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Development of an immunoglobulin-fortified milk replacer and a purified, injectable immunoglobulin solution… Crowley, Margaret L. 1990

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DEVELOPMENT OF AN IMMUNOGLOBULIN-FORTIFIED MILK REPLACER AND A PURIFIED, INJECTABLE IMMUNOGLOBULIN SOLUTION AS ALTERNATIVE METHODS OF ACHIEVING PASSIVE IMMUNITY IN COLOSTRUM-DEPRIVED NEONATAL CALVES. By M a r g a r e t L. Crowley B. Sc. ( A g r . ) , The U n i v e r s i t y o f B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1990 ® Margaret L. Crowley  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  A M I (V\  AL  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  An  immunoglobulin-fortified milk  replacer  and a  subcutaneous (SC) i n j e c t a b l e s o l u t i o n o f immunoglobulins (Ig)  were  examined  as methods  immunity i n n e o n a t a l  calves.  of achieving  passive  Bovine I g , from a b a t t o i r  b l o o d , were p u r i f i e d by p o l y p h o s p h a t e f r a c t i o n a t i o n and ion-exchange chromatography.  I n experiment 1, c a r r i e d  out a t A g r i c u l t u r e Canada Research S t a t i o n , A g a s s i z , 37 colostrum-deprived  Holstein-Friesian bull  a l l o t t e d t o one o f f o u r t r e a t m e n t s . fed  colostrum  on day 1 and whole  calves  were  Col/WM c a l v e s were milk,  days  2 - 42.  MR-Nolg c a l v e s ( c o n t r o l ) were f e d m i l k r e p l a c e r w i t h no I g , days 1 - 4 2 .  MR-Hi/Lo c a l v e s were f e d m i l k r e p l a c e r  w i t h I g a t 50 mg/ml on day 1, and a t 10 mg/ml, days 2 21.  MR-Hi/No c a l v e s were f e d m i l k r e p l a c e r w i t h I g a t 50  mg/ml, day 1,'and w i t h no I g , days 2 - 21. From days 2 2 - 42, MR-Hi/Lo and MR-Hi/No t r e a t m e n t c a l v e s m i l k r e p l a c e r w i t h no I g . at  I n experiment 2, c a r r i e d o u t  t h e U n i v e r s i t y Research  colostrum-deprived allotted  Farm a t O y s t e r R i v e r ,  Holstein-Friesian bull  t o one o f t h r e e  received  treatments.  calves  The f i r s t  24  were two  t r e a t m e n t s were t h e same as f o r experiment 1, Col/WM and MR-Nolg f e d f o r days 1 - 2 1 .  MR-Lo I n j c a l v e s were f e d  m i l k r e p l a c e r w i t h I g a t 10 mg/ml, days 1 - 2 1 , and were a l s o g i v e n a SC i n j e c t i o n o f I g s o l u t i o n w i t h i n t h e f i r s t 6 hours o f l i f e .  F o r days 2 - 4 2 , c a l v e s were f e d WM o r  iii MR-Nolg, as p e r experiment  1.  For both  experiments,  b l o o d samples and c a l f w e i g h t s were t a k e n a t b i r t h , 24 & 48 hours weeks.  o f age, day 7 and weekly  for six  D i a r r h e a ( s c o u r s ) l e v e l s , r e c t a l t e m p e r a t u r e s and  general health  o f c a l v e s were r e c o r d e d d a i l y  f i r s t t h r e e weeks as w e l l . weeks  thereafter  o f age was  11  Experiment  out of  11  f o r the  1 survival at 6  calves  f o r Col/WM  t r e a t m e n t , 8 o u t o f 8 c a l v e s f o r MR-Hi/Lo t r e a t m e n t , 7 out o f 8 c a l v e s f o r MR-Hi/No and a s i g n i f i c a n t l y  lower  (P>0.05) 7 o u t o f 9 c a l v e s f o r MR-Nolg. I n experiment 2, s u r v i v a l was 7 o u t o f 8 c a l v e s f o r b o t h Col/WM and MR-LoI n j t r e a t m e n t s and a s i g n i f i c a n t l y l o w e r (P>0.05) 4 o u t of  8 c a l v e s f o r MR-Nolg t r e a t m e n t .  C a l v e s on MR-Hi/No  had s i g n i f i c a n t l y h i g h e r d i a r r h e a l e v e l s t h a n t h e o t h e r t h r e e t r e a t m e n t s over weeks one and f o u r i n experiment 1. In experiment 2, c a l v e s which d i d n o t r e c e i v e any I g had s i g n i f i c a n t l y h i g h e r d i a r r h e a l e v e l s over weeks t h r e e and f o u r t h a n c a l v e s which r e c e i v e d I g . Experiment 1 average d a i l y g a i n s (ADG) were s i g n i f i c a n t l y h i g h e r f o r c a l v e s on Col/WM, MR-Hi/Lo and MR-Hi/No t r e a t m e n t s t h a n f o r c a l v e s on MR-Nolg a t s i x weeks o f age.  I n experiment  2, s i x  week ADG were s i g n i f i c a n t l y h i g h e r f o r c a l v e s on Col/WM and  MR-Lo I n j t r e a t m e n t s t h a n  experiments,  f o r MR-Nolg.  For both  serum I g l e v e l s o f c a l v e s on Col/WM were  s i g n i f i c a n t l y h i g h e r t h a n c a l v e s on t h e o t h e r t r e a t m e n t s a t 24 and 48 hours o f age. MR-Hi/Lo, MR-Hi/No and MR-Lo I n j c a l v e s t r e n d e d t o h i g h e r serum I g l e v e l s t h a n MR-Nolg calves  b u t were n o t s i g n i f i c a n t l y  different.  Calves  iv w h i c h r e c e i v e d I g , from c o l o s t r u m , replacer  or  a  subcutaneous  Ig  s u r v i v a l r a t e s , lower d i a r r h e a  the I g - f o r t i f i e d m i l k injection,  levels,  less  had  higher  antibiotic  t r e a t m e n t and h i g h e r average d a i l y g a i n s t h a n c a l v e s r e c e i v i n g any I g . administered effective,  hot  I t was c o n c l u d e d t h a t i m m u n o g l o b u l i n s ,  either  orally  or  a l t e r n a t i v e method,  immunity i n n e o n a t a l  calves.  parenterally,  are  for  passive  providing  an  V  TABLE OF CONTENTS Page Abstract  i i  L i s t of Tables  vi  L i s t of Figures  v i i  Acknowledgements  ix  Introduction  •.  1  L i t e r a t u r e Review  The Immune System Stem C e l l D i f f e r e n t i a t i o n P r i m a r y and Secondary Responses The M a j o r H i s t o c o m p a t i b i l i t y Complex The Complement System Phagocytosis Immunoglobulins Immunoglobulin G Immunoglobulin M Immunoglobulin A The Immune Response Development o f Immunocompetence i n the F e t a l C a l f A b s o r p t i o n o f Immunoglobulins Mechanism o f A b s o r p t i o n Experimental S e c t i o n Experiment One  Introduction M a t e r i a l s and Methods Results Discussion  Experiment  Two  Introduction M a t e r i a l s and Methods Results Discussion  4 4 7 9 10 11 12 14 16 17 18 18 20 25  27 28 35 46 51 51 59 69  Conclusions  73  References  74  vi LIST OF TABLES Page  Table 1.  Table 2.  O n t o g e n e t i c development o f b o v i n e immune system d u r i n g g e s t a t i o n C o n c e n t r a t i o n o f IgG, IgM and IgA i n b o v i n e c o l o s t r u m and m i l k  20  ...22  Table 3.  Experimental design f o r studying e f f e c t of a d m i n i s t r a t i o n o f immunoglobulins t o c a l v e s - (experiment 1) 29  Table 4.  Concentration of freeze dried b o v i n e IgG  36  The e f f e c t o f I g s u p p l e m e n t a t i o n on survival of calves - (experiment 1)  38  Table 5.  Table 6.  Experimental design f o r studying e f f e c t of a d m i n i s t r a t i o n o f immunoglobulins t o c a l v e s - (experiment 2) 54  Table 7.  IgG l e v e l s i n p u r i f i e d s o l u t i o n , c o l o s t r u m and m i l k  60  The e f f e c t o f I g s u p p l e m e n t a t i o n on survival of calves - (experiment 2)  60  Table 8.  vii LIST OF FIGURES Page  F i g u r e 1.  Hemopoietic stem c e l l d i f f e r e n t i a t i o n ... 5  F i g u r e 2.  Immune responses t o an a n t i g e n o v e r t i m e as r e f l e c t e d by serum antibody l e v e l s  8  F i g u r e 3a. B a s i c s t r u c t u r e o f IgG m o l e c u l e showing heavy and l i g h t c h a i n s  15  F i g u r e 3b. S t r u c t u r e o f IgG.,, IgG , IgA and IgM m o l e c u l e c l a s s e s  15  2  F i g u r e 3c. P a p a i n t r e a t m e n t o f I g m o l e c u l e breaking into F 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.  ab  and F fragments c  A b s o r p t i o n mechanism by p i n o c y t o s i s  .... 15 26  G e l e l e c t r o p h o r e s i s of polyphosphate f r a c t i o n s from b o v i n e serum The e f f e c t o f I g s u p p l e m e n t a t i o n on r e c t a l temperatures f o r t h e f i r s t s i x weeks o f l i f e  38  The e f f e c t o f I g s u p p l e m e n t a t i o n on diarrhea levels (experiment 1)  39  The e f f e c t o f I g s u p p l e m e n t a t i o n on c a l f weights (experiment 1)  40  The e f f e c t o f I g s u p p l e m e n t a t i o n on average d a i l y g a i n - • .' . (experiment 1)  41  F i g u r e 10. The  e f f e c t o f I g s u p p l e m e n t a t i o n on feed e f f i c i e n c y (experiment 1)  37  42  viii F i g u r e 11. The e f f e c t o f I g s u p p l e m e n t a t i o n on serum IgG l e v e l s (experiment 1)  43  F i g u r e 12. The e f f e c t o f I g s u p p l e m e n t a t i o n on blood glucose l e v e l s (experiment 1)  44  F i g u r e 13. The e f f e c t o f I g s u p p l e m e n t a t i o n on blood urea nitrogen l e v e l s (experiment 1) .  ....45  F i g u r e 14. The e f f e c t o f I g s u p p l e m e n t a t i o n on r e c t a l t e m p e r a t u r e s for, t h e f i r s t s i x weeks o f l i f e (experiment 2)  61  F i g u r e 15. The e f f e c t o f I g s u p p l e m e n t a t i o n on diarrhea levels (experiment 2)  62  F i g u r e 16. The e f f e c t o f I g s u p p l e m e n t a t i o n on c a l f weights (experiment 2)  63  F i g u r e 17. The e f f e c t o f I g s u p p l e m e n t a t i o n on average d a i l y g a i n (experiment 2)  64  F i g u r e 18. The e f f e c t o f I g s u p p l e m e n t a t i o n on feed e f f i c i e n c y (experiment 2)  65  F i g u r e 19. The e f f e c t o f I g s u p p l e m e n t a t i o n on serum IgG l e v e l s (experiment 2)  66  F i g u r e 20. The e f f e c t o f I g s u p p l e m e n t a t i o n on blood glucose l e v e l s (experiment 2)  67  F i g u r e 21. The e f f e c t o f I g s u p p l e m e n t a t i o n on  blood urea n i t r o g e n l e v e l s (experiment 2)  68  ix ACKNOWLEDGEMENTS I w i s h t o thank Dr. Bruce Owen f o r h i s e x p e r t g u i d a n c e ; h i s s t e a d y encouragement and s u p p o r t were most a p p r e c i a t e d and h e l p f u l t h r o u g h t h e t h r e e y e a r s o f my r e s e a r c h . The members o f my committee, Dr. L o m e F i s h e r , Dr. J i m S h e l f o r d , Dr. C.R. K r i s h n a m u r t i and Dr. B. J . Skura were a l s o v e r y h e l p f u l . There a r e many p e o p l e who contributed t o the completion of t h i s t h e s i s i n c l u d i n g t h o s e a t t h e A g r i c u l t u r e Canada A g a s s i z Research S t a t i o n , i n p a r t i c u l a r , Dr. L o m e F i s h e r , S u s i S o t h and Ruby Schmidt, as w e l l as Susan H a i n s t o c k and t h e d a i r y workers. As w e l l , my thanks goes t o t h e p e o p l e a t U n i v e r s i t y o f B r i t i s h Columbia's O y s t e r R i v e r Research Farm. A l s o , I a p p r e c i a t e d t h e e x c e l l e n t t e c h n i c a l h e l p and a d v i c e from I r e n e Bevandick, Murray Drew, Frances Newsome and G i l l e s G a l z y . Financial assistance was provided by t h e P r o d u c t i v i t y Enhancement Program o f t h e C a n a d a / B r i t i s h Columbia Agri-Food Regional Development S u b s i d i a r y Agreement. I thank Dr. R. B l a i r f o r h i s a s s i s t a n c e as well. F i n a l l y , I am i n d e b t e d t o my f a m i l y f o r t h e i r l o y a l s u p p o r t , i n a l l ways, w i t h o u t whom I would n o t have s u c c e s s f u l l y achieved t h i s goal.  INTRODUCTION  The  importance  of colostrum  as  the source  of  immunoglobulin p r o t e c t i o n f o r t h e newborn c a l f has been well  established  (Corley  e t a l . , 1977; K r u s e ,  McEwan e t a l . , 1970; Smith & L i t t l e ,  1922).  immunoglobulins ( I g ) , absorbed from t h e s m a l l into the blood c i r c u l a t i o n ,  197 0;  Colostral intestine  g i v e p a s s i v e immunity and  p r o t e c t t h e c a l f a g a i n s t s e p t i c e m i a s (Boyd, 1972; B u r t o n , 1986) . However, t h e amount o f I g absorbed v a r i e s w i d e l y and t e n t o t h i r t y p e r c e n t o f c a l v e s may have l i t t l e o r no Ig  i n their  serum,  and  thus  no  passive,  systemic  immunity. One c r u c i a l f a c t o r i n a b s o r p t i o n o f c o l o s t r a l I g i n the c a l f i s t h e p e r i o d of i n t e s t i n a l p e r m e a b i l i t y .  This  p e r i o d o f t i m e t h a t a c a l f i s a b l e t o absorb I g from t h e intestine hours  has been e s t i m a t e d t o be between 24 and 48  o f age.  Closure,  which  a b s o r p t i o n o f macromolecules bloodstream  i n neonates,  i s the termination of  from t h e i n t e s t i n e t o t h e has  been  shown  to  occur  spontaneously, a t a p r o g r e s s i v e l y i n c r e a s i n g r a t e a f t e r 12 hours p o s t - p a r t u m ( S t o t t e t a l . , 1979).  I g remaining  i n t h e s m a l l i n t e s t i n e ( i . e n o t absorbed) p r o v i d e a l o c a l protective  effect,  termed  copro-immunity,  against  b a c t e r i a c a u s i n g d i a r r h e a ( s c o u r s ) o r c o l i b a c i l l o s i s and other  enteric  pathogens.  However,  these  protective  e f f e c t s appear t o be independent o f one a n o t h e r as h i g h  2 serum immunoglobulin l e v e l s do not p r e v e n t d i a r r h e a nor do  high  intestinal  levels  prevent  death  from  (Logan Se Pearson, 1978) .  colisepticemia  P a s s i v e immunity i n t h e n e o n a t a l c a l f i s dependent upon c o l o s t r a l absorption  immunoglobulin t r a n s f e r  for  two  main  reasons:  via  1)  intestinal  due  to  the  s y n d e s m o c h o r i a l p l a c e n t a t i o n i n r u m i n a n t s , i n which t h e r e are  f i v e t i s s u e l a y e r s between m a t e r n a l and f e t a l b l o o d  circulation,  there  macromolecules,  is  such  as  no  placental  Ig, during  transfer  gestation;  and  of 2)  a l t h o u g h t h e a c t i v e immune system o f a c a l f i s f u n c t i o n a l a t b i r t h , i t i s not f u l l y mature f o r t h e f i r s t few weeks of l i f e .  A c a l f i s t h u s b o r n w i t h v i r t u a l l y no p a s s i v e  p r o t e c t i o n and i s dependent upon t h e cow's c o l o s t r u m f o r its  survival. P r e v i o u s s t u d i e s showed t h a t adequate q u a n t i t i e s o f  c o l o s t r u m f e d p r i o r t o 24 hours o f age a r e n e c e s s a r y f o r a calf's survival 1977) .  (Michanek e t a l . . 1989a & 1989b; P a t t ,  Colostrum  substitutes,  such  as  a  powdered  immunoglobulin p r o d u c t s e p a r a t e d from c o l o s t r u m o r whey, were a l s o e v a l u a t e d .  Success i n a c h i e v i n g an  adequate  l e v e l o f I g i n t h e c a l f ' s serum has been v a r i a b l e (Haines e t a l . . 1990). A c a l f s w i t c h e d t o m i l k r e p l a c e r w i t h i n a few days after  birth  associated  may  develop  problems,  due  diarrhea to  lack  or of  scours,  and  copro-immunity.  C o n t i n u e d f e e d i n g of c o l o s t r u m has been shown t o reduce i n c i d e n c e of s c o u r s  (Logan  & Pearson, 1978).  A  milk  3 r e p l a c e r f o r t i f i e d w i t h I g which c o u l d g i v e t h i s needed immunity may reduce t h e i n c i d e n c e o f e n t e r i c d i s e a s e s and the a s s o c i a t e d morbidity. N e o n a t a l c a l v e s s o l d t o be r a i s e d f o r d a i r y - b e e f o r v e a l may  be d e p r i v e d o f c o l o s t r u m  which would  t h e l o s s e s due t o s y s t e m i c d i s e a s e s . receive  enough  colostrum.  Ig  via  increase  Some c a l v e s may not  absorption  even  though  fed  A p u r i f i e d immunoglobulin s o l u t i o n , g i v e n as  a subcutaneous i n j e c t i o n which would be absorbed i n t o t h e b l o o d s t r e a m , would p r o v i d e t h e s e  hypo-gammaglobulinemic  calves  the  necessary  immunity,  and  reduce  the  incidence  of  associated mortality.  would  septicemic  disease  presumably and  the  An i n t r a - p e r i t o n e a l o r i n t r a v e n o u s  r o u t e u s i n g b o v i n e plasma was t e s t e d and shown t o be an effective levels  method  of  increasing  (Anderson e t a l . .  This  thesis,  then,  serum  immunoglobulin  1987). examines  t h e development  and  t e s t i n g o f an i m m u n o g l o b u l i n - f o r t i f i e d m i l k r e p l a c e r and a purified,  i n j e c t a b l e immunoglobulin  solution,  using  b o v i n e b l o o d from s e v e r a l a b a t t o i r s as a s o u r c e o f I g , t o improve p a s s i v e immunity i n n e o n a t a l  calves.  4  LITERATURE REVIEW  THE IMMUNE SYSTEM  To g a i n a c l e a r u n d e r s t a n d i n g r e s u l t s reported the  of the experimental  i n t h i s t h e s i s , a general overview o f  immune system  i s i n order.  In a f u l l y  mature,  f u n c t i o n i n g immune system, t h e r e a r e b o t h n o n - s p e c i f i c d e f e n s e f a c t o r s and s p e c i f i c responses.  The n o n - s p e c i f i c  d e f e n s e f a c t o r s i n c l u d e s u r f a c e b a r r i e r s , such as s k i n and e p i t h e l i a l t i s s u e s , i n t e r n a l and e x t e r n a l s e c r e t i o n s , normal body f l o r a and o t h e r i n t e r a c t i n g systems such as the  c o a g u l a t i o n , and complement systems.  The s p e c i f i c  r e s p o n s e s can be c h a r a c t e r i z e d and s u b d i v i d e d major components, a t h y m i c - d e r i v e d bursa-derived  ( B - c e l l ) system.  i n t o two  ( T - c e l l ) system and a  These two components a r e  d i s t i n g u i s h e d from each o t h e r i n t h a t t h e t h y m i c - d e r i v e d system p r o v i d e s c e l l - m e d i a t e d immunity w h i l e t h e b u r s a d e r i v e d system p r o v i d e s humoral immunity. Stem C e l l  Differentiation  Both t h e c e l l - m e d i a t e d and humoral immune systems c o n s i s t o f some s p e c i a l i z e d organs and s e v e r a l d i f f e r e n t types  of c e l l s .  The s p e c i a l i z e d organs i n c l u d e bone  marrow, thymus, l i v e r and lymph nodes.  The c e l l s a r e a l l  d e r i v e d from p l u r i p o t e n t stem c e l l s i n t h e bone marrow. These stem c e l l s  are very  important  i n t h a t they a r e  s e l f - r e n e w i n g and a r e c a p a b l e o f d i f f e r e n t i a t i n g i n t o a l l  5  t h e c e l l groups of t h e body which are d e r i v e d from t h e bone marrow. The  p l u r i p o t e n t stem c e l l s d i f f e r e n t i a t e i n t o  •main l i n e a g e s o f c e l l s includes  ( F i g u r e 1) .  erythrocytes,  precursors.  The  differentiates  into  The  platelets  granulocyte  myeloid  and  two line  granulocyte  precursor  n e u t r o p h i l s , monocytes  further (which  are  immature macrophages) and macrophages. The lymphoid l i n e includes  the  T-lymphocytes  lymphocytes ( B - c e l l s ) . cells  are  F i g u r e 1.  and  the  The g r a n u l o c y t e s , T - c e l l s and  involved  Immunoglobulins  (T-cells)  in  cell-mediated  differentiation  P l u r i p o t e n t Stem C e l l Myeloid Lineage  /  M y e l o i d Stem C e l l  Neutrophils  \  Lymphoid Lineage  Lymphoid Stem C e l l  Macrophages  B—  immunity.  ( a n t i b o d i e s ) , which are produced by  Hematopoietic stem c e l l (Cooper et a l , 1984).  B-  B-  c e l l s , and t h e complement system f a c t o r s a r e i n v o l v e d i n humoral immunity. The  lymphoid l i n e a g e of t h e p l u r i p o t e n t stem c e l l s  c o n s i s t s o f two p o p u l a t i o n s and t h e T - c e l l s . same group antigens  of  Both of t h e s e c e l l l i n e s come from t h e  cells  on t h e i r  The  and  have r e c e p t o r s  for  specific  are,  however,  surfaces.  functions  completely  of lymphocytes, the B - c e l l s  of  these  different.  two  lines  T - c e l l s , which  regulate  f u n c t i o n i n g of the immune system, have t h r e e c e l l each, w i t h  one  major f u n c t i o n .  Cytotoxic  the types  T-cells  are  r e s p o n s i b l e f o r d e s t r o y i n g abnormal o r f o r e i g n c e l l s such as c a n c e r o u s c e l l s o r v i r u s - i n f e c t e d c e l l s . c e l l s promote immune r e s p o n s e s , and  Helper  T-  suppressor T - c e l l s  s u p p r e s s immune r e s p o n s e s . B - c e l l s d i f f e r e n t i a t e i n t o plasma c e l l s and memory cells.  Plasma c e l l s produce and s e c r e t e immunoglobulins  o r s o l u b l e a n t i b o d i e s which can b i n d t o an a n t i g e n , i . e . , any  foreign  production.  substance  that  stimulates  antibody  Memory c e l l s are produced when an a n t i g e n i s  e n c o u n t e r e d t h e f i r s t t i m e but t h e y do not go t h e step  i n producing  and  secreting antibodies.  final  When  an  a n t i g e n i s encountered f o r a second t i m e , t h e y 'remember' t h e a n t i g e n , t h a t i s , t h e y are s p e c i f i c f o r i t and produce  antibodies  to  because  they  already  have  differentiation  steps.  the  antigen gone  much more  through  the  can  quickly primary  7  P r e c u r s o r T- and B - c e l l s a r e produced i n t h e bone marrow and a r e r e l e a s e d i n t o t h e b l o o d s t r e a m .  Precursor  T - c e l l s m i g r a t e t o t h e thymus where they a r e p r o c e s s e d t o an u n s p e c i a l i z e d T - c e l l which can t h e n d i f f e r e n t i a t e i n t o h e l p e r T-, s u p p r e s s o r  T- o r c y t o t o x i c T - c e l l s .  B-cells  were named f o r t h e f a c t t h a t they mature i n t h e b u r s a o f F a b r i c u s i n b i r d s (where they were f i r s t d i s c o v e r e d ) . there  i s no bursa  i n mammals,  precursor  As  B - c e l l s are  t h o u g h t t o mature.in bone marrow, g u t - a s s o c i a t e d lymphoid tissue  (GALT),  activation,  liver  and/or  differentiate  synthesize  and s e c r e t e  Individual  class  to  have on  upon  cells  which  plasma  a  receptor-specific  their  c o r r e l a t e s with the antibody also are c r i t i c a l  B-cells,  immunoglobulins o r a n t i b o d i e s .  B-cells  immunoglobulin  spleen.  cell  surfaces  c l a s s produced.  f o r the d e t o x i f i c a t i o n  that  B-cells  of proteins,  p o l y s a c c h a r i d e s and t o x i n s . Primary  and Secondary Responses  When  an  antigen  i s encountered  i n an  animal,  immunoglobulins o r a n t i b o d i e s w i l l be produced t h a t can combine w i t h ,  or bind  t o , that  antigen.  Antibodies  u s u a l l y bind only t o the antigen that stimulated production.  T h i s i s termed a n t i g e n  their  specificity.  The amount o f a n t i b o d i e s produced can be measured i n serum and i s used as a measure o f t h e immune response (Figure  2).  Following  the f i r s t  encounter  antigen there i s a l a g period of approximately  with  an  one week  b e f o r e a n t i b o d i e s a r e d e t e c t a b l e i n serum". T h e i r  levels  8 Amount of antibody in serum (titer)  Secondary immune response  Primary immune response  14 Second dose of antigen administered  Antigen administered F i g u r e  in  2.  Days  Immune responses t o an a n t i g e n over t i m e as r e f l e c t e d by serum a n t i b o d y l e v e l s ( T i z a r d , 1984).  serum w i l l  c l i m b t o a peak i n 10 t o 14 days b e f o r e  d e c l i n i n g and d i s a p p e a r i n g i n a few weeks.  The amount o f  a n t i b o d y s y n t h e s i z e d , and hence t h e amount o f p r o t e c t i o n , d u r i n g t h i s f i r s t response i s r e l a t i v e l y s m a l l .  Thisi s  termed a " p r i m a r y " response. If  this  a n t i g e n i s encountered a second  time the  a n t i b o d y response i s s i m i l a r t o a p r i m a r y r e s p o n s e , b u t the  l a g p e r i o d i s o n l y two t o t h r e e days.  The amount o f  a n t i b o d y produced t h e n r i s e s r a p i d l y t o a h i g h e r l e v e l before d e c l i n i n g months a f t e r .  slowly  and may be d e t e c t e d f o r many  T h i s i s termed a "secondary"  response.  A t h i r d e n c o u n t e r r e s u l t s i n an even s h o r t e r l a g p e r i o d , a  still  higher antibody t i t e r  prolonged response.  i n serum,  and a more  g  The M a j o r H i s t o c o m p a t i b i l i t y T-cells  and B - c e l l s need t o be a b l e t o r e c o g n i z e  t h e i r target c e l l s bacteria  Complex  (which may be macrophages, B - c e l l s ,  or viruses)  i n order t o i n t e r a c t with  These a c t i v i t i e s a r e r e g u l a t e d  by p r o t e i n s  surfaces of these target c e l l s . called histocompatibility  found on t h e  These p r o t e i n s  (MHC) a n t i g e n s .  are  Cytotoxic  T - c e l l s may be needed t o d e s t r o y abnormal c e l l s p a r t o f t h e body. all  potential  The p r o t e i n s target  histocompatibility therefore,  cells  are  the  foreign  called  class  Cytotoxic  recognize t h e i r target  a n t i g e n s on t h e c e l l  i n any  found on t h e s u r f a c e o f  antigens.  MHC  them.  I  T-cells,  c e l l s by t h e c l a s s I  surfaces,  i n combination  with  antigens.  The p r o t e i n s  found on t h e s u r f a c e s o f B - c e l l s and  a n t i g e n - p r e s e n t i n g c e i l s , such as macrophages, a r e c a l l e d class  I I histocompatibility  interact with  B-cells  antigens.  Helper  and a n t i g e n - p r e s e n t i n g  T-cells cells in  o r d e r t o promote an immune response by r e c o g n i z i n g c l a s s I I MHC antigen.  antigens, i n combination with  Class  I I MHC  antigens are also  s e l f - r e c o g n i t i o n or s e l f - t o l e r a n c e . protective attack  mechanism  that  prevents  these  the foreign involved  in  T h i s i s an e s s e n t i a l an  indiscriminate  on normal body t i s s u e s and c e l l s .  The h i s t o c o m p a t i b i l i t y a n t i g e n s o f b o t h c l a s s e s a r e coded by genes l o c a t e d c l o s e t o g e t h e r on one chromosome and  form  the  gene  Histocompatibility  complex  Complex.  known  as  the  Major  10 The Complement System One o f t h e many i n t e r a c t i n g systems i n t h e body i s t h e complement system, so termed because i t complements the a n t i b o d y system. all  There a r e a t l e a s t 15 components,  serum p r o t e i n s ,  percent  w h i c h t o g e t h e r make up about t e n  of the globular  protein  fraction  o f serum.  Complement components a r e s y n t h e s i z e d a t v a r i o u s s i t e s i n the,body; f o r example, t h e C l components a r e s y n t h e s i z e d i n macrophages and f i b r o b l a s t s ; C2, C3, C4, C5, H, P, D, and  B i n macrophages  (Tizard,  1987) .  controlled  L e v e l s o f C l , C2 and C4 i n serum a r e  by  Class  Histocompatibility complement  and C3, C6 and C9 i n t h e l i v e r  III  Complex  system r e s u l t s  membranes and i s r e g u l a t e d  genes  (MHC).  in  the  Activation  Major of the  i n the disruption  of  cell  by mechanisms t h a t i n v o l v e a  s e r i e s o f i n t e r l i n k e d enzyme r e a c t i o n s . The complement r e a c t i o n pathways. is  initiated  membranes.  by  system c o n s i s t s  of three  distinct  One o f t h e s e , t h e c l a s s i c a l pathway, antigen-antibody  reactions  on  cell  The second, o r a l t e r n a t i v e pathway, p r o v i d e s  a r o u t e by w h i c h p o t e n t i a l i n v a d e r s , such as b a c t e r i a , f u n g i , o r p a r a s i t e s may a c t i v a t e t h e complement in  t h e absence o f a n t i g e n - a n t i b o d y  f i r s t two pathways a r e i n v o l v e d  complexes.  factors These  i n the production of the  two C3 c o n v e r t a s e s (enzymes) t h r o u g h a s e r i e s o f c a s c a d e l i k e reactions.  The t h i r d , o r t e r m i n a l  a true cascade-like  pathway, i s n o t  reaction but involves  combining t h e  C3 c o n v e r t a s e and t h e o t h e r complement components i n t o a  l a r g e m a c r o m o l e c u l a r complex. surfaces  forming  a  T h i s complex b i n d s t o c e l l  structure  with  membrane-damaging  p r o p e r t i e s ( T i z a r d , 1984). Phagocytosis N e u t r o p h i l s are the f i r s t l i n e of d e f e n s e d u r i n g an infection.  They  are  .found  i n large  numbers  in  the  b l o o d s t r e a m and are r a p i d l y m o b i l i z e d t o a s i t e of t i s s u e injury  by  various  chemotactic  stimuli.  They  act  by  p h a g o c y t i z i n g f o r e i g n m a t e r i a l o r a n t i g e n s ; i n g e s t i n g the p a r t i c l e , which i s t h e n k i l l e d and d i g e s t e d . maintain  by o x i d a t i v e m e t a b o l i s m  N e u t r o p h i l s have l i m i t e d energy and cannot  t h e i r a t t a c k f o r v e r y l o n g so macrophages are  r e q u i r e d t o s u s t a i n t h e response. Macrophages a r i s e from bone marrow as promonocytes, enter  and  circulate  in  the  bloodstream  macrophages, termed monocytes, and where t h e y mature t o macrophages. in  these  t i s s u e s at  strategic  as  migrate  immature  to tissues  Some become r e s i d e n t  points  i n the  body  to  remove f o r e i g n m a t e r i a l and m i c r o o r g a n i s m s from t h e b l o o d stream.  The  t h r e e main f u n c t i o n s of macrophages are  a) e l i m i n a t i o n of f o r e i g n m a t e r i a l by  phagocytosis,  b) a n t i g e n p r e s e n t a t i o n t o lymphocytes, c)  and  s e c r e t i o n of b i o a c t i v e m o l e c u l e s i m p o r t a n t  in  host  defense. Macrophages chemotactic complement dying  migrate  to  infection  response t o b a c t e r i a l system  f a c t o r s , and  sites  breakdown  in  products,  factors released  n e u t r o p h i l s , to phagocytize  b a c t e r i a or  a  from  foreign  particles  in  macrophages  a  similar  mature,  manner  they  as  neutrophils.  develop  m o l e c u l e s ) t o immunoglobulins and  receptors  one  As  (protein  o f the complement  system fragments, C3, on t h e i r membranes which a i d i n t h e i r physiological functions. opsonins  to  b a c t e r i a by  enhance  the  binding  These r e c e p t o r s  adherence  of  act  as  macrophages  to  f i r m l y t o the b a c t e r i a l membranes.  I n a d d i t i o n t o b e i n g p h a g o c y t i c , macrophages s e c r e t e factors  that  tissues.  cause f e v e r ,  A wide v a r i e t y  macrophages  including  i n f l a m m a t i o n and of  p r o d u c t s are  healing  of  secreted  by  antibacterial proteins  such  as  lysozyme, l a c t o f e r r i n , some of the complement components and  immunoregulatory f a c t o r s such as i n t e r l e u k i n - 1 . Macrophages  presentation.  are  also  involved  Foreign material  such  t a k e n i n by macrophages, degraded by  as  in  antigen  bacteria  l y s o z y m a l enzymes  and the b a c t e r i a l a n t i g e n s are passed back t o t h e membrane (involved  in  association  in  self  with  Class  recognition).  are  II  MHC  T-cells  surface  molecules which  are  s p e c i f i c f o r t h i s a n t i g e n - C l a s s I I complex can b i n d t o i t and b e g i n the f i r s t s t e p i n a c t i v a t i o n of h e l p e r T - c e l l s . Immunoglobulins Humoral  immune  immunoglobulins.  The  responses  are  mediated  f u n c t i o n of immunoglobulins i s t o  n e u t r a l i z e the e x t r a c e l l u l a r phase of b a c t e r i a , and p a r a s i t e s . receptors and  by  viruses  Immunoglobulins s e r v e as B - c e l l membrane  as w e l l as b e i n g s e c r e t e d  i n t o the b l o o d s t r e a m  o t h e r body f l u i d s t o b i n d t o s o l u b l e  antigens.  Immunoglobulins  are  protein  molecules  ( g l y c o p r o t e i n s ) produced by plasma c e l l s as a r e s u l t o f the  interaction  between a n t i g e n - s e n s i t i v e B - c e l l s and  s p e c i f i c antigen.  They c o n s i s t o f f o u r p r o t e i n c h a i n s  c o v a l e n t l y l i n k e d together.  There a r e two l i g h t  and two heavy c h a i n s i n each m o l e c u l e .  chains  They a r e made up  o f b l o c k s o f a p p r o x i m a t e l y 110 amino a c i d s c a l l e d domains (Hood e t a l . , 1984) .  The l i g h t  domains and t h e heavy c h a i n s domains  depending  (Figure  3-a).  upon  chains  c o n s i s t of four or f i v e  the class  The domains  consist of 2  of  can be  immunoglobulin  subdivided  into  v a r i a b l e and c o n s t a n t domains.  The v a r i a b l e domains a r e  the  of  antigen-binding  molecule. class  regions  immunoglobulin  The heavy c h a i n c o n s t a n t domains d e t e r m i n e t h e  of t h e molecule.  (termed  the  isotypes)  There a r e t h r e e main c l a s s e s  o f immunoglobulins  i n the bovine,  d e s i g n a t e d a l p h a , gamma, and mu which c o r r e s p o n d s t o IgA, IgG and.IgM, r e s p e c t i v e l y . I t i s p o s s i b l e t h a t IgE may be produced as w e l l as t h e r e have been IgE-type r e a c t i o n s found i n cows ( T i z a r d , 1987). The microscopy, used  IgG  molecule,  looks l i k e  as a model  ( F i g u r e 3-b) .  as  revealed  by  a Y-shaped m o l e c u l e  f o r the other  electron  and can be  i s o t y p e s , IgM and IgA  The 'arms' o f t h e Y, made up o f one l i g h t  c h a i n and one heavy c h a i n j o i n e d by a d i s u l p h i d e bond, are t h e antigen-binding ( v a r i a b l e ) regions.  The ' t a i l '  ( c o n s t a n t ) r e g i o n , made up o f two heavy c h a i n s j o i n e d by two  d i s u l p h i d e bonds,  determines  the isotype  of the  14 immunoglobulin.  The p r o t e o l y t i c  enzyme, p a p a i n ,  break t h e m o l e c u l e i n t o a p p r o x i m a t e l y  can  three equal-sized  fragments, c o r r e s p o n d i n g t o t h e two 'arms' and t h e ' t a i l ' (Figure 3-c).  The two arm fragments a r e i d e n t i c a l i n  amino a c i d sequence and can s t i l l b i n d a n t i g e n . called  the F  fragments.  gb  The t h i r d  They a r e  fragment, t h e  ' t a i l ' , cannot b i n d a n t i g e n b u t i s c r y s t a l l i z a b l e and i s therefore  called  the F  c  fragment.  There  are  some  b i o l o g i c a l a c t i v i t i e s which a r e mediated by t h e c o n s t a n t r e g i o n o f t h e heavy c h a i n s activation  of  immunoglobulin  the  (F region).  complement  has bound  These i n c l u d e  c  cascade  antigen,  once  binding  the  o f immune  complexes t o p h a g o c y t i c c e l l s ( o p s o n i z a t i o n ) and b i n d i n g to  cell  surfaces  epithelium during The  such  as macrophages  structural  sites  levels  d i f f e r e n c e s among t h e c l a s s e s o f  of production  of production  responses,  intestinal  absorption.  immunoglobulins c o r r e s p o n d their  and  to functional and a c t i o n ,  i n primary  and t h e i r p h y s i o l o g i c a l  differences i n their  and secondary  relative immune  roles.  Immunoglobulin G IgG  is  the  immunoglobulin  found  in  highest  c o n c e n t r a t i o n i n t h e b l o o d , making up about 75-85 p e r c e n t o f t h e t o t a l serum immunoglobulins, and i n t h e c o l o s t r u m produced by ruminants.  IgG has a r e l a t i v e l y s m a l l s i z e ,  w i t h a m o l e c u l a r weight o f 180,000 d a l t o n s . as  I t i s found  a monomer i n b o t h t h e membrane-bound and s e c r e t e d  form.  IgG has two s u b - c l a s s e s o r i s o t y p e s : IgG. and  F i g u r e 3-a.  heavy chain  Schematic d r a w i n g o f immunoglobulin m o l e c u l e showing main s t r u c t u r a l f e a t u r e s . V and C are t h e v a r i a b l e and c o n s t a n t regions, respectively, of t h e heavy (H) and l i g h t (L) c h a i n s . (Hood e t al..1984) .  light chain  cfb Gp igG2  F i g u r e 3-b.  Structures of thethree immunoglobulin c l a s s e s , IgG, IgM and IgA and t h e two s u b - c l a s s e s o f IgG. (Hood e t a l . , 1984).  IgM  IgA dimer  F i g u r e 3-c.  Papain  treatment  Pepsin  treatment  Schematic drawing o f enzyme t r e a t m e n t on immunoglobulin molecule, breaking i n t o F . and F„ ab  Fab  Fab  F(ab)'2  c  fragments. (Hood e t a l . , 1984).  IgG .  Because o f i t s s m a l l s i z e , i t can move i n and out  2  of  blood  vessels  more  easily  than  the  other  immunoglobulin m o l e c u l e s and p l a y s a major r o l e  i n the  d e f e n s e o f t i s s u e spaces and body s u r f a c e s . main  immunoglobulin  response.  synthesized  during  IgG i s t h e a  secondary  IgG can o p s o n i z e , a g g l u t i n a t e and p r e c i p i t a t e  a n t i g e n , b u t can a c t i v a t e t h e complement cascade o n l y i f enough IgG m o l e c u l e s have accumulated  on t h e a n t i g e n  surface. Immunoglobulin M IgM i s found i n second h i g h e s t c o n c e n t r a t i o n i n t h e blood.  IgM has a m o l e c u l a r w e i g h t o f 900,000 d a l t o n s and  i s made up o f f i v e i d e n t i c a l s u b - u n i t s o r monomers, each 180,000 d a l t o n s . an  Each monomer i s s t r u c t u r a l l y s i m i l a r t o  IgG m o l e c u l e .  IgM m o l e c u l e s  a r e bound t o B - c e l l  membranes as a monomer, where they f u n c t i o n as r e c e p t o r s for antigen,  or are secreted  i n the blood  stream as a  pentamer, w i t h f i v e m o l e c u l e s o f IgM arranged with the antigen-binding  radially  s i t e s p o i n t i n g outwards.  The  IgM monomers a r e l i n k e d by d i s u l p h i d e bonds i n a c i r c u l a r f a s h i o n w i t h a p o l y p e p t i d e c a l l e d t h e J - c h a i n j o i n i n g two of  t h e monomers.  Because  m o l e c u l e s a r e found m a i n l y the  immunoglobulin  ontogeny.  of t h e i r  large  size,  i n t h e .blood stream.  synthesized  first  during  IgM  IgM i s B-cell  I t i s a l s o t h e main immunoglobulin s y n t h e s i z e d  and s e c r e t e d d u r i n g a p r i m a r y immune r e s p o n s e .  A primary  response o c c u r s t h e f i r s t time a h o s t comes i n c o n t a c t w i t h an a n t i g e n .  IgM m o l e c u l e s a r e more e f f i c i e n t t h a n  17 IgG a t complement a c t i v a t i o n , o p s o n i z a t i o n , a g g l u t i n a t i o n and n e u t r a l i z a t i o n o f . v i r u s e s . Immunoglobulin A The daltons  IgA monomer has a m o l e c u l a r with  a similar  w e i g h t o f 160,000  s t r u c t u r e t o IgG.  I t usually  o c c u r s as a monomer i n t h e membrane-bound form and as a dimer  i n the secreted  form.  The  two monomers o f  s e c r e t o r y IgA a r e j o i n e d by t h e J - c h a i n as f o r IgM. I t does n o t b i n d t o macrophages o r enhance p h a g o c y t o s i s b u t can n e u t r a l i z e some v i r u s e s and some v i r a l and b a c t e r i a l enzymes.  I t s most i m p o r t a n t mode o f a c t i o n i s p r e v e n t i o n  of t h e adherence o f b a c t e r i a and v i r u s e s t o e p i t h e l i a l surfaces. the  A f t e r IgA i s s y n t h e s i z e d by plasma c e l l s i n  intestinal  interior  mucosa  i t binds  surface of e p i t h e l i a l  t o a r e c e p t o r , on t h e cells.  T h i s complex o f  IgA dimer and r e c e p t o r i s t h e n e n g u l f e d i n a v e s i c l e and transported across the epithelium.  When i t r e a c h e s t h e  e x t e r n a l s u r f a c e o f t h e e p i t h e l i a l c e l l (lumen s i d e ) , t h e v e s i c l e f u s e s w i t h t h e membrane e x p o s i n g the  lumen.  release  The complex  t h e IgA dimer  i s cleaved  with  about  t h e complex t o  by a p r o t e a s e 75 p e r c e n t  to  of the  r e c e p t o r s t i l l a t t a c h e d t o i t . T h i s r e c e p t o r fragment i s termed t h e s e c r e t o r y component and p r o t e c t s t h e IgA dimer from d i g e s t i o n by i n t e s t i n a l  enzymes.  The IgA dimer-  s e c r e t o r y component complex i s known as s e c r e t o r y IgA (SIgA) .  SIgA  i s t h e main  s e c r e t i o n s and on e p i t h e l i a l  immunoglobulin surfaces.  found i n  18 The Immune Response When an a n t i g e n i s encountered, other  antigen-presenting cells)  macrophages ( o r  ingest,  degrade,  or  p r o c e s s , and p r e s e n t i t on t h e i r s u r f a c e i n a s s o c i a t i o n w i t h c l a s s I I MHC m o l e c u l e s . An a n t i g e n - s p e c i f i c h e l p e r T - c e l l b i n d s t o t h e a n t i g e n - c l a s s I I complex c a u s i n g t h e r e l e a s e o f i n t e r l e u k i n - 1 from t h e h e l p e r T - c e l l . helper  T-cell  (a  clone)  is  then  Another  activated  (by  i n t e r l e u k i n - 1 ) which i n t u r n a c t s on a n t i g e n - s p e c i f i c Bc e l l s by c e l l  to cell  i n t e r a c t i o n s and t h e r e l e a s e o f  other a c t i v a t i n g factors. and  form  plasma  The B - c e l l b e g i n s t o d i v i d e  cells.  The  plasma  cells  produce  immunoglobulins, IgM f o r a p r i m a r y response and memory c e l l s produce IgA o r IgG f o r a secondary r e s p o n s e . B  cells  also  specificity  differentiate  into  memory  cells  Some (with  f o r t h a t a n t i g e n ) which enhance t h e immune  response i f t h e same a n t i g e n i s encountered a g a i n i n t h e future.  When t h e f o r e i g n a n t i g e n has been e l i m i n a t e d ,  suppressor T - c e l l s  s t o p t h e immune response by a c t i n g  upon, o r s u p p r e s s i n g , h e l p e r T - c e l l s .  DEVELOPMENT OF IMMUNOCOMPETENCE IN THE FETAL CALF Immunocompetence i s d e f i n e d as t h e a b i l i t y t o mount an immune response. virtually  A l t h o u g h t h e newborn c a l f i s born  agammaglobulinaemic,  several  studies  have  c o n f i r m e d t h a t t h e immune system b e g i n s t o d e v e l o p f a i r l y e a r l y i n f e t a l l i f e and t h e c a l f i s immunocompetent a t birth.  However, t h e c a l f ' s immune system i s immature a t  b i r t h and i s slow t o respond t o a n t i g e n i c , c h a l l e n g e .  The  n e o n a t a l immune system r e q u i r e s a few weeks t o mature t o t h e s t a g e where a q u i c k e r p r i m a r y response can o c c u r as w e l l as a secondary response. O n t o g e n e t i c development is  largely  length  o f immune system  dependent upon two v a r i a b l e s :  of gestation.  Schultz  (1972)  functions  s p e c i e s and  reviewed  many  s t u d i e s which have r e s u l t e d i n an u n d e r s t a n d i n g o f when immunocompetence d e v e l o p s i n t h e b o v i n e f e t u s and neonate (Table 1 ) . These s t u d i e s have d e a l t w i t h o b s e r v i n g t h e development  o f v a r i o u s components o f t h e immune system  t h r o u g h o u t t h e g e s t a t i o n a l p e r i o d as w e l l as e x p e r i m e n t s where f e t u s e s were i n f e c t e d w i t h s p e c i f i c organisms t o measure t h e response o f t h e f e t a l immune system.  Because  t h e t y p e o f p l a c e n t a l attachment i n t h e b o v i n e does n o t a l l o w passage o f m a t e r n a l I g t o t h e f e t u s , any I g found in  the fetus  would  presumably  be  of  fetal  origin.  However, i f p a t h o l o g i c a l l e s i o n s o c c u r i n t h e p l a c e n t a as a result of microbial  i n f e c t i o n o r from o t h e r c a u s e s ,  l e a k a g e o f m a t e r n a l I g may o c c u r ( B r a m b e l l , 1970).  In  n e a r l y a l l samples c o l l e c t e d a f t e r 200 days o f g e s t a t i o n , low l e v e l s o f IgM and IgG were p r e s e n t . R e s u l t s o f t h e s e s t u d i e s show t h a t t h e b o v i n e f e t u s and p r e - c o l o s t r a l c a l f a r e immunocompetent b u t t h e l e v e l s o f IgG and IgM a r e w e l l below a d u l t v a l u e s and t h e l e v e l s r e q u i r e d t o meet a challenge i n practice. still  Lymphoid t i s s u e development i s  a t an immature s t a g e , as w e l l .  20 Table l . Ontogeny of development of immune system i n the bovine f e t u s (as compiled by Schultz, 1972)  Gestation Day 42  ; Event lymphocytes f i r s t r e c o g n i z e d i n thymus  45  lymphocytes f i r s t r e c o g n i z e d i n peripheral blood  55  lymphocytes f i r s t r e c o g n i z e d i n bone marrow  59  IgM p r e s e n t i n s p l e e n  60-100  lymph nodes were observed i n v a r i o u s areas  13 0  IgM i n f e t a l serum  145  IgG i n f e t a l serum  150-175  lymphoid t i s s u e i n t o n s i l and GI t r a c t  280  birth  ABSORPTION OF IMMUNOGLOBULINS Failure  to  immunoglobulins  absorb  adequate  amounts  of  (Ig) has been a s s o c i a t e d w i t h t h e h i g h  r a t e o f m o r b i d i t y and m o r t a l i t y found i n c a l f p r o d u c t i o n (Boyd, 1972 ; Logan & Penhale, 1971; McEwan e t a l . , 1970; McGuire  et a l . ,  surveys  have  1976; Penhale  shown  a  positive  et a l . ,  1973).  relationship  Many between  absorbed c o l o s t r a l I g and c a l f s u r v i v a l r a t e s ( K l a u s e t a l . , 1969; McEwan e t a l . , 1970; McGuire e t a l . , 1976; Penhale e t a l . , 1973; Smith & L i t t l e , 1979).  1922; S t o t t e t a l . ,  The amount o f I g absorbed v a r i e s w i d e l y , however.  From t e n t o t h i r t y p e r c e n t o f c a l v e s may have l i t t l e o r  no I g i n t h e i r serum and t h u s no p a s s i v e immunity a g a i n s t septicemic disease.  A low serum I g c o n c e n t r a t i o n , termed  hypo-gammaglobulinemia, can r e s u l t from a v a r i e t y of factors:  a) an inadequate amount of c o l o s t r u m  b)  c o n c e n t r a t i o n of I g i n c o l o s t r u m ,  a low  delayed  ingested, c)  late  or  f e e d i n g of c o l o s t r u m , d) e a r l y c l o s u r e , o r l o s s  of a b s o r p t i v e c a p a b i l i t y of the i n t e s t i n e ( S t o t t et a l . , 1979)  and  e)  p h y s i o l o g i c a l s t r e s s due  environment ( S t o t t e t a l . , Vermorel•et a l . , of  undesirable  1975)..  (1989a; 1989b) s t u d i e d t h e  c a l v i n g c o n d i t i o n on  calves.  to  energy metabolism  effect  i n newborn  They found t h a t c a l v e s which were b o r n i n c o l d ,  wet c o n d i t i o n s , o r c a l v e s which had a d e l a y e d  parturition  or d i f f i c u l t b i r t h s , had a d e l a y e d a b s o r p t i o n of I g significantly  lower  serum I g l e v e l s a t s i x , 12  hours of age t h a n do c a l v e s born Straub  and  concentrations  Matthaeus  of c o l o s t r u m  measured  Ig  found t h e amount of I g  r a p i d l y d e c r e a s e s w i t h time a f t e r b i r t h .  Kruse  also  of  found a r a p i d d e c l i n e i n I g c o n t e n t  (40% d e c r e a s e a f t e r 16 h o u r s ) .  24  normally. (1978)  and  and  and  Age of dam  (1970)  colostrum  and number of  l a c t a t i o n s are f a c t o r s c o r r e l a t e d w i t h amounts of I g i n colostrum.  A  study  done by  D e v e r y - P o c i u s and  (1983) showed cows a t 30 months o f age,  Larson  in their  first  l a c t a t i o n , produced l e s s t o t a l I g w h i l e o l d e r cows had more IgG  1  i n t h e i r colostrum.  T o t a l amounts of IgG  2  and  IgM r o s e i n second l a c t a t i o n and l e v e l l e d o f f w h i l e t o t a l IgG. r o s e t o a maximum i n the f o u r t h l a c t a t i o n ,  double  22 the  amount  i n the  first  lactation.  Table  2  shows  c o n c e n t r a t i o n s o f IgG, IgM and IgA i n c o l o s t r u m and m i l k .  Table 2.  Mean C o n c e n t r a t i o n s of IgG, IgM Colostrum and M i l k  and IgA i n  Concentration IgG  IgM  IgA  C o l o s t r u m (mg/ml) at p a r t u r i t i o n  45.6 - 62.9  a t 24 h r s p o s t partum Milk  4.4  32.4  4.0  13.0 - 22.2  (jig/ml)  3.2 2.9  0.5  1.7  ( c o m p i l e d from 155 c o l o s t r u m and m i l k samples, B u r t o n e t a l . . 1989; S t r a u b & Matthaeus, 1978).  B e s s e r and Gay,  (1987),  between c o n c e n t r a t i o n serum,  found  that  immunoglobulin  studying the r e l a t i o n s h i p  of Ig i n colostrum  calves  fed colostrum  concentrations  developed  and  in calf  with  higher  higher  serum  immunoglobulin c o n c e n t r a t i o n s , although the e f f i c i e n c y of absorption  decreased  colostrum.  Calves  with fed  increasing  concentration i n  colostrum  with  a  lower  immunoglobulin c o n c e n t r a t i o n absorbed a h i g h e r p r o p o r t i o n of  the Ig than  calves  fed the colostrum  immunoglobulin concentrations.' separate  with  higher  Work c a r r i e d out i n t h r e e  s t u d i e s showed t h a t i n g e s t i o n o f c o l o s t r u m  s u c k l i n g u s u a l l y r e s u l t s i n h i g h e r serum  by  concentrations  23 t h a n i n g e s t i o n of c o l o s t r u m by b u c k e t f e e d i n g al..  1984;  (1979),  Selman e t a l . . 1970  & 1971).  studying c l o s u r e times  and  (Nocek e t  Stott et a l . .  delayed  feeding  of  c o l o s t r u m , e s t i m a t e d c l o s u r e o c c u r r e d a t 21 hours of age, f o r c a l v e s f e d a t b i r t h , and t h a t a d e l a y e d  feeding  of  c o l o s t r u m d e l a y e d the c l o s u r e t i m e up t o a maximum of 3 3 hours'of of  age.  time  W i t h d e l a y e d f e e d i n g , however, t h e l e n g t h  the  calves  actually  had  to  absorb  was  d r a m a t i c a l l y d e c r e a s e d from 21 hours f o r c a l v e s f e d a t b i r t h t o about 8 hours f o r c a l v e s f e d a t 24 hours of Michanek e t a l . ,  age.  (1989a & 1989b), showed t h a t i f  c a l v e s were d e p r i v e d of c o l o s t r u m t h e a b i l i t y t o absorb macromolecules d i d not hours of pooled  life.  colostrum  decline during  Calves  were g i v e n  a t one,  the  first  a first  eight  feeding  e i g h t , 16 o r 24 hours of  w i t h t h r e e more subsequent f e e d i n g s a t 8 hour  of age  intervals.  There were no d i f f e r e n c e s between groups i n a b s o r p t i o n of Ig f o r t h e f i r s t f e e d i n g , but t h e c a l v e s t h a t were f e d a t one hour a f t e r b i r t h absorbed s i g n i f i c a n t l y more t h a n t h e other  three  feedings.  groups The  i n the  similarity  second, in  third  amount  of  and Ig  fourth absorbed  between a l l f o u r groups a t the f i r s t f e e d i n g suggested a base l e v e l of a b s o r p t i o n c a p a c i t y t h a t may d u r i n g t h e f i r s t 24 hours o r l o n g e r . that  an  achieved  acceptable  level  of  be  maintained  T h e i r r e s u l t s imply  passive  immunity  may  i n c a l v e s t h a t get t h e i r f i r s t c o l o s t r u m  t h a n 8 h o u r s , i f a q u a n t i t y of I g i s f e d t h a t w i l l  be  later give  the c a l f an o p p o r t u n i t y t o f u l l y u t i l i z e i t s a b s o r p t i o n  capacity a t i t s f i r s t feeding.  For calves that are fed  e a r l y , t h e f i r s t feeding i s l e s s important  because t h e y  can absorb c o n s i d e r a b l e amounts o f I g a t l a t e r previously,  the l e v e l  feedings.  However,  as s t a t e d  of Ig i n  colostrum  by t h i s t i m e has d e c r e a s e d s u b s t a n t i a l l y , so  t h i s would o n l y be o f b e n e f i t i f c a l v e s were b e i n g f e d a h i g h q u a l i t y ( i . e . h i g h Ig) c o l o s t r u m . A study done by McCoy e t a l . , finding  that  absorption.  delayed  feeding  (197 0),  reduced  supported the the period  In addition, of the calves  f e e d i n g was d e l a y e d  whose  of  first  t o 12, 16, 20 o r 24 hours o f age,  t h r e e , 17, 3 0 and 57 p e r c e n t o f t h e c a l v e s , r e s p e c t i v e l y , were u n a b l e t o absorb any I g a t a l l . were f e d b e f o r e Stott  et  A l l calves  that  12 hours o f age were a b l e t o absorb I g .  a l . , (1979),  also  found  no  significant  d i f f e r e n c e i n mean c l o s u r e t i m e between t h e 3 c l a s s e s o f Ig  (IgG, IgM, I g A ) . The amounts o f c o l o s t r u m  f e d (0.5,  1.0 & 2.0 l i t r e s ) a l s o had no i n f l u e n c e on t h e a b s o r p t i v e period.  A n o t h e r s e p a r a t e study s u p p o r t e d t h e s e f i n d i n g s  ( P a t t , 1977). Previous feedings  s t u d i e s showed t h e importance f o r i n i t i a l  of colostrum  t o be w i t h i n f o u r t o s i x hours  a f t e r b i r t h and subsequent f e e d i n g s up t o 24 - 36 hours after 1974;  birth Staley  (Logan e t a l . , 1972; P a t t , e t a l . , 1972),  1977 ;  f o r calves  to  Petrie, obtain  s u f f i c i e n t l e v e l s o f serum I g ( i . e . >12 mg/ml) t o f i g h t bacterial  septicemias  (colisepticemia)  (Stott et a l . ,  25 1979) .  Earlier  feeding of colostrum  does s h o r t e n t h e  p e r i o d o f a b s o r p t i o n b u t spontaneous c l o s u r e i n c a l v e s d e p r i v e d o f c o l o s t r u m o c c u r r e d a t a mean o f 24 hours so e a r l y c o l o s t r a l f e e d i n g may s h o r t e n a b s o r p t i o n p e r i o d by only  two t o t h r e e  suggested  that  hours.  Corley  shortening  et a l . ,  the absorptive  (1977),  period  by  f e e d i n g early.may a l s o have an added b e n e f i c i a l e f f e c t by protecting  the  calf  from  prolonged  i n d i s c r i m i n a t e a b s o r p t i o n o f pathogens.  exposure  to  As w e l l , i t has  never been shown t h a t c l o s u r e i n t h e c a l f i s a f f e c t e d by n u t r i t i o n a l f a c t o r s (macromolecular) a l t h o u g h t h e y a f f e c t c l o s u r e i n t h e p i g l e t and lamb (Lecce and Morgan, 1962) and  i n l a b o r a t o r y animals  ( C l a r k and Hardy 1970; Lecce  and Broughton 1973). MECHANISM OF ABSORPTION Uptake  or absorption  o f macromolecules  such  as  immunoglobulins by i n t e s t i n a l e p i t h e l i u m i s an energydependent p r o c e s s w i t h macromolecules b e i n g absorbed by pinocytosis  and t r a n s m i t t e d ,  within  a membrane-bound  v e s i c l e , t h r o u g h t h e c y t o p l a s m o f the e n t e r o c y t e s 4).  Absorption  s e l e c t i v e process probably  by p i n o c y t o s i s appears t o be a h i g h l y depending upon by s p e c i f i c  l o c a t e d on t h e e n t e r o c y t e m i c r o v i l l i  a l . , 1984).  (Figure  receptors (Hood e t  C o m p e t i t i o n f o r attachment t o t h e r e c e p t o r s  by m o l e c u l e s o f a s i m i l a r n a t u r e has been shown t o o c c u r and t h e mechanism o f a b s o r p t i o n i s s u b j e c t t o s a t u r a t i o n ( T i z a r d , 1987).  26  F i g u r e 4.  G e n e r a l mechanism f o r uptake and t r a n s p o r t of macromolecules by the i n t e s t i n e . (Walker, 1987).  n i t ©^© .  lysosomes  Phagolysosome  Immunoglobulins  absorbed from t h e s m a l l  intestine  i n t o t h e b l o o d c i r c u l a t i o n g i v e p a s s i v e immunity o r p r o t e c t t h e c a l f a g a i n s t s e p t i c e m i a s (Boyd, 1972; B u r t o n , 1986).  Ig remaining i n the small i n t e s t i n e  t h a t a r e n o t absorbed)  can p r o v i d e a l o c a l  ( i . e those protective  e f f e c t , termed copro-immunity, a g a i n s t b a c t e r i a c a u s i n g diarrhea  (scours) o r c o l i b a c i l l o s i s .  These p r o t e c t i v e  e f f e c t s , however, appear t o be independent o f one a n o t h e r as  high  serum  immunoglobulin  levels  do  not prevent  d i a r r h e a n o r do h i g h i n t e s t i n a l l e v e l s p r e v e n t d e a t h from c o l i s e p t i c e m i a (Logan & Pearson, 1978). to  milk  replacer  within  a few days  Calves switched after  birth  may  d e v e l o p s c o u r s o r d i a r r h e a , and a s s o c i a t e d problems, due to  lack  of  copro-immunity.  Continued  feeding  of  c o l o s t r u m has been shown t o reduce i n c i d e n c e o f s c o u r s (Logan & Pearson, 1978).  27  EXPERIMENTAL EXPERIMENT ONE INTRODUCTION  Previous  studies  have  shown  t h e importance  c o l o s t r u m as a source o f immunoglobulin  of  protection for  t h e newborn c a l f (Boyd, 1972; Kruse, 1970; McCoy, 1970). Calves  switched  after  birth  associated Continued  t o a m i l k r e p l a c e r w i t h i n a few days  may  develop  problems,  scours  o r d i a r r h e a , and  due t o l a c k  o f copro-immunity.  f e e d i n g o f c o l o s t r u m has been shown t o reduce  incidence o f scours  (Logan and Pearson,  1978).  A milk  r e p l a c e r f o r t i f i e d w i t h I g , which would g i v e t h i s needed immunity, c o u l d reduce t h e i n c i d e n c e o f e n t e r i c d i s e a s e and a s s o c i a t e d m o r b i d i t y . 1990,  compared  supplements. variable high  A study done by Haines e t a l . ,  some c o m m e r c i a l l y  low and  I g c o n c e n t r a t i o n s compared t o t h o s e  found i n  (i.e.,  supplements  colostral  contained  quality  These  available  high  Ig) c o l o s t r u m .  While  these  p r o d u c t s may be o f some b e n e f i t a f t e r c l o s u r e , t h e y a r e unlikely  t o be v e r y  effective  i n colostrum-deprived  calves. The  o b j e c t i v e o f experiment  1 was t o d e v e l o p and  t e s t a milk r e p l a c e r f o r t i f i e d with Ig, separated b o v i n e a b a t t o i r b l o o d by polyphosphate a  method  of providing  systemic  f r a c t i o n a t i o n , as  and e n t e r i c  immunity t o c o l o s t r u m - d e p r i v e d c a l v e s .  from  passive  28 MATERIALS AND METHODS P r e p a r a t i o n o f Bovine  Immunoglobulins  C i t r a t e d b o v i n e b l o o d was o b t a i n e d from two s o u r c e s , Intercontinental  P a c k e r s , Vancouver,  I n d u s t r i e s , A b b o t s f o r d , B.C. all  B.C.  Throughout  or  Coastpac  the procedure  m a t e r i a l s were k e p t a t 4° C t o m i n i m i z e  growth.  bacterial  Plasma was s i p h o n e d o f f a f t e r c e n t r i f u g a t i o n of  the  whole b l o o d (3 000 r e v o l u t i o n s p e r minute  ten  minutes, at 5°C).  F i b r i n was  (rpm) f o r  p r e c i p i t a t e d by t h e  a d d i t i o n o f d i c a l c i u m phosphate t o t h e plasma  (50 c c / L ) ,  a l l o w e d t o e q u i l i b r a t e o v e r n i g h t i n 4°C and removed by c h o p p i n g up. and supernatant  f i l t e r i n g through c h e e s e c l o t h w i t h the  serum  retained.  The  serum  was  then  f r a c t i o n a t e d by a m o d i f i c a t i o n o f t h e method by Lee e t a l . , (1988), u s i n g sodium p o l y p h o s p h a t e " g l a s s " s o l u t i o n c o n t a i n i n g Na ml  of  a  solution  15  P  13  O  A0  - Na  containing  20  P  18  0 .  114.4  55  (Calgon)  One  hundred  of  sodium  g/L  p o l y p h o s p h a t e g l a s s (Sigma Chemical Co., S t . L o u i s , and 84.85 g/L  NaCl were added p e r l i t r e  constant s t i r r i n g . HCl, to  MO)  o f serum w i t h  A f t e r pH adjustment t o 3.95 w i t h 3N  t h e m i x t u r e was s t i r r e d f o r t e n minutes and a l l o w e d equilibrate  overnight.  centrifuged  a t 5000 rpm  supernatant  retained.  supernatant  were  The  f o r t e n minutes The  was and  the  immunoglobulins  in  the  using  .a  Pellicon  ( M i l l i p o r e Corporation, Bedford,  MA) w i t h a 100,000 nominal m o l e c u l a r w e i g h t l i m i t pack.  The  then  (5°C)  concentrated  u l t r a f i l t r a t i o n system  mixture  concentrated  fraction,  filter  containing  29 a p p r o x i m a t e l y 130 mg Ig/ml s o l u t i o n , was l y o p h i l i z e d and s t o r e d a t -20° C i n s e a l e d p l a s t i c c o n t a i n e r s . E x p e r i m e n t a l Design A completely newborn  randomized d e s i g n was used i n which  c a l v e s were randomly a l l o c a t e d t o one o f f o u r  treatments,  w i t h a minimum o f 8 c a l v e s p e r t r e a t m e n t  (Table 3 ) .  Treatment A c a l v e s were f e d c o l o s t r u m on day  1 and whole m i l k on days 2 t o 21 (Col/WM).  Treatment B  c a l v e s were f e d m i l k r e p l a c e r a t a l e v e l o f 50 mg/ml I g on day 1, and 10 mg/ml I g on days 2 t o 21 (MR-Hi/Lo) . These l e v e l s a r e based on p u b l i s h e d l i t e r a t u r e v a l u e s f o r m i l k and c o l o s t r u m  (Straub & Matthaeus, 1978; F l e e n o r &  S t o t t , 1980). The h i g h l e v e l was i n t e n d e d t o approximate colostrum  levels  and t h e low l e v e l was i n t e n d e d t o be  i n t e r m e d i a t e between  colostrum  and m i l k .  Treatment C  c a l v e s were f e d m i l k r e p l a c e r w i t h t h e h i g h l e v e l (50  T a b l e 3. Experimental p r o t o c o l f o r s t u d y i n g a d m i n i s t r a t i o n o f immunoglobulins t o c a l v e s .  A  TREATMENTS Cmg Ig/ml d i e t l i g u i d ) C B D  DAYS OF AGE 1  Col  50  2-21  WM  10  22 - 42  WM  No I g  No. o f animals  11  8  50 No I g No I g  8  No I g No I g No I g  9  30 mg/ml) of I g on day days 2 - 2 1  (MR-  1, and m i l k r e p l a c e r w i t h no I g  Hi/No).  on  Treatment D ( c o n t r o l ) c a l v e s  were f e d m i l k r e p l a c e r w i t h no I g added from b i r t h t o day 21  (MR-No  Ig).  Animals A t o t a l of 37 newborn, c o l o s t r u m - d e p r i v e d H o l s t e i n F r i e s i a n b u l l c a l v e s were o b t a i n e d from A g a s s i z R e s e a r c h S t a t i o n , A g a s s i z , B.C.,  where t h e t r i a l was r u n , and from  d a i r y p r o d u c e r s i n the s u r r o u n d i n g a r e a .  The c a l v e s were  o b t a i n e d s h o r t l y a f t e r b i r t h , b e f o r e they had n u r s e d , and randomly a l l o t t e d t o Each  calf  was  before the i n i t i a l in t i e s t a l l s .  treatments. weighed  feeding.  and  a  blood  C a l v e s were housed  S t a l l s were a p p r o x i m a t e l y  3 f e e t deep w i t h bedding of sawdust and Each c a l f  given  a  indoors  2 f e e t wide x straw. four  hours of b i r t h , w i t h a second f e e d i n g a p p r o x i m a t e l y  four  s i x hours a f t e r t h e f i r s t ,  first  taken  feeding w i t h i n  to  was  sample  t o ensure t h e y r e c e i v e d a  minimum of f o u r t o f i v e l i t r e s o f t h e i r d i e t . 21  of  treatment  post-treatment total  of  10%  period  and  days  22  On days 2 -  42  of  p e r i o d , c a l v e s were f e d t w i c e d a i l y , a t a of  their  body w e i g h t .  A l l calves  were  weighed a t b i r t h , 24 hours of age, day 7 and once a week thereafter. of w e i g h t .  Feed i n t a k e was a d j u s t e d weekly on the b a s i s C a l v e s were b o t t l e f e d on days 1 and 2,  s w i t c h e d t o p a i l f e e d i n g from Day  3 onward.  and  31 Milk replacer The  b a s a l d i e t was  a commercial, non-medicated a l l  m i l k p r o t e i n c a l f m i l k r e p l a c e r o b t a i n e d from Van Waters &  Rogers  contained  Ltd., 21%  Vancouver,  p r o t e i n , 12%  phosphorus and  B.C.  The  f a t , 1%  f o r t i f i e d w i t h NRC  mixed  calcium  water  and  0.8%  Precise milk replacer  i s proprietary information. . with  replacer  r e q u i r e m e n t l e v e l s of  a l l o t h e r v i t a m i n s and m i n e r a l s . formula  milk  according  recommendations t o p r o v i d e a 14%  The  dry d i e t  to  was  manufacturers  solids milk replacer.  F r e e z e d r i e d immunoglobulins were mixed w i t h t h e d r y m i l k r e p l a c e r t o provide the appropriate c o n c e n t r a t i o n .  Daily  4 ml samples of c o n t r o l m i l k r e p l a c e r and m i l k r e p l a c e r fortified  with  I g were  frozen  at  -20°  C.  in  plastic  c r y o v i a l s and kept f o r l a t e r I g a n a l y s i s . Colostrum Colostrum  (first  & second m i l k i n g s ) was  collected  from mature cows a t t h e A g a s s i z Research S t a t i o n , p o o l e d to  g i v e a u n i f o r m c o n c e n t r a t i o n of I g and  l i t r e p l a s t i c f r e e z e r bags.  frozen i n 2  I t was thawed as needed, i n  a p a i l of hot t a p w a t e r , and f e d t o c a l v e s on Treatment A (Col/WM). was  A 4 ml sample of each f e e d i n g of c o l o s t r u m  f r o z e n a t -20°  C.  i h p l a s t i c c r y o v i a l s and k e p t f o r  l a t e r a n a l y s i s f o r immunoglobulin c o n c e n t r a t i o n . Whole M i l k Whole m i l k was  obtained and  fed  fresh daily,  milkings,  pooled  to  calves  (Col/WM).  A d a i l y 4 ml m i l k sample was  on  from r e g u l a r Treatment  f r o z e n a t -20°  A C.  32 in  plastic  c r y o v i a l s and  kept  for later  analysis for  immunoglobulin c o n c e n t r a t i o n . G r a i n and An  Hay 18%  p r o t e i n , commercial, t e x t u r e d  calf  grower  (East C h i l l i w a c k A g r i c u l t u r a l Co-op, C h i l l i w a c k ,  B.C.)  was  a v a i l a b l e t o t h e c a l v e s from about 5 days o f  age.  Hay  was  p r o v i d e d t o a maximum 2.0  kg t w i c e d a i l y .  B l o o d Samples Blood  samples were t a k e n by  jugular  venipuncture,  from a l l c a l v e s a t b i r t h , 24 h r s (day, 1 ) , 48 h r s (day and  on days 7,  14,  21,  28,  35 and  42.  One  4 ml  2)  blood  sample was c o l l e c t e d i n a h e p a r i n i z e d v a c u t a i n e r tube and one 4 ml sample i n a s o d i u m - f l u o r i d a t e d tube.  (NaF ) v a c u t a i n e r 2  B l o o d was c e n t r i f u g e d immediately,  a t 3 000 rpm f o r  t e n mins., and t h e serum s e p a r a t e d i n t o p l a s t i c c r y o v i a l s and  frozen  analyzed urea  (-20°  C.)  f o r later- a n a l y s i s .  Samples were  f o r serum I g c o n c e n t r a t i o n , b l o o d g l u c o s e  and  nitrogen.  Diarrhea  scores  S e v e r i t y of d i a r r h e a was d e s c r i b e d by Nocek e t a l . .  estimated  using  a  scale  (1984):  1 - normal, no f l u i d  4 - watery, a l l f l u i d  2 - s o f t , mostly  5 - watery, w i t h  solid  3 - runny, m o s t l y Fecal  samples  determination.  blood  fluid were  taken  daily  for  dry  matter  33 Therapy A l l c a l v e s r e c e i v e d a 2.0 ml i n j e c t i o n o f V i t a m i n A and D  1  (Poten A.D., S t e e r e E n t e r p r i s e s , Vancouver, B.C.)  a t b i r t h and a t 2 weeks o f age. t h e c a l f b a r n t o o t h e r animals  Due t o t h e p r o x i m i t y o f a t the research  station,  c a l v e s on t h e experiment were t r e a t e d f o r d i a r r h e a . I f a c a l f had a d i a r r h e a index l e v e l o f 3 o r more, t h e c a l f was g i v e n  approximately  (Kaolin  and  Pectin)  15 ml/kg body w e i g h t o f S t a t plus  electrolytes  (Steere  2  E n t e r p r i s e s , Vancouver, B.C.) i n i t s m i l k p l u s antibacterial (Furalean  b o l u s e s depending upon d i a r r h e a l s e v e r i t y  - E. c o l i  3  MTC P h a r m a c e u t i c a l s ,  bolus,  o r Sulkamycin - S  Cambridge, ON) .  1  V i t a m i n A - 500,000 I.U. Vitamin D 75,000 I.U. plus Vitamin E anti-oxidant  2  Kaolin 129.00 mg/ml Pectin 5.00 mg/ml Aluminum Oxide 1.66 mg/ml Potassium Acetate 3.30 mg/ml Mg-Cl-hexahydrate 1.00 mg/ml Na-acetatetrihydrate 19.80 mg/ml NaCl 18.00 mg/ml Ionic concentration K Mg Na CI 34 10 454 .318 +  1 or 2  ++  +  -  4  Bolus,  I f d i a r r h e a was  (meq/L) V 180  HC(  3  Nifuraldezone 1 gm Bismuth 260 mg Subsalicylate 260 mg Vitamin A 25000 I.U. 1 b o l u s / 7 0 kg body weight - t w i c e d a i l y f o r 2 days  4  Sulfamethazine 2 gm Neomycin-S0 250 mg 2 b o l u s e s / 5 0 kg body w e i g h t - t w i c e d a i l y , 1 s t day 1 b o l u s / 5 0 kg body w e i g h t - t w i c e d a i l y , n e x t 2-3 days 3  s e v e r e , c a l v e s were t a k e n o f f m i l k t r e a t m e n t 2.0 l i t r e s  electrolyte  solution  5  (Ionolyte -  E n t e r p r i s e s , Vancouver, B.C.) f o r 2 - 3 boluses.  R e c t a l temperature  and g i v e n Steere  days p l u s t h e  and g e n e r a l c o n d i t i o n were  r e c o r d e d d a i l y , f o r t h e f i r s t t h r e e weeks, as w e l l . Post-Mortem  Examinations  Post-mortem e x a m i n a t i o n s  were performed w i t h i n 48  hours o f death on a l l b u t one c a l f . complications  from  treatment p e r i o d . Veterinary Tissue  a twisted Examinations  Pathology  and f l u i d  intestine  i n the post-  were done by P r o v i n c i a l  Laboratory  samples were  s t a n d a r d post-mortem  This c a l f died of  i n Abbotsford, analyzed  B.C.  according t o  procedure.  A s s a y s o f a d m i n i s t e r e d and serum immunoglobulins Q u a n t i t a t i v e a n a l y s e s o f b o v i n e IgG was done u s i n g a  double  sandwich  enzyme-linked  immunosorbent  (ELISA) d e s c r i b e d by V o l l e r e t a l . . for  standards  and r e a g e n t s  Chemical Co., S t . L o u i s , MO. in  order  t o obtain  standard  and  were  (1976). obtained  assay  Antibodies from  Sigma  P r e l i m i n a r y t e s t s were r u n  t h e range  f o r t h e serum  of d i l u t i o n s samples.  for  Bovine  the IgG  c o n c e n t r a t i o n s were a n a l y z e d i n each sample o f serum from each c a l f , and i n d a i l y samples o f t h e c o l o s t r u m , m i l k , m i l k r e p l a c e r and I g - f o r t i f i e d m i l k r e p l a c e r .  5  I o n i c c o n c e n t r a t i o n - meq/L K Mg Na C l " HC0 " 34 10 454 318 180 +  ++  +  3  35 Blood Glucose . Blood  Determination  glucose  was determined  using  an e n z y m a t i c  ( g l u c o s e o x i d a s e ) p r o c e d u r e (Sigma D i a g n o s t i c s K i t #510, Sigma Chemical Co., S t . L o u i s , MO). B l o o d Urea N i t r o g e n Blood  urea  determination nitrogen  was  determined  using  a  u r e a s e / b e r t h e l o t p r o c e d u r e (Sigma D i a g n o s t i c s K i t #640, Sigma Chemical Co., S t . L o u i s , MO). STATISTICAL  ANALYSIS  R e s u l t s were a n a l y z e d  using General  L i n e a r Models  p r o c e d u r e o f SAS ( S t a t i s t i c a l A n a l y s i s System I n s t i t u t e I n c . , 1985).  Repeated measures on l e a s t squares means  were done f o r serum I g , b l o o d  glucose  and b l o o d  urea  n i t r o g e n and weight g a i n s .  A n a l y s i s o f c o v a r i a n c e was  performed  gains  weight  on average d a i l y  as t h e c o v a r i a t e .  using  Survival  initial  birth  was a n a l y z e d  by  a s s i g n i n g 1 t o c a l v e s t h a t s u r v i v e d t h e experiment and 0 to those t h a t d i e d . were a n a l y z e d  D i f f e r e n c e s between t r e a t m e n t  using orthogonal  contrasts.  means  A l l results  r e p o r t e d a r e l e a s t squares means f o r t r e a t m e n t s .  RESULTS  Measurement o f Bovine Immunoglobulins' Concentration of I g i n the freeze dried preparations from  bovine  serum  i s shown  i n Table  4.  Gel  e l e c t r o p h o r e s i s , using Corning u n i v e r s a l e l e c t r o p h o r e s i s  f i l m a g a r o s e , was c a r r i e d o u t on b o t h p r e c i p i t a t e and 6  supernatant f r a c t i o n s  from p o l y p h o s p h a t e  fractionation  and shown i n F i g u r e 5.  L e v e l o f I g measured i n p o o l e d  sample o f f r e e z e  dried  Ig preparation  mg/ml o f serum.  L e v e l s i n c o l o s t r u m f e d t o c a l v e s on  averaged 136.6  t r e a t m e n t one averaged 23.1 mg/ml ( r a n g i n g from 18 t o 28 mg/ml); i n h i g h l e v e l d i e t f e d t o c a l v e s , l e v e l s averaged 25.4  mg/ml; and i n low l e v e l d i e t , l e v e l s averaged  mg/ml.  9.4  L e v e l s i n b o t h whole m i l k and m i l k r e p l a c e r base  d i e t ranged from 0.0 t o 0.1 mg/ml.  T a b l e 4. C o n c e n t r a t i o n s of IgG in F r e e z e Dried P r e p a r a t i o n , M R - H i , M R - L o , C o l o s t r u m and Milk F e d to C a l v e s C o n c e n t r a t i o n of IgG  Range  (mg/ml)  (mg/ml) 84.0  -  150.0  Freeze Dried IgG  136.6  M R - H i diet  25.4  M R - L o diet  9.4  6.0  -  12.0  Colostrum  23.1  18.0  -  30.0  Milk  0.1  15.0 -- 4 1 . 0  c o n t a i n i n g 1% (w/v) agarose, 5% (w/v) s u c r o s e , and 0.035% (w/v) EDTA d i s o d i u m s a l t i n a 0.065 M b a r b i t a l b u f f e r , pH 8.6. American S c i e n t i f i c P r o d u c t s , McGraw P a r k , I L  37 Bovine  Serum  . J  Fraction  1  Fraction  2  Fraction  3  Fraction  4  Pure IgG sample  IgG  Figure  5.  Albumen  Gel electrophoresis of precipitate (fraction 4) a n d s u p e r n a t a n t ( f r a c t i o n s 1, 2 & 3) from p o l y p h o s p h a t e fractionation.  Survival Table survival Col/WM  5  rates  shows  effect  of calves  (11 o u t o f  11)  of  treatment  on  percent  f o r s i x weeks.  A l l c a l v e s on  a n d MR-Hi/Lo  (8 o u t o f 8)  t r e a t m e n t s s u r v i v e d t o s i x weeks o f a g e . o u t o f 8 c a l v e s s u r v i v e t o s i x weeks.  MR-Hi/No h a d 7  MR-Nolg  (control)  h a d s i g n i f i c a n t l y l o w e r s u r v i v a l t h a n Col/WM a n d MR-Hi/Lo w i t h 7 o u t o f 9 c a l v e s s u r v i v i n g t o s i x weeks  (P<0.05).  Temperature Rectal  temperatures  range and averaged  (Figure  6) w e r e i n t h e n o r m a l  38.5° C f o r c a l v e s on a l l t r e a t m e n t s .  C a l v e s w h i c h were s c o u r i n g had e l e v a t e d t e m p e r a t u r e s and  38  r e a c h e d t e m p e r a t u r e s above 3 9 . 0 ° C f o r t h o s e w h i c h were s c o u r i n g most  severely.  Table 5. T h e E f f e c t of Ig S u p p l e m e n t a t i o n  on  Calf Survival Rates EXPERIMENT 1 No. of c a l v e s s u r v i v i n g at TREATMENT  Birth  Day  42  Col/WM  11  11  MR-Nolg  9  7  MR-Hi/Lo  8  8  MR-Hi/No  8  7  Figure 6. E f f e c t of Ig S u p p l e m e n t a t i o n on R e c t a l T e m p e r a t u r e for the F i r s t T h r e e W e e k s of L i f e 39.4  -I  -  39.3-  38.2  i  1  0  7  1  14 DAYS O F A G E  '  —  i  21  39  Diarrhea Average weekly d i a r r h e a s c o r e s a r e shown i n F i g u r e 7.  Control calves  (MR-Nolg) had s i g n i f i c a n t l y more  d i a r r h e a t h a n c a l v e s on Col/WM and MR-Hi/Lo d u r i n g week one  (P<0.05). MR-Nolg c a l v e s a l s o had s i g n i f i c a n t l y more  diarrhea four  than  (P<0.01).  a l l three  other  treatments during  There were no s i g n i f i c a n t  week  differences  between t h e f o u r t r e a t m e n t s d u r i n g weeks two, t h r e e , f i v e and s i x .  F i g u r e 7. 0>  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on D i a r r h e a L e v e l s (bacterial  scours)  3 B a s e d on N o c e k  scale:  1 - normal  2.5  2 - soft, mostly solid 3 - runny, mostly fluid 4 - watery, all fluid 5 - watery, with blood  1.5 Treatments 1H  -  Col/WM MR-No Ig  0.5  -MR-Hi/Lo -a1  2 3 4 5 PERIOD OF TREATMENT - WEEK  6  MR-Hi/No  40 C a l f Weights Calf  weights  (Figure  8) were  not  significantly  d i f f e r e n t between any o f t h e t r e a t m e n t s a t b i r t h , 24 hours and days 7, 14 and 28.  MR-Nolg w e i g h t s tended t o  be l o w e r t h a n t h e o t h e r t h r e e t r e a t m e n t s t h r o u g h o u t t h e experiment  and were s i g n i f i c a n t l y  c a l f w e i g h t s a t day 21 (P<0.05).  l o w e r t h a n MR-Hi/Lo MR-Nolg w e i g h t s were  a l s o s i g n i f i c a n t l y l o w e r t h a n Col/WM and MR-Hi/Lo a t days 35 and 42 (P<0.05).  F i g u r e 8.  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on C a l f Weight  DAYS OF AGE  Average D a i l y G a i n s Figure  9 shows average d a i l y  gains  (ADG) f o r t h e  t h r e e t r e a t m e n t p e r i o d s . MR-Nolg c a l v e s had s i g n i f i c a n t l y lower ADG t h a n c a l v e s on Col/WM, MR-Hi/Lo o r MR-Hi/No f o r the three treatment age  p e r i o d s , from zero t o t h r e e weeks o f  (P<0.05), zero t o s i x weeks o f age (P<0.02) and t h r e e  t o s i x weeks o f age (P<0.05).  There were no s i g n i f i c a n t  d i f f e r e n c e s i n ADG between c a l v e s on Col/WM, MR-Hi/Lo o r MR-Hi/No.  F i g u r e 9.  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on Average Daily Gain  1 ro  0-3  weeks 0 - 6 weeks 3 - 6 weeks PERIOD OF EXPERIMENT  42  Feed E f f i c i e n c y Figure  10  supplementation  shows on f e e d  effect  of  efficiency  immunoglobulin  (kgs d r y feed/kgs  g a i n ) o f c a l v e s f o r t h e f i r s t s i x weeks o f l i f e .  MR-Nolg  c a l v e s had s i g n i f i c a n t l y p o o r e r (P<0.05) f e e d e f f i c i e n c y than t h e other three treatment c a l v e s .  F i g u r e 10.  3.00  T h e E f f e c t of Ig S u p p l e m e n t a t i o n o n F e e d E f f i c i e n c y for the F i r s t S i x W e e k s of Life  ro  UJ T3 Q)  0.50 -  U.  0.00  Col/WM  MR-Hi/Lo  MR-Hi/No  MR-Nolg  43 Serum IgG L e v e l s Serum IgG l e v e l s a r e shown i n F i g u r e 11. There were no s i g n i f i c a n t d i f f e r e n c e s i n serum IgG l e v e l s any o f t h e t r e a t m e n t s a t b i r t h o r day 21. hours  of  age,  and  at  day  7,  between  A t 24 and 48  Col/WM  calves  had  s i g n i f i c a n t l y h i g h e r serum IgG l e v e l s t h a n c a l v e s on MRNolg,  MR-Hi/Lo and MR-Hi/No  (P<0.01).  There were no  s i g n i f i c a n t d i f f e r e n c e s between MR-Nolg, MR-Hi/Lo and MRHi/No t r e a t m e n t s . significantly calves  higher  (P<0.02).  significantly  A t day 14 and 28, c a l v e s on Col/WM had serum  IgG l e v e l s  than  MR-Hi/No  A t day 35 and 42, MR-Hi/No c a l v e s had  lower  serum  IgG  levels  than  (P<0.01), MR-Nolg and MR-Hi/Lo c a l v e s (P<0.05).  F i g u r e 11. T h e E f f e c t of Ig S u p p l e m e n t a t i o n on S e r u m IgG L e v e l s 20  DAYS OF  AGE  Col/WM  Blood Glucose L e v e l s Blood  glucose  levels  (Figure  12)  were  not  s i g n i f i c a n t l y d i f f e r e n t between any o f t h e t r e a t m e n t s , a t b i r t h , 48 hours o f age, and a t days 7, 14, 21, 28 and 42. A t 24 hours o f age, b l o o d g l u c o s e l e v e l s f o r c a l v e s on Col/WM and MR-Nolg were s i g n i f i c a n t l y h i g h e r t h a n c a l v e s on  MR-Hi/No  (P<0.05), w i t h  a trend t o higher  between MR-Nolg and MR-Hi/Lo. lower  than  the other  significantly.  MR-Hi/Lo  treatments  a t day  tended  levels t o be  14 b u t n o t  B l o o d Urea N i t r o g e n B l o o d Urea N i t r o g e n (BUN) l e v e l s ( F i g u r e 13) tended to  be h i g h e r i n t h e f i r s t  week and showed a s h a l l o w  d e c l i n e o v e r t h e n e x t f i v e week p e r i o d .  BUN l e v e l s i n  MR-Nolg c a l v e s were s i g n i f i c a n t l y h i g h e r t h a n MR-Hi/Lo and MR-Hi/No c a l v e s a t b i r t h  (P<0.05) and s i g n i f i c a n t l y  h i g h e r t h a n MR-Hi/No c a l v e s a t 24 hours o f age (P<0.05). There were no s i g n i f i c a n t d i f f e r e n c e s between any o f t h e t r e a t m e n t s a t 48 hours o f age. BUN v a l u e s f o r c a l v e s on MR-Hi/No t r e a t m e n t tended  t o be lower t h a n t h e o t h e r  t h r e e t r e a t m e n t s t h r o u g h o u t t h e s i x week p e r i o d o f t h e experiment,  and were  significantly  lower  (P<0.05).  Figure 13. T h e E f f e c t of Ig S u p p l e m e n t a t i o n on  0  7  14 21 DAYS OF  28 AGE  35  42  a t day  14  46 DISCUSSION  Although  t h e r e was a low m o r t a l i t y o v e r a l l ,  there  was a s i g n i f i c a n t d i f f e r e n c e i n s u r v i v a l between Col/WM (11/11) o r MR-Hi/Lo (7/9) .  The  calves  same s u r v i v a l  (8/8), rate  and MR-Nolg f o r both  calves  Col/WM  and  MR-Hi/Lo c a l v e s shows t h a t f e e d i n g an I g - f o r t i f i e d m i l k r e p l a c e r can s e r v e calves passive,  as an a l t e r n a t i v e method o f g i v i n g  systemic  immunity and can reduce t h e  m o r t a l i t y a s s o c i a t e d w i t h hypo-gammaglobulinemia. D i a r r h e a l e v e l s (scours) were f a i r l y low t h r o u g h o u t the experiment.  The a n t i b i o t i c t h e r a p y g i v e n as soon as  a c a l f was n o t i c e d s c o u r i n g may have reduced t h e numbers of i n t e s t i n a l b a c t e r i a c a u s i n g s c o u r i n g .  T h i s may have  tended  t o decrease  d i f f e r e n c e s due t o t r e a t m e n t s ,  as  well.  There was, however, a s i g n i f i c a n t d i f f e r e n c e i n  week one and week f o u r between MR-Hi/No c a l v e s and Col/Wm and MR-Hi/Lo c a l v e s . which  received  episodes  no  These r e s u l t s i n d i c a t e t h a t c a l v e s I g had  of scours.  more  severe  The e l e v a t e d  and  rectal  recurring  temperatures  d u r i n g s c o u r i n g were i n d i c a t i v e o f f e v e r a s s o c i a t e d w i t h a bacterial  infection.  MR-Nolg c a l v e s tended t o s c o u r  l e s s t h a n e i t h e r MR-Hi/Lo o r Col/WM c a l v e s , a l t h o u g h t h e d i f f e r e n c e was n o t s i g n i f i c a n t .  T h i s may have been due  t o t h e i n c r e a s e d a n t i b i o t i c t h e r a p y g i v e n t o t h e MR-Nolg calves.  These r e s u l t s suggest t h a t c o n t i n u e d f e e d i n g o f  I g i n m i l k r e p l a c e r (MR-Hi/Lo) t o t h r e e weeks o f age may have  had  a  protective  effect  i n the  gut,  reducing  b a c t e r i a l - r e l a t e d d i a r r h e a . These f i n d i n g s a r e s u p p o r t e d  by  other  colostrum  work which showed t h a t c o n t i n u e d reduced t h e i n c i d e n c e  feeding of  of scours  (Logan and  P e a r s o n , 1978). Treatments had a s i g n i f i c a n t e f f e c t on w e i g h t and average d a i l y calves.  gains,  as w e l l as f e e d  e f f i c i e n c i e s of  MR-Nolg c a l f w e i g h t s were s i g n i f i c a n t l y  lower  t h a n t h e o t h e r t h r e e t r e a t m e n t s by day 21 and t h e r e were no  compensatory  post-treatment  gains  in  the  three  p e r i o d o f t h e experiment.  to  s i x week  Weekly w e i g h t s  o f Col/WM c a l v e s were not s i g n i f i c a n t l y d i f f e r e n t  from  MR-Hi/Lo o r MR-Hi/No c a l v e s t h r o u g h o u t t h e e x p e r i m e n t . The o v e r a l l growth r a t e s o f c a l v e s on Col/WM (587 g/d) were n o t s i g n i f i c a n t l y d i f f e r e n t from c a l v e s r e c e i v i n g MR-Hi/Lo  (544 g/d) o r MR-Hi/No (575 g / d ) , however, a l l  t h r e e were s i g n i f i c a n t l y h i g h e r t h a n MR-Nolg c a l v e s (377 g/d) .  The  feed  efficiency  f o r MR-Nolg  calves  was  s i g n i f i c a n t l y p o o r e r (P<0.01) t h a n f o r Col/WM o r MR-Hi/No calves.  These r e s u l t s show t h a t c a l v e s r e c e i v i n g I g ,  e i t h e r from c o l o s t r u m  or i n a f o r t i f i e d milk replacer,  w i l l do b e t t e r over t h e c r i t i c a l  f i r s t t h r e e weeks t h a n  c a l v e s t h a t do n o t r e c e i v e any I g . They w i l l s c o u r  less,  g a i n more and be a t a h i g h e r weight a t weaning ( s i x weeks of age). Passive colostrum peaked decline.  immunity  from  absorption  o r MR-Hi I g t r e a t m e n t s ,  a t about  36  to 4 8  hours,  seen  of  Ig  from  i n Figure  and t h e n  11,  began t o  T h i s normal d e c r e a s e i n serum I g l e v e l s  occurs  because, a f t e r c l o s u r e , t h e l e v e l s o f p a s s i v e I g d e c r e a s e  48 as t h e y a r e c a t a b o l i z e d . The h a l f - l i f e o f I g range from 14 - 21 days  i n the blood.  Development  of active  immunity c a n be seen i n the- serum l e v e l s o f t h e MR-No I g c a l v e s w h i c h n o r m a l l y b e g i n s some time between day 7 t o day  14.  These  therefore,  calves  received  no  passive  the increase  i n their  serum  I g and  immunogloblin  l e v e l s would r e f l e c t t h e i r own immune system development and  maturation.  The serum  immunoglobulin  levels for  MR-Hi/Lo and MR-Hi/No c a l v e s d i d n o t r i s e Col/WM c a l v e s (16.3 v s 8.3 mg/ml).  as h i g h as  However, t h e r e was  s i g n i f i c a n t a b s o r p t i o n o f t h e I g from t h e f o r t i f i e d m i l k replacer.  The a b s o r p t i o n e f f i c i e n c y f o r t h e s e  treatments  (27 - 32%) i s c o n s i s t e n t w i t h l i t e r a t u r e v a l u e s o f 10 t o 46%. due  The h i g h e r l e v e l s found i n Col/WM c a l v e s c o u l d be to factors  immunoglobulin Hardy, level  19 69).  i n the colostrum absorption The lower  which  may  enhance  ( B a l f o u r and Comline, serum l e v e l s  1962;  from t h e MR-Hi  f e e d i n g s c o u l d a l s o be due t o t h e f a c t t h a t t h e  actual  levels  measured  (25.4 mg/ml) were  half  that  c a l c u l a t e d f o r a d d i t i o n t o t h e m i l k r e p l a c e r (50 mg/ml). Problems were encountered when r u n n i n g t h e i n i t i a l ELISA assays.  A b o v i n e IgG s t a n d a r d i s done a t t h e same time  as t h e unknown samples f o r a s t a n d a r d c u r v e t o be used i n c a l c u l a t i n g c o n c e n t r a t i o n o f immunoglobulins.  The v a l u e s  o b t a i n e d f o r t h e s t a n d a r d c u r v e were n o t c o n s i s t e n t b u t due t o a time c o n s t r a i n t a b e s t f i t c u r v e was used.  This  may have r e s u l t e d i n i n c o r r e c t v a l u e s c a l c u l a t e d f o r t h e c o n c e n t r a t i o n s i n t h e immunoglobulin  s o l u t i o n which then  c o u l d have r e s u l t e d i n a m i s c a l c u l a t i o n f o r amounts t o be added t o t h e m i l k r e p l a c e r i n t h e MR-Hi d i e t s . serum l e v e l s degradation tissue  could  The lower  a l s o be due t o some d e n a t u r i n g  i n the i n t e s t i n a l  absorption.  This  lumen, as w e l l a s , some  could  be  dealt  with  i n c r e a s i n g t h e amount o f I g added t o m i l k r e p l a c e r . MR-Hi/No t r e a t m e n t  or  c a l v e s had q u i t e d i s s i m i l a r  compared w i t h t h e o t h e r t h r e e t r e a t m e n t s .  by The  results  These c a l v e s  s h o u l d have responded s i m i l a r l y t o t h e Col/WM c a l v e s i n t h e i r serum I g l e v e l s , w i t h t h e i r a c t i v e immune l e v e l s slowly increasing.  They a r e n o t i n agreement w i t h t h e  o t h e r r e s u l t s and no d e f i n i t e e x p l a n a t i o n i s a p p a r e n t . However,  this  treatment,  may  have  been  due t o t h e  antibiotic  which may have reduced t h e l e v e l o f b a c t e r i a  t o a p o i n t where t h e r e was n o t enough o f an a n t i g e n i c challenge  f o r the c a l f ' s  immune system t o respond t o .  F u r t h e r s t u d i e s a r e needed t o u n d e r s t a n d i f t h e r e a r e any factors  involved  which  would  possibly  suppress t h e  development o f t h e immune response. B l o o d g l u c o s e and b l o o d urea n i t r o g e n r e s u l t s a r e i n general  agreement w i t h  work done  levels  from o t h e r  experimental  ( B a z i n and B r i s s o n , 1975; Drevjany e t a l . .  1982; P e t i t e t a l . . 1988a & 1988b; W i l l i a m s and Smith, 1975).  B l o o d g l u c o s e and b l o o d urea n i t r o g e n l e v e l s were  i n t h e normal range f o r a p r e - r u m i n a n t c a l f ' s development from b i r t h t o s i x weeks o f age. glucose  levels  The v a r i a t i o n i n b l o o d  was n o t n e c e s s a r i l y due t o  e f f e c t s b u t may have been due t o t i m e o f b l o o d  treatment sampling  i n r e l a t i o n t o feeding.  Depending upon when c a l v e s a r e  f e d , b l o o d g l u c o s e and BUN l e v e l s r i s e t o a peak and f a l l a g a i n t o average l e v e l s over s i x t o e i g h t hours p o s t feeding  ( P e t i t e t a l . . 1988a & 1988b) .  T h i s a l s o may  have been due t o t h e b l o o d samples n o t b e i n g c e n t r i f u g e d as soon as p o s s i b l e a f t e r c o l l e c t i o n .  I f t h e b l o o d was  l e f t t o s i t , t h e b l o o d c e l l s may have c o n t i n u e d t o use up t h e g l u c o s e and t h i s would r e s u l t i n lowered v a l u e s . The  calves  fed the I g - f o r t i f i e d  milk  replacer  s c o u r e d l e s s , needed l e s s a n t i b i o t i c t h e r a p y , and g a i n e d more w e i g h t .  C a l v e s n o t r e c e i v i n g any o r enough p a s s i v e  I g s c o u r e d more s e v e r e l y , had h i g h e r r e c t a l and r e c e i v e d more a n t i b i o t i c t h e r a p y .  temperatures  T h e i r growth r a t e  was l e s s t h a n t h e c a l v e s which r e c e i v e d I g and t h e r e f o r e , they  weighed  weeks.  less  than  The r e s u l t s  the others  of t h i s  a t t h e end o f s i x  experiment  show t h a t an  I g - f o r t i f i e d m i l k r e p l a c e r can produce an adequate l e v e l o f p a s s i v e immunity t o reduce s y s t e m i c d i s e a s e s and, w i t h c o n t i n u e d f e e d i n g , can reduce b a c t e r i a l s c o u r s .  51  EXPERIMENT 2 INTRODUCTION  C a l v e s , f o r v a r i o u s r e a s o n s , may not r e c e i v e enough I g v i a a b s o r p t i o n even though a l l o w e d t o n u r s e o r consume colostrum.  N e o n a t a l c a l v e s s o l d t o be r a i s e d f o r v e a l o r  b e e f may be d e p r i v e d o f c o l o s t r u m which would l o s s e s due t o s y s t e m i c d i s e a s e s . occurs,  these  calves  passive  immunity  increase  Once i n t e s t i n a l c l o s u r e  have no chance t o a c h i e v e  levels.  A purified,  good  injectable Ig  s o l u t i o n , which would be absorbed i n t o t h e b l o o d s t r e a m , could  this  needed  immunity and reduce t h e i n c i d e n c e o f s e p t i c e m i c  disease  and  give  hypo-gammaglobulinemic  the associated mortality.  calves  The e f f e c t i v e n e s s o f an  i n t r a p e r i t o n e a l o r i n t r a v e n o u s r o u t e u s i n g b o v i n e plasma f o r i n c r e a s i n g serum immunoglobulin l e v e l s has been shown by Anderson e t a l . , The  (1987).  o b j e c t i v e o f t h i s experiment was t o d e v e l o p an  i n j e c t a b l e form o f I g , s e p a r a t e d and p u r i f i e d from b o v i n e abattoir calves systemic  blood,  and t e s t  this  as an a l t e r n a t e method  in  colostrum-deprived  of achieving  passive,  immunity.  MATERIALS AND METHODS  P r e p a r a t i o n o f B o v i n e Immunoglobulins B o v i n e I g p r e p a r a t i o n by p o l y p h o s p h a t e f r a c t i o n a t i o n f o l l o w e d t h e same p r o c e d u r e as e x p l a i n e d i n experiment 1.  52 Preparation The  o f i n j e c t a b l e Icr s o l u t i o n liquid  fractionation  Ig  method  solution  obtained  described  from  i n experiment  the  1  was  p u r i f i e d , b e f o r e l y o p h i l i z a t i o n , by ion-exchange column chromatography t e c h n i q u e al.,  (Friesen  e t a l . , 1985; Lee e t  1987) u s i n g DEAE-Sephacel beads  Picataway, NJ).  (Pharmacia, I n c . ,  The p u r i f i c a t i o n p r o c e d u r e was done a t  a t e m p e r a t u r e o f 4°C t o r e t a r d  b a c t e r i a l growth.  method was  volumes  adapted f o r l a r g e r  of Ig  The  solution  needed as f o l l o w s : A 11.5 cm d i a . x 11.0 cm h i g h column was assembled w i t h an i n l e t tube e n t e r i n g a t t h e t o p o f t h e column and an o u t l e t t u b e coming from t h e bottom o f t h e column and c o n n e c t e d t o a d r i p r a t e c o n t r o l l e r (stopcock) and drop c o u n t e r on an automated f r a c t i o n c o l l e c t o r .  A s l u r r y of  1500 ml o f t h e DEAE-Sephacel beads was poured i n t o t h e column and a l l o w e d t o s e t t l e . The o t h e r end o f t h e i n l e t tube was i n s e r t e d i n an 0.9% s a l i n e s o l u t i o n and s e t t o d r i p a t a r a t e o f 10 d r o p s p e r minute. The DEAE-Sephacel was e q u i l i b r a t e d o v e r n i g h t , by washing w i t h a volume o f T r i s - H C L b u f f e r s o l u t i o n (0.02 M, pH 7.4) w h i c h was f i v e t i m e s t h a t o f t h e column (7500 m l ) . then applied  t o t h e column  p u r i f i e d per elution)  The I g s o l u t i o n was  (100 ml o f I g s o l u t i o n  and a l l o w e d t o p e r c o l a t e  was  through  the column, by opening t h e s t o p c o c k a t t h e bottom, u n t i l serum s o l u t i o n i s f l u s h w i t h t h e t o p o f t h e g e l .  Tris-  HCl b u f f e r was t h e n poured onto t h e g e l s l o w l y t o t h e t o p of  t h e column.  The  l i d was  put  on  t h e column  and  tightened  s o i t was a i r t i g h t .  The i n l e t  tube  was  i n s e r t e d i n t h e T r i s - H C l b u f f e r s o l u t i o n and a n o t h e r tube was  inserted  saline  between  solution.  the buffer  solution  and a 0.9%  As t h e immunoglobulin  p u l l e d through the g e l , Tris-HCl  solution i s  i s then p u l l e d i n t o t h e  column and t h r o u g h t h e g e l , and t h e n t h e s a l t s o l u t i o n i s pulled  into  the Tris-HCl  buffer  solution;  pulled  into  t h e column and t h r o u g h  then  the gel.  i t is A  salt  g r a d i e n t from 0 - 0.5 M was o b t a i n e d by t h i s method. The drip  rate  was s e t a t 6 drops  p e r minute  f r a c t i o n s were c o l l e c t e d i n 10 ml t u b e s .  The f r a c t i o n s  were measured f o r s a l t c o n c e n t r a t i o n on a meter  (Solu-Bridge, I n d u s t r i a l  and 8 ml  conductivity  Instruments  Inc.) and  a n a l y z e d f o r IgG c o n c e n t r a t i o n by g e l e l e c t r o p h o r e s i s , as well  as by ELISA  (as d e s c r i b e d on page  fractions containing  57) .  The  t h e p u r i f i e d IgG were p o o l e d and p u t  i n t o S p e c t r a / p o r m o l e c u l a r p o r o u s d i a l y s i s membrane t u b i n g with  a molecular  weight  cut-off  o f 12,000  -  14,000  (Spectrum M e d i c a l Ind. I n c . , Los A n g e l e s , CA) , suspended in  0.9% s a l i n e  and brought  to a physiological  saline  concentration  (0.9%).  T h i s s o l u t i o n was p u t i n t o 4 1-  litre  bottles  and s e a l e d w i t h r u b b e r  sterile  w h i c h had an a r e a f o r s t e r i l e bottles  o f IgG s o l u t i o n  radiation. irradiated  A gamma c e l l a t 24,000  samples o f each  removal  by n e e d l e .  were t h e n s t e r i l i z e d was used  stoppers The  by gamma  and each b o t t l e was  r a d f o r 13.3 m i n u t e s .  of the four bottles  Two ml  were checked f o r  s t e r i l i t y by p l a t i n g on l a b media agar.  Nutrient  54 media  agar  was  dissolved  a u t o c l a v e d f o r 2 0 minutes. round  in distilled  water  and  The h o t agar was poured  into  c u l t u r e p l a t e s and a l l o w e d t o c o o l i n a s t e r i l e  environment hood.  The gamma r a d i a t e d samples o f IgG  s o l u t i o n were p l a t e d on t h e agar and i n c u b a t e d a t 37°C f o r 72 h o u r s . bacterial  The p l a t e s were checked e v e r y 24 hours f o r  growth.  The IgG s o l u t i o n b o t t l e s were t h e n  s t o r e d a t 4°C u n t i l needed f o r i n j e c t i o n . Experimental  Design:  Experiment which  2 was a c o m p l e t e l y randomized d e s i g n i n  colostrum-deprived  newborn  bull  calves  were  randomly a s s i g n e d t o one o f t h r e e t r e a t m e n t s , as shown i n T a b l e 6, w i t h 8 c a l v e s p e r t r e a t m e n t .  Treatment A c a l v e s  were f e d c o l o s t r u m on day 1 and whole m i l k on days 2 t o 21  (Col/WM).  subcutaneous possible.  Treatment injection  B  calves  were  given  a  o f i g as soon  after  birth  as  I n a l l c a s e s , t h i s was b e f o r e 6 hours o f age.  Treatment C c a l v e s were f e d m i l k r e p l a c e r w i t h no I g T a b l e 6.  Experimental p r o t o c o l f o r s t u d y i n g e f f e c t o f a d m i n i s t r a t i o n o f immunoglobulins t o c a l v e s .  TREATMENTS A DAYS OF AGE 1  Col  B  C  Subcu I n j . of I g  No I g  2-21  WM  10 mg/ml I g  No I g  22-42  WM  No I g  No I g  NO. Of animals  8  8  8  added on day  1, and on days 2 t o 21 t h e y were f e d m i l k  r e p l a c e r w i t h I g added a t a l e v e l Inj). was  of  10 mg/ml (MR-Lo  T h i s l e v e l , as used i n t h e p r e v i o u s i n t e n d e d t o be  milk.  Treatment  experiment,  i n t e r m e d i a t e between c o l o s t r u m C  (control)  calves  r e p l a c e r w i t h no I g added from b i r t h  were  and  fed  milk  (day 1) t o day  21  (MR-Nolg) . Animals A t o t a l o f 24 newborn c o l o s t r u m - d e p r i v e d Friesian  bull  Research  Farm, where t h e t r i a l  producers  c a l v e s were o b t a i n e d  i n the  surrounding  was area.  Holstein-  from O y s t e r  River  r u n , o r from  dairy  The  calves  were  o b t a i n e d s h o r t l y a f t e r b i r t h , b e f o r e t h e y had n u r s e d , randomly  allotted  to  c a l v e s per t r e a t m e n t .  one  of  three  treatments  and  with  8  Each c a l f was weighed, and a b l o o d  sample was t a k e n b e f o r e the i n i t i a l f e e d i n g .  C a l v e s were  a s s i g n e d a t random t o a pen.  Pens were 4' deep x 3" wide  x  of sawdust and  4.5'  allowed  h i g h w i t h a bedding contact  on  Treatment C were t h e n g i v e n a subcutaneous i n j e c t i o n  of  stalls.  v i a a 60 cc s y r i n g e i n 2 - 3 l o c a t i o n s  the shoulder area. concentration  of  over  The amount a d m i n i s t e r e d , based on t h e purified  weight o f t h e c a l f , was circulating  adjacent  and  Calves  Ig s o l u t i o n  between  straw,  Ig  solution,  and  the  birth  i n t e n d e d t o p r o v i d e an adequate  l e v e l o f I g ( i . e . >12  mg/ml).  Each c a l f was g i v e n i t s f i r s t f e e d i n g w i t h i n 2 hours a f t e r b i r t h , w i t h subsequent f e e d i n g s a p p r o x i m a t e l y 4 6 h o u r s a f t e r t h e f i r s t t o ensure t h e y r e c e i v e d a minimum  of 4 - 5 l i t r e s treatment  o f c o l o s t r u m o r MR.  period  and days  22 - 42 o f  p e r i o d , c a l v e s were f e d t w i c e d a i l y t h e i r body w e i g h t .  basis.  post-treatment  a t o t a l o f 10% o f  A l l c a l v e s were weighed a t b i r t h , 24  h o u r s o f age, day 7 - and intake  F o r days 2 - 21 o f  was a d j u s t e d  once a week t h e r e a f t e r .  according  t o weight  Feed  on a weekly  C a l v e s were n u r s i n g - b o t t l e f e d on days 1 and 2,  and were s w i t c h e d t o p a i l f e e d i n g from Day 3 onward. Milk replacer The  basal  diet  was  that  used  i n experiment  1.  F r e e z e d r i e d immunoglobulins were mixed w i t h t h e d r y m i l k replacer t o provide the appropriate concentration. 4 ml samples  of milk  Daily  r e p l a c e r and I g - f o r t i f i e d  milk  r e p l a c e r were f r o z e n a t -20°C i n s e a l e d p l a s t i c t u b e s and k e p t f o r l a t e r immunoglobulin a n a l y s i s . Colostrum Colostrum  (first  & second m i l k i n g s ) was c o l l e c t e d  from mature cows a t O y s t e r  R i v e r Research Farm,  pooled  and f r o z e n a t -20°C i n 2 l i t r e p l a s t i c f r e e z e r bags. I t was thawed as needed i n a p a i l o f h o t t a p w a t e r , and f e d t o c a l v e s on Treatment A. of c o l o s t r u m  A 4 ml sample o f each f e e d i n g  was f r o z e n a t -20°C i n s e a l e d p l a s t i c and  k e p t f o r l a t e r immunoglobulin a n a l y s i s . Whole M i l k Whole m i l k was o b t a i n e d  fresh daily,  from r e g u l a r  m i l k i n g s , p o o l e d and f e d t o c a l v e s on Col/WM  treatment.  A 4 ml d a i l y sample o f m i l k was f r o z e n a t -2 0°C i n s e a l e d p l a s t i c and k e p t f o r l a t e r immunoglobulin a n a l y s i s .  57 G r a i n and  Hay  An 18% p r o t e i n commercial c a l f s t a r t e r / g r o w e r ( O t t e r Farm & Home Co-op, A l d e r g r o v e , B.C.) c a l v e s from about 5 days of age. maximum of 2.5  kg t w i c e  was a v a i l a b l e t o t h e Hay was  provided to a  daily.  B l o o d Samples Blood  samples were t a k e n v i a j u g u l a r  venipuncture  from a l l c a l v e s a t b i r t h , 24 h r s (day 1 ) , 48 h r s and  on Days 7,  14,  21, 28,  35 and  42.  One  (day  4 ml  2)  blood  sample was c o l l e c t e d i n a h e p a r i n i z e d v a c u t a i n e r tube and one 4 ml sample i n a s o d i u m - f l u o r i d a t e d (NaF ) v a c u t a i n e r 2  tube.  B l o o d was c e n t r i f u g e d immediately,  t e n mins., and t h e serum s e p a r a t e d t u b e s and samples  frozen were  (-20°  C.)  for  c o n c e n t r a t i o n , b l o o d urea blood glucose Diarrhea  (NaF  the  analysis.  serum  nitrogen  plastic Blood  immunoglobulin  ( h e p a r i n tubes)  and  tubes).  scores  D i a r r h e a was by  2  into sealed  for later  analyzed  a t 3 000 rpm f o r  same  e s t i m a t e d , w i t h a l l o b s e r v a t i o n s done  observer,  using  a  scale  described  in  experiment 1. Therapy A l l c a l v e s r e c e i v e d 2.0 and D (POTEN A.D.,  ml i n j e c t i o n  of V i t a m i n s  S t e e r e E n t e r p r i s e s , Vancouver,  a t b i r t h and a t 2 weeks of age.  I f d i a r r h e a was  c a l v e s were t a k e n o f f m i l k t r e a t m e n t and g i v e n 2.0  A  B.C.)  severe, litres  58 electrolyte  solution  (Ionolyte  7  Vancouver, B.C.) f o r 2 - 3 days.  - Steere  Enterprises,  R e c t a l t e m p e r a t u r e and  g e n e r a l c o n d i t i o n was r e c o r d e d as w e l l .  One c a l f was  t r e a t e d w i t h 2 ml o f a n t i b a c t e r i a l T r i v e t r i n f o r 3 days f o r a b a c t e r i a l i n f e c t i o n i n an i n j u r e d r i g h t h i n d l e g . Post-Mortem  Examinations  Post-mortem e x a m i n a t i o n s were performed w i t h i n 24 hours o f d e a t h on a l l b u t one o f t h e c a l v e s t h a t d u r i n g t h e experiment. Veterinary samples  Clinic sent  Laboratory  E x a m i n a t i o n s were done by a Comox  veterinarian  to  died  Provincial  i n A b b o t s f o r d , B.C.  with  tissue  Veterinary  and  fluid  Pathology  A post-mortem was n o t  done on one c a l f t h a t d i e d o f apparent b l o a t . Assay o f a d m i n i s t e r e d and serum  immunoglobulins  Q u a n t i t a t i v e a n a l y s e s o f b o v i n e IgG was done u s i n g a c o m p e t i t i v e , d i r e c t e n z y m e - l i n k e d immunosorbent a s s a y (ELISA)  (Kiriyama  et  a l . . 1989).  Antibodies f o r  s t a n d a r d s and r e a g e n t s were o b t a i n e d from H e l i x B i o t e c h I n d u s t r i e s , Richmond, B.C. T e s t s were r u n t o compare t h e r e s u l t s between t h e d o u b l e - a n t i b o d y sandwich t e c h n i q u e (used  i n experiment  technique.  Both  1) and t h e c o m p e t i t i v e , gave  similar  results  direct  f o r IgG  c o n c e n t r a t i o n s i n s t a n d a r d s and i n serum samples.  Bovine  IgG c o n c e n t r a t i o n s were a n a l y z e d i n serum from each c a l f ,  C o n c e n t r a t i o n - meq/L K Mg Na C l " . HC0 " 34 10 454 318 180 +  ++  +  3  and  daily  samples  of the colostrum,  milk  and  milk  followed  the  same  replacer with Ig. Blood Glucose Blood  Determination  glucose  determination  p r o c e d u r e as f o r experiment 1. B l o o d Urea N i t r o g e n  determination  B l o o d u r e a n i t r o g e n d e t e r m i n a t i o n f o l l o w e d t h e same p r o c e d u r e as f o r experiment 1.  RESULTS  Measurement o f Bovine Immunoglobulins Analysis  of  level  of  immunoglobulins  l y o p h i l i z e d p r e p a r a t i o n from p o l y p h o s p h a t e of b o v i n e b l o o d  i s shown i n T a b l e 7.  i n the  fractionation  The p u r i f i e d I g  s o l u t i o n used f o r subcutaneous i n j e c t i o n c o n t a i n e d 513.0 3  m<  Ig/<? °f  dry matter.  This  was  added  to  sterile  b u f f e r e d s a l i n e t o g i v e a 6% p r o t e i n c o n c e n t r a t i o n i n t h e solution.  L e v e l s measured i n c o l o s t r u m and m i l k f e d t o  c a l v e s a r e a l s o shown i n T a b l e 7. Survival T a b l e 8. shows e f f e c t o f t r e a t m e n t on s u r v i v a l r a t e s of  calves  treatments  f o r s i x weeks.  Both Col/WM and MR-Lo I n j  had 7 o u t o f 8 c a l v e s s u r v i v e t o s i x weeks o f  age b u t , o n l y 4 o u t o f 8 MR-Nolg c a l v e s s u r v i v e d , which was  significantly  treatments.  lower  (P<0.05)  than  the other  One c a l f from Col/WM t r e a t m e n t s  two  died i n the  p o s t - t r e a t m e n t p e r i o d from causes u n r e l a t e d t o t r e a t m e n t . One. c a l f on MR-Lo I n j t r e a t m e n t  d i e d as a r e s u l t o f a  T a b l e 7.  C o n c e n t r a t i o n of IgG in P u r i f i e d Injectable S o l u t i o n , M R - L o diet, C o l o s t r u m a n d Milk C o n c e n t r a t i o n of IgG (mg/ml)  P u r i f i e d IgG solution  2488.3  IgG s o l u t i o n as injected  12.7  M R - L o diet  10.3  Colostrum  27.8  Milk  T a b l e 8.  0.1  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on Calf Survival Rates EXPERIMENT 2 No. of c a l v e s s u r v i v i n g at  TREATMENT  Birth  Day 42  Col/WM  8  7  MR-Nolg  8  4  MR-Lo Inj  8  7  61 r o t a v i r u s i n f e c t i o n i n t h e f i r s t 24 hours o f l i f e .  The  deaths o f c a l v e s from MR-Nolg t r e a t m e n t were caused by E. c o l i septicemia w i t h severe dehydration. R e c t a l Temperature F i g u r e 14 shows r e c t a l t e m p e r a t u r e s f o r t h e f i r s t t h r e e weeks o f age. higher three  MR-Nolg c a l v e s had s i g n i f i c a n t l y  (P<0.05) r e c t a l than  Col/WM  temperatures  calves,  and  i n weeks one and  significantly  higher  (P<0.05) t h a n MR-Lo I n j c a l v e s i n week t h r e e .  F i g u r e 14. E f f e c t of Ig S u p p l e m e n t a t i o n on R e c t a l T e m p e r a t u r e for the F i r s t T h r e e W e e k s of Life  Treatments —  Col/WM  —I—  MR-Nolg MR-Lo Inj  14  DAYS OF AGE  21  Diarrhea Diarrhea  levels  ( F i g u r e 15) f o r c a l v e s on MR-Nolg  t r e n d e d h i g h e r t h a n e i t h e r Col/WM o r MR-Lo I n j t h r o u g h o u t the experiment. diarrhea  than  MR-Nolg c a l v e s had s i g n i f i c a n t l y more MR-Lo  I n j calves  during  weeks  three  (P<0.05) and f o u r (P<0.01), and s i g n i f i c a n t l y more t h a n Col/WM d u r i n g week f o u r  F i g u r e 15. 3  T h e E f f e c t of Ig S u p p l e m e n t a t i o n o n D i a r r h e a L e v e l s (bacterial s c o u r s )  a>  co o  (P<0.01).  -i  CO  B a s e d on Nocek  a> o 2.5 O  Z  c o  3 - runny, mostly fluid 4 - watery, all fluid  2 -  5 - watery, with blood  TJ  0) CO CO  _Q  scale:  1 T normal . 2 - soft, mostly solid  1.5  I  co m  1  co  0.5 -  Treatments Col/WM  CD  —I—  MR-No Ig  — M R - L o Inj  CO  Q  1  2 3 4 5 PERIOD OF TREATMENT - WEEK  Calf  Weights F i g u r e 16 shows t h e e f f e c t o f t r e a t m e n t s on w e i g h t  gain.  There were no s i g n i f i c a n t d i f f e r e n c e s i n w e i g h t  between any o f t h e t r e a t m e n t s a t b i r t h , 24 hours o f age," and a t days 7, 14, 21 and 28. A t day 35, Col/WM and MRN o l g were a p p r o a c h i n g s i g n i f i c a n c e (P<0.05) and a t day 42, w e i g h t o f Col/WM c a l v e s was s i g n i f i c a n t l y h i g h e r t h a n weight  o f MR-Nolg  calves  (P<0.05).  There  were  no  s i g n i f i c a n t d i f f e r e n c e s (P>0.05) between Col/WM and MR-Lo I n j c a l f w e i g h t s a t b o t h days 35 and 42.  F i g u r e 16. „  ^  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on Calf Weight  DAYS OF AGE  Average D a i l y G a i n s F i g u r e 17 shows average d a i l y g a i n s three treatment  periods.  (ADG) f o r t h e  ADG o f c a l v e s on MR-Nolg were  s i g n i f i c a n t l y lower t h a n ADG o f c a l v e s on t h e o t h e r two treatments  f o r the treatment  p e r i o d s from z e r o t o t h r e e  weeks o f age and from z e r o t o s i x weeks o f age (P<0.01). From  three  t o s i x weeks  o f age, Col/WM  calves  had  s i g n i f i c a n t l y h i g h e r ADG t h a n c a l v e s on MR-Nolg (P<0.05) , however t h e r e was no s i g n i f i c a n t d i f f e r e n c e between MRN o l g and MR-Lo I n j c a l v e s .  ADG were n o t s i g n i f i c a n t l y  d i f f e r e n t between c a l v e s on Col/WM and MR-Lo I n j f o r any of t h e t r e a t m e n t  Figure 17.  periods.  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on Average Daily Gain  1 -i  0-3 weeks  0-6 weeks 3-6 weeks PERIOD OF TRIAL  Figure  18  supplementation  shows on  feed  effect efficiency  of  immunoglobulin  (kgs d r y  feed/kgs  g a i n ) o f c a l v e s f o r t h e f i r s t s i x weeks o f l i f e .  MR-Nolg  c a l v e s had s i g n i f i c a n t l y p o o r e r (P<0.05) f e e d e f f i c i e n c y t h a n t h e c a l v e s i n t h e o t h e r two t r e a t m e n t s .  Figure 18. 3.00  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on F e e d E f f i c i e n c y for the First S i x W e e k s of  Life  2.50 ro A  | 2.00 ro E  •o  1,50  1.00 Col/WM  M R - L o Inj  MR-Nolg  66  Serum IgG L e v e l s Figure  19 shows serum IgG l e v e l s .  There was no  s i g n i f i c a n t d i f f e r e n c e i n serum I g l e v e l s between any o f the  t r e a t m e n t s a t b i r t h , and days 21, 28, 35 and 42. A t  24 h o u r s and 48 h o u r s o f age, and a t days 7 and 14, c a l v e s on Col/WM had s i g n i f i c a n t l y h i g h e r serum t h a n c a l v e s on MR-Nolg (P<0.05).  levels  (P<0.01) and c a l v e s on MR-Lo I n j  From day 28 t o t h e end o f t h e e x p e r i m e n t a l  p e r i o d , serum I g l e v e l o f c a l v e s on MR-Nolg c o n t i n u e d t o r i s e and t e n d e d t o be h i g h e r t h a n b o t h Col/WM and MR-Lo Inj .  67 Blood Glucose Levels B l o o d g l u c o s e l e v e l s a r e shown i n F i g u r e 20. glucose  levels  f o r Col/WM  calves  were  Blood  significantly  higher  (P<0.05) t h a n MR-Nolg and MR-Lo  Inj calves at  birth.  A t day 7, Col/WM c a l v e s were h i g h e r t h a n MR-Nolg  and MR-Lo I n j c a l v e s b u t d i d n o t d i f f e r Blood  glucose  levels  significantly.  between t r e a t m e n t s  t i m e s were n o t s i g n i f i c a n t l y d i f f e r e n t  at a l l other  from each o t h e r  (P>0.05).  Figure 2 0 .  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on Blood Glucose  180 -i  Levels  160 -  Treatments  o o  CD  —  MR-Nolg  60 ^-  40  0  Col/WM  7  14i 21 DAYS OF  28 : AGE  35  42  M R - L o Inj  68 B l o o d Urea N i t r o g e n  Levels  F i g u r e 21 shows b l o o d urea n i t r o g e n  (BUN) l e v e l s .  BUN l e v e l s were n o t s i g n i f i c a n t l y d i f f e r e n t between any of the treatments  a t b i r t h , 2 4 h r s , day 7 and day 35.  Col/WM c a l v e s had s i g n i f i c a n t l y h i g h e r BUN l e v e l s  than  MR-Nolg c a l v e s a t 48 h r s and days 14 (P<0.05), 28 and 42 (P<0.01), and s i g n i f i c a n t l y h i g h e r t h a n MR-Lo I n j c a l v e s a t 48 h r s and day 14 (P<0.01) . MR-Nolg c a l v e s BUN l e v e l s were s i g n i f i c a n t l y lower t h a n MR-Lo I n j c a l v e s a t day 42 (P<0.05).  21.  T h e E f f e c t of Ig S u p p l e m e n t a t i o n on Blood Urea Nitrogen Levels  -o E 0) > C OI  o Treatments  CO Q)  Col/WM MR-Nolg  •o O O  — M R - L o Inj  m 14  21  DAYS OF AGE  28  35  42  69 DISCUSSION Survival  during  the  first  few  days  of  life  is  r e l a t e d t o t h e amount o f p a s s i v e I g o b t a i n e d by t h e c a l f . The  importance of t h i s i s shown by t h e s u r v i v a l r a t e s i n  the  second experiment.  S u r v i v a l o f MR-Nolg  (control)  c a l v e s were s i g n i f i c a n t l y l o w e r t h a n t h e Col/WM o r MR-Lo I n j c a l v e s (P<0.05) and had r e l a t i v e l y low s u r v i v a l (4/8) compared t o t h e MR-Nolg c a l v e s i n t h e f i r s t (7/9) .  This  calves  be  p a r t l y due  i n experiment 2 d i d not  therapy died  may  for scouring.  succumbed  dehydration.  to  One  t o the  experiment  fact that  r e c e i v e any  the  antibiotic  Most of t h e MR-Nolg c a l v e s E.  coli  septicemia  with  c a l f each from Col/WM and  treatments a l s o died.  The  that  severe  MR-Lo I n j  Col/WM c a l f d i e d w i t h i n t h e  f i r s t 24 hours o f age due t o a r o t a v i r u s i n f e c t i o n w h i c h was  i s o l a t e d a t post-mortem.  alleviated that  the  feeding  colostrum  specific obtained  even by  for  this  from one  been p r e s e n t developed  a  rotavirus.  did  T h i s d i d not  colostrum.  not  have a  rotavirus.  As  seem t o  I t i s possible high  the  level  level  farm, and of  specific  was  have  to  this  a good l e v e l  of  a n t i b o d i e s s p e c i f i c t o t h i s v i r u s i n the colostrum,  it  may  However, even i f t h e r e was  Ig  not have  t h e cows would not  antibodies  of  colostrum  farm o n l y , t h e r o t a v i r u s may  on t h e  be  not have been enough as t h e c a l f d i e d t o o soon t o be  a b l e t o have absorbed enough I g . t h e o n l y way  Research i n d i c a t e s t h a t  t o f i g h t r o t a v i r u s e s may  be t o immunize a t  b i r t h (Dr. P. O ' B r i e n , Comox V e t e r i n a r y C l i n i c ,  personal  70 communication).  The MR-Lo I n j c a l f  b l o a t which was n o t r e l a t e d Diarrhea l e v e l s  died  o f apparent  t o the treatments.  (scours) o f MR-Nolg c a l v e s  trended  h i g h e r t h a n e i t h e r Col/WM o r MR-Lo I n j c a l v e s t h r o u g h o u t t h e s i x week p e r i o d o f t h e experiment. during  week  four,  MR-Nolg  By week t h r e e and  calves  were  scouring  s i g n i f i c a n t l y more t h a n MR-Lo I n j c a l v e s (P<0.05).  For  t h e f i r s t t h r e e weeks o f t h e e x p e r i m e n t , Col/WM c a l v e s scoured  more  than  significantly.  the  MR-Lo  Inj  calves  but  not  C o n t i n u e d f e e d i n g o f an I g - f o r t i f i e d m i l k  r e p l a c e r reduced s c o u r s  levels  over t h e c r i t i c a l  first  t h r e e weeks. R e c t a l t e m p e r a t u r e s were a l s o s i g n i f i c a n t l y h i g h e r (P<0.05) i n t h e MR-Nolg c a l v e s d u r i n g week one and three.  As  in  experiment  t e m p e r a t u r e s were i n d i c a t i v e were  above  3 9°C  i n the  1,  the  higher  of b a c t e r i a l calves  which  rectal  infection scoured  and more  severely. Weekly  weights  were  not s i g n i f i c a n t l y  between any o f t h e t h r e e t r e a t m e n t s MR-Nolg c a l v e s were s i g n i f i c a n t l y either for  Col/WM o r MR-Lo I n j c a l v e s .  MR-Nolg  and  they  higher  (P<0.05)  Average d a i l y lower  f o r t h e whole experiment  than gains  than the (P<0.01)  t h e MR-Nolg c a l v e s had t h e p o o r e s t  f e e d e f f i c i e n c i e s as w e l l . first  lower  f a i l e d t o g a i n any w e i g h t i n t h e second week.  As would be expected,  the  u n t i l day 35, when  c a l v e s were s i g n i f i c a n t l y  o t h e r two t r e a t m e n t s  different  I n agreement w i t h r e s u l t s i n  e x p e r i m e n t , experiment 2 MR-Nolg c a l v e s had  rectal  temperatures, gained  p o o r l y and d i d n o t  c a t c h up t o t h e Col/WM o r MR-Lo I n j of  c a l v e s by s i x weeks  age. Serum I g l e v e l s were i n d i c a t i v e of t h e  g i v e n and  s i m i l a r to r e s u l t s obtained  treatments  i n experiment  1.  Col/WM c a l v e s had s i g n i f i c a n t l y h i g h e r l e v e l s o f I g a t 24 and 48 hours and a t day 7 (P<0.01) , t h a n b o t h MR-Nolg and MR-Lo I n j c a l v e s .  A g a i n , t h e p o s s i b i l i t y of f a c t o r s  t h e c o l o s t r u m which c o u l d enhance t h e I g a b s o r p t i o n be t h e r e a s o n ( B a l f o u r and Comline, 1962;  in may  Hardy, 1969).  Serum I g l e v e l s of t h e MR-Nolg c a l v e s show development of t h e a c t i v e immune system from about day 14 onward. was  a good l e v e l o f a b s o r p t i o n  from t h e  injectable  s o l u t i o n w i t h an a b s o r p t i o n e f f i c i e n c y of 42%.  Serum I g l e v e l s  r i s e as h i g h as expected. t h e r e was  mg/ml) but d i d  The r e a s o n may  IgG i s t h e s m a l l e s t immunoglobulin m o l e c u l e  spaces as w e l l  since  endothelial tissues.  i t can  through  T i s s u e a b s o r p t i o n was not a c c o u n t e d This f a c t should  account when c a l c u l a t i n g  amount o f I g s o l u t i o n t o be i n j e c t e d . to  in tissue  pass more e a s i l y  f o r p r i o r t o s t a r t of experiment. into  not  have been t h a t  i s found not o n l y i n t h e b l o o d s t r e a m but  taken  higher  some t i s s u e a b s o r p t i o n of I g a t t h e s i t e s o f  injection. and  (6.3 vs 3.0  Ig  approximately  i n MR-Lo I n j c a l v e s r o s e  t h a n t h e MR-Nolg c a l v e s  There  i n c r e a s e serum I g l e v e l s would be  concentration Another  be and  solution  t o g i v e a second  group o f i n j e c t i o n s s i x - t w e l v e hours a f t e r t h e f i r s t t o boost  serum  levels.  Further  work  is  required  to  d e t e r m i n e o p t i m a l amounts and c o n c e n t r a t i o n s t o be used,  however, t h e s e  results  i n d i c a t e t h a t an i n j e c t a b l e I g  s o l u t i o n given subcutaneously  i s an e f f e c t i v e method o f  p r o v i d i n g systemic p a s s i v e I g i n the bloodstream. B l o o d g l u c o s e and b l o o d u r e a n i t r o g e n l e v e l s were i n t h e normal range f o r newborn c a l v e s up t o s i x weeks o f age.  There were s i g n i f i c a n t d i f f e r e n c e s a t some t i m e s  but t h e s e were p r o b a b l y due t o d i u r n a l v a r i a t i o n s r e l a t e d t o f e e d i n g r a t h e r t h a n due t o t r e a t m e n t s .  Blood glucose  l e v e l s showed a s l i g h t upward t r e n d and BUN l e v e l s showed a s h a l l o w downward t r e n d , however, f u r t h e r r e s e a r c h i s needed  to  determine  i f these  were  true  treatment  differences. In  conclusion,  these  results  indicate-  that  a d m i n i s t r a t i o n o f an i n j e c t a b l e s o l u t i o n o f I g can be used  as  an  alternative  gammaglobulinemic independent mortality  calves  method  to  give  level  of  passive  a  o f i n t e s t i n a l a b s o r p t i o n which w i l l due  to septicemias.  Continued  hypoIg  reduce  feeding of  I g - f o r t i f i e d m i l k r e p l a c e r reduced s e v e r i t y o f s c o u r s and s u p p o r t s r e s u l t s o b t a i n e d i n experiment  1.  73  CONCLUSIONS An  immunoglobulin-fortified  injectable  form  alternatives  to  survival  2)  less  severe  3)  improved weight  4)  adequate  Continued  and  immunoglobulins  c o l o s t r u m and r e s u l t  1) i n c r e a s e d  milk  of  replacer  are  and  an  effective  in  rate  gain  serum IgG of  levels an  an e f f e c t i v e  improving weight  replacer  scours  feeding is  milk  gain.  immunoglobulin-fortified method o f  reducing  scours  74  REFERENCES Anderson, K.L., Hunt, E. & F l e m i n g , S.A. 1987. Plasma transfusions in failure of colostral immunoglobulin transfer (1) . The Bovine P r a c t i t i o n e r - November 22:129. B a l f o u r , W.E. and Comline, R.S. 1962. 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