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Artificial rearing of low birth-weight pigs 1982

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ARTIFICIAL REARING OF LOW BIRTH-WEIGHT PIGS by RICHARD JOHN WHITING Sc. Ag., The Univ e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 © Richard John Whiting, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of A/"Wy*»( ^ <X~*ir~L*t The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Four exper iments were conducted to eva lua te a crude immunoglobul in p r e p a r a t i o n f r a c t i o n a t e d from a b a t t o i r po rc ine serum w i t h ammonium s u l p h a t e . The p r e p a r a t i o n was used as an a d d i t i v e to m i l k r e p l a c e r f o r co los t rum d e p r i v e d , low b i r t h - w e i g h t p igs reared i n a n o n - i s o l a t e d envi ronment . S i x p igs per t reatment were used i n exper iments 1 and 3 . E igh t p igs per t reatment were used i n e x p e r i - ments 2 and 4 . In the f i r s t exper iment , members of the negat i ve c o n t r o l group d i d not s u r v i v e and m o r t a l i t y o f the o ther th ree groups which r e c e i v e d some immunity was h i g h . In the second exper iment , the nega t i ve c o n t r o l group was e l i m i n a t e d from the t r i a l , so those r e c e i v - i ng o n l y co los t rum f o r 12 hours d i e d , but the two groups r e c e i v i n g immunoglobul in t reatment showed improved s u r v i v a l (63 , 50%). In the t h i r d exper iment , h igher l e v e l s o f immunoglobul in ( 1 5 g . / k g . body we igh t , i n i t i a l l y f o l l owed by 5 g . / k g / d a y ) d i d not show a s i g n i f i c a n t e f f e c t on s u r v i v a l i n comparison w i th the p rev ious l e v e l s o f l O g . / k g . to 2 g . / k g . . However, r a t e o f ga in i n body weight was s i g n i f i c a n t l y h igher i n the h igh dose l e v e l o f immunoglobul in . In the f ou r t h exper iment the p igs were main ta ined on immunoglobul in f o r 10, 15 , and 21 days and i t was found t ha t 21 day t reatment e l i m i n a t e d dea ths . The h ighes t r a t e o f ga in was ach ieved w i t h those on 21 day t reatment i n exper iment 4 . However, these ra tes o f ga in were c o n s i d e r a b l y below those ach ieved with Digs on the sow. The causes o f m o r t a l i t y were predominate ly E. c o l i s c o u r s , sep t i caemia due to E. c o l i and o ther b a c t e r i a , pneumonia, and 1n the \n i i two experiments, Salmonellosis. The prevention of death in experiment 4 by the immunoglobulin extract, indicated the success of the preparation against the Salmonella species encountered. i i i TABLE OF CONTENTS Page 1. INTRODUCTION 1 2. LITERATURE REVIEW 2 2.1 The Mammalian Immune System 2 2.1.1 The Development of Immunocytes 2 2.1.2 The Intestinal Secretory Immune System . . . . 4 2.1.3 Antibody 5 2.1.3.1 The Structure of Antibodies 5 2.1.4 Complement 10 2.1.5 Antibody Reactions in the Gut Associated Lymphoid Tissue (GALT) 13 2.2 Prenatal Development of the Porcine Immune System . 16 2.3 Postnatal Development of the Porcine Immune System . 18 2.4 The "Runt" Phenomenon . 23 2.5 Mechanisms of Action of Bacteria in the Intestine. . 26 2.6 Diarrhea in Piglets 28 2.7 Non-Specific Gastro-Intestinal Immune Mechanisms . . 30 2.7.1 The Gastric Trap 30 2.7.2 Intestinal Motility 30 2.7.3 Intestinal Microflora 31 2.7.4 Non-Immunological Factors in the Secretions of the G. I. Tract 32 2.8 Humoral and Cellular Immunity in Porcine Mammary Secretions 34 2.9 Preweaning Mortality 36 2.9.1 Causes of Preweaning Mortality 36 iv 2 . 9 . 2 E f f e c t o f Low B i r t h -We igh t on S u r v i v a l . . . 38 3 . EXPERIMENTAL 42 3.1 I n t r o d u c t i o n 42 3.2 Exper iment I 44 3 .2 .1 O b j e c t i v e 44 3 . 2 . 2 M a t e r i a l s and Methods 44 3 . 2 . 2 . 1 Exper imenta l Animals 44 3 . 2 . 2 . 2 P r e p a r a t i o n o f P o r c i n e Immunoglobulins 44 3 . 2 . 2 . 3 D i e t a r y Treatments 44 3 . 2 . 2 . 4 Feeding Regimen . 45 3 . 2 . 3 Measurments and Observa t ions 45 3 .2 .4 Resu l t s and D i s c u s s i o n 46 3.3 Exper iment II 49 3 .3 .1 Ob jec t i ve 49 3 . 3 . 2 M a t e r i a l s and Methods 49 3 . 3 . 2 . 1 Exper imenta l Animals 49 3 . 3 . 2 . 2 D i e t a r y Treatments 49 3 . 3 . 2 . 3 Feeding Regimen 50 3 . 3 . 3 Measurments and Observa t ions 50 3 .3 .4 Resu l t s and D i s c u s s i o n 51 3.4 Exper iment I I I 54 3 .4 .1 O b j e c t i v e 54 3 . 4 . 2 M a t e r i a l s and Methods 54 3 . 4 . 2 . 1 Exper imenta l Animals 54 3 . 4 . 2 . 2 D i e t a r y Treatments 54 v 3 . 4 . 2 . 3 Feeding Regimen 55 3 . 4 . 3 Measurments and Observa t ions 55 3 .4 .4 R e s u l t s and D i s c u s s i o n 56 3.5 Experiment IV 64 3 .5 .1 O b j e c t i v e 64 3 . 5 . 2 M a t e r i a l s and Methods 64 3 . 5 . 2 . 1 Exper imenta l Animals 64 3 . 5 . 2 . 2 D i e t a r y Treatments 64 3 . 5 . 2 . 3 Feeding Regimen 65 3 . 5 . 3 Measurments and Observa t ions . . . 65 3 .5 .4 R e s u l t s and D i s c u s s i o n 65 3.6 General D i s c u s s i o n 70 3.7 Conc lus ions 75 4 . BIBLIOGRAPHY 77 v i LIST OF TABLES Tab le Page 2.1 Immunoglobulin S t r u c t u r e 9 2.2 Mechanisms o f Ant ibody A n t i - B a c t e r i a l A c t i v i t y 14 2 . 3 P rena ta l Development o f the Po rc ine Immune System . . . 17 2.4 A Summary o f Major Causes C o n t r i b u t i n g to Death i n Baby P igs 37 2 .5 E f f e c t o f B i r t h Weight on M o r t a l i t y 39 3.1a Exper iment I: Exper imenta l Animals 47 3.1b Exper iment I: Resu l t s 47 3.2a Exper iment I I : Exper imenta l Animals 52 3.2b Experiment I I : Resu l t s 52 3.3a Exper iment I I I : Exper imenta l Animals 57 3.3b Exper iment I I I : R e s u l t s 58 3.4a Exper iment IV : Exper imenta l Animals 66 3.4b Exper iment IV : Resu l t s 66 v i i LIST OF FIGURES F igu re Page 2.1 The IgG Ant ibody M o l e c u l e : a Schemat ic P r e s e n t a t i o n o f S t r u c t u r e 6 2 .2 A c t i v a t i o n Pathways and P h y s i o l o g i c a l Funct ions o f Complement Components 12 2 .3 Serum Immunoglobulin P r o f i l e s o f the Neonatal P i g . . . 20 3.1a Growth i n Experiment I I I , R e p l i c a t e 1 60 3.1b Growth i n Experiment I I I , R e p l i c a t e 2 61 3.2 Scou r ing Frequency 63 3 .3 Cumulat ive Weight G a i n : Exper iment IV 68 3.4 Comparat ive Growth Curves 71 v i i i ACKNOWLEDGMENTS I would l i k e to g r a t e f u l l y acknowledge the wonderful patience and understanding of my project supervisor, Dr.B.D. Owen who provided strong support during some d i f f i c u l t periods. I should also l i k e to thank Dr. R.M. Beames f o r his assistance toward completion of the study. Dr. R. Peterson provided assistance with s t a t i s t i c a l analyses. I am indebted to the people at B.C. Veterinary Laboratory i n Abbotsford f o r carrying out autopsies and b a c t e r i o l o g i c a l assess- ments. F i n a l l y , I would l i k e to express thanks to the three summer students, B r i g i t t e Sonendrucher, Larry Nault, and Don Roberts, who helped during odd hours of the night and day to carry out the experiments. ix 1 1. INTRODUCTION Severa l s t u d i e s have been done on the a r t i f i c i a l r e a r i n g o f neonatal p igs us ing supplemental immunoglobul ins added to the d i e t (Owen, B e l l , and W i l l i a m s , 1961; S c o o t , 1972; McCal lum, 1977; Lodge and E l l i o t , 1979) . The exper iments repor ted he re in were at tempts to r a i s e low b i r t h w e i g h t p i g s , i n c l u d i n g s o - c a l l e d r u n t s , i n a farm environment us ing p r e v i o u s l y s t u d i e d l e v e l s and pe r iods o f serum-der ived immuno- g l o b u l i n supp lementa t ion . Below a b i r t hwe igh t o f 900 to 1000 grams, p i g m o r t a l i t y dur ing the f i r s t week of l i f e d r a m a t i c a l l y i nc reases (Pomeroy, 1960; Sharpe , 1966; L e f c e , 1971; Fahmy and Be rna rd , 1971; Be resk in e t a l . , 1973; E n g l i s h and Smi th , 1975) . Prev ious workers have found t h a t , g i ven an environment removed from compe t i t i on and t rauma, low b i r t h - weight p igs can be a r t i f i c i a l l y reared w i th low m o r t a l i t y and more s a t i s f a c t o r y growth ra tes ( L e c c e , 1971; Eng land , 1974) . .However, these e a r l i e r s t u d i e s u t i l i z e d a s e m i - i s o l a t e d environment and s t e r i l e food . The cu r ren t p r o j e c t i nvo l ved at tempts to r a i s e these low b i r t h w e i g h t p igs i n a n o n - i s o l a t e d environment us ing added immunoglobul ins to e l i m i n a t e the need f o r an i s o l a t e d envi ronment . 2 2 . LITERATURE REVIEW 2.1 The Mammalian Immune System 2.1.1 The Development o f Immunocytes The major e f f e c t o r c e l l s i n immunity a re the lymphocytes, o f which there are two c l a s s e s . There are T lymphocytes where "T" r e f e r s to t h e i r d e r i v a t i o n i n the thymus g land and there are B lymphocytes where the " B " r e f e r s to the major d i f f e r e n t i a t i o n reg ion i n the b i r d c a l l e d the Bursa o f Fab r i cus (Cooper and Lawton, 1974) . The bursa has no analogous reg ion y e t demonstrated i n the mammal a l though s i m i l a r development o f B c e l l s here seems to occur i n the f e t a l l i v e r or bone marrow. (Cooper & Lawton 1974) . These lymphocytes are de r i ved from a common stem c e l l f o r hemopoiesis from which o ther c i r c u l a t i n g c e l l s are d e r i v e d . Thus, lymphocytes, monocytes, p o l y - morphonuclear l e u c o c y t e s , macrophage? megakaryocytes ( from which p l a t e l e t s are d e r i v e d ) , mast c e l l s , e o s i n o p h i l s and e r y t h r o c y t e s are a l l de r i ved from the same stem c e l l . A l l , except e r y t h r o c y t e s , are i n v o l v e d i n the immune system. The p r i m o r d i a l stem c e l l f i r s t develops i n the b lood i s l a n d s of the embryonic y o l k sac and then migra tes to the l i v e r o f the f e t u s . La te r i n g e s t a t i o n and du r ing adu l t l i f e these stem c e l l s are found i n the bone marrow, hav ing migrated from the f e t a l l i v e r , and they con t inue the very a c t i v e p ro - cess o f hemopoiesis throughout the l i f e o f the i n d i v i d u a l (Hood e_t a_K, 1978). In the hemopoiet ic t i s s u e s a s u b t l e and as ye t undetermined d i f f e r e n t i a t i o n occurs g i v i n g r i s e to c e l l s which w i l l i n d i v i d u a l l y home to a s p e c i f i c t i s s u e microenv i ronment . Here f u r t h e r d i f f e r e n t i a t i o n and a commitment occurs which means t ha t the c e l l i s s e t on a u n i - d i r e c t i o n a l path o f development. T c e l l p recu rso rs become committed i n the microenvironment o f the thymus under the c o n t r o l o f thymus hormone; B c e l l s are committed i n the fetal l iver, bone marrow and, possibly Peyer's patches (Bienstock, 1979); macrophages and polymorphonuclear leucocytes in the bone marrow and spleen; and mast cells in the bone marrow (Hood et aj_., 1978). The lymphocytes develop within these specific tissues in an antigen independent differentiation until they are capable of responding to antigens. At this stage both B and T cells carry antigen receptor molecules on their membranes. The B cell receptor molecule is an immunoglobulin. The T cell receptor molecule is as yet unidentified but does not seem to be immunoglobulin (Benaceraf and Unanue, 1979). The B cell immunoglobulin is f irst IgM but later bears the isotype to be produced by the end cells. (Hood et a l . , 1978). If these lymphocytes bind a cognate antigen to these surface receptors they become activated; they enlarge with the formation of polysomes, microtubules and macromolecules in a process called blast transformation (Hood et a l . , 1978). These activated cells proliferate and continue to differentiate. Thus clones of T cells are formed containing both effector cells and memory T cells. Similarly, clones of effector and memory B cells are formed. The memory cells remain in circulation and provide a continuously reactive clone of cells which respond in a secondary immune response upon later antigen stimulation. The major effector cells are directly involved in antibody production as with T helper and T suppressor and plasma cells (B end cells) or in cell mediated immunity as with T kil ler and T - delayed hypersensitivity cells. (Hood et a l . , 1978). The function of the lymphoid system is to provide maximal contact of any antigen with its repertoire of antigen specific lymphocytes. Foreign material is phagocytized by macrophages which present antigen from 4 the f o r e i g n m a t e r i a l to the T c e l l s . These T c e l l s (T he lpe rs ) are then a c t i v a t e d and s t i m u l a t e maximal an t ibody p roduc t ion by the plasma c e l l s . Any an t igen which en te rs t i s s u e i s gathered i n t o the l ymphat i c system by the lymph f l u i d and c a r r i e d to a nearby f i l t e r i n g lymph node o r the sp leen to s t i m u l a t e ant ibody p r o d u c t i o n . An t igens which en te r the upper r e s p i r a t o r y t r a c t o r g a s t r o - i n t e s t i n a l t r a c t encounter l o c a l lymph nodes as w e l l as s p e c i a l i z e d o rgans : t o n s i l s , adeno ids , P e y e r ' s pa t ches , and the appendix . B lood borne an t igens are d e a l t w i th ma in ly by the s p l e e n . (Hood e t a l . , 1978), 2 . 1 . 2 The I n t e s t i n a l Sec re to r y Immune System A major a rea o f con tac t w i t h a n t i g e n i c m a t e r i a l i n the e n v i r o n - ment i s a t the mucosal l i n i n g o f the g a s t r o - i n t e s t i n a l sys tem. Consequent to the development o f gut m i c r o f l o r a , the lamina o f the gut becomes i n f i l t r a t e d w i t h immunocytes produc ing predominate ly IgM and IgA which are t r anspo r ted ac ross the gut e p i t h i l i u m i n t o the lumen ( P o r t e r e t a l _ . , 1976) . I n i t i a l l y i n the development o f a young animal IgM producing c e l l s predominate but l a t e r there i s a g rea te r p r o l i f e r a t i o n o f IgA c e l l s ( A l l e n and P o r t e r , 1973) . The i n t e s t i n a l t r a c t i s e s p e c i a l l y adapted f o r p roduc t ion o f an t ibody to the lumena l l y o c c u r r i n g an t igens o f b a c t e r i a , v i r u s e s , and o ther food borne m a t e r i a l s . The mucosa o v e r l y i n g the P e y e r ' s patches appears to be adapted to the uptake o f l a r g e molecu les and even i n t a c t microorganisms ( M c C l e l l a n d , 1979) . In t h i s way p recu rso r lymphocytes are s e n s i t i z e d i n the P e y e r ' s patches and a re r e l e a s e d to c i r c u l a t e and home i n t o the i n t e s t i n a l lamina p r o p r i a where they develop i n t o IgA s e c r e t i n g plasma c e l l s ( P i e r c e and Cowans, 1975; Hasland e t a l _ . , 1976) . The development o f a sys temic an t ibody p roduc t ion due to the g a s t r o - i n t e s t i n a l immunizat ion rou te may a l s o occur from IgG or IgM forming 5 c e l l s which are seeded by P e y e r ' s patches i n e x t r a - i n t e s t i n a l lymphoid t i s s u e s . A l s o there i s the p o s s i b i l i t y o f an t igen reach ing lymphocytes i n the sp leen o r p e r i p h e r a l lymph nodes from the gut (C ra ig and Cebra , 1971) . Serum IgA a l s o o r i g i n a t e s from g a s t r o i n t e s t i n a l secretion (Tomasi , 1976) . Mammary s e c r e t i o n s o f an t ibody a re found to be s e n s i t i z e d to i n t e s t i n a l an t igens (Chid low and P o r t e r , 1979) . Th is i s the g u t - t o mammary a x i s r e f e r r e d to by Parmely e t a K , (1976) . IgA s e c r e t i n g lympho- c y t e s , p o s s i b l y o r i g i n a t i n g i n P e y e r ' s patches and t r a v e l l i n g through the mesenter ic lymph nodes i n t o c i r c u l a t i o n are t rapped by a c t i v e mammary t i s s u e under the i n f l u e n c e o f mammotropic hormones (Lamm e t a l _ . , 1978) . 2 . 1 . 3 Ant ibody Ant ibody i s produced by plasma c e l l s which are end B c e l l s ( N o s s a l , 1976) . The an t igen i s he ld by the immunoglobul in recep to r i n the c e l l membrane and i s processed by i n t e r n a l i z a t i o n o r e n d o c y t o s i s . The b ind ing o f an t i gen by B c e l l s i s e s s e n t i a l f o r the development o f p roduc t ion o f an t ibody (Benaceraf and Unanue, 1979) . The an t ibody i s o f f i v e i s o t y p e s : M, G, A , D, o r E . M, G , or A are r e s p o n s i b l e f o r humoral and sec re to r y defense w h i l e IgD i s p r e s e n t l y on l y known as a membrane bound recep to r on many lymphocytes, and IgE i s concerned w i th the a l l e r g i c response (Benaceraf and Unanue, 1979) . 2 . 1 .3 .1 The S t r u c t u r e o f A n t i b o d i e s ( f i g u r e 2 .1 ) A n t i b o d i e s belong to a c l a s s o f p r o t e i n s c a l l e d immuno- g l o b u l i n s . The b a s i c u n i t o f s t r u c t u r e i s a complex o f f o u r p o l y p e p t i d e s , two i d e n t i c a l " l i g h t " ( low mo lecu la r weight) cha ins and two i d e n t i c a l "heavy" (h igh mo lecu la r weight ) cha ins l i n k e d toge ther by d i s u l p h i d e 6 L Choin Hypervoriable Regions CH3 Figure 2.1. The IgG Antibody Molecule: a' Schematic Presentation of Structure (Benaceraf and Unanue, 1979) 7 bonds (Hood e t . a l . , 1978) . The f i v e c l a s s e s o f a n t i b o d i e s are c a l l e d i s o t y p e s and t h e i r d i f f e r e n t s t r u c t u r e s are determined by t h e i r heavy c h a i n s . Thus , IgG has gamma heavy c h a i n s , IgM has mu, IgA has a l p h a , IgD has d e l t a , and IgE has e p s i l o n heavy c h a i n s . A l l i s o t y p e s may have one o f two types o f l i g h t c h a i n , e i t h e r kappa o r 1 ambda. The heavy cha ins have th ree cons tan t reg ions and one v a r i a b l e reg ion (a t the amino- te rm ina l end) w h i l e the l i g h t cha ins have one cons tan t w i t h one v a r i a b l e r e g i o n . The v a r i a b l e reg ions are f u r t h e r composed o f framework r e g i o n s , and h y p e r v a r i a b l e reg ions which are the l e a s t homogenous reg ions o f the immunoglobul in (Beneceraf and Unanue, 1979) . The v a r i a b l e reg ions o f the heavy and l i g h t cha ins form a c l e f t which i s the s p e c i f i c b ind ing s i t e o f the an t ibody f o r an an t i gen (Searhar t e t a l _ . , 1981) . The b ind ing s i t e i s p r e c i s e l y complementary to the s t r u c t u r e o f the an t igen and b inds to the an t igen by weak e l e c t r o s t a t i c a t t r a c t i o n , hydrogen bond ing , and Van der Waal f o r c e s (Bach , 1978) . The s y n t h e s i s o f these immunoglobul ins i s c o n t r o l l e d by gene c l u s t e r s (Hood e t a_K, 1978) . For each heavy or l i g h t cha in a d i v e r s e c l u s t e r o f V ( v a r i a b l e ) reg ion genes o f unknown number i s l i n k e d w i t h a c l u s t e r o f C (cons tan t ) r eg ion genes on one chromosome. Thus, heavy cha in c l u s t e r s o f V and C reg ions may be on one chromosome, a kappa C reg ion w i th a c l u s t e r o f V reg ions on ano the r , and a lambda C reg ion w i th a c l u s t e r o f V reg ions on another . A s i n g l e V reg ion on the chromosome f o r the heavy cha in can be a s s o c i a t e d w i t h two o r more C reg ion genes du r i ng the d i f f e r e n t - i a t i o n o f an t ibody produc ing c e l l s (Hood e t . a l . , 1978) r e s u l t i n g i n i d e n t i c a l i d i o t y p e s ( i . e . i d e n t i c a l v a r i a b l e reg ions ) among d i f f e r e n t i s o t y p e s . Fu r the r work by Tonegawa e t a]_., (1978) found the presence o f J ( j o i n i n g ) 8 l o c i on l i g h t cha in chromosomes, and both J and D ( d i v e r s i t y ) l o c i on heavy cha in chromosomes. D i v e r s i t y o f an t ibody p roduc t ion to deal w i th the many p o s s i b l e an t igens i n the env i ronment , i n t heo ry , r e s u l t s from two p o s s i b l e mechanisms (Hood e t a l _ . , (1978) . The f i r s t i s d i v e r s i t y w i t h i n the same germ l i n e ( i . e . d i v e r s i t y o f V reg ion genes) . Thus, a combinat ion from the s e l e c t i o n o f v a r i a b l e l i g h t cha in genes w i t h v a r i a b l e heavy cha in genes produces a m u l t i p l e o f them i n an t ibody c l e f t s . In combinat ion w i th J and D l o c i there i s a f u r t h e r m u l t i p l i c a t i o n o f d i v e r s i t y (Gearhar t e t a l _ . , 1981). The second mechanism o f an t ibody d i v e r s i t y cou ld r e s u l t from somat ic mutat ion or recombinat ion which cou ld occur dur ing an i n d i v i d u a l ' s immune development. The consequence o f these mechanisms i s a ve ry d i v e r s e r e p e r t o i r e o f an t ibody s p e c i f i c i t i e s w h i c h , i n the presence o f an t i gen s t i m u l a t i o n , leads to a c o n t i n u o u s l y c i r c u l a t i n g a r ray o f an t i gen s p e c i f i c lymphocytes ready to p r o - l i f e r a t e under heav ie r an t i gen s t i m u l a t i o n . The newborn i s i nexper ienced i n the environment which i s c o n s t a n t l y p resen t i ng d i v e r s e an t igens and consequent- l y has an undeveloped r e p e r t o i r e o f lymphocytes to respond. The an t ibody c l a s s e s d i f f e r i n gross fo rmat ion ( Table 2.1) IgG i s a monomer o f two heavy and two l i g h t s cha ins o f immunoglobul in . IgM i s a pentamer which i s he ld together by a j o i n i n g (J) cha in w i th d i s u l p h i d e bonds (Tomasi , 1976) . IgM may a l s o be a s s o c i a t e d i n d i s u l p h i d e o r non-cova len t bonds w i th another p r o t e i n , the s e c r e t o r y component (Brandtzaeg & B a k l i e n , 1977) a t l e a s t i n human IgM. IgA may e x i s t as a monomer, o r as a dimer w i t h a J cha in as i n serum. I t may a l s o e x i s t as s e c r e t o r y IgA (S- IgA) which i s a dimer w i t h J cha in and s e c r e t o r y compoent (SC) . The b i nd ing o f SC i n IgM i s weaker (more non-cova len t bonding) than i n S-IgA (Brandtzaeg & B a k l i e n , 1977) . IgD Immunoglobulin Light Chain Type Heavy Chain Type Other Components Structure 10 and IgE both e x i s t as monomers. Both J cha in and Sec re to r y Component a re po l ypep t i des (Tomasi , 1976) . Accord ing to Brandtzaeg & B a k l i e n (1977) J cha in i s s y n t h e s i z e d i n the plasma c e l l w i t h immunoglobul in where i t po lymer izes the IgA o r IgM. SC i s produced by serous type e p i t h i l i a l c e l l s and a c t s as a s p e c i f i c r ecep to r f o r the immunoglobul in . Cova len t and non-cova len t i n t e r a c t i o n between SC and immunoglobul in a re completed du r ing the passage o f the complex through the e p i t h i l i a l c e l l s and , con jugated IgA (o r IgM) w i th S C , p lus f r e e SC i s t r anspo r ted i n t o the lumen by e x o c y t o s i s (Benaceraf and Unanue, 1979) . S ince S-IgA i s the predominate immunoglobul in i n a l l body f l u i d s o u t s i d e the v a s c u l a r system (w i th the excep t i on o f co los t rum which con ta ins most ly IgG) ( P o r t e r , 1976) , t h i s model probably a p p l i e s to a l l e xosec re t i ons o f s e c r e t o r y IgA. SC conjugated IgA i s r e s i s t a n t to p r o t e o l y s i s by g a s t r o - i n t e s t i n a l enzymes (Tomasi and C a l v a n i c o , 1968) . SC i s a l s o thought to f a c i l i t a t e b ind ing o f the immunoglobul in to the mucosa coat o f the i n t e s t i n a l e p i t h i l i u m ( P o r t e r e t a l _ . , 1972) . 2 . 1 . 4 Complement The term complement r e f e r s to a complex group o f enzymes i n normal b lood serum (Mayer, 1973) . Thus , the complement system c o n s i s t s o f 17 plasma p r o t e i n s (Benaceraf and Unanue, 1979) which comprise a s i g n i f i c a n t p o r t i o n o f the serum g l o b u l i n f r a c t i o n (Hood e t a j_ . , 1978; Mayer , 1973) . These p r o t e i n s f a l l i n t o two f u n c t i o n a l g roups . One group i s the c l a s s i c a l com- ponents which a re symbol ized w i t h a c a p i t a l C and a number, one to n i n e . The o the r group i s the components o f the a l t e r n a t e pathway which a re denoted w i t h a c a p i t a l l e t t e r and are B , D, and P ( p r o p e r d i n ) . Many o f these p r o t e i n s a re c leaved du r ing complement r e a c t i o n s and c leavage fragments are s u f f i x e d w i th \ 11 lower case l e t t e r s , f o r example, C3a and C3b, w i th the s u f f i x b denot ing the l a r g e r o f the two fragments (Beneceraf and Unanue, 1979) . The f u n c t i o n s o f complement are s e v e r a l - f o l d . Complet ion o f the complement sequence on an a t tacked c e l l leads to c y t o l y s i s . The c leavage product o f C 3 , C3B, b inds to microorganism c e l l su r faces producing immune adherence which f a c i l i t a t e s phagocytos is (Mayer, 1973; Benaceraf and Unanue, 1979) . C3a and C5a c leavage fragments are anaphy lo tox ins which are noted f o r caus ing l e t h a l bronchospasm i n guinea p igs and wheal and f l a r e r e a c t i o n s i n human s k i n by deg ranu la t i ng mast c e l l s (Benaceraf and Unanue, 1979) . Anaphy lo tox ins are a c t i v e i n o the r mammals a l s o , caus ing acute in f lammat ion (Hood e t a l _ . , 1978) . C5a and f r e e C5b-6-7 complex are chemo- t a c t i c f a c t o r s which a t t r a c t polymorphonuclear l eucocy tes (PMN), which are phagocytes , to a s i t e o f complement a c t i v i t y (Hood e t a l _ . , 1978) . The sequence o f the complement cascade i s most s imp ly demonstrated i n f i g u r e 2 . 2 . E s s e n t i a l l y , there i s an e f f e c t o r pathway (C5 to C9) which "punches" a hole i n the a f f e c t e d c e l l and can be a c t i v a t e d by e i t h e r the c l a s s i c a l sequence i n v o l v i n g C l , C2 , C4 and ca l c ium or the a l t e r n a t e sequence i n v o l v i n g B, D, P and magnesium. The c l a s s i c a l sequence requ i r es s p e c i f i c an t ibody which must be e i t h e r IgM o r IgG. A s i n g l e molecu le o f IgM bound to an an t igen on a c e l l membrane w i l l i n i t i a t e the c l a s s i c a l sequence whereas two ad jacent IgG molecu les are necessary . S ince an t ibody i s s c a t t e r e d over the c e l l su r face q u i t e randomly, the p r o b a b i l i t y o f two IgG molecu les occupying ad jacen t s i t e s i s q u i t e smal l and t he re fo re the f requency w i th which IgG a c t i v a t e s the c l a s s i c a l sequence i s low (Mayer, 1973) . The a l t e r n a t e pathway i s a n o n - s p e c i f i c mechanism o f immune defense (Mayer, 1977) s i n c e i t d o e s n ' t r e q u i r e an t ibody f o r i n i t i a t i o n . I t i s impor tant i n defense aga ins t 12 activation antigen-antibody complex (classical pathway) physiological ^ functions endotoxin (alternative pathway) properdin factors B and D immune adherence and phagocytosis , CS freeCSb.6,7 C7 complex CB OB | holes in membrane (lysis) | Acute inflammation blood-vessel dilation transudation P M N chemotaxls phagccytosii lysosomal activation necrosis repair regeneration Figure 2.2 A c t i v a t i o n Pathways and P h y s i o l o g i c a l Functions of Complement Components (Hood et al_., 1978) 13 gram-negative bacteria that inhabit the gut. Lipopolysaccharide from the c e l l walls of these organisms (endotoxins) combine d i r e c t l y with the serum f a c t o r , properdin (Hood et al_., 1978). There i s i n d i c a t i o n that S-IgA w i l l also act with lysozyme to ac t i v a t e complement f i x a t i o n by the alternate pathway ( H i l l and Porter, 1974). Both the c l a s s i c a l a c t i v a t i o n sequence and the alternate pathway lead to cleavage of C3 which i n i t i a t e s the e f f e c t o r sequence. 2.1.5 Antibody Reactions in the Gut Associated Lymphoid Tissue (GALT) Table 2.2 l i s t s most of the known a c t i v i t i e s of antibody both systemically and e x t e r n a l l y as "secretory antibody". Within the gut associated lymphoid tissue (GALT) there are e s s e n t i a l l y two l i n e s of antibody defense as interpreted by Tomasi (1976). The f i r s t l i n e of defense involves reactions within the i n t e s t i n a l lumen and the second l i n e of defense involves reactions in the tissues of the GALT a l l i e d to systemic immunity. The f i r s t l i n e of defense i s mainly c a r r i e d out by S-IgA (as copra antibodies). Miler et aK (1975), using l i g a t e d porcine i n t e s t i n a l segments and l i v e E . c o l i cultures as challenge i n j e c t i o n s , found that l o c a l protective e f f e c t s of immunoglobulins IgG, IgM, and IgA were d i f f e r e n t , the minimum e f f e c t i v e concentrations being 0.5, 0.05, andO.005 mg/ml resp e c t i v e l y for IgG, M, and A. Miler et al_. (.1975) further stated that IgA does not possess enterotoxin n e u t r a l i z i n g a c t i v i t y and that the mechanisms of action might be due to i n h i b i t i o n of absorption of bacteria or by blocking of binding of enterotoxin to receptors on e p i t h e l i a l c e l l s . The e f f i c a c y of IgA was further demonstrated by Steele et al_. (1974). They found that of the immuno- globulins, S-IgA was most e f f e c t i v e in reducing mortality in rabbits infected with l i v e cholera organisms. 14 Table 2.2 Mechanisms of Ant ibody A n t i - b a c t e r i a l A c t i v i t y C lass o f Ant ibody A c t i v i t y IgG Tox in n e u t r a l i z a t i o n A g g l u t i n a t i o n 1 IgM O p s o n i z a t i o n 1 B a c t e r i o l y s i s by complement f i x a t i o n I n h i b i t i o n o f b a c t e r i a l adhe rence* Tox in n e u t r a l i z a t i o n 1 A g g l u t i n a t i o n * O p s o n i z a t i o n J i B a c t e r i o l y s i s by complement f i x a t i o n IgA A n t i - t o x i n a c t i v i t y * A g g l u t i n a t i o n M I n h i b i t i o n o f b a c t e r i a l m o t i l i t y B a c t e r i o s t a s i s 5 I n h i b i t i o n o f b a c t e r i a l adherence* Complement f i x i n g by the a l t e r n a t e pathway * B ind ing to mucin o f mucosa* I n h i b i t i o n o f b a c t e r i a l enzymes I n h i b i t i o n o f f o r e i g n an t igen uptake J 1. Benaceraf and Unanue, 1979 2. Tomasi , 1976 3 . Brandenburg and W i l s o n , 1974 4 . M c C l e l l a n d , 1979 5. P o r t e r , e t a l _ . , 1976 15 The second l i n e o f de fense , o r sys temic immunity, depends l a r g e l y on IgG and IgM. The a c t i v i t i e s o f IgG and IgM are q u i t e d i f f e r e n t . Due to i t s pentameric fo rm, IgM has more a c t i v e an t ibody b ind ing s i t e s than IgG (Bach, 1978) . Thus, the a c t i v i t y o f IgM i s cons ide red to be g rea te r i n ant ibody r e a c t i o n s . Rowley and Turner (1966) found the 8 IgM mo lecu les /bac te r i um were necessary as opsonins whereas 2200 IgG mo lecu les / bac ter ium were necessary . Brandenbourg and Wi lson (1974) s t a t e d tha t IgM was 500 to 1000 t imes more e f f e c t i v e as an opsonin than IgG. They a l s o s t a ted tha t IgM was much more e f f e c t i v e i n complement f i x a t i o n (See Sec t i on 2 . 1 . 4 ) . S t e e l e e t a l _ . , (1974) developed data suppor t ing the g rea te r a c t i v i t y o f IgM us ing r a b b i t Ig aga ins t V. c h o l e r a e . P o r t e r e t a l ^ , (1977) say tha t the dominant immunoglobul ins found i n the lamina p r o p r i a are IgM and IgA. S ince IgM may form on ly a smal l pa r t o f the sec re ted I g , i t may form a s t rong second l i n e o f defense i n t i s s u e s behind the e p i t h i l i u m ( A l l e n and P o r t e r , 1973) where i t can c a r r y out complement f i x 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 t o x i n n e u t r a l i z a t i o n aga ins t pathogenic i n v a d e r s . 16 2.2 P rena ta l Development o f the Po rc ine Immune System (see Table 2.3) Working w i t h the f e t a l lamb and f e t a l mouse i t was found tha t development o f competence i n the mammalian f e tus occurs i n a c o n t r o l l e d , s t ep -w i se f a s h i o n . "A h i e r a r c h y o f an t igens e x i s t s to which the f e t u s develops competence a t d i f f e r e n t s tages o f g e s t a t i o n " ( S i l v e r s t e i n , 1977) . The immunologic competence matura t ion i s independent o f lymphoid deve lop - ment. The f e t u s be fore having o rgan ized lymphoid t i s s u e i s ab le to respond to some an t igens presumably u t i l i z i n g lymphoid c e l l s i n the f e t a l l i v e r . Once i t i s competent to a c e r t a i n a n t i g e n , the fe tus i s ab le to produce comparable ant ibody t i t r e s to an a d u l t , and forms them i n the usual sequence o f immunoglobul in c l a s s e s (IgM fo l l owed by IgG) ( S i l v e r s t e i n , 1977) . How- e v e r , i n c o n t r a d i c t i o n , there may be some r e s t r i c t i o n s to f e t a l response. The l e v e l o f an t igen needed f o r s t i m u l a t i o n may be h igher ( S t e r z l , 1963) , o r there may be fewer stem c e l l s , o r there may be c l o n a l r e s t r i c t i o n i n the i n i t i a l response (Kl inman and P r e s s , 1975) . 17 Table 2.3 Prenatal Development of the Porcine Immune System Gestational Time of Occurrence Event Day 28 Day 32 Day 38 Day 40 Day 51 Before 65 days Day 77 After 70 - 80 days Aft e r 80 days At b i r t h Lymphoid c e l l s are detected i n the region of the thymus' The lymph nodes begin forming without germinal centres (unless the animal i s infected/ , Lymphoid c e l l s are detected i n the l i v e r and blood Components of the complement system are present * Number of lymphocytes gradually begin to increase in the spleen 1 Less than 1% of the lymphocytes have surface immunoglobulin^ H i s t o l o g i c a l l y , the thymus i s i d e n t i c a l to development at b i r t h ' In the lamina propria of the terminal ileum lymphocytes occur in f o l l i c u l a r aggregations' Approximately 10% of blood lymphocytes have surface immunoglobulin i n d i c a t i n g development of immunocompetence* A more generalized f o l l i c u l a r lymphoid structure i s developing with larger populations of lympho- cytes. Immune responses can be shown against several antigens: formalized Salmonella* , sheep red blood c e l l s * and phage 6 . Transplantation immunity, to allogeneic c e l l s was induced a f t e r 80 days ; i f injected at 60 days, allogeneic c e l l s induce tolerance to skin g r a f t s . 7 Peyer's patches are present' 1. Kruml et al_., 1970 2. Pestana e t al_., 1965 3. Day et al_., 1969 4. Binns, 1973 5. Binns and Symons, 1973 6. Tlaskalova e t al_., 1970 7. Binns, 1967 18 2.3 Pos tna ta l Development o f the Porc ine Immune System In germ-f ree neonatal p i g l e t s , no immunoglobul in i s found w i th the excep t ion o f a smal l " h a l f mo lecu le " o f IgG type which has been shown to c o n s i s t o f one heavy and one l i g h t cha in and i s not an t igen respons ive (Prokesova e t a l _ . , 1970) . Th is fragment has been t h e o r i z e d as a r e l e a s e d , membrane-associated immune recep to r molecu le from lymphocytes ( P o r t e r , 1979). Due to an e p i t h i l i o c h o r i a l p l a c e n t a , the p ig f e tus i s prevented from r e c e i v i n g ant ibody from the dam's c i r c u l a t i o n . The reason f o r t h i s i s u n c l e a r , because, a l though i n the e a r l y embryonic s tages as many as s i x h i s t o l o g i c a l l y d i s t i n c t l a y e r s separa te the two b loods t reams, i n l a t e r pregnancy the embryonic c a p i l l a r i e s develop more i n s u b - e p i t h i l i a l p o s i t i o n s and invade the embryonic t r o p h o b l a s t so tha t maternal and f e t a l b loods are i n c l o s e p rox im i t y (Mar rab le , 1971) . Whatever the s i t u a t i o n o f the c i r c u l a t o r y s u p p l y , no maternal a n t i b o d i e s (or immeasurable q u a n t i t i e s ) pass (Kim e t a l _ . , 1967) . The p lacen ta i s a l s o thought to b lock an t igen s t i m u l a t i o n (Prokesova e t a l _ . , 1970) . Immunization o f a newborn p i g l e t w i t h a l a rge dose o f an t igen r e s u l t s i n a r e l a t i v e l y r a p i d onset o f ant ibody fo rmat ion i n the b l ood . An t i bod ies are produced w i t h i n 36 hours as IgM, and a f t e r 6 days both IgM and IgG which were an t igen respons ive are found (Prokesova e t a l _ . , 1970). However, i f c e r t a i n an t igens ( e . g . Hog Cho lera V i r u s ) are used , i n j e c t i o n i n neonates induces t o l e r a n c e to t ha t a n t i g e n . (Weide e t a j_. , 1962). Th is neonatal i n d u c t i o n o f t o l e r a n c e to c e r t a i n an t igens imp l i es an immatur i ty o f development and i s commonly demonstrated i n mice (Beneceraf and Unanue, 1979) . A f t e r b i r t h , lymphocytes are a n t i g e n i c a l l y s t imu la ted a t the 19 P e y e r ' s patches and/or the mesenter ic lymph nodes to precommitment f o r IgA p r o d u c t i o n ; they then en ter the c i r c u l a t i o n and home main ly to the lamina p r o p r i a o f the smal l i n t e s t i n e . Thus, a response o f the i n t e s t i n a l s e c r e t o r y immune system requ i r es a n t i g e n i c s t i m u l a t i o n . In the duodenum, Brown and Bourne (1976) found cons ide rab le numbers o f c e l l s s t a i n i n g f o r each o f the th ree c l a s s e s o f immnuglobul in a f t e r the f i r s t week. The presence o f IgM c e l l s was g r e a t e r than IgA c e l l s f o r the f i r s t 3 weeks; IgA exceeded IgM c e l l s a f t e r 3 weeks dur ing which t ime IgG c e l l s were p resen t i n c o n s i d e r a b l e but s m a l l e r numbers. A f t e r about 1 month, the plasma c e l l popu la t i on o f the ducdenum was l i k e t ha t o f an a d u l t . Humoral immunity a l s o begins w i th the presence o f syn thes i zed IgM f i r s t du r ing the end o f the f i r s t week; IgA i s found i n the serum a f t e r 7 - 1 2 days ; IgG i s found i n the serum a f t e r 14 days (Bourne and C u r t i s , 1973), The immune mechanism o f an adu l t can be con t ras ted w i th t ha t o f a neonate. The a d u l t has been p r imed, du r i ng i t s an t igen e x p e r i e n c e , to a g rea t d i v e r s i t y o f an t igens from b a c t e r i a l f l o r a , and t h i s response may be c r o s s - r e a c t i v e to an even l a r g e r a r ray o f i n v a d e r s . Thus , a response which appears pr imary i n a d u l t s may r e a l l y be a secondary and more r a p i d l y o c c u r r i n g response , whereas i n the young or germ-f ree animal a pr imary response i s t r u l y p r imary ; (Solomon, 1971) . I t i s p u z z l i n g why a p i g l e t which i s immunocompetent a t b i r t h should be so s u s c e p t i b l e to i n f e c t i o n p a r t i c u l a r l y when depr ived o f maternal a n t i b o d y . But t h i s may be answered tha t because o f a n t i g e n i c v i r g i n i t y , there may be a reasonab le de lay i n response. E s s e n t i a l l y a race occurs between the m u l t i p l i c a t i o n o f an t igen s t imu la ted c e l l s and pathogens (Solomon, 1971) . 20 21 The general development o f the immune system a f t e r b i r t h i s a cont inuous matura t ion due to s t i m u l a t i o n by na tu ra l a n t i g e n s . Th is may be observed i n the r a p i d enlargement o f lymphoid organs such as the sp leen and the s lower development o f lymph nodes w i th age (Solomon, 1971) . These s t r u c t u r e s do not develop n e a r l y as f a s t i n germ-f ree an ima l s . In the a l imen ta ry t r a c t o f germ-f ree p igs there i s v i r t u a l l y no development o f lymphoid t i s s u e s by 31 days (Kenworthy, 1970) . C o n t r a s t i n g r e s u l t s have been found w i th regard to the e f f e c t o f co los t rum ant ibody i n the newborn p i g . Segre and Myers (1964) i n t h e i r t r i a l s found tha t p i g l e t s seemed to respond b e t t e r to an t i gen -an t i body complexes where the ant ibody i s s u p p l i e d by co los t rum. Adu l t p igs respond j u s t as w e l l to an t i gen a lone w h i c h , accord ing to these wo rke rs , i s p r e - sumably because there i s s u f f i c i e n t na tu ra l an t ibody present i n a d u l t s . However, S t e r z l , e t a l _ . , (1965) found tha t co los t rum tended to i n h i b i t the a c t i v e p roduc t ion o f an t i body . La te r r e s u l t s tend not to r e s o l v e t h i s c o n t r a d i c t i o n except t ha t low l e v e l s o f an t ibody w i l l sometimes enhance an immunological response whereas h igh l e v e l s o f an t ibody w i l l cause a poor response (Solomon, 1970) . The l a t e r case may be understood e i t h e r to be a case o f removal o f a n t i g e n i c s t i m u l a t i o n by the ant ibody (Dixon e t a l . , 1967) o r a case o f nega t i ve feedback. Low l e v e l s o f an t ibody may f a c i l i t a t e phagocytos is by macrophages and enhance an t igen p resen ta t i on to T -he lpe r c e l l s (Kim e t a l _ . , 1966) . Sera from p r e - c o l o s t r a l , n e o n a t a l , germ-f ree p i g l e t s i s b a c t e r i c i d a l f o r rough s t r a i n s o f E . c o l i , and t h i s a c t i v i t y can be c o r r e l a t e d w i t h the presence o f complement ( S t e r z l e t a l _ . , 1964) . Th i s i s a mechanism which can f u n c t i o n n o n - s p e c i f i c a l l y aga ins t gram negat ive 22 b a c t e r i a w i thou t the presence o f an t ibody and cou ld be e f f e c t i v e a g a i n s t any gut f l o r a which become i n v a s i v e . 23 2.4 The "Runt" Phenomenon As b i r t h w e i g h t dec reases , m o r t a l i t y i nc reases ( C a r r o l l e t a J L , 1962; Sharpe , 1966; Fahmy and Be rna rd , 1971) . Sma l l e r p igs o f ten r e q u i r e manual a s s i s t a n c e to suck le and a d d i t i o n a l warmth to s u r v i v e . F a i l u r e to suck le can l ead to hypoglycaemia and death (Newland e t a l ^ . , 1952) ; l a c k o f warmth can l ead to hypothermia and dea th . E x c e p t i o n a l l y smal l p igs a t b i r t h are termed " r u n t s " and are d i s t i n g u i s h e d from normal p igs by both t h e i r l i g h t weight and d i s t i n c t i v e p h y s i c a l c h a r a c t e r i s t i c s . Cooper e t al_.» (1970) de f i ned run ts as being the l i g h t e s t 5% o f the t o t a l popu la t i on a t b i r t h . Per ry and Rowel 1 (1969) took a run t to have a p rena ta l weight o f two - t h i r ds o f the average f o r i t s u t e r i n e horn . P h y s i c a l l y , runts are s m a l l , t h i n , and have a d i s p r o p o r t i o n a t e l y l a r g e r and more domed head than normal progeny. The p h y s i c a l c h a r a c t e r i s t i c s o f a run t are the r e s u l t o f i n t r a - u t e r i n e growth r e t a r d a t i o n which i s not the r e s u l t o f f e t a l anomoly or maternal i l l n e s s (Cooper e t a l _ . , 1978) . The run t i ng phenomenon may be a r e s u l t o f i n t r i n s i c f e t a l growth f a c t o r s o r be secondary to p l a c e n t a l f u n c t i o n (Cooper e t a l _ . , 1978) but the cause i s not c l e a r . When f i v e o r fewer f e t i are i n each horn o f the u t e r u s , the p o s i t i o n o f the embryo has no e f f e c t on s i z e . B u t , as the number i n c r e a s e s , those a t e i t h e r end tend to have an i n c r e a s i n g advantage over those i n the middle o f the horn , and those a t the o v a r i a n end have a g r e a t e r advantage than those a t the c e r v i c a l end (Per ry and Rowel 1 , 1969) . Marrab le (1971) suggested tha t even w i th smal l l i t t e r s i z e s , the p o s i t i o n a l e f f e c t w i l l be o p e r a t i v e l a t e r i n 24 pregnancy. Waldorf et al_., (1957) suggested that the uterine a r t e r i e s provided higher blood pressure at terminal s i t e s but, although t h i s may be relevant i n other multiparous mammals, the porcine vascular system i s such that supply to the cervix and uterine body i s augmented by a branch from the urogenital artery (Marrable, 1971). Several studies have been done about prenatal appearance of runting. Pomeroy (1960) found s i g n i f i c a n t runting appearance at 74 days of gestation but not at 51 days. Perry and Rowel 1 (1969) found f e t i of less than two-thirds the weight f o r the average of the uterine horn at 31 to 49 days of gestation and i d e n t i f i e d these as runts appearing(y>Cooper et al_., (1978) found runts at gestational ages of 44, 53, 56 and 75 days of gestation. Obviously these data do not pin-point a conclusive stage of gestation at which f e t a l growth retardation occurs. However, since 99 per cent of f e t a l growth (by weight) occurs a f t e r day 40 of gestation (Marrable, 1971), the occurrence of runting t h i s early suggests that i n t r a - u t e r i n e retardation may be determined before i t becomes recognizable and may occur throughout f e t a l development i n some cases. Widdowson (1971) compared the development of organs in the runt with that of a fetus taken at an equal si z e and with that of a l i t t e r - mate of normal s i z e . Two outstanding differences were noted: f i r s t l y , muscle development, as measured by quadriceps s i z e was s i g n i f i c a n t l y less in the runt than in e i t h e r the fetus of equal weight or the normal l i t t e r - mate; secondly, the l i v e r s of the fetus and runt were comparable in size yet very much smaller than the l i v e r of the normal lit t e r m a t e with the added c h a r a c t e r i s t i c of a very low carbohydrate content in the runt. Heart, spleen and stomach of the runt were intermediate i n weight between those 25 o f the f e tus and normal l i t t e r m a t e . The b ra i n o f the run t was nearer to normal weight than any o the r pa r t o f the body. G e n e r a l l y , Widdowson (1971) found tha t the decrease i n organ s i z e o f the run t was due to both hypop las ia (decreased c e l l s i z e ) and hypotrophy (decreased c e l l number). Widdowson (1971) went on to compare the growths o f a run t and a normal l i t t e r m a t e f o r th ree years and found tha t the runt was 60 kg . l i g h t e r a t the end o f t h i s term. S ince there were no r e s t r i c t i o n s i n d i e t o r as a r e s u l t o f envi ronmental e f f e c t s a f t e r weaning, i t may i n d i c a t e tha t i n t r a - u t e r i n e growth r e t a r d a t i o n may p rov ide a permanent s e t - b a c k . 26 2.5 Mechanisms o f A c t i o n o f B a c t e r i a i n the I n t e s t i n e Wi th in the i n t e s t i n e there are th ree major ways tha t b a c t e r i a may a s s o c i a t e w i th the i n t e s t i n a l mucosa to produce d i s e a s e . F i r s t l y , they can a t t ach to the mucosa w i thout pene t ra t i on and induce d i s e a s e by m u l t i - 3 p l y i n g and producing exo tox in . Th i s occurs i n E. c o l i e n t e r i t i s o f the smal l i n t e s t i n e and i s caused by e n t e r o t o x i n producing E. c o l i . Second ly , b a c t e r i a can a t t ach to and penet ra te the mucosa but not the s u b e p i t h i l i a l t i s s u e s and induce d i sease by damaging e p i t h i l i a l c e l l s and a l s o produce e x o t o x i n . Th is can occur i n E. c o l i e n t e r i t i s o f the l a rge i n t e s t i n e and i s caused by i n v a s i v e s t r a i n s o f E . c o l i . T h i r d l y , b a c t e r i a can a t t ach t o , and penet ra te the mucosa to reach the s u b e p i t h e l i a l t i s s u e s , m u l t i p l y i n the submucosa, and spread s y s t e m a t i c a l l y a l s o growing i n t r a c e l l u l a r ^ i n phagocytes. Th is mode o f a c t i o n occurs w i th Sa lmone l la e n t e r i t i s caused by S . t y p h i . ( M c C l e l l a n d , 1979) . A major phenomenon i n v o l v e d i n b a c t e r i a l i n vas i on o f e p i - t h e l i a l su r faces i s b a c t e r i a l adherence. S ince p e r i s t a l s i s and f l u s h i n g w i th s e c r e t i o n s tends to remove b a c t e r i a and ma in ta in a con t i nuous l y lower l e v e l o f growth and prevent invaders from p r o l i f e r a t i n g , attachment to the gut e p i t h e l i u m by adherence i s a major mechanism a f f e c t i n g the a b i l i t y o f a pathogen to p r o l i f e r a t e . Bes t known i n t h i s connect ion i s E. c o l i w i th i t s va ry ing adhesion f i m b r i a e . The most s t ud ied i s the K88 an t igen which has n o n - f l a g e l l a r f i l amentous su r face p i l l i c a r r i e d on the genome o f a p l asm id . (Smith and L inggood, 1971) . Other adherence f a c t o r s o f E. c o l i i n the neonatal po rc ine i n t e s t i n e are the an t igens K99, s t r a i n 987 t y p e , and type 1 common p i l l i o f which each has a d i f f e r e n t r ecep to r s i t e on the i n t e s t i n a l e p i t h e l i u m (Dupond and P i c k e r i n g , 1980) . A l l o f these su r face 27 s t r u c t u r e s are a n t i g e n i c and e l i c i t ant ibody responses where the ant ibody ac t s as an an t i -adherence f a c t o r p o s s i b l y by masking the adherence s i t e o f the bac te r ium. In re fe rence to the K88 a n t i g e n , the po rc ine recep to r s i t e f o r i t i s c o n t r o l l e d by a dominant gene and t he re fo re animals which are double r e c e s s i v e are immune to K88 s t r a i n s o f b a c t e r i a un less o ther f a c t o r s are present de termin ing v i r u l e n c e . ( R u t t e r , 1975; Ru t te r e t a l _ . , 1976) . The mechanism o f en te ro tox i ns i s not f u l l y unders tood. However two en te ro tox i ns have been shown to be produced by s t r a i n s o f E. c o l i patho- gen ic f o r sw ine : one i s a n t i g e n i c and heat l a b i l e (LT) and the o ther i s non -an t i gen i c and heat s t a b l e (ST) (Dupont and P i c k e r i n g 1980). Both t o x i n s are t r ansm i t t ed by a p lasmid and both may be produced by the same s t r a i n o f E. c o l i . I t has been suggested t ha t the non -an t i gen i c ST was impor tant i n the d i a r r h e a o f swine ( G y l e s , 1971) . 28 2.6 Diarrhea in P i g l e t s In a normal, healthy i n t e s t i n a l mucosa, s a l t s and water are absorbed by an energy requiring mechanism. The d r i v i n g force of water absorption i s an active transport of e l e c t r o l y t e s , p a r t i c u l a r l y sodium and chlorine. Acute enteritis,which causes diarrhea i n newborn animals^produced watery stools from fecal f l u i d p r i m a r i l y o r i g i n a t i n g in the small i n t e s t i n e (Tennant and Hornbuckle, 1980). The rapid de- hydration causes hemoconcentration and along with the movement of ions, leads to metabolic acidosis caused by renal f a i l u r e to excrete hydrogen ions and by increased production of organic acids r e s u l t i n g from decreased tiss u e oxygenation. Hyperkalemia (hiigh potassium) also r e s u l t s as does hypoglycemia due to decreased gluconeogenesis and increased anaerobic g l y c o l y s i s (Tennant and Hornbuckle, 1980). In the s p e c i f i c case of the enterotoxic mechanism of E. c o l i , the toxin (LT only) acts by binding to receptors on the mucosal c e l l membranes to ac t i v a t e adenyl cyclase which converts ATP to c y c l i c AMP. The cAMP acts as a second messenger to influence permeability of the membrane. The net r e s u l t i s that sodium absorption i s blocked and chlorine i s secreted. The chlorine ion p u l l s water and cations with i t into the gut lumen to maintain osmotic equilibrium. This i s the cause of f l u i d loss (Dupont and Pickering, 1980). E. c o l i heat stable toxin (ST) does not activa t e adenyl cyclase however, as has been discovered recently, i n mice and rabbi t s , i t s action may be mediated through the build-up of guanyl cyclase which produces cGMP (Hughes et al_., 1978). In the case of Salmonella i n f e c t i o n , s i m i l a r dehydration and hydrogen ion and e l e c t r o l y t e disturbances are the r e s u l t . Salmonella 29 i s an i n v a s i v e organism and penet ra tes the mucosa and p r o l i f e r a t e s i n the gut a s s o c i a t e d lymphoid t i s s u e (Dupont and P i c k e r i n g , 1980) . I t s e f f e c t can occur throughout the i n t e s t i n e a l though i t u s u a l l y invades the lower gut mucosa more r e a d i l y . Using the r a t , Powell e t a l _ . , (1971) showed d i a r r h e a inc reased e s p e c i a l l y the i l e a l s e c r e t i o n o f wa te r , sodium and c h l o r i n e and po tass ium. They found an i nc reased h y d r o s t a t i c pressure i n the lamina p r o p r i a , perhaps caused by venous or l ymphat ic o b s t r u c t i o n , w i th r e s u l t a n t i nc reased membrane p e r m e a b i l i t y . 30 2.7 N o n - S p e c i f i c G a s t r o - i n t e s t i n a l Immune Mechanisms There i s a s e r i e s o f mechanisms which opera te i n defense a g a i n s t pathogenic b a c t e r i a and i n some c a s e s , v i r u s e s and p a r a s i t e s . 2 .7 .1 The G a s t r i c Trap A g a s t r i c t rap i s p rov ided by the HCl which i s a n t i - b a c t e r i a l and i s h i g h l y e f f e c t i v e aga ins t a lmost a l l b a c t e r i a . However, i f the g a s t r i c mucosa i s not f u n c t i o n a l , a s , p o s s i b l y , i n the neonate, o r i f the cha l l enge dose i s l a r g e enough, o r i f b a c t e r i a are not reached by the a c i d i t y o f the stomach, b a c t e r i a may pass t h i s t rap to p r o l i f e r a t e l a t e r i n the g a s t r o - i n t e s t i n a l t r a c t ( G i a n n e l l a e t a l _ . , 1971) . In neonates , the work on g a s t r i c a c i d s e c r e t i o n seems q u i t e incomplete and c o n t r a d i c t o r y . Cranwel l e t a l _ . , (1968) found tha t p i g l e t s i n a c l ean environment had a c i d s e c r e t i o n a t one week o f age, wh i l e those r a i s e d i n a convent iona l environment d i d n ' t sec re te u n t i l 30 days o f age. L a t e r , Cranwel l and T i t chen (1974) d i scove red tha t s e c r e t i o n cou ld begin from 2 days o f age us ing a s u r g i c a l l y separated pouch from the stomach and t ha t the s e c r e t i o n was s i g n i f i c a n t . H i l l (1970) s t a ted tha t a c i d s e c r e t i o n was very minimal dur ing the f i r s t 2 weeks. Jones (1972) r e f e r r e d to work which s ta ted t ha t HCl s e c r e t i o n does not begin u n t i l 20 days o f age o f m i l k f ed or 14 days i f g i ven c e r e a l s . The l i k e l y e x p l a n a t i o n o f d i s c r e p a n c i e s i s t ha t the p i g l e t has a competent g a s t r i c mucosa a t l e a s t w i t h i n a few days o f b i r t h , but environmental o r n u t r i t i o n a l f a c t o r s c o n t r o l i t s development (Cranwel l and T i t c h e n , 1974) . 2 . 7 . 2 I n t e s t i n a l M o t i l i t y The ra te o f passage o f chyme w i l l determine to a l a rge ex ten t whether l o c a l propagat ion w i l l o c c u r . There i s ev idence tha t the t ime o f 31 con tac t between the e p i t h i l i a l l i n i n g o f the gut and an i n v a s i v e pathogen such as a Sa lmone l la s t r a i n o f b a c t e r i a determines the development o f d i sease ( S p r i n z , 1969) . I t has been shown by the use o f a gang l ion b l o c k i n g drug which slowed the ra te o f p e r i s t a l s i s i n r a t s t ha t there was a s u b s t a n t i a l i n c r e a s e i n the number o f c o l i f o r m s i n the smal l i n t e s t i n e . (Dixon and P a u l l e y , 1963) . D ia r rhea i s a s s o c i a t e d w i th decreased m o t i l i t y ( C h r i s t i a n s e n e t a l . , 1972) and gut s t a s i s ( R u t t e r , 1975; White e t . a l . , 1972; K o h l e r , 1972) . Re la ted to i n t e s t i n a l m o t i l i t y i s the l e v e l o f s e c r e t i o n o f l a r g e amounts o f s t e r i l e f l u i d by the duodenum which washes out b a c t e r i a (N ie l son e_t aj_-, 1968) . 2 . 7 . 3 I n t e s t i n a l M i c r o f l o r a In the deve lop ing p i g l e t r a i s e d c o n v e n t i o n a l l y , wh i l e g a s t r i c a c t i v i t y i s low, b a c t e r i a are inges ted from the environment and s u c c e s s - f u l l y adapted s t r a i n s p r o l i f e r a t e . P r o b a b l y , the es tab l i shment o f a hea l thy and na tu ra l m i c r o f l o r a occurs here and evo lves as the d i e t changes from m i l k to s o l i d f o o d . In a hea l thy young p i g the eco logy o f the gut ma in ta ins a homeostasis which i s f u r t h e r ensconced i n a more mature a n i m a l . In the nurs ing p i g l e t l a c t o b a c i l l i p r o l i f e r a t e producing l a c t i c a c i d which i n i t s a c i d i t y i n h i b i t s o ther b a c t e r i a . L a t e r , the r e s i d e n t b a c t e r i a c rea te a n t i b a c t e r i a l c a t a b o l i t e s i n c l u d i n g l a c t i c a c i d and sho r t cha in v o l a t i l e f a t t y a c i d s to lower the pH. In a d d i t i o n , the r e s i d e n t f l o r a dominate the demand f o r space and n u t r i e n t s to prevent o ther forms from ga in i ng access to grow ( F r e t e r and Abrams, 1972; F r e t e r , 1974) . Working w i th cont inuous f low c u l t u r e s , Ozawa and F r e t e r (1964) found tha t r e s i d e n t s t a i n s used nea r l y a l l energy sources and any invader s t r a i n c o u l d n ' t p r o l i f e r a t e un less a source not used by the r e s i d e n t s was s u p p l i e d . 32 Another f a c t o r o f the environment o f the lumen i s t ha t i t has a low redox p o t e n t i a l which a l l o w s on l y c e r t a i n s t r a i n s o f b a c t e r i a to s u r v i v e ( M c C l e l l a n d , 1979) . A f u r t h e r f a c t o r i n f l u e n c i n g b a c t e r i a l eco logy i n the gut i s t ha t some b a c t e r i a can produce s p e c i f i c a n t i m i c r o b i a l s o f l i m i t e d spectrum which appear to i n f l u e n c e the a b i l i t y o f a p a r t i c u l a r s t r a i n to s u r v i v e (Branche e t a K , 1963) . In t r e a t i n g w i th a n t i b i o t i c s a g a i n s t an i n f e c t i o n ( e . g . Sa lmone l la ) the magnitude o f the cha l l enge dose may be reduced and the i n f e c t i o n may be pro longed by reduc ing the t o t a l i n t e s t i n a l f l o r a and a l l o w i n g the i n f e c t i v e s t r a i n to p r o l i f e r a t e (Ase rko f f and Benne t t , 1969) . The s t a b i l i t y o f the m i c r o f l o r a suggests t ha t immunity i s o p e r a t i v e a g a i n s t the e f f e c t s o f i n v a s i v e or mucosal a s s o c i a t e d b a c t e r i a and tha t low grade pathogens ( i . e . the i n t e s t i n a l f l o r a ) e i t h e r are r e s t r i c t e d to the gut lumen where immunological i n t e r - a c t i o n s are prevented or d i s p l a y a low degree o f a n t i g e n i c i t y . (Shed lo fsky and F r e t e r , 1974) . 2 . 7 . 4 Non-Immunological Fac to rs i n the S e c r e t i o n s o f the G . I . T r a c t Severa l n o n - s p e c i f i c i n h i b i t o r s o f b a c t e r i a l growth are present i n the duodenal s e c r e t i o n s and i n m i l k o f the nu rs ing i n d i v i d u a l . B i l e s a l t s are metabo l i zed by the r e s i d e n t b a c t e r i a and may form a n t i - me tabo l i t es to c e r t a i n b a c t e r i a ( M c C l e l l a n d , 1979) . L a c t o f e r r i n and lysozyme are both present i n i n t e s t i n a l s e c r e t i o n s and m i l k and , w i th s e c r e t o r y IgA, are b a c t e r i c i d a l ( H i l l and P o r t e r , 1974; Knopf e t a l . , 1971) . L a c t o f e r r i n , which i s commonly present i n m i l k c h e l a t e s i r o n which r e s t r i c t s b a c t e r i a l growth (Orson and R e i t e r , 1981; B u l l e n e t a l . , 1972) . Complement may be i n v o l v e d i n r e a c t i o n s i n o r near the mucosa. The a l t e r n a t e o r p roperd in pathway o f complement a c t i v a t i o n i s accepted 33 as important in the defense against gram negative bacteria of the gut (Hood et a l . , 1978). This is supported by the discovery that some complement components are synthesized by the intestinal mucosa (Lai A Fat et a i . , 1976). 34 2.8 Humoral and C e l l u l a r Immunity i n Po rc i ne Mammary S e c r e t i o n s Colost rum i s the f l u i d sec re ted by the mammary g land dur ing the f i r s t 24 hours ( K a r l s s o n , 1966) and m i l k i s sec re ted du r ing the remainder o f l a c t a t i o n . IgG i s the pr imary immunoglobul in component o f serum and c o l o s t r u m ; IgA i s the pr imary component o f m i l k ; and IgM i s a minor component o f bo th . In the f i r s t 24 hours IgG decreases f i v e - f o l d and w i t h i n the f i r s t week i t decreases t h i r t y - f o l d . Thus i t drops from forming 80% o f t o t a l c o l o s t r a l immunoglobul in to 25% o f t o t a l m i l k immunoglobul in ( C u r t i s and Bourne, 1971) . IgA on l y decreases t h r e e - f o l d dur ing the f i r s t week and hence emerges as the major immunoglobul in i n sow m i l k , accoun t ing f o r 50 to 60% o f m i l k immunoglobul in ( C u r t i s and Bourne, 1971) . Co los t rum f u n c t i o n s ma in ly i n p r o t e c t i o n a g a i n s t sys temic i n f e c t i o n because the immunoglobul ins i t con ta ins are absorbed through the gut mucosa i n t o the c i r c u l a t i o n . Th i s occurs i n a pe r i od o f t ime du r ing which the p i g l e t i n t e s t i n e absorbs macromolecules i n t a c t before gut c l o s u r e a f t e r 24 to 36 hours (Payne and Marsh , 1962) . M i l k p rov ides c o p r a - a n t i b o d i e s which f u n c t i o n w i t h i n the i n t e s t i n a l lumen dur ing the pre-weaning pe r iod ( W i l s o n , 1974; B rand tzaeg , 1973) . 125 Bourne and C u r t i s (1973) us ing I l a b e l l e d immunoglobul in determined the p ropo r t i ons o f serum d e r i v e d and l o c a l l y produced immuno- g l o b u l i n i n mammary s e c r e t i o n s . They found tha t a l l c o l o s t r a l IgG came from the serum and tha t 60% o f c o l o s t r a l IgA was syn thes i zed by the mammary g land (a sma l l p o r t i o n o f t o t a l c o l o s t r a l immunog lobu l ins ) , and , conse- q u e n t l y , cons ide red tha t co los t rum was a serum t ransudate (Bourne, 1973). They found t ha t m i l k , on the o the r hand, was a t r ue mammary s e c r e t i o n s ince 35 more than 90% o f the IgA and IgM and 70% o f IgG was produced l o c a l l y . In con junc t i on w i t h immunoglobul ins, po rc ine m i l k con ta ins a t r y p s i n i n h i b i t o r which occurs f i r s t l y a t h igh l e v e l s and f a l l s to a very low l e v e l by the f i f t h day (Laskowski e t a l _ . , 1957) . T h i s probably f u n c t i o n s to prevent degradat ion o f c o l o s t r a l immunoglobul ins before they can be absorbed i n the i n t e s t i n e ( S c o o t , 1972) . A l though no i n fo rma t i on e x i s t s f o r po rc ine c o l o s t r u m , human co los t rum shows a r e l a t i v e l y h igh l e v e l o f T lymphocytes and macrophages (Parmely and B e e r , 1977) . Parmely and Beer suggested t h a t i t was p o s s i b l e , but not p roven , tha t these c e l l s cou ld s u r v i v e i n the g a s t r o - i n t e s t i n a l t r a c t . They f u r t h e r suggested tha t the mammary t i s s u e may "package" s p e c i f i c c e l l u l a r components i n the co los t rum to c o n t r i b u t e to immuno- competency i n the neonate and tha t t h i s c e l l mediated immunity (CMI) from the mammary g land depended upon i n d u c t i v e events a t d i s t a n t mucosal sur face (b ronch ia l a s s o c i a t e d and gut a s s o c i a t e d lymphoid t i s s u e s ) . 36 2.9 Preweaning M o r t a l i t y In a survey o f the l i t e r a t u r e from 1937 to 1976 McCallum (1977) found t h a t l e v e l s o f preweaning m o r t a l i t y have c o n s i s t e n t l y ranged between 18 and 30 per cent o f p igs born ( i n c l u d i n g s t i l l b i r t h s ) . Two s t u d i e s (Fahmy and B e r n a r d , 1971; E n g l i s h and S m i t h , 1974) recorded t h a t 50 per cent o f the t o t a l preweaning m o r t a l i t y occur red be fore 3 days o f age. Pomeroy (1960) noted t ha t 70.2 per cent o f t o t a l preweaning m o r t a l i t y occur red be fore 3 days of age , and Jones (1972) recorded 63.1 per c e n t . Thus , any at tempt to reduce m o r t a l i t y must a f f e c t the e a r l i e s t days o f pos t na ta l e x i s t e n c e . 2.9.1 Causes o f Preweaning M o r t a l i t y The causes were f a i r l y c o n s i s t e n t among the authors c i t e d and are summarized i n Tab le 2 .3 These are t rauma, s t a r v a t i o n , general weakness, d i s e a s e , congen i t a l a b n o r m a l i t i e s , and o ther causes . Trauma i n v o l v e s c rush ing o r t ramp l ing o f p igs by the sow. Some o f the data may r e f l e c t the e f f e c t o f o ther pr imary causes p red i spos ing trauma such as m a l n u t r i t i o n , genera l weakness, or c h i l l i n g . S t a r v a t i o n , a major pr imary cause o f d e a t h , cou ld r e s u l t from two d i f f e r e n t c o n t r i b u t i n g f a c t o r s . The f i r s t i s a g a l a c t i a o f the sow w h i c h , i n the s t u d i e s o f E n g l i s h and Smith (1974) c o n t r i b u t e d to 27 per cent o f the s t a r v a t i o n . The o ther f a c t o r , c o n t r i b u t i n g 73 per c e n t , i s severe compe t i t i on w i t h i n l i t t e r s due e i t h e r to supernumary p i gs i n r e l a t i o n to a v a i l a b l e t e a t s or to d i s - p a r i t y i n b i r t h w e i g h t s which r e s u l t s i n unequal compe t i t i ve advantage f o r heav ie r p igs ( E n g l i s h and S m i t h , 1974) . The common r e s u l t o f s t a r v a t i o n i s hypoglycaemia (Sharpe, 1966; Edwards, 1972) which may be aggravated by c h i l l i n g ( C u r t i s , 1974) . General weakness, which c o n t r i b u t e s to h igh Table 2 . 4 A Summary of Major Causes C o n t r i b u t i n g to Death i n Baby P igs (per cent c o n t r i b u t i o n ) Reference Trauma S t a r v a t i o n R e l a t i v e Disease Congen i ta l Other Weakness Abnormal i ty Sharpe (1966) 21.2 17.1 9.6 9.6 2.1 40 .4 Fahmy and Bernard 19.2 — 26.9 17.5 14.2 22.2 (1971) E n g l i s h and Smith 18.2 42.8 14.9 — 12.3 11.8 (1974) Rodef fe r e t a l . , 30.9 17.6 14.7 18.2 — 13.1 (1975F 38 losses, is most clearly associated with parturition aberrations of which the most common result is prenatal anoxia or hypoxia (English and Smith, 1974). "Parturition aberrations" include premature umbilical cord rupture and a prolonged farrowing interval between births (Rodeffer e_t aJL. 1975) Disease, as a cause of death, includes different diseases depending on the study. Sharpe (1966) listed the major causes as gastroenteritis and septicaemia. Fahmy and Bernard (1971) listed scours and pneumonia as major disease factors. Rodeffer et al_., (1975) listed transmissable gastroenteritis (T.G.E.) , other diarrheas, and pneumonia. Congenital abnormalities varied among the studies. English and Smith (1974) listed the major traits including atresi ani, cardiac abnormality, congenital splay-leg, cleft palate, and hypoplasia kidney. 2.9.2 Effect of Low Birthweight on Survival "The effect of birth weight on survival is essentially linear within a wide range of values" (Bereskin et al_.» 1973). Thus, low birth- weight is a major contributing factor to reducing survival (Sharpe, 1966;; Fahmy and Bernard, 1971; Bereskin_et al_., 1973). Table 2.4 l ists mortality values found for low birthweight pigs by several researchers. These high mortality rates may vary with strain of pig or environment which, in turn, may include temperature and milking ability of the sow (Pomeroy, 1960). These high mortality rates reflect a greater susceptibility to the major causes of death. There was a higher incidence of trauma in low birthweight pigs (Sharpe, 1966); a markedly higher incidence of starvation (Sharpe, 1966; English and Smith, 1974); and low birthweight had some effect on the incidence of septicaemia (Sharpe, 1966). Also, low birth- weight pigs are much more susceptible to chilling. Newland et al_., (1952) Table 2 . 5 E f f e c t o f B i r t h Weight on M o r t a l i t y Reference Weight Range Per Cent M o r t a l i t y Pomeroy (1960) <900g 83 .0 (before 3 days) Sharpe (1966) <800g 82.1 Fahmy and Bernard <910g 60 .0 (1971) ( E n g l i s h and Smith <907g 74.6 (1974) VO 40 found that the drop in body temperature after chilling was inversely related to body weight. Pomeroy (1960) cited three factors contributing to higher mortality in low birth weight pigs: 1. they may be less vigorous and active, and less competitive at the teats during nursing. 2. they have a larger surface area in relation to body weight. Therefore there is a relatively greater heat loss per unit of body weight. 3. they may be physiologically immature. 41 Foot-Notes 1. Immunocytes include all cells of the immune system. 2. The failure of the immune system, as a result of previous contact with antigen, to respond to the same antigen, although capable of responding to tohers. Tolerance is best established by neonatal injection of an antigen (Benaceraf and Unanue, 1979). 3. An exotoxin is a soluble poisonous substance passing into the medium during growth of a microorganism. An endotoxin is a poisonous substance present in bacteria but separable from the cell body only on its disintegration. An enterotoxin is a toxin of bacteria produced within and affecting the intestine. (Websters 7th New Collegiate dictionary, 1965) 42 3 . Exper imenta l 3.1 I n t r o d u c t i o n Co los t rum-depr i ved p igs have been s u c c e s s f u l l y rea red i n a n o n - i s o l a t e d environment s i n c e 1961 (Owen e t a l _ . , 1961) . A f t e r work by Owen and B e l l (1964) , Scoot (1972) , and McCallum (1977) , McCallum (1977) conc luded tha t i t was p o s s i b l e " t o r a i s e co los t rum-depr i ved neonata l p igs i n an o r d i n a r y swine barn environment and ach ieve s u r v i v a l r a t es comparable to those p r e s e n t l y t o l e r a t e d under na tu ra l c o n d i t i o n s " us ing a b a t t o i r serum-der ived immunoglobul ins i n m i l k r e p l a c e r s . Both Scoot (1972) and McCallum (1977) concluded tha t o r a l a d m i n i s t r a t i o n o f 10 grams per k i l og ram o f body weight o f immunoglobul ins on the f i r s t day f o l l o w e d by 2 grams per k i l og ram on succeed ing days cou ld con fe r adequate pass i ve immunity on co los t rum-depr i ved p i g s . Scoot (1972) found tha t a d m i n i s t r a t i o n o f immunoglobul ins over 21 days i n c r e a s e d s u r v i v a l compared to 10 day t reatment ( from 75 per cent to 88 per cent ) and McCallum (1977) observed a sharp i n c r e a s e i n m o r t a l i t y f o l l o w i n g removal o f immunoglobul in a f t e r 10 days compared to those cont inued on t reatment f o r 20 days . The purpose o f exper iments repor ted he re in was to eva lua te the e f f e c t o f o r a l l y admin is te red immunoglobul ins on co los t rum-depr i ved p igs o f low b i r t h w e i g h t . Exper iments o f Lodge and E l l i o t (1979) , us ing the a r t i f i c i a l r e a r i n g techn ique o f McCallum e t a l . , (1977) , found tha t 21 day w e i g h t , 56 day weight and age a t 90kg. were h i g h l y c o r r e l a t e d w i t h b i r t h w e i g h t , but t ha t low b i r t h w e i g h t was l e s s i n h i b i t i n g under a r t i f i c i a l r e a r i n g than under na tu ra l r e a r i n g . Lodge and E l l i o t (1979) a l s o found t ha t a r t i f i c i a l r e a r i n g reduced m o r t a l i t y o f low b i r t h w e i g h t p i g s . 43 The t r i a l s described herein were c a r r i e d out in a barn environment on a commercial farm 1 in an environmentally c o n t r o l l e d room adjacent to the farrowing rooms. A series of 4 experiments was car r i e d out to determine e f f e c t s of low b i r t h weight on surviv a l and growth. 44 3 .2 Exper iment I 3.2.1 O b j e c t i v e The o b j e c t i v e o f t h i s exper iment .was t o assess the e f f i c a c y o f a b a t t o i r d e r i v e d po rc ine immunoglobul in e x t r a c t from serum as a m i l k r e p l a c e r a d d i t i v e f o r r e a r i n g low b i r t h w e i g h t p i g s . 3 . 2 . 2 M a t e r i a l s and Methods 3 .2 .2 .1 Exper imenta l Animals Twenty- four low b i r t h weight p igs weigh ing l e s s than lOOOg o f Y o r k s h i r e X Landrace breed ing were a l l o t t e d w i t h i n a ten day p e r i o d . Farrowings were at tended and exper imenta l p igs were removed from the sow a t b i r t h o r 12 hours l a t e r . P igs were weighed a t b i r t h , ea r tagged , and i n d i v i d u a l l y penned i n a nursery room. P igs were randomly ass igned to t rea tmen ts . Cages were a d j o i n i n g w i r e mesh 46cm x 25cm x 20cm i n two t i e r s . P r i o r to the t r i a l the room and cages were thorough ly c leaned and d i s - i n f e c t e d . Cages were washed p e r i o d i c a l l y du r i ng the t r i a l . Temperature i n the nursery room was main ta ined between 30°C and 33°C. A l l p igs rece i ved i r o n dext ran i n j e c t i o n s and had t h e i r needle tee th and t a i l s c l i p p e d a t two days o f age. A second i r o n dex t ran i n j e c t i o n was g iven a t 10 days o f age. 3 . 2 . 2 . 2 P r e p a r a t i o n o f Po rc i ne Immunoglobulins Immunoglobulins used i n a l l t r i a l s were prepared by Canada Packers L t d . , To ron to , O n t a r i o . The immunoglobul in - f r a c t i o n de r i ved a t 40 per cent s a t u r a t i o n w i t h ( N H 4 ) 2 SO^ was washed, d i a l y z e d , mixed w i th condensed whole m i l k and spray d r i e d (Owen, personal communication). 3 . 2 . 2 . 3 D i e t a r y Treatments In a l l t r ea tmen ts , p igs r e c e i v e d a non-medicated commercial 2 m i l k r e p l a c e r . Feed i n t a k e was ad jus ted to 7 .5 per cen t o f t h e i r body 45 weight i n a i r dry mat ter which was d i l u t e d 1 : 4 w i t h wa te r . For the f i r s t 3 days d i e t s were formula ted w i th a 10 per cent dex t rose s o l u t i o n . P igs were weighed every o the r day and feed a l lowances were ad jus ted a c c o r d i n g l y . Immunoglobulin was mixed w i t h the m i l k r e p l a c e r and fed a t l e v e l s s e t out i n the f o l l o w i n g schedu le : 1. No c o l o s t r u m , m i l k r e p l a c e r o n l y . 2 . Colost rum f o r 12 hou rs , f o l l owed by m i l k r e p l a c e r o n l y . 3 . Co los t rum f o r 12 h o u r s , p lus immunoglobul in a t 2g/kg body we igh t /day f o r 9 .5 days . 4 . Immunoglobulin e x t r a c t on f i r s t day ( lOg /kg body w e i g h t ) , p lus immunoglobul in (2g/kg body we igh t /day) f o r 9 days . Scour ing an imals were t r e a t e d , as necessa ry , on an i n d i v i d u a l 3 p i g b a s i s w i t h Furoxone . The t r i a l was o f 21 days d u r a t i o n . 3 . 2 . 2 . 4 Feeding Regimen S i n c e the p igs would not feed from sha l l ow bowls immediate ly a f t e r b i r t h , they were f o r c e fed w i th sy r i nges u n t i l they would n i p p l e f e e d . In l a t e r t r i a l s n i p p l e feed ing was s u c c e s s f u l from b i r t h but dur ing t h i s t r i a l some d i f f i c u l t y was encountered. At 10 days o f age a l l p igs were swi tched to sha l l ow bowls . Feeding was every 2 hours f o r the f i r s t 4 days , every 4 hours on days 5 to 8 and every 6 hours from days 9 to 21 . Fresh water was made a v a i l a b l e from day 5 and creep feed was o f f e r e d f r e s h d a i l y a f t e r 10 days o f age. 3 . 2 . 3 Measurements and Observa t ions P i g s were weighed every 2 days . M o r t a l i t y was recorded and post mortems were c a r r i e d out on a l l dead p igs by the P r o v i n c i a l Ve te r i na r y L a b o r a t o r y , A b b o t s f o r d , B . C . I n f e c t i o u s agents and c a u s a t i v e f a c t o r s were 46 i d e n t i f i e d whenever possible. Mortality was analyzed s t a t i s t i c a l l y by giving a survivor a zero value and a dead pig a 1 value. The analysis used was analysis of variance (ANOVA) with the design model being: Y^j = p + T- + e^j where T.. = Treatment E f f e c t s y = Population Mean £.. = Experimental Error Rate of gain was also analyzed with t h i s model. The mean birth-weights of survivors and dead pigs were compared with a " t " t e s t (Choi, 1978). 3.2.4 Results and Discussion Mortality was high (Table 3.1.b) with a l l pigs in the negative control group dying before 10 days of age. Those which received colostrum plus supplemental immunoglobulin extract f o r 10 days showed the highest s u r v i v a l . The differences between treatments were not s i g n i f i c a n t (P 0.05). The mean birth-weight of the s u r v i v i o r s was s i g n i f i c a n t l y higher than that of the dead pigs (P 0.01). This difference suggests that higher birth-weight pigs had an advantage in th i s t r i a l . The cause of death was predominantly a s p i r a t i o n pneumonia complicated by E. c o l i septicaemia. The pneumonia was in v a r i a b l y caused by the presence of foreign material in the bronchi and lungs introduced while force feeding with syringes. A major c h a r a c t e r i s t i c of dead pigs, regardless of treatment, was a f l a c c i d , d i l a t e d i n t e s t i n e or distended stomach. The cause of th i s state could have been over-feeding and could have involved E. c o l i e n t e r i t i s . A l l pigs were d i a r r h e t i c befor death. Most pigs which died showed a marked lack of depot f a t in d i c a t i n g no energetic reserves f o r s u r v i v a l , and 47 Tab le 3.1a Experiment 1: Exper imenta l Animals Treatment Number i n Treatment Mean B i r t h -We igh t Group t S . D . No c o l o s t r u m ; no immuno- 6 765 ± 157 g g l o b u l i n Co los t rum; no immuno- 6 854 + 121 g g l o b u l i n Colost rum + immunoglobul in 6 843 + 190 g (2g /kg /day) Immunoglobulin ( lOg /kg /day 6 752 + 190 g to 2g /kg /day) Tab le 3.1b Experiment 1: Resu l t s Treatment % S u r v i v a l % S u r v i v a l Rate o f Gain to Day 10 to Day 21 ( q/day ) No c o l o s t r u m ; no immuno- 0 g l o b u l i n Co los t rum; no immuno- g l o b u l i n 33 33 46 .5 + 24.2 Colos t rum + immunoglobul in 50 50 42.1 + 23.5 (2g /kg /day) Immunoglobulin ( lOg /kg /day 16 16 7.5 (1 p i g ) - to 2g /kg /day) 48 dehydration, no doubt a result of the diarrhea. A higher level of disease carrying microbes in the environ- ment might have been present due to the reservoir of infection carried by the negative control animals which were all i l l from the beginning of the t r i a l . Sanitation and high moisture levels were difficult to control by washing the cages. The resulting moist, humid, and non-sterile conditions may have been ideal for the growth of microbes. It was assumed that a 12 hour nursing period was adequate for attaining sufficient immunoglobulin levels in the plasma. This is supported by work of Scoot (1972) and Carlson and Lecce (1973). 49 3 .3 Exper iment I I 3.3.1 Ob jec t i ve The o b j e c t i v e o f t h i s experiment was to f u r t h e r assess the e f f i c a c y o f a b b a t o i r d e r i v e d po rc i ne serum immunoglobul in e x t r a c t as a m i l k r e p l a c e r a d d i t i v e f o r r e a r i n g low b i r t h weight p i g s . To reduce the pool o f i n f e c t i o n , nega t i ve c o n t r o l s r e c e i v i n g no immunoglobul ins ( a r t i f i c i a l o r co los t rum de r i ved ) were not i nc luded i n t h i s t r i a l . 3 . 3 . 2 M a t e r i a l s and Methods 3 .3 .2 .1 Exper imenta l Animals Twenty- four low b i r t h weight p igs weigh ing l e s s than lOOOg o f Y o r k s h i r e X Landrace breeding were a l l o t t e d w i t h i n a ten day p e r i o d . Farrowings were a t tended and exper imenta l an imals were removed a t b i r t h o r 12 hours l a t e r . P igs were weighed a t b i r t h , ear tagged , and i n d i v i d u a l l y penned i n a nursery room. P i g s were randomly ass igned to t rea tmen ts . Cages were a d j o i n i n g w i re mesh 46cm x 25cm x 20cm i n two t i e r s . P r i o r to the t r i a l the room and cages were thorough ly c leaned and d i s i n f e c t e d . Cages were l e f t unwashed throughout the t r i a l p e r i o d . Temperature i n the nursery room was main ta ined between 30°C and 33°C. A l l p igs r e c e i v e d i r o n dext ran i n j e c t i o n s and had t h e i r needle t ee th and t a i l s c l i p p e d a t two days o f age. A second i r o n dex t ran i n j e c t i o n was g i ven a t 10 days o f age. 3 . 3 . 2 . 2 D i e t a r y Treatments 2 P i g s r e c e i v e d a non-medicated commercial m i l k r e p l a c e r . Feed i n t ake was reduced from the l e v e l i n exper iment I to 6 per cent o f body weight i n a i r dry mat ter which was d i l u t e d 1 : 4 w i th wate r . For the f i r s t 3 days d i e t s were fo rmu la ted w i t h a 10 per cent dex t rose s o l u t i o n . P igs were weighed every o the r day and feed a l lowances were ad jus ted acco rd - 50 ingly. Immunoglobulin extract was mixed with the milk replacer and fed at l e v e l s set out in the following schedule of treatments: 1. Colostrum f o r 12 hours, followed by milk replacer only. 2. Colostrum f o r 12 hours, followed by immunoglobulin at 2g/kg body weight/day f o r 9.5 days. 3. Immunoglobulin (lOg/kg body weight) on the f i r s t day followed by immunoglobulin at 2g/kg body weight/day f o r 9.5 days. Scouring animals were treated, as necessary, on an 4 individual pig basis with Anistat , a broad spectrum a n t i b i o t i c . The t r i a l was of 21 days duration. 3.3.2.3 Feeding Regimen Pigs were nipple fed dor one week then changed to bowl feeding f o r the remainder of the t r i a l . Feeding was every 2 hours f o r the f i r s t 4 days, every 4 hours on days 5 to 8, and every 6 hours from days 9 to 21. Fresh water was ava i l a b l e to them from day 5 and creep feed was offered fresh d a i l y a f t e r 10 days of age. 3.3.3 Measurements and Observations Pigs were weighed every 2 days. M o r t a l i t y was recorded and post mortems were conducted on a l l dead pigs by the Provincial Veterinary Laboratory, Abbotsford, B.C.. Infectious agents and causative factors were i d e n t i f i e d whenever possible. Mortality and rate of gain were analysed as in Experiment 1, using analysis of variance. The mean b i r t h weights of survivors and dead pigs were compared with a " t " t e s t (Choi, 1978). 51 3.3.4 Results and Discussion Mortality (Table 3.2.b) decreased from the level achieved in Experiment 1. This may be attributed to improved technique, elimination of the negative control group (as a pool of i n f e c t i o n ) , and improved environmental conditions. The group receiving only colostrum f o r 12 hours showed a mortality rate at 21 days s i g n i f i c a n t l y higher than the other two groups (P 0.05). There was no s i g n i f i c a n t difference in mortality between thos receiving colostrum with immunoglobulin and those receiving immunoglobulin extract instead of colostrum. Since i t was observed in Experiment 1 that feeding at 7.5 per cent of body weight led to a distended stomach and possible g a s t r i c s t a s i s producing scours, the level of feeding was reduced to 6 per cent of body weight in a i r dry matter, and t h i s problem was consequently reduced. White et al_., (1969) found in t h e i r t r i a l s , that scour was always preceded by g a s t r i c s t a s i s . They found that a d i r e c t r e l a t i o n s h i p existed between pH and growth of bacteria in the stomach. A lack of l a c t i c acid producing organisms in the stomach of the achlorhydric pig , with resultant high pH in the stomach, might lead to rapid growth conditions for coliforms which could then pass to the i n t e s t i n e and cause a scour syndrome. Endotoxin release in the stomach by coliform bacteria could also lead to reduced g a s t r i c acid secretion exacerbating the conditions. There was no s i g n i f i c a n t difference between b i r t h weights of surviving versus dead pigs. 52 Table 3.2a Experiment 2; Experimental Animals Treatment Number in Treatment Mean Birth-weight Group ± S.D. Colostrum; no immuno- 8 677 ± 111 g globulin Colostrum + immunoglobulin 8 707 ± 170 g (2g/kg/day) Immunoglobulin (lOg/kg/day 8 736 ± 143 g to 2g/kg/day) Table 3.2b Experiment 2: Results Treatment % Survival % Survival Rate of Gain to Day 10 to Day 21 ( g/day ) Colostrum; no immuno- 37 0 globulin Colostrum + immunoglobulin 75 63^ 35.2 + 15.4 g (2g/kg/day) Immunoglobulin (lOg/kg/day 63 50 b 39.8 ± 16.9 g to 2g/kg/day) (differing superscripts denote s t a t i s t i c a l l y different values at P= .05 ) 53 Causes o f m o r t a l i t y were genera l sep t i caemia f o r 6 p igs probably due to c o l i f o r m i n f e c t i o n , d e f i n i t e c o l i f o r m sep t i caemia f o r 3 p i g s , and c o l i f o r m e n t e r i t i s w i t h sep t i caemia f o r 7 p i g s . A l l dead p igs showed c h a r a c t e r i s t i c s o f emac ia t ion and dehydra t ion which may be a r e s u l t o f the d i a r r h e a which occu r red i n a l l an imals which d i e d . Rate o f ga in d i d not d i f f e r s i g n i f i c a n t l y between the two groups o f s u r v i v i n g p igs (Table 3.2 b ) and o v e r a l l remained a t a very low l e v e l . Consumption o f c reep feed and water s u p p l i e d ad l i b i t u m cou ld not be weighed a c c u r a t e l y because o f wastage. 54 3.4 Exper iment I I I 3 .4 .1 O b j e c t i v e The o b j e c t i v e o f t h i s exper iment was to compare a lower l e v e l o f immunoglobul in e x t r a c t a d m i n i s t r a t i o n , ( lOg /kg body weight f o r the f i r s t day fo l l owed by 2g/kg body we ight /day f o r 9 days) w i th a hioher immunoglobul in l e v e l (15g/kg body weight f o r the f i r s t day f o l l owed by 5g/kg body w e i g h t / d a y ) . Treatment groups o f p igs nu rs ing co los t rum f o r 12 hours i n s t e a d o f h igh i n i t i a l doses o f immuno- g l o b u l i n were a l s o i nc l uded f o r compar ison. 3 . 4 . 2 M a t e r i a l s and Methods 3 .4 .2 .1 Exper imenta l Animals Two consecu t i ve r e p l i c a t e s were c a r r i e d o u t . To each r e p l i c a t e 24 low b i r t h w e i g h t p igs o f l e s s than lOOOg o f Y o r k s h i r e X Landrace breed ing were a l l o t t e d w i t h i n a 10 day p e r i o d . Farrowings were a t tended and exper imenta l p igs were removed a t b i r t h o r 12 hours l a t e r . P igs were weighed a t b i r t h , ear tagged, and i n d i v i d u a l l y penned i n a nursery room. P igs were randomly ass igned to t rea tments . Cages were a d j o i n i n g w i re mesh 46cm x 25cm x 20cm i n two t i e r s . P r i o r to the t r i a l the room and cages were thorough ly c leaned and d i s i n f e c t e d . Cages were l e f t unwashed throughout the t r i a l p e r i o d . Temperature i n the nursery was main ta ined between 30°C and 33°C. A l l p igs r e c e i v e d i r o n dex t ran i n j e c t i o n s and had t h e i r needle tee th and t a i l s c l i p p e d a t two days o f age. A second i r o n dex t ran i n j e c t i o n was g iven a t 10 days o f age. 3 . 4 . 2 . 2 D i e t a r y Treatments 2 P i g s r e c e i v e d a non-medicated commercial m i l k r e p l a c e r . Feed i n t ake was se t a t 6% o f body weight of a i r dry mat ter which was d i l u t e d 55 1 : 4 with water. For the f i r s t 3 days diets were formulated with a 10 per cent dextrose sol u t i o n . Pigs were weighed every other day and fed accordingly. Immunoglobulin extract was mixed with the milk replacer and fed at le v e l s set out in the following schedule of treatments: 1. 15g/kg bodyweight immunoglobulin on the f i r s t day followed by 5g/kg body weight/day f o r 9 days. 2. lOg/kg body weight immunoglobulin on the f i r s t day followed by 2g/kg body weight/day f o r 9 days. 3. 12 hours of colostrum nursing followed by 9.5 days of immunoglobulin at 5g/kg body weight/day. 4. 12 hours of colostrum nursing followed by 9.5 days of immunoglobulin at 2g/kg/day. 4 In r e p l i c a t e 1 the a n t i b i o t i c A n i stat was used. In 3 r e p l i c a t e 2 the a n t i b a c t e r i a l Furoxone was used. 3.4.2.3 Feeding Regimen Pigs were nipple fed for one week then changed to bowl feeding f o r the remainder of the t r i a l . Feeding was every 2 hours f o r the f i r s t 4 days, every 4 hours on days 5 to 8, and every 6 hours from days 9 to 21. Fresh water was ava i l a b l e from day 5 and creep feed was offered fresh d a i l y a f t e r 10 days of age. 3.4.3 Measurments and Observations Measurments and s t a t i s t i c a l analyses were the same as in the two previous experiments except that another dependant variable was included, frequency of scouring. The design was a randomized block design since two r e p l i c a t e s were ca r r i e d out. 56 3.4.4 Results and Discussion Mortality (Table 3.3..b) was s i m i l a r to that in Experiment 2 where i t was approximately 50 per cent in the treated groups. There was no s i g n i f i c a n t difference between treatments f o r mortality. Thus, the r e s u l t s indicated no s i g n i f i c a n t difference between those receiving colostrum and those receiving immunoglobulin extract, or between those receiving a higher level of immunoglobulin and thowe receiving a lower l e v e l . The assumption that 12 hours of nursing was adequate was based on the findings of Scoot (1972) and Carlson and Lecce (1973). This assumption r e l i e s on the pig receiving adequate colostrum during nursing. However, low b i r t h weight pigs are frequently disadvantaged in t h i s respect, not being as competitive or not being able to reach a teat. Thus, in some cases within t h i s t r i a l , i n d i v i d u a l s may not receive an adequate level of systemic passive immunity which may lead to s u s c e p t i b i l i t y to i n f e c t i o n a f f e c t i n g the mortality rate. As was the case, those receiving a r t i f i c i a l immunoglobulin extract instead of colostrum, showed a high survival before day 10, but the difference was not s i g n i f i c a n t . The difference between mean b i r t h weights of survivors and dead pigs was not s i g n i f i c a n t . I t was observed that Table 3.3a Experiment 3: Experimental Animals 57 Replicate Treatment Number i n Treat- Mean Birth-Weight ment Group ± S.D. Immunoglobulin (15g/kg 6 761 ± 166 g /day to 5g/kg/day) Immunoglobulin (lOg/kg 6 729 ± 227 g /day to 2g/kg/day) Colostrum + immuno- 6 835 + 99 g glo b u l i n (5g/kg/day) Colostrum + immuno- 6 746 ± 192 g glo b u l i n (2g/kg/day) 2 Immunoglobulin (15 to 6 814 ± 83 g 5 g/kg/day) Immunoglobulin (10 to 6 671 + 96 g 2 g/kg/day) Colostrum + immuno- 6 704 ± 218 g glo b u l i n (5g/kg/day) Colostrum + immuno- 6 gl o b u l i n (2g/kg/day) 834 i 59 g Table 3.3b Experiment 3: Experimental Results Replicate Treatments % Survival % Survival Rate of Gain Scouring Frequency - to Day 10~~~ to Day 21 (g/day) days scouring/survivor) Mean Scouring • Frequency 1 15 g/kg for Day 1 then 50 46.3 + 4.0 a 7 5 g/kg/day for 9 days 10 g/kg for Day 1 the 2 g/kg/day for 9 days Colostrum for 12 hrs. 5g/kg/day for 9.5 days Colostrum for 12 hrs. ; 2g/kg/day for 9.5 days 15 g/kg for Day 1 then 50 55.7 + 3.5 a 9 5 g/kg/day for 9 days 10 g/kg for Day.1 then 83 55.7 ± 3.5 a 8.8 n 50 39.7 ± 1.2b 5.3 . ; 33 51.5 ± 0.7 a 5 66 55.3 + 8.8 a 3.3 5.0 + 2.6C 2 g/kg/day for 9 days Colostrum for 12 hrs. ; 17 59.0(1 only)" 15 5 g/kg/day for 9.5 days Colostrum for 12 hrs. ; 50 60.0 ± 8.7 a 7 8.9 + 3.5 d 2 g/kg/day for 9.5 days Combined 15 g/kg for Day 1 then 84 50 51.0 ± 6 . l a 5 g/kg/day for 9 days 10 g/kg for Day 1 ther 2 g/kg/day for 9 days Colostrum for 12 hrs.; 5 g/kg/day for 9.5 days Colostrum for 12 hrs.; 2 g/kg/day for 9.5 days n 84 66 41.0 ± 6.5 b 75 25 54.0 + 4.4 a 75 58 57.3 ± 8.4 a c o 59 under500g b i r t h weight there was no s u r v i v a l , however the number o f obse rva t i ons was few. Cause o f death dur ing the f i r s t r e p l i c a t e , i n 11 cases was S a l m o n e l l o s i s caus ing d i a r r h e a and s e p t i c a e m i a . The ac tua l sero type was i d e n t i f i e d as Group B Sa lmone l l a i n 4 cases w h i l e i n the remaining cases sero type was u n i d e n t i f i e d . In one case death was due to E. c o l i e n t e r i t i s . Dur ing the second r e p l i c a t e , Sa lmone l la were h e a v i l y i m p l i c a t e d , bu t , due to the i n t e r f e r e n c e o f a n t i b i o t i c s g iven dur ing t rea tment , cou ld not be i s o l a t e d i n a l l c a s e s . K l e b s i e l l a was a l s o i s o l a t e d from two p i g s . Rate o f ga in (Table 3 .3 .b ) was i nc reased over t ha t found i n Exper iment I I . Those r e c e i v i n g immunoglobul in a t the lower l e v e l showed a s i g n i f i c a n t l y lower r a t e o f ga in i n both r e p l i c a t e s (P = 0.05) (F igu res 3 . 1 . a and 3 . 1 . b ) . S ince the p igs r e c e i v i n g 2g/kg body we igh t / day o f immunoglobul in a f t e r r e c e i v i n g co los t rum showed a s i g n i f i c a n t l y h igher (P = 0.05) growth r a t e than those which r e c e i v e d a r t i f i c i a l immuno- g l o b u l i n ( lOg /kg body weight on the f i r s t day) i n s t e a d o f c o l o s t r u m , the e f f e c t may be due to an i n s u f f i c i e n t i n i t i a t i n g dose o f lOg /kg body weight f o r pass i ve sys temic immunity. However, even i f t h i s t reatment a f f e c t e d growth r a t e , i t d i d not show any s i g n i f i c a n t a f f e c t on m o r t a l i t y r a t e . Both Scoot (1972) and McCallum (1977) found tha t the h igher dosage o f immunoglobul ins (15 and 5g/kg body we igh t /day) i nc reased ra te o f ga in s l i g h t l y . Scoot (1972) i n d i c a t e s t ha t b lood g l o b u l i n l e v e l s were not as h igh w i th a r t i f i c i a l l y supplemented as w i th sow nursed p i g s . Frequency o f scou r i ng o f the s u r v i v o r s showed 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 scheduled t rea tmen ts , however, the f requency o f scou r ing was s i g n i f i c a n t l y d i f f e r e n t between the two r e p l i c a t e s (Table F i g u r e 3.1.a Growth i n E x p e r i m e n t 3, R e p l i c a t e 1 Fiqure 3.1.b Growth in Experiment 3, Rep 2000 1500 A -Colostrum + m - " + 2g/kg/day Ig 5g/kg/day Ig • -10 to 2 g/kg/day Ig O -15 to 5 g/kg/day Ig 1000 U If IB 20 62 3 . 3 . b ) . The obv ious reason f o r t h i s d i f f e r e n c e seems to have been tha t d i f f e r e n t a n t i - s c o u r drugs were used i n each r e p l i c a t e sugges t ing t ha t 4 the a n t i b i o t i c A n i s t a t was more e f f e c t i v e i n c o n t r o l l i n g scours than 3 the a n t i b a c t e r i a l Furoxone . In both r e p l i c a t e s , the scou r i ng f requency o f s u r v i v o r s i nc reased the h ighes t l e v e l a f t e r removal o f p igs from immunoglobul in supplementat ion a t 10 days o f age (F igu re 3 . 2 ) . Th i s suggests t ha t the p igs r equ i r ed f u r t h e r pass i ve p r o t e c t i o n w i th immuno- g l o b u l i n s beyond the ten th day. Figure 3 .2 . Scour ing Frequency (per cent o f s u r v i v o r s scou r i nq ) 100 A Experiment 3 ; R e p l i c a t e 1 A Experiment 3 ; R e p l i c a t e 2 ° Experiment 4 A A A O A A 2 _L_ A A A A A 8 JL A A L_ o o J 1 L_ A A O A J I 1 3 5 Days on T r i a l 11 13 15 17 19 21 64 3 .5 Exper iment IV 3.5.1 O b j e c t i v e The o b j e c t i v e o f t h i s exper iment was to compare per iods o f immunoglobul in f e e d i n g . The pe r iods s e l e c t e d f o r comparison were 10 , 15 and 21 days on immunoglobul in e x t r a c t a t a l e v e l o f lOg /kg body weight f o r the f i r s t day f o l l owed by 2g/kg body we igh t /day f o r 9 days . 3 . 5 . 2 M a t e r i a l s and Methods 3 .5 .2 .1 Exper imenta l Animals Twenty- four low b i r t hwe igh t p igs o f l e s s than lOOOg o f Y o r k s h i r e X Landrace breed ing were a l l o t t e d w i t h i n a ten day p e r i o d . Farrowings were at tended and exper imenta l p igs were removed a t b i r t h . P i g s were weighed a t b i r t h , ea r tagged , and i n d i v i d u a l l y penned i n a nursery room. P igs were randomly ass igned to t rea tments . Cages were a d j o i n i n g w i r e mesh 46cm x 25cm x 20cm i n two t i e r s . P r i o r to the t r i a l the room and cages were thorough ly c leaned and d i s i n f e c t e d . Cages were l e f t unwashed throughout the t r i a l p e r i o d . Temperature i n the nursery room was main ta ined between 30°C and 33°C. A l l p igs r e c e i v e d i r o n dext ran i n j e c t i o n s and had t h e i r needle tee th and t a i l s c l i p p e d a t two days o f age. A second i r o n dex t ran i n j e c t i o n was g iven a t 10 days o f age. 3 . 5 . 2 . 2 D i e t a r y Treatments 2 P i g s r e c e i v e d a non-medicated commercial m i l k r e p l a c e r . Feed i n take was ad jus ted to 6 per cent o f body weight i n a i r dry mat ter which was d i l u t e d 1 : 4 w i th wate r . For the f i r s t 3 days d i e t s were fo rmula ted w i th a 10 per cent dex t rose s o l u t i o n . P igs were weighed every o the r day and feed a l lowances were ad jus ted a c c o r d i n g l y . Immunoglobulin was mixed w i th the m i l k r e p l a c e r and fed a t one l e v e l : lOg /kg body weight 65 on the f i r s t day followed by 2g/kg body weight/day for succeeding days. Three periods of immunoglobulin feeding were applied: 10 days, 15 days, and 21 days. Scouring was treated, as necessary, on an in d i v i d u a l pig 3 basis, with Furoxone . 3.5.2.3 Feeding Regimen Pigs were nipple-fed throughout the 21 day t r i a l period. Creep feed was offered from day 10 but water was offered p e r i o d i c a l l y , only, by b o t t l e , to prevent s p i l l a g e . Feeding was every 2 hours for the f i r s t 4 days, every 4 hours on days 5 to 8 and every 6 hours from days 9 to 21. 3.5.3 Measurements and Observations Pigs were weighed every 2 days. Mortality was recorded and post mortems were conducted on a l l dead pigs by the Provincial Veterinary Laboratory, Abbotsford, B.C. Infectious agents and causative factors were i d e n t i f i e d whenever possible. M o r t a l i t y , rate of gain, and frequency of scouring (for survivors) were s t a t i s t i c a l l y analysed with analysis of variance using the same model as in Experiment 1. Also the mean b i r t h weights of ; survivors and dead pigs were compared with a " t " t e s t . 3.5.4 Results and Discussion Mortality rate (Table 3.4.b) improved compared to previous 66 Tab le 3.4a Experiment 4 : Exper imenta l Animals Treatment Number i n Treatment Mean B i r t h - w e i g h t Group t S .D . 10 days on immunoglobul in 8 674 + 148 g . 15 days on immunoglobul in 8 708 + 111 g . 21 days on immunoglobul in 8 749 + 116 g . Tab le 3.4b Experiment 4 : Resu l t s Treatment % S u r v i v a l % S u r v i v a l Rate o f Gain Frequency o f to Day 10 to Day 21 (g/day) Scour ing (Days s c o u r i n g / s u r v i v o r ) 10 days 63 5 0 a 40.8 + 2 3 . 2 c d 1.0 15 days 100 8 R a b 37.4 + 1 4 . 5 C 1.1 21 days 100 100 b 60.3 i 1 0 . 6 d 0.3 67 t r i a l s . M o r t a l i t y was s i g n i f i c a n t l y h igher (P = 0.05) on the 10 day t reatment compared to the 21 day t rea tment . However, 3 out o f 4 which d i e d i n the 10 day t reatment group d i d so w h i l e r e c e i v i n g immunoglobul in be fo re 10 days o f age. T h e r e f o r e , the r e s u l t i s s p u r i o u s . The e f f e c t o f pro longed feed ing o f immunoglobul in d e f i n i t e l y reduced m o r t a l i t y by m a i n t a i n i n g b e t t e r hea l t h and p reven t ing scours i n those p igs r e c e i v i n g immunoglobul in a f t e r 10 days . Death was due to b a c t e r i a l i n f e c t i o n caus ing d i a r r h e a i n 4 cases and pneumonia i n 1 case . Sa lmone l la was i s o l a t e d from 4 o f the 5 dead and presumed present a l s o i n the f i f t h . Thus the i n f e c t i o n caus ing death was d iagnosed as S a l m o n e l l o s i s i n a l l c a s e 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 between mean b i r t h weights o f s u r v i v o r s and dead P i g s . Rate o f ga in (Tab le 3 .4 .b and F igu re 3.3) was h ighes t i n the group r e c e i v i n g immunoglobul ins f o r 21 d a y s , but o n l y s i g n i f i c a n t l y h igher (P = 0.05) than the group r e c e i v i n g immunoglobul ins f o r 15 days . The 21 day group had the h i ghes t mean r a t e o f ga in o f a l l groups i n the f o u r exper iments c a r r i e d o u t . Frequency o f scou r i ng (Tab le 3 .4 .b ) was very markedly reduced du r i ng t h i s t r i a l compared to Experiment I I I where i t was a l s o r eco rded . The group r e c e i v i n g immunoglobul in f o r 21 days showed no d i a r r h e a a f t e r 4 days o f age and the o the r two groups showed a much reduced f requency between 13 and 20 days o f age (F i gu re 3 . 2 ) . S i nce bowl f eed ing was e l i m i n a t e d and n i p p l e - f e e d i n g was cont inued throughout , chances o f sp read ing contamina t ion i n the feed were l i k e l y markedly reduced. Water p r o v i s i o n was l i m i t e d to p e r i o d i c b o t t l e f eed ing s i n c e i t c o u l d n ' t be prov ided ad l i b i t u m w i thou t s p i l l i n g l e a d i n g to a wet Figure 3.3. Cumulative Weight Gain. Experiment 4 1400 f 1200 1000 800 600 400 200 A - 10 days on Immunoglobulin O - 15 days on Immunoglobulin • - 21 days on Immunoglobulin 11 13 15 Days of Age 69 environment which seemed to be deterimental to the pigs from experience in the preceding t r i a l s . 70 3.6 General D i s c u s s i o n The approach o f t h i s s tudy was t w o - f o l d . F i r s t l y , a r t i - f i c i a l r e a r i n g was seen as a mean f o r i n c r e a s i n g s u r v i v a l o f low b i r t h - we igh t p i g s . S e c o n d l y , t h i s a r t i f i c i a l r e a r i n g techn ique w i t h immuno- g l o b u l i n e x t r a c t was a p p l i e d i n a n o n - i s o l a t e d envi ronment . Thus , the r e s u l t s can be d i s c u s s e d from both p e r s p e c t i v e s . An impor tant obse rva t i on i n these t r i a l s was a lower ra te o f ga in than p r e v i o u s l y found by McCallum (1977) us ing a very s i m i l a r d i e t , and by o the r workers (F igu re 3 . 4 ) . The ra te was h ighes t w i th the 21 day t reatment i n Exper iment IV where scou r i ng was markedly reduced but was s t i l l w e l l below ra tes found w i t h sow reared p igs o f low b i r t h weight w i t h i n the same herd (60g/day vs 125g/day) . Two ou ts tand ing reasons f o r t h i s are e v i d e n t . F i r s t l y , the p i gs on t r i a l may have been r e s t r i c t e d i n t h e i r food i n t a k e too much f o r optimum growth. Cons ide r i ng the d i e t to be adequate, the main c o n t r i b u t i n g f a c t o r cou ld be too i n f r equen t feed ing s i n c e t h e i r i n t a k e a t each feed ing was l i m i t e d by t h e i r c a p a c i t y . However, w i thou t automat ic f e e d i n g , more f requen t feed ing would be d i f f i c u l t . Second l y , the lower r a t e o f ga in may have been due to t h e i r low b i r t h we igh t s . Low b i r t h w e i g h t seems to have a d e f i n i t e e f f e c t on r a t e o f g a i n . However, Lodge and E l l i o t (1979) i n d i c a t e d t ha t a lower b i r t h - weight was l e s s i n h i b i t i n g on growth under a r t i f i c i a l r e a r i n g than under na tu ra l r e a r i n g . Widdowson (1971) , i n a t r i a l , showed t ha t run t p igs never reach the p o t e n t i a l o f l a r g e l i t t e r mates. One must d i s t i n g u i s h between low b i r t h w e i g h t p igs which are normal a n a t o m i c a l l y , but s m a l l , and t rue r u n t s . Not a l l p igs under lOOOg are n e c e s s a r i l y r u n t s . The Figure 3.4 Comparative Growth Curves A - A - o -: Perry and Lecce (1968); a r t i f i c i a l rearing » " " " ; sow rearing Siers et a l . (1977); a r t i f i c i a l rearing ; sow rearing O - This t r i a l : Experiment IV (21 days on Ig) 10 12 14 16 18 20 22 Days of Age 72 m a n i f e s t a t i o n o f run t c h a r a c t e r i s t i c s i s l a r g e l y i n d i c a t e d by s i z e , however p igs above 600g d o n ' t seem to show any r u n t i n g appearance. True run ts seem underdeve loped, p o s s i b l y due to p rena ta l undernour ishment. T h e i r p h y s i c a l appearance o f very smal l s i z e and d i s p r o p o r t i o n a t e l y l a r g e domed heads suggests t h i s . S m a l l , but non-runt p igs may not be as d isadvantaged as t rue run ts when prov ided adequate n u t r i t i o n and hence may show adequate compensatory growth to make up f o r low b i r t h w e i g h t . Indeed, i n a l l but the f i r s t t r i a l , b i r t h w e i g h t d i d not a f f e c t s u r v i v a l . However, a l l those below 500g a t b i r t h , d i e d . I t i s p o s s i b l e t ha t the s u r v i v a l o f ve ry smal l t r ue run ts ( e . g . below 500g b i r t h w e i g h t ) r e q u i r e s more e x t r a o r d i n a r y a t t e n t i o n to hygiene and envi ronment . Dur ing sampl ing o f the sow-reared low b i r t h w e i g h t p i g s , v a r i a b i l i t y on growth r a t e was found to be high ( c o e f f i c i e n t o f v a r i a t i o n = 30.4 per c e n t ) . Th i s i s unders tandable i f the o p e r a t i v e f a c t o r s d e t e r - min ing growth a t t h i s pre-weaning age are c o n s i d e r e d . Such f a c t o r s i n c l u d e : 1. Nurs ing success which i s determined by a g g r e s s i v e n e s s , m i l k p roduc t ion o f the sow, and access to a p roduc t i ve t e a t . 2. Freedom from scours or o ther d i sease m a l a d i e s . 3 . Genet ic p o t e n t i a l o f the i n d i v i d u a l . S ince low b i r t h w e i g h t p igs a re l e s s compe t i t i ve du r ing nu rs ing and are more s u s c e p t i b l e to c h i l l i n g (as exp la i ned by the r e l a t i o n s h i p between we igh t , su r f ace a r e a , and heat l o s s (Monte i th and Mount, 1974 ) ) , a r t i f i c i a l r e a r i n g i n a c o n t r o l l e d environment would e l i m i n a t e many o f the s t r e s s - f u l f a c t o r s and a l l o w a more e q u i t a b l e s i t u a t i o n . Runts may be even 73 f u r t h e r d isadvantaged f o r s u r v i v a l because o f a low rese rve o f carbohydrate i n the l i v e r and muscles a t b i r t h f o r energy (Widdowson, 1971) . The high s u r v i v a l l e v e l s i n Exper iment IV p rov ide support f o r the advantage o f a r t i f i c i a l r e a r i n g o f low b i r t h w e i g h t p i g s . The f a c t o r s c o n t r i b u t i n g to the e f f i c a c y o f the immuno- g l o b u l i n e x t r a c t can be a n a l y s e d . Th i s e x t r a c t , which i s a p r e c i p i t a t e " s a l t e d out " w i t h a 40 per cent ( N H 4 ) 2 SO^ s o l u t i o n , con ta ins a l l o f the gamma-globul in and much o f the b e t a - g l o b u l i n f r a c t i o n s o f serum (Owen, 1961). L i k e c o l o s t r u m , which i s cons ide red a serum t ransudate (Bourne, 1973) , i t would c o n t a i n over 80 per cent IgG, l e s s IgA then co los t rum (8 vs 13 per c e n t ) , and more IgM than co los t rum (10 vs 4 per cent ) (Bourne, 1973) . A l s o , u n l i k e c o l o s t r u m , serum d e r i v e d immungolobul in con ta ins no sec re to r y IgA ( S - I g A ) . One cou ld presume tha t the e f f i c a c y o f the e x t r a c t was predom- i n a t e l y due to IgG. However, the presence o f IgM and non -sec re to ry IgA cannot be i g n o r e d . M i l e r e t a l _ . , (1975) found tha t the minimum e f f e c t i v e concen t ra t i ons f o r l o c a l p r o t e c t i v e e f f e c t i n l i g a t e d loop t e s t s w i th p igs were 0 . 5 , 0 . 0 5 , and 0.005 mg/ml o f IgG, IgM, and IgA r e s p e c t i v e l y . There- f o r e , even lower l e v e l s o f IgM and IgA may have a s i g n i f i c a n t p r o t e c t i v e e f f e c t . IgG i t s e l f , has been found to be o f s i g n i f i c a n t p r o t e c t i o n aga ins t e n t e r i c c o l i b a c i l l o s i s (Brandenburg and W i l s o n , 1972) . How IgG func t i ons w i t h i n the gut lumen i s open to ques t i on but i t has a n t i t o x i n and a g g l u - t i n a t i n g p r o p e r t i e s (Beneceraf and Unanue, 1979) . A l though i t may not be ab le to adhere to the gut l i n i n g as S-IgA does , a g g l u t i n a t i o n o f b a c t e r i a would f a c i l i a t e t h e i r p e r i s t a l t i c remova l . F i n a l l y , the e f f i c a c y o f t h i s e x t r a c t a g a i n s t Sa lmone l la b a c t e r i a i s o f c o n s i d e r a b l e i n t e r e s t . To d a t e , t h i s techn ique has been 74 used to prevent m o r t a l i t y due to E. c o l i e n t e r i t i s (Owen e_t aj_., 1961; Scoot, 1972; McCallum, 1977; and Kenelley et_ al_., 1979). Since i t protects against Salmonella, as shown i n the present study, many of the herds from which the blood f o r immunoglobulin extraction was taken must have antigenic quantities of Salmonella i n f e s t i n g them. The contribution of these herds must have been great enough to provide a s u f f i c i e n t l y high antibody t i t r e against Salmonella to be preventive in these experiments. 75 Conclusions 1. The a r t i f i c i a l r earing technique with a r t i f i c i a l immunoglobulins used i n these t r i a l s w i l l save low birthweight pigs i f weaned at b i r t h . However, growth rate i s r e s t r a i n e d , possibly by r e s t r i c t i v e feeding or by t h e i r low b i r t h weight. 2. The l e v e l of immunoglobulin of 10 g/kg./body weight on the f i r s t day followed by 2 g/kg/day on succeeding days seems to be e f f e c t i v e as a dosing l e v e l . 3. Longer periods of immunoglobulin administration may be necessary i n a non-isolated environment. Whether t h i s i s a r e s u l t of low birthweight i s not known. 4. This immunoglobulin extract used was e f f e c t i v e protectio against the Salmonella bacteria encountered in these t r i a l s . Certain types of Salmonella may be present at endemic l e v e l s i n many swine herds. 5. An increased understanding of the causes of runting i s necessary i n order to j u s t i f y the e f f o r t of attempting to save the smallest ones. 76 Foot -Notes 1. R and H Farms, A l d e r g r o v e , B .C . 2 . Federated Coopera t i ves L t d . M i l k Rep lacer - 30 . 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