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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 Margaret L. Crowley B. Sc. (Agr.), The U n i v e r s i t y of 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 i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1990 ® Margaret L. Crowley In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying 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) A B S T R A C T 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 subcutaneous (SC) i n j e c t a b l e s o l u t i o n of immunoglobulins (Ig) were examined as methods of ac h i e v i n g passive immunity i n neonatal c a l v e s . Bovine I g , from a b a t t o i r blood, were p u r i f i e d by polyphosphate f r a c t i o n a t i o n and ion-exchange chromatography. In experiment 1, c a r r i e d out at 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 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 a l l o t t e d t o one of four treatments. Col/WM c a l v e s were fed colostrum on day 1 and whole m i l k , days 2 - 42. MR-Nolg c a l v e s ( c o n t r o l ) were fed m i l k r e p l a c e r w i t h no Ig, days 1 - 4 2 . MR-Hi/Lo cal v e s were fed m i l k r e p l a c e r w i t h Ig at 50 mg/ml on day 1, and at 10 mg/ml, days 2 -21. MR-Hi/No cal v e s were fed m i l k r e p l a c e r w i t h I g at 50 mg/ml, day 1,'and w i t h no Ig , days 2 - 21. From days 2 2 - 42, MR-Hi/Lo and MR-Hi/No treatment c a l v e s r e c e i v e d m i l k r e p l a c e r w i t h no Ig. In experiment 2, c a r r i e d out at the U n i v e r s i t y Research Farm at Oyster R i v e r , 24 colostrum-deprived 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 a l l o t t e d t o one of three treatments. The f i r s t two treatments were the same as f o r experiment 1, Col/WM and MR-Nolg fed f o r days 1 - 2 1 . MR-Lo In j ca l v e s were fed mi l k r e p l a c e r w i t h Ig at 10 mg/ml, days 1 - 2 1 , and were a l s o given a SC i n j e c t i o n of Ig s o l u t i o n w i t h i n the f i r s t 6 hours of l i f e . For days 2 - 4 2 , cal v e s were fed WM or i i i MR-Nolg, as per experiment 1. For both experiments, blood samples and c a l f weights were taken at b i r t h , 24 & 48 hours of age, day 7 and weekly t h e r e a f t e r f o r s i x weeks. Diarrhea (scours) l e v e l s , r e c t a l temperatures and general h e a l t h of ca l v e s were recorded d a i l y f o r the f i r s t three weeks as w e l l . Experiment 1 s u r v i v a l at 6 weeks of age was 11 out of 11 ca l v e s f o r Col/WM treatment, 8 out of 8 calves f o r MR-Hi/Lo treatment, 7 out of 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 out of 9 ca l v e s f o r MR-Nolg. In experiment 2, s u r v i v a l was 7 out of 8 ca l v e s f o r both Col/WM and MR-Lo-In j treatments and a s i g n i f i c a n t l y lower (P>0.05) 4 out of 8 c a l v e s f o r MR-Nolg treatment. Calves on MR-Hi/No had s i g n i f i c a n t l y higher d i a r r h e a l e v e l s than the other three treatments over weeks one and four i n experiment 1. In experiment 2, calves which d i d not r e c e i v e any Ig had s i g n i f i c a n t l y higher d i a r r h e a l e v e l s over weeks three and four than c a l v e s which received Ig. Experiment 1 average d a i l y gains (ADG) were s i g n i f i c a n t l y higher f o r c a l v e s on Col/WM, MR-Hi/Lo and MR-Hi/No treatments than f o r c a l v e s on MR-Nolg at s i x weeks of age. In experiment 2, s i x week ADG were s i g n i f i c a n t l y higher f o r c a l v e s on Col/WM and MR-Lo I n j treatments than f o r MR-Nolg. For both experiments, serum Ig l e v e l s of calves on Col/WM were s i g n i f i c a n t l y higher than calves on the other treatments at 24 and 48 hours of age. MR-Hi/Lo, MR-Hi/No and MR-Lo Inj c a l v e s trended t o higher serum Ig l e v e l s than MR-Nolg ca l v e s but were not s i g n i f i c a n t l y d i f f e r e n t . Calves i v which r e c e i v e d I g , from colostrum, the I g - f o r t i f i e d m i l k r e p l a c e r or a subcutaneous Ig i n j e c t i o n , had higher s u r v i v a l r a t e s , lower d i a r r h e a l e v e l s , l e s s a n t i b i o t i c treatment and higher average d a i l y gains than c a l v e s hot r e c e i v i n g any Ig. I t was concluded t h a t immunoglobulins, administered e i t h e r o r a l l y or p a r e n t e r a l l y , are an e f f e c t i v e , a l t e r n a t i v e method, f o r p r o v i d i n g passive immunity i n neonatal c a l v e s . V TABLE OF CONTENTS Page Abstract i i L i s t of Tables v i L i s t of Figures v i i Acknowledgements i x Introduction • . 1 L i t e r a t u r e Review The Immune System 4 Stem C e l l D i f f e r e n t i a t i o n 4 Primary and Secondary Responses 7 The Major H i s t o c o m p a t i b i l i t y Complex 9 The Complement System 10 Phagocytosis 11 Immunoglobulins 12 Immunoglobulin G 14 Immunoglobulin M 16 Immunoglobulin A 17 The Immune Response 18 Development of Immunocompetence i n the F e t a l C a l f 18 Absorption of Immunoglobulins 2 0 Mechanism of Absorption 25 Experimental Section Experiment One I n t r o d u c t i o n 27 M a t e r i a l s and Methods 28 Res u l t s 3 5 Dis c u s s i o n 46 Experiment Two I n t r o d u c t i o n 51 M a t e r i a l s and Methods 51 Res u l t s 59 Disc u s s i o n 69 Conclusions 7 3 References 74 v i LIST OF TABLES Page Table 1. Ontogenetic development of bovine immune system during g e s t a t i o n 20 Table 2. Concentration of IgG, IgM and IgA i n bovine colostrum and m i l k ...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 of immunoglobulins to c a l v e s - (experiment 1) 29 Table 4. Concentration of freeze d r i e d bovine IgG 3 6 Table 5. The e f f e c t of Ig supplementation on s u r v i v a l of calves - (experiment 1) 38 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 of immunoglobulins to 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 , colostrum and m i l k 60 Table 8. The e f f e c t of Ig supplementation on s u r v i v a l of calves - (experiment 2) 60 v i i Figure 1. Hemopoietic stem c e l l d i f f e r e n t i a t i o n ... 5 LIST OF FIGURES Page Figure 2. Immune responses t o an antigen over time as r e f l e c t e d by serum antibody l e v e l s 8 Figure 3a. B a s i c s t r u c t u r e of IgG molecule showing heavy and l i g h t chains 15 Figure 3b. S t r u c t u r e of IgG.,, IgG 2, IgA and IgM molecule c l a s s e s 15 Figure 3c. Papain treatment of Ig molecule breaking i n t o F a b and F c fragments .... 15 Figure 4. Absorption mechanism by p i n o c y t o s i s 26 'Figure 5. Gel 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 bovine serum 37 Figure 6. The e f f e c t of Ig supplementation on r e c t a l temperatures f o r the f i r s t s i x weeks of l i f e 38 Figure 7. The e f f e c t of Ig supplementation on di a r r h e a l e v e l s -(experiment 1) 39 Figure 8. The e f f e c t of Ig supplementation on c a l f weights -(experiment 1) 40 Figure 9. The e f f e c t of Ig supplementation on average d a i l y gain - • .' . (experiment 1) 41 Figure 10. The e f f e c t of Ig supplementation on feed e f f i c i e n c y -(experiment 1) 42 v i i i Figure 11. The e f f e c t of Ig supplementation on serum IgG l e v e l s -(experiment 1) 43 Figure 12. The e f f e c t of Ig supplementation on blood glucose l e v e l s -(experiment 1) 44 Figure 13. The e f f e c t of Ig supplementation on blood urea n i t r o g e n l e v e l s -(experiment 1) . ....45 Figure 14. The e f f e c t of Ig supplementation on r e c t a l temperatures for, the f i r s t s i x weeks of l i f e -(experiment 2) 61 Figure 15. The e f f e c t of Ig supplementation on di a r r h e a l e v e l s -(experiment 2) 62 Figure 16. The e f f e c t of Ig supplementation on c a l f weights -(experiment 2) 63 Figure 17. The e f f e c t of Ig supplementation on average d a i l y gain -(experiment 2) 64 Figure 18. The e f f e c t of Ig supplementation on feed e f f i c i e n c y -(experiment 2) 65 Figure 19. The e f f e c t of Ig supplementation on serum IgG l e v e l s -(experiment 2) 66 Figure 20. The e f f e c t of Ig supplementation on blood glucose l e v e l s -(experiment 2) 67 Figure 21. The e f f e c t of Ig supplementation on blood urea n i t r o g e n l e v e l s -(experiment 2) 68 i x ACKNOWLEDGEMENTS I wish to thank Dr. Bruce Owen f o r h i s expert guidance; h i s steady encouragement and support were most appreciated and h e l p f u l through the three years of my research. The members of my committee, Dr. Lome F i s h e r , Dr. Jim S h e l f o r d , Dr. C.R. Krishnamurti and Dr. B. J . Skura were a l s o very h e l p f u l . There are many people who c o n t r i b u t e d t o the completion of t h i s t h e s i s i n c l u d i n g those at the A g r i c u l t u r e Canada Agassiz Research S t a t i o n , i n p a r t i c u l a r , Dr. Lome F i s h e r , S u s i Soth and Ruby Schmidt, as w e l l as Susan Hainstock and the d a i r y workers. As w e l l , my thanks goes t o the people at U n i v e r s i t y of B r i t i s h Columbia's Oyster R i v e r Research Farm. A l s o , I appreciated the e x c e l l e n t t e c h n i c a l help and advice from Irene Bevandick, Murray Drew, Frances Newsome and G i l l e s Galzy. F i n a n c i a l a s s i s t a n c e was provided by the P r o d u c t i v i t y Enhancement Program of the Canada/British 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 w e l l . F i n a l l y , I am indebted t o my f a m i l y f o r t h e i r l o y a l support, i n a l l ways, without whom I would not have s u c c e s s f u l l y achieved t h i s g o a l . INTRODUCTION The importance of colostrum as the source of immunoglobulin p r o t e c t i o n f o r the newborn c a l f has been w e l l e s t a b l i s h e d (Corley et a l . , 1977; Kruse, 197 0; McEwan et a l . , 1970; Smith & L i t t l e , 1922). C o l o s t r a l immunoglobulins ( I g ) , absorbed from the small i n t e s t i n e i n t o the blood c i r c u l a t i o n , give passive immunity and p r o t e c t the c a l f against septicemias (Boyd, 1972; Burton, 1986) . However, the amount of Ig absorbed v a r i e s widely and ten t o t h i r t y percent of calves may have l i t t l e or no Ig i n t h e i r serum, and thus no passive, systemic immunity. One c r u c i a l f a c t o r i n absorption of c o l o s t r a l Ig i n the c a l f i s the pe 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 of time t h a t a c a l f i s able t o absorb Ig from the i n t e s t i n e has been estimated t o be between 24 and 48 hours of age. Closure, which i s the t e r m i n a t i o n of absorption of macromolecules from the i n t e s t i n e t o the bloodstream i n neonates, has been shown t o occur spontaneously, at 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 post-partum ( S t o t t et a l . , 1979). Ig remaining i n the small i n t e s t i n e ( i . e not absorbed) provide a l o c a l p r o t e c t i v e e f f e c t , termed copro-immunity, against b a c t e r i a causing d i a r r h e a (scours) or c o l i b a c i l l o s i s and other e n t e r i c pathogens. However, these p r o t e c t i v e e f f e c t s appear t o be independent of one another as high 2 serum immunoglobulin l e v e l s do not prevent d i a r r h e a nor do high i n t e s t i n a l l e v e l s prevent death from c o l i s e p t i c e m i a (Logan Se Pearson, 1978) . Passive immunity i n the neonatal c a l f i s dependent upon c o l o s t r a l immunoglobulin t r a n s f e r v i a i n t e s t i n a l a b s orption f o r two main reasons: 1) due t o the syndesmochorial p l a c e n t a t i o n i n ruminants, i n which there are f i v e t i s s u e l a y e r s between maternal and f e t a l blood c i r c u l a t i o n , there i s no p l a c e n t a l t r a n s f e r of macromolecules, such as Ig, during g e s t a t i o n ; and 2) although the a c t i v e immune system of a c a l f i s f u n c t i o n a l at b i r t h , i t i s not f u l l y mature f o r the f i r s t few weeks of l i f e . A c a l f i s thus born w i t h v i r t u a l l y no passive p r o t e c t i o n and i s dependent upon the cow's colostrum f o r i t s s u r v i v a l . Previous s t u d i e s showed th a t adequate q u a n t i t i e s of colostrum fed p r i o r to 24 hours of age are necessary f o r a c a l f ' s s u r v i v a l (Michanek et a l . . 1989a & 1989b; P a t t , 1977) . Colostrum s u b s t i t u t e s , such as a powdered immunoglobulin product separated from colostrum or whey, were a l s o evaluated. Success i n a c h i e v i n g an adequate l e v e l of Ig i n the c a l f ' s serum has been v a r i a b l e (Haines et a l . . 1990). A c a l f switched t o m i l k r e p l a c e r w i t h i n a few days a f t e r b i r t h may develop d i a r r h e a or scours, and a s s o c i a t e d problems, due t o l a c k of copro-immunity. Continued feeding of colostrum has been shown to reduce incidence of scours (Logan & Pearson, 1978). A m i l k 3 r e p l a c e r f o r t i f i e d w i t h Ig which could g i v e t h i s needed immunity may reduce the incidence of e n t e r i c diseases and the a s s o c i a t e d morbidity. Neonatal c a l v e s s o l d t o be r a i s e d f o r dairy-beef or v e a l may be deprived of colostrum which would increase the l o s s e s due t o systemic diseases. Some cal v e s may not r e c e i v e enough Ig v i a absorption even though fed colostrum. A p u r i f i e d immunoglobulin s o l u t i o n , given as a subcutaneous i n j e c t i o n which would be absorbed i n t o the bloodstream, would provide these hypo-gammaglobulinemic c a l v e s the necessary immunity, and would presumably reduce the incidence of septicemic disease and the a s s o c i a t e d m o r t a l i t y . An i n t r a - p e r i t o n e a l or intravenous route u s i n g bovine plasma was t e s t e d and shown t o be an e f f e c t i v e method of i n c r e a s i n g serum immunoglobulin l e v e l s (Anderson et a l . . 1987). This t h e s i s , then, examines the development and t e s t i n g of 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 p u r i f i e d , i n j e c t a b l e immunoglobulin s o l u t i o n , using bovine blood from s e v e r a l a b a t t o i r s as a source of I g , t o improve passive immunity i n neonatal c a l v e s . 4 LITERATURE REVIEW THE IMMUNE SYSTEM To gain a c l e a r understanding of the experimental r e s u l t s reported i n t h i s t h e s i s , a general overview of the 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, there are both n o n - s p e c i f i c defense 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 defense f a c t o r s i n c l u d e surface 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 other 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 responses can be c h a r a c t e r i z e d and subdivided i n t o two major components, a thymic-derived ( T - c e l l ) system and a bursa-derived ( B - c e l l ) system. These two components are d i s t i n g u i s h e d from each other i n t h a t the thymic-derived system provides c e l l - m e d i a t e d immunity wh i l e the bursa-d e r i v e d system provides humoral immunity. Stem C e l l D i f f e r e n t i a t i o n Both the cell - m e d i a t e d and humoral immune systems c o n s i s t of 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 are 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 the bone marrow. These stem c e l l s are very important i n t h a t they are self-renewing and are capable of d i f f e r e n t i a t i n g i n t o a l l 5 the c e l l groups of the body which are d e r i v e d from the 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 two •main li n e a g e s of c e l l s (Figure 1) . The myeloid l i n e i n c l u d e s e r y t h r o c y t e s , p l a t e l e t s and granulocyte p r e c u r s o r s . The granulocyte precursor f u r t h e r d i f f e r e n t i a t e s i n t o n e u t r o p h i l s , monocytes (which are immature macrophages) and macrophages. The lymphoid l i n e i n c l u d e s the T-lymphocytes ( T - c e l l s ) and the B-lymphocytes ( B - c e l l s ) . The granulocytes, T - c e l l s and B— c e l l s are i n v o l v e d i n c e l l - m e d i a t e d immunity. Immunoglobulins ( a n t i b o d i e s ) , which are produced by B-Figure 1. Hematopoietic stem c e l l d i f f e r e n t i a t i o n (Cooper et a l , 1984). P l u r i p o t e n t Stem C e l l Myeloid Lineage / Myeloid Stem C e l l \ Lymphoid Lineage Lymphoid Stem C e l l N e u t r o p h i l s Macrophages c e l l s , and the complement system f a c t o r s are i n v o l v e d i n humoral immunity. The lymphoid li n e a g e of the 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 of two populations of lymphocytes, the B - c e l l s and the T - c e l l s . Both of these c e l l l i n e s come from the same group of c e l l s and have receptors f o r s p e c i f i c antigens on t h e i r surfaces. The f u n c t i o n s of these two l i n e s are, however, completely d i f f e r e n t . T - c e l l s , which r e g u l a t e the f u n c t i o n i n g of the immune system, have three c e l l types each, w i t h one major f u n c t i o n . C y t o t o x i c T - c e l l s 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 or f o r e i g n c e l l s such as cancerous c e l l s or v i r u s - i n f e c t e d c e l l s . Helper T-c e l l s promote immune responses, and suppressor T - c e l l s suppress immune responses. 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 c e l l s . Plasma c e l l s produce and secrete immunoglobulins or s o l u b l e a n t i b o d i e s which can bind to an antigen, i . e . , any f o r e i g n substance t h a t s t i m u l a t e s antibody production. Memory c e l l s are produced when an antigen i s encountered the f i r s t time but they do not go the f i n a l step i n producing and s e c r e t i n g a n t i b o d i e s . When an antigen i s encountered f o r a second time, they 'remember' the antigen, t h a t i s , they are s p e c i f i c f o r i t and can produce a n t i b o d i e s to the antigen much more q u i c k l y because they have already gone through the primary d i f f e r e n t i a t i o n steps. 7 Precursor T- and B - c e l l s are produced i n the bone marrow and are relea s e d i n t o the bloodstream. Precursor T - c e l l s migrate t o the thymus where they are processed to an u n s p e c i a l i z e d T - c e l l which can then d i f f e r e n t i a t e i n t o helper T-, suppressor T- or c y t o t o x i c T - c e l l s . B - c e l l s were named f o r the f a c t t h a t they mature i n the bursa of Fabricus 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 ) . As there i s no bursa i n mammals, precursor B - c e l l s are thought t o mature.in bone marrow, gut-ass o c i a t e d lymphoid t i s s u e (GALT), l i v e r and/or spleen. B - c e l l s , upon a c t i v a t i o n , d i f f e r e n t i a t e t o plasma c e l l s which sy n t h e s i z e and secrete immunoglobulins or a n t i b o d i e s . I n d i v i d u a l B - c e l l s have a r e c e p t o r - s p e c i f i c immunoglobulin c l a s s on t h e i r c e l l surfaces t h a t c o r r e l a t e s w i t h the antibody c l a s s produced. B - c e l l s a l s o are c r i t i c a l f o r the d e t o x i f i c a t i o n of p r o t e i n s , polysaccharides and t o x i n s . Primary and Secondary Responses When an antigen i s encountered i n an animal, immunoglobulins or 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 , t h a t antigen. A n t i b o d i e s u s u a l l y bind only t o the antigen t h a t s t i m u l a t e d t h e i r production. This i s termed antigen s p e c i f i c i t y . The amount of a n t i b o d i e s produced can be measured i n serum and i s used as a measure of the immune response (Figure 2 ) . Following the f i r s t encounter w i t h an antigen there i s a l a g p e r i o d of approximately one week before a n t i b o d i e s are detectable i n serum". Their l e v e l s 8 Amount of antibody in serum (titer) Primary immune response Secondary immune response Antigen administered Second dose of antigen administered 14 Days F i g u r e 2. Immune responses t o an antigen over time as r e f l e c t e d by serum antibody l e v e l s ( T i z a r d , 1984). i n serum w i l l climb t o a peak i n 10 t o 14 days before d e c l i n i n g and disappearing i n a few weeks. The amount of antibody synthesized, and hence the amount of p r o t e c t i o n , during 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 . This i s termed a "primary" response. I f t h i s antigen i s encountered a second time the antibody response i s s i m i l a r t o a primary response, but the l a g p e r i o d i s only two to three days. The amount of antibody produced then r i s e s r a p i d l y t o a higher l e v e l before d e c l i n i n g s l o w l y and may be detected f o r many months a f t e r . This i s termed a "secondary" response. A t h i r d encounter 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 s t i l l higher antibody t i t e r i n serum, and a more prolonged response. g The Major H i s t o c o m p a t i b i l i t y Complex T - c e l l s and B - c e l l s need t o be able t o recognize t h e i r t a r g e t c e l l s (which may be macrophages, B - c e l l s , b a c t e r i a or v i r u s e s ) i n order t o i n t e r a c t w i t h them. These a c t i v i t i e s are regulated by p r o t e i n s found on the surfaces of these t a r g e t c e l l s . These p r o t e i n s are c a l l e d h i s t o c o m p a t i b i l i t y (MHC) antigens. C y t o t o x i c T - c e l l s may be needed t o destroy abnormal c e l l s i n any p a r t of the body. The p r o t e i n s found on the surface of a l l p o t e n t i a l t a r g e t c e l l s are c a l l e d c l a s s I h i s t o c o m p a t i b i l i t y antigens. C y t o t o x i c T - c e l l s , t h e r e f o r e , recognize t h e i r t a r g e t c e l l s by the c l a s s I MHC antigens on the c e l l s u rfaces, i n combination with the f o r e i g n antigens. The p r o t e i n s found on the surfaces of B - c e l l s and antigen-presenting c e i l s , such as macrophages, are c a l l e d c l a s s I I h i s t o c o m p a t i b i l i t y antigens. Helper T - c e l l s i n t e r a c t w i t h B - c e l l s and antigen-presenting c e l l s i n order to promote an immune response by r e c o g n i z i n g these c l a s s I I MHC antigens, i n combination wi t h the f o r e i g n antigen. Class I I MHC antigens are a l s o i n v o l v e d i n 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 . This i s an e s s e n t i a l p r o t e c t i v e mechanism th a t prevents an i n d i s c r i m i n a t e a t t a c k 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 antigens of both c l a s s e s are coded by genes l o c a t e d c l o s e together on one chromosome and form the gene complex known as the Major H i s t o c o m p a t i b i l i t y Complex. 10 The Complement System One of the many i n t e r a c t i n g systems i n the body i s the complement system, so termed because i t complements the antibody system. There are at l e a s t 15 components, a l l serum p r o t e i n s , which together make up about ten percent of the g l o b u l a r p r o t e i n f r a c t i o n of serum. Complement components are synthesized at v a r i o u s s i t e s i n the,body; f o r example, the C l components are synthesized 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 and C3, C6 and C9 i n the l i v e r ( T i z a r d , 1987) . Levels of C l , C2 and C4 i n serum are c o n t r o l l e d by Class I I I genes i n the Major H i s t o c o m p a t i b i l i t y Complex (MHC). A c t i v a t i o n of the complement system r e s u l t s i n the d i s r u p t i o n of c e l l membranes and i s regulated by mechanisms t h a t i n v o l v e a s e r i e s of i n t e r l i n k e d enzyme r e a c t i o n s . The complement system c o n s i s t s of three d i s t i n c t r e a c t i o n pathways. One of these, the c l a s s i c a l pathway, i s i n i t i a t e d by antigen-antibody r e a c t i o n s on c e l l membranes. The second, or a l t e r n a t i v e pathway, provides a route by which p o t e n t i a l invaders, such as b a c t e r i a , f u n g i , or p a r a s i t e s may a c t i v a t e the complement f a c t o r s i n the absence of antigen-antibody complexes. These f i r s t two pathways are inv o l v e d i n the production of the two C3 convertases (enzymes) through a s e r i e s of cascade-l i k e r e a c t i o n s . The t h i r d , or t e r m i n a l pathway, i s not a t r u e c a s c a d e - l i k e r e a c t i o n but i n v o l v e s combining the C3 convertase and the other complement components i n t o a l a r g e macromolecular complex. This complex binds t o c e l l s urfaces forming a s t r u c t u r e w i t h 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 defense during an i n f e c t i o n . They are .found i n l a r g e numbers i n the bloodstream 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 i n j u r y by v a r i o u s chemotactic s t i m u l i . 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 or antigens; i n g e s t i n g the p a r t i c l e , which i s then k i l l e d by o x i d a t i v e metabolism and digested. N e u t r o p h i l s have l i m i t e d energy and cannot maintain t h e i r a t t a c k f o r very long so macrophages are r e q u i r e d t o s u s t a i n the response. Macrophages a r i s e from bone marrow as promonocytes, enter and c i r c u l a t e i n the bloodstream as immature macrophages, termed monocytes, and migrate t o t i s s u e s where they mature to macrophages. Some become r e s i d e n t i n these t i s s u e s at s t r a t e g i c p o i n t s i n the body to remove f o r e i g n m a t e r i a l and microorganisms from the blood stream. The three 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) antigen p r e s e n t a t i o n t o lymphocytes, and c) s e c r e t i o n of b i o a c t i v e molecules important i n host defense. Macrophages migrate to i n f e c t i o n s i t e s i n a chemotactic response to b a c t e r i a l breakdown products, complement system f a c t o r s , and f a c t o r s r e l e a s e d from dying n e u t r o p h i l s , t o phagocytize b a c t e r i a or f o r e i g n p a r t i c l e s i n a s i m i l a r manner as n e u t r o p h i l s . As macrophages mature, they develop receptors ( p r o t e i n molecules) to immunoglobulins and one of the complement system fragments, C3, on t h e i r membranes which a i d i n t h e i r p h y s i o l o g i c a l f u n c t i o n s . These receptors act as opsonins to enhance the adherence of macrophages to b a c t e r i a by b i n d i n g f i r m l y to the b a c t e r i a l membranes. In a d d i t i o n t o being phagocytic, macrophages secrete f a c t o r s t h a t cause fever, inflammation and h e a l i n g of t i s s u e s . A wide v a r i e t y of products are secreted by macrophages i n c l u d i n g a n t i b a c t e r i a l p r o t e i n s 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 are a l s o i n v o l v e d i n antigen p r e s e n t a t i o n . Foreign m a t e r i a l such as b a c t e r i a are taken i n by macrophages, degraded by lysozymal enzymes and the b a c t e r i a l antigens are passed back to the surface membrane i n a s s o c i a t i o n w i t h Class I I MHC molecules (i n v o l v e d i n s e l f r e c o g n i t i o n ) . T - c e l l s which are s p e c i f i c f o r t h i s antigen-Class I I complex can bind to i t and begin the f i r s t step i n a c t i v a t i o n of h elper T - c e l l s . Immunoglobulins Humoral immune responses are mediated by immunoglobulins. The f u n c t i o n of immunoglobulins i s to 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 , v i r u s e s and p a r a s i t e s . Immunoglobulins serve as B - c e l l membrane receptors as w e l l as being secreted i n t o the bloodstream and other body f l u i d s to bind t o s o l u b l e antigens. I m m u n o g l o b u l i n s a r e p r o t e i n m o l e c u l e s (gl 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 i n t e r a c t i o n 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 of four p r o t e i n chains c o v a l e n t l y l i n k e d together. There are two l i g h t chains and two heavy chains i n each molecule. They are made up of blocks of approximately 110 amino aci d s c a l l e d domains (Hood et a l . , 1984) . The l i g h t chains c o n s i s t of 2 domains and the heavy chains c o n s i s t of four or f i v e domains depending upon the c l a s s of immunoglobulin (Figure 3-a). The domains can be subdivided i n t o v a r i a b l e and constant domains. The v a r i a b l e domains are the antigen-binding regions of the immunoglobulin molecule. The heavy chain constant domains determine the c l a s s of the molecule. There are three main c l a s s e s (termed isotypes) of immunoglobulins i n the bovine, designated alpha, gamma, and mu which corresponds 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 there 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 IgG molecule, as revealed by e l e c t r o n microscopy, looks l i k e a Y-shaped molecule and can be used as a model f o r the other i s o t y p e s , IgM and IgA (Figure 3-b) . The 'arms' of the Y, made up of one l i g h t c hain and one heavy chain j o i n e d by a d i s u l p h i d e bond, are the antigen-binding ( v a r i a b l e ) regions. The ' t a i l ' (constant) r e g i o n , made up of two heavy chains j o i n e d by two d i s u l p h i d e bonds, determines the isot y p e of the 14 immunoglobulin. The p r o t e o l y t i c enzyme, papain, can break the molecule i n t o approximately three e q u a l - s i z e d fragments, corresponding t o the two 'arms' and the ' t a i l ' (Figure 3-c). The two arm fragments are 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 ind antigen. They are c a l l e d the F g b fragments. The t h i r d fragment, the ' t a i l ' , cannot bind antigen but i s c r y s t a l l i z a b l e and i s t h e r e f o r e c a l l e d 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 are mediated by the constant region of the heavy chains (F c r e g i o n ) . These i n c l u d e a c t i v a t i o n of the complement cascade once the immunoglobulin has bound antigen, b i n d i n g of immune complexes t o phagocytic c e l l s (opsonization) and bi n d i n g to c e l l surfaces such as macrophages and i n t e s t i n a l e p i t h e l i u m during absorption. The s t r u c t u r a l d i f f e r e n c e s among the c l a s s e s of immunoglobulins correspond t o f u n c t i o n a l d i f f e r e n c e s i n t h e i r s i t e s of production and a c t i o n , t h e i r r e l a t i v e l e v e l s of production i n primary and secondary immune responses, and t h e i r p h y s i o l o g i c a l r o l e s . Immunoglobulin G IgG i s the immunoglobulin found i n highest c o n c e n t r a t i o n i n the blood, making up about 75-85 percent of the t o t a l serum immunoglobulins, and i n the colostrum produced by ruminants. IgG has a r e l a t i v e l y small s i z e , w i t h a molecular weight of 180,000 dalton s . I t i s found as a monomer i n both the membrane-bound and secreted form. IgG has two sub-classes or i s o t y p e s : IgG. and heavy chain light chain cfb G p igG2 Figure 3-b. S t r u c t u r e s of the three immunoglobulin c l a s s e s , IgG, IgM and IgA and the two sub-classes of IgG. (Hood et a l . , 1984). Figure 3-a. Schematic drawing of immunoglobulin molecule showing main s t r u c t u r a l f e a t u r e s . V and C are the v a r i a b l e and constant regions, r e s p e c t i v e l y , of the heavy (H) and l i g h t (L) chains. (Hood et al..1984) . IgM IgA dimer P a p a i n t r e a t m e n t P e p s i n t r e a t m e n t F a b F a b F (ab ) '2 Figure 3-c. Schematic drawing of enzyme treatment on immunoglobulin molecule, breaking i n t o F . and F„ ab c fragments. (Hood et a l . , 1984). IgG 2. Because of i t s small s i z e , i t can move i n and out of blood v e s s e l s more e a s i l y than the other immunoglobulin molecules and plays a major r o l e i n the defense of t i s s u e spaces and body surfaces. IgG i s the main immunoglobulin synthesized during a secondary response. IgG can opsonize, a g g l u t i n a t e and p r e c i p i t a t e antigen, but can a c t i v a t e the complement cascade only i f enough IgG molecules have accumulated on the antigen s u r f a c e . Immunoglobulin M IgM i s found i n second highest c o n c e n t r a t i o n i n the blood. IgM has a molecular weight of 900,000 daltons and i s made up of f i v e i d e n t i c a l sub-units or monomers, each 180,000 da l t o n s . Each monomer i s s t r u c t u r a l l y s i m i l a r to an IgG molecule. IgM molecules are bound t o B - c e l l membranes as a monomer, where they f u n c t i o n as receptors f o r antigen, or are secreted i n the blood stream as a pentamer, wi t h f i v e molecules of IgM arranged r a d i a l l y w i t h the antigen-binding s i t e s p o i n t i n g outwards. The IgM monomers are 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 polypeptide c a l l e d the J-chain j o i n i n g two of the monomers. Because of t h e i r l a r g e s i z e , IgM molecules are found mainly i n the .blood stream. IgM i s the immunoglobulin synthesized f i r s t during B - c e l l ontogeny. I t i s a l s o the main immunoglobulin synthesized and secreted during a primary immune response. A primary response occurs the f i r s t time a host comes i n contact w i t h an antigen. IgM molecules are more e f f i c i e n t than 17 IgG at 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 IgA monomer has a molecular weight of 160,000 daltons w i t h a s i m i l a r s t r u c t u r e t o IgG. I t u s u a l l y occurs as a monomer i n the membrane-bound form and as a dimer i n the secreted form. The two monomers of sec r e t o r y IgA are j o i n e d by the J-chain as f o r IgM. I t does not bind t o macrophages or enhance phagocytosis but 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 important mode of a c t i o n i s prevention of the adherence of 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 s u r f a c e s . A f t e r IgA i s synthesized by plasma c e l l s i n the i n t e s t i n a l mucosa i t binds t o a receptor, on the i n t e r i o r surface of e p i t h e l i a l c e l l s . This complex of IgA dimer and receptor i s then engulfed i n a v e s i c l e and tr a n s p o r t e d across the ep i t h e l i u m . When i t reaches the e x t e r n a l surface of the e p i t h e l i a l c e l l (lumen s i d e ) , the v e s i c l e fuses w i t h the membrane exposing the complex t o the lumen. The complex i s cleaved by a protease to re l e a s e the IgA dimer w i t h about 75 percent of the receptor s t i l l attached to i t . This receptor fragment i s termed the se c r e t o r y component and p r o t e c t s the 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 se c r e t o r y IgA (SIgA) . SIgA i s the main immunoglobulin found i n s e c r e t i o n s and on e p i t h e l i a l surfaces. 18 The Immune Response When an antigen i s encountered, macrophages (or other antigen-presenting c e l l s ) i n g e s t , degrade, or process, and present i t on t h e i r surface 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 molecules. An a n t i g e n - s p e c i f i c helper T - c e l l binds t o the a n t i g e n - c l a s s I I complex causing the re l e a s e of i n t e r l e u k i n - 1 from the helper T - c e l l . Another helper T - c e l l (a clone) i s then a c t i v a t e d (by i n t e r l e u k i n - 1 ) which i n t u r n acts on a n t i g e n - s p e c i f i c B-c e l l s by c e l l to c e l l i n t e r a c t i o n s and the r e l e a s e of other a c t i v a t i n g f a c t o r s . The B - c e l l begins t o d i v i d e and form plasma c e l l s . The plasma c e l l s produce immunoglobulins, IgM f o r a primary response and memory c e l l s produce IgA or IgG f o r a secondary response. Some B c e l l s a l s o d i f f e r e n t i a t e i n t o memory c e l l s (with s p e c i f i c i t y f o r t h a t antigen) which enhance the immune response i f the same antigen i s encountered again i n the fu t u r e . When the f o r e i g n antigen has been e l i m i n a t e d , suppressor T - c e l l s stop the immune response by a c t i n g upon, or suppressing, helper T - c e l l s . DEVELOPMENT OF IMMUNOCOMPETENCE IN THE FETAL CALF Immunocompetence i s defined as the a b i l i t y t o mount an immune response. Although the newborn c a l f i s born v i r t u a l l y agammaglobulinaemic, s e v e r a l s t u d i e s have confirmed t h a t the immune system begins t o develop f a i r l y e a r l y i n f e t a l l i f e and the c a l f i s immunocompetent at b i r t h . However, the c a l f ' s immune system i s immature at b i r t h and i s slow t o respond t o antigenic, challenge. The neonatal immune system r e q u i r e s a few weeks to mature to the stage where a quicker primary response can occur as w e l l as a secondary response. Ontogenetic development of immune system f u n c t i o n s i s l a r g e l y dependent upon two v a r i a b l e s : species and length of g e s t a t i o n . Schultz (1972) reviewed many st u d i e s which have r e s u l t e d i n an understanding of when immunocompetence develops i n the bovine fe 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 observing the development of v a r i o u s components of the immune system throughout the g e s t a t i o n a l p e r i o d as w e l l as experiments where fetuses were i n f e c t e d w i t h s p e c i f i c organisms to measure the response of the f e t a l immune system. Because the type of p l a c e n t a l attachment i n the bovine does not all o w passage of maternal Ig t o the f e t u s , any Ig found i n the f e t u s would presumably be of f e t a l o r i g i n . However, i f p a t h o l o g i c a l l e s i o n s occur i n the plac e n t a as a r e s u l t of m i c r o b i a l i n f e c t i o n or from other causes, leakage of maternal Ig may occur (Brambell, 1970). In ne a r l y a l l samples c o l l e c t e d a f t e r 200 days of g e s t a t i o n , low l e v e l s of IgM and IgG were present. R e s u l t s of these s t u d i e s show t h a t the bovine fetus and p r e - c o l o s t r a l c a l f are immunocompetent but the l e v e l s of IgG and IgM are w e l l below a d u l t values and the l e v e l s r e q u i r e d t o meet a challenge i n p r a c t i c e . Lymphoid t i s s u e development i s s t i l l at an immature stage, as w e l l . 20 Table l . Ontogeny of development of immune system i n the bovine fetus (as compiled by Schultz, 1972) G e s t a t i o n Day ; Event  42 lymphocytes f i r s t recognized i n thymus 45 lymphocytes f i r s t recognized i n p e r i p h e r a l blood 55 lymphocytes f i r s t recognized i n bone marrow 59 IgM present i n spleen 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 b i r t h ABSORPTION OF IMMUNOGLOBULINS F a i l u r e to absorb adequate amounts of immunoglobulins (Ig) has been a s s o c i a t e d w i t h the high r a t e of mo r b i d i t y and m o r t a l i t y found i n c a l f production (Boyd, 1972 ; Logan & Penhale, 1971; McEwan et a l . , 1970; McGuire et a l . , 1976; Penhale et a l . , 1973). Many surveys have shown a p o s i t i v e r e l a t i o n s h i p between absorbed c o l o s t r a l Ig and c a l f s u r v i v a l r a t e s (Klaus et a l . , 1969; McEwan et a l . , 1970; McGuire et a l . , 1976; Penhale et a l . , 1973; Smith & L i t t l e , 1922; S t o t t et a l . , 1979). The amount of Ig absorbed v a r i e s widely, however. From ten t o t h i r t y percent of ca l v e s may have l i t t l e or no Ig i n t h e i r serum and thus no passive immunity against septicemic disease. A low serum Ig 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 f a c t o r s : a) an inadequate amount of colostrum ingested, b) a low c o n c e n t r a t i o n of Ig i n colostrum, c) l a t e or delayed feeding of colostrum, d) e a r l y c l o s u r e , or 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 t o u n d e s i r a b l e environment ( S t o t t et a l . , 1975).. Vermorel•et a l . , (1989a; 1989b) s t u d i e d the e f f e c t of c a l v i n g c o n d i t i o n on energy metabolism i n newborn c a l v e s . They found t h a t calves which were born i n c o l d , wet c o n d i t i o n s , or c a l v e s which had a delayed p a r t u r i t i o n or d i f f i c u l t b i r t h s , had a delayed absorption of Ig and s i g n i f i c a n t l y lower serum Ig l e v e l s at s i x , 12 and 24 hours of age than do calves born normally. Straub and Matthaeus (1978) measured Ig concentrations of colostrum and found the amount of Ig r a p i d l y decreases w i t h time a f t e r b i r t h . Kruse (1970) a l s o found a r a p i d d e c l i n e i n Ig content of colostrum (40% decrease a f t e r 16 hours). Age of dam 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 Ig i n colostrum. A study done by Devery-Pocius and Larson (1983) showed cows at 30 months of age, i n t h e i r f i r s t l a c t a t i o n , produced l e s s t o t a l Ig 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 rose 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. rose to 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 f i r s t l a c t a t i o n . Table 2 shows concentrations of IgG, IgM and IgA i n colostrum and milk. Table 2. Mean Concentrations of IgG, IgM and IgA i n Colostrum and Milk Concentration  IgG IgM IgA Colostrum (mg/ml) at p a r t u r i t i o n 45.6 - 62.9 4.4 3.2 at 24 hrs post-partum 32.4 4 . 0 2.9 M i l k (jig/ml) 13.0 - 22.2 0.5 1.7 (compiled from 155 colostrum and m i l k samples, Burton et a l . . 1989; Straub & Matthaeus, 1978). Besser and Gay, (1987), studying the r e l a t i o n s h i p between co n c e n t r a t i o n of Ig i n colostrum and i n c a l f serum, found t h a t c a l v e s fed colostrum w i t h higher immunoglobulin concentrations developed higher serum immunoglobulin concentrations, although the e f f i c i e n c y of absorption decreased with i n c r e a s i n g c o n c e n t r a t i o n i n colostrum. Calves fed colostrum w i t h a lower immunoglobulin co n c e n t r a t i o n absorbed a higher p r o p o r t i o n of the Ig than calves fed the colostrum w i t h higher immunoglobulin concentrations.' Work c a r r i e d out i n three separate s t u d i e s showed t h a t i n g e s t i o n of colostrum by s u c k l i n g u s u a l l y r e s u l t s i n higher serum concentrations 23 than i n g e s t i o n of colostrum by bucket feeding (Nocek et  a l . . 1984; Selman et a l . . 1970 & 1971). S t o t t et a l . . (1979), studying c l o s u r e times and delayed feeding of colostrum, estimated c l o s u r e occurred at 21 hours of age, f o r c a l v e s fed at b i r t h , and t h a t a delayed feeding of colostrum delayed the c l o s u r e time up t o a maximum of 3 3 hours'of age. With delayed feeding, however, the length of time the calves a c t u a l l y had t o absorb was d r a m a t i c a l l y decreased from 21 hours f o r c a l v e s fed at b i r t h t o about 8 hours f o r calves fed at 24 hours of age. Michanek et a l . , (1989a & 1989b), showed t h a t i f c a l v e s were deprived of colostrum the a b i l i t y t o absorb macromolecules d i d not d e c l i n e during the f i r s t e i g h t hours of l i f e . Calves were given a f i r s t feeding of pooled colostrum at one, e i g h t , 16 or 24 hours of age w i t h three more subsequent feedings at 8 hour i n t e r v a l s . There were no d i f f e r e n c e s between groups i n absorption of Ig f o r the f i r s t feeding, but the calves t h a t were fed at 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 than the other three groups i n the second, t h i r d and f o u r t h feedings. The s i m i l a r i t y i n amount of Ig absorbed between a l l four groups at the f i r s t feeding suggested a base l e v e l of absorption c a p a c i t y t h a t may be maintained during the f i r s t 24 hours or longer. Their r e s u l t s imply t h a t an acceptable l e v e l of passive immunity may be achieved i n c a l v e s t h a t get t h e i r f i r s t colostrum l a t e r than 8 hours, i f a q u a n t i t y of Ig i s fed t h a t w i l l g ive the c a l f an opportunity to f u l l y u t i l i z e i t s absorption c a p a c i t y at i t s f i r s t feeding. For cal v e s t h a t are fed e a r l y , the f i r s t feeding i s l e s s important because they can absorb considerable amounts of Ig at l a t e r feedings. However, as s t a t e d p r e v i o u s l y , the l e v e l of Ig i n colostrum by t h i s time has decreased s u b s t a n t i a l l y , so t h i s would only be of b e n e f i t i f ca l v e s were being fed a high q u a l i t y ( i . e . high Ig) colostrum. A study done by McCoy et a l . , (197 0), supported the f i n d i n g t h a t delayed feeding reduced the p e r i o d of absorptio n . In a d d i t i o n , of the cal v e s whose f i r s t feeding was delayed t o 12, 16, 20 or 24 hours of age, three, 17, 3 0 and 57 percent of the c a l v e s , r e s p e c t i v e l y , were unable t o absorb any Ig at a l l . A l l c a l v e s t h a t were fed before 12 hours of age were able to absorb Ig. S t o t t et a l . , (1979), a l s o found no s i g n i f i c a n t d i f f e r e n c e i n mean c l o s u r e time between the 3 c l a s s e s of Ig (IgG, IgM, IgA). The amounts of colostrum fed (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 the absorptive p e r i o d . Another separate study supported these f i n d i n g s ( P a t t , 1977). Previous s t u d i e s showed the importance f o r i n i t i a l feedings of colostrum t o be w i t h i n four t o s i x hours a f t e r b i r t h and subsequent feedings up t o 24 - 36 hours a f t e r b i r t h (Logan et a l . , 1972; P a t t , 1977 ; P e t r i e , 1974; S t a l e y et a l . , 1972), f o r calves t o obta i n s u f f i c i e n t l e v e l s of serum Ig ( i . e . >12 mg/ml) t o f i g h t b a c t e r i a l septicemias ( c o l i s e p t i c e m i a ) ( S t o t t et a l . , 25 1979) . E a r l i e r feeding of colostrum does shorten the p e r i o d of absorption but spontaneous c l o s u r e i n cal v e s deprived of colostrum occurred at a mean of 24 hours so e a r l y c o l o s t r a l feeding may shorten absorption p e r i o d by only two t o three hours. Corley et a l . , (1977), suggested t h a t shortening the absorptive p e r i o d by feeding 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 p r o t e c t i n g the c a l f from prolonged exposure t o i n d i s c r i m i n a t e absorption of pathogens. As w e l l , i t has never been shown th a t c l o s u r e i n the 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) although they a f f e c t c l o s u r e i n the 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 (Clark and Hardy 1970; Lecce and Broughton 1973). MECHANISM OF ABSORPTION Uptake or absorption of 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 energy-dependent process w i t h macromolecules being absorbed by p i n o c y t o s i s and t r a n s m i t t e d , w i t h i n a membrane-bound v e s i c l e , through the cytoplasm of the enterocytes (Figure 4). Absorption by p i n o c y t o s i s appears t o be a h i g h l y s e l e c t i v e process depending upon by s p e c i f i c receptors probably l o c a t e d on the enterocyte m i c r o v i l l i (Hood et  a l . , 1984). Competition f o r attachment t o the receptors by molecules of a s i m i l a r nature has been shown t o occur and the mechanism of absorption i s subject t o s a t u r a t i o n ( T i z a r d , 1987). 26 n i t © ^  © . lysosomes Phagolysosome Figure 4. General 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). Immunoglobulins absorbed from the small i n t e s t i n e i n t o the blood c i r c u l a t i o n give passive immunity or p r o t e c t the c a l f against septicemias (Boyd, 1972; Burton, 1986). Ig remaining i n the small i n t e s t i n e ( i . e those t h a t are not absorbed) can provide a l o c a l p r o t e c t i v e e f f e c t , termed copro-immunity, against b a c t e r i a causing d i a r r h e a (scours) or 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 of one another as high serum immunoglobulin l e v e l s do not prevent d i a r r h e a nor do high i n t e s t i n a l l e v e l s prevent death from c o l i s e p t i c e m i a (Logan & Pearson, 1978). Calves switched to m i l k r e p l a c e r w i t h i n a few days a f t e r b i r t h may develop scours or d i a r r h e a , and ass o c i a t e d problems, due to l a c k of copro-immunity. Continued feeding of colostrum has been shown t o reduce incidence of scours (Logan & Pearson, 1978). 27 EXPERIMENTAL EXPERIMENT ONE  INTRODUCTION Previous s t u d i e s have shown the importance of colostrum as a source of immunoglobulin p r o t e c t i o n f o r the newborn c a l f (Boyd, 1972; Kruse, 1970; McCoy, 1970). Calves switched t o a m i l k r e p l a c e r w i t h i n a few days a f t e r b i r t h may develop scours or d i a r r h e a , and as s o c i a t e d problems, due t o l a c k of copro-immunity. Continued feeding of colostrum has been shown t o reduce incidence of 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 Ig, which would give t h i s needed immunity, could reduce the incidence of e n t e r i c disease and a s s o c i a t e d morbidity. A study done by Haines et a l . , 1990, compared some commercially a v a i l a b l e c o l o s t r a l supplements. These supplements contained low and v a r i a b l e Ig concentrations compared t o those found i n high q u a l i t y ( i . e . , high Ig) colostrum. While these products may be of some b e n e f i t a f t e r c l o s u r e , they are u n l i k e l y t o be very e f f e c t i v e i n colostrum-deprived c a l v e s . The o b j e c t i v e of experiment 1 was t o develop and t e s t a m i l k r e p l a c e r f o r t i f i e d w i t h Ig, separated from bovine a b a t t o i r blood by polyphosphate f r a c t i o n a t i o n , as a method of p r o v i d i n g systemic and e n t e r i c passive immunity t o colostrum-deprived c a l v e s . 28 MATERIALS AND METHODS Pre p a r a t i o n of Bovine Immunoglobulins C i t r a t e d bovine blood was obtained from two sources, I n t e r c o n t i n e n t a l Packers, Vancouver, B.C. or Coastpac I n d u s t r i e s , Abbotsford, B.C. Throughout the procedure a l l m a t e r i a l s were kept at 4° C t o minimize b a c t e r i a l growth. Plasma was siphoned 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 blood (3 000 r e v o l u t i o n s per minute (rpm) f o r ten minutes, at 5°C). F i b r i n was p r e c i p i t a t e d by the a d d i t i o n of d i c a l c i u m phosphate t o the plasma (50 c c / L ) , allowed t o e q u i l i b r a t e overnight i n 4°C and removed by chopping up. and f i l t e r i n g through cheesecloth w i t h the supernatant serum r e t a i n e d . 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 of the method by Lee et a l . , (1988), using sodium polyphosphate " g l a s s " (Calgon) s o l u t i o n c o n t a i n i n g Na 1 5 P 1 3 OA0 - Na 2 0 P 1 8 0 5 5. One hundred ml of a s o l u t i o n c o n t a i n i n g 114.4 g/L of sodium polyphosphate g l a s s (Sigma Chemical Co., St. L o u i s , MO) and 84.85 g/L NaCl were added per l i t r e of serum wi t h constant s t i r r i n g . A f t e r pH adjustment t o 3.95 w i t h 3N HCl, the mixture was s t i r r e d f o r ten minutes and allowed to e q u i l i b r a t e overnight. The mixture was then c e n t r i f u g e d at 5000 rpm f o r ten minutes (5°C) and the supernatant r e t a i n e d . The immunoglobulins i n the supernatant were concentrated using . a P e l l i c o n u l t r a f i l t r a t i o n system ( M i l l i p o r e Corporation, Bedford, MA) w i t h a 100,000 nominal molecular weight l i m i t f i l t e r pack. The concentrated f r a c t i o n , c o n t a i n i n g 29 approximately 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 stor e d at -20° C i n sealed p l a s t i c c o n t a i n e r s . Experimental Design A completely randomized design was used i n which newborn c a l v e s were randomly a l l o c a t e d t o one of four treatments, w i t h a minimum of 8 cal v e s per treatment (Table 3). Treatment A calves were fed colostrum on day 1 and whole m i l k on days 2 t o 21 (Col/WM). Treatment B calv e s were fed m i l k r e p l a c e r at a l e v e l of 50 mg/ml Ig on day 1, and 10 mg/ml Ig on days 2 t o 21 (MR-Hi/Lo) . These l e v e l s are based on published l i t e r a t u r e values f o r m i l k and colostrum (Straub & Matthaeus, 1978; Fleenor & S t o t t , 1980). The high l e v e l was intended t o approximate colostrum l e v e l s and the low l e v e l was intended t o be intermediate between colostrum and milk. Treatment C cal v e s were fed mi l k r e p l a c e r with the high l e v e l (50 Table 3. Experimental protocol for studying administration of immunoglobulins to calves. TREATMENTS Cmg Ig/ml d i e t l i g u i d ) A B C D DAYS OF AGE 1 Col 50 50 No Ig 2 - 2 1 WM 10 No Ig No Ig 22 - 42 WM No Ig No Ig No Ig No. of animals 11 8 8 9 30 mg/ml) of Ig on day 1, and m i l k r e p l a c e r w i t h no Ig on days 2 - 2 1 (MR- Hi/No). Treatment D ( c o n t r o l ) c a l v e s were fed m i l k r e p l a c e r w i t h no Ig added from b i r t h t o day 21 (MR-No Ig). Animals A t o t a l of 37 newborn, colostrum-deprived H o l s t e i n -F r i e s i a n b u l l c alves were obtained from Agassiz Research S t a t i o n , Agassiz, B.C., where the t r i a l was run, and from d a i r y producers i n the surrounding area. The calves were obtained s h o r t l y a f t e r b i r t h , before they had nursed, and randomly a l l o t t e d t o treatments. Each c a l f was weighed and a blood sample taken before the i n i t i a l feeding. Calves were housed indoors i n t i e s t a l l s . S t a l l s were approximately 2 f e e t wide x 3 f e e t deep w i t h bedding of sawdust and straw. Each c a l f was given a f i r s t feeding w i t h i n four hours of b i r t h , w i t h a second feeding approximately four to s i x hours a f t e r the f i r s t , t o ensure they r e c e i v e d a minimum of four t o f i v e l i t r e s of t h e i r d i e t . On days 2 21 of treatment p e r i o d and days 22 - 42 of post-treatment p e r i o d , calves were fed twice d a i l y , at a t o t a l of 10% of t h e i r body weight. A l l c a l v e s were weighed at b i r t h , 24 hours of age, day 7 and once a week t h e r e a f t e r . Feed intake was adjusted weekly on the b a s i s of weight. Calves were b o t t l e fed on days 1 and 2, and switched t o p a i l feeding from Day 3 onward. 31 M i l k r e p l a c e r 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 obtained from Van Waters & Rogers L t d . , Vancouver, B.C. The m i l k r e p l a c e r contained 21% p r o t e i n , 12% f a t , 1% calcium and 0.8% phosphorus and f o r t i f i e d w i t h NRC requirement l e v e l s of a l l other vitamins and minerals. P r e c i s e m i l k r e p l a c e r formula i s p r o p r i e t a r y i n f o r m a t i o n . The dry d i e t was mixed . w i t h water according to manufacturers recommendations to provide a 14% s o l i d s m i l k r e p l a c e r . Freeze d r i e d immunoglobulins were mixed wi t h the dry 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 . D a i l y 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 f o r t i f i e d w i t h Ig were frozen at -20° C. i n p l a s t i c c r y o v i a l s and kept f o r l a t e r Ig a n a l y s i s . Colostrum Colostrum ( f i r s t & second milkings) was c o l l e c t e d from mature cows at the Agassiz Research S t a t i o n , pooled to give a uniform conc e n t r a t i o n of Ig and frozen 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 of hot tap water, and fed t o c a l v e s on Treatment A (Col/WM). A 4 ml sample of each feeding of colostrum was frozen at -20° C. i h p l a s t i c c r y o v i a l s and kept 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 f r e s h d a i l y , from r e g u l a r m i l k i n g s , pooled and fed to c a l v e s on Treatment A (Col/WM). A d a i l y 4 ml m i l k sample was frozen at -20° C. 32 i n p l a s t i c c r y o v i a l s and kept f o r l a t e r a n a l y s i s f o r immunoglobulin co n c e n t r a t i o n . Grain and Hay An 18% p r o t e i n , commercial, t e x t u r e d c a l f 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 the c a l v e s from about 5 days of age. Hay was provided t o a maximum 2.0 kg twice d a i l y . Blood Samples Blood samples were taken by j u g u l a r venipuncture, from a l l c a l v e s at b i r t h , 24 hrs (day, 1), 48 hrs (day 2) and on days 7, 14, 21, 28, 35 and 42. One 4 ml 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 sodium-fluoridated (NaF2) v a c u t a i n e r tube. Blood was c e n t r i f u g e d immediately, at 3 000 rpm f o r ten mins., and the serum separated i n t o p l a s t i c c r y o v i a l s and frozen (-20° C.) f o r l a t e r - a n a l y s i s . Samples were analyzed f o r serum Ig c o n c e n t r a t i o n , blood glucose and urea n i t r o g e n . Diarrhea scores S e v e r i t y of d i a r r h e a was estimated using a s c a l e described by Nocek et a l . . (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 s o l i d 5 - watery, w i t h blood 3 - runny, mostly f l u i d F e cal samples were taken d a i l y f o r dry matter determination. 33 Therapy A l l c a lves received a 2.0 ml i n j e c t i o n of Vitamin A and D1 (Poten A.D., Steere E n t e r p r i s e s , Vancouver, B.C.) at b i r t h and at 2 weeks of age. Due t o the p r o x i m i t y of the c a l f barn t o other animals at the research s t a t i o n , c a l v e s on the 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 of 3 or more, the c a l f was given approximately 15 ml/kg body weight of S t a t ( K a o l i n and Pectin) plus e l e c t r o l y t e s 2 (Steere E n t e r p r i s e s , Vancouver, B.C.) i n i t s m i l k plus 1 or 2 a n t i b a c t e r i a l boluses depending upon d i a r r h e a l s e v e r i t y (Furalean 3 - E. c o l i bolus, or Sulkamycin - S 4 Bolus, MTC Pharmaceuticals, Cambridge, ON) . I f d i a r r h e a was 1 Vitamin A - 500,000 I.U. Vitamin D - 75,000 I.U. plus Vitamin E a n t i - o x i d a n t 2 K a o l i n 129.00 mg/ml P e c t i n 5.00 mg/ml Aluminum Oxide 1.66 mg/ml Potassium Acetate 3.30 mg/ml Mg-Cl-hexahydrate 1.00 mg/ml Na - a c e t a t e t r i h y d r a t e 19.80 mg/ml NaCl 18.00 mg/ml I o n i c c o n c e n t r a t i o n - (meq/L) K+ Mg++ Na+ CI - HC(V 34 10 454 .318 180 3 Nifuraldezone 1 gm Bismuth 260 mg S u b s a l i c y l a t e 260 mg Vitamin A 25000 I.U. 1 bolus/70 kg body weight - twice d a i l y f o r 2 days 4 Sulfamethazine 2 gm Neomycin-S0 3 250 mg 2 boluses/50 kg body weight - twice d a i l y , 1st day 1 bolus/50 kg body weight - twice d a i l y , next 2-3 days severe, c a l v e s were taken o f f m i l k treatment and given 2.0 l i t r e s e l e c t r o l y t e 5 s o l u t i o n (Ionolyte - Steere E n t e r p r i s e s , Vancouver, B.C.) f o r 2 - 3 days plus the boluses. R e c t a l temperature and general c o n d i t i o n were recorded d a i l y , f o r the f i r s t three weeks, as w e l l . Post-Mortem Examinations Post-mortem examinations were performed w i t h i n 48 hours of death on a l l but one c a l f . This c a l f died of compli c a t i o n s from a t w i s t e d i n t e s t i n e i n the post-treatment p e r i o d . Examinations were done by P r o v i n c i a l V e t e r i n a r y Pathology Laboratory i n Abbotsford, B.C. Tissue and f l u i d samples were analyzed according t o standard post-mortem procedure. Assays of administered and serum immunoglobulins Q u a n t i t a t i v e analyses of bovine IgG was done using a double sandwich enzyme-linked immunosorbent assay (ELISA) described by V o l l e r et a l . . (1976). A n t i b o d i e s f o r standards and reagents were obtained from Sigma Chemical Co., St. Louis, MO. P r e l i m i n a r y t e s t s were run i n order t o obta i n the range of d i l u t i o n s f o r the standard and f o r the serum samples. Bovine IgG concentrations were analyzed i n each sample of serum from each c a l f , and i n d a i l y samples of the colostrum, milk, 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 + Cl" HC03" 34 10 454 318 180 35 Blood Glucose Determination . Blood glucose was determined using an enzymatic (glucose oxidase) procedure (Sigma Diagnostics K i t #510, Sigma Chemical Co., St. Louis, MO). Blood Urea Nitrogen determination Blood urea n i t r o g e n was determined u s i n g a urease/ b e r t h e l o t procedure (Sigma Diagnostics K i t #640, Sigma Chemical Co., St. Louis, MO). STATISTICAL ANALYSIS R e s u l t s were analyzed using General L i n e a r Models procedure of 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 Inc., 1985). Repeated measures on l e a s t squares means were done f o r serum I g , blood glucose and blood urea n i t r o g e n and weight gains. A n a l y s i s of covariance was performed on average d a i l y gains using i n i t i a l b i r t h weight as the c o v a r i a t e . S u r v i v a l was analyzed by as s i g n i n g 1 t o cal v e s t h a t survived the experiment and 0 to those t h a t d i e d . D i f f e r e n c e s between treatment means were analyzed using orthogonal c o n t r a s t s . A l l r e s u l t s reported are l e a s t squares means f o r treatments. RESULTS Measurement of Bovine Immunoglobulins' Concentration of Ig i n the freeze d r i e d 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 agarose 6, was c a r r i e d out on both p r e c i p i t a t e and supernatant f r a c t i o n s from polyphosphate f r a c t i o n a t i o n and shown i n Figure 5. Level of Ig measured i n pooled sample of freeze d r i e d Ig pr e p a r a t i o n averaged 136.6 mg/ml of serum. Levels i n colostrum fed t o cal v e s on treatment one averaged 23.1 mg/ml (ranging from 18 t o 28 mg/ml); i n high l e v e l d i e t fed 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 9.4 mg/ml. Levels i n both 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. Table 4. Concen t ra t i ons of IgG in Freeze Dried Prepara t ion , M R - H i , M R - L o , Co los t r um and Milk Fed to Ca l ves Concent ra t ion of IgG Range (mg/ml) (mg/ml) Freeze Dried IgG 136.6 84.0 - 150.0 M R - H i diet 25.4 15.0 -- 41.0 M R - L o diet 9.4 6.0 - 12.0 Co los t rum 23.1 18.0 - 30.0 Milk 0.1 c o n t a i n i n g 1% (w/v) agarose, 5% (w/v) sucrose, and 0.035% (w/v) EDTA disodium 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 Products, McGraw Park, IL Bovine Serum F r a c t i o n 1 F r a c t i o n 2 F r a c t i o n 3 F r a c t i o n 4 Pure IgG sample IgG Albumen . J 37 F i g u r e 5. G e l e l e c t r o p h o r e s i s o f p r e c i p i t a t e ( f r a c t i o n 4) and s u p e r n a t a n t ( f r a c t i o n s 1, 2 & 3) f r o m 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 . S u r v i v a l T a b l e 5 shows e f f e c t of t r e a t m e n t on p e r c e n t s u r v i v a l r a t e s o f c a l v e s f o r s i x weeks. A l l c a l v e s on Col/WM (11 out o f 11) and MR-Hi/Lo (8 out 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 age. MR-Hi/No had 7 out o f 8 c a l v e s s u r v i v e t o s i x weeks. MR-Nolg ( c o n t r o l ) had 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 and MR-Hi/Lo w i t h 7 out 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 R e c t a l t e m p e r a t u r e s ( F i g u r e 6) were i n t h e normal range and aver a g e d 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 3 8 reached temperatures above 3 9 . 0 ° C f o r those which were scouring most sev e r e l y . Table 5. The Ef fect of Ig Supplementat ion on Cal f Surv iva l Rates E X P E R I M E N T 1 No. of ca lves surv iv ing at T R E A T M E N T Birth Day 42 C o l / W M 11 11 MR-No lg 9 7 M R - H i / L o 8 8 M R - H i / N o 8 7 Figure 6. E f fec t of Ig Supplementat ion on Rec ta l Temperature for the F i rs t Three W e e k s of L i fe 39.4 - I -3 9 . 3 -38.2 i 1 1 ' — i 0 7 14 21 DAYS OF AGE 39 Diarrhea Average weekly d i a r r h e a scores are shown i n Figure 7. C o n t r o l c a l v e s (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 than c a l v e s on Col/WM and MR-Hi/Lo during week one (P<0.05). MR-Nolg calves a l s o had s i g n i f i c a n t l y more d i a r r h e a than a l l three other treatments during week four (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 the four treatments during weeks two, three, f i v e and s i x . 0> Figure 7. The Ef fect of Ig Supplementat ion on Diar rhea Levels (bacter ial scours ) 3 -2.5 1.5 -1H 0.5 Based on Nocek scale: 1 - normal 2 - soft, mostly solid 3 - runny, mostly fluid 4 - watery, all fluid 5 - watery, with blood Treatments - Col/WM MR-No Ig - M R - H i / L o - a - MR-Hi/No 1 2 3 4 5 6 PERIOD OF TREATMENT - WEEK 40 C a l f Weights C a l f weights (Figure 8) 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 of the treatments at b i r t h , 24 hours and days 7, 14 and 28. MR-Nolg weights tended t o be lower than the other three treatments throughout the experiment and were s i g n i f i c a n t l y lower than MR-Hi/Lo c a l f weights at day 21 (P<0.05). MR-Nolg weights were a l s o s i g n i f i c a n t l y lower than Col/WM and MR-Hi/Lo at days 35 and 42 (P<0.05). Figure 8. The Ef fec t of Ig Supplementat ion on Cal f Weight DAYS OF AGE Average D a i l y Gains Figure 9 shows average d a i l y gains (ADG) f o r the three treatment periods. MR-Nolg calves had s i g n i f i c a n t l y lower ADG than c a l v e s on Col/WM, MR-Hi/Lo or MR-Hi/No f o r the three treatment p e r i o d s , from zero t o three weeks of age (P<0.05), zero t o s i x weeks of age (P<0.02) and three t o s i x weeks of 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 ca l v e s on Col/WM, MR-Hi/Lo or MR-Hi/No. Figure 9. The Ef fec t of Ig Supplementat ion on Average Dai ly 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 shows e f f e c t of immunoglobulin supplementation on feed e f f i c i e n c y (kgs dry feed/kgs gain) of ca l v e s f o r the f i r s t s i x weeks of l i f e . MR-Nolg ca l v e s had s i g n i f i c a n t l y poorer (P<0.05) feed e f f i c i e n c y than the other three treatment c a l v e s . Figure 10. The Ef fect of Ig Supp lementa t ion on F e e d Ef f ic iency for the F i rs t S i x W e e k s of Li fe 3.00 ro UJ T3 Q) U. 0.50 -0.00 Col/WM MR-Hi/Lo MR-Hi/No MR-Nolg 43 Serum IgG Levels Serum IgG l e v e l s are shown i n Figure 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 between any of the treatments at b i r t h or day 21. At 24 and 48 hours of age, and at day 7, Col/WM cal v e s had s i g n i f i c a n t l y higher serum IgG l e v e l s than c a l v e s on MR-Nolg, 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 MR-Hi/No treatments. At day 14 and 28, cal v e s on Col/WM had s i g n i f i c a n t l y higher serum IgG l e v e l s than MR-Hi/No cal v e s (P<0.02). At day 35 and 42, MR-Hi/No ca l v e s had s i g n i f i c a n t l y lower serum IgG l e v e l s than Col/WM (P<0.01), MR-Nolg and MR-Hi/Lo calves (P<0.05). Figure 11. The Ef fec t of Ig Supplementat ion on Serum IgG Levels 20 DAYS OF AGE Blood Glucose Levels Blood glucose l e v e l s (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 of the treatments, at b i r t h , 48 hours of age, and at days 7, 14, 21, 28 and 42. At 24 hours of age, blood glucose 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 higher than c a l v e s on MR-Hi/No (P<0.05), w i t h a trend t o higher l e v e l s between MR-Nolg and MR-Hi/Lo. MR-Hi/Lo tended t o be lower than the other treatments at day 14 but not s i g n i f i c a n t l y . Blood Urea Nitrogen Blood Urea Nitrogen (BUN) l e v e l s (Figure 13) tended to be higher i n the f i r s t week and showed a shallow d e c l i n e over the next 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 higher than MR-Hi/Lo and MR-Hi/No calves at b i r t h (P<0.05) and s i g n i f i c a n t l y higher than MR-Hi/No ca l v e s at 24 hours of 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 of the treatments at 48 hours of age. BUN values f o r ca l v e s on MR-Hi/No treatment tended t o be lower than the other three treatments throughout the s i x week p e r i o d of the experiment, and were s i g n i f i c a n t l y lower at day 14 (P<0.05). Figure 13. The Ef fec t of Ig Supplementat ion on 0 7 14 21 28 35 42 DAYS OF AGE 46 DISCUSSION Although there 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) or MR-Hi/Lo ca l v e s (8/8), and MR-Nolg ca l v e s (7/9) . The same s u r v i v a l r a t e f o r both Col/WM and MR-Hi/Lo c a l v e s shows t h a t feeding 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 serve as an a l t e r n a t i v e method of g i v i n g c a l v e s p a s s i v e , systemic immunity and can reduce the 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. Diarrhea l e v e l s (scours) were f a i r l y low throughout the experiment. The a n t i b i o t i c therapy given as soon as a c a l f was n o t i c e d scouring may have reduced the numbers of i n t e s t i n a l b a c t e r i a causing scouring. This may have tended t o decrease d i f f e r e n c e s due t o treatments, as w e l l . 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 four between MR-Hi/No cal v e s and Col/Wm and MR-Hi/Lo c a l v e s . 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 which r e c e i v e d no Ig had more severe and r e c u r r i n g episodes of scours. The elevated r e c t a l temperatures during scouring were i n d i c a t i v e of fever a s s o c i a t e d w i t h a b a c t e r i a l i n f e c t i o n . MR-Nolg calves tended t o scour l e s s than e i t h e r MR-Hi/Lo or Col/WM c a l v e s , although the d i f f e r e n c e was not s i g n i f i c a n t . This may have been due to the increased a n t i b i o t i c therapy given t o the MR-Nolg c a l v e s . These r e s u l t s suggest t h a t continued feeding of Ig i n m i l k r e p l a c e r (MR-Hi/Lo) t o three weeks of age may have had a p r o t e c t i v e e f f e c t 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 are supported by other work which showed t h a t continued feeding of colostrum reduced the incidence of scours (Logan and Pearson, 1978). Treatments had a s i g n i f i c a n t e f f e c t on weight and average d a i l y g ains, as w e l l as feed e f f i c i e n c i e s of c a l v e s . MR-Nolg c a l f weights were s i g n i f i c a n t l y lower than the other three treatments by day 21 and there were no compensatory gains i n the three t o s i x week post-treatment p e r i o d of the experiment. Weekly weights of Col/WM cal 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 or MR-Hi/No calves throughout the experiment. The o v e r a l l growth r a t e s of calves on Col/WM (587 g/d) 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 c a l v e s r e c e i v i n g MR-Hi/Lo (544 g/d) or MR-Hi/No (575 g/d), however, a l l three were s i g n i f i c a n t l y higher than MR-Nolg ca l v e s (377 g/d) . The feed e f f i c i e n c y f o r MR-Nolg c a l v e s was s i g n i f i c a n t l y poorer (P<0.01) than f o r Col/WM or MR-Hi/No c a l v e s . These r e s u l t s show th 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 colostrum or i n a f o r t i f i e d m i l k r e p l a c e r , w i l l do b e t t e r over the c r i t i c a l f i r s t three weeks than c a l v e s t h a t do not r e c e i v e any Ig. They w i l l scour l e s s , g a i n more and be at a higher weight at weaning ( s i x weeks of age). Passive immunity from absorption of Ig from colostrum or MR-Hi Ig treatments, seen i n Figure 11, peaked at about 3 6 t o 4 8 hours, and then began t o d e c l i n e . This normal decrease i n serum Ig l e v e l s occurs because, a f t e r c l o s u r e , the l e v e l s of passive Ig decrease 48 as they are c a t a b o l i z e d . The h a l f - l i f e of Ig range from 14 - 21 days i n the blood. Development of a c t i v e immunity can be seen i n the- serum l e v e l s of the MR-No Ig cal v e s which normally begins some time between day 7 t o day 14. These ca l v e s received no pass i v e Ig and t h e r e f o r e , the increase i n t h e i r serum 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 l e v e l s f o r MR-Hi/Lo and MR-Hi/No calves d i d not r i s e as high as Col/WM c a l v e s (16.3 vs 8.3 mg/ml). However, there was s i g n i f i c a n t absorption of the Ig from the f o r t i f i e d m i l k r e p l a c e r . The absorption e f f i c i e n c y f o r these 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 values of 10 t o 46%. The higher l e v e l s found i n Col/WM cal v e s could be due t o f a c t o r s i n the colostrum which may enhance immunoglobulin absorption (Balfour and Comline, 1962; Hardy, 19 69). The lower serum l e v e l s from the MR-Hi l e v e l feedings could a l s o be due t o the f a c t t h a t the a c t u a l l e v e l s measured (25.4 mg/ml) were h a l f t h a t c a l c u l a t e d f o r a d d i t i o n t o the mi l k r e p l a c e r (50 mg/ml). Problems were encountered when running the i n i t i a l ELISA assays. A bovine IgG standard i s done at the same time as the unknown samples f o r a standard curve 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 of immunoglobulins. The values obtained f o r the standard curve were not c o n s i s t e n t but due t o a time c o n s t r a i n t a best f i t curve was used. This may have r e s u l t e d i n i n c o r r e c t values c a l c u l a t e d f o r the concentrations i n the immunoglobulin s o l u t i o n which then could 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 the mi l k r e p l a c e r i n the MR-Hi d i e t s . The lower serum l e v e l s could a l s o be due t o some denaturing or degradation i n the i n t e s t i n a l lumen, as w e l l as, some t i s s u e absorption. This could be d e a l t w i t h by i n c r e a s i n g the amount of Ig added t o m i l k r e p l a c e r . The MR-Hi/No treatment c a l v e s had q u i t e d i s s i m i l a r r e s u l t s compared w i t h the other three treatments. These ca l v e s should have responded s i m i l a r l y to the Col/WM cal v e s i n t h e i r serum Ig 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 s l o w l y i n c r e a s i n g . They are not i n agreement w i t h the other r e s u l t s and no d e f i n i t e e xplanation i s apparent. However, t h i s may have been due t o the a n t i b i o t i c treatment, which may have reduced the l e v e l of b a c t e r i a to a p o i n t where there was not enough of 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 . Further s t u d i e s are needed t o understand i f there are any f a c t o r s i n v o l v e d which would p o s s i b l y suppress the development of the immune response. Blood glucose and blood urea n i t r o g e n r e s u l t s are i n general agreement wi t h l e v e l s from other experimental work done (Bazin and B r i s s o n , 1975; Drevjany et a l . . 1982; P e t i t et a l . . 1988a & 1988b; W i l l i a m s and Smith, 1975). Blood glucose and blood urea n i t r o g e n l e v e l s were i n the normal range f o r a pre-ruminant c a l f ' s development from b i r t h t o s i x weeks of age. The v a r i a t i o n i n blood glucose l e v e l s was not n e c e s s a r i l y due t o treatment e f f e c t s but may have been due t o time of blood sampling i n r e l a t i o n t o feeding. Depending upon when c a l v e s are fed, blood glucose and BUN l e v e l s r i s e t o a peak and f a l l again t o average l e v e l s over s i x t o e i g h t hours post-feeding ( P e t i t et a l . . 1988a & 1988b) . This a l s o may have been due t o the blood samples not being 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 the blood was l e f t t o s i t , the blood c e l l s may have continued t o use up the glucose and t h i s would r e s u l t i n lowered values. The c a l v e s fed the I g - f o r t i f i e d m i l k r e p l a c e r scoured l e s s , needed l e s s a n t i b i o t i c therapy, and gained more weight. Calves not r e c e i v i n g any or enough passive Ig scoured more sev e r e l y , had higher r e c t a l temperatures and r e c e i v e d more a n t i b i o t i c therapy. T h e i r growth r a t e was l e s s than the cal v e s which r e c e i v e d Ig and t h e r e f o r e , they weighed l e s s than the others at the end of s i x weeks. The r e s u l t s of t h i s 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 of passive immunity t o reduce systemic diseases and, w i t h continued feeding, can reduce b a c t e r i a l scours. 51 EXPERIMENT 2  INTRODUCTION Calves, f o r v a r i o u s reasons, may not r e c e i v e enough Ig v i a absorption even though allowed t o nurse or consume colostrum. Neonatal calves s o l d t o be r a i s e d f o r v e a l or beef may be deprived of colostrum which would increase l o s s e s due t o systemic diseases. Once i n t e s t i n a l c l o s u r e occurs, these calves have no chance t o achieve good pass i v e immunity l e v e l s . A p u r i f i e d , i n j e c t a b l e Ig s o l u t i o n , which would be absorbed i n t o the bloodstream, could g i v e hypo-gammaglobulinemic ca l v e s t h i s needed immunity and reduce the incidence of septicemic disease and the a s s o c i a t e d m o r t a l i t y . The e f f e c t i v e n e s s of an i n t r a p e r i t o n e a l or intravenous route using bovine 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 et a l . , (1987). The o b j e c t i v e of t h i s experiment was t o develop an i n j e c t a b l e form of Ig, separated and p u r i f i e d from bovine a b a t t o i r blood, and t e s t t h i s i n colostrum-deprived c a l v e s as an a l t e r n a t e method of ac h i e v i n g p a s s i v e , systemic immunity. MATERIALS AND METHODS P r e p a r a t i o n of Bovine Immunoglobulins Bovine Ig p r e p a r a t i o n by polyphosphate f r a c t i o n a t i o n f o llowed the same procedure as explained i n experiment 1. 52 Pr e p a r a t i o n of i n j e c t a b l e Icr s o l u t i o n The l i q u i d Ig s o l u t i o n obtained from the f r a c t i o n a t i o n method described i n experiment 1 was p u r i f i e d , before l y o p h i l i z a t i o n , by ion-exchange column chromatography technique (Friesen et a l . , 1985; Lee et  a l . , 1987) using DEAE-Sephacel beads (Pharmacia, Inc., Picataway, NJ). The p u r i f i c a t i o n procedure was done at a temperature of 4°C t o r e t a r d b a c t e r i a l growth. The method was adapted f o r l a r g e r volumes of Ig s o l u t i o n needed as f o l l o w s : A 11.5 cm d i a . x 11.0 cm high column was assembled wi t h an i n l e t tube e n t e r i n g at the top of the column and an o u t l e t tube coming from the bottom of the column and connected t o a d r i p r a t e c o n t r o l l e r (stopcock) and drop counter 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 of the DEAE-Sephacel beads was poured i n t o the column and allowed t o s e t t l e . The other end of the 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 set t o d r i p at a r a t e of 10 drops per minute. The DEAE-Sephacel was e q u i l i b r a t e d overnight, by washing w i t h a volume of Tris-HCL b u f f e r s o l u t i o n (0.02 M, pH 7.4) which was f i v e times t h a t of the column (7500 ml). The Ig s o l u t i o n was then a p p l i e d t o the column (100 ml of Ig s o l u t i o n was p u r i f i e d per e l u t i o n ) and allowed t o p e r c o l a t e through the column, by opening the stopcock at the 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 the top of the g e l . T r i s -HCl b u f f e r was then poured onto the g e l s l o w l y t o the top of the column. The l i d was put on the column and t i g h t e n e d so 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 the T r i s - H C l b u f f e r s o l u t i o n and another tube was i n s e r t e d between the b u f f e r s o l u t i o n and a 0.9% s a l i n e s o l u t i o n . As the immunoglobulin s o l u t i o n i s p u l l e d through the g e l , T r i s - H C l i s then p u l l e d i n t o the column and through the g e l , and then the s a l t s o l u t i o n i s p u l l e d i n t o the T r i s - H C l b u f f e r s o l u t i o n ; then i t i s p u l l e d i n t o the column and through the g e l . A s a l t g r a d i e n t from 0 - 0.5 M was obtained by t h i s method. The d r i p r a t e was set at 6 drops per minute and 8 ml f r a c t i o n s were c o l l e c t e d i n 10 ml tubes. 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 c o n d u c t i v i t y meter (Solu-Bridge, I n d u s t r i a l Instruments Inc.) and analyzed f o r IgG co 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 w e l l as by ELISA (as des c r i b e d on page 57) . The f r a c t i o n s c o n t a i n i n g the p u r i f i e d IgG were pooled and put i n t o Spectra/por molecularporous d i a l y s i s membrane tu b i n g w i t h a molecular weight c u t - o f f of 12,000 - 14,000 (Spectrum Medical Ind. Inc., Los Angeles, CA) , suspended i n 0.9% s a l i n e and brought t o a p h y s i o l o g i c a l s a l i n e c o n c e n t r a t i o n (0.9%). This s o l u t i o n was put i n t o 4 1-l i t r e s t e r i l e b o t t l e s and sealed with rubber stoppers which had an area f o r s t e r i l e removal by needle. The b o t t l e s of IgG s o l u t i o n were then s t e r i l i z e d by gamma r a d i a t i o n . A gamma c e l l was used and each b o t t l e was i r r a d i a t e d at 24,000 rad f o r 13.3 minutes. Two ml samples of each of the four b o t t l e s were checked f o r s t e r i l i t y by p l a t i n g on lab media agar. N u t r i e n t 54 media agar was d i s s o l v e d i n d i s t i l l e d water and autoclaved f o r 2 0 minutes. The hot agar was poured i n t o round c u l t u r e p l a t e s and allowed 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 of IgG s o l u t i o n were p l a t e d on the agar and incubated at 37°C f o r 72 hours. The p l a t e s were checked every 24 hours f o r b a c t e r i a l growth. The IgG s o l u t i o n b o t t l e s were then s t o r e d at 4°C u n t i l needed f o r i n j e c t i o n . Experimental Design: Experiment 2 was a completely randomized design i n which colostrum-deprived newborn b u l l c a l v e s were randomly assigned t o one of three treatments, as shown i n Table 6, w i t h 8 cal v e s per treatment. Treatment A c a l v e s were fed colostrum on day 1 and whole m i l k on days 2 t o 21 (Col/WM). Treatment B cal v e s were given a subcutaneous i n j e c t i o n of i g as soon a f t e r b i r t h as p o s s i b l e . In a l l cases, t h i s was before 6 hours of age. Treatment C cal v e s were fed mi l k r e p l a c e r w i t h no Ig Table 6. Experimental protocol for studying e f f e c t of administration of immunoglobulins to calves. TREATMENTS A B C DAYS OF AGE 1 Col Subcu I n j . of Ig No Ig 2 - 2 1 WM 10 mg/ml Ig No Ig 2 2 - 4 2 WM No Ig No Ig NO. Of animals 8 8 8 added on day 1, and on days 2 to 21 they were fed m i l k r e p l a c e r w i t h Ig added at a l e v e l of 10 mg/ml (MR-Lo I n j ) . T h i s l e v e l , as used i n the previous experiment, was intended t o be intermediate between colostrum and m i l k . Treatment C (control) calves were fed m i l k r e p l a c e r w i t h no Ig added from b i r t h (day 1) t o day 21 (MR-Nolg) . Animals A t o t a l of 24 newborn colostrum-deprived 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 obtained from Oyster R i v e r Research Farm, where the t r i a l was run, or from d a i r y producers i n the surrounding area. The c a l v e s were obtained s h o r t l y a f t e r b i r t h , before they had nursed, and randomly a l l o t t e d t o one of three treatments w i t h 8 c a l v e s per treatment. Each c a l f was weighed, and a blood sample was taken before the i n i t i a l feeding. Calves were assigned at random to a pen. Pens were 4' deep x 3" wide x 4.5' high w i t h a bedding of sawdust and straw, and allowed contact between adjacent s t a l l s . Calves on Treatment C were then given a subcutaneous i n j e c t i o n of Ig s o l u t i o n v i a a 60 cc syringe i n 2 - 3 l o c a t i o n s over the shoulder area. The amount administered, based on the c o n c e n t r a t i o n of p u r i f i e d Ig s o l u t i o n , and the b i r t h weight of the c a l f , was intended t o provide an adequate c i r c u l a t i n g l e v e l of Ig ( i . e . >12 mg/ml). Each c a l f was given i t s f i r s t feeding w i t h i n 2 hours a f t e r b i r t h , w i t h subsequent feedings approximately 4 -6 hours a f t e r the f i r s t t o ensure they r e c e i v e d a minimum of 4 - 5 l i t r e s of colostrum or MR. For days 2 - 21 of treatment p e r i o d and days 22 - 42 of post-treatment p e r i o d , c a l v e s were fed twice d a i l y a t o t a l of 10% of t h e i r body weight. A l l c a l v e s were weighed at b i r t h , 24 hours of age, day 7 - and once a week t h e r e a f t e r . Feed i n t a k e was adjusted according t o weight on a weekly b a s i s . Calves were n u r s i n g - b o t t l e fed on days 1 and 2, and were switched t o p a i l feeding from Day 3 onward. M i l k r e p l a c e r The b a s a l d i e t was t h a t used i n experiment 1. Freeze d r i e d immunoglobulins were mixed w i t h the dry 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 . D a i l y 4 ml samples of 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 were frozen at -20°C i n sealed p l a s t i c tubes and kept f o r l a t e r immunoglobulin a n a l y s i s . Colostrum Colostrum ( f i r s t & second mi l k i n g s ) was c o l l e c t e d from mature cows at Oyster R i v e r Research Farm, pooled and f r o z e n at -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 of hot tap water, and fed to c a l v e s on Treatment A. A 4 ml sample of each feeding of colostrum was frozen at -20°C i n sealed p l a s t i c and kept 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 obtained f r e s h d a i l y , from r e g u l a r m i l k i n g s , pooled and fed t o calves on Col/WM treatment. A 4 ml d a i l y sample of m i l k was frozen at -2 0°C i n sealed p l a s t i c and kept f o r l a t e r immunoglobulin a n a l y s i s . 57 Grain and Hay An 18% p r o t e i n commercial c a l f s tarter/grower (Otter Farm & Home Co-op, Aldergrove, B.C.) was a v a i l a b l e t o the c a l v e s from about 5 days of age. Hay was provided t o a maximum of 2.5 kg twice d a i l y . Blood Samples Blood samples were taken v i a j u g u l a r venipuncture from a l l c a l v e s at b i r t h , 24 hrs (day 1), 48 hrs (day 2) and on Days 7, 14, 21, 28, 35 and 42. One 4 ml 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 sodium-fluoridated (NaF2) v a c u t a i n e r tube. Blood was c e n t r i f u g e d immediately, at 3 000 rpm f o r ten mins., and the serum separated i n t o sealed p l a s t i c tubes and frozen (-20° C.) f o r l a t e r a n a l y s i s . Blood samples were analyzed f o r serum immunoglobulin c o n c e n t r a t i o n , blood urea n i t r o g e n (heparin tubes) and blood glucose (NaF2 tubes). Diarrhea scores Diarrhea was estimated, w i t h a l l observations done by the same observer, using a s c a l e d e s c r i b e d i n experiment 1. Therapy A l l c a l v e s received 2.0 ml i n j e c t i o n of Vitamins A and D (POTEN A.D., Steere E n t e r p r i s e s , Vancouver, B.C.) at b i r t h and at 2 weeks of age. I f d i a r r h e a was severe, c a l v e s were taken o f f m i l k treatment and given 2.0 l i t r e s 58 e l e c t r o l y t e s o l u t i o n 7 (Ionolyte - Steere E n t e r p r i s e s , Vancouver, B.C.) f o r 2 - 3 days. R e c t a l temperature and general c o n d i t i o n was recorded as w e l l . One c a l f was t r e a t e d w i t h 2 ml of 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 ind l e g . Post-Mortem Examinations Post-mortem examinations were performed w i t h i n 24 hours of death on a l l but one of the cal v e s t h a t d i e d during the experiment. Examinations were done by a Comox V e t e r i n a r y C l i n i c v e t e r i n a r i a n w i t h t i s s u e and f l u i d samples sent t o P r o v i n c i a l V e t e r i n a r y Pathology Laboratory i n Abbotsford, B.C. A post-mortem was not done on one c a l f t h a t d i e d of apparent b l o a t . Assay of administered and serum immunoglobulins Q u a n t i t a t i v e analyses of bovine IgG was done using a c o m p e t i t i v e , d i r e c t enzyme-linked immunosorbent assay (ELISA) (Kiriyama et a l . . 1989). A n t i b o d i e s f o r standards and reagents were obtained 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. Tests were run t o compare the r e s u l t s between the double-antibody sandwich technique (used i n experiment 1) and the competitive, d i r e c t technique. Both gave s i m i l a r r e s u l t s f o r IgG concentrations i n standards and i n serum samples. Bovine IgG concentrations were analyzed i n serum from each c a l f , Concentration - meq/L K+ Mg++ Na + C l " . HC03" 34 10 454 318 180 and d a i l y samples of the colostrum, m i l k and m i l k r e p l a c e r w i t h Ig. Blood Glucose Determination Blood glucose determination followed the same procedure as f o r experiment 1. Blood Urea Nitrogen determination Blood urea n i t r o g e n determination followed the same procedure as f o r experiment 1. RESULTS Measurement of Bovine Immunoglobulins A n a l y s i s of l e v e l of immunoglobulins i n the l y o p h i l i z e d p r e p a r a t i o n from polyphosphate f r a c t i o n a t i o n of bovine blood i s shown i n Table 7. The p u r i f i e d Ig s o l u t i o n used f o r subcutaneous i n j e c t i o n contained 513.0 m<3 Ig/<? °f dry matter. This was added t o s t e r i l e b u f f e r e d s a l i n e t o give a 6% p r o t e i n c o n c e n t r a t i o n i n the s o l u t i o n . Levels measured i n colostrum and m i l k fed t o ca l v e s are a l s o shown i n Table 7. S u r v i v a l Table 8. shows e f f e c t of treatment on s u r v i v a l r a t e s of c a l v e s f o r s i x weeks. Both Col/WM and MR-Lo I n j treatments had 7 out of 8 calves s u r v i v e t o s i x weeks of age but, only 4 out of 8 MR-Nolg cal v e s s u r v i v e d , which was s i g n i f i c a n t l y lower (P<0.05) than the other two treatments. One c a l f from Col/WM treatments d i e d i n the post-treatment p e r i o d from causes u n r e l a t e d t o treatment. One. c a l f on MR-Lo Inj treatment died as a r e s u l t of a Table 7. Concen t ra t i on of IgG in Pur i f ied Injectable So lu t ion , M R - L o diet, Co los t rum and Mi lk Concent ra t ion of IgG (mg/ml) Pur i f ied IgG solut ion 2488 .3 IgG solut ion as in jected 12.7 M R - L o diet 10.3 Co los t rum 27.8 Milk 0.1 Table 8. The Ef fec t of Ig Supp lementa t ion on Cal f Surv iva l Rates E X P E R I M E N T 2 No. of ca lves surv iv ing at TREATMENT Bir th 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 the f i r s t 24 hours of l i f e . The deaths of cal v e s from MR-Nolg treatment were caused by E.  c o l i septicemia w i t h severe dehydration. R e c t a l Temperature Figure 14 shows r e c t a l temperatures f o r the f i r s t t h ree weeks of age. MR-Nolg cal v e s had s i g n i f i c a n t l y h igher (P<0.05) r e c t a l temperatures i n weeks one and three than Col/WM c a l v e s , and s i g n i f i c a n t l y higher (P<0.05) than MR-Lo I n j calves i n week three. Figure 14. E f fec t of Ig Supp lementa t ion on Rec ta l Temperature for the F i rs t Three W e e k s of Li fe 14 21 Treatments — Col/WM —I— MR-Nolg MR-Lo Inj DAYS OF AGE Diarrhea Diarrhea l e v e l s (Figure 15) f o r cal v e s on MR-Nolg trended higher than e i t h e r Col/WM or MR-Lo I n j throughout the experiment. MR-Nolg calves had s i g n i f i c a n t l y more d i a r r h e a than MR-Lo I n j cal v e s during weeks three (P<0.05) and four (P<0.01), and s i g n i f i c a n t l y more than Col/WM during week four (P<0.01). a> co o CO a> o O Z c o T J 0) CO CO _Q I co Figure 3 -i 2.5 co CD CO Q 2 -1.5 m 1 0.5 -15. The Ef fec t of Ig Supp lementa t ion on Diar rhea Levels (bacter ia l scou rs ) 1 2 3 4 5 PERIOD OF TREATMENT - WEEK Based on Nocek scale: 1 T normal . 2 - soft, mostly solid 3 - runny, mostly fluid 4 - watery, all fluid 5 - watery, with blood Treatments Col/WM — I — MR-No Ig — M R - L o Inj C a l f Weights Figure 16 shows the e f f e c t of treatments on weight g a i n . 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 weight between any of the treatments at b i r t h , 24 hours of age," and at days 7, 14, 21 and 28. At day 35, Col/WM and MR-Nolg were approaching s i g n i f i c a n c e (P<0.05) and at day 42, weight of Col/WM calves was s i g n i f i c a n t l y higher than weight of MR-Nolg cal v e s (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 Inj c a l f weights at both days 35 and 42. Figure 16. The Ef fec t of Ig Supplementat ion on „ ^ Cal f Weight DAYS OF AGE Average D a i l y Gains Figure 17 shows average d a i l y gains (ADG) f o r the three treatment periods. ADG of cal v e s on MR-Nolg were s i g n i f i c a n t l y lower than ADG of calves on the other two treatments f o r the treatment periods from zero t o three weeks of age and from zero t o s i x weeks of age (P<0.01). From three t o s i x weeks of age, Col/WM ca l v e s had s i g n i f i c a n t l y higher ADG than c a l v e s on MR-Nolg (P<0.05) , however there was no s i g n i f i c a n t d i f f e r e n c e between MR-Nolg and MR-Lo In j c a l v e s . ADG 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 cal v e s on Col/WM and MR-Lo In j f o r any of the treatment periods. Figure 17. The E f fec t of Ig Supp lementa t ion on Average Dai ly Gain 1 -i 0-3 weeks 0-6 weeks 3-6 weeks PERIOD OF TRIAL Figure 18 shows e f f e c t of immunoglobulin supplementation on feed e f f i c i e n c y (kgs dry feed/kgs gain) of c a l v e s f o r the f i r s t s i x weeks of 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 poorer (P<0.05) feed e f f i c i e n c y than the c a l v e s i n the other two treatments. Figure 18. The Ef fect of Ig Supplementat ion on F e e d 3.00 2.50 -ro A | 2.00 ro E •o 1,50 1.00 Ef f ic iency for the Fi rst S ix W e e k s of L i fe C o l / W M M R - L o Inj M R - N o l g 66 Serum IgG Levels 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 Ig l e v e l s between any of the treatments at b i r t h , and days 21, 28, 35 and 42. At 24 hours and 48 hours of age, and at days 7 and 14, ca l v e s on Col/WM had s i g n i f i c a n t l y higher serum l e v e l s than c a l v e s on MR-Nolg (P<0.01) and cal v e s on MR-Lo I n j (P<0.05). From day 28 t o the end of the experimental p e r i o d , serum Ig l e v e l of calves on MR-Nolg continued t o r i s e and tended t o be higher than both Col/WM and MR-Lo Inj . 67 Blood Glucose Levels Blood glucose l e v e l s are shown i n Figure 20. Blood glucose l e v e l s f o r Col/WM calves were s i g n i f i c a n t l y h igher (P<0.05) than MR-Nolg and MR-Lo I n j c a l v e s at b i r t h . At day 7, Col/WM cal v e s were higher than MR-Nolg and MR-Lo I n j cal v e s but d i d not d i f f e r s i g n i f i c a n t l y . Blood glucose l e v e l s between treatments at a l l other times 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 each other (P>0.05). Figure 20 . The Ef fec t of Ig Supp lementa t ion on 180 -i Blood G l u c o s e Levels 160 -Treatments o o CD — C o l / W M 60 - M R - N o l g ^ - M R - L o Inj 40 0 7 14 i 21 : DAYS OF AGE 28 35 42 68 Blood Urea Nitrogen Levels Figure 21 shows blood urea n i t r o g e n (BUN) l e v e l s . BUN l e v e l 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 between any of the treatments at 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 higher BUN l e v e l s than MR-Nolg c a l v e s at 48 hrs 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 higher than MR-Lo I n j cal v e s at 48 hrs 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 than MR-Lo I n j cal v e s at day 42 (P<0.05). -o E 0) > C OI o CO Q) •o O O m 21. The Ef fec t of Ig Supplementat ion on B lood Urea Ni t rogen Leve ls 14 21 28 DAYS OF AGE 35 42 Treatments Col/WM MR-Nolg — M R - L o Inj 69 DISCUSSION S u r v i v a l during the f i r s t few days of l i f e i s r e l a t e d t o the amount of passive Ig obtained by the c a l f . The importance of t h i s i s shown by the 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 of MR-Nolg (c o n t r o l ) c a l v e s were s i g n i f i c a n t l y lower than the Col/WM or MR-Lo Inj 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 the MR-Nolg calves i n the f i r s t experiment (7/9) . This may be p a r t l y due t o the f a c t t h a t the c a l v e s i n experiment 2 d i d not r e c e i v e any a n t i b i o t i c therapy f o r scouring. Most of the MR-Nolg c a l v e s t h a t d i e d succumbed to E. c o l i septicemia w i t h severe dehydration. One c a l f each from Col/WM and MR-Lo I n j treatments a l s o died. The Col/WM c a l f d i e d w i t h i n the f i r s t 24 hours of age due t o a r o t a v i r u s i n f e c t i o n which was i s o l a t e d at post-mortem. This d i d not seem t o be a l l e v i a t e d even by feeding colostrum. I t i s p o s s i b l e t h a t the colostrum d i d not have a high l e v e l of Ig s p e c i f i c f o r t h i s r o t a v i r u s . As the colostrum was obtained from one farm only, the r o t a v i r u s may not have been present on the farm, and the cows would not have developed a 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 r o t a v i r u s . However, even i f there was 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, i t may not have been enough as the c a l f d i e d too soon t o be able t o have absorbed enough Ig. Research i n d i c a t e s t h a t the only way t o f i g h t r o t a v i r u s e s may be t o immunize at b i r t h (Dr. P. O'Brien, Comox V e t e r i n a r y C l i n i c , personal 70 communication). The MR-Lo In j c a l f d i e d of apparent b l o a t which was not r e l a t e d t o the treatments. Diarrhea l e v e l s (scours) of MR-Nolg ca l v e s trended higher than e i t h e r Col/WM or MR-Lo I n j c a l v e s throughout the s i x week p e r i o d of the experiment. By week three and during week four, MR-Nolg ca l v e s were scouring s i g n i f i c a n t l y more than MR-Lo In j calves (P<0.05). For the f i r s t three weeks of the experiment, Col/WM ca l v e s scoured more than the MR-Lo I n j c a l v e s but not s i g n i f i c a n t l y . Continued feeding of 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 scours l e v e l s over the c r i t i c a l f i r s t t hree weeks. R e c t a l temperatures were a l s o s i g n i f i c a n t l y higher (P<0.05) i n the MR-Nolg ca l v e s during week one and three. As i n experiment 1, the higher r e c t a l temperatures were i n d i c a t i v e of b a c t e r i a l i n f e c t i o n and were above 3 9°C i n the calves which scoured more se v e r e l y . Weekly weights 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 of the three treatments u n t i l day 35, when MR-Nolg c a l v e s were s i g n i f i c a n t l y lower (P<0.05) than e i t h e r Col/WM or MR-Lo In j c a l v e s . Average d a i l y gains f o r MR-Nolg ca l v e s were s i g n i f i c a n t l y lower than the other two treatments f o r the whole experiment (P<0.01) and they f a i l e d t o gain any weight i n the second week. As would be expected, the MR-Nolg ca l v e s had the poorest feed e f f i c i e n c i e s as w e l l . In agreement wi t h r e s u l t s i n the f i r s t experiment, experiment 2 MR-Nolg ca l v e s had higher r e c t a l temperatures, gained p o o r l y and d i d not catch up t o the Col/WM or MR-Lo Inj c a l v e s by s i x weeks of age. Serum Ig l e v e l s were i n d i c a t i v e of the treatments given and s i m i l a r t o r e s u l t s obtained 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 higher l e v e l s of Ig at 24 and 48 hours and at day 7 (P<0.01) , than both MR-Nolg and MR-Lo I n j c a l v e s . Again, the p o s s i b i l i t y of f a c t o r s i n the colostrum which could enhance the Ig absorption may be the reason (Balfour and Comline, 1962; Hardy, 1969). Serum Ig l e v e l s of the MR-Nolg calves show development of the a c t i v e immune system from about day 14 onward. There was a good l e v e l of absorption from the i n j e c t a b l e Ig s o l u t i o n w i t h an absorption e f f i c i e n c y of approximately 42%. Serum Ig l e v e l s i n MR-Lo Inj c a l v e s rose higher than the MR-Nolg cal v e s (6.3 vs 3.0 mg/ml) but d i d not r i s e as high as expected. The reason may have been t h a t there was some t i s s u e absorption of Ig at the s i t e s of i n j e c t i o n . IgG i s the s m a l l e s t immunoglobulin molecule and i s found not only i n the bloodstream but i n t i s s u e spaces as w e l l s i n c e i t can pass more e a s i l y through e n d o t h e l i a l t i s s u e s . Tissue absorption was not accounted f o r p r i o r t o s t a r t of experiment. This f a c t should be taken i n t o account when c a l c u l a t i n g c o n c e n t r a t i o n and amount of Ig s o l u t i o n t o be i n j e c t e d . Another s o l u t i o n t o i n crease serum Ig l e v e l s would be to g i v e a second group of i n j e c t i o n s s i x - twelve hours a f t e r the f i r s t t o boost serum l e v e l s . Further work i s r e q u i r e d t o determine optimal amounts and concentrations t o be used, however, these r e s u l t s i n d i c a t e t h a t an i n j e c t a b l e Ig s o l u t i o n given subcutaneously i s an e f f e c t i v e method of p r o v i d i n g systemic passive Ig i n the bloodstream. Blood glucose and blood urea n i t r o g e n l e v e l s were i n the normal range f o r newborn calves up t o s i x weeks of 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 at some times but these were probably 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 feeding r a t h e r than due t o treatments. Blood glucose l e v e l s showed a s l i g h t upward trend and BUN l e v e l s showed a shallow downward trend, however, f u r t h e r research i s needed t o determine i f these were t r u e treatment d i f f e r e n c e s . In c o n c l u s i o n , these r e s u l t s i n d i c a t e - t h a t a d m i n i s t r a t i o n of an i n j e c t a b l e s o l u t i o n of Ig can be used as an a l t e r n a t i v e method t o gi v e hypo-gammaglobulinemic calves a l e v e l of passive Ig independent of i n t e s t i n a l absorption which w i l l reduce m o r t a l i t y due t o septicemias. Continued 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 of scours and supports r e s u l t s obtained i n experiment 1. 73 CONCLUSIONS 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 an i n j e c t a b l e form o f immunoglobul ins a r e e f f e c t i v e a l t e r n a t i v e s t o c o l o s t r u m and r e s u l t i n 1) i n c r e a s e d s u r v i v a l r a t e 2) l e s s s evere s c o u r s 3) improved weight g a i n 4) adequate serum IgG l e v e l s C o n t i n u e d f e e d 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 i s an e f f e c t i v e method o f r e d u c i n g s c o u r s and i m p r o v i n g weight g a i n . 74 REFERENCES Anderson, K.L., Hunt, E. & Fleming, S.A. 1987. Plasma t r a n s f u s i o n s i n f a i l u r e of c o l o s t r a l immunoglobulin t r a n s f e r (1) . The Bovine P r a c t i t i o n e r - November 22:129. 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