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Early embryonic surgical bursectomy : an investigation of some aspects of the avian immune system 1973

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EARLY EMBRYONIC SURGICAL BURSECTOMY: AN INVESTIGATION OF SOME ASPECTS OF THE AVIAN IMMUNE SYSTEM v ' by ESTA M..F. sS.PSRKS >• BSc. (Hons.), University of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Poultry Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of fOUA 77*? 1/ Oc^f/^CcT The University of B r i t i s h Columbia Vancouver 8, Canada 1 ABSTRACT S u r g i c a l removal of presumptive b u r s a l t i s s u e a t 72 h r i n ovo r e s u l t s i n h i g h m o r t a l i t y b e f o r e h a t c h i n g and low s u r v i v a l i n the f i r s t week t h e r e a f t e r . The v i a b l e c h i c k s are underweight and u s u a l l y remain so. Both the s p l e e n and thymus of the bursectomized (Bx) c h i c k s are reduced i n s i z e and lymphoid p o p u l a t i o n . The e f f e c t on the thymus may be r e l a t e d to s t r e s s and subsequent a d r e n a l c o r t i c a l a c t i v i t y o r may r e f l e c t some endocrine f u n c t i o n o f the b u r s a necessary f o r the m a t u r a t i o n o f thymic lymphoid t i s s u e . The e f f e c t on the s p l e e n seems to be more d i r e c t . In the absence o f the primary l e v e l organ which induces m a t u r a t i o n of the stem c e l l s , the number of g e r m i n a l c e n t r e s i n the secondary l e v e l organ i s reduced. Some of the remaining g e r m i n a l c e n t r e s may be of T - c e l l o r i g i n , although these c e l l s are more commonly found i n d i f f u s e lymphoid a r e a s . The b u l k of the g e r m i n a l c e n t r e s found i n the Bx s p l e e n must be of B - c e l l o r i g i n . Repeated s t i m u l a t i o n of the humoral immune system w i t h antigens and mitogens e l i c i t s a response i n some Bx b i r d s . The response i s g e n e r a l l y not found i n the primary s t i m u l a t i o n and i s r a r e l y o f the magnitude o f a normal response even a f t e r s e v e r a l s t i m u l a t i o n s . Antibody p r o d u c t i o n may be l i m i t e d to IgM type immunoglobulin; f u r t h e r s t u d i e s are n e c e s s a r y to v e r i f y t h i s . Autopsy and h i s t o l o g i c a l examination of the t i s s u e s o f the r e s p o n s i v e b i r d s f a i l e d to show any evidence of r e s i d u a l b u r s a l t i s s u e . Whether these f i n d i n g s c o n s t i t u t e p r o o f of the r o l e o f the b u r s a i n the humoral immune response or whether they r e f l e c t the broader consequences o f bursectomy i n the development o f the b i r d r e q u i r e s f u r t h e r study. The i n d u c t - i v e c a p a c i t i e s of the b u r s a and the source and autonomous c a p a c i t y of the stem c e l l s thought to be induced i n the bursa should a l s o be i n v e s t i g a t e d . The e f f e c t s o f e a r l y hormonal and l a t e s u r g i c a l bursectomy •11 are s i m i l a r to those found i n t h i s study, where an e a r l y embryonic s u r g i c a l technique was used t o a v o i d the p o s s i b l e c o m p l i c a t i o n s of these o t h e r methods. i i i TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW A. The Bursa of F a b r i c i u s . 4 B. S t r u c t u r e o f the Bursa. 4 C. Development o f the Bursa. 6 D. Immunoglobulin S y n t h e s i s . 8 E. Bursectomy. 10 F. E f f e c t s o f Bursectomy. 10 G. The Endocrine F u n c t i o n o f the Bursa. 12 PART I : S u r g i c a l and Hormonal Bursectomy Techniques, H i s t o l o g y and S k i n G r a f t i n g . A. Methods i ) E a r l y embryonic s u r g i c a l bursectomy. 13 i i ) E a r l y embryonic hormonal bursectomy. 14 i i i ) P r e l i m i n a r y examinations. 14 i v ) H i s t o l o g i c a l examination. 16 v) S k i n g r a f t i n g . 16 B. Re s u l t s and D i s c u s s i o n 17 PART I I : T e s t s o f the Humoral Immune System A. Methods i ) Serum samples. 2 5 i i ) P r e p a r a t i o n o f a n t i s e r a . 25 i i i ) Immunoelectrophoresis. 2 5 i v ) T e s t f o r s p e c i f i c a n t i b o d y p r o d u c t i o n . 26 B. R e s u l t s and D i s c u s s i o n 26 PART I I I : S t i m u l a t i o n o f Spleen, Thymus and Bursa C e l l s w i t h Mitogens. A. I n t r o d u c t i o n to Mitogens 34 B. Methods 3 5 C. Re s u l t s and D i s c u s s i o n 36 GENERAL DISCUSSION 41 SUMMARY AND CONCLUSION 48 BIBLIOGRAPHY 49 APPENDIX 53 i v LIST OF TABLES Table Table I Table I I T a b l e I I I Table IV Table V T a b l e VI T a b l e V I I T i t l e Page Average weights of normal and 20 s u r g i c a l l y b ursectomized c h i c k s from hatch to 8 weeks of age. Spleen and body weights f o r day o l d 21 normal and s u r g i c a l l y b u r s e ctomized c h i c k s . Prognosis o f s k i n g r a f t s a t 7, 14.and 24 28 days a f t e r g r a f t i n g . Haemagglutination t i t r e s f o r normal, 33 SBx and HBx c h i c k e n s e r a f o l l o w i n g s u c c e s s i v e s t i m u l a t i o n s w i t h SRBC. Average s t i m u l a t i o n o f normal (N) and 38 SBx c h i c k e n s p l e e n c e l l s w i t h mitogens. Average s t i m u l a t i o n of normal (N) and 39 SBx c h i c k e n thymus c e l l s w i t h mitogens. Average s t i m u l a t i o n o f normal c h i c k e n 40 b u r s a c e l l s w i t h mitogens. V LIST OF FIGURES F i g u r e F i g u r e 1. F i g u r e 2. F i g u r e 3. F i g u r e 4, F i g u r e 5. F i g u r e 6. F i g u r e 7. F i g u r e 8. F i g u r e 9. F i g u r e 10. F i g u r e 11. T i t l e Page Bursa development shown as a per- 5 centage o f body weight. The times of i n i t i a t i o n o f immunoglobulin s y n t h e s i s are shown. Cross s e c t i o n o f the b u r s a o f 7 F a b r i c i u s , showing p l i c a e and lymphoid f o l l i c l e s (from Romanoff, 10). Chick embryo a t 3 days (72hrs) o f 15 i n c u b a t i o n — d o r s a l v i e w a n d l o n g i t u d i n a l s e c t i o n through t a i l r e g i o n . T a i l r e g i o n ( d o r s a l view and l o n g - 15 i t u d i n a l s e c t i o n ) showing i n c i s i o n through membranes and t i p o f t a i l l y i n g f r e e . T a i l r e g i o n ( d o r s a l view o n l y ) f o l l o w i n g 15 l i g a t i o n and removal o f presumptive b u r s a l t i s s u e . H i s t o l o g i c a l s e c t i o n s of normal (A) 22 and bursectomized (B) c h i c k s p l e e n s . H i s t o l o g i c a l s e c t i o n s o f normal (A) 23 and bursectomized (B) c h i c k thymic l o b e s . P r e c i p i t i n bands from immunoelectro- 29 p h o r e s i s o f normal 5 day c h i c k serum (from Tureen ^ t a l . , 33). P r e c i p i t i n bands from immunoelectro- 30 p h o r e s i s o f 14 day normal (A) and bursectomized (B) c h i c k s e r a a g a i n s t r a b b i t a n ti-whole c h i c k e n serum. P r e c i p i t i n bands from immunoelectro- 31 p h o r e s i s o f 14 day normal (A) and bursectomized (B) c h i c k s e r a a g a i n s t r a b b i t a n t i - c h i c k e n ; I g M . M i c r o t i t r e h a e m a g g l u t i n a t i o n o f SRBC 32 by normal (N), s u r g i c a l l y b u r s e c t o m i z e d (SBx) and hormonally bursectomized (HBx) c h i c k e n s e r a f o l l o w i n g primary s t i m u l a t i o n w i t h SRBC. v i ACKNOWLEDGEMENT I would l i k e to express my a p p r e c i a t i o n to my s u p e r v i s o r , Dr. R.C. Fitzsimmons, to Dr. D.G. K i l b u r n of M i c r o b i o l o g y and Dr. R.J. Hudson of Animal S c i e n c e f o r t h e i r a s s i s t a n c e i n o r g a n i z i n g and p e r s u i n g t h i s r e s e a r c h . I am a l s o very g r a t e f u l to Donna Mason f o r h er a i d i n the performance of 1,466 bursectomies. Thank you. 1 INTRODUCTION The immune system functions to protect an animal against i n f e c t i o n , disease, foreign c e l l s and neoplastic c e l l s of s e l f o r i g i n . These functions may be f u l f i l l e d e i t h e r by the production of antibodies or by d i r e c t c e l l - t o - c e l l i n t e r - actions and are c a r r i e d out by two f u n c t i o n a l l y d i s t i n c t l i n e s of lymphocytes. The c e l l u l a r immune system provides s u r v e i l l a n c e against neoplastic c e l l s , functions i n g r a f t r e j e c t i o n and i s involved i n delayed h y p e r s e n s i t i v i t y and graft-vs-host reactions. The c e l l s of t h i s s y s t e m a r e a l s o n e c e s s a r y f o r e f f i c i e n t antibody production i n some cases. The lymphocytes involved i n these reactions appear to be derived l a r g e l y from the thymus and are termed " T - c e l l s " . These c e l l s do not themselves produce immunoglobulin molecules or s p e c i f i c antibodies. They are capable of k i l l i n g foreign c e l l s on contact and also produce a var i e t y of chemical fa c t o r s . These include cytotoxin,which lyses c e l l s , chemotaxin and migration i n h i b i t i o n f a c t o r f o r c o n t r o l l i n g the movements of nearby lymphocytes and macro- phages, blastogen, which stimulates the maturation of lympho- cytes, and factors f o r s e n s i t i z i n g b a c t e r i a and viruses. The humoral immune system as the source of s p e c i f i c immunoglobulin molecules termed antibodies (Ab). The Ab- producing c e l l s are thought to be of bone marrow o r i g i n i n mammals and from the bursa of Fabricius i n aves. These "B - c e l l s " produce s p e c i f i c antibodies to a wide v a r i e t y of substances including chemicals such as dinitrophenol (DNP), c e l l surface products including proteins and b a c t e r i a l l i p o - polysaccharides, v i r a l coat proteins and such substances as bovine serum albumin (BSA) and human chorionic gonadotrophin (HCG). B - c e l l s carry immunoglobulin molecules on t h e i r surfaces and do not appear to be d i r e c t contact k i l l e r s (1). Lymphocytes are found i n a v a r i e t y of adult mammalian tissues such as the spleen, thymus, bone marrow, lymph nodes, t o n s i l s , appendix and peyer's patches of the i n t e s t i n a l w a l l . 2 In aves, the caecae and bursa of Fabricius are a d d i t i o n a l sources of lymphocytes, but the lymph nodes, t o n s i l s and appendix are absent. The organs of primary i n t e r e s t i n the developing embryo are the thymus, which i s the source of T - c e l l s , and the bursa of Fabricius, which i s the source of B - c e l l s . The spleen i s also of i n t e r e s t as i t i s the s i t e of greatest accumulation of mature lymphocytes of both classes. The existance of the bursa, a s i n g l e organ postulated to control the development of the humoral immune system, i s of great convenience to the Immunologist. In most species t h i s function i s att r i b u t e d to widely dispersed t i s s u e . Removal of t h i s primary l e v e l organ should enable the investigator to examine the nature and function of th i s branch of the immune system during i t s development. Removal of the bursa can be accomplished by eith e r s u r g i c a l or hormonal techniques. Hormonal bursectomy, which i s performed early i n development i s , however, somewhat complicated by the side e f f e c t s of introducing large quantities of s t e r o i d hormones into a d i f f e r e n t i a t i n g system. Surgical removal of the bursa at the time of hatching also has d i s - advantages. Late removal allows a period of time between the early appearance of stem c e l l s i n the bursa and the removal of the organ during which maturing stem c e l l s may escape to seed other lymphoid organs. The sequential seeding of the secondary l e v e l organs with c e l l s capable of producing f i r s t one then another class of immunoglobulins may explain the s e l e c t i v e effects of bursectomy near the time of hatching. Removal of the bursa or presumptive bursal tis s u e very early i n development should circumvent the problems of the side effects of steroids and of seeding of other organs p r i o r to bursectomy. Such an operation i s possible only during a short period between the i n i t i a t i o n of the t a i l f o l d and the time at which the f r a g i l i t y of the surrounding membranes and blood vessels makes the operation p h y s i c a l l y impossible. In this investigation, a technique for s u r g i c a l removal of the 3 presumptive bursal tissue at 72 hrs in o v o was used to produce birds d e f i c i e n t i n B- c e l l s and therefore incapable of mounting an humoral immune response (2). These s u r g i c a l l y bursectomized (SBx) birds were raised for periods of up to 10 months and a series of tests to investigate the status of t h e i r immune systems was c a r r i e d out. Their lymphoid tissues were h i s t o l o g i c a l l y examined f o r the presence of germinal centres, the s i t e s of lymphocyte pro- l i f e r a t i o n . Their sera were examined by Immunoelectrophoresis for the presence of immunoglobulin; they were challenged w i t h s p e c i f i c antigens and t e s t e d f o r antibody production; and t h e i r lymphocytes were cultured in v i t r o i n the presence of mitogens. The re s u l t s of these tests were compared with s i m i l a r tests on normal b i r d s . In addition to these s u r g i c a l bursectomies, some hormonal bursectomies were performed using testosterone propionate. These HBx chicks were subjected to many of the same tests as the SBx chicks. They were also tested f o r t h e i r a b i l i t y to r e j e c t skin g r a f t s . Their responses were compared to those of normal and SBx b i r d s . The data obtained from t h i s study should supplement present knowledge and perhaps a i d i n c l a r i f y i n g the ro l e of the bursa of Fabricius i n the developing avian immune system. 4 LITERATURE REVIEW A. The Bursa of F a b r i c i u s . The b u r s a of F a b r i c i u s , d i s c o v e r e d i n 1621 by Hieronymus F a b r i c i u s (3), i s a s m a l l o v a l sac o f l a r g e l y lymphoid t i s s u e connected by a s h o r t s t a l k t o the d o r s a l r e g i o n of the c l o a c a l - c o l o n i c j u n c t i o n o f the l a r g e i n t e s t i n e . The bur s a forms e a r l y i n embryonic l i f e , i s f u l l y developed i n the immature b i r d and r e g r e s s e s around the time of s e x u a l m a t u r i t y (4). The time of maximum mean weight v a r i e s among breeds o f c h i c k e n s , r a n g i n g from A\ - 6 weeks i n White Leghorns to 10 -12 weeks i n Barred c r o s s e s . In the White Leghorn, the maximum burs a to body weight r a t i o a t 3 weeks o f age i s 0.05%. F i g u r e 1 shows burs a development as a percentage o f body weight o f White Leghorn c h i c k e n s . The b u r s a s t a r t s t o r e g r e s s a t about 7 weeks o f age; the r i s i n g l e v e l o f androgens a t the onset o f s e x u a l m a t u r i t y hastens the process o f i n v o l u t i o n so the organ n e a r l y d i s a p p e a r s by the end o f the f i r s t y e a r . The s i z e o f the b u r s a tends t o correspond w i t h t h a t o f the s p l e e n and p a r a l l e l s the growth and i n v o l u t i o n o f the thymus d u r i n g the f i r s t few months o f development (5). The b u r s a o f F a b r i c i u s was long thought t o be i n v o l v e d e i t h e r i n the d i g e s t i v e process, due to i t s a s s o c i a t i o n w i t h the gut, or i n the onset o f s e x u a l m a t u r i t y , as i t begins t o re g r e s s a t t h i s time. The nature o f i t s r o l e i n the immune system was a c c i d e n t a l l y d i s c o v e r e d by G l i c k ^ t a_l. (6). B i r d s which had been s u r g i c a l l y b u r s e ctomized a t 2 weeks o f age were found to be i n c a p a b l e o f mounting an immune response when used i n a demonstration o f a n t i b o d y p r o d u c t i o n . B. S t r u c t u r e o f the Bursa. The i n n e r s u r f a c e of the bur s a i s f o l d e d i n t o 11 - 13 primary p l i c a e , each o f which has 6 - 7 secondary f o l d s . The p l i c a e a re l i n e d w i t h columnar e p i t h e l i u m , below which i s l o c a t e d the t u n i c a p r o p r i a . Each p l i c a c o n t a i n s a v e i n and an a r t e r y . The p l i c a e and the w a l l s of the b u r s a are t h i c k e n e d 0.06 u J D Z 0.05 ' !• i 0.04 0.03 / 0.02 0.01 IgA| i 1 i i • i i 1 1 i i F i g u r e 1. Age 3 4 (weeks) Bursa development shown as a percentage of body weight, The times of i n i t i a t i o n of immuno- g l o b u l i n s y n t h e s i s are shown. N. 6 by numerous l a r g e lymphoid f o l l i c l e s which n e a r l y f i l l the t u n i c a p r o p r i a (Figure 2 ) . The f o l l i c l e s a re found i n p o l y - h e d r a l formations, s e p a r a t e d from one another by l o o s e c o n n e c t i v e t i s s u e , s m a l l b l o o d v e s s e l s and c a p i l l a r i e s . Each f o l l i c l e i s d i v i d e d i n t o a c e n t r a l medulla and a p e r i p h e r a l c o r t e x by a basement membrane which i s continuous w i t h the basement membrane o f the s u r f a c e e p i t h e l i u m . The medulla i s the main lymphocytopoetic area and c o n t a i n s d e v e l o p i n g and mature lymphocytes (7). C. Development of the Bursa. The b u r s a l primordium f i r s t appears a t about 4 days of i n c u b a t i o n as an e p i t h e l i a l p r o l i f e r a t i o n a long the v e n t r a l c audal area o f c o n t a c t between the c l o a c a and the e x t e r n a l ectodermal e p i t h e l i u m (8). By day 6 an e p i t h e l i a l anlage p r o j e c t s from the d o r s o - c a u d a l margin of the u r o d a e l membrane. T h i s anlage c o n t a i n s numerous lacunae which, a t 7 days, b e g i n to c o a l e s c e to form a lumen which becomes continuous w i t h t h a t of the proctodaeum. The b u r s a then r o t a t e s foreward to l i e v e r t i c a l l y and grows out on a c y l i n d r i c a l s t a l k . The f i r s t p l i c a e appear a t 10 or 11 days o f i n c u b a t i o n and by 15 days the lumen c o n t a i n s about 12 p l i c a e . From the 12th to 16th days the p r i m a t i v e e p i t h e l i u m along the basement membrane o f the medulla t h i c k e n s and buds b e g i n to p r o j e c t i n t o the t u n i c a p r o p r i a . The bud f o r m a t i o n i s a s s o c i a t e d w i t h v a s c u l a r i z a t i o n and an i n c r e a s e i n the a l k a l i n e phosphatase a c t i v i t y o f the s u b e p i t h e l i a l mesenchyme. A t the s i t e s of bud f o r m a t i o n the e p i t h e l i a l l a y e r becomes detached from the basement membrane; the buds form s o l i d masses of c e l l s and c o n t i n u e to e n l a r g e . Lymphoblasts b e g i n to appear i n the b u r s a when the buds reach about 60^ i n diameter (about 16 d a y s ) . Although Ackerman and Knouff (7) c o n s i d e r these c e l l s to o r i g i n a t e i n the e p i t h e l i a l bud i t s e l f , c u r r e n t o p i n i o n i s t h a t they are o f y o l k sac o r i g i n . Moore and Owen (9), working w i t h p a r a b i o t i c c h i c k embryos of o p p o s i t e sexes w i t h a sex chromosome marker, 7 Figure 2. Cross section of the bursa of Fabricius, showing p l i c a e and lymphoid f o l l i c l e s , (from Romanoff, 10). 8 demonstrated t h a t up to 50% of the d i v i d i n g c e l l s i n the b u r s a are d e r i v e d from the p a r t n e r . They take t h i s t o i n d i c a t e an e x t r i n s i c source of p r o b a b l y blood-borne c e l l s . The b u r s a l lymphocytes are i n d i s t i n g u i s h a b l e from those found i n o t h e r lymphoid tissues-. The appearance o f lymphoblasts throughout the m e d u l l a r y p o r t i o n of the f o l l i c l e s c o n t i n u e s u n t i l the f o l l i c l e s e n l a r g e to 100 - 300 y . The lymphoblasts c o n t i n u e c h a r a c t e r i s t i c d i f f e r e n t i a t i o n through decrease i n c e l l s i z e , i n n u c l e a r s i z e and b a s o p h i l i a and i n a c o n d e n s a t i o n of chromatin. Small lymphocytes appear i n t h e b u r s a b e t w e e n 16 and 18 days of i n c u b a t i o n . The p r e v i o u s l y u n d i f f e r e n t i a t e d c e l l s a long the basement membrane become l e s s b a s o p h i l i c and e x h i b i t a decrease i n n u c l e a r s i z e a t the same time as the s m a l l lymphocytes appear. Two t i s s u e s c o n t r i b u t e to the f o r m a t i o n of the c o r t e x : the mesenchymal c e l l s o f the t u n i c a p r o p r i a and some o f the u n d i f f e r e n t i a t e d e p i t h e l i a l c e l l s which f i n d t h e i r way i n t o the t u n i c a d u r i n g the e a r l y stages o f bud f o r m a t i o n . As the bud p r o j e c t s i n t o the t u n i c a the subadjacent mesenchymal c e l l s l o s e t h e i r c o n t i n u i t y w i t h the basement membrane. E n l a r g e - ment o f the bud crowds the mesenchymal c e l l s i n t o a r e l a t i v e l y compact c e l l u l a r zone about the e p i t h e l i a l bud. A t the s t a r t o f lymphoblast appearance (16 days) a number of u n d i f f e r e n t i a t e d e p i t h e l i a l and b l a s t c e l l s from the p e r i p h e r y of the medulla may pass through the basement membrane i n t o the c o r t e x . The c o r t e x then a l s o transforms i n t o a lymphocytopoetic area w i t h r e t i c u l a r c e l l s and immature lymphocytes. D. Immunoglobulin S y n t h e s i s . Immunofluorescent s t a i n i n g o f b u r s a l c r o s s s e c t i o n s (with f l u o r e s c e i n l a b e l l e d anti-immunoglobulin sera) shows no immunoglobulin producing c e l l s a t 15 days o f i n c u b a t i o n (11). More s e n s i t i v e t e s t s f o r s p e c i f i c immunoglobulins i n d i c a t e t h a t a s m a l l q u a n t i t y of IgM i s produced by 18 days o f 9 i n c u b a t i o n (12). Large numbers o f competent c e l l s appear i n the f o l l i c l e s by 4 - 7 days a f t e r h a t c h i n g . By t h i s time immunofluorescent c e l l s a re a l s o found i n the s p l e e n and i n the g e r m i n a l c e n t r e s o f the i n t e s t i n e . IgG s y n t h e s i s i n the bu r s a and s p l e e n f o l l o w s the appearance o f IgM. S i m i l a r l y , i n the response o f the mature immunized c h i c k e n , IgM type a n t i - body i s formed p r i o r t o IgG type a n t i b o d y (13). Kincade and Cooper (14) used s p e c i f i c a n t i b o d i e s to the IgM (yu) type and IgG ( v) type o f heavy c h a i n s and to l i g h t c hains o f immunoglobulin molecules to demonstrate the presence of IgM c o n t a i n i n g c e l l s i n the lymphoid f o l l i c l e s o f the. bursae o f 14 day embryos. They a l s o found t h a t IgG c o n t a i n i n g c e l l s do not appear u n t i l 21 days of i n c u b a t i o n . Expansion of the IgM and IgG c o n t a i n i n g c e l l p o p u l a t i o n s was observed i n the bursae o f 5 day c h i c k s and the spleens o f 8 day c h i c k s . S i n g l e c e l l s from the b u r s a c o n t a i n e d b o t h types o f heavy cha i n s w h i l e s i n g l e c e l l s from the s p l e e n c o n t a i n e d c h a i n s o f o n l y one o r the o t h e r c l a s s o f immunoglobulins. T h i s suggest- ed a developmental s w i t c h from IgM to IgG p r o d u c t i o n o c c u r i n g w i t h i n the b u r s a . T h i s t h e o r y i s f u r t h e r supported by the o b s e r v a t i o n t h a t treatment _in ovo w i t h a n t i - l g M serum f o l l o w e d by n e o n a t a l bursectomy r e s u l t e d i n s u p r e s s i o n o f both IgM and IgG s y n t h e s i s . When the a n t i - l g M treatment was dela y e d u n t i l a f t e r the ne o n a t a l bursectomy however, o n l y IgM s y n t h e s i s was depressed. The authors suggest t h a t the e f f e c t o f _in ovo treatment w i t h a n t i - l g M serum was the d e s t r u c t i o n o f a l l IgM producing c e l l s i n c l u d i n g those which were t o s w i t c h t o IgG s y n t h e s i s . As a l l lymphocytes are i n the b u r s a a t t h i s time, the secondary lymphoid organs have not been seeded. By the time o f h a t c h i n g , however, the s w i t c h i n p r o d u c t i o n has a l r e a d y occured and o n l y the IgM producing c e l l s i n the p e r i p h e r a l t i s s u e s are a f f e c t e d by the treatment. The s w i t c h i n immuno- g l o b u l i n s y n t h e s i s appears to occur e x c l u s i v e l y i n the bursa; some Bx c h i c k s having supernormal l e v e l s o f IgM never r e p a i r t h e i r IgG s y n t h e s i s mechanism, even over a prolonged period;.. (15). . 10 Immunoglobulin p r o d u c t i o n i n the b u r s a d e c l i n e s by about 3 months. That o f the s p l e e n does not d e c l i n e over time; the s p l e e n i s the major source o f competent lymphocytes i n the mature b i r d . The thymus appears to produce no IgM. IgG appears i n s m a l l q u a n t i t i e s a t 2 weeks and strengthens by 3 months. Both immunoglobulins appear i n the i n t e s t i n a l lymphoid c e l l s a f t e r 2 weeks (12). E. Bursectomy. The b u r s a can e a s i l y be s u r g i c a l l y removed from 18 days o f i n c u b a t i o n onwards, although t h e r e are l e s s c o m p l i c a t i o n s a f t e r h a t c h i n g (16,17,18,19). Premature r e g r e s s i o n o f the b u r s a can be induced by the i n j e c t i o n o f ACTH, a d r e n a l c o r t - i c o i d s , sex s t e r o i d s , and t h i o u r a c i l o r by such s t r e s s e s as fowl t y p h o i d , muscular e x e r c i s e o r s t a r v a t i o n (5). I n j e c t i o n o f 0.65 mg o f 1 9 - n o r t e s t o s t e r o n e on the 5th day o f i n c u b a t i o n causes complete i n h i b i t i o n o f b u r s a l development. S m a l l e r doses cause p a r t i a l i n h i b i t i o n . A t 11 - 13 days of i n c u b a t i o n , the time of e p i t h e l i a l bud form- a t i o n , a s i m i l a r dose o f t e s t o s t e r o n e may cause p a r t i a l or complete i n h i b i t i o n o f lymphoid d i f f e r e n t i a t i o n i n the e p i t h - e l i a l bud. T h i s may be due to i n h i b i t i o n o f a l k a l i n e phosphatase a c t i v i t y i n the s u b e p i t h e l i a l mesenchyme (20). T e s t o s t e r o n e p r o p i o n a t e , i n j e c t e d i n t o the albumin o r absorbed through the s h e l l i n an a l c o h o l s o l u t i o n i s a l s o an e f f e c t i v e agent f o r hormonal bursectomy. The b u r s a can a l s o be caused to r e g r e s s a t the time o f h a t c h i n g by i n j e c t i o n of the immunosupressant drug c y c l o p h o s - phamide (21). F. E f f e c t s o f Bursectomy. Bursectomy, whether by s u r g i c a l or hormonal means, has been performed by a l a r g e number o f i n v e s t i g a t o r s i n the attempt to e l u c i d a t e i t s r o l e i n the immune system. The e f f e c t s 11 o f bursectomy a t v a r i o u s times d u r i n g embryonic development and j u s t a f t e r h a t c h i n g p r o v i d e evidence t h a t the b u r s a i s e s s e n t i a l i n the development o f the stem c e l l s o f the humoral immune system. These are the c e l l s which g i v e r i s e t o a p o p u l a t i o n o f c e l l s c a pable o f m i g r a t i o n and of d i f f e r e n t i a t i o n i n t o a n t i b o d y producing c e l l s . The e f f e c t s of bursectomy on the immune system appear to depend on the time and method o f bursectomy. The d i r e c t e f f e c t s of bursectomy g e n e r a l l y i n c l u d e the f o l l o w i n g : a) s u p r e s s i o n o f the a n t i b o d y response. The primary response i s u s u a l l y absent or reduced (22, 23) but the secondary response may be normal or n e a r l y so (24). b) r e d u c t i o n o f immunoglobulin s y n t h e s i s , u s u a l l y more r e s t r i c t i v e o f igG than o f igM p r o d u c t i o n . c) r e d u c t i o n o f the d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n t o h e r e d i t a r y immune t h y r o i d i t i s , b u t not t o t u b e r c u l i n or d i p t h e r i a . d) i n c r e a s e o f m o r t a l i t y due to a v i a n d i s e a s e s such as i n f e c t i o n by S a l m o n e l l a typhimurium and to m y e l o b l a s t o s i s . e) changes i n the b l o o d and t i s s u e c e l l p o p u l a t i o n s . Plasma c e l l s and g e r m i n a l c e n t r e s are reduced i n the s p l e e n and c a e c a l t o n s i l s , a lthough the number o f lymphocytes i n the i n t e s t i n a l e p i t h e l i u m i s i n c r e a s e d . The white c e l l count f o r lymphocytes and h e t e r o p h i l s may be near to normal. Bursectomy combined w i t h x - i r r a d i a t i o n enhances the above e f f e c t s to the p o i n t o f aggammaglobulinemia and a l s o r e s u l t s i n a d e f i c i e n c y i n red b l o o d c e l l s and haemoglobin (25). The time of bursectomy appears to be c r i t i c a l . E a r l y bursectomy (at 5 days _in ovo f o r example) w i l l p r event or reduce the p r o d u c t i o n o f both IgM and IgG type a n t i b o d i e s . Neonatal bursectomy appears to have a major e f f e c t on the p r o d u c t i o n o f IgG w h i l e IgM p r o d u c t i o n i s l e s s i n f l u e n c e d . S e v e r a l weeks a f t e r h a t c h i n g , removal o f the b u r s a does not a f f e c t immunoglobulin p r o d u c t i o n . 12 G. The Endocrine F u n c t i o n o f the Bursa. The e f f e c t s o f bursectomy extend beyond the immune system and have l e d to the p r o p o s i t i o n o f an endocrine f u n c t i o n f o r the b u r s a . M o d i f i c a t i o n o f the a d r e n a l response to s t r e s s f o l l o w i n g bursectomy was f i r s t d e s c r i b e d by Perek and E i l a t (26) who noted t h a t a d r e n a l a s c o r b i c a c i d (AAA) became d e p l e t e d . Freeman (27) p o s t u l a t e d t h a t the b u r s a produces an hormonal f a c t o r which f a c i l i t a t e s the development and m a t u r a t i o n of the a d r e n a l ' s a s c o r b i c a c i d r e p l e t i o n mechanism. Bursectomy, then, would render the animal i n c a p a b l e o f m a i n t a i n i n g s t o r e s o f AAA f o l l o w i n g s t i m u l u s d e p l e t i o n . P i n t e a and Pethes (28) noted t h a t the r a t e o f r a d i o - i o d i n e uptake by the t h y r o i d and the r a t e of t h y r o i d hormone s y n t h e s i s are depressed by bursectomy. The b u r s a i s a l s o p o s t u l a t e d as the source o f an hormonal f a c t o r i n f l u e n c i n g e r y t h r o p o e s i s , as bursectomized and x - i r r a d i a t e d c h i c k s develop severe anaemia when compared to x - i r r a d i a t e d c o n t r o l c h i c k s (29). The e x i s t a n c e o f any b u r s a l hormonal p r o d u c t has not been c o n c l u s i v e l y demonstrated. I f such a p r o d u c t does e x i s t , i t would have to be c l a s s i f i e d as a 'developmental hormone' to accomodate the range of i t s e f f e c t s . 13 PART I : S u r g i c a l and Hormonal Bursectomy Techniques, H i s t o l o g y and S k i n G r a f t i n g . Methods: i ) E a r l y embryonic s u r g i c a l bursectomy. F e r t i l e eggs were o b t a i n e d from White Leghorn s t o c k main- t a i n e d on the P o u l t r y Farm o f the U n i v e r s i t y o f B r i t i s h Columbia. A l l eggs were i n c u b a t e d f o r 3 days (72 h r s ) i n a Jamesway Model 252 Incubator under s t a n d a r d c o n d i t i o n s . The eggs were p l a c e d s m a l l end up f o r 1 h r p r e c e e d i n g the o p e r a t i o n . The eggs were s u r f a c e s t e r i l i z e d w i t h 70% e t h a n o l and the o p e r a t i o n was c a r r i e d out under s t e r i l e c o n d i t i o n s . One egg was i n i t i a l l y s a c r i f i c e d t o p r o v i d e albumin and s h e l l membranes f o r f i l l i n g and s e a l i n g the opened eggs. The empty s h e l l s were s t o r e d open end down i n an egg c a r t o n to prevent d r y i n g o f the membranes. A s m a l l h o l e was made i n the narrow end o f the egg, thus p r e s e r v i n g the a i r c e l l , and e n l a r g e d w i t h f o r c e p s u n t i l the whole embryo was exposed (about 1 cm i n d i a m e t e r ) . Albumin was added from a s y r i n g e to f l o a t the embryo to the top o f the h o l e . The embryos were then staged a c c o r d i n g to Hamburger and Hamilton (30) and o n l y those o f stage 17 - 18 were used ( F i g u r e 3 ) . Operations on a c c e p t a b l e embryos were c a r r i e d out under a d i s s e c t i o n microscope (mag. 120x). A c a t a r a c t k n i f e was used to make a s m a l l s l i t i n the c h o r i o n p e r p e n d i c u l a r to the embryonic a x i s j u s t p o s t e r i o r to the l e g buds. The t i p o f the k n i f e was i n s e r t e d to one s i d e o f the m i d l i n e and the opening was extended to the o p p o s i t e s i d e . The amnion was c u t i n the same manner. Care was taken not t o puncture the v i t e l l i n e c i r c u l a t i o n or the y o l k sac. The t i p o f the t a i l was then drawn out to l i e on top o f the c h o r i o n ( F i g u r e 4 ) . Albumin or c h i c k r i n g e r (Appendix) was added to p r e v e n t d e s i c c a t i o n o f the embryo. A l o o p o f c h i l d ' s h a i r , r i n s e d i n 70% e t h a n o l and s t e r i l e c h i c k r i n g e r s o l u t i o n was then passed over the f r e e end o f the t a i l and drawn up almost to the l e g buds. The knot was t i g h t - ened s u f f i c i e n t l y to l i g a t e the p a i r e d d o r s a l a o r t a e but not 14 to c u t the t a i l . The t a i l and the f r e e ends o f the h a i r were trimmed o f f w i t h i r i s s c i s s o r s and removed ( F i g u r e 5). The stump was tucked back under the c h o r i o n and a l i t t l e albumin was added to f i l l the egg. A p i e c e o f the double s h e l l membrane was c u t to f i t and p l a c e d , i n s i d e down, over the opening i n the s h e l l . T h i s was allowed t o d r y f o r a few minutes then a l a y e r o f h i g h m e l t i n g p o i n t p a r a f f i n (mp 65 - 70 C) was p a i n t e d s p a r i n g l y over the edges o f the patch. The eggs were r e t u r n e d to the i n c u b a t o r s m a l l end down and checked a f t e r 1 h r f o r le a k s which were patched w i t h a d d i t i o n a l wax. I f both s h e l l membranes were used f o r the patch few leaks o c c u r r e d . The eggs were then i n c u b a t e d under s t a n d a r d cond- i t i o n s f o r the remainder o f t h e i r term (2). I n i t i a l l y , a number o f sham o p e r a t i o n s were a l s o performed but as the c h i c k s hatched appeared normal t h i s p r a c t i c e was d i s c o n t i n u e d . C o n t r o l b i r d s were normal White Leghorn c h i c k s i n c u b a t e d under s t a n d a r d c o n d i t i o n s w i t h the bur s e c t o m i z e d c h i c k s . i i ) E a r l y embryonic hormonal bursectomy. F e r t i l e White Leghorn eggs were i n c u b a t e d f o r 3 days as above. They were s u r f a c e s t e r i l i z e d w i t h 70% e t h a n o l and a p a i r o f h o l e s was d r i l l e d through the s h e l l and s h e l l membranes. One p e n e t r a t e d i n t o the a i r c e l l a t the l a r g e end o f the egg, the o t h e r allowed access to the albumin through the s i d e of the egg. Each egg was i n j e c t e d through t h i s l a t t e r h o l e w i t h 0.1 ml o f a 20 mg/ml s o l u t i o n o f t e s t o s t e r o n e p r o p i o n a t e i n peanut o i l ( t h i s i s a f i n a l dose o f 2 mg/egg). The h o l e s were then s e a l e d w i t h a drop o f p a r a f f i n and the eggs were r e t u r n e d t o the i n c u b a t o r f o r the remainder o f t h e i r term, i i i ) P r e l i m i n a r y examinations. A l l c h i c k s were weighed on the day o f h a t c h i n g and a t weekly i n t e r v a l s t h e r e a f t e r . Those which d i e d were examined f o r the presence o f b u r s a l remnants. Chicks were b l e d and s a c r i f i c e d a t weekly i n t e r v a l s and the s p l e e n , thymus and lower l a r g e i n t e s t i n e were e x c i s e d and prepared f o r h i s t o l o g i c a l 15 F i g u r e 3 F i g u r e 4 3. Chick embryo a t 3 days (72 h r s ) i n c u b a t i o n — d o r s a l view and l o n g i t u d i n a l s e c t i o n through t a i l r e g i o n . 4. T a i l r e g i o n ( d o r s a l view and l o n g i t u d i n a l s e c t i o n ) showing i n c i s i o n through membranes and t i p o f t a i l l y i n g f r e e . 5. T a i l r e g i o n ( d o r s a l view only) f o l l o w i n g l i g a t i o n and removal of presumptive b u r s a l t i s s u e . TAbbreviations: am : amnion; ch : c h o r i o n ; h i : h a i r loop; l b : limb bud; pv : p o s t e r i o r v i t e l - l i n e v e i n ; ys : y o l k sac. Figure Figure F i g u r e 16 examination. Four normal and 4 bursectomized chicks were s a c r i f i c e d on the day a f t e r hatching and t h e i r spleen and body weights were recorded. Records were kept of h a t c h a b i l i t y and mortality of normal, SBx and HBx chicks. Serum was c o l l e c t e d from the blood samples and stored at -20 C u n t i l analysed f o r serum proteins and immunoglobulins, iv) H i s t o l o g i c a l examination. Tissues were stored i n buffered formalyn (Appendix) u n t i l prepared f o r h i s t o l o g i c a l sectioning. Standard wax embedded s e r i a l sections were prepared from the spleen, thymus, caecae a n d t r i d e n t a r e a o f t h e l a r g e i n t e s t i n e f o r b o t h n o r m a l and SBx chicks. Sections were also prepared from the bursa of normal chicks and the c l o a c a l area of SBx chicks. The sections were stained with hematoxylin and eosin and were examined microscopically f o r the presence of germinal centres and lymphoid c e l l s . v) Skin g r a f t i n g . The capacity for g r a f t r e j e c t i o n by HBx, a few SBx and normal White Leghorn (WL) chicks was tested by r e c i p r o c a l g r a f t i n g . Patches of dorsal skin were exchanged between the above mentioned groups and New Hampshire Red (NH) chicks. Grafting was done as soon as possible a f t e r the hatching chicks had dried. The down was plucked from the backs of the chicks and the exposed skin was painted with c o l l o i d o n to dry and stretch the skin. When thi s was dry, 0.1 ml of chick ringer s o l u t i o n was injected subcutaneously near the midline over the p e l v i s , l i f t i n g the epidermis free from the under- l y i n g tissue and bone (31). A small c i r c u l a r biopsy punch was used to cut out a c i r c l e of skin about 5 mm i n diameter. Patches of skin were prepared simultaneously from WL and NH chicks and were exchanged between the two. The g r a f t s were placed i n t h e i r normal ant e r i o r - p o s t e r i o r o r i e n t a t i o n and a drop of c o l l o i d o n was added to help dry the g r a f t i n place. The wound was protected with an E l a s t o p l a s t spot bandaid. The grafts were examined at weekly i n t e r v a l s (or at the time o f death) and the p r o g n o s i s o f the g r a f t was noted. R e s u l t s and D i s c u s s i o n : A t o t a l o f 1466 embryos were s u r g i c a l l y b u r s e c t o m i z e d . Of these, 176 (12%) hatched and 46 (27% o f hatch) s u r v i v e d p a s t one week o f age. S i m i l a r l y f o r the hormonally bursectomized embryos, 51 of 239 (21%) hatched and 16 (31%) s u r v i v e d p a s t one week. M o r t a l i t y d u r i n g the i n c u b a t i o n p e r i o d appeared to be g r e a t e s t j u s t a f t e r the o p e r a t i o n , p r o b a b l y due to wounding d u r i n g the o p e r a t i o n . There was another peak o f m o r t a l i t y a t 18 days o f i n c u b a t i o n . These embryos u s u a l l y had mal- formed limbs and the y o l k sac was remaining o u t s i d e the body c a v i t y . Some o f the c h i c k s were a b l e to p i p the s h e l l b u t c o u l d not break out o f the s h e l l w i t h o u t h e l p . The m o r t a l i t y peaks o f the Bx embryos cor r e s p o n d to m o r t a l i t y peaks i n normal d e v e l o p i n g embryos, i n d i c a t i n g c r i t i c a l requirements which must be met a t t h i s time. During the f i r s t week a f t e r h a t c h i n g the h i g h m o r t a l i t y o f the Bx c h i c k s was l a r g e l y due to compaction o f the l a r g e i n t e s t i n e owing to poor development of the a n a l s p h i n c t e r . Those c h i c k s which s u r v i v e d the f i r s t week c o n t i n u e d to develop r e l a t i v e l y n o r m a l l y u n t i l the time they were s a c r i f i c e d . Four b i r d s d i e d a f t e r 6 - 8 weeks, a l l s u f f e r i n g from acute n e p h r i t i s . The SBx c h i c k s were about 6 gm l i g h t e r a t h a t c h i n g than normal c h i c k s and by 8 weeks were about 13 0 gm l i g h t e r (Table I ) . Autopsy i n d i c a t e d t h a t the Bx b i r d s l a c k e d the b u r s a , about 1/3 o f the l a r g e i n t e s t i n e and the l a s t few v e r t e b r a e . The caecae were not a f f e c t e d and the c l o a c a appeared normal i n b i r d s s e v e r a l weeks o f age. Development o f the o v i d u c t i n female c h i c k s appeared normal. S e v e r a l hens were kept f o r over 10 months and produced normal eggs. The spleens o f newly hatched SBx c h i c k s weighed an average o f 0.012 g (0.040 % o f body w e i g h t ) . Those o f normal c h i c k s 18 weighed an average o f 0.22 g (0.054% o f body w e i g h t ) . The d i f f e r e n c e i n s p l e e n to body weight r a t i o s i n d i c a t e s t h a t the spleens o f the Bx c h i c k s were r e a l l y s m a l l e r than those of normal c h i c k s (Table I I ) . The spleens o f the Bx c h i c k s appeared darker than those o f normal c h i c k s , perhaps because of the r e l a t i v e l y h i g h e r p r o p o r t i o n o f r e d c e l l s . H i s t o l o g i c s e c t i o n s showed the Bx spleens to have fewer g e r m i n a l c e n t r e s than normal c h i c k s ( F i g u r e 6). The thymus glands of the Bx c h i c k s a l s o appeared s m a l l e r than those o f the normal WL c h i c k s , but the d i f f u s e nature of the t i s s u e and the abundance of a s s o c i a t e d f a t deposits made r e l i a b l e weight data d i f f i c u l t t o o b t a i n . H i s t o l o g i c a l s e c t i o n s , however, r e v e a l t h a t the s m a l l s i z e o f the thymic lobes i s due to incomplete development o f the c o r t e x ( F i g u r e 7 ) . The p r o g n o s i s o f the s k i n g r a f t s on SBx, HBx, normal White Leghorn and normal New Hampshire c h i c k s was r e c o r d e d a t 7, 14 and 28 days a f t e r g r a f t i n g (Table I I I ) . The g r a f t s were observed to pass through a s e r i e s o f stages i n the r e j e c t i o n p r o c e s s . Reddening and s l i g h t s w e l l i n g o f the area was f o l l o w e d by s w e l l i n g o f the g r a f t s i t e t o about 5 mm i n h e i g h t . The edges o f the g r a f t e d patch then s e p a r a t e d from the h o s t t i s s u e and f i n a l l y the g r a f t b l a c k e n e d and f e l l o f f . Success- f u l g r a f t s d i d not pass beyond the s l i g h t s w e l l i n g stage; t h i s soon su b s i d e d and the g r a f t patch appeared to be i n c o r p o r a t e d i n t o the s k i n of the h o s t . G r a f t s were r e c o r d e d as r e j e c t e d when the edges o f the patch s e p a r a t e d from the h o s t t i s s u e as r e j e c t i o n seemed to be i r r e v e r s i b l e from t h i s p o i n t . As a l a r g e number o f the Bx c h i c k s d i e d w i t h i n the f i r s t week, the complete h i s t o r y o f the g r a f t was o b t a i n e d i n o n l y about 20% of the cases. These r e s u l t s do, however, show some tre n d s . The NH b i r d s r e j e c t e d the g r a f t s w i t h i n the f i r s t week i n 80% o f the t r a n s f e r s . Normal WL c h i c k s were somewhat slower, but a l l r e j e c t e d the g r a f t s w i t h i n 28 days. Both the SBx and HBx groups, however, c o n t a i n e d a few b i r d s which showed no s i g n s of g r a f t r e j e c t i o n a t the end o f 28 days. A t 8 weeks 19 of age, as feathering neared completion, the grafts s t i l l appeared i n t a c t . Observation of the thymus gland through the skin of the neck of these birds showed them to be smaller than the thymus glands of normal birds observed i n the same way. If this smaller s i z e was due to the absence or decrease of the thymic cortex as was found i n other Bx bir d s , the poor g r a f t r e j e c t i o n may be explained as a lack of competent lymphocytes. It i s also possible that some B- c e l l s are necessary i n the process of g r a f t r e j e c t i o n and that some Bx birds were unable to b u i l d up a s u f f i c i e n t population of these c e l l s to carry out r e j e c t i o n . 20 Table I Average weights o f normal and s u r g i c a l l y b ursectomized c h i c k s from h a t c h to 8 weeks of age. Normal SBx Age Weight (g) E r r o r Sample S i z e Weight (g) E r r o r Samp Siz< Hatch 41.2 4.1 29 36.3 4.4 29 1 wk. 63.8 7.3 8 47.9 2.2 6 2 wk. 197.6 25.3 8 93.3 14.4 6 3 wk. 288.1 30.8 8 141.3 22.9 3 4 wk. 435.0 17.7 2 210.0 40.6 2 5 wk. 433.8 85.5 6 247.8 60.5 2 6 wk. - - - 310.7 94.1 3 8 wk. 808.3 265.7 3 675.3 124.8 3 21 Tab l e I I Spl e e n and body weights f o r day o l d normal and s u r g i c a l l y b u r s e c t o m i z e d c h i c k s . S p l e e n Wt. Body Wt. R a t i o Normal 0.0183 g 41.9 g 0.00043 Normal 0.0217 43.1 0.00050 Normal 0.0213 36.2 0.00058 Normal 0.0246 38.0 0.00064 Average 0.0227 36.8 0.00054 SBx 0.0123 g 29.8 g 0.00041 SBx 0.0160 36.3 0.00044 SBx 0.0144 37.0 0.00038 SBx 0.0079 19.8 0.00039 Average 0.0126 30.7 0.00040 22 F i g u r e 6. H i s t o l o g i c a l s e c t i o n s o f normal (A) and b u r s e ctomized (B) s p l e e n s ; note the reduced number of g e r m i n a l c e n t r e s i n Bx s p l e e n . H & E stain.. M a g n i f i c a t i o n 1 2 0 x . 23 Figure 7. H i s t o l o g i c a l sections of normal (A) and bursectomized (B) thymic lobes; note the reduced thymic cortex i n Bx thymus. H & E s t a i n . Magnification 120 x. 24 Table I I I Prognosis o f s k i n g r a f t s a t a f t e r g r a f t i n g . 7, 14, and 28 days Host (No) Donor 7 (-) days (+) d i e d b e f o r e 14 days 14 days (-) (+) 28 days (-) ( + ) WL (16) NH 4 12 5 2 5 5 0 SBx ( 5) NH 0 5 3 0 2 0 2 HBx (26) NH 6 20 16 1 3 1 2 NH (16) WL 9 7 2 4 1 1 0 NH (26) HBX 23 3 1 1 1 1 0 Those grafts scored as a (-) i n one observation are not included i n the next observation. 25 PART I I : T e s t s of the Humoral Immune System. Methods: i ) Serum samples. Blood was c o l l e c t e d a t weekly i n t e r v a l s by c a r d i a c punc- t u r e or from the j u g u l a r v e i n of young b i r d s and from the wing v e i n of b i r d s over 5 weeks of age. At l e a s t 0.5 ml o f b l o o d was c o l l e c t e d from c h i c k s and up to 5.0 ml was taken from the o l d e r b i r d s . The b l o o d was a llowed to c l o t a t room temperature f o r 3 - 4 h r s , then r e f r i g e r a t e d o v e r n i g h t . The serum was then removed w i t h a p a s t e u r p i p e t t e and s t o r e d i n covered t e s t tubes a t -20 C u n t i l used. i i ) P r e p a r a t i o n of a n t i s e r a . A d u l t c h i c k e n serum was used i n the p r e p a r a t i o n o f r a b b i t a n t i - c h i c k e n s e r a . A m o d i f i c a t i o n o f the method of Hudson e t a l . (32) f o r the s e p a r a t i o n o f ovine serum immunoglobulin was used to o b t a i n p u r i f i e d c h i c k e n IgM. Whole a d u l t c h i c k e n serum was a p p l i e d to a G-200 Sephadex column and e l u t e d w i t h a 0.1 M T r i s - H C l b u f f e r o f pH 8.2 (Appendix). The f i r s t p r o t e i n peak d e t e c t e d by UV spectrophotometry (Pharmacia UV Monitor Model 100) was c o l l e c t e d and c o n c e n t r a t e d by n i t r o g e n p r e s s u r e d i a l y s i s . F i v e mg o f p r o t e i n ( e i t h e r whole serum o r the IgM p u r i f i e d above) i n a 2% s o l u t i o n i n c h i c k r i n g e r was mixed 1:1 w i t h Freunds complete adjuvant and i n j e c t e d subcutaneously i n t o r a b b i t s a t 2 one week i n t e r v a l s . R a b b it b l o o d was c o l l e c t e d from an ear v e i n one week a f t e r the second i n j e c t i o n . The b l o o d was allowed to c l o t as above and the serum c o n t a i n i n g a n t i - c h i c k e n a n t i b o d i e s was s t o r e d a t -20 C. i i i ) Immunoelectrophoresis. A s t a n d a r d immunoelectrophoresis procedure ( c h i c k e n serum i n w e l l s , r a b b i t antiserum i n trough) was used to o b t a i n serum p r o t e i n p a t t e r n s . Serum samples from d a y - o l d c h i c k s through to those s e v e r a l weeks of age were t e s t e d i n t h i s way f o r a l l serum p r o t e i n s and s p e c i f i c a l l y f o r IgM. The p r e c i p i t i n curves were i d e n t i f i e d a c c o r d i n g to Tureen et a l . (33). 26 i v ) T e s t f o r s p e c i f i c a n t i b o d y p r o d u c t i o n . Sheep r e d b l o o d c e l l s (SRBC) were used to s t i m u l a t e s p e c i f i c a n tibody f o r m a t i o n i n normal, SBx and HBx b i r d s . F i v e week o l d b i r d s were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 0.5 ml o f a 2% s o l u t i o n o f SRBC i n c h i c k r i n g e r s o l u t i o n . F i v e days l a t e r the b i r d s were b l e d from the wing v e i n and g i v e n a second i n j e c t i o n o f SRBC. These s e r i a l b l e e d i n g s and i n j e c t - i ons were repeated 3 - 4 times. Serum was c o l l e c t e d from the c l o t t e d b l o o d as above and s t o r e d a t -20 C. The serum from these s t i m u l a t e d b i r d s was t e s t e d f o r the presence o f s p e c i f i c anti-SRBC antibody by m i c r o t i t r e haem- a g g l u t i n a t i o n o f SRBC. Twenty-five JA.1 o f c h i c k e n serum was p l a c e d i n the f i r s t w e l l o f a m i c r o t i t r e p l a t e and d o u b l i n g d i l u t i o n s were made through 11 more w e l l s each c o n t a i n i n g 2 5 ^ i l o f c h i c k r i n g e r . The serum d i l u t i o n s were incubated w i t h 2 5 p i o f 5% SRBC f o r 30 min a t 37 C. The end p o i n t t i t r e was rec o r d e d as t h a t d i l u t i o n of serum which l a s t caused a g g l u t i n - a t i o n o f the SRBC. Re s u l t s and D i s c u s s i o n : A c c o r d i n g to Tureen et al. (33) Immunoelectrophoresis o f s e r a from growing c h i c k s and o l d e r b i r d s shows a changing p a t t e r n o f serum p r o t e i n s as the b i r d s mature. F i g u r e 8 shows t h e i r serum p a t t e r n f o r a 5 day o l d c h i c k . The pre - albumin and b e t a - l i p o p r o t e i n bands show the g r e a t e s t changes w i t h age. Both are p r e s e n t from the time o f h a t c h i n g u n t i l 8 days when the b e t a - l i p o p r o t e i n band d i s a p p e a r s . On the 10th day the prealbumin band s t a r t s to become f a i n t e r and di s a p p e a r s by the 20th day. The gamma g l o b u l i n s appear as t h r e e curves c o r r e s p o n d i n g to IgA, IgM and IgG. A l l t h r e e a re p r e s e n t by 5 days i n normal c h i c k s . A f o u r t h g l o b u l i n f r a c t i o n , perhaps IgD, i s a l s o known to occur b ut i s not c o n s t a n t l y found. T r a n s f e r r i n appears as a prominant band near the o r i g i n i n the younger b i r d s and sometimes d i s a p p e a r s i n o l d e r b i r d s . The b e t a g l o b u l i n s appear more p r o m i n a n t l y i n o l d e r b i r d s . 2 7 The serum samples from the normal White Leghorn c h i c k s showed the expected immunoelectrophoretic p a t t e r n s f o r t r a n s f e r r i n , albumin, l i p o p r o t e i n and the serum g l o b u l i n s . IgM was p r e s e n t from the time o f h a t c h i n g and IgG appeared i n the f i r s t few days. F i g u r e s 9A and 10A show the immuno- e l e c t r o p h o r e t i c p a t t e r n s f o r normal 14 day c h i c k s e r a a g a i n s t anti-whole serum and anti-IgM r e s p e c t i v e l y . The anti- I g M p l a t e s show s e v e r a l bands because the heavy c h a i n s o f immuno- g l o b i n molecules are common to a l l i m m u n o g l o b u l i n s , r e s u l t i n g i n c r o s s r e a c t i v i t y . The t r a n s f e r r i n band i s p r e s e n t because o f i r u c o m p l e t e p u r i f i c a t i o n . T h e s e e x t r a b a n d s c o u l d have been removed by i n c u b a t i o n o f the antiserum w i t h embryonic c h i c k serum p r i o r t o e l e c t r o p h o r e s i s . The immunoelectrophoretic p a t t e r n s f o r 14 day SBx c h i c k serum a g a i n s t r a b b i t anti-whole c h i c k e n serum and a n t i - I g M (Figures 9B and 10B r e s p e c t i v e l y ) show the normal p a t t e r n s f o r t r a n s f e r r i n , l i p o p r o t e i n and albumin. The serum g l o b u l i n s , however, are incomplete; IgM and IgG immunoglobulin bands are absent. In some cases (not shown here) t h e r e were t r a c e s o f IgM appearing s e v e r a l days a f t e r h a t c h i n g , b u t IgG was not found. The h a e m a g g l u t i n a t i o n t e s t s f o r s p e c i f i c a n t i b o d y t o SRBC (Fi g u r e 11) i n d i c a t e t h a t the SBx b i r d s do not produce d e t e c t - a b l e Ab f o l l o w i n g a primary c h a l l e n g e . However, as the pro-- d u c t i o n o f antibody to SRBC r e q u i r e s c o - o p e r a t i o n between B- and T - c e l l s , the absence o f the response may not be e n t i r e l y due t o an absence or r e d u c t i o n o f b u r s a - d e r i v e d c e l l s . T a b l e IV g i v e s the r e s u l t s o f a s e r i e s o f c h a l l e n g e s f o r normal, SBx and HBx b i r d s . Some o f the HBx b i r d s respond t o the primary c h a l l e n g e , b ut the degree of the response i s f a r below t h a t o f the normal b i r d s . Hormonal bursectomy does not always impale the development of the thymic c o r t e x . The a n t i b o d y t i t r e s o f both Bx groups s l o w l y r i s e f o l l o w i n g s u c c e s s i v e c h a l l e n g e s and would p r o b a b l y reach normal l e v e l s i f the s e r i e s were c o n t i n u e d . The reduced lymphocyte p o p u l a t i o n s o f the Bx b i r d s r e q u i r e a 2 8 longer time i n which to b u i l d up s u f f i c i e n t c e l l s producing a s p e c i f i c antibody than do those of normal b i r d s . The HBx birds appear to approach normal l e v e l s more r a p i d l y than the SBx bir d s , perhaps suggesting that the effects of hormonal bursectomy are less severe than those of early s u r g i c a l bursec- tomy. 29 Figure 8 . P r e c i p i t i n bands from Immunoelectrophoresis of normal 5 day chick serum (from Tureen et a l . , 3 3 ) . Key: A. prealbumin B. albumin C. alpha l i p o p r o t e i n D. beta,lipoprotein E. t r a n s f e r r i n F. IgA gamma g l o b u l i n G. IgM gamma g l o b u l i n H. IgG gamma g l o b u l i n 30 A M | B Figure 9. P r e c i p i t i n bands from immunoelectr- phoresis of 14 day normal (A) and bursectomized (B) chick sera against ra b b i t anti-whole chicken serum. 31 Figure 10. P r e c i p i t i n bands from Immunoelectro- phoresis of 14 day normal (A) and bursectomized (B) chick sera against r a b b i t anti-chicken IgM. 32 000000000000 S8" ©©©©©©©©00© < oooooooooooo * © ® ® © • • • • • • 0 © * ©©0©0©©00©0O * ©0000©©0©©0Q) * ©©©©©@©©@®@© » F i g u r e 11. M i c r o t i t r e h a e m a g g l u t i n a t i o n o f SRBC by normal (N), s u r g i c a l l y b u r s e c t o m i z e d (SBx) and hormonally b u r s e c t o m i z e d (HBx) c h i c k e n s e r a f o l l o w i n g primary s t i m u l a t i o n w i t h SRBC. Table IV Haemagglutination t i t r e s f o r normal, SBx and HBx chicken sera following successive stimulations with SRBC. Birds (No) Primary Secondary Tertary Normal (10) 128* 256 - 512 1024 - 2 048 SBx (6) - 8 - 32 256 - 512 HBx (8) 4 8 - 64 512 - 1024 * a t i t r e of 128 indicates that a d i l u t i o n of serum 1/128 i n chick ringer i s the greatest d i l u t i o n which s t i l l contains s u f f i c i e n t anti-SRBC antibody to agglutinate 2 5 ul of 5% SRBC. 34 PART I I I : S t i m u l a t i o n o f s p l e e n , t h y m u s a n d b u r s a c e l l s w i t h m i t o g e n s . I n t r o d u c t i o n t o m i t o g e n s : S e v e r a l p l a n t p r o t e i n s k n o w n t o b e r e d b l o o d c e l l a g g l u t i n i n s a n d t h e b a c t e r i a l p r o d u c t e n d o t o x i n h a v e b e e n s h o w n t o s t i m u l a t e t h e t r a n s f o r m a t i o n o f l e u k o c y t e s _ i n v i t r o . Some o f t h e s e m i t o g e n s a r e t h o u g h t t o s p e c i f i c a l l y s t i m u l a t e T - c e l l s a s t h e y h a v e n o e f f e c t o n m o u s e l y m p h o c y t e s t r e a t e d w i t h a n t i - t h e t a t o k i l l a l l t h y m u s - d e r i v e d c e l l s ( 3 4 ) . O t h e r s , w h i c h h a v e l i t t l e e f f e c t o n T - c e l l s a r e t h o u g h t t o b e s p e c i f i c s t i m u l a n t s o f B - c e l l s , a l t h o u g h t h e e v i d e n c e f o r t h i s i s l e s s c o n c r e t e t h a n t h a t f o r t h e T - c e l l s p e c i f i c m i t o g e n s . P h y t o h a e m a g g l u t i n i n (PHA) i s d e r i v e d f r o m t h e r e d k i d n e y b e a n , P h a s e o l u s v u l g a r i s , b y s a l i n e e x t r a c t i o n . I t s m i t o g e n i c p r o p e r t i e s w e r e d i s c o v e r e d i n 19 59 b y H u n g e r f o r d ^ t a l . ( 3 5 ) . C o n c a n a v a l i n A (ConA) i s a s i m i l a r e x t r a c t f r o m t h e j a c k b e a n . T h e s e t w o m i t o g e n s s t i m u l a t e d i v i s i o n o f m i x e d l e u k o c y t e c u l t u r e s b u t f a i l t o s t i m u l a t e a n t i - t h e t a t r e a t e d c e l l s a n d a r e t h u s c o n s i d e r e d t o b e T - c e l l s p e c i f i c m i t o g e n s . T h e y s t i m u l a t e r a p i d c e l l d i v i s i o n a n d t h e t r a n s f o r m a t i o n o f u n d i f - f e r e n t i a t e d l y m p h o c y t e s i n t o b l a s t c e l l s . P H A - i n d u c e d t r a n s - f o r m a t i o n d o e s n o t a p p e a r t o r e q u i r e t h e p r e s e n c e o f m a c r o - p h a g e s n e c e s s a r y f o r a n t i g e n - i n d u c e d t r a n s f o r m a t i o n ( 3 4 ) . E n d o t o x i n i s a l i p o p o l y s a c c h a r i d e p r o d u c t o f b a c t e r i a , u s u a l l y o b t a i n e d f r o m c u l t u r e s o f g r a m n e g a t i v e b a c t e r i a s u c h a s j E ^ c o l i . I t h a s b e e n s h o w n t o i n d u c e DNA s y n t h e s i s i n B l y m p h o c y t e s a n d h a s n o e f f e c t o n t h y m u s - d e r i v e d l y m p h o c y t e s ( 3 6 , 3 7 ) . E n d o t o x i n i s c a p a b l e o f s t i m u l a t i n g B - c e l l s i n t h e a b s e n c e o f T - c e l l s — i t i s a T - c e l l - i n d e p e n d a n t a n t i g e n ( 3 8 ) . T h e r e s p o n s e o f a n _ i n v i t r o c e l l p o p u l a t i o n t o a v a r i e t y o f c e l l - s p e c i f i c m i t o g e n s a l l o w s f o r s p e c u l a t i o n a s t o t h e r e l a t i v e p r o p o r t i o n s o f B - a n d T - c e l l s i n t h a t p o p u l a t i o n . F o r t h i s p u r p o s e , s p l e e n a n d t h y m u s c e l l s f r o m b u r s e c t o m i z e d b i r d s a n d s p l e e n , t h y m u s a n d b u r s a c e l l s f r o m n o r m a l b i r d s w e r e c u l t u r e d i n t h e p r e s e n c e o f m i t o g e n s . 35 Methods: Spleen and thymus c e l l s from 5 to 8 week o l d SBx c h i c k e n s and s p l e e n , thymus and b u r s a c e l l s from normal c h i c k e n s were c u l t u r e d i n the w e l l s o f m i c r o t i t r e p l a t e s and were s t i m u l a t e d w i t h 2 T - c e l l s p e c i f i c (PHA and ConA) mitogens and one B - c e l l s p e c i f i c (endotoxin) mitogen i n an e f f o r t t o determine the e f f e c t of bursectomy on the B- and T - c e l l p o p u l a t i o n s . C e l l s f o r c u l t u r e were prepared i n the f o l l o w i n g manner. The b i r d s were b l e d to p r o v i d e autologous serum f o r the c u l t u r e medium then were k i l l e d by i n t r a c a r d i a c i n j e c t i o n o f 10 cc o f a i r . The a p p r o p r i a t e organs were removed a s c e p t i c - a l l y , trimmed o f f a t and washed i n c h i c k r i n g e r . Each organ was then p l a c e d i n 5 ml o f c h i c k r i n g e r and 1 ml o f autologous serum and the c e l l s were g e n t l y teased from the organ w i t h two p a i r s o f f o r c e p s . A s i n g l e c e l l s u s p e n s i o n was o b t a i n e d by p a s s i n g the t i s s u e fragments through a 1 ml s y r i n g e equipped w i t h a 26 guage needle. The c e l l s were washed 2 times i n c h i c k r i n g e r and f i n a l l y resuspended i n RPMI 1640 c u l t u r e medium (Appendix) s u p p l e m e n t e d w i t h 1% p e n i c i l l i n / s t r e p t o m y c i n . A sample of the s u s p e n s i o n was then d i l u t e d 1/100 i n c u l t u r e medium and the v i a b l e lympho- c y t e s counted w i t h a hemocytometer i n a 1/1 mixture w i t h t r y p a n b l u e . The c e l l s u spension was then d i l u t e d t o o b t a i n 5 ml samples 7 6 o f lymphocyte c o n c e n t r a t i o n s o f 2.5 x 10 and 2.5 x 10 c e l l s per ml i n b o t h serum f r e e and 5% autologous serum supplemented c u l t u r e medium. The c e l l s were d i s p e n s e d w i t h a Hamilton s y r i n g e i n t o the w e l l s o f m i c r o t i t r e p l a t e s to f i n a l concen- 6 5 t r a t i o n s of 5 x 10 and 5 x 10 c e l l s per w e l l f o r both media. T r i p l i c a t e w e l l s were made f o r c o n t r o l s and f o r each type o f mitogen; a l l c e l l types were s t i m u l a t e d w i t h a l l mitogens. The mitogens were added i n the f o l l o w i n g c o n c e n t r a t i o n s : PHA - 10 g/ml, ConA - 20 g/ml, endotoxin - 20 g/ml each i n 0.05 ml o f s a l i n e . The t o t a l volume o f each w e l l was made up to 0.2 5 ml w i t h the a p p r o p r i a t e medium and the c u l t u r e s were 36 i n c u b a t e d a t 37 C f o r 48 h r . A t t h i s time 0.05 ml o f HTdr ( t r i t i a t e d thymidine) or 20 j/ici/m.1 °f i s o t o p e was added to each w e l l and the c u l t u r e s were in c u b a t e d f o r a f u r t h e r 12 h r s . The c e l l s were h a r v e s t e d by a vacuum c o l l e c t i o n machine, c o n s t r u c t e d f o r t h i s purpose, onto g l a s s f i b r e f i l t e r s . The c e l l s were washed 5 times w i t h tap water to remove excess r a d i o a c t i v e m a t e r i a l , t o l y s e the c e l l s and to p r e c i p i t a t e the DNA a l l i n one s t e p . The f i l t e r s were then d r i e d under a h e a t lamp, p l a c e d i n s c i n t i l l a t i o n v i a l s w i t h 3 ml o f s c i n t i l l a t i o n f l u i d ( L i q u i f l u o r and t o l u e n e , no methanol) and counted i n a s c i n t i l l a t i o n c o u n t e r . Dpms were not determined as the r e s u l t s were expressed as a r a t i o o f two counts. S t i m u l a t i o n by a mitogen = CPM o f mitogen s t i m u l a t e d c u l t u r e . CPM o f a n o n - s t i m u l a t e d c u l t u r e R e s u l t s and D i s c u s s i o n : A s e r i e s of p r e l i m i n a r y t e s t s w i t h v a r i o u s combinations of c e l l d e n s i t y , serum types and serum c o n c e n t r a t i o n s determin- ed t h a t homologous, p r e f e r a b l y autologous, serum a t a con- 5 c e n t r a t i o n of 5 - 10% and a lymphocyte d e n s i t y o f 5 x 10 to g 5 x 10 produced the most c o n s i s t a n t r e s u l t s . Among these, 5 c u l t u r e s o f 5 x 10 c e l l s per w e l l c u l t u r e d w i t h o u t serum appeared to be the h e a l t h i e s t and were l e a s t o f t e n i n f e c t e d w i t h b a c t e r i a . The serum was most o f t e n the c a r r i e r o f con- t a m i n a t i o n . The u n s t i m u l a t e d o r c o n t r o l c u l t u r e s gave cpms i n the range of s e v e r a l hundred, those o f the mitogen s t i m u l a t e d c u l t u r e s were 3 to 300 times g r e a t e r . Normal s p l e e n c e l l s a t 5 x 10 c e l l s per w e l l w i t h o u t serum were s t i m u l a t e d 25 times w i t h PHA, 17 times w i t h ConA and 40 times w i t h endotoxin. Spleen c e l l s from an SBx c h i c k e n were s t i m u l a t e d 50 times w i t h PHA, 15 times w i t h ConA and o n l y 15 times w i t h endotoxin. The s p l e e n of the SBx b i r d , however, was expected to be d e f i c i e n t i n B - c e l l s . I f t h i s were the 5 case, a sample o f 5 x 10 lymphocytes from such a s p l e e n would 37 c o n t a i n a r e l a t i v e l y s m a l l e r p r o p o r t i o n o f B - c e l l s and a r e l a t i v e l y l a r g e r p r o p o r t i o n o f T - c e l l s . T h i s appears to be f u r t h e r supported by the l a r g e s t i m u l a t i o n by PHA, a T- c e l l mitogen. The r e s u l t s o f the ConA s t i m u l a t i o n do not g i v e support t o t h i s i d e a , b ut are not s u f f i c i e n t l y c o n s i s t e n t to d i s p r o v e i t e i t h e r (Table V ) . 5 A t 5 x 10 c e l l s per w e l l i n the absence of serum the s t i m u l a t i o n o f normal and SBx c h i c k e n thymus c e l l s was very s i m i l a r . Although the thymus glands o f the SBx b i r d s were much s m a l l e r than those of the normal b i r d s , a standar d s i z e sample of c e l l s showed the same a b i l i t y as c e l l s from a normal b i r d to respond to mitogens. The response to endotoxin was remarkably h i g h f o r a predominantly T - c e l l p o p u l a t i o n (Table V I ) . The b u r s a c e l l s responded o n l y to endotoxin, s u p p o r t i n g the view t h a t the b u r s a c o n t a i n s o n l y B - c e l l s (Table V I I ) . 38 Table V Average stimulation* of normal (N) and SBx chicken spleen c e l l s with mitogens. No. of c e l l s Serum Endotoxin ConA PHA N Bx N Bx N Bx 5 X 10 5 — 40 15 17 15 25 50 5 X 10 5 + 11 3 14 15 18 26 5 X 10 6 - 5 7 65 15 44 10 5 X 10 6 + 5 20 26 30 41 58 * Stimulation = CPM of mitogen stimulated cultures CPM of non-stimulated (control) cultures 39 Table VI Average s t i m u l a t i o n o f normal (N) and SBx c h i c k e n thymus c e l l s w i t h mitogens. No. o f c e l l s Serum Endotoxin ConA PHA N Bx N BX N BX 5 x 10 5 - 120 125 20 24 17 20 5 x 10 5 + 5 25 - 10 13 5 x 10 6 - 25 16 133 26 30 30 5 x 10 6 + 14 8 10 13 40 T a b l e V I I Average s t i m u l a t i o n o f normal c h i c k e n b u r s a c e l l s w i t h mitogens. No. o f c e l l s Serum Endotoxin ConA PHA 5 x 10 5 5 x 10 5 + 5 x 10 6 5 x 10 6 + 5 8 11 41 GENERAL DISCUSSION Present evidence favours the view t h a t the b u r s a o f F a b r i c i u s i s an anatomical and p h y s i o l o g i c a l s i t e where u n d i f - f e r e n t i a t e d stem c e l l s o f y o l k sac o r i g i n s erve as p r e c u r s o r s of a d e v e l o p i n g lymphocyte p o p u l a t i o n t h a t d i f f e r e n t i a t e s i n t o immunoglobulin producing c e l l s l a t e i n embryonic l i f e . In the ontogeny o f the immune system, c e l l s p r o d u c i n g IgG appear to develop e x c l u s i v e l y from c e l l s which p r e v i o u s l y s y n t h e s i z e d IgM. Once seeded from the bursa, c e l l s seem to be i r r e v o c a b l y committed to the p r o d u c t i o n of a s i n g l e c l a s s and s p e c i f i c i t y o f antibody. T h i s implys a r o l e f o r t h e b u r s a i n d i r e c t i n g the g e n e t i c events l e a d i n g to a n t i b o d y h e t e r o g e n e c i t y (3 9). Removal of the b u r s a a t the time o f h a t c h i n g or s u p r e s s i n g i t s growth by i n j e c t i o n of hormones d u r i n g i t s development r a r e l y produces t o t a l l y agammaglobulinemic b i r d s . Neonatal bursectomy f o l l o w e d by t o t a l body i r r a d i a t i o n r e s u l t s i n agammaglobulinemia and an i n a b i l i t y t o produce Ab a f t e r r e p e a t - ed c h a l l e n g e s i n o n l y about 40% o f the b i r d s t r e a t e d (25). Hormonal bursectomy _in ovo can r e s u l t i n agammaglobulinemia i n 40 - 50% of t r e a t e d b i r d s (40) b u t t h i s can be r a i s e d t o more than 90% e f f e c t i v e i f coupled w i t h repeated c y c l o p h o s - phamide (an immunosupressant) treatments d u r i n g the f i r s t few days a f t e r h a t c h i n g (41). T h i s treatment, although h i g h l y e f f e c t i v e , r e s u l t s i n h i g h m o r t a l i t y . The technique developed here f o r e a r l y embryonic s u r g i c a l bursectomy produced v i a b l e b u r s a - l e s s b i r d s i n o n l y about 2% o f a l l embryos t r e a t e d . T h i s low s u r v i v a l r a t e has been noted by o t h e r workers u s i n g b o t h s u r g i c a l and hormonal treatments (42). E a r l y treatment w i t h hormones r e s u l t s i n abnormal development of the c l o a c a . Meyer e t a l . (40) noted s t u n t e d growth o f the v e n t r a l a n a l l i p , i n c r e a s e d musculature of the d o r s a l l i p and a t t e n u a t i o n o f the proctodaeum. These abnor- m a l i t i e s , s i m i l a r to those noted f o l l o w i n g e a r l y s u r g i c a l bursectomy, r e s u l t e d i n i n t r a c l o a c a l a ccumulation of f a e c e s , u s u a l l y t e r m i n a t i n g i n death. I t would appear, then, t h a t 42 e a r l y m o r t a l i t y i s the r e s u l t of p h y s i c a l a b n o r m a l i t i e s r e s u l t i n g from the treatment r a t h e r than the r e s u l t o f any developmental requirement f o r the b u r s a . Although the body weight data i n Table I are i n s u f f i c i e n t f o r growth r a t e a n a l y s i s , the weight d i f f e r e n c e s between the normal and Bx c h i c k s appear to be g r e a t e r than can be a t t r i b - u ted to l o s s o f the t a i l s t r u c t u r e and lower l a r g e i n t e s t i n e . I t i s p o s s i b l e t h a t the s t r e s s of the o p e r a t i o n r e t a r d e d the growth of the embryo, but one would not expect these e f f e c t s t o p e r s i s t 8 weeks a f t e r h a t c h i n g . Freeman (43!) noted t h a t b ursectomized b i r d s showed a reduced response to s t r e s s from h a n d l i n g . The e f f e c t o f bursectomy on the g e n e r a l p h y s i o - l o g i c a l s t a t e of the b i r d i s not c l e a r and may extend beyond the immune system. Spleen to body weight r a t i o s f o r day o l d c h i c k s (Table II) i n d i c a t e t h a t the spleens o f Bx c h i c k s r e p r e s e n t a some- what s m a l l e r p r o p o r t i o n o f the body weight than do those o f normal c h i c k s . T h i s , again, may r e f l e c t a g e n e r a l p h y s i o l o g i c e f f e c t of bursectomy as the lymphoid t i s s u e s appear to be the most s e n s i t i v e to such s t r e s s e s . H i s t o l o g i c a l s e c t i o n s o f the s p l e e n , however, i n d i c a t e t h a t the reduced s i z e of the Bx s p l e e n may be the r e s u l t o f a reduced lymphoid p o p u l a t i o n ( F i g u r e 6 ) . The reduced number o f g e r m i n a l c e n t r e s i s an i n d i c a t i o n o f a d e f i c i e n c y of humoral lymphocytes. The o r i g i n o f the B - c e l l s forming g e r m i n a l c e n t r e s i n the Bx b i r d s i s not known. No b u r s a l remnants were found i n these b i r d s , nor were any ger- m i n a l c e n t r e s found i n the h i s t o l o g i c a l s e c t i o n s of the caecae and l a r g e i n t e s t i n e . A u x i l l i a r y bursae are o c c a s i o n a l l y found a t the j u n c t i o n of the b u r s a l s t a l k and the c l o a c a , b u t were not found i n t h i s study. I t i s p o s s i b l e t h a t the B - c e l l s i n the s p l e e n are of d i r e c t y o l k sac o r i g i n and have migrated here i n the absence o f the b u r s a . The g e r m i n a l c e n t r e s o f the g u t - a s s o c i a t e d t i s s u e s do not appear u n t i l about 10 days a f t e r h a t c h i n g and so are u n l i k e l y to be i n v o l v e d i n the s e e d i n g o f o t h e r lymphoid t i s s u e s . J a n k o v i c e t a l . (24) noted t h a t 43 n e o n a t a l bursectomy reduces the number o f plasma c e l l s and g e r m i n a l c e n t r e s i n the caecae. Thymus weights were not o b t a i n e d as, i n aves, t h i s g l a n d i s formed as a s e r i e s o f lobes embedded i n f a t along the l e n g t h o f the neck extending almost to the l e v e l o f the t h y r o i d s . The thymic lobes o f the Bx c h i c k s appeared s m a l l e r than those o f normal b i r d s ; c r o s s - s e c t i o n s a t the same mag- n i f i c a t i o n ( F igure 7) support t h i s o b s e r v a t i o n . These s e c t i o n s a l s o show t h a t the reduced s i z e o f the lobes i s due to a d e f i c i e n c y i n the thymic c o r t e x , the main area of lympho- c y t o p o e s i s . The same s t r e s s e s , such as ACTH and a d r e n a l c o r t - i c o i d s , which cause premature r e g r e s s i o n o f the b u r s a have a s i m i l a r e f f e c t on the thymus. The p a t t e r n o f e a r l y growth and m a t u r i t y f o l l o w e d by r e g r e s s i o n i n the a d u l t animal found f o r the b u r s a i s a l s o t r u e f o r the thymus. The removal o f the b u r s a c o u l d a f f e c t the development o f the thymus i n s e v e r a l ways. The s t r e s s o f the o p e r a t i o n i t s e l f i s u n l i k e l y to be a major f a c t o r as sham operated b i r d s showed a p p a r e n t l y normal thymic development. The e f f e c t o f bursectomy on the g e n e r a l p h y s i o l o g i c a l s t a t e o f the b i r d may r e s u l t i n incomplete c o r t i c a l development. I t i s a l s o p o s s i b l e t h a t the b u r s a may p l a y a d i r e c t or hormone mediated r o l e i n the development o f the thymic branch o f the immune system. Atrophy o f the thymic c o r t e x f o l l o w i n g hormonal bursectomy a t 13 days _in ovo was noted by Warner _et a l . (44). They observed a v a r i e t y o f e f f e c t s r a n g i n g from v e r y l i t t l e change i n 60% o f the c h i c k s to severe r e d u c t i o n o f the c o r t e x and an i n a b i l i t y t o r e j e c t s k i n g r a f t s i n 10% o f the c h i c k s t r e a t e d . These data are d i f f i c u l t t o i n t e r p r e t as bursectomy was accomplished by the i n j e c t i o n o f s t e r o i d hormone r a t h e r l a t e i n development, when the e f f e c t s may have been wide and v a r i e d . The o b s e r v a t i o n of s i m i l a r e f f e c t s f o l l o w i n g s u r g i c a l bursectomy suggest t h a t the phenomenon may be the r e s u l t o f a b u r s a l e f f e c t r a t h e r than a s i d e e f f e c t o f hormonal treatment. The c a p a c i t y f o r s k i n g r a f t r e j e c t i o n was t e s t e d i n a 44 s m a l l number of SBx c h i c k s and a group o f c h i c k s hormonally bursectomized a t 72 h r _in ovo. The r e s u l t s of t h i s study (Table I I I ) i n d i c a t e t h a t the SBx b i r d s were l e a s t capable o f s k i n g r a f t r e j e c t i o n . The time r e q u i r e d f o r the edges of the g r a f t t o separate from the h o s t t i s s u e was a t l e a s t twice t h a t r e q u i r e d f o r a normal c h i c k . Hormonally bursectomized c h i c k s a l s o showed a r e t a r d e d time course o f g r a f t r e j e c t i o n b u t most o f t h i s group showed s i g n s o f r e j e c t i o n w i t h i n the t e s t p e r i o d . The b i r d s used i n t h i s t e s t were not k i l l e d and a u t o p s i e d as they were r e q u i r e d f o r o t h e r t e s t s . S u p e r f i c i a l examination o f the lobes o f the thymus through the s k i n o f the neck i n d i c a t e d t h a t the glands were c o n s i d e r a b l y s m a l l e r i n those b i r d s which r e q u i r e d an extended time course f o r g r a f t r e j e c t i o n . I t i s prob a b l e t h a t these b i r d s , s i m i l a r l y t o those o f Warner _et a l . (44), were l a c k i n g i n thymic c o r t e x . The data to t h i s p o i n t , then, do not c l e a r l y i n d i c a t e the r o l e o f the b u r s a i n the immune system. Removal of the organ o b v i o u s l y has e f f e c t s extending beyond the immune system to the g e n e r a l p h y s i o l o g i c a l s t a t e o f the b i r d . The d i r e c t e f f e c t s on the immune system appear to be a r e d u c t i o n o f the germ i n a l c e n t r e s o f the p e r i p h e r a l lymphoid t i s s u e s and perhaps an e f f e c t on the lymphocyte p o p u l a t i o n of the thymus. Immunoelectrophoresis o f serum samples through the f i r s t few weeks a f t e r h a t c h i n g showed the normal (33) p a t t e r n s f o r prealbumin, albumin, l i p o p r o t e i n and b e t a g l o b u l i n f r a c t i o n s . The t r a n s f e r r i n band remained s t r o n g throughout the f o u r week sampling p e r i o d . The s e r a o f the bursectomized b i r d s , however, showed r e d u c t i o n or absence o f the gamma g l o b u l i n bands. The presence o f these bands immediately a f t e r h a t c h i n g can be a t t r i b u t e d to the presence o f maternal immunoglobulin, b u t by the l a t t e r p a r t o f the t e s t p e r i o d c o u l d o n l y have been pro- duced by the c h i c k . The s e r a from most o f the Bx c h i c k s were e n t i r e l y devoid o f IgG. In some cases an IgM band was found b u t tended to be r a t h e r weak. I t i s p o s s i b l e t h a t the g l o b u l i n s were b e i n g produced i n such s m a l l q u a n t i t i e s as to be 45 u n d e t e c t a b l e by t h i s method. The immunoglobulin bands o f the normal c h i c k s were e a s i l y d e t e c t a b l e . S t i m u l a t i o n o f the humoral immune system by the i n t r o - d u c t i o n o f a n t i g e n i c substances i n c r e a s e s the amount of c i r c u l a t i n g immunoglobulin. T h i s s t i m u l a t i o n d i d e l i c i t the p r o d u c t i o n o f s p e c i f i c a n tibody i n some of the bur s e c t o m i z e d b i r d s . S u r g i c a l l y and hormonally bursectomized c h i c k e n s , s t i m - u l a t e d f o r the f i r s t time a t 5 weeks of age w i t h SRBC produced l i t t l e or no antibody. M i c r o t i t r e h a e m a g g l u t i n a t i o n a n a l y s i s (Table IV) of the s e r a i n d i c a t e d a t i t r e o f 128 f o r normal chi c k e n s and t i t r e s o f 0 to 4 f o r the bursectomized b i r d s . N e o n a t a l l y b ursectomized b i r d s a re a l s o known to have reduced a n t i b o d y t i t r e s f o l l o w i n g the primary c h a l l e n g e (22,23,24). A second c h a l l e n g e w i t h the same a n t i g e n may r e s u l t i n n e a r l y normal a n t i b o d y p r o d u c t i o n i n some Bx b i r d s (24). The r e s u l t s i n Table IV i n d i c a t e d t h a t some of the Bx b i r d s d i d produce antibody i n the 2° response although the l e v e l o f Ab d i d not approach normal l e v e l s . F u r t h e r s t i m u l a t i o n w i t h the same a n t i g e n d i d cause the Ab t i t r e s t o r i s e toward normal l e v e l s ; the HBx b i r d s appeared to respond t o the s e r i a l s t i m - u l a t i o n s more r a p i d l y than d i d the SBx b i r d s . The low number of g e r m i n a l c e n t r e s found i n the spleens o f Bx b i r d s suggests t h a t the g r a d u a l response i s the r e s u l t o f a g r a d u a l i n c r e a s e i n humoral c e l l numbers. As the i n c r e a s e i n numbers i s l o g - a r i t h m i c f o l l o w i n g s t i m u l a t i o n , and as o n l y a s m a l l number of the lymphocytes p r e s e n t are capable o f responding t o any one antig e n , the process o f b u i l d i n g up the c e l l p o p u l a t i o n takes some time. T h i s i s p r o b a b l y not the o n l y f a c t o r i n v o l v e d i n the slow humoral response; the p r o d u c t i o n o f a n t i b o d y t o a number of antigens r e q u i r e s the c o o p e r a t i o n o f T- and B- c e l l s . A d e f i c i e n c y i n the thymic c o r t e x and thus i n T - c e l l s would a l s o a f f e c t the p r o d u c t i o n o f a n t i b o d y f o l l o w i n g s t i m u l a t i o n w i t h SRBC. Jankovic and I s a k o v i c (24), working w i t h n e o n a t a l l y Bx 46 b i r d s , found t h a t no antibody was produced f o l l o w i n g the primary c h a l l e n g e . A f t e r a secondary c h a l l e n g e these b i r d s produced a s m a l l amount of 19S (IgM) type antibody. In a normal response, IgM i s produced i n the primary r e a c t i o n and i s o f much l e s s importance i n the subsequent r e a c t i o n s . IgG, which i s n o r m a l l y u n d e t e c t a b l e i n the primary response, r i s e s to much h i g h e r l e v e l s i n the secondary response. In the Bx b i r d s mentioned above, no IgG was found. These r e s u l t s may i n p a r t be due to the time o f bursectomy more than the f a c t o f bursectomy as n e o n a t a l removal seems to m a i n l y impair IgG p r o d u c t i o n . E a r l y s u r g i c a l bursectomy seems to have a s i m i l a r e f f e c t on the c l a s s of immunoglobulin produced. Immunoelectrophoresis of serum from Bx b i r d s twice c h a l l e n g e d w i t h b o v i n e serum albumin (BSA) shows the presence o f s m a l l amounts of IgM (2). Other workers a l s o r e p o r t low Ab t i t r e s i n Bx b i r d s f o l l o w i n g s t i m u l a t i o n w i t h a v a r i e t y o f a n t i g e n s , some o f which r e q u i r e B-T c e l l c o o p e r a t i o n , o t h e r s which do not. Warner _et a l . (44) found primary a n t i b o d y t i t r e s o f l e s s than 10 f o l l o w i n g c h a l l e n g e w i t h B r u c e l l a s u i s , i n f l u e n z a type A v i r u s , human c h o r i o n i c gonadotrophin or BSA. Primary t i t r e s f o r normal b i r d s s t i m u l a t e d w i t h these a n t i g e n s ranged from 320 t o 20480. The r e s i d u a l c a p a c i t y t o s y n t h e s i z e Ab f o l l o w i n g n e o n a t a l bursectomy can be removed by t o t a l body x - i r r a d i a t i o n . T h i s treatment i n d i s c r i m i n a n t l y k i l l s a l l r a p i d l y d i v i d i n g c e l l s i n c l u d i n g those lymphocytes which are s e e d i n g the s p l e e n and o t h e r p e r i p h e r a l lymphoid t i s s u e s (22). Warner e t a l . ( 4 4 ) noted t h a t even though c y t o t o x i c a n t i - body was absent, s u r g i c a l l y and hormonally bursectomized b i r d s were capable of r e j e c t i n g s k i n g r a f t s i n the normal time. In a l a t e r p u b l i c a t i o n however, they found t h a t a s m a l l percentage of t h e i r hormonally bursectomized b i r d s e x h i b i t e d atrophy of the thymic c o r t e x and were i n c a p a b l e of r e j e c t i n g g r a f t s (43). Others, w i t h i n t e r m e d i a t e damage to the thymic c o r t e x , showed 47 a graded a b i l i t y p r o p o r t i o n a l to the e x t e n t o f damage. The e f f e c t s o f bursectomy on the humoral immune system cannot be e n t i r e l y d i v o r c e d from the e f f e c t s o f bursectomy on the r e s t o f the immune system and on the b i r d g e n e r a l l y . S u r g i c a l and hormonal bursectomy appear to have s i m i l a r e f f e c t s on the p r o d u c t i o n of immunoglobulins and the s y n t h e s i s o f s p e c i f i c antibody, although the e f f e c t o f e a r l y s u r g i c a l bursectomy appears to be more severe. Bursectomy a t any time seems to be more damaging to the IgG p r o d u c i n g c e l l s than to the IgM producing c e l l s . I t may be t h a t the most c r i t i c a l p a r t of the r o l e o f the b u r s a i n the humoral immune system i s i n the s w i t c h from IgM to IgG p r o d u c t i o n i n some c e l l s b e f o r e they are seeded to the p e r i p h e r a l t i s s u e . The B- and T - c e l l s u b p o p u l a t i o n s of a mixed lymphocyte p o p u l a t i o n can be i n d i v i d u a l l y e s t i m a t e d by s t i m u l a t i o n w i t h s p e c i f i c mitogens:. These substances appear to be capable o f s t i m u l a t i n g a l l of one on these s u b p o p u l a t i o n s r a t h e r than j u s t a few s e l e c t c e l l s as i n the case of a normal a n t i g e n . A comparison of the s t i m u l a t i o n of normal and Bx c h i c k e n s p l e e n c e l l s w i t h B- and T - c e l l mitogens leads t o the s p e c u l a t i o n t h a t the s p l e e n of the Bx b i r d c o n t a i n s r e l a t i v e l y fewer B - c e l l s and r e l a t i v e l y more T - c e l l s per stand a r d sample than does the s p l e e n o f a normal b i r d . The c e l l s o f the thymus, although reduced i n number, do not appear to be impared i n t h e i r a b i l i t y to respond to mitogens. 4 8 SUMMARY AND CONCLUSION The g e n e r a l e f f e c t s of e a r l y embryonic s u r g i c a l bursectomy are s i m i l a r to those found by o t h e r workers employing both s u r g i c a l and hormonal techniques f o r bursectomy. Many of the e f f e c t s of bursectomy cannot be c o n s i d e r e d o n l y i n terms o f a reduced humoral lymphoid p o p u l a t i o n b u t must a l s o be examined i n terms of the e f f e c t s on the thymus, on B-T c e l l c o o p e r a t i o n and on the p h y s i o l o g i c a l s t a t e of the b i r d . The d e p l e t i o n o f lymphoid p o p u l a t i o n s , e s p e c i a l l y those of the humoral system, and the r e d u c t i o n of immunoglobulin s y n t h e s i s support the p o s t u l a t e d r o l e of the b u r s a i n the ontogeny o f the immune response. There appear to be some lymphoid c e l l s which are capable of d i f f e r e n t i a t i o n i n t o c e l l s a t l e a s t c apable of producing IgM type a n t i b o d y even i n the absence o f the b u r s a . The source and degree of autonomy i n the d i f f e r e n t i a t i o n o f 'these c e l l s i s u n c l e a r . I t i s p o s s i b l e t h a t they are o f y o l k sac o r i g i n or o f bone marrow o r i g i n and they appear to be capable of s e l f d e t e r m i n a t i o n to the p o i n t o f IgM s y n t h e s i s but unable to s w i t c h to IgG s y n t h e s i s . I t may be t h a t the major r o l e o f the b u r s a i s i n the d i v i s i o n o f the humoral lymphocytes i n t o IgM and IgG p r o d u c i n g p o p u l a t i o n s . F u r t h e r s t u d i e s on the nature of the a n t i b o d y producing c e l l s i n the bursectomized b i r d are n e c e s s a r y to answer t h i s q u e s t i o n . A more e x t e n s i v e i n v e s t i g a t i o n of the source of the stem c e l l s e n t e r i n g the b u r s a and the c a p a c i t y of these c e l l s to develop i n t o competent lymphocytes i n the absence of the b u r s a l environment would be of v a l u e . 49 BIBLIOGRAPHY 1. Gordon, B.L. and D.K. Ford, 1971, " E s s e n t i a l s of Immuno- logy", F.A. 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APPENDIX : Formulae f o r s o i u t i A. Chick Ringer NaCl KC1 C a C l 2 d i s t . H 20 B. B u f f e r e d Formalyn commercial formaldehyde Na 2HP0 4 (anhydrous) NaH 2P0 4 (anhydrous) d i s t . H 20 C. T r i s - H C l B u f f e r T r i s (Tham) NaCl HC1 d i s t . H 20 D. RPMI 1640 T i s s u e C u l t u r e ] Ca(N0 3) 2". 4H 20 Glucose MgS0 4 • 7H 20 KC1 Na 2HP0 4 • 7H 20 NaCl L - A r g i n i n e ( f r e e base) L-Asparagine L - A s p a r t i c a c i d L - C y s t i n e L-Glutamic a c i d L-Glutamine G l u t a t h i o n e (reduced) G l y c i n e L - H i s t i d i n e ( f r e e base) 53 and c u l t u r e media. 7.00 g 0.42 g 0.24 g to 1000 ml 100 ml 6.5 g 3.5 g to 1000 ml 15.75 g 117.90 g 7.0 ml to 1000 ml ium (Gibco) 100.0 mg/1 2000.0 100.0 400.0 1512.0 6000.0 200.0 50.0 20.0 50.0 20.0 •300.0 1.0 10.0 15.0 cont. RPMI 1640 L-Hydroxyproline 20. 0 L - I s o l e u c i n e ( a l i o f r e e ) 50. 0 L-Leucine (methionine f r e e ) 50. 0 L - L y s i n e HCl 40. 0 L-Metionine 15. 0 L - P h e n y l a l a n i n e 15. 0 L - P r o l i n e (Hydroxy f r e e ) 20. 0 L - S e r i n e 30. 0 L-Threonine ( A l i o f r e e ) 20. 0 L-Tryptophane 5. 0 L - T y r o s i n e 20. 0 L - V a l i n e 20. 0 B i o t i n 0. 2 V i t a m i n 0. 00 D-Ca pantothenate 0. 25 C h o l i n e C l 3. 0 F o l i c a c i d 1. 0 i - I n o s i t o l 35. 0 N i c o t i n a m i d e 1. 0 Para-Aminobenzoic a c i d 1. 0 P y r i d o x i n e HCl 1. 0 R i b o f l a v i n 0. 2 Thiamine HCl 1. 0 Phenol Red 5. 0 NaHC0 3 2000. 0

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