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Early embryonic surgical bursectomy : an investigation of some aspects of the avian immune system Sparks, Esta Madeleine Frances 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.),  U n i v e r s i t y o f 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 o f Poultry  We accept t h i s  Science  t h e s i s as  to the r e q u i r e d  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA October,  1973  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree the  L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  that  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be granted by  the Head of my  I t i s understood t h a t copying or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be written  permission.  Department of  fOUA  77*? 1/  The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada  Department or  Oc^f/^CcT Columbia  allowed without  my  1  ABSTRACT Surgical in  ovo  removal  results  survival  of presumptive  i n t h e f i r s t week t h e r e a f t e r . and  usually  remain  thymus o f t h e b u r s e c t o m i z e d lymphoid  related o r may for on  to stress  organ  The  so.  effect  subsequent  some e n d o c r i n e  level  Both  on  the s p l e e n  the  in size  thymus may  centres  found  antigens  and  The  response  and  is rarely  several  lymphoid  necessary  The  effect  In the absence  mitogens  tissues  these c e l l s  areas.  stimulation  elicits not  Autopsy  of r e s i d u a l b u r s a l  found  Antibody  and  The  of B - c e l l  origin.  i n some Bx  i n the primary  studies  histological failed  constitute  with  birds. stimulation  response be  even  after  limited  are necessary  examination t o show any  of  to  to the  evidence  proof of the r o l e  immune r e s p o n s e  consequences  development o f the b i r d  requires  o f t h e b u r s a and  o f t h e stem c e l l s  a l s o be  germinal  may  tissue.  i n the humoral  the b r o a d e r  capacity  b u l k of the  p r o d u c t i o n may  further  Whether t h e s e f i n d i n g s  ive capacities  commonly  a response  o f the r e s p o n s i v e b i r d s  reflect  The  a r e more  o f the magnitude o f a normal  immunoglobulin;  the b u r s a  stem level  o f t h e h u m o r a l immune s y s t e m  i s generally  this.  of  induces m a t u r a t i o n of the  s p l e e n must be  stimulations.  type  verify  although  i n t h e Bx  Repeated  be  activity  f u n c t i o n o f the b u r s a tissue.  and  Some o f t h e r e m a i n i n g g e r m i n a l c e n t r e s  origin,  in diffuse  should  low  t h e number o f g e r m i n a l c e n t r e s i n t h e s e c o n d a r y  of T - c e l l  hr  viable chicks  adrenal c o r t i c a l  lymphoid  organ which  i s reduced.  found  IgM  and  The  t h e s p l e e n seems t o be more d i r e c t .  cells,  a t 72  (Bx) c h i c k s a r e r e d u c e d  the m a t u r a t i o n o f thymic  the primary  be  population.  reflect  tissue  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  are underweight  and  bursal  or whether  o f bursectomy further  thought  t o be  they  i n the  study.  t h e s o u r c e and  of  The  induct-  autonomous  induced  i n the  bursa  investigated.  effects  o f e a r l y h o r m o n a l and  late  surgical  bursectomy  •11  are  similar  embryonic  t o those found  surgical  complications  i n this  t e c h n i q u e was  s t u d y , where  used  o f t h e s e o t h e r methods.  an  early  to avoid the p o s s i b l e  iii  TABLE OF CONTENTS Page INTRODUCTION  1  LITERATURE REVIEW A. B. C. D. E. F. G. PART  I A.  B. PART  The B u r s a o f F a b r i c i u s . S t r u c t u r e o f the Bursa. Development o f t h e B u r s a . Immunoglobulin S y n t h e s i s . Bursectomy. E f f e c t s o f Bursectomy. The E n d o c r i n e F u n c t i o n o f t h e B u r s a . :  A.  B.  S u r g i c a l and H o r m o n a l B u r s e c t o m y T e c h n i q u e s , 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. i i ) E a r l y embryonic hormonal bursectomy. i i i ) Preliminary examinations. iv) H i s t o l o g i c a l examination. v) S k i n g r a f t i n g . R e s u l t s and D i s c u s s i o n  II  :  Tests  :  13 14 14 16 16 17  o f t h e H u m o r a l Immune S y s t e m  Methods i ) Serum s a m p l e s . i i ) Preparation of antisera. i i i ) Immunoelectrophoresis. iv) Test f o r s p e c i f i c antibody R e s u l t s and D i s c u s s i o n  PART I I I  4 4 6 8 10 10 12  S t i m u l a t i o n o f Spleen, C e l l s with Mitogens.  A. B.  Introduction Methods  C.  Results  t o Mitogens  and D i s c u s s i o n  production.  25 25 25 26 26  Thymus and B u r s a 34 35 36  GENERAL DISCUSSION  41  SUMMARY AND CONCLUSION  48  BIBLIOGRAPHY APPENDIX  49 53  iv  L I S T OF  Title  Table Table  TABLES  I  Page  A v e r a g e w e i g h t s o f n o r m a l and s u r g i c a l l y bursectomized chicks h a t c h t o 8 weeks o f age.  20 from  Table  II  S p l e e n and b o d y w e i g h t s f o r d a y o l d n o r m a l and s u r g i c a l l y b u r s e c t o m i z e d chicks.  21  Table  III  Prognosis of skin g r a f t s 28 d a y s a f t e r g r a f t i n g .  24  Table  IV  Haemagglutination t i t r e s f o r normal, 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.  33  Table  V  Average s t i m u l a t i o n o f normal SBx c h i c k e n s p l e e n c e l l s w i t h  (N) and mitogens.  38  Table  VI  Average s t i m u l a t i o n of normal SBx c h i c k e n thymus c e l l s w i t h  (N) and mitogens.  39  Table  VII  Average s t i m u l a t i o n o f normal c h i c k e n bursa c e l l s w i t h mitogens.  a t 7,  14.and  40  V  LIST Figure  OF  FIGURES  Title  Page  Figure  1.  B u r s a d e v e l o p m e n t shown as a p e r c e n t a g e o f body w e i g h t . The t i m e s of i n i t i a t i o n o f immunoglobulin s y n t h e s i s a r e shown.  5  Figure  2.  Cross s e c t i o n o f the b u r s a o f F a b r i c i u s , s h o w i n g p l i c a e and l y m p h o i d f o l l i c l e s ( f r o m Romanoff, 10).  7  Figure  3.  Chick  embryo a t 3 days  incubation—dorsal  section  through  view  tail  (72hrs) o f and  longitudinal  15  region.  Figure  4,  T a i l r e g i o n ( d o r s a l v i e w and l o n g i t u d i n a l s e c t i o n ) showing incision t h r o u g h membranes and t i p o f t a i l lying free.  15  Figure  5.  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 l i g a t i o n and r e m o v a l o f p r e s u m p t i v e bursal tissue.  15  Figure  6.  H i s t o l o g i c a l s e c t i o n s o f n o r m a l (A) and b u r s e c t o m i z e d (B) c h i c k s p l e e n s .  22  Figure  7.  H i s t o l o g i c a l s e c t i o n s o f n o r m a l (A) and b u r s e c t o m i z e d (B) c h i c k t h y m i c lobes.  23  Figure  8.  P r e c i p i t i n bands from immunoelectrop h o r e s i s o f n o r m a l 5 d a y c h i c k serum (from T u r e e n ^ t a l . , 3 3 ) .  29  Figure  9.  P r e c i p i t i n bands from immunoelectrop h o r e s i s o f 14 d a y n o r m a l (A) and b u r s e c t o m i z e d (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 - w h o l e c h i c k e n serum.  30  Figure  10.  P r e c i p i t i n bands from immunoelectrop h o r e s i s o f 14 day n o r m a l (A) and b u r s e c t o m i z e d (B) c h i c k s e r a a g a i n s t rabbit anti-chicken IgM.  31  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 b y n o r m a l (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 h o r m o n a l l y b u r s e c t o m i z e d (HBx) chicken sera following primary s t i m u l a t i o n w i t h SRBC.  32  ;  Figure  11.  vi  ACKNOWLEDGEMENT I would  like  t o e x p r e s s my a p p r e c i a t i o n  Dr.  R.C. F i t z s i m m o n s ,  and  D r . R . J . Hudson o f A n i m a l S c i e n c e  in  organizing  the  this  grateful  f o rtheir  assistance  research.  t o Donna Mason f o r h e r a i d i n  p e r f o r m a n c e o f 1,466 b u r s e c t o m i e s . Thank y o u .  supervisor,  t o D r . D.G. K i l b u r n o f M i c r o b i o l o g y  and p e r s u i n g  I am a l s o v e r y  t o my  1 INTRODUCTION The immune system f u n c t i o n s t o p r o t e c t an animal a g a i n s t i n f e c t i o n , d i s e a s e , f o r e i g n c e l l s and n e o p l a s t i c c e l l s o f self origin.  These f u n c t i o n s may be f u l f i l l e d  e i t h e r by  the p r o d u c t i o n o f a n t i b o d i e s o r by d i r e c t c e l l - t o - c e l l a c t i o n s and are c a r r i e d out by two f u n c t i o n a l l y  inter-  distinct  l i n e s o f lymphocytes. The c e l l u l a r immune system p r o v i d e s s u r v e i l l a n c e a g a i n s t neoplastic c e l l s ,  f u n c t i o n s i n g r a f t r e j e c t i o n and i s i n v o l v e d  i n d e l a y e d h y p e r s e n s i t i v i t y and g r a f t - v s - h o s t r e a c t i o n s . of t h i s  cells  system  are  p r o d u c t i o n i n some c a s e s .  also  necessary  for  The lymphocytes  efficient  The  antibody  i n v o l v e d i n these  r e a c t i o n s appear t o be d e r i v e d l a r g e l y from the thymus and are termed  "T-cells".  These c e l l s do n o t themselves produce  immunoglobulin molecules o r s p e c i f i c a n t i b o d i e s . capable of k i l l i n g  f o r e i g n c e l l s on c o n t a c t and a l s o produce  a v a r i e t y of chemical f a c t o r s . lyses c e l l s ,  They a r e  These i n c l u d e c y t o t o x i n , w h i c h  chemotaxin and m i g r a t i o n 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 o f nearby lymphocytes and macrophages, b l a s t o g e n , which s t i m u l a t e s the m a t u r a t i o n o f lymphoc y t e s , and f a c t o r s f o r s e n s i t i z i n g b a c t e r i a and The humoral immune system as the source immunoglobulin molecules termed a n t i b o d i e s  viruses.  of s p e c i f i c  (Ab).  The  Ab-  p r o d u c i n g c e l l s a r e thought t o be o f bone marrow o r i g i n i n mammals and from the b u r s a o f F a b r i c i u s i n aves.  These  " B - c e l l s " produce s p e c i f i c a n t i b o d i e s t o a wide v a r i e t y o f substances i n c l u d i n g c h e m i c a l s such as d i n i t r o p h e n o l c e l l s u r f a c e products i n c l u d i n g p r o t e i n s and b a c t e r i a l  (DNP), lipo-  p o l y s a c c h a r i d e s , v i r a l c o a t p r o t e i n s and such substances as b o v i n e serum albumin (HCG).  (BSA) and human c h o r i o n i c gonadotrophin  B - c e l l s c a r r y immunoglobulin molecules on t h e i r  s u r f a c e s and do not appear t o be d i r e c t c o n t a c t k i l l e r s (1). Lymphocytes  a r e found i n a v a r i e t y o f a d u l t mammalian  t i s s u e s such as the s p l e e n , thymus, bone marrow, lymph nodes, t o n s i l s , appendix and peyer's patches o f the i n t e s t i n a l w a l l .  2  In  aves, the caecae and b u r s a o f F a b r i c i u s are a d d i t i o n a l  sources o f lymphocytes, b u t the lymph nodes, appendix are absent.  tonsils  and  The organs o f primary i n t e r e s t i n the  d e v e l o p i n g embryo a r e the thymus, which i s the source o f T-cells,  and the b u r s a o f F a b r i c i u s , which i s the source o f  B-cells.  The s p l e e n i s a l s o o f i n t e r e s t as i t i s the s i t e  of  g r e a t e s t accumulation o f mature lymphocytes o f b o t h c l a s s e s . The e x i s t a n c e o f the b u r s a , a s i n g l e organ p o s t u l a t e d t o  c o n t r o l the development  o f the humoral  g r e a t convenience to the Immunologist.  immune system, i s o f In most s p e c i e s  function i s a t t r i b u t e d to widely dispersed t i s s u e . of  this  Removal  t h i s primary l e v e l organ s h o u l d enable the i n v e s t i g a t o r t o  examine the n a t u r e and f u n c t i o n o f t h i s branch o f the immune system d u r i n g i t s development. Removal o f the b u r s a can be accomplished by s u r g i c a l o r hormonal  techniques.  i s performed e a r l y i n development  either  Hormonal bursectomy,  which  i s , however, somewhat  c o m p l i c a t e d by the s i d e e f f e c t s o f i n t r o d u c i n g l a r g e q u a n t i t i e s of  s t e r o i d hormones i n t o a d i f f e r e n t i a t i n g system.  Surgical  removal o f the b u r s a a t the time o f h a t c h i n g a l s o has advantages. the  Late removal a l l o w s a p e r i o d o f time between  e a r l y appearance o f stem c e l l s  i n the b u r s a and the  removal o f the organ d u r i n g which maturing stem c e l l s escape t o seed o t h e r lymphoid organs. of  may  The s e q u e n t i a l s e e d i n g  the secondary l e v e l organs w i t h c e l l s c a p a b l e o f p r o d u c i n g  f i r s t one then another c l a s s o f immunoglobulins may the  dis-  explain  s e l e c t i v e e f f e c t s o f bursectomy near the time o f h a t c h i n g . Removal o f the b u r s a o r presumptive b u r s a l t i s s u e v e r y  e a r l y i n development  s h o u l d circumvent the problems o f the  s i d e e f f e c t s o f s t e r o i d s and o f s e e d i n g o f o t h e r organs to  bursectomy.  prior  Such an o p e r a t i o n i s p o s s i b l e o n l y d u r i n g a  s h o r t p e r i o d between the i n i t i a t i o n o f the t a i l  f o l d and the  time a t which the f r a g i l i t y o f the s u r r o u n d i n g membranes and b l o o d v e s s e l s makes the o p e r a t i o n p h y s i c a l l y i m p o s s i b l e . this investigation,  In  a t e c h n i q u e f o r s u r g i c a l removal o f the  3 presumptive b u r s a l t i s s u e a t 72 h r s in  o  v  o  was used t o  produce b i r d s d e f i c i e n t i n B - c e l l s and t h e r e f o r e i n c a p a b l e o f mounting an humoral immune response ( 2 ) . These s u r g i c a l l y b u r s e c t o m i z e d  (SBx) b i r d s were r a i s e d  f o r p e r i o d s o f up t o 10 months and a s e r i e s o f t e s t s t o i n v e s t i g a t e the s t a t u s o f t h e i r immune systems was c a r r i e d out. T h e i r lymphoid  t i s s u e s were h i s t o l o g i c a l l y examined f o r the  presence o f g e r m i n a l c e n t r e s , the s i t e s o f lymphocyte liferation.  T h e i r s e r a were examined by  f o r the presence o f immunoglobulin; with  specific  a n t i g e n s and t e s t e d  and t h e i r lymphocytes o f mitogens.  pro-  Immunoelectrophoresis  they were c h a l l e n g e d f o r antibody production;  were c u l t u r e d in v i t r o i n the presence  The r e s u l t s o f these t e s t s were compared w i t h  s i m i l a r t e s t s on normal  birds.  In a d d i t i o n t o these s u r g i c a l b u r s e c t o m i e s , some hormonal bursectomies were performed  using testosterone propionate.  These HBx c h i c k s were s u b j e c t e d t o many o f t h e same t e s t s as the SBx c h i c k s .  They were a l s o t e s t e d f o r t h e i r a b i l i t y t o  reject skin grafts. o f normal  T h e i r responses were compared t o those  and SBx b i r d s .  The data o b t a i n e d from t h i s study should supplement p r e s e n t knowledge and perhaps  aid in clarifying  the r o l e o f  the b u r s a o f F a b r i c i u s i n t h e d e v e l o p i n g a v i a n immune  system.  4 LITERATURE REVIEW A. The B u r s a The  bursa  Fabricius  early bird The  of Fabricius,  by a s h o r t s t a l k  junction  life,  o f maximum mean w e i g h t f r o m A\  - 6 weeks  The b u r s a  developed  t h e time  varies  among b r e e d s  i n White Leghorns  a t 3 weeks o f age i s 0.05%.  Leghorn  chickens.  weeks o f age;  The b u r s a  the r i s i n g  sexual maturity hastens  level  t h e maximum b u r s a t o F i g u r e 1 shows  o f body weight  starts  of chickens,  t o 10 -12 weeks i n  body w e i g h t  d e v e l o p m e n t as a p e r c e n t a g e  forms  of sexual maturity (4).  In the White Leghorn,  bursa  tissue  i n t h e immature  Barred crosses. ratio  lymphoid  region of the c l o a c a l -  intestine.  is fully  and r e g r e s s e s a r o u n d  ranging  to the dorsal  of the large  i n embryonic  time  d i s c o v e r e d i n 1621 b y H i e r o n y m u s  (3), i s a small o v a l sac o f l a r g e l y  connected colonic  of Fabricius.  o f White  t o r e g r e s s a t about  7  o f androgens a t the onset o f  the process  of involution  n e a r l y d i s a p p e a r s by t h e end o f t h e f i r s t  The s i z e o f  the bursa  tends  parallels  t h e g r o w t h and i n v o l u t i o n o f t h e thymus d u r i n g t h e  first  to correspond with  year.  so t h e o r g a n  t h a t o f t h e s p l e e n and  few months o f d e v e l o p m e n t ( 5 ) . The  either  b u r s a o f F a b r i c i u s was  i n the d i g e s t i v e  process,  long thought  t o be i n v o l v e d  due t o i t s a s s o c i a t i o n  with  t h e g u t , o r i n t h e o n s e t o f s e x u a l m a t u r i t y , as i t b e g i n s regress system  at this was  time.  The n a t u r e o f i t s r o l e  accidentally  when u s e d  i n t h e immune  d i s c o v e r e d b y 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 bursectomized age w e r e f o u n d  to  a t 2 weeks o f  t o b e i n c a p a b l e o f m o u n t i n g a n immune  i n a demonstration  response  of antibody production.  B. S t r u c t u r e o f t h e B u r s a . The primary plicae located  inner surface of the bursa plicae,  each  i s folded  o f which has 6 - 7  into  secondary  11 - 13 folds.  The  a r e l i n e d w i t h columnar e p i t h e l i u m , below which i s the tunica  an a r t e r y .  propria.  The p l i c a e  Each p l i c a  c o n t a i n s a v e i n and  and t h e w a l l s o f t h e b u r s a  are thickened  0.06  u D J Z  '  0.05  !• i  0.04  0.03  /  0.02  0.01  IgA| i  1  i  i  •  i  i  3 Age  F i g u r e 1.  1  1  i  i  4 (weeks)  B u r s a d e v e l o p m e n t shown as a p e r c e n t a g e o f body weight, The t i m e s o f i n i t i a t i o n o f immunog l o b u l i n s y n t h e s i s a r e shown.  N.  6 by  numerous l a r g e l y m p h o i d  tunica  propria  hedral  formations,  connective follicle  (Figure 2).  tissue,  i s divided  c o r t e x by  into  an  projects  epithelium  from  anlage  and  the proctodaeum.  vertically  and  first  appears  with  The  the  medulla  a t about 4 days  proliferation  (8).  along  By  day  6 an  the  and  the e x t e r n a l  epithelial  margin of  The  bursa on  o r 11  then  anlage  t h e u r o d a e l membrane. a t 7 days,  stalk.  d a y s o f i n c u b a t i o n and plicae.  From t h e 1 2 t h  first  15 to  that  to l i e  The by  begin  with  r o t a t e s foreward  a cylindrical  lumen c o n t a i n s a b o u t 12  of  ventral  a lumen w h i c h becomes c o n t i n u o u s  grows o u t  a p p e a r a t 10  a peripheral  contains developing  c o n t a i n s numerous l a c u n a e w h i c h ,  of  Each  Bursa.  the d o r s o - c a u d a l  c o a l e s c e t o form  loose  i s continuous  a r e a and  i n poly-  capillaries.  surface epithelium.  epithelial  to  the  by  a r e a o f c o n t a c t b e t w e e n t h e c l o a c a and  ectodermal  plicae  another  a c e n t r a l medulla  b u r s a l primordium  i n c u b a t i o n as  This  one  found  the  (7).  Development o f the  caudal  from  are  s m a l l b l o o d v e s s e l s and  the main lymphocytopoetic  The  which n e a r l y f i l l  follicles  a b a s e m e n t membrane w h i c h  mature lymphocytes C.  The  separated  b a s e m e n t membrane o f t h e is  follicles  days 16th  days the p r i m a t i v e e p i t h e l i u m a l o n g  t h e b a s e m e n t membrane o f  the medulla  to p r o j e c t  propria. and  an  t h i c k e n s and b u d s b e g i n The  bud  formation  epithelial t h e buds  form  to appear  i n diameter  Knouff  the e p i t h e l i a l  chick  the s i t e s from  s o l i d masses o f c e l l s  about 60^  A c k e r m a n and  yolk  At  bud  sac o r i g i n .  and  of  the  formation  the  t h e b a s e m e n t membrane; continue  to  enlarge.  i n t h e b u r s a when t h e b u d s  (about  16  (7) c o n s i d e r t h e s e itself,  o f bud  the t u n i c a  vascularization  phosphatase a c t i v i t y  l a y e r becomes d e t a c h e d  Lymphoblasts b e g i n reach  i s associated with  i n c r e a s e i n the a l k a l i n e  s u b e p i t h e l i a l mesenchyme.  into  days). cells  Although to o r i g i n a t e  in  c u r r e n t o p i n i o n i s t h a t they are  Moore and  Owen  embryos o f o p p o s i t e s e x e s  (9), working w i t h  with  a sex  of  parabiotic  chromosome m a r k e r ,  7  Figure  2.  Cross s e c t i o n o f the bursa o f 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).  8 demonstrated  t h a t up  t o 50% o f t h e d i v i d i n g  b u r s a a r e d e r i v e d from indicate The  an e x t r i n s i c  bursal  throughout the The through and in  are i n d i s t i n g u i s h a b l e  tissues-.  The  lymphoblasts decrease  of the  e n l a r g e t o 100  bursa  in cell  between  16  become l e s s b a s o p h i l i c t h e same t i m e as Two  tissues  size,  and  18  bud  and  lose their continuity with ment o f t h e b u d  At  the s t a r t  the c o r t e x .  lymphocytopoetic  appear  The  i n nuclear  size  formation of the c o r t e x : propria  and  find  some o f  the  t h e i r way  into  formation.  As  t h e s u b a d j a c e n t mesenchymal  t h e b a s e m e n t membrane.  the e p i t h e l i a l  of lymphoblast epithelial  The  basophilia  appear.  s t a g e s o f bud  p e r i p h e r y o f t h e m e d u l l a may into  and  incubation.  c e l l s which  zone about  of undifferentiated  continues  lymphocytes  crowds t h e m e s e n c h y m a l c e l l s  compact c e l l u l a r  found  differentiation  a decrease  of the t u n i c a  the t u n i c a  those  a l o n g t h e b a s e m e n t membrane  to the  epithelial  into  of  cells.  .  Small  days  exhibit  d u r i n g the e a r l y  projects  follicles  the s m a l l lymphocytes  t h e mesenchymal c e l l s  the t u n i c a  to  lymphoblasts  i n nuclear size  cells  contribute  undifferentiated  of  - 300 y  i n a condensation of chromatin. the  this  from  continue c h a r a c t e r i s t i c  previously undifferentiated  at  appearance  the medullary p o r t i o n follicles  They t a k e  i n the  source of probably blood-borne  lymphocytes  i n o t h e r lymphoid  until  the p a r t n e r .  cells  pass  blast  c o r t e x then a l s o  area with r e t i c u l a r  Enlargea  relatively  (16 d a y s ) a number  cells  through  cells  bud.  appearance  and  into  the  from  the  t h e b a s e m e n t membrane  transforms  cells  and  into  a  immature  lymphocytes.  D.  Immunoglobulin S y n t h e s i s . Immunofluorescent  fluorescein  labelled  staining  of bursal  anti-immunoglobulin  immunoglobulin  producing c e l l s  More s e n s i t i v e  tests  a t 15  for specific  t h a t a s m a l l q u a n t i t y o f IgM  cross sections s e r a ) shows  (with  no  days o f i n c u b a t i o n (11).  immunoglobulins  i s produced  by  18  indicate  days  of  9 incubation  (12).  the f o l l i c l e s  Large  by 4 - 7  immunofluorescent  numbers o f c o m p e t e n t c e l l s  days a f t e r h a t c h i n g .  cells  are also  found  in  the response  b o d y i s formed Kincade IgM  the appearance  o f IgM. chicken,  and C o o p e r  (14) u s e d  IgM c o n t a i n i n g c e l l s  cells  specific  molecules  anti-  antibodies to the  o f 14 d a y embryos. do n o t a p p e a r  They a l s o  until  in  the bursae  follicles  found  from  cells  from  within  This theory i s further  the bursa. that  synthesis.  The a u t h o r s  cells  suggest  including  that  the e f f e c t  organs  the destruction  some Bx c h i c k s h a v i n g  o f a l l IgM  are i n the bursa a t t h i s have n o t been seeded.  time,  By t h e  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  a r e a f f e c t e d by the treatment. s y n t h e s i s appears  o f _in o v o  t h o s e w h i c h w e r e t o s w i t c h t o IgG  As a l l l y m p h o c y t e s lymphoid  until  however, o n l y IgM s y n t h e s i s was  o c c u r e d a n d o n l y t h e IgM p r o d u c i n g c e l l s  their  IgM a n d  When t h e a n t i - l g M t r e a t m e n t was d e l a y e d  t i m e o f h a t c h i n g , however,  globulin  supported by the  i n supression o f both  t r e a t m e n t w i t h a n t i - l g M serum was  tissues  suggest-  IgM t o IgG p r o d u c t i o n o c c u r i n g  resulted  the neonatal bursectomy  the secondary  This  t r e a t m e n t _in o v o w i t h a n t i - l g M serum f o l l o w e d  n e o n a t a l bursectomy  synthesis.  observed  types o f heavy  o f immunoglobulins.  s w i t c h from  producing  Expansion  the spleen contained chains of  ed a d e v e l o p m e n t a l  depressed.  IgG c o n t a i n i n g  p o p u l a t i o n s was  the bursa contained both  o n l y one o r t h e o t h e r c l a s s  after  that  o f the.  o f 5 day c h i c k s and t h e s p l e e n s o f 8 day c h i c k s .  chains while single  observation  the presence  21 d a y s o f i n c u b a t i o n .  t h e IgM a n d I g G c o n t a i n i n g c e l l  Single cells  t o demonstrate  i n the lymphoid  of  IgG  IgM t y p e  t o IgG t y p e a n t i b o d y ( 1 3 ) .  o f immunoglobulin  bursae  by  Similarly,  (yu) t y p e a n d IgG ( v) t y p e o f h e a v y c h a i n s a n d t o l i g h t  chains of  time  IgG s y n t h e s i s i n t h e  o f t h e m a t u r e immunized prior  By t h i s  i n t h e 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 . b u r s a and s p l e e n f o l l o w s  appear i n  i n the peripheral T h e s w i t c h i n immuno-  to occur e x c l u s i v e l y  supernormal  levels  i n the bursa;  o f IgM n e v e r  IgG s y n t h e s i s mechanism, e v e n o v e r a p r o l o n g e d  repair period;.. ( 1 5 ) . .  10 Immunoglobulin 3 months.  i s the major  thymus a p p e a r s  small quantities immunoglobulins 2 weeks  not d e c l i n e  over time;  s o u r c e o f competent  lymphocytes  to produce  IgG a p p e a r s i n  a t 2 weeks and appear  no  IgM.  i n the  s t r e n g t h e n s b y 3 months.  i n the i n t e s t i n a l  lymphoid  cells  Both after  (12).  Bursectomy. The b u r s a c a n e a s i l y be  of  does  about  bird.  The  E.  i n the b u r s a d e c l i n e s by  That o f the s p l e e n  the s p l e e n mature  production  surgically  removed  i n c u b a t i o n onwards, a l t h o u g h t h e r e a r e l e s s  after hatching b u r s a can be icoids, fowl  (16,17,18,19).  induced by  sex s t e r o i d s ,  typhoid,  the i n j e c t i o n  and  muscular  I n j e c t i o n o f 0.65  11 - 13 ation,  mg  elial  inhibition  bud.  phosphatase  complete  of incubation,  a s i m i l a r dose  agent  activity  b e due  phamide  inhibition.  into  At form-  of  i n the  or epith-  alkaline (20).  the albumin or absorbed  solution  i s a l s o an  effective  bursectomy.  injection  of  caused to regress  o f the immunosupressant  a t the time o f drug cyclophos-  Bursectomy.  Bursectomy, performed by  attempt  of bursal  (21).  F. E f f e c t s  been  the 5th  cause p a r t i a l  to i n h i b i t i o n  as  (5).  i n t h e s u b e p i t h e l i a l mesenchyme  The b u r s a c a n a l s o be h a t c h i n g by  inhibition  o f t e s t o s t e r o n e may  cort-  such s t r e s s e s  t h e time o f e p i t h e l i a l bud  i n an a l c o h o l  f o r hormonal  o r by  o f lymphoid d i f f e r e n t i a t i o n  T h i s may  the s h e l l  o f ACTH, a d r e n a l  cause p a r t i a l  Testosterone propionate, injected through  complications  o f 1 9 - n o r t e s t o s t e r o n e on  S m a l l e r doses  days  complete  thiouracil  days  r e g r e s s i o n of the  exercise or starvation  day o f i n c u b a t i o n causes development.  Premature  f r o m 18  whether a large  to elucidate  by  surgical  or hormonal  number o f i n v e s t i g a t o r s  i t s role  means,  has  i n the  i n t h e immune s y s t e m .  The  effects  11 of bursectomy and  just  at various  after hatching  essential  provide  i n the development  immune s y s t e m .  These  population of c e l l s into  times d u r i n g  embryonic  evidence that  development  the b u r s a i s  o f the stem c e l l s  o f the  are the c e l l s which g i v e  rise  c a p a b l e o f m i g r a t i o n and o f  humoral  to a  differentiation  antibody producing c e l l s . The  depend  effects  of bursectomy  on t h e immune s y s t e m a p p e a r  o n t h e t i m e and method o f b u r s e c t o m y .  effects  of bursectomy  generally  include  the  The  absent or reduced  s e c o n d a r y r e s p o n s e may b)  be  The p r i m a r y  (22, 23) b u t  normal o r n e a r l y  so  o f i g G t h a n o f igM  usually  more  production.  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 hereditary  the  (24).  r e d u c t i o n o f immunoglobulin s y n t h e s i s ,  restrictive  direct  following:  a) s u p r e s s i o n o f t h e a n t i b o d y r e s p o n s e . response i s u s u a l l y  to  immune t h y r o i d i t i s ,  reaction  but not to t u b e r c u l i n  to  or  diptheria. d)  i n c r e a s e o f m o r t a l i t y due  to avian diseases  i n f e c t i o n b y S a l m o n e l l a t y p h i m u r i u m and e) c h a n g e s Plasma  cells  i n the blood  tonsils,  intestinal  epithelium  effects  The  may  be  cells  ( a t 5 d a y s _ i n ovo  appears  t o be  and  and  results  also  (25).  critical.  IgG t y p e  to have a major  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  immunoglobulin  count f o r  the above  f o r example) w i l l  S e v e r a l weeks a f t e r h a t c h i n g ,  i n the  enhances  and h a e m o g l o b i n  appears  r e d u c e t h e p r o d u c t i o n o f b o t h IgM  affect  spleen  near to normal.  o f aggammaglobulinemia  i n red blood  Neonatal bursectomy  The w h i t e c e l l  with x-irradiation  time o f bursectomy  bursectomy  are reduced i n the  i s increased.  combined  to the p o i n t  in a deficiency  populations.  a l t h o u g h t h e number o f l y m p h o c y t e s  and h e t e r o p h i l s  Bursectomy  to myeloblastosis.  tissue c e l l  and g e r m i n a l c e n t r e s  and c a e c a l  lymphocytes  and  s u c h as  prevent or antibodies.  effect  i s less  Early  on  the  influenced.  r e m o v a l o f t h e b u r s a does  production.  not  12 G.  The  Endocrine Function  The and  have l e d to  the  proposition  Modification  of  f o l l o w i n g b u r s e c t o m y was who  the  noted  depleted.  that  Freeman  an h o r m o n a l  adrenal (27)  the  adrenal's  maintaining  stores  Pethes  uptake by  synthesis  are  postulated  o f AAA  the  (28)  the  erythropoesis,  noted  thyroid  s e v e r e a n a e m i a when c o m p a r e d The  existance  conclusively  of  any  accomodate the  classified  range of  produces and  the  rate  depletion.  rate of  The  of  of  radio-  thyroid bursa  is  hormone also  influencing  x - i r r a d i a t e d chicks  develop  to x - i r r a d i a t e d c o n t r o l chicks  bursal  demonstrated.  w o u l d h a v e t o be  and  Eilat  became  incapable  an h o r m o n a l f a c t o r  bursectomized  stress  development  stimulus  that the  for  a c i d r e p l e t i o n mechanism.  bursectomy.  source of  as  and  to  the b u r s a  the  system  function  P e r e k and  (AAA)  animal  following  depressed by  as  the  by  acid  that  ascorbic  immune  response  described  facilitates  the  endocrine  adrenal  postulated  then, would render  and  an  ascorbic  Bursectomy,  Pintea  of  the  first  f a c t o r which  maturation of  iodine  Bursa.  e f f e c t s o f bursectomy extend beyond  the b u r s a .  (26)  of  h o r m o n a l p r o d u c t has  not  been  I f s u c h a p r o d u c t does e x i s t , i t as  a  ' d e v e l o p m e n t a l hormone'  its effects.  (29).  to  13 PART I  :  S u r g i c a l and H o r m o n a l B u r s e c t o m y H i s t o l o g y and S k i n G r a f t i n g .  Techniques,  Methods: i)  E a r l y embryonic  Fertile tained  on  eggs w e r e o b t a i n e d  the  Columbia.  P o u l t r y Farm o f  eggs w e r e p l a c e d  Incubator  small  eggs w e r e s u r f a c e  o p e r a t i o n was One shell  bursectomy. from White Leghorn s t o c k  the  U n i v e r s i t y of  A l l eggs w e r e i n c u b a t e d  Jamesway M o d e l 252  The  surgical  egg  under standard  up  for 1 hr  s t e r i l i z e d with  carried was  end  out  initially  and  the  the  was  made i n t h e  air cell,  w h o l e embryo was  and  exposed  The  Hamilton 3).  and  Operations  only on  t o make a s m a l l  those  (mag.  slit  axis  k n i f e was  i n s e r t e d t o one  extended to the  same manner. circulation drawn o u t or chick of  the  tail  of  C a r e was or the y o l k  t o l i e on ringer  operation.  and  the  albumin  carton  and The to  forceps  egg,  thus  until  the  i n diameter).  Albumin  embryo t o t h e  according 17  120x).  top of  the  t o Hamburger  and  - 18 w e r e u s e d  taken not sac.  The  the  ( A p p e n d i x ) was  l e g buds.  The  The and  a m n i o n was  to puncture the t i p of  chorion added  out  perpendicular  the m i d l i n e  side.  (Figure under  A cataract knife  chorion  side of  top of  the  embryos w e r e c a r r i e d  i n the  opposite  with  the  p o s t e r i o r to the  the  to  the  t i p of  the  the  opening  cut  in  the  vitelline  t a i l was  (Figure 4).  to prevent  was  then Albumin  desiccation  embryo.  A loop of c h i l d ' s chick  n a r r o w end  of stage  acceptable  embryonic  was  just  the  opened eggs. egg  1 cm  to f l o a t  a d i s s e c t i o n microscope used  The  conditions.  down i n an  embryos w e r e t h e n s t a g e d  (30)  ethanol  to provide  enlarged  (about  added from a s y r i n g e  hole.  in a  membranes.  A s m a l l h o l e was preserving  (72 h r s )  conditions.  s e a l i n g the  empty s h e l l s w e r e s t o r e d o p e n end drying of  70%  sacrificed  British  preceeding  under s t e r i l e  membranes f o r f i l l i n g  prevent  f o r 3 days  main-  hair,  r i n g e r s o l u t i o n was and  drawn up  ened s u f f i c i e n t l y  r i n s e d i n 70%  then passed over  ethanol  and  the  f r e e end  of  The  k n o t was  tight-  almost to the  l e g buds.  to l i g a t e  paired dorsal aortae  the  sterile  but  the  not  14 to  c u t the t a i l .  The t a i l  and t h e f r e e ends o f t h e h a i r  trimmed o f f w i t h  iris  stump was  back under t h e c h o r i o n and a l i t t l e  was  tucked  added t o f i l l  membrane was  t h e egg.  i n the s h e l l .  minutes  then  painted  If  shell  occurred. itions  allowed  s p a r i n g l y over  to the incubator  The eggs w e r e t h e n  discontinued. incubated  albumin  shell  down, o v e r  the  t o d r y f o r a few  point paraffin  small  (mp 65 -  with  incubated  a d d i t i o n a l wax. few  leaks  under s t a n d a r d  cond-  term ( 2 ) .  a number o f sham o p e r a t i o n s appeared normal  were a l s o  this  performed  p r a c t i c e was  C o n t r o l b i r d s were normal White Leghorn  under s t a n d a r d  The  e n d down and c h e c k e d  f o r the patch  f o r the remainder o f t h e i r  as t h e c h i c k s h a t c h e d  The  t h e edges o f t h e p a t c h .  membranes w e r e u s e d  Initially, but  T h i s was  inside  1 h r f o r l e a k s which were patched  both  (Figure 5).  A piece o f the double  a layer of high melting  eggs w e r e r e t u r n e d after  a n d removed  c u t t o f i t and p l a c e d ,  opening  70 C) was  scissors  were  conditions with  the  chicks  bursectomized  chicks. ii)  E a r l y embryonic hormonal  bursectomy.  F e r t i l e W h i t e L e g h o r n eggs w e r e i n c u b a t e d above. pair  They were s u r f a c e s t e r i l i z e d w i t h  o f h o l e s was  One p e n e t r a t e d the other the  into  allowed  egg.  drilled  through  the a i r c e l l  access  E a c h egg was  f o r 3 d a y s as  70% ethanol  the s h e l l  and s h e l l  and a membranes.  a t t h e l a r g e end o f t h e egg,  to the albumin through the s i d e of injected  through  this  l a t t e r hole  0.1 m l 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 peanut o i l ( t h i s were then returned iii) All  sealed with  Preliminary  d o s e o f 2 mg/egg).  a drop o f p a r a f f i n  to the incubator  sacrificed  holes  term,  examinations.  thereafter.  a t weekly  in  a n d t h e eggs w e r e  f o r the remainder o f t h e i r  Those w h i c h d i e d were  the presence o f b u r s a l remnants.  large  The  c h i c k s were w e i g h e d on t h e day o f h a t c h i n g  weekly i n t e r v a l s for  is a final  with  intervals  Chicks  examined  w e r e b l e d and  and t h e s p l e e n ,  i n t e s t i n e w e r e e x c i s e d and p r e p a r e d  and a t  thymus and l o w e r  for histological  15  Figure 3  Figure  4  F i g u r e 3.  C h i c k embryo a t 3 d a y s (72 h r s ) i n c u b a t i o n — d o r s a l v i e w and l o n g i t u d i n a l s e c t i o n t h r o u g h t a i l region.  F i g u r e 4.  T a i l r e g i o n ( d o r s a l v i e w and l o n g i t u d i n a l s e c t i o n ) s h o w i n g i n c i s i o n t h r o u g h membranes and t i p o f t a i l l y i n g f r e e .  Figure  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 l i g a t i o n and r e m o v a l o f p r e s u m p t i v e b u r s a l tissue.  5.  TAbbreviations: am : amnion; ch loop; l b : l i m b bud; l i n e vein; ys : y o l k  : chorion; hi : hair pv : p o s t e r i o r v i t e l sac.  16 examination.  Four normal and  s a c r i f i c e d on  the day  4 bursectomized chicks  a f t e r h a t c h i n g and  body weights were r e c o r d e d . and  mortality  of normal, SBx  a n a l y s e d f o r serum p r o t e i n s  and  HBx  and  chicks. stored  Serum a t -20  was C  s e r i a l sections and  trident  SBx  chicks.  i n buffered  formalyn  of  the  large  for  both  normal  and  S e c t i o n s were a l s o prepared from the b u r s a and  microscopically  the c l o a c a l area o f SBx  chicks.  The  of  sections  e o s i n and were examined  f o r the presence o f g e r m i n a l c e n t r e s  and  cells.  v)  Skin g r a f t i n g .  The  capacity  f o r g r a f t r e j e c t i o n by  normal White Leghorn grafting.  (WL)  c h i c k s was  Patches of d o r s a l  above mentioned groups and G r a f t i n g was chicks  had  dried.  chicks  and  the  HBx,  The  New  Hampshire Red  down was  reciprocal  (NH)  l y i n g t i s s u e and bone  (31).  a f t e r the  dry,  0.1  ml  of  epidermis f r e e from the  and were exchanged between the  two.  p l a c e d i n t h e i r normal a n t e r i o r - p o s t e r i o r added to h e l p dry  protected  the dry  chick midline under-  A s m a l l c i r c u l a r b i o p s y punch i n diameter.  Patches of s k i n were prepared s i m u l t a n e o u s l y from WL  The wound was  hatching  p a i n t e d w i t h c o l l o i d o n to  a c i r c l e of s k i n about 5 mm  drop o f c o l l o i d o n was  the  chicks.  i n j e c t e d subcutaneously near the the  and  p l u c k e d from the backs of  When t h i s was  over the p e l v i s , l i f t i n g used to c u t out  SBx  s k i n were exchanged between  exposed s k i n was  r i n g e r s o l u t i o n was  a few  t e s t e d by  done as soon as p o s s i b l e  s t r e t c h the s k i n .  The  embedded  s p l e e n , thymus, caecae  intestine  were s t a i n e d w i t h h e m a t o x y l i n and lymphoid  (Appendix) u n t i l  Standard wax  were prepared from the  area  normal c h i c k s  chicks  until  immunoglobulins,  prepared f o r h i s t o l o g i c a l s e c t i o n i n g .  was  and  H i s t o l o g i c a l examination.  T i s s u e s were s t o r e d  and  t h e i r spleen  Records were kept o f h a t c h a b i l i t y  c o l l e c t e d from the b l o o d samples and iv)  were  The  and  NH  g r a f t s were  o r i e n t a t i o n and  the g r a f t i n  a  place.  w i t h an E l a s t o p l a s t s p o t bandaid.  g r a f t s were examined a t weekly i n t e r v a l s  (or a t  the  t i m e o f d e a t h ) and  Results  and  these,  past  one  o f 1466  176  (12%)  hatched  week o f age.  survived  one  past  at  18  just  formed limbs  could  a f t e r the  and  the  not break out the  Bx  yolk  of  Bx  met  s a c was  16  (31%)  to p i p  birds  after 6 - 8  be  mortality malthe  shell The  body but  mortality  t o m o r t a l i t y peaks  indicating critical  in  requirements  time.  Those c h i c k s which s u r v i v e d until  the  s h e l l without help.  l a r g e l y due  normally  to  t o wounding  remaining outside  f i r s t week a f t e r h a t c h i n g  relatively  due  a n o t h e r peak o f  the  the  the h i g h  to compaction of  owing t o p o o r development o f  died  and  appeared  probably  embryos c o r r e s p o n d  c h i c k s was  intestine  hatched  T h e s e embryos u s u a l l y h a d  the  at this  the  survived  hormonally  incubation period  c h i c k s were a b l e  w h i c h must be  of  bursectomized.  (27% o f h a t c h )  (21%)  T h e r e was  embryos,  the  46  operation,  normal d e v e l o p i n g  During  and  o f 239  incubation.  Some o f t h e  peaks o f  51  the  operation.  days o f  cavity.  noted.  week.  Mortality during  the  t h e g r a f t was  S i m i l a r l y f o r the  embryos,  greatest  of  embryos w e r e s u r g i c a l l y  bursectomized  during  prognosis  Discussion:  A total Of  the  first  the  anal  mortality the  large  sphincter.  week c o n t i n u e d  to  develop  time t h e y were s a c r i f i c e d .  weeks, a l l s u f f e r i n g f r o m  Four  acute  nephritis. The  SBx  c h i c k s w e r e a b o u t 6 gm  normal c h i c k s I).  by  of  the  large  caecae were not  birds  the  o v e r 10  a f f e c t e d and  The o f 0.012  appeared normal.  months and spleens g  Bx  birds  i n t e s t i n e and  s e v e r a l weeks o f age.  female chicks  the  the  last  cloaca  Several  produced normal  o f body w e i g h t ) .  than  lighter  lacked  Development o f  few  the  (Table  bursa,  vertebrae.  appeared normal the  oviduct  hens were k e p t  in  in for  eggs.  o f n e w l y h a t c h e d SBx  (0.040 %  at hatching  8 weeks w e r e a b o u t 13 0 gm  Autopsy i n d i c a t e d t h a t  a b o u t 1/3 The  and  lighter  c h i c k s w e i g h e d an Those o f normal  average chicks  18 w e i g h e d an  average  difference  i n s p l e e n t o body w e i g h t r a t i o s  the spleens of  of  o f t h e Bx  normal c h i c k s  appeared  o f 0.22  darker  (0.054% o f body w e i g h t ) .  c h i c k s were r e a l l y  (Table I I ) . than  those  The  indicates  showed t h e Bx  s m a l l e r than  spleens  o f t h e Bx  than normal c h i c k s The than  because  to have fewer g e r m i n a l  o f t h e Bx  o f t h e n o r m a l WL  chicks also  chicks, but  made r e l i a b l e w e i g h t d a t a d i f f i c u l t sections,  however, r e v e a l t h a t t h e  i s due The  28  and  through  a series slight  smaller nature  grafts  soon subsided  and  beyond  the g r a f t  As  seemed t o b e  HBx,  The  thymic  separated  the s l i g h t  irreversible  a l a r g e number o f t h e Bx  14  were  area  was  5 mm  i n height.  the  host  off.  Success-  s w e l l i n g stage;  from  from  a t 7,  rejection  from  fell  appeared  separated  normal White  i n the  to about  and  (Figure 7).  grafts  t o be  as  the h o s t  this  this  incorporated  G r a f t s were r e c o r d e d  edges o f t h e p a t c h  rejection  site  then  patch  the s k i n of the h o s t .  when t h e  o f the  recorded  of stages  the g r a f t blackened  d i d not pass  Histological  s w e l l i n g o f the  s w e l l i n g o f the g r a f t  finally  on SBx,  (Table I I I ) .  edges o f t h e g r a f t e d p a t c h  tissue  small size  H a m p s h i r e c h i c k s was  R e d d e n i n g and  f o l l o w e d by  into  the d i f f u s e  to obtain.  of the s k i n g r a f t s  n o r m a l New  to pass  process.  ful  appeared  development o f the c o r t e x  days a f t e r g r a f t i n g  observed  The  to incomplete  prognosis  L e g h o r n and and  centres  t i s s u e and t h e abundance o f a s s o c i a t e d f a t d e p o s i t s  the  lobes  Histologic  (Figure 6).  thymus g l a n d s  those  those chicks  o f normal c h i c k s , perhaps  spleens  The that  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 of red c e l l s .  sections  of  g  rejected tissue  as  point.  c h i c k s d i e d w i t h i n the  first  week, t h e c o m p l e t e h i s t o r y o f t h e g r a f t was  obtained  i n only  about 20%  of the cases.  however,  show some  trends.  The  NH  birds  These r e s u l t s rejected  week i n 8 0 %  o f the  slower,  but  a l l rejected  SBx  HBx  groups,  no  and  signs of g r a f t  transfers.  do,  the g r a f t s w i t h i n the N o r m a l WL  c h i c k s w e r e somewhat  t h e g r a f t s w i t h i n 28  however, c o n t a i n e d a few  rejection  a t t h e end  first  o f 28  days.  Both  b i r d s which days.  the showed  A t 8 weeks  19 of age, as f e a t h e r i n g neared completion, appeared  intact.  the g r a f t s  O b s e r v a t i o n o f the thymus g l a n d  still  through  the s k i n o f the neck o f these b i r d s showed them to be s m a l l e r than the thymus glands o f normal b i r d s observed way.  i n the same  I f t h i s s m a l l e r s i z e was due t o the absence o r decrease  o f the thymic c o r t e x as was found i n o t h e r Bx b i r d s , the poor g r a f t r e j e c t i o n may be e x p l a i n e d as a l a c k o f competent lymphocytes.  I t i s a l s o p o s s i b l e t h a t some B - c e l l s a r e  necessary i n the process o f g r a f t r e j e c t i o n and t h a t some Bx b i r d s were unable t o b u i l d up a s u f f i c i e n t p o p u l a t i o n o f these cells  to c a r r y out r e j e c t i o n .  20  Table  I  Average w e i g h t s o f 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 f r o m h a t c h t o 8 weeks o f age.  Normal Error Sample Size  SBx Error  Age  Weight (g)  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  Weight (g)  Samp Siz<  21  Table  II  S p l e e n and body w e i g h t s 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 .  Spleen  Wt.  B o d y Wt.  Ratio  Normal Normal Normal Normal  0.0183 g 0.0217 0.0213 0.0246  41.9 g 43.1 36.2 38.0  0.00043 0.00050 0.00058 0.00064  Average  0.0227  36.8  0.00054  SBx SBx SBx SBx  0.0123 g 0.0160 0.0144 0.0079  29.8 g 36.3 37.0 19.8  0.00041 0.00044 0.00038 0.00039  Average  0.0126  30.7  0.00040  22  Figure  6.  H i s t o l o g i c a l s e c t i o n s o f n o r m a l (A) and b u r s e c t o m i z e d (B) s p l e e n s ; note the reduced number o f 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.. Magnification 120 x .  23  F i g u r e 7.  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 c t o m i z e d (B) thymic l o b e s ; note the reduced thymic c o r t e x i n Bx thymus. H & E stain. M a g n i f i c a t i o n 120 x.  24  Table  III  Prognosis of s k i n g r a f t s after grafting.  7 days (-) (+)  died 14  before days  14, and 28  14 d a y s (-) (+)  days  Host  (No)  WL  (16)  NH  4  12  5  2  5  5  0  SBx  (  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  5)  Donor  a t 7,  Those g r a f t s s c o r e d as a (-) i n one i n c l u d e d i n the next o b s e r v a t i o n .  o b s e r v a t i o n are  28 d a y s (-) (+ )  not  25  PART  II  :  Tests  of  the  Humoral  Immune S y s t e m .  Methods: i)  Serum  samples.  B l o o d was ture  or  c o l l e c t e d at weekly  from the  v e i n of b i r d s was  jugular  over  v e i n o f young b i r d s  5 weeks o f  c o l l e c t e d from c h i c k s  older birds. for 3 - 4  The  hrs,  blood  i n t e r v a l s by  age.  and  was  up  At  to  a t -20  ii)  C until  chicken  anti-chicken al.  (32)  used  to obtain  0.1  of  s e r u m was  M o d e l 100)  purified  by  was  used  blood the  a t room  temperature  The  was  serum  i n covered  of  of  preparation  the  test  o v i n e serum  chicken  IgM.  o f pH  8.2  c o l l e c t e d and  (Appendix).  was  eluted with  The  by  et  chicken  first  ( P h a r m a c i a UV  concentrated  rabbit  immunoglobulin  Whole a d u l t  spectrophotometry  of  method o f H u d s o n  t o a G-200 S e p h a d e x c o l u m n and  UV  of  wing  taken from  stored  i n the  A modification  M Tris-HCl buffer  peak d e t e c t e d  and  ml  the  antisera.  separation  applied  from  punc-  used.  sera.  f o r the  s e r u m was  was  then r e f r i g e r a t e d overnight.  Preparation  Adult  ml  to c l o t  t h e n removed w i t h a p a s t e u r p i p e t t e tubes  and  l e a s t 0.5  5.0  allowed  cardiac  a  protein  Monitor  nitrogen  pressure  dialysis. F i v e mg  of p r o t e i n  a b o v e ) i n a 2%  ( e i t h e r whole serum o r  solution i n chick  F r e u n d s c o m p l e t e a d j u v a n t and rabbits  a t 2 one  f r o m an  ear  blood  was  iii)  v e i n one  antibodies  as  was  1:1  second  a b o v e and stored  the  a t -20  was  purified with into  collected  injection. serum  The  containing  C.  Immunoelectrophoresis.  A standard in wells,  immunoelectrophoresis  r a b b i t antiserum  protein patterns. to  Rabbit blood  week a f t e r t h e  to c l o t  mixed  IgM  i n j e c t e d subcutaneously  week i n t e r v a l s .  allowed  anti-chicken  r i n g e r was  the  serum p r o t e i n s  and  i n t r o u g h ) was  Serum s a m p l e s  t h o s e s e v e r a l weeks o f  age  according  used  from d a y - o l d  were t e s t e d  specifically  c u r v e s were i d e n t i f i e d  procedure  f o r IgM.  (chicken  serum  to obtain  serum  chicks  through  i n t h i s way  for a l l  The  t o T u r e e n et  precipitin al.  (33).  26 iv) Test  for specific  Sheep r e d b l o o d specific  antibody  antibody  cells  (SRBC) w e r e u s e d  formation  i n normal,  F i v e week o l d b i r d s w e r e i n j e c t e d ml  production.  later  a second  t h e b i r d s were b l e d  i n j e c t i o n o f SRBC.  ions were repeated 3 - 4 c l o t t e d blood The  placed  ringer  times.  o f SRBC.  ringer.  and  given  and i n j e c t -  a t -20 C. b i r d s was t e s t e d  T w e n t y - f i v e JA.1 o f c h i c k e n of a microtitre plate  f o r the  s e r u m was  and  doubling  each c o n t a i n i n g  T h e serum d i l u t i o n s w e r e i n c u b a t e d  2 5 p i o f 5% SRBC f o r 30 m i n a t 37 C. r e c o r d e d as t h a t  Five  a n t i - S R B C a n t i b o d y b y m i c r o t i t r e haem-  i n the f i r s t w e l l  of chick  0.5  Serum was c o l l e c t e d f r o m t h e  d i l u t i o n s w e r e made t h r o u g h 11 more w e l l s ^il  solution.  These s e r i a l b l e e d i n g s  serum f r o m t h e s e s t i m u l a t e d  agglutination  SBx and HBx b i r d s .  from t h e wing v e i n  as a b o v e a n d s t o r e d  presence o f s p e c i f i c  stimulate  intraperitoneally with  o f a 2% s o l u t i o n o f SRBC i n c h i c k  days  to  dilution  The e n d p o i n t  o f serum w h i c h l a s t  25  with  titre  caused  was  agglutin-  a t i o n o f t h e SRBC.  Results  and D i s c u s s i o n :  According sera  t o T u r e e n et al. (33)  from growing c h i c k s  pattern  o f serum p r o t e i n s  shows t h e i r serum p a t t e r n  Immunoelectrophoresis o f  and o l d e r b i r d s as t h e b i r d s  shows a c h a n g i n g  mature.  Figure  f o r a 5 day o l d c h i c k .  The p r e -  a l b u m i n and b e t a - l i p o p r o t e i n b a n d s show t h e g r e a t e s t with  age.  Both a r e p r e s e n t  by  until  On t h e 1 0 t h  t h e p r e a l b u m i n b a n d s t a r t s t o become f a i n t e r and d i s a p p e a r s t h e 20th day.  corresponding 5 days IgD,  changes  from t h e time o f h a t c h i n g  8 d a y s when t h e b e t a - l i p o p r o t e i n b a n d d i s a p p e a r s . day  8  The gamma g l o b u l i n s  t o IgA, IgM and IgG.  i n normal c h i c k s .  A fourth  a p p e a r as t h r e e  A l l three  are present by  g l o b u l i n f r a c t i o n , perhaps  i s a l s o known t o o c c u r b u t i s n o t c o n s t a n t l y  Transferrin  appears as a p r o m i n a n t band n e a r  younger b i r d s beta globulins  curves  and s o m e t i m e s d i s a p p e a r s a p p e a r more p r o m i n a n t l y  found.  the o r i g i n i n the  i n older birds. i n older  birds.  The  27  The  serum s a m p l e s  showed t h e e x p e c t e d transferrin,  albumin,  the f i r s t  anti-whole plates  lipoprotein the time  few d a y s .  electrophoretic  patterns f o r  and t h e serum  appeared  F i g u r e s 9A a n d 10A show t h e immuno-  respectively.  The a n t i - I g M  show s e v e r a l b a n d s b e c a u s e t h e h e a v y c h a i n s o f immunoa r e common t o 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  irucomplete  The t r a n s f e r r i n b a n d  purification.  These  serum p r i o r The  could  have  embryonic  been  chick  to electrophoresis.  immunoelectrophoretic  serum a g a i n s t r a b b i t (Figures  i s present because  e x t r a bands  removed b y i n c u b a t i o n o f t h e a n t i s e r u m w i t h  p a t t e r n s f o r 14 d a y SBx c h i c k  anti-whole  c h i c k e n s e r u m and a n t i - I g M  9B and 10B r e s p e c t i v e l y )  transferrin,  lipoprotein  I n some c a s e s  appearing  several  show t h e n o r m a l p a t t e r n s f o r  and albumin.  however, a r e i n c o m p l e t e ;  IgM  globulins.  o f h a t c h i n g a n d IgG  serum a n d a n t i - I g M  in cross reactivity.  absent.  chicks  p a t t e r n s f o r n o r m a l 14 d a y c h i c k s e r a a g a i n s t  g l o b i n molecules  of  the normal White Leghorn  immunoelectrophoretic  IgM was p r e s e n t f r o m in  from  The serum  globulins,  IgM and I g G i m m u n o g l o b u l i n  ( n o t shown h e r e )  bands a r e  t h e r e were t r a c e s o f  d a y s a f t e r h a t c h i n g , b u t IgG was n o t  found. The h a e m a g g l u t i n a t i o n ( F i g u r e 11) i n d i c a t e a b l e Ab f o l l o w i n g  that  tests  forspecific  t h e SBx b i r d s  do n o t p r o d u c e  a primary challenge.  duction of antibody  a n t i b o d y t o SRBC detect-  However, a s t h e pro--  t o SRBC r e q u i r e s c o - o p e r a t i o n b e t w e e n B-  and  T-cells,  the absence o f the response  due  t o an absence o r 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 .  IV g i v e s t h e r e s u l t s and  HBx b i r d s .  challenge,  of a series  the normal b i r d s .  reduced  Table  cortex.  following  reach normal l e v e l s  SBx  to the primary  i s f a r below t h a t o f  H o r m o n a l b u r s e c t o m y does n o t a l w a y s  b o t h Bx g r o u p s s l o w l y r i s e  The  respond  of the response  the development o f the thymic  would probably  entirely  o f c h a l l e n g e s f o r normal,  Some o f t h e HBx b i r d s  b u t t h e degree  may n o t b e  The a n t i b o d y  impale  titres of  s u c c e s s i v e c h a l l e n g e s and  i f t h e s e r i e s were  l y m p h o c y t e p o p u l a t i o n s o f t h e Bx b i r d s  continued.  require a  28  l o n g e r time i n which t o b u i l d up s u f f i c i e n t c e l l s a s p e c i f i c a n t i b o d y than do those o f normal b i r d s . b i r d s appear  t o approach  normal  producing The HBx  l e v e l s more r a p i d l y than the  SBx b i r d s ,  perhaps  bursectomy  a r e l e s s severe than those o f e a r l y s u r g i c a l b u r s e c -  tomy.  s u g g e s t i n g t h a t the e f f e c t s o f hormonal  29  Figure 8 .  P r e c i p i t i n bands from Immunoelectrophoresis of normal 5 day c h i c k serum (from Tureen et a l . , 3 3 ) .  Key:  A. B. C. D. E. F. G. H.  prealbumin albumin alpha l i p o p r o t e i n beta,lipoprotein transferrin IgA gamma g l o b u l i n IgM gamma g l o b u l i n IgG gamma g l o b u l i n  30  A  M  F i g u r e 9.  |  B  P r e c i p i t i n bands from immunoelectrp h o r e s i s o f 14 day normal (A) and b u r s e c t o m i z e d (B) c h i c k s e r a a g a i n s t r a b b i t anti-whole c h i c k e n serum.  31  F i g u r e 10.  P r e c i p i t i n bands from Immunoelectrop h o r e s i s o f 14 day normal (A) and b u r s e c t o m i z e d (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 IgM.  32  000000000000 " ©©©©©©©©00© < oooooooooooo * S8  ©®®©••••••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 b y n o r m a l (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 h o r m o n a l l y b u r s e c t o m i z e d (HBx) c h i c k e n sera following primary s t i m u l a t i o n with SRBC.  Haemagglutination t i t r e s f o r normal, SBx and HBx chicken sera following successive stimulations w i t h SRBC.  Table IV  Birds  (No)  Normal  (10)  SBx  (6)  HBx  (8)  Primary 128*  -  4  Secondary 256  - 512  Tertary 1024  - 2 048  8 - 32  256  - 512  8 - 64  512  - 1024  * a t i t r e o f 128 i n d i c a t e s t h a t a d i l u t i o n o f serum 1/128 i n c h i c k r i n g e r i s the g r e a t e s t d i l u t i o n which s t i l l c o n t a i n s s u f f i c i e n t anti-SRBC antibody t o a g g l u t i n a t e 2 5 u l o f 5% SRBC.  34 PART  III  :  S t i m u l a t i o n of with mitogens.  Introduction  to  Several agglutinins  plant and  of  these mitogens  as  they have  have  the no  are  concrete  than  thought  effect  a l l  of  the  for  were  (ConA)  formation  to  cell not  usually  is  obtained  into  B  and has  to  has been no  absence of  T - c e l l s — i t is of  is  proportions  For  purpose,  this  birds  and  spleen,  were c u l t u r e d  in  thymus  on  allows  B-  cells  and b u r s a  presence of  and  They undif-  PHA-induced  trans-  macro-  of  (34).  bacteria,  bacteria  DNA s y n t h e s i s  antigen  population  such in  lymphocytes  stimulating B-cells to  s p e c u l a t i o n as  and T - c e l l s  bean.  of  thymus-derived  for  thymus  mitogenic  jack  transformation  cell  kidney  a l . ( 3 5).  presence of  induce  less  leukocyte  treated  a T-cell-independant  an _in v i t r o of  Its  gram n e g a t i v e  capable of  s p l e e n and the  of  shown t o  c e l l - s p e c i f i c mitogens  relative  red  transformation the  is  mitogens.  mixed  cells.  anti-  which  this  Hungerford ^ t  the  require  effect  Endotoxin  of  for  from the  anti-theta  T-cells  specific  extraction.  antigen-induced  (36,37).  response  evidence  derived  blast  from c u l t u r e s  It  The  to be  a lipopolysaccharide product  as jE^ c o l i . lymphocytes  Others,  T - c e l l s p e c i f i c mitogens.  appear  necessary for  Endotoxin  the  d i v i s i o n and  lymphocytes  does  thought  1 9 59 b y  stimulate  Some  treated with  (34).  stimulate d i v i s i o n of  c o n s i d e r e d to be rapid  cells  is  shown  l e u k o c y t e s _in v i t r o .  saline  in  cell  a s i m i l a r e x t r a c t from the  are  phages  fail  is  but  ferentiated  by  discovered  cultures stimulate  cells  endotoxin have been  T-cell specific  (PHA)  vulgaris,  red blood  lymphocytes  on T - c e l l s are  that  two m i t o g e n s  thus  and b u r s a  specifically stimulate  thymus-derived although  Concanavalin A These  to  B-cells,  Phaseolus  properties  to be  of  on mouse  Phytohaemagglutinin bean,  known  transformation  l i t t l e effect  stimulants  proteins  the b a c t e r i a l product  stimulate  to k i l l  thymus  mitogens:  to  theta  spleen,  a  in  the  (38). variety  to  the  in  that  population.  cells  from  bursectomized  cells  from normal  mitogens.  birds  35 Methods: Spleen and  and  thymus c e l l s  from  s p l e e n , thymus and b u r s a  cultured with  cells  from  i n the w e l l s o f m i c r o t i t r e  2 T-cell  specific effect  5 t o 8 week o l d SBx  specific  (PHA  and  (endotoxin) mitogen  o f b u r s e c t o m y on  Cells  normal c h i c k e n s were  plates  t h e B- and  effort  ally,  of autologous  in chick  pairs  of forceps.  with  a 26 g u a g e  i n RPMI 1640  then d i l u t e d  cytes  injection  of  1/100  counted w i t h  ringer. of chick ringer  A  single  fragments  cell  through  i n chick ringer  c u l t u r e medium  1% p e n i c i l l i n / s t r e p t o m y c i n .  with  serum f o r t h e  and from  suspension a 1  ml  needle.  c e l l s were washed 2 times  resuspended  manner.  the c e l l s were g e n t l y t e a s e d  passing the t i s s u e  equipped  The  following  intracardiac  t h e n p l a c e d i n 5 ml serum and  two  obtained by  syringe  was  by  trimmed o f f a t and w a s h e d  the organ w i t h was  the  The a p p r o p r i a t e organs were removed a s c e p t i c -  E a c h o r g a n was 1 ml  B-cell  to determine  i n the  The b i r d s w e r e b l e d t o p r o v i d e a u t o l o g o u s c u l t u r e medium t h e n w e r e k i l l e d  one  T-cell populations.  f o r c u l t u r e were p r e p a r e d  10 c c o f a i r .  and w e r e s t i m u l a t e d  ConA) m i t o g e n s and  i n an  chickens  and  supplemented  (Appendix)  A sample o f the  i n c u l t u r e medium and  a h e m o c y t o m e t e r i n a 1/1  finally  suspension  the v i a b l e  lympho-  mixture with  trypan  blue. The of  cell  s u s p e n s i o n was  t h e n d i l u t e d t o o b t a i n 5 ml 7 6  l y m p h o c y t e c o n c e n t r a t i o n s o f 2.5  p e r ml  i n both  s e r u m f r e e and  c u l t u r e medium. syringe  into  trations  The  x 10  5% a u t o l o g o u s  and  5 x 10  cells  plates  a l l cell  mitogens were added  10  g/ml,  ConA - 20  of saline.  0.2 5 ml w i t h  The  and  i n the  g/ml, total  following  a  cells  supplemented Hamilton concen-  f o r both  f o r each  media.  type  of  a l l mitogens.  c o n c e n t r a t i o n s : PHA  e n d o t o x i n - 20  g/ml  volume o f e a c h w e l l was  t h e a p p r o p r i a t e medium and  x 10  to f i n a l  types were s t i m u l a t e d w i t h  The  ml  serum  per w e l l  T r i p l i c a t e w e l l s w e r e made f o r c o n t r o l s mitogen;  2.5  c e l l s were d i s p e n s e d w i t h  the w e l l s o f m i c r o t i t r e 6 5  o f 5 x 10  and  samples  each  in  made up  the c u l t u r e s  were  -  0.05 to  36  incubated  a t 37  (tritiated each w e l l The  C f o r 48 h r .  thymidine) and  o r 20  as  purpose,  to lyse  the c e l l s  The  fluid  filters  (Liquifluor  in a scintillation  HTdr  a mitogen  and  =  as  CPM CPM  filters.  t o remove and  to  The  excess  precipitate  were t h e n d r i e d  under of  t o l u e n e , no m e t h a n o l ) Dpms w e r e n o t  a ratio  hrs.  machine,  v i a l s w i t h 3 ml  counter.  the r e s u l t s were e x p r e s s e d  fibre  tap water  lamp, p l a c e d i n s c i n t i l l a t i o n  Results  o f two  and  determined  counts.  o f mitogen s t i m u l a t e d c u l t u r e of a non-stimulated culture  .  and D i s c u s s i o n :  A series of c e l l ed  a vacuum c o l l e c t i o n  onto g l a s s  5 times w i t h  step.  i s o t o p e was  of  12  a l l i n one  S t i m u l a t i o n by  ml  the c u l t u r e s were i n c u b a t e d f o r a f u r t h e r  radioactive material,  counted  0.05  to  for this  scintillation  time  added  c e l l s were washed  a heat  this  j/ici/m.1 °f  c e l l s were h a r v e s t e d by  constructed  t h e DNA  At  of preliminary  density,  serum types  tests with various and  t h a t homologous, p r e f e r a b l y  combinations  serum c o n c e n t r a t i o n s d e t e r m i n -  autologous,  serum a t a  con5 5 x 10 to  c e n t r a t i o n o f 5 - 1 0 % and a l y m p h o c y t e d e n s i t y o f 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 5 cultures  o f 5 x 10  appeared  t o be  cells  per w e l l  the h e a l t h i e s t  with bacteria.  The  s e r u m was  cultured without  and w e r e l e a s t most o f t e n  often  these,  serum infected  the c a r r i e r  of  con-  tamination. The range  unstimulated or control  of s e v e r a l hundred,  c u l t u r e s w e r e 3 t o 300  serum w e r e s t i m u l a t e d 25 40  times w i t h  w e r e s t i m u l a t e d 50 15  times w i t h  was  expected  case,  those o f the mitogen  a t 5 x 10  cells  times w i t h  PHA,  endotoxin. times w i t h  endotoxin. t o be  g a v e cpms i n t h e stimulated  times g r e a t e r .  Normal s p l e e n c e l l s  and  cultures  The  per w e l l 17  Spleen c e l l s PHA,  15  times w i t h from  times w i t h  s p l e e n o f t h e SBx  deficient in B-cells. 5 a sample o f 5 x 10 lymphocytes from  without  an SBx  chicken  ConA and bird,  I f t h i s were such  ConA  only  however, the  a s p l e e n would  37  contain a relatively relatively  smaller  larger proportion  be f u r t h e r s u p p o r t e d cell  mitogen.  give  support  to d i s p r o v e At  o f B - c e l l s and a  of T-cells.  This  appears t o  b y t h e l a r g e s t i m u l a t i o n b y PHA,  The r e s u l t s to this  a T-  o f t h e ConA s t i m u l a t i o n do n o t  idea, but are not s u f f i c i e n t l y  i teither  consistent  (Table V ) .  5  5 x 10  proportion  cells  per well  i n t h e a b s e n c e o f serum t h e  s t i m u l a t i o n o f n o r m a l and SBx c h i c k e n  thymus c e l l s  similar.  o f t h e SBx b i r d s w e r e  Although  much s m a l l e r  t h e thymus g l a n d s  than those showed  bird  to mitogens.  remarkably high  very  o f the normal b i r d s , a standard  sample o f c e l l s t o respond  was  t h e same a b i l i t y  as c e l l s  The r e s p o n s e  f o r a predominantly T - c e l l  size  from a normal  to endotoxin population  was  (Table  VI). The the  bursa  cells  responded o n l y  view t h a t t h e b u r s a  contains  to endotoxin,  only  B-cells  supporting  (Table V I I ) .  38  Table V  Average s t i m u l a t i o n * o f normal s p l e e n c e l l s w i t h mitogens.  No. o f c e l l s  Serum  Endotoxin Bx N  (N) and SBx  chicken  N  ConA Bx  N  PHA Bx  5  X  10  5  —  40  15  17  15  25  50  5  X  10  5  +  11  3  14  15  18  26  X  10  6  -  5  7  65  15  44  10  X  10  6  +  5  20  26  30  41  58  5 5  * Stimulation =  CPM o f mitogen s t i m u l a t e d c u l t u r e s CPM o f n o n - s t i m u l a t e d ( c o n t r o l ) c u l t u r e s  39  Table VI  No. o f c e l l s  Average s t i m u l a t i o n o f normal thymus c e l l s w i t h m i t o g e n s .  Serum  Endotoxin N Bx  5 x 10  5  -  120  5 x 10  5  125  +  5  5 x 10  6  -  25  16  5 x 10  6  +  14  8  (N) a n d SBx  chicken  N  ConA BX  N  PHA BX  20  24  17  20  25  -  10  13  133  26  30  30  10  13  40  Table VII  No. o f c e l l s  5 x 10  5  5 x 10  5  5 x 10 5 x 10  Average s t i m u l a t i o n with mitogens.  Serum  6  Endotoxin  5 + 8  6  +  o f normal c h i c k e n b u r s a  11  ConA  cells  PHA  41  GENERAL DISCUSSION Present Fabricius  evidence  i s an  ferentiated  favours  anatomical  stem c e l l s  of a developing  cells  from  from  This  s e r v e as  i n embryonic cells  a role  injection  produces  producing  cells  totally  time  o f hormones d u r i n g  inability  i n o n l y about 40%  phamide  effective  effective, The  results  Early  This  developed  growth o f the l i p and  malities,  using both  treatment  ventral  bursectomy,  can be  repeat-  (25).  during  raised  to  cyclophosthe  first  few  although h i g h l y  here  for early  survival  surgical  and  embryonic  r a t e has  hormonal  w i t h hormones r e s u l t s M e y e r e t a l . (40)  been  noted  terminating i n death.  noted  accumulation  I t would  noted  i n abnormal  following early  in intracloacal  2%  treatments  stunted  anal l i p , increased musculature  to those  resulted  surgical  i n o n l y about  a t t e n u a t i o n o f the proctodaeum.  similar  in  after  treated  repeated  treatment,  T h i s low  development of the c l o a c a .  usually  this  viable bursa-less birds  treated.  other workers  dorsal  (40) b u t  Neonatal  i n agammaglobulinemia  i f coupled with  (41).  bursectomy produced  (42).  o f the b i r d s result  (3 9 ) .  i n high mortality.  technique  o f a l l embryos  directing  results  t o p r o d u c e Ab  (an i m m u n o s u p r e s s a n t ) t r e a t m e n t s  days a f t e r h a t c h i n g  by  can  of treated b i r d s  more t h a n 9 0 %  specificity  agammaglobulinemic b i r d s .  an  50%  in  irrevocably  i t s development  a g a m m a g l o b u l i n e m i a and  -  appear  of hatching or supressing  t o t a l body i r r a d i a t i o n  i n 40  and  f o r the bursa  a t the  H o r m o n a l b u r s e c t o m y _ i n ovo  into In  IgG  seem t o b e  bursectomy f o l l o w e d by  ed c h a l l e n g e s  life.  leading to antibody heterogenecity  Removal o f t h e b u r s a growth by  precursors  c e l l s which p r e v i o u s l y synthesized  the bursa,  implys  the g e n e t i c events  rarely  s i t e where u n d i f -  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  of antibody.  its  late  immune s y s t e m ,  exclusively  Once s e e d e d  committed  physiological  of  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  the ontogeny o f the  IgM.  and  view t h a t the b u r s a  of y o l k sac o r i g i n  immunoglobulin producing  to develop  the  appear,  of  These  the  abnor-  surgical of  faeces,  then,  that  42  early mortality resulting  from  developmental  i s the r e s u l t  the treatment r a t h e r requirement  Although  uted It  and Bx  to loss  analysis,  chicks  is possible  that  data i n Table I are  the weight  appear  of the t a i l  than the r e s u l t  of  insufficient  d i f f e r e n c e s between  t o be g r e a t e r t h a n c a n be  structure  the s t r e s s  and  lower  large  to p e r s i s t  Freeman  8 weeks a f t e r h a t c h i n g .  logical the  The  showed a r e d u c e d  effect  o f bursectomy  s t a t e of the b i r d  immune  indicate  i s not c l e a r  that  ratios  and  f o r day  t h e s p l e e n s o f Bx  normal  chicks.  effect  of bursectomy  most s e n s i t i v e  This,  may  be  The  reduced  a g a i n , may  as  indicate  the r e s u l t  that  of a reduced  of humoral  at  the reduced lymphoid  of the b u r s a l  i n this  study.  and  i n the absence  gut-associated hatching other  and  tissues  do  not appear  so a r e u n l i k e l y  lymphoid  tissues.  The  t o be  the  of  o f t h e Bx  the spleen  indication of a o f the  B-cells  i s n o t known. n o r w e r e any  sections  of the  No gercaecae  are o c c a s i o n a l l y the c l o a c a ,  It is possible  of the bursa.  of  t o be  sections  origin  bursae  stalk  those  appear  size  i n these b i r d s ,  Auxilliary  (Table  p o p u l a t i o n (Figure 6).  The  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 here  beyond  a general physiologic  tissues  i n the h i s t o l o g i c a l  intestine.  the j u n c t i o n  not found  t h a n do  Histological  lymphocytes.  remnants were found  large  from  r e p r e s e n t a some-  number o f g e r m i n a l c e n t r e s i s a n  m i n a l c e n t r e s found and  extend  old chicks  reflect  f o r m i n g g e r m i n a l c e n t r e s i n t h e Bx b i r d s bursal  to stress  may  chicks  the lymphoid  to such s t r e s s e s .  however,  deficiency  that  the g e n e r a l p h y s i o -  what s m a l l e r p r o p o r t i o n o f the body w e i g h t  spleen,  (43!) n o t e d  response on  effects  system.  S p l e e n t o body w e i g h t II)  attrib-  of the o p e r a t i o n r e t a r d e d the not expect these  handling.  the  intestine.  g r o w t h o f t h e embryo, b u t one w o u l d  bursectomized birds  any  f o r the b u r s a .  the body weight  f o r growth r a t e normal  of physical abnormalities  that  found  b u t were  the B - c e l l s  and h a v e  migrated  germinal centres of  until  involved  about  in  10  days  the  after  i n the seeding o f  J a n k o v i c e t a l . (24) n o t e d  that  43  neonatal bursectomy reduces germinal centres  i n the  the  f o r m e d as  length  of  a series of  the  thyroids.  obtained  lobes  neck e x t e n d i n g  The  thymic  lobes  than those of normal b i r d s ; nification sections  (Figure  also  7)  show t h a t  cytopoesis.  The  the  as, i n aves, t h i s  embedded  almost  to  the  of  Bx  chicks  the  this  thymic c o r t e x ,  same s t r e s s e s ,  the  s u c h as  e f f e c t on  and  maturity  for  the b u r s a  bursa could ways.  The  the  thymus.  followed  by  is also  a f f e c t the stress of  a m a j o r f a c t o r as  cortical play the  development.  thymic branch of  13  the  d a y s _in ovo  60%  of  inability  the  chicks  The  the  suggest that rather  the  capacity  to  in  the  several  i s u n l i k e l y to  the  be normal  general  incomplete  that  r o l e i n the  the b u r s a  development  may of  system. f o l l o w i n g hormonal bursectomy  from very  i n 10%  of  i n t e r p r e t as  i n j e c t i o n of  a  removal of  thymus  result in  to severe r e d u c t i o n  of  the  the  They  little  change  cortex  and  chicks  bursectomy  was  h a v e b e e n w i d e and  similar effects following be  the  e f f e c t of hormonal  an  treated.  s t e r o i d hormone r a t h e r  e f f e c t s may  phenomenon may  than a s i d e  The  immune  cort-  found  showed a p p a r e n t l y  e f f e c t s ranging  d e v e l o p m e n t , when t h e of  animal  n o t e d b y W a r n e r _et a l . ( 4 4 ) .  difficult  observation  The  itself  lympho-  e a r l y growth  thymus.  It i s also possible  to r e j e c t s k i n g r a f t s  accomplished by  of  of  due  b u r s a have  adult  may  thymic c o r t e x  was  the  is  adrenal  e f f e c t o f b u r s e c t o m y on  the b i r d  the  a v a r i e t y of  These d a t a are  in  The  a d i r e c t o r hormone m e d i a t e d  observed in  operation  same mag-  lobes  ACTH and  the  smaller  These  i n the  sham o p e r a t e d b i r d s  state of  Atrophy of at  f o r the  the  main area  pattern  development of  the  thymic development. physiological  The  regression  true  of  the  the  of  the  appeared  at  s i z e of  i c o i d s , which cause premature r e g r e s s i o n similar  level  observation.  reduced  gland  i n f a t along  cross-sections  support  to a d e f i c i e n c y i n the  and  caecae.  Thymus w e i g h t s w e r e n o t is  number o f p l a s m a c e l l s  late varied.  s u r g i c a l bursectomy  r e s u l t of  a bursal  treatment.  f o r s k i n g r a f t r e j e c t i o n was  tested  in a  effect  44 s m a l l number o f SBx c h i c k s and a g r o u p bursectomized (Table of  a t 72 h r _in o v o .  III) indicate  skin graft  the g r a f t that  neck  chick.  group  The b i r d s  Hormonally  used  i n this  that  f o r other tests. through  an extended  I t i s probable  data  organ to  that  to this  of the bursa  point,  these b i r d s ,  then,  the general p h y s i o l o g i c a l  state  on t h e immune s y s t e m  effect  on t h e l y m p h o c y t e  Immunoelectrophoresis  appear  The  albumin,  t r a n s f e r r i n band  sampling  period.  presence  lipoprotein remained  the l a t t e r  part o f the t e s t  by the c h i c k .  entirely but  t h e immune  system  The d i r e c t  t o be a r e d u c t i o n o f t h e lymphoid  tissues  and  perhaps  through  the f i r s t  (33) p a t t e r n s f o r  and b e t a g l o b u l i n  strong throughout  fractions.  t h e f o u r week birds,  however,  o f t h e gamma g l o b u l i n b a n d s . a f t e r h a t c h i n g can be  o f maternal period  The  immunoglobulin,  c o u l d o n l y have been  b u t by pro-  The s e r a f r o m m o s t o f t h e Bx c h i c k s w e r e  d e v o i d o f IgG.  tended  indicate  o f the b i r d .  t h e normal  o f these bands i m m e d i a t e l y to the presence  cortex.  Removal o f t h e  The s e r a o f t h e b u r s e c t o m i z e d  attributed  duced  do n o t c l e a r l y  o f serum s a m p l e s  showed r e d u c t i o n o r a b s e n c e  for graft  p o p u l a t i o n o f t h e thymus.  few weeks a f t e r h a t c h i n g showed prealbumin,  the s k i n o f the  i n thymic  e x t e n d i n g beyond  germinal centres o f the p e r i p h e r a l an  Superficial  similarly to  i n t h e immune s y s t e m .  o b v i o u s l y has e f f e c t s  effects  rejection  time course  t h o s e o f W a r n e r _et a l . ( 4 4 ) , w e r e l a c k i n g  the r o l e  bursectomized  t h e g l a n d s 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 rejection.  twice  t e s t w e r e n o t k i l l e d and  o f t h e l o b e s o f t h e thymus  indicated  The  f o r t h e edges o f  showed s i g n s o f r e j e c t i o n w i t h i n t h e  as t h e y w e r e r e q u i r e d  examination  required  study  capable  showed a r e t a r d e d t i m e c o u r s e o f g r a f t  period.  autopsied  of this  t h e h o s t t i s s u e was a t l e a s t  f o r a normal  b u t most o f t h i s test  The t i m e  t o s e p a r a t e from  also  The r e s u l t s  hormonally  t h e SBx b i r d s w e r e l e a s t  rejection.  required  chicks  that  of chicks  I n some c a s e s  t o b e r a t h e r weak.  g l o b u l i n s were b e i n g produced  a n IgM b a n d was  I t i s possible i n such  found  that the  small quantities  as t o b e  45  undetectable by t h i s  method.  The i m m u n o g l o b u l i n b a n d s 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 . Stimulation  o f the humoral  immune s y s t e m b y t h e i n t r o -  d u c t i o n o f a n t i g e n i c substances circulating  immunoglobulin.  production of specific  i n c r e a s e s t h e amount o f  This s t i m u l a t i o n d i d e l i c i t the  antibody  i n some o f t h e b u r s e c t o m i z e d  birds. Surgically  and h o r m o n a l l y  ulated  f o r the f i r s t  little  o r no a n t i b o d y .  (Table  IV) o f t h e s e r a i n d i c a t e d  chickens  and t i t r e s  time  bursectomized  titres  A second nearly  birds  challenge with  more r a p i d l y  of germinal that  centres  arithmic  antigen,  numbers.  present  the process  some t i m e .  This  A deficiency also affect with  reduced  (22,23,24).  ( 2 4 ) . The  the l e v e l  to rise  to respond  i s the r e s u l t  toward  normal  to the s e r i a l  i s probably  T h e low number  o f Bx b i r d s  suggests  of a gradual  increase i s log-  o f responding  up t h e c e l l  t o a n y one  population  not the only f a c t o r  takes  involved i n  the production of antibody  to a  r e q u i r e s t h e c o o p e r a t i o n o f T- a n d B-  i n t h e thymic  stim-  and a s o n l y a s m a l l number o f  are capable  of building  o f Ab  Further s t i m u l a t i o n with the  As t h e i n c r e a s e i n numbers  the slow humoral response; number o f a n t i g e n s  although  i n the spleens  following stimulation,  lymphocytes  challenge  t h a n d i d t h e SBx b i r d s .  the gradual response  i n humoral c e l l  the  appeared  found  birds.  t h a t some o f t h e Bx b i r d s d i d  same a n t i g e n d i d c a u s e t h e Ab t i t r e s  ulations  analysis  o f 128 f o r n o r m a l  p r o d u c t i o n i n some Bx b i r d s  n o t approach normal l e v e l s .  t h e HBx b i r d s  produced  a r e a l s o known t o h a v e  i n the 2° response  levels;  stim-  t h e same a n t i g e n may r e s u l t i n  IV i n d i c a t e d  produce antibody did  a titre  f o l l o w i n g the primary  i n Table  SRBC  of 0 to 4 f o r the bursectomized  normal a n t i b o d y  results  a t 5 weeks o f age w i t h  M i c r o t i t r e haemagglutination  Neonatally bursectomized antibody  chickens,  c o r t e x and thus  the production o f antibody  i n T-cells  cells.  would  following stimulation  SRBC. Jankovic  and I s a k o v i c  (24), w o r k i n g w i t h  n e o n a t a l l y Bx  46  birds,  found  that  no  a n t i b o d y was  primary challenge. produced normal is  to  a secondary  a s m a l l amount o f 19S  response,  o f much l e s s  which  After  IgM  produced  (IgM)  i s produced  importance  following  challenge these  type antibody.  i n the primary  i n the subsequent  birds  mentioned  in  p a r t be  due  of  bursectomy  i n the secondary  above,  no  IgG was  to the time o f bursectomy as  n e o n a t a l removal  In a  reaction  and IgG,  response,  response.  found.  birds  reactions.  i s normally u n d e t e c t a b l e i n the primary  much h i g h e r l e v e l s  the  rises  In the  These r e s u l t s more t h a n  seems t o m a i n l y  the  Bx may  fact  impair  IgG  production. Early  s u r g i c a l bursectomy  on  the c l a s s  of  s e r u m f r o m Bx b i r d s  albumin  (BSA)  of immunoglobulin  also  cell  W a r n e r _et a l . (44)  found  following  virus, for  normal  320  t o 20480.  low Ab  primary  birds  c a n be  capacity  removed b y  (2).  birds  those  lymphocytes lymphoid  absent,  surgically  were c a p a b l e o f r e j e c t i n g  t h e i r hormonally  the thymic  influenza  o r BSA.  Primary  following  a l l rapidly  dividing  that  even though  and h o r m o n a l l y  they  A  titres from  neonatal This cells and  (22). cytotoxic  i n the normal  found  bursectomized birds  anti-  bursectomized  that  a small  exhibited  c o r t e x and w e r e i n c a p a b l e o f r e j e c t i n g  Others, w i t h  than  type  these antigens ranged  skin grafts  p u b l i c a t i o n however,  of less  which are seeding the s p l e e n  tissues  Warner e t a l . ( 4 4 ) n o t e d  not.  t o t a l body x - i r r a d i a t i o n .  including  other peripheral  suis,  t o s y n t h e s i z e Ab  indiscriminantly k i l l s  of  i n Bx  antibody t i t r e s  stimulated with  treatment  a later  titres  c o o p e r a t i o n , o t h e r s w h i c h do  challenge with Brucella  residual  bursectomy  b o d y was  serum  o f s m a l l amounts o f IgM  human c h o r i o n i c g o n a d o t r o p h i n  The  Immunoelectrophoresis  twice challenged with bovine  report  effect  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  w h i c h r e q u i r e B-T  10  produced.  shows t h e p r e s e n c e  Other workers following  seems t o h a v e a s i m i l a r  i n t e r m e d i a t e damage t o t h e t h y m i c  birds  time.  In  percentage  atrophy grafts  of (43).  c o r t e x , showed  47 a graded a b i l i t y The  effects  c a n n o t be  effects of  divorced  appears  p a r t of the r o l e the switch  t o b e more s e v e r e .  and  stimulating just  a few  I t may  These  a l l o f one select  the  of early  be  that  synthesis  surgical a t any  t h e most  critical  immune s y s t e m i s  lymphocyte  stimulation with  substances appear  t o be  as i n t h e c a s e o f a n o r m a l o f normal  and T - c e l l m i t o g e n s  the  s p l e e n o f t h e Bx b i r d  and  r e l a t i v e l y more T - c e l l s  s p l e e n o f a normal b i r d .  leads  contains  capable of  reduced  i n number, do  ability  to respond to mitogens.  antigen.  Bx c h i c k e n  r e l a t i v e l y fewer  cells  not appear  and  than  spleen  to the s p e c u l a t i o n  per standard The  before  tissue.  on t h e s e s u b p o p u l a t i o n s r a t h e r  cells  time  than to  i n some c e l l s  e s t i m a t e d by  A comparison of the s t i m u l a t i o n c e l l s w i t h B-  and  s u b p o p u l a t i o n s o f a mixed  individually  mitogens:.  similar  t o t h e IgG p r o d u c i n g c e l l s  to the p e r i p h e r a l  p o p u l a t i o n c a n be  generally.  Bursectomy  t o IgG p r o d u c t i o n  T-cell  system  bursectomy  t o have  o f the b u r s a i n the humoral  f r o m IgM  they are seeded  specific  of  o f immunoglobulins  IgM p r o d u c i n g c e l l s .  B-  appear  antibody, although the e f f e c t  seems t o be more damaging  The  from the e f f e c t s  immune  o f t h e immune s y s t e m and o n t h e b i r d  on t h e p r o d u c t i o n  bursectomy  in  on t h e h u m o r a l  and h o r m o n a l b u r s e c t o m y  specific  the  t o t h e e x t e n t o f damage.  of bursectomy  entirely  on t h e r e s t Surgical  proportional  that  B-cells  s a m p l e t h a n does  the  o f t h e thymus, a l t h o u g h  t o be  impared  i n their  48  SUMMARY AND The  CONCLUSION  general effects  are s i m i l a r surgical  to those  and  of early  found by  of bursectomy cannot  reduced  humoral  in and  terms o f t h e e f f e c t s on  the  state  role  capable  There  IgM  source  'these c e l l s sac o r i g i n  major r o l e lymphocytes on  and  degree  cell  of the b i r d . those  The  system, the  immune  c e l l s which  at least  It is possible  o r o f b o n e marrow o r i g i n  and  that  capable  o f the b u r s a into  IgM  synthesis.  I t may  i s i n the d i v i s i o n  and  IgG  producing  IgM  of  the b u r s a  are necessary  and  of  be  to  that  populations.  t o answer t h i s  be  i n the absence of the  the  Further  cells  i n the  question.  o f t h e s o u r c e o f the stem  value.  of  humoral  the c a p a c i t y of these c e l l s  lymphocytes  w o u l d be  bursa.  synthesis  of the  the nature of the a n t i b o d y producing bird  are  they are of yolk  they appear  d e t e r m i n a t i o n to the p o i n t of  t o s w i t c h t o IgG  i n t o competent  depletion of  synthesis support  cells  a  cooperation  o f the humoral  some l y m p h o i d  into  more e x t e n s i v e i n v e s t i g a t i o n  environment  B-T  o f autonomy i n t h e d i f f e r e n t i a t i o n  i s unclear.  bursectomized  entering  on  i n the ontogeny o f the  t o be  the  examined  type a n t i b o d y even i n the absence o f the  of s e l f  unable  studies  appear  of d i f f e r e n t i a t i o n  producing  capable  o f the b u r s a  Many o f  b u t must a l s o be  thymus,  the r e d u c t i o n o f immunoglobulin  response.  but  on  both  c o n s i d e r e d o n l y i n terms o f  populations, especially  postulated  The  be  bursectomy  employing  f o r bursectomy.  population  the p h y s i o l o g i c a l  lymphoid and  lymphoid  surgical  other workers  hormonal techniques  effects  embryonic  to  A  cells develop  bursal  49  BIBLIOGRAPHY 1.  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B u r n e t t , 1969, The immunological r o l e o f d i f f e r e n t lymphoid organs o f t h e c h i c k e n , A u s t . J . e x p t l B i o l , med S c i . 40:373 388.  45.  Warner, N.L. and A. S z e n b e r g , 1964, The i m m u n o l o g i c a l c t i o n o f the b u r s a o f F a b r i c i u s i n the c h i c k e n , Rev. M i c r o b i o l . 18:253 - 268.  B.,  1973,  personnal  communication.  funAnn.  53 APPENDIX  : Formulae  and c u l t u r e  for soiuti  media.  A. C h i c k R i n g e r NaCl  7.00  g  KC1  0.42  g  0.24  g  1000  ml  100  ml  CaCl  2  dist.  H 0  to  2  B. B u f f e r e d  Formalyn  commercial  formaldehyde  Na HP0  4  (anhydrous)  6.5  g  NaH P0  4  (anhydrous)  3.5  g  1000  ml  2  2  dist.  H 0  to  2  C. T r i s - H C l B u f f e r Tris  (Tham)  NaCl  15.75  g  117.90  g  HC1 dist.  H 0  to  2  D. RPMI 1640 T i s s u e C u l t u r e ] Ca(N0 ) ". 4H 0 3  2  ium  7.0  ml  1000  ml  (Gibco) 100.0  2  Glucose  2000.0  MgS0 • 7 H 0  100.0  KC1  400.0  4  Na HP0 2  2  4  • 7H 0  1512.0  2  NaCl L-Arginine  6000.0 (free  base)  200.0  L-Asparagine  50.0  L-Aspartic  20.0  acid  50.0  L-Cystine L-Glutamic  20.0  acid  •300.0  L-Glutamine Glutathione  (reduced)  10.0  Glycine L-Histidine  1.0  (free  base)  15.0  mg/1  cont.  RPMI  1640 20. 0  L-Hydroxyproline L-Isoleucine L-Leucine L-Lysine  (alio  free)  (methionine  free)  HCl  50. 0 50. 0 40. 0  L-Metionine  15. 0  L-Phenylalanine  15. 0  L-Proline  (Hydroxy  free)  20. 0 30. 0  L-Serine L-Threonine  (Alio  free)  20. 0 5. 0  L-Tryptophane L-Tyrosine  20. 0  L-Valine  20. 0  Biotin  0. 2  Vitamin  0. 00  D-Ca  0. 25  pantothenate  Choline C l  3. 0  Folic  1. 0  acid  i-Inositol  35. 0  Nicotinamide Para-Aminobenzoic Pyridoxine  HCl  1. 0 acid  1. 0 1. 0  Riboflavin  0. 2  Thiamine  1. 0  Phenol NaHC0  3  HCl  Red  5. 0 2000. 0  

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