band-24 (B-24). The other wattle served as a control. The degree of swelling was observed at 20 minutes, and periodically thereafter at 2, 5, 24, 48, 72 and 96 hours post injection. The skin reactions were recorded on a relative scale from 0 to 4+. A reading of 1+ indicated that the induration and/or inflam- mation of the wattle was twice as thick and a 4+ indicated that it was five times as thick as the uninoculated control wattle. In Vitrg Techniqges A. Leucocytes for Migration Inhibition Test The procedure for the MIF test was an adaption of that described by Mallmann 93 g. (1971) . Ten m1 of heparinized blood was poured through glass wool (Corning Pyrex Brand 3950 Fibre Glass No. 7220, Corning, New York), centrifuged at 1,000xg for 20 minutes and the leucocyte rich buffy coat was drawn into sterile heparinized capillary tubes. The tubes were sealed with wax, centrifuged and broken at the cell-plasma interphase. The leucocytes were removed with a 8 cells/ml in syringe and 26 gauge needle and diluted to 10 tissue culture medium. Drops approximately 4 mm in diameter were placed on 35 mm diameter plastic petri dishes (Falcon Plastics, Los Angeles, Calif.). After 10 minutes the non- attached cells were rinsed off and 2 ml of antibiotic-free 55 Flo-M199 tissue culture medium (Witter gg_gl., 1969) con- taining 15% serum from SPF chickens instead of calf serum, and antigen as indicated, was added. Duplicate plates usually containing 8 spots per plate were prepared for each antigen and control culture. The diameters of the spots of attached leucocytes were measured with an ocular micrometer before and after 24 hours of incubation in a C02 incubator, 5% CO2 in air at 37 C. The distance migrated was calculated by subtracting the diameter of the spot prior to incubation from the diameter of each spot after incubation. Viability of cells inhibited in migration was shown by Trypan Blue exclusion. B. Antigens Old Tuberculin (OT, serial number 9537, USDA) was used undiluted, for skin testing at 145 mg protein in a volume of 0.05 ml and i_x; vi_t_:_:9_ at either 25 ug/ml or 50 ug/ml cul- ture medium. B-24, a protein isolated from culture filtrates of BCG (Roszman gg'gl., 1968; Fauser £2.2l-r 1969) (supplied by V. H. Mallmann, Assoc. Prof., Department Microbiology and Public Health, M. S. U., East Lansing, Mich.), was used. The skin testing dose was diluted in sterile PBS to contain 3.5 ug protein in 0.05 ml PBS. Ig_yi3£g}2 Hg protein/ml culture medium was used. Crude MDV antigen was prepared as previously described (Chubb and Churchill, 1968). The GA strain of MDV was 56 propagated in chick embryo fibroblasts in a mixture of FlO-M199 tissue culture medium supplemented with SPF serum. Crude MDV antigen was used for skin testing undiluted and EE.X£E£2 diluted 1/24 in tissue culture medium. Antigen A (Churchill g2pgl., 1969; Purchase, 1970) was obtained from Dr. P. A. Long prepared as previously described (Long, 1973). The DEAE sephadex A-25 eluate was. dialyzed against PBS and filtered through 0.45 p Millipore filter pretreated with chicken serum (Ver g£_gl., 1968). Antigen A was used undiluted, 0.05 ml/chicken, for skin testing. The antigen was used ig,yig£g at a 1/8 dilution, the dilution at which a strong precipitin line was found with standard MDV antiserum in double diffusion in agar coated slides. C. Antibodies Precipitating antibodies were detected by double diffusion in agar coated slides with a two-fold concentra- tion of second strength purified protein derivative contain- ing 0.01 mg protein (PPD, Parke-Davis, Detroit, Mich., #NDC 71-1298-1 Bio. 484) as antigen. A 1% agar was prepared in an 8% NA C1 solution and poured onto clean glass slides. Holes were punched in the solidified agar, filled with test sera or plasma, according to the method of Cho and Kramer (1970), and incubated in a humidified chamber for at least 48 hours to allow precipitin lines to develop. 57 The indirect fluorescent antibody method was performed according to the procedure described by Purchase (1970) and Solomon ggflgl. (1971). Chick kidney cells were grown on coverslips until a monolayer had formed (Calnek and Madin, 1969). Monolayers were infected with either HVT or MDV (GA strain). During primary foci formation, the coverslips were removed from the culture medium, washed in saline, and fixed in acetone. The air dried coverslips were stored at -20 C until used. The coverslips were divided by latex paint into 4 equal areas. Test serums, all SPF serums, and standard positive and negative control serums were randomly assigned by code, 4 per each coverslip. After incubation of cover- slips with a 1/20 dilution of coded test sera for one-half hour, in a humidified chamber, the coverslips were washed for 15 minutes in FTA Hemagglutination buffer (Bioquest, Cockeysville, Maryland). Thereafter, the buffer was removed, and coverslips were covered with fluorescein labeled horse anti-chicken serum and incubated one-half hour. They were then washed for 15 minutes with FTA buffer. The coverslips were rinsed in distilled water, mounted on glass slides with Elvanol (Polyvinyl Alcohol Grade 51-05, DuPont, de Nemours and Co., Wilmington, Delaware; Rodriguez and Deinhardt, 1960), monolayer surface down, and viewed in a fluorescence microscope. The degree of fluorescence was scored by a relative scale from 0 to 4+. 58 D. Bacteriological Techniqges Bacteriological techniques were used on liver and spleens from one lot of chickens to isolate acid fast bacilli. Fragments of the liver and spleen were ground, in a mortor with a pestle, in nutrient broth. An equal amount of 1N NaOH was added to the tissue homogenate and after 15 minutes at room temperature, was neutralized with 1N HCl. The samples were centrifuged and the sediment was seeded on 5 tubes each of Dubos oleic agar and Lowenstein- Jenson media (Difco Lab., Detroit, Mich.). The tubes were examined for growth on the first of each month thereafter. Before being discarded, acid fast stains were made on smears taken from the surface of the tubes to confirm no growth. Postmortem Examinations At the termination of each experiment, chickens were carefully examined for thymic remnants when appropriate and scored as normal thymus or as either complete or incomplete for thymectomy. All the viscera and brachial, sciatic and celiac nerves were examined for gross lesions indicative of MD. Viscera were examined for tuberculosis-like lesions when appropriate. Liver, spleen and serosa samples from all BCG and CFA inoculated chickens were collected, prepared for histo- ‘pathologic examination, and stained with new fuchsin-hema- toxylin-eosin (Willigan gg,gl., 1961) according to methods 59 used by Panigrahi (1970). Tissues were examined for acid fast bacilli, for the presence of giant cells, and granulo- matous lesions. Statistical Analyses Data were analyzed by the basic analysis of variance technique aided by the USDA-ARS data Systems Application Division. To accommodate missing data or unequal sample sizes, the least-square analysis (Kirk, 1968) was employed. Where possible, Bartlett's test for homogeneity of variance (program BMDP9D) was used. The analysis of leucocyte migration of the thymecto- mized or bursectomized chickens utilized a split plot model in a random design with repeated measurements of animals (Gill, 1971). Alpha levels, the probability of incorrectly inferring the existence of a true effect of treatment were established for each experiment to reflect the biologically justifiable expectations within each experiment and, unless otherwise specified, were alpha 0.05. Experimental Designs A. Experiment I The objectives of this experiment were (1) to determine if mdgration of leucocytes from control, CFA (containing (Mycobacterium butyricum) sensitized and BCG (M, bgyig) sensitized chickens are inhibited by two concentrations of 60 OT and by B-24 (prepared from BCG), and, (2) to determine the concentration of antigen needed to inhibit leucocyte migration but retain cell viability ig’yigrg, Day-old male chicks, line 15 x line 7 offspring, were identified by wing bands assigned randomly and housed in Horsfall/Bauer“stainless steel isolators (Hartford Metal Products, Inc., Aberdeen, Md.) at the United States Depart- ment of Agriculture Regional Poultry Research Laboratory (USDA-RPL) at East Lansing, Michigan. Leucocytes from the chickens, when two months old, and 2 months after sensitization, were tested for MIF pro- duction. Leucocytes from each chicken were cultured in duplicate plates for each, no antigen, 25 ug OT/ml culture medium, and 50 ug OT/ml culture medium. Twenty adult chickens were randomly assigned to one of three experimental groups. Serum samples were collected. One group of 8 chickens received viable BCG in IFA, another group of 8 chickens were injected with CFA and 4 chickens served as controls. No more than 4 adult chickens, each with the same inoculum, were housed in each isolator. Two months after sensitization, leucocytes were obtain- ed two times from chickens in all lots and tested for migra- tion inhibition with two concentrations of OT used in tissue culture. Two additional test plates with B-24 at 2 pg pro- tein/m1 culture medium were prepared from each chicken's leucocytes during the second test. 61 Three months after sensitization, all chickens were skin tested with B-24. One wattle was injected with 0.05 ml sterile PBS, as the control. Two weeks later skin test- ing was repeated with OT. At the termination of the experiment all chickens were bled for serum, and killed by intracardiac injection with air. The presence or absence of lesions involving internal organs was recorded. Samples of lesions, liver, and spleen were taken for histopathologic examination and bacteriologic reisolation. All serums were tested for precipitating antibody with PPD. B. Experiment II The objective of this experiment was to determine whether MD can induce delayed sensitivity to a soluble anti- gen of MDV. Six-month old chickens housed as in Experiment I, were bled for serum and their leucocytes tested for inhibition of migration with crude MDV antigen and OT. Each chicken was randomly assigned to one of three treatment groups, 6 chickens each. The three lots consisted of (1) Controls, (2) MDV in CFA injected, and (3) MDV injected chickens housed with noninoculated chickens to simulate conditions for the natural spread of MDV by contact exposure. 62 One to three months later, leucocytes from surviving chickens were tested for migration inhibition with crude and partially purified A-antigen preparations and OT. They were skin tested with both MDV antigens, and later with OT. At 11 months of age, chickens were bled for serum and the sera were tested for the presence of MDV antibody by the "“1 indirect fluorescent antibody test. 1mm I. At the termination of the experiment the brachial, sciatic, and celiac nerves were collected and prepared for histopathologic examination if no gross MD lesions were evident. C. Experiment III The objectives of this experiment were to determine whether the potential to initiate GVH disease was eliminated from leucocytes derived from.thymectomized donors and to determine whether iglzggrg production of MIF was thymic- dependent. The study included intact, bursectomized and thymecto- mized White Leghorn progeny of the USDA-RPL line 6 x 7 6’65 3 x line 6 99. Surgical bursectomies and thymectomies were performed within 24 hours after hatching. The chickens were housed initially in a battery brooder, then in cages until they were transferred to Horsfall/Bauer isolators (USDA-RPL) at 6 months of age. 63 The GVH assay was performed as described above.’ Recipient embryos were either line 15 subline l or subline 4. Eight-month-old donor chickens belonged to the follow- ing treatment groups: intact, bursectomized, or thymecto- mized. The GVH assay was repeated 6 weeks later using six thymectomized and six intact donors. Five recipient embryos each of line 15 subline l and line 15 subline 4 were injected with each of the donor's leucocytes. Each time cells were prepared for a GVH assay, the plasma was tested for MDV anti- body by the fluorescent antibody test. In addition, leuco- cytes from dams of the same subline as the embryo recipients were tested for GVH reactivity as a control. Prior to sensitization at 9 months of age, blood leuco- cytes were tested twice at 3 week intervals for inhibition of migration. The serum was tested for precipitating anti- body to PPD and MDV and all chickens were skin tested with 3.5 mg protein of B-24. The same chickens were subsequently sensitized intra-abdominally with 5 mg wet weight BCG in IFA. Three weeks after sensitization their leucocytes were again, twice, at 3 week intervals, tested for migration in- hibition. Antibody determinations were made as above, and chickens were skin tested with B-24 and with OT. One week after skin testing with B-24, all chickens were vaccinated with HVT, and tested for HVT antibody 6 weeks later at the time of skin testing with OT. H—m‘ J 64 Chickens were asphyxiated by CO At necropsy, com- 2. pleteness of thymectomy was determined, and sections of liver, spleen, and any grossly visible granulomatous . lesions were processed for histopathologic examination. RESULTS “A. Experiment I The results of ig_yi£rg migration of leucocytes from control (nonsensitized) chickens in the absence of antigen and with the addition of 25 and 50 ug OT were analyzed using the complete block design for a random model (Steel and Torrie, 1960). The data was first transformed by adding the value five to each migration measure and the analysis was performed using the data from six chickens with no miss- ing data. Table 1 enumerates the transformed measurements of the distance migrated in micrometer units after 24 hours of $3.!iEEQ culture by leucocytes with and without antigen added to the culture medium. The analysis of variance ob- tained from the data is presented in Table 2. The analysis indicated no detectable effect by antigen on leucocyte migra- tion. The distance of migration varied among individual chickens. The lack of detectable interaction between chick- ens and antigens indicated that the variability for the individual chickens was not antigen dependent. There were no significant differences between duplicate plates. The greatest variability was among spots within duplicate plates. The variation of total migration for all spots in all plates 65 66 Hun—«.511 IL I .oumam mom muomm m .muHcs HmuoEOHOHE mm Ummmmumxm .soHumnsosH ouomon pom GOHumnsosH musos em Hmumm nonwoooswa Ho womm on» ma umumEMHv on» soo3uon mosmummmHo can an tosflaumumo mm topmnmfifi oosmumHo H mH om mm mm mm mH om om ma on ma 0H mH on ma om ma mH.om ma ma ma mH CH "HH madam mH.mH ma mH ma ma ma ma mH om om om ma ma ma ma mH ma ma om mm on ma mH " H oumam 0mm mH 0H mm om mH mH mH ma om om on ma ma ma mm ma ma mm mo mo ON ON mH mH "HH «swam mo mH mm mN 0H mH om mm mH om mm ma ma om ma ma ma mm ma mo ma om mH om u H mumam mmm mm on om mH mH mH ma om 0H ma om mH mm mH 0H mH mH ma mH mH mm om mm om uHH oumam 0H CH mH mH ma ma 0H ma OH on ma 0H mH mm ma mm ma ma ma ma ma mH om mH u H oumam mmw 0H ma mH mo om ma mH ma ma mm 0H ma ma ma ma ma ma mm mH 0H om ma mm 0H uHH madam 0H oH.mo ma ma ma mo mH ma ma mH mH 0H ma om mH mH mo mH mo 0H mH mm 0H u H madam 0mm mH om om mm mm om mH om mm om mm ma mm ON ON mH mH mm mm ON ON mH mH ma uHH oumam mm om mH 0H mH 0H mo mo mm ma mN mm mH on on mm Cu om ON on om om mm mm " H mumam mvm ma mH mm mH mH mH mH mH om mH om mm mm mm mH mm ma mm mH ma ON on ma mH uHH oumHm 0H mH mH mH mH mH mH 0H om om mH ma om ma mm mm ma 0H mH mH mm mm mm mNAHvu H madam «mm 90 on om so mm mm somwusm oz soxowno .EDmeE stuHso one :H comHucm usonuHs one :uHs mcmeHno Houucoo scum manhooosoa mo ouuH> mm sOHumumHz .H manna 67 Table 2. Analysis of variance of data in Table 1. Source of Degrees of Sums of Mean Variation Freedom Squares Square F Value (1) Chickens 5 745.23 149.05 4.32* (2) Antigens 2 224.13 112.07 3.25 (3) Chickens x Anti— gens 10 344.62 34.46 0.89 (4) Plates/Chickens x Antigens 18 695.31 38.63 1.93 (5) Spots/Plates 252 5,034.38 19.98 Total (N-l) 287 7,043.67 (1) (2) (3) (4) (5) Significant at a = 0.05 Block effect Treatment effect Error term, Chickens by Antigens interaction Sampling error, Plates within Chickens by Antigens interaction Subsampling error d 68 was significant among chickens, and ranged from 112.5 to 155 micrometer units. The least square means were obtained for the leucocyte migration from chickens sensitized with BCG, CFA and control chickens, following sensitization, and ig|yi£rg_testing of leucocyte migration with the antigens OT and B-24. The least square means represent weighted average migration of leucocytes adjusted for missing data and unequal sample sizes. The least square means are given in Table 3. On the basis that inhibition of migration in the pre- sence of the antigen was used to detect ig’yigrg_sensitivity, the results indicated the following: (1) OT at both concen- trations tested inhibited migration of leucocytes from sensi- tized as well as control chickens. (2) 8-24 enhanced the migration of leucocytes from the control group and CFA sensi- tized group, resulting in migration as far or further than leucocytes with no antigen. (3) B-24 inhibited $2.!iEEQ migration of leucocytes from the BCG sensitized group. (4) 8-24 appeared to eliminate the possibility of a false positive migration inhibition. The data were further ana- lyzed to determine the contribution of chickens, sensitiza- tion, antigen, plates, and spots in affecting significantly the migration of leucocytes. The statistical model is a split plot, random model design with sampling and subsampling. Results of the analysis are in Table 4. ”'1. 51' 69 sOHumuHuHmsmm nouns nausea N .ummu mHS Aev hHmsoosmusonsm wmuooflsH .mmH Saws GOHmHsao sH sHHosormuumEHmo oHHHomm Amy hHmsomcmusonsm wouoonsH .mmm guH3 sowmasfim sH usm>snpm m.©ssmum muoameoo “my vouHuHmsomsoz AHV HB.MH mo.mH h¢.wH No.HN . m 00m Amy hm.om mH.mm mm.vm vm.m~ m 4WD ANV Hm.>~ mm.~H vo.mH mm.mH w Houusou AHV vmlm 90 m: om so on mm smmwusm oz .msoum\msmxowso mdouw GOHumumHE mo memos mumsvm swoon Ave «0 nmnasz .msmos chosen ammoa mm tmuHumsEsm soHumanz .msmmHusm nuHs msmeHgo Ho mmsoum owns» an GOHuMHmHH ouwooosoq .m magma 70 wsHHmammnsm .Honno msHHmEMm can OmHm mH one shop m House on» mm poms mH mammHucm .mcoxoweo .coHumuwaHmsmm\moumam mHthmsm mHnu sH d Houum mo.o n d unmoHuHsmHm Ame Amy AHV .mo.mmm.mmH mvNH Hence mm.oe om.mam.mv omoH mumaa\muomm Am. .om.m om.mmm mm.mve.ea we qupmuHuemcom \msox0H30 x msomHus<\mOHMHm Ame Hm.o vv.mmH mo.oao.m Hm msowuuNHuHmsmm \msmeHso x mammwusm .om.m me.0Hm om.ewe.m o mammauqe x meoflumueuememm «NN.HH hm.o~o.m oo.~om.b m msomHus¢ mm.omb.m mm.mme.hv SH mGOHumnwuHmsmm\msmx0Hso AHV .mH.o NH.vom.eH NH.mmm.¢m m mcoeumueuemcmm msHm> m mmumswm monmsvm socmmum GOHHMHHM> new: no wssm Ho moonmoo mo mousom .Hopoa sounds uon uHHmm m we touhflmsw use m magma cw ooNHHMEEsm sump Mom mosmwwm> mo mehHmsm .v magma 71 The error term usually used to construct the test F ratio is the value represented by antigen by chickens with- in sensitization effect (Steel and Torrie, 1960). However, because there were significant variations between chickens, and the chicken effect was confounded with sensitization, the error term used in this analysis was plates within sensitization, chickens, antigen. It is a more conservative statistic and less likely to result in an inflated P value. Sensitization and antigen significantly affected leucocyte migration as shown by the analysis in Table 4. Because sensitization itself affected migration signifi— cantly, and there was a sensitization by antigen interac- tion, the independent effect of antigen on migration could not validly be compared among sensitization groups. Therefore, supplementary testing was conducted to determine whether antigen inhibited leucocyte migration in the re- spective sensitization groups. The supplementary statistic- al analyses comparing least square migration means of leuco- cytes incubated with the antigens are given in Table 5 by sensitization groups. The analysis in Table 4 indicates that the variability of migration of individual spots was great, and that the leucocyte migration was significantly different between duplicate plates. The results summarized in Table 5 indicate that both concentrations of OT inhibited migration of leucocytes from sensitized and control chickens, and did not discriminate ’1'.“ 1)) I n I; 72 r .8 : .I'I. .. z — In wanes meocuoom mom Ame um manna ovosuoom wow Amy st0 m mm poms mH osommuwnu new swmsusm HQ GOHumumHE oumooosoa mo cOHanHssH um ashes ouoquoom mom has umou omHemu mom mummy owumHumum B mo.o n e .uemosoeemem . mom.o . . IIIIII I .m> some cm on .HH m me Ho meo.v em m .u . . 5mm.o m: .w> some so on ewe o lam Ho oHo.o so om .u . . mom.o . .mm N “Hm He eem.m so a: mm m> sausage on oom In. . . .Nmmwm I .m> some so 0: mo H me Ho omm.o em m .u .om.o me.Ho esm.o so on om .m> someuem 0: emu o . . esm.o . «mm N lam Ho eom.m so on mm m> cmmsuee 0: «so Am. . I . mmN.H I .m> some so as Hm o me Ho mmm.mI «N m .u «Ho.e me.HV mmmwmm so m: cm .m> amassed 0: omH m .me.~ Ism.ac mmwmnm so a: mm .m> emeeuee oe Houpeoo Ase sowuannsH Boommum ovau COmHHmmEoo GOHumumHS sOHumuwuHmsmm Mom osHummu mo mmoumma ”mos usumsumumus .v wands as woumHmsm memo mom mcHumou humusosoammsm .somwuso saws mcoonso ooNHuHmsom mom can pmNHuHmsom «mu .Honvsou mo memos nosumumfia muaooosma mo somwummsoo .m OHQMB 73 immunologic sensitivity from nonsensitivity on the basis of leUcocyte migration at the antigen concentrations tested. The results of a pilot study were no inhibition of leucocyte migration by 10 and 20 ug OT and leucocyte mortality at con- centrations of 100 ug OT. In contrast, B-24 did not inhibit migration of leucocytes from control chickens or CFA sensi- m! tized chickens. Rather, it enhanced migration in controls 'F‘oi 6‘ and inhibited migration in the BCG group. The activity of B-24 ig yi2£g_was immunologically more specific than OT. This selective inhibition in migration of leucocytes by B-24 from BCG sensitized chickens but not from control or CFA chickens also contributed to the significant interaction between sensitization and antigen, as given in Table 4. As previously reported (Fauser g; gl., 1973c), the cells attaching to the petri dish were approximately 40% mononuclear and 60% polymorphonuclear leucocytes when stained with Wrights stain. The morphology of leucocytes at the end of\24‘hours of incubation was altered by cytoplasmic vac- uoles so that a differential analysis of cell type was not possible. The results of skin reactions, antibody determinations and pathologic involvement of organs and tissues are summar- ized in Table 6. The centrol group remained negative for antibody, skin reactions and pathologic reactions. Both sensitized groups had positive skin reactions to OT: 5/8 74 m\s m\m mxm m\o HmuouInsm Inn. Inn. Ian. I mmm + + + + emm + + + + mmm + + + + omm + + + + new + + + + mmm I + + + mvm + + + I mmm 00m Amy MKW me mRM m\m Honoulnsm + + I I mmm + + + Ixso smm + + + it , vvm + + + I new + + + + omm + + I + mmm + + I + 0mm + + + + 3m 58 A3 .|\0 E B. E 3...... I I I I mmm I I I I mmm I I I I neon oz I I I I smm Houucoo 3v mEmHsmmso omm vam nusz Bo auH3 smeHno msouw poms osoo so on aponsusm cosuomos coHuomou \eee meoemoq loo meeumueesoeue Ame seem Ive seem rec .H uses Iwnmmxm CH msmxosno mo mmsosw m Ho mGOHuommH osmoHonumm use noncommmu atonHusm .msoHuommH stm .o mHQms 75 0>Hummos n I 0>HuHmom u + .ossom mums mEmHsmmHo ummm oHom Hos msOHmmH HosuHms moumowosH AIV m>Hummos a .0>H9Hmom A+v mm vmtuooon ohms machmmHo ummu oHom nuHs meow .onoomouoHB H0\psm .mmoum .msowmma msommmo H0\osm msoumsoassonu .cosumuHuHmsom on uoHsm o>Hummmc moonHusm mum3 msoxosno HHm .mooHHm pounce soon as cosmsmmHo oHAsoo as poms mm3 A.n0Hz .usouuoo .mH>mnonHmm .nmmv o>Hum>Huoo :Hmuosm pwHMHHsm numsmuum vsoomm mo AsHouonm ma Ho.ov sowumuusmosoo oHoonauIfi .ooswmuum mm: mauumz pmumHsoochs on» nuw3 venom Iaoo mm mmmchHnu oHuuma sH mmmmuosw tHOHIo3u m we ESEHGHE M NH 0>Husmom omomnm ouo3 wGOHuomoH stm .m magma muosuooM 00m .m wands ouosuoom mom .m wands. ouofioom 0mm any Amy Amy Ava Ame Amy AHV 76 CFA and 6/8 BCG. More BCG sensitized chickens than CFA chickens responded with skin reactions to B-24: 8/8 as com- pared to 5/8. Precipitating antibody to PPD was present in sera from all sensitized chickens. Lesions, some with acid fast organisms, were found in 8/8 CFA sensitized and 7/8 BCG sensitized chickens. All BCG injected chickens had caseous granulomas of the serosa of the gizzard. Lesions in chickens injected with CFA were either granulomatous or granulomatous with caseation necrosis. No acid fast organ- isms were recovered by bacteriologic culture. B. Experiment II Leucocyte migration was tested in nine chickens to determine whether the antigens OT and crude MDV inhibited migration prior to sensitization. The statistical model is a randomized complete block design with sampling and sub- sampling. The results are given in Table 7. As in Experiment I, the individual variation in leuco- cyte migration among chickens was statistically significant. The presence of antigen in the culture medium had a signifi- cant effect on leucocyte migration and the effect of antigen was similar for leucocytes of all chickens tested. This is reflected by no significant interaction between chickens and antigens. Leucocyte migration of spots in duplicate plates under identical culture conditions were significantly differ- ent. 77 .m wanes .mouoeuoom mom Amy I mo.o I a an “censusemsm “Ho mm.Hmm.am Hme Heuos Hm.Hm .mmqmmmqmm .mmm oumam\muomm Ame .mm.~ Hm.vm ms.mem.~ em meomsuqe x meoxusao\mmumam rec mm.s mo.mes ms.msm.~ we mammauee x meaxoeeo Ame .e~.os oe.-m.s ms.eeo.m N mcomsuaa Awe .mo.mm os.~am.v mo.mom.mm m meoxosao Ase msHm> m museum mmnmswm Becomes coquHHm> and: mo masm Ho mmwsmoo mo mousom so can do mouse mammsusm on» HSO£UH3 new :uH3 mmumooosoa mo soHumumHE .msoxOHno Houusoo scum Ho oosmwum> mo mwmaHmsd .h manna 78 The mean migrations by individual chicken's leucocytes with and without antigen are summarized in Table 8. The table presents mean migration in micrometer units of all . leucocyte spots from nine control chickens with no antigen, OT, and crude GA antigen. The variation among chickens was reflected in the range of mean (arithmetic) migration for all spots with each of three antigens: (1) with no antigen the range of mean migration was 13.4 to 43.4 micrometer units, (2) with 50 ug OT 7.2 to 36.6 and (3) with crude GA antigen 8.1 to 36.9. The effect of antigen on migration was reflected in the mean migration of leucocytes from all chick- ens with no antigen as a mean of 23.4, 50 ug OT a mean of 19.0 and crude GA a mean of 17.0. Inspection of these mean values with the Wilcoxon matched-pairs signed-ranks test (Siegel, 1956) showed that each antigen significantly (a = 0.02) inhibited mean migration of leucocytes as compared with no antigen. Incubation of leucocytes with an extract prepared from the same cells used to culture the GA strain of MDV resulted in no inhibition of leucocyte migration. The results of the statistical analysis of migration of leucocytes from the same chickens, but following sensitiza- tion are given in Table 9. No significant effect of sensi- tization or antigen Egg gg was detected. However, there was a significant antigen sensitization interaction, which is tabulated as least square means in Table 10. As in the control (presensitization) analysis, antigen per gg did not 79 Table 8. .Mean migration in micrometer units by all leucocyte spots from 9 control chickens with no antigen, OT, and crude GA. Chicken number No antigen 50 ug OT Crude GA 1594 41.6 36.6 30.3 1595 43.4 32.5 36.9 1597 16.6 17.5 10.9 1604 24.7 19.1 15.0 1606 15.6 14.1 10.6 1607 23.8 18.4 13.4 1612 15.0 9.1 16.9 1613 16.3 16.6 10.9 1614 .L3-_4. _Zi _u 23.4 19.0 17.0 xI 3295.33 2: m House was mcHHmsmm Amy poms maamsms m Houum ANV a house AHV mo.o I e um unmoeusemem . 80 eo.mos.om mHe Hmuos om.oe sm.mmm.sH son mumaa\muomm Ive mm.H m¢.mo mv.¢mm.a mm msomfluse x memxoeao .eoeueueuemeom\emumse Ame se.~ mv.emH os.mmm.s NH eosumueusmemmxmaoxosno x smashes Ame .ms.s ms.omv mm.oem.s e meoeumususmemm x memosuee eH.m om.mmH Hm.smm N meemsuea ~m.eeo oa.som.m o measumneusmeom\meoxoeno 1H1 em.~ os.emm.H mm.mms.m m meoHuMNHuemeom wsHm> m msmsvm mmuosvm socoosm _ soquHhm> saw: no mfism mo newsman Ho.oousom .Hoooa sounds uon psHmm o no oswusou manage ummoa may nuH3 wouhHoso msoxOHno owususmsmm >oz use Houusoo scum mophooosoH.Ho sOHumumHa ouuH> mm mo mosmwum> Ho mHmemsd .m mHnms 81. Table 10. Antigen by sensitization interaction of data on Table 9 represented by least square means migration with and without antigen. Number of Least square means of migration Group chicken/group .No antigen 50 ug OT Crude GA #1: Control 3 11.98 13.63 15.80 g (1) GA 3 13.32 13.02 8.23 i..- (2) GA + CFA 3 22.71 15.83 17.29 (1) GA strain MDV injected and/or chickens in contact exposure to GA strain MDV injected chickens (2) GA strain MDV injected in emu1sion with complete Freund's adjuvant ‘82 significantly affect migration from one chicken to the next. The analysis also indicated that no significant differences could be detected between duplicate plates. The least square means tabulated in Table 10 indicate the control group had no detectable inhibition of leucocyte migration with either OT or crude GA antigen. The GA group was inhibited significantly by GA antigen and not by OT and the GA+CFA group was inhibted by both OT and crude GA. The results of leucocyte migration of MDV infected and control chickens with A-antigen in the culture medium are given in Table 11. In this experiment, one plate with seven spots was prepared for each culture condition being tested. A-antigen inhibited radial leucocyte migration in cul- tures from all MDV infected chickens by 16 to 66%, with a mean of 44.4%. It enhanced migration of leucocytes from the normal chickens by a mean of 9.7%. Results of skin tests and antibody determinations are given in Table 12. Control chickens had no skin reactions with OT, crude GA and A-antigen. They had no detectable antibody to MDV or PPD. Sensitization of chickens with GA- MDV in CFA induced sensitivity to tuberculin as detected by skin tests to OT and antibody responses to PPD, but did not induce sensitivity to GArMDV as determined by antibody and skin reactions. Chickens sensitized with viable GA-MDV and those in contact exposure to MD had positive skin reactions to crude GA and A-antigens and had fluorescent antibody to 83 comwucm sadness woumsmws mosmumwo 2: x 53...... fit. 83.3.. 853.8“— I 83323 3588 E m.mH H.NN H.NN >nz bmmH H.mv m.s~ o.mv >nz mmmH m.ov v.HN v.ov >nz. mmmH H.ow m.h «.mm >nz vmma H.5HI v.0m H.Hm Houusoo vaoH m.o I m.mv m.m¢ Houusoo mama m.HHI b.m¢ H.Hv Houusou «Hod soHu somwusMIfl. advance oz usosumoua (Hogans I325 confine? e335 unmouom momsm>< AHV muomm b m0 sowumumda ommuo>< .smmsucMIm new smmsusm 0c guHs mcmxosno >Dz was Houusoo scum manhooosoH mo sOHumumHE some 0Huoanusum .HH manna um soaumusmsa mam msaaamsm ca movaSmmn comchMIm on soasooou caxm_ umou moonausm pcoomouosam Amy m wands muonuoom 00m any ooaHEumuoo uoz Io. o wanes ouoeuoom mom Ame .musog mv an amsuoc on msanmasasam muse: mm .mnson me an amsuoc on meanmasasam mason mm um mavens Ho maasoxoanu ca mouasmou comausd mm ensue on soauommu caxm .m wanes muosuoom mom Avv mcwxoano mouoonca m0 samuum >az on musmomxo uomucoo ca u0\msm monomnsa mm cannum >nz..mv uso>sflmm mmesoss.muonfioo ca «0 samnum >nz any mmuauamsomsoz Aav 84 Oz ozxmo + + + + I + + Q + + + E + + + + + IABV hmma mmma mmma #mma moma moma homa moma voma mama mama mama aama cw Ame emu as aw Am. Houuaoo AH. one on 68.35 Ame soonsucm >oz_xoo :moHu:MI¢ on .aoauomom cme Hey cw mmsnu on coauomom saxm Amy so on eosuomom eexm Ave cmxoseo macaw .HH peasanmmxm sa msmxoano mo mmmcommou hmonausm use mcoauomos :me .Na manna 85 MDV. With crude GA antigen, the thickening of thefwattlejw commenced at 25 hours and had mostly disappeared by 48 hours. With A-antigen a small amount of swelling and in- duration was detected at 36 hours, but had disappeared by 48 hours. At necropsy none of the chickens had lesions. All MDV infected chickens were emaciated. No neural lesions were found on histopathologic examination of those chickens in- jected with and/or exposed to MDV. C. Experiment III The least square mean weight in grams of 19 day chicken embryo spleens from embryos injected at 14 days embryonation with leucocytes from intact, bursectomized, and thymectomized donors is shown in Table 13. The variability of spleen weights was great among those embryos receiving leucocytes from donors belonging to differ- ent treatment groups. The least square spleen weights of embryos injected with leucocytes from the three treatment groups were as follows: (1) Intact 0.1059 grams, (2) Bursec- tdmized 0.1101 grams, (3) Thymectomized 0.1527 grams. The mean spleen weights of the controls were as follows: (1) Maternal leucocytes 0.0442, (2) Tissue culture medium 0.0142, and (3) Uninoculated 0.0141. The recorded spleen weight means in some cases are from fewer than 10 embryos 86 Table 13. Least square mean spleen weights in grams of 19-day old embryos injected at 14 days embryonation with leucocytes from intact, bursectomized and thymectomized donors and mean spleen weights of control spleens. Intact Bursectomized Thymectomized Control (1) 0.1093 0.1342 0.1187 (2) 0.0442 (10) 0.1687 0.0748 0.1125 (3) 0.0141 ( 9) 0.1214 (1) 0.1844 0.1540 (3) 0.0143 (10) 0.0688 0.1241 0.1244 (4) 0.0141 ( 9) 0.1331 0.0978 0.0911 g; m 0.1381 0.1206 0.1251 0.0902 0.0694 0.1347 0.1692 0.1689 T FITS—2'7 0.1060 0.1739 0.1367 0.0808 0.1135 50—71—161 0.1199 32' 671655 (1) (2) (3) (4) Least square mean spleen weights from embryos injected with leuco- cytes from 1 donor chicken, less than 10 embryos survived to 19 days embryonation. Spleen weights from embryos injected with leucocytes from one of the dams, number in ( ) is number of embryos surviving to 19 days embryonation. .Mean spleen weights of embryos injected with tissue culture medium. .Mean spleen weights of uninoculated embryos. 87 due to embryo mortalities. In determining if thymectomy or bursectomy of the donors affected the GVH capability of their leucocytes the data were analyzed using regression analysis, a weighting procedure to compensate for unequal numbers of replicates and missing data. The analysis is shown in Table 14. The greatest splenomegaly was elicited by the leucocytes from the thymectomized group of donors. Ira-14'? law All donor chickens were negative for MDV antibody with the indirect fluorescent antibody test. Because there was in- creased instead of the expected decreased splenomegaly the experiment was repeated. To reduce the variability of spleen weight response to the individual donor leucocytes by recipient embryos, five each of sublines 1 and 4 were injected and recorded separately as recipients of each donor leucocyte inoculum. Also, the bursectomized donor group was omitted.- The least square means of embryo spleen weights resulting from injection with leucocytes, are pre- sented in Table 15. All donors had no detectable MDV anti- body. With one exception, there was greater splenomegaly in embryos of subline 1 than subline 4 injected with leuco- cytes from the same donor. This is significant in indicat- ing that both sublines responded in a parallel manner to the injection of leucocytes from the same donor. Thymecto- :mized donor leucocytes elicited greater splenomegaly 0.1249 grams, than intact donors leucocytes, 0.0667 grams. The mean spleen weights of embryos inoculated with the leucocytes 88 Table 14. Analysis of variance for data summarized in Table 13. Source of Degrees of Variation Freedom Mean Square F Value Surgery 2 5,298,146.8931 20.57l6* Chickens 26 1,036,198.7629 4.0233* Error 2§£_ 257,546.1480 Total 282 #LA *Significant a = 0.05 89 Table 15. Least square means of spleen weight in grams by embryos of two sublines of line 15 injected with leucocytes from in- tact and thymectomized donors. Intact Thymectomized Subline l Subline 4 Subline l Subline 4. 0.1089 0.0827 0.1691 0.0224 (1) 0.0711 (1) 0.1205 0.1168 0.0748 0.1310 0.0606 0.0962 0.0953 0.1274 0.0642 0.1430 0.0486 0.1196 '0.0720 0.1292 0.0624 0.1277 0.0638 0.1388 0.0527 0.0667 0.1249 (1) Only case where least square mean of spleen weight of subline 1 was less than that of subline 4. 90 of the same subline and the heterologous subline, embryos inoculated with the culture medium used as the diluent for donor leucocytes, and uninoculated embryo controls are given in Table 16. In all controls the mean spleen weights of line 15 subline 1 were greater than those of subline 4. The embryos receiving syngeneic donor leucocytes had slight splenomegaly but less than embryos which received leucocytes from the other subline. The analysis of variance of the data summarized in Table 15 is given in Table 17. The greater splenomegaly of embryos inoculated with leucocytes from thymectomized chick- ens was significant. Donor chickens differed significantly in the capacity of their leucocytes to induce splenomegaly. The differences in splenomegaly by each of the sublines is significant. The analysis of the results obtained from the study to determine whether thymectomized sensitized chickens have impaired MIF production $2.21EEE is summarized in Table 18. There were no missing data, and all sample sizes were equal. The analysis indicates that sensitization with BCG as nested in time (times 1 and 2 are prior to sensitization and times 3 and 4 are after sensitization) does not, in and of itself, detectably influence leucocyte migration £2.2iEEQ! This also indicates the validity of repeated measurements in time. The response of leucocyte migration £2.2iEEQ does not appear to change appreciably during the time frame measured. 91 Table 16. .Mean spleen weights of controls for data on Table 15. Donor Recipient Spleen weight ‘1) 1s subline 4 (i) 15 subline 1 (2) 0.1122 (5) 15 subline 1 15 subline 4 0.0930 (4) 15 subline 1 15 subline 1 0.0281 (5) 15 subline 4 15 subline 4 0.0198 (5) F10-199+15% SPF serum 15 subline 1 0.0169 (5) F10-199+15% SPF serum 15 subline 4 0.0168 (5) no donor 15 subline 1 0.0171 (5) no donor 15 subline 4 0.0162 (5) (1) Line 15 (2) Number in ( ) is number of embryos surviving to day 19 of embryona- tion. 92 Table 17. Analysis of variance for data summarized in Table 15. Source of Degrees of Mean Variation Freedom Square F Value Surgery 1 8 .713 ,870.7056 139. 9806* “1‘ Chickens 10 1,708,627.6925 27.4476* 1 Line (Chicken x Surgery) 12 1,135,994.1719 l8.2487* L~-‘ Error _8; 62 ,250. 5764 Total 105 *Significant a = 0.05 93 .umou muueauuem .msoosemoaos ones mooseaue> Amy .soauenauamsem House vam no oosemne use oosemoum sa msoaueumaa euhooosea one; v use m mesa» use vam mo mosemne use eosemesm sa soauesmafi oumoousea scavenauamsemeum ouo3 N use a nosaa mo.o u 5 ye useoamasmam “av mm.HH«.sov ..Ismo.~ Heuos mm.em ms.mem.~ms mmn.~ oumamxmuosm .me.~ mm.meH om.mem.v~ meH censure x macroeno x hummusm x msas\ueueam eo.H mH.emH -.v~m.m em suomusm \msexoaso x esas x somausx. .Hm.H mm.em~ mm.mm~.m as mucousmxmemroeno x smashes .He.~ mm.Hsm mm.am«.~ e suomusm x mess x sausage m~.o Hm.ev me.mm m Hummusm x eemsuee .sm.m ~m.smm se.mos.H m mess x.eoeaue< m~.o He.mm H>.mm H cemeuqe Ad HOHHm—J ev.Hsm.H os.sm~.om em suomuem\meorosao x mass ms.som.e mo.mmm.em ma summu8m\meoxueso me.H vs.mme.m ee.~mm.vH o snowman x mass mm.m ee.om~.ea mm.oom.mm m sausage mv.H e~.emm.~ ss.mms.s m mass AH. enae> m muesvw mesesum soueesm soaueaue> Ho eonsom “my see: so warm «0 awesome .osua>.mm soaueumas eumooosoa sa semause use .soaueuauamsem .hsomHSn Ho moseaue> Ho mamhaesm .ma oaQeB 94 The surgical manipulation of thymectomy or bursectomy does not detectably influence migration; however, there was a suggestive effect of surgery (a = 0.055) considering the mean migration of all leucocyte spots for all 4 times of measure- ment were as follows: (1) Intact, 36.0; (2) Bursectomized, 41.9; and (3) Thymectomized, 33.6 micrometer units. There was also no interaction of time (sensitization) with surgery so the effect on migration by surgery only remained similar for all groups. Antigen, in this case B-24, did not detectably affect migration at a significant level. The overall means for migration without antigen, over all time periods and sur- gery groups was 37.4 micrometer units and with B-24, 37.0 micrometer units. There is a statistically significant interaction be- tween antigen and time, and to represent this interaction, the means are given in micrometer units in Table 19. Inspection of the means of leucocyte spots, regardless of individual chicken and surgery effects indicates leucocyte migration was greater with B-24 than with no antigen when assayed prior to sensitization. B-24 inhibited migration after'sensitization. The absence of a Significant interaction of antigen and surgery indicated that the presence or absence of B-24 was associated with Similar migration for intact, thymecto- miZed and bursectomized chickens. 95 Table 19. Migration means for antigen by time interaction (1) Table 18. Incubation with Presensitization PostsenSitization Time 1 Time 2 Time 3 Time 4 No antigen “33.6 38.3 37.8 39.8 B-24 35.6 38.6 36.3 37.7 (1) At both presensitization times, there was no migration inhibition, as summarized by means, rather enhancement by the antigen B-24. At both postsensitization times, the means indicated inhibition of migration by the antigen B-24. 96 The antigen by time by surgery interaction is most noteworthy, and of greatest interest in determining whether migration inhibition by the sensitizing antigen in sensi- tized thymectomized chickens was diminished or absent as predicted. A summary, using group means, of the data comprising the interaction is given in Table 20. The parallel response trend of the normal and bursec- tomized groups is evident. During both presensitization assays, B—24 enhanced migration in the intact and bursec- tomized groups, and inhibited migration during both post- sensitization assays. In contrast, the thymectomized group means indicate possible enhancement during the first pre- sensitization assay and inhibition during the second pre- sensitization assay. Following sensitization, there was inhibition during the first postsensitization assay and enhancement during the second. The response of the thymec- tomized group did not parallel that of the intact and bur- sectomized groups. In this experiment there was no Significant interac- tion of antigen by time by chickens within respective sur- gery, meaning that respective chickens varied in their leucocyte migration with and without antigen during the four time frames sampled in a Similar manner. When the data were expressed in terms of mean percent increase in migration inhibition of the postsensitization versus presensitization group levels, the intact group was characterized by an 97 FI-L._ .mmsosm usuasouoomssn use uoeusa esp Ho sen» aeaaeuem nos uau msosm ueuasouoeshsu on» no emIm use semanse.aséauIeasomwouxsedueumaaIdea -.Imuoasem. asap scavenauamsew Iumom use Imam can esp. msausu semause 0s nuaawudnemsaa..me:ss maeuouQSm msouw ueuaeopoemusm maeuoun5m msosw HUMUGH E + + + + + + +| nee ewsmsmo Insane omsmsoo Ave Amy Amy 80 vmlm msoauoeem stm Aav senses msosu meoemms Ame seonsuca :mroeeo .HHH usefiasmmxm sa msmxoaso pasue mo msoamea use .emsommeu huonause .msoauoees saxm .am wanes 101 .eanesOHHmmsv n m .uossouu msexoaso m ewes» museums uosasueuou uoz e>auemes u I m>auamom u + .usaou mums mamasemuo umem uaoe Hos msonea Hesuaes moueoausa HIV o>auemms e .e>auamom A+V me uouuooeu ones mamasemuo umem uaoe spas seem .oamoumouoaa;s0\use .wmoum .msOHmea msoemeo u0\use msoueEOasseuu . .msexoaso aae sa e>apemes mes s0aueuauamsmm on Haasm emsommos muonauss .wuonause msaueuamaomsm use useomeHosam .soapeuauamsem on noaum e>auemes muonause 0se3 msexoaso Has .mouaam ueueoo heme sa scamsmmau mansou sa uews mes A.soaz .uaonumn ema>eolexuem .ommv e>aue>aseu saeuonm ueamausm.sumsenum,usooem Ho Asaeuonm ma ao.ov soHueHusmosoo uaomIosu s Ase ssosx nos ma magnum so ueaeaeu menu mes muses mv pe soauoees seasons osaumeu sense meson N we wanes me manepueueu soauoees saxm .msmxoaso aae sa e>auemws ones sOHueuauamsmm on noasm vam seas memes saxm .umsaeuue mes masses ueuowflsass es» seas uouem I500 me mmesxoasu masses sH emeouosa uHOMIosu e no essasas e ma e>auamom umueum ewes msoauueeu saxm Amy any Amy Ame Ave Amy Amy «as DISCUSSION Factors Influencing Weucocyte Migration Some of the designated variables in the experimental design influenced leucocyte migration of control and sensi- tized chickens. Thus, the variables relating to the experi- mental procedure and biologic assumptions of the MIF test and the Significance of the results of the various hypothesis testing experimental objectives are discussed herein. The primary objective was to determine whether an MIF test using chicken blood leucocytes could be developed. Prior to testing whether leucocytes from sensitive chickens were inhibited by antigen during ig yiggg migration, con- trols necessary for biologic reliability had to be tested. The innate variability in migration by individual control chickens in Experiments I and II (Tables 2 and 7), indicated that a standardized value in micrometer units to differen- tiate migration from inhibition was not possible. Had it Jbeen, the design of the MIF test as used with chicken leuco- cytes could have been simplified to require no control (nonantigen containing) plates and statistical analyses of