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The provision of passive immunity to colostrum-deprived piglets by bovine or porcine serum immunoglobulins,… Drew, Murray D. 1989

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THE  PROVISION OF PASSIVE IMMUNITY TO COLOSTRUM-DEPRIVED PIGLETS BY BOVINE OR PORCINE SERUM  IMMUNOGLOBULINS,  IRON CHELATORS AND VIABLE LEUKOCYTES By Murray D. Drew B.Sc.  (Agr), The University of Guelph, 1983  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1989 ©  Murray D. Drew, 1989  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  publication of this thesis  or  her  the  I agree  requirements  for  an  Department of The University of British Columbia Vancouver, Canada  advanced  that the Library shall make it  I further agree that permission for extensive  purposes  may  representatives.  It  be is  granted  by the head  understood  that  for financial gain shall not be allowed without  permission.  DE-6 (2/88)  of  of  my  copying  or  my written  ii ABSTRACT This thesis examines the effect of supplementing sow milk replacers fed to colostrum deprived p i g l e t s with 1) bovine or porcine immunoglobulins; 2) synthetic  iron chelators and 3) viable leukocytes.  Piglets require dietary immunoglobulins during the f i r s t day after b i r t h to provide passive systemic immunity. Piglets that did not receive immunoglobulins during this period had survival rates of 19% and 0% i n two different  exper-  iments. Bovine immunoglobulins on day 1 after b i r t h were poorly absorbed from the  diet  resulting  i n inadequate plasma immunoglobulin  concentrations, low  survival and low weight gains compared to p i g l e t s that received porcine immunoglobulins  on day 1. During the days 2-14, however, p i g l e t s receiving  either  bovine or porcine immunoglobulins were equal i n survival and growth rates. The  milk protein  l a c t o f e r r i n protects p i g l e t s from enteric  infections by  binding ionic iron making, i t unavailable to bacteria and preventing b a c t e r i a l growth. Two synthetic  iron  chelators,  ethylenediamine-di-orthohydroxyphenyl  acetic acid (EDDA) and N,N'-bis(o-hydroxybenzyl)-ethylenediamine diacetic acid (HBED) have a f f i n i t i e s  f o r iron similar  to l a c t o f e r r i n ' s and are potential  substitutes for l a c t o f e r r i n in sow milk replacers. The a n t i b a c t e r i a l properties of  l a c t o f e r r i n , EDDA and HBED were compared i n v i t r o . Lactoferrin  and EDDA  inhibited the growth of E^ c o l i 0 157 K88 over a 12 hour period while HBED had no effect on the growth of this organism. When EDDA or HBED were fed to p i g l e t s on  days 2-14, those that  received HBED had low survival  and growth  rates.  Piglets that received EDDA had growth rates similar to those receiving porcine immunoglobulins from days 2-14. When EDDA was fed during days 1-14 however p i g l e t growth rates were depressed. This was probably due to higher absorption  of EDDA during day 1. Dietary EDDA also increased the excretion of iron i n the urine and feces and decreased  the incorporation of iron into hemoglobin.  The feeding of viable leukocytes derived from abattoir blood to a r t i f i c i a l l y reared p i g l e t s fed porcine immunoglobulins resulted i n increased c e l l mediated immune responses i n 2 of 4 l i t t e r s . The major histocompatibility complex types of  the donor  and recipient  of the leukocytes  may be responsible  for  the  inconsistent r e s u l t s . Porcine immunoglobulins (25 mg mir on day 1, followed by either bovine or 1  porcine  immunoglobulins  (5 mg mir ) on days 2-14 provided 1  adequate  immunity, survival and growth rates i n colostrum deprived p i g l e t s .  passive  iv  T A B L E OF CONTENTS  Page ABSTRACT  i i  L I S T OF TABLES  vi  L I S T OF FIGURES  X  ACKNOWLEDGEMENTS  xii  INTRODUCTION  1  A REVIEW OF THE IMMUNE SYSTEM  2  The Major Histocompatability Complex Neutrophils and Macrophages T Lymphocytes B Lymphocytes and Immunoglobulins Complement The Ontogeny of the Piglet's Immune System Host Defences i n the Neonatal Piglet PROTECTIVE FACTORS I N SOW'S COLOSTRUM  AND M I L K  Viable C e l l s Immunoglobulins Immunoglobulin F o r t i f i e d Milk Replacers Lactoferrin Synthetic Iron Chelators Vitamin B12 Binding Protein Lysozyme The Lactoperoxidase System Glycoconjugate and Oligosaccharide Receptor Analogues EXPERIMENT  1  Introduction Materials and Methods Results Discussion EXPERIMENT  2  Introduction Materials and Methods Results Discussion EXPERIMENT  3  Introduction Materials and Methods Results  4 6 8 12 20 23 25 28  30 33 41 43 52 55 56 57 58 60  60 60 65 67 79  79 79 83 84 90  90 90 94  V  Discussion EXPERIMENT 4  Introduction Materials and Methods Results Discussion EXPERIMENT 5  Introduction Materials and Methods Results Discussion EXPERIMENT 6  Introduction Materials and Methods Results Discussion  •••  96  109  109 109 112 115 131  131 131 134 135  144  144 144 148 149  GENERAL D I S C U S S I O N  161  CONCLUSIONS  164  LITERATURE CITED  165  vi LIST OF TABLES  Page  Table 1.  C l a s s i f i c a t i o n of placentation according to the intervening tissues and correlation with time of transfer of immunity from mother to offspring  Table 2. Nonspecific host defense factors Table 3. Ontogeny of the immune system of the f e t a l p i g l e t  1 3 24  Table 4.  Approximate chemical analysis of sow's colostrum and milk  Table 5 .  D i f f e r e n t i a l c e l l counts for the mammary secretions of sows in which b a c t e r i a l i n f e c t i o n was absent  32  Immunoglobulin levels i n serum, colostrum, milk and i n t e s t i n a l juice of pigs  34  Concentration of l a c t o f e r r i n i n various secretions of the bovine mammary gland  50  Comparison of the s t a b i l i t y constants of HBED and EDDA chelates  55  Experimental protocol for studying the administration of immunoglobulins to p i g l e t s ( Experiment 1)  61  Table 6. Table 7 . Table 8. Table 9.  ... 28  Table 10. Composition of freeze dried bovine and porcine immunoglobulin concentrates  72  Table 11. The e f f e c t of bovine or porcine immunoglobulins on the survival of colostrum deprived piglets (Experiment 1)  73  Table 12. The e f f e c t of bovine or porcine immunoglobulins on the average weekly diarrhea scores of colostrum deprived p i g l e t s (Experiment 1)  73  Table 13. The effect of bovine or porcine immunoglobulins on the average d a i l y gains of colostrum deprived p i g l e t s (Experiment 1)  74  Table 14. The effect of bovine or porcine immunoglobulins on the body weights of colostrum deprived p i g l e t s (Experiment 1)  74  Table 15. The effect of bovine or porcine immunoglobulins on the plasma immunoglobulin concentrations of colostrum deprived p i g l e t s levels (Experiment 1)  75  vii Table 16. Experimental protocol for testing the effect of l a c t o f e r r i n , EDDA and HBED on the growth rate of E^. c o l i 0 157 K88 (Experiment 2)  80  Table 17. The effect of l a c t o f e r r i n , EDDA and HBED with and without porcine immunoglobulins, on the mean Log(CFU) mir of E ^ c o l i 0 157 K88 (Experiment 2)  88  Table 18. Experimental protocol for testing the effect of EDDA and HBED on the performance of a r t i f i c i a l l y reared p i g l e t s (Experiment 3)  91  1  Table 19. The effect of EDDA or HBED, with or without porcine immunoglobulins on the survival of colostrum deprived p i g l e t s (Experiment 3)  102  Table 20. The effect of EDDA or HBED, with or without porcine immunoglobulins on the average weekly diarrhea scores of colostrum deprived p i g l e t s (Experiment 3)  103  Table 21. The effect of EDDA or HBED, with or without porcine immunoglobulins on the average d a i l y gains of colostrum deprived p i g l e t s (Experiment 3)  104  Table 22. The effect of EDDA or HBED, with or without porcine immunoglobulins on the body weights of colostrum deprived p i g l e t s (Experiment 3)  105  Table 23. The effect of EDDA or HBED, with or without porcine immunoglobulins on the plasma iron and t o t a l iron binding capacity (TIBC) of colostrum deprived p i g l e t s (Experiment 3)  106  Table 24. The effect of EDDA or HBED, with or without porcine immunoglobulins on the packed c e l l volume and hemoglobin concentrations of colostrum deprived p i g l e t s (Experiment 3)  107  Table 25. The effect of EDDA or HBED, with or without porcine immunoglobulins on the plasma immunoglobulin concentrations of colostrum deprived piglets (Experiment 3)  108  Table 26. Experimental protocol for studying the administration of EDDA with porcine or bovine immunoglobulins (Experiment 4)  111  Table 27. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the survival of colostrum deprived piglets (Experiment 4)  123  viii  Table 28. The effect of EDDA , with and without bovine or porcine immunoglobulins, on the average weekly diarrhea scores of colostrum deprived p i g l e t s (Experiment 4)  124  Table 29. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the average d a i l y gains of colostrum deprived p i g l e t s (Experiment 4)  125  Table 30. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the body weights of colostrum deprived piglets (Experiment 4)  126  Table 31. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the plasma iron and t o t a l iron binding capacity (TIBC) of colostrum deprived p i g l e t s (Experiment 4)  127  Table 32. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the packed c e l l volume and hemoglobin concentrations of colostrum deprived p i g l e t s (Experiment 4)  128  Table 33. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the plasma immunoglobulin concentrations of colostrum deprived piglets (Experiment 4)  129  Table 34. The e f f e c t of EDDA on individual piglets on the day of " F e i n j e c t i o n (Experiment 5)  139  Table 35. The effect of EDDA on excretion and d i s t r i b u t i o n of F e (Experiment 5) 3 9  140  Table 36. Experimental protocol used to study the effect of sow or a r t i f i c i a l rearing on colostrum deprived p i g l e t s (Experiment 6)  145  Table 37. The effect of sow or a r t i f i c i a l rearing on p i g l e t survival (Experiment 6)  156  Table 38. The effect of sow or a r t i f i c i a l rearing on p i g l e t average d a i l y gains (Experiment 6)  157  Table 39. The effect of sow or a r t i f i c i a l rearing on mean p i g l e t weights (Experiment 6)  157  Table 40. The effect of sow or a r t i f i c i a l rearing on plasma PIgG levels (Experiment 6)  158  ix Table 41. The effect of sow or a r t i f i c i a l rearing on piglet intradermal response to PHA at 3 weeks of age (Experiment 6)  159  Table 42. The effect of sow or a r t i f i c i a l rearing on p i g l e t intradermal response to PHA at 24 hours for treatment x block interaction (Experiment 6)  160  Table 43. The average d a i l y gains and survival of p i g l e t s i n Experiments 1, 3, 4 and 6  164  X  LIST OF FIGURES Page Figure 1-  Hemopoietic stem c e l l d i f f e r e n t i a t i o n  Figure 2.  Structure of an IgG molecule  13  Figure 3.  The basic structure of the immunoglobulin classes  15  Figure 4.  The ontogeny of B c e l l s  17  Figure 5.  The genetic basis of antibody production  18  Figure 6 .  The c l a s s i c a l , alternate and terminal pathways of the complement cascade  21  The concentration of IgG, IgM and IgA i n the sera of naturally reared p i g l e t s  36  Figure 7 . Figure 8 .  Figure 9 .  5  The covalent structure of human monomeric, dimeric and secretory IgA  38  The preparation of bovine l a c t o f e r r i n from milk  44  Figure 10. The molecular structure of EDDA and HBED Figure 11. Gel electrophoresis of polyphosphate fractions from porcine serum Figure 12. Gel electrophoresis of polyphosphate fractions from bovine serum  53  76 77  Figure 13. The effect of bovine or porcine immunoglobulins on the plasma immunoglobulin concentration of colostrum deprived p i g l e t s (Experiment 1)  78  Figure 14. The effect of l a c t o f e r r i n , EDDA and HBED with and without porcine immunoglobulins, on the mean Log (CPU) mL- of J L c o l i 0 157 K88 (Experiment 2)  89  1  Figure 15. The effect of EDDA or HBED, with or without porcine immunoglobulins on the plasma immunoglobulin concentrations of colostrum deprived p i g l e t s (Experiment 3)  109  Figure 16. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the plasma immunoglobulin concentrations of colostrum deprived p i g l e t s (Experiment 4)  130  xi Figure 17. The effect of EDDA on plasma iron disappearance (Experiment 5)  141  Figure 18. The effect of EDDA on the incorporation of " F e into red blood c e l l s (Experiment 5)  142  Figure 19. The effect of EDDA on urinary and f e c a l excretion of F e (Experiment 5) 3 9  Figure 2 0 . The effect of sow or a r t i f i c i a l rearing on p i g l e t plasma IgG (Experiment 6)  143 161  xii ACKNOWLEDGEMENTS I would l i k e to thank Dr. Bruce Owen for his ideas, support  and kindness  over the l a s t four years. The members of my committee, Dr. R. B l a i r , Dr. C. R. Krishnamurti, Dr. J.A. Shelford and Dr. B.J. Skura have a l l given me excellent advice and encouragement throughout study. Many  sleepless nights  were  spent  feeding  piglets  by Richard  Whiting,  Margaret Crowley, Shane Gumprich and T r i c i a Arnold. I thank them for their dedication. G i l l e s Galzy and Frances Newsome were always there with help and good ideas when problems arose. In p a r t i c u l a r ,  I would l i k e to thank Irene  Bevandick for her commitment and excellent technical assistance. Financial assistance provided by the Productivity Enhancement Program of the Canada/British Columbia Agri-Food Regional Development Subsidiary Agreement and by  the Natural  Science  and Engineering  Research Council i s g r a t e f u l l y ac-  knowledged. F i n a l l y , the love and support of my wife Bev have made the l a s t four years possible and enjoyable.  1  INTRODUCTION The newborn p i g l e t i s a p h y s i o l o g i c a l l y mature and capable creature compared to  the young of many other species. I t can walk, see and feed i t s e l f soon after  b i r t h . A l l of the elements of the adult immune system are present and the piglet is  able to mount an immune response, but only a primary immune response (Salmon  1987). The primary response of the immune system on f i r s t exposure to a pathogen is  slow to develop and provides inadequate protection against i n f e c t i o n . In  of  addition to inadequate active immunity, the e p i t h e l i o c h o r i a l placentation  the pig does not allow placental transfer of immunoglobulins  (see Table 1).  The p i g l e t i s thus born with no passive protection and i s completely dependent on sow's colostrum and milk for i t s s u r v i v a l .  Table 1.  C l a s s i f i c a t i o n of placentation according to the intervening tissues and the c o r r e l a t i o n with  tiae of transfer of immunity froa mother to offspring (Sterzl and S i l v e r s t e i n 19671 . L  Uterine tissues Type of Placentation  Aniaal  Fetal tissues  Tiae of Transmission  EndoConnec- Epithe- Tropho- Connec- Endothe theliua tive liua blast tive liua Prenatal Postnatal  E p i t h e l i o c h o r i a l Horse, Pig  +  +  +  0  +++ (36 hrs)  Syndesaochorial  +  +  0  0  +++ (36 hrs)  Endotheliochorial Cat, Dog  +  0  0  +  ++ (10 Days)  Heaoendothelial  Rat, House  0  0  0  +  ++ (20 Days)  Heaoendothelial  Rabbit  0  0  0  +++  0  Heaochorial  Man, Monkey  0  0  0  +++  0  1  Sheep, Cow  Symbols employed i n t h i s table are: 0 = absence; + = pres nee; ++ = major contribution;  +++ = sole contribution.  2 Fortunately  f o r the p i g l e t , sow colostrum i s usually available to provide  this needed protection. However, the death of the sow, mastitis, agalactia, and large l i t t e r s can mean that p i g l e t s do not receive adequate c o l o s t r a l passive immunity. A method of replacing sow's milk and colostrum would be a valuable tool to the swine producer. To develop such a system requires understanding the development of the p i g l e t ' s immune system and i t s regulation by the sow's milk and colostrum. A REVIEW OF THE IMMUNE SYSTEM The Webster New American Dictionary  (1972) defines the word immune as  "exempt, as from a disease". The words immunity and immune system immediately conjure up the idea of a large host destroying  a small parasite  (bacteria,  virus etc.) that has invaded i t s body. In a larger sense, immunity also affects the host's reaction to food proteins, insect stings and tissue  transplants.  The immune system consists of a l l of the physical structures and physiological responses that affect interactions between the host and the rest of the universe. Non-specific the  aspects of immunity are usually overlooked i n discussions of  immune system  (see Table 2). Non-specific  host defense i s at least as  important as s p e c i f i c immunity, however. Intact body surfaces, normal b a c t e r i a l f l o r a etc. reduce the number of interactions the s p e c i f i c immune system has with the bacteria and viruses. In addition, i t i s d i f f i c u l t to t e l l where nons p e c i f i c immunity ends and s p e c i f i c responses begin as non-specific defenses interact with s p e c i f i c ones. The s p e c i f i c immune system i s responsible for distinguishing self from nons e l f and mounting an appropriate normal body f l o r a  or food  response. The response may be tolerance for  proteins.  I t may be destructive  f o r pathogenic  bacteria or virus infected c e l l s . This i s a complex task, so complex that the immune system i s comparable to the human brain i n i t s i n t r i c a c y . The immune system i s l i k e the brain i n many respects. I t can process information, memory and i s able to respond to antigens  i t has  i t has never encountered before.  Table 2. Nonspecific host defense factors (Beisel, 1984).  Actively responsive Factors  Host system Microbiological  Passive Factors Normal body f l o r a  Physical  Anatomical surface barriers Anatomical pathways C i l i a r y cleansing motion  Cough r e f l e x Vomiting r e f l e x Intestinal peristalsis  Secretions (internal and external)  Gastric and urinary a c i d i t y Mucins and their enzymes Fatty acids i n secretions Lysozyme i n secretions Lactoferrin Salivary enzymes H2O2 Surfactants  Interferons Tuftsin C i r c u l a t i n g S-lysins Mast c e l l products e.g., Histamine Heparin, Serotonin, Anaphylactic factors, Enzymes Lymphokines and Thymic hormones, B i o l o g i c a l l y active small molecules  Physiological  Humoral  Age related influences Sex related influences Genetic e f f e c t s Circadian rhythms Tissue pH  Plasma transport proteins Plasma redox potential Divalent cations: C a and Mg 2+  2+  Inflammatory reactions C e l l u l a r phagocytic and b a c t e r i c i d a l activity Fever generation Metabolic responses eg., amino acids, carbohydrates,lipids, electrolytesminerals, Coagulation system Complement system Kinin system Plasma acute-phase reactant proteins  4 Antigens are the basic unit to which the immune system responds. To i n i t i a t e an immune response an antigen must have several properties. They are normally large complex molecules with stable 3-dimensional structures (Tizard 1982). This makes most protein molecules ideal  antigens. Carbohydrates and  lipids  are  usually too small and f l e x i b l e to make good antigens. The immune system consists of several different specialized organs, for example  types of c e l l s  the thymus. The c e l l s  and some  are a l l derived from  pluripotent stem c e l l s found i n the bone marrow. These stem c e l l s have several important properties. They are self-renewing and are capable of d i f f e r e n t i a t i o n into a l l c e l l groups derived from the bone marrow. Because the stem c e l l s are continually renewing themselves, they are extremely sensitive to radiation. Their destruction i s the primary cause of death from exposure to radiation. The stem c e l l s d i f f e r e n t i a t e into two lineages of c e l l s (see Figure 1). The c e l l s of the myeloid lineage include erythrocytes, p l a t e l e t s , granulocytes and macrophages. The lymphoid lineage includes T-lymphocytes and B-lymphocytes. The macrophages, T c e l l s and B c e l l s are the c e l l u l a r components of the immune system. The Major Histocompatibility Complex The major histocompatibility complex (MHC) i s a group of genes that encode the MHC antigens. The MHC i n swine i s termed the Swine Lymphocyte Antigen (SLA). It occurs on chromosome 7 (Vaiman 1987) and consists of three different classes of genes. Class I SLA genes are the A, B and C l o c i (Vaiman et a l . 1986). These genes code for the class I SLA antigens. These antigens are glycoproteins with a molecular weight of about 45 Kd. A l l nucleated c e l l s i n the body express these  5  Figure 1. Hemopoietic stem c e l l d i f f e r e n t i a t i o n (based on Cooper et a l . 1984).  Pluripotent Stem C e l l Myeloid  \  /  Lineage  Myeloid Stem C e l l  /1  Erythrocyte  Platelets  Lymphoid Stem C e l l  Granulocyte Progenitor  /  Neutrophils  Lymphoid Lineage  B  Lymphocytes  \  Lymphocytes  Plasma C e l l  Macrophages  antigens. The class I antigens are released into the cytosol of the c e l l where they bind with peptides present there (Maryanski et a l . 1986). These peptides may be normal c e l l constituents or abnormal products of viruses or cancer c e l l s . The complex of class I molecule and peptides then migrate to the surface of the cell.  Cytotoxic T c e l l s  recognize  foreign polypeptides  and lyse  the c e l l .  Cytotoxic T c e l l s also lyse c e l l s that display foreign class I MHC molecules. This i s the mechanism that causes the rejection of transplants between noncompatible donors. I t i s also responsible for graft-versus-host disease. Graftversus-host disease occurs when immunocompetent c e l l s transferred to a MHC class I incompatible host react against the host tissues. Normally the immune system of the host can r e s i s t this attack. In immunodeficient  hosts such as neonatal  6  p i g l e t s ,however, the graft can cause considerable damage to host tissues. More than 30 d i f f e r e n t class I SLA antigens have been characterized i n swine (Vaiman 1987)  with approximately  10-12  a l l e l e s per locus. This means that over  1000 d i f f e r e n t haplotypes are possible. Only 62 d i f f e r e n t haplotypes have been found however. This i s due to high linkage disequilibrium between the class I genes. The  class I haplotype  of pigs affects a number of production  traits  including ovulation rate (Rothschild et a l . 1984), embryonic survival (Vaiman et a l . 1986)  and b i r t h and weaning weights (Rothschild et a l . 1986).  There are two class II l o c i i n the SLA: the DQ and the DR. Class II antigens occur on lymphocytes and macrophages. They control interactions among these cellular  components of  the  immune system. Macrophages phagocytize  foreign  p a r t i c l e s i n the body and digest them. These fragments are bound by class II antigens and the complex migrates to the surface of the macrophage (Babbitt  et  a l . 1985). The class II haplotype of pigs controls their a b i l i t y to respond  to  different antigens and s u s c e p t i b i l i t y to disease The  class III SLA  (Vaiman et a l . 1986).  genes code for several components of  the  complement  cascade. This class of SLA genes influences hemolytic complement a c t i v i t y and responsiveness  to antigens  (Vaiman 1987).  Neutrophils and Macrophages Neutrophils are the f i r s t l i n e of defence during an i n f e c t i o n . They occur in large numbers i n the blood stream and are rapidly mobilized to a s i t e tissue injury by various chemotactic  of  stimuli (Klebanoff and Clark 1978) . Here  they rapidly phagocytize foreign material. Once the p a r t i c l e i s ingested i t i s k i l l e d by oxidative metabolites and digested. Neutrophils have limited energy however and cannot maintain their attack for very long. Macrophages are required to sustain the response to i n f e c t i o n .  7  Macrophages have 3 major functions: (i) they are important i n the elimination of foreign material v i a phagocytosis; ( i i ) they present antigen to lymphocytes and ( i i i ) they secrete important bioactive molecules that are important i n host defense  (Kende 1982;  Shevach 1984).  Macrophages consist of several d i f f e r e n t types of c e l l s . After  differen-  t i a t i o n i n the bone marrow, immature macrophages enter the blood stream where they c i r c u l a t e  (Kende 1982). These c e l l s migrate  to the tissues where they  become mature macrophages. Some types of macrophages become resident i n body tissues. These include Kupffer's c e l l s i n the l i v e r and alveolar c e l l s i n the lung. These c e l l s  are stationed at strategic points of the body to remove  foreign material and microorganisms from the blood stream. Macrophages migrate to s i t e s of i n f e c t i o n i n response to b a c t e r i a l products, complement cascade products and factors released by dying neutrophils (Tizard 1986a). Once at the s i t e , macrophages bind to foreign bacteria, ingest them and k i l l them. The production of hydroxyl r a d i c a l s , singlet oxygen and hypochloride ion during the respiratory burst k i l l s the ingested microbe. As the macrophages mature they develop membrane receptors that aid i n their physiological functions (Silberberg-Sinakin et a l . 1980). For phagocytosis of bacteria to take place there must be adherence between the macrophage and the bacteria. B a c t e r i a l slime coats, capsules and carbohydrates decrease adherence so phagocytosis  i s less  efficient.  Immunoglobulins  and  the C3b  complement  fragment can bind to these b a c t e r i a l surfaces. The receptors for the Fc portion of  IgG  and  IgM  and  for the C3b  between macrophages and  complement fragment increase the adherence  bacteria.  Factors  phagocytic c e l l s and bacteria are c a l l e d  that increase adherence between  opsonins.  Macrophages also have very high levels of membrane Class II MHC  antigens.  8 This relates to their role i n presenting antigens to T c e l l s . After foreign antigens are ingested, they are denatured and cleaved i n lysosomes (Babbitt et a l . 1985). These fragments are then bound by class II MHC molecules  and the  complex migrates to the c e l l membrane. T c e l l s s p e c i f i c for this complex bind to i t . This i s the f i r s t step i n the activation of the T helper c e l l . T c e l l s are incapable of responding to many antigens unless macrophages f i r s t process and present the antigen (Waldron et a l . 1973). Only macrophages that express Class II MHC on their c e l l surfaces can present antigen to T c e l l s  (Yamashita  and Schevach 1977). The more Class II antigen present on the c e l l surface, the more e f f i c i e n t  the macrophage  i s i n stimulating an immune  macrophages of neonates have low levels of Class II antigens and this i s p a r t i a l l y responsible for their immunodeficient The  presentation of antigen to a T c e l l  response.  (Tizard 1986a)  condition.  by a macrophage results i n the  release of Interleukin-1 by the macrophage. This stimulates T c e l l and also stimulates B c e l l s of  The  activity  (Kende 1982). Macrophages secrete a wide variety  different products which include a n t i b a c t e r i a l proteins l i k e lysozyme and  lactoferrin,  complement components and immunoregulatory factors l i k e  inter-  leukin-1. The functions of the macrophage include roles i n non-specific host  defense  and i n the immune system. It coordinates the a c t i v i t y of both systems so  they  operate e f f i c i e n t l y i n the protection of the host. T Lymphocytes The lymphoid lineage of the pluripotent stem c e l l s consists of 2 populations of  lymphocytes, the B lymphocytes and the T lymphocytes. The two populations  arise from the same group of c e l l s and are indistinguishable i n appearance. Both have  receptors  for s p e c i f i c  antigens  and the DNA  of these  receptors i s  9 rearranged two  i n somatic  c e l l s . Here the s i m i l a r i t i e s  groups of lymphocytes are completely  end. The functions of the  d i f f e r e n t . B c e l l s produce soluble  antibodies which bind to antigen. T c e l l s regulate the functioning of the immune system and are responsible for destroying virus infected c e l l s and cancer c e l l s . T cells  can be divided into subpopulations  several markers. The terminology  based upon the expression of  of these markers i s d i f f e r e n t for different  species. Most studies of T c e l l s use mice so the terminology for murine T c e l l s i s used here with appropriate references to terminology for pigs. A l l mature murine T c e l l s express the CD3 complex (Samuelson et a l . 1985). This complex consists of 6 or more proteins associated with the T c e l l antigen receptor. Other important markers of T c e l l s include the CD4 and CD8 antigens. These are functional markers (von Boehmer 1988). T c e l l s expressing the CD4 antigen are T helper c e l l s . These CD4  +  c e l l s activate other components of the  immune system including B c e l l s and cytotoxic T c e l l s . The CD8 T c e l l s include +  these cytotoxic T c e l l s . In addition to these functional differences, the and CD8  +  CD4  +  T c e l l s also d i f f e r i n the class of MHC molecules  cells  recognize  class II MHC molecules  CD4  +  they recognize. The  while the CD8  +  cells  recognize  class I molecules. Markers for these populations i n pigs have been characterized (Salmon 1987) . The  CD4 antigen i n mice corresponds  antigen corresponds  to the PT4 antigen i n pigs and the CD8  to the PT8 antigen. T c e l l s bearing the PT4 antigen are  helper c e l l s while the PT8 T c e l l s are suppressor or effector T C e l l s . In addition several markers that d i f f e r e n t i a t e porcine T c e l l s from B c e l l s e x i s t . Porcine T c e l l s w i l l form rosettes with sheep red blood c e l l s . This effect i s due to a receptor s p e c i f i c to porcine T c e l l s . A monoclonal antibody (MSA4) to this receptor has been synthesized (Pescowitz et a l . 1985).  10  The antigen receptor of T c e l l s d i f f e r s from immunoglobulin molecules that serve as B c e l l antigen receptors. T c e l l receptors are not secreted by T c e l l s and  they  do not bind  characterizing  soluble antigens  (von Boehmer 1988). This  them much more d i f f i c u l t  than  immunoglobulins.  The T  receptor does not recognize native antigen but i t recognizes processed associated with class I or class II MCH The T c e l l  has made cell  antigen  molecules.  receptor i s a heterodimer containing an a and a S chain which  are joined by d i s u l f i d e bridges  (Yague et a l . 1985). Both proteins have mole-  cular weights of about 40 Kd. The d i v e r s i t y of T c e l l receptor antigen specif i c i t i e s i s generated by the rearrangement of the genes that code for the a and fi chains  (Snodgrass et a l . 1985). The same genes code f o r the antigen  receptors of both CD4  +  and CD8+ c e l l s (Dembic et a l . 1986) . This i s surprising  because the two populations  of c e l l s recognize d i f f e r e n t MHC molecules. The  answer to this i s that the recognition of antigen by T c e l l requires a whole complex of proteins of which the T c e l l receptor i s one part (von Boehmer 1986; Emmrich et a l . 1986). The CD3 complex and the T c e l l receptor are part of the antigen receptor f o r both CD4 .and CD8 +  +  T c e l l s . The CD4 molecule i s thought  to cross l i n k with the a and fi chains of the T c e l l receptor on Class II MHC molecules.  The same i s true f o r the CD8  +  T c e l l s except that here the CDS  protein cross l i n k s with the a and fi chains on a class I MHC molecule. The s p e c i f i c i t y for the antigen bound by the MHC molecule i s provided by the a and fi chains. The s p e c i f i c i t y for either class I or II molecules i s due to the CD4 or CD8 molecules. T lymphocytes are produced i n the bone marrow and migrate to the thymus. These immature T c e l l s are CD3" • CD4~ • CD8- and the genes that code for the T c e l l receptor have not been rearranged  (von Boehmer 1988). The f i r s t event i s  11 the rearrangement of the genes for the a and 2 chains (von Boehmer 1988). After this the c e l l s become CD3+,CD4 ,CD8 +  +  or CD3~ • CD4 ,CD8 . These c e l l s then undergo +  +  thymic processing i n which a l l CD3- c e l l s are destroyed. In addition CD3  +  that react with s e l f antigens are also destroyed.  cells  Less than 1% of the T c e l l s  that enter the thymus leave i t (Scollay et a l . 1980). The c e l l s that leave the thymus  are CD3 ,CD4 ,CD8 . +  +  +  These  cells  are unspecialized.  They  can be  transformed into either CD3 ,CD4 ,CD8 or CD3+ ,CD4 • CD8 c e l l s as required (Shen +  +  _  -  +  et a l . 1980). The  CD3 ,CD4 ,CD8~ +  +  cells  are T helper  cells.  As mentioned  previously,  macrophages process and present antigen i n the context of class II MHC molecule. When a T helper c e l l that i s s p e c i f i c f o r this presented antigen binds to the macrophage interleukin-1 i s released. Interleukin-1  activates the T c e l l and  i t begins to divide. The clone of T c e l l s i n turn activates B c e l l s . B c e l l activation may be antigen s p e c i f i c or non-specific (Tada 1984) . Antigen s p e c i f i c help involves the binding of the T c e l l to determinants on an antigen bound to the  B cells  antigen  receptor.  These T c e l l s  also  recognize  class  II MHC  molecules on the surface of the B c e l l . The recognition of both s p e c i f i c antigen and  the class II molecule causes the T c e l l to release interleukin-2. Inter-  leukin 2 causes the T c e l l s to divide and to secrete lymphokines that activate B c e l l s (Miedema and Melief 1985) . T c e l l s can also help B c e l l s by the release of soluble factors. These factors are not antigen s p e c i f i c . They non-selectively stimulate B c e l l s to produce immunoglobulins. Cytotoxic T c e l l s destroy virus infected and cancerous target c e l l s . These T c e l l s are CD3 ,CD4"-CD8 +  recognize cell  +  and are MHC class I r e s t r i c t e d .  Cytotoxic T c e l l s  non-self peptides bound to the class I molecules. This causes the T  to secrete perforins  (Herberman and Ortaldo  1981). Perforins aggregate  12 together and form hollow tubes. These tubes insert into the membrane of the target c e l l and cause l y s i s . Suppressor  T cells  are another population  of CD3 ,CD4 > CD8 +  _  +  cells  (von  Boehmer 1988). These c e l l s suppress immune responses by i n h i b i t i n g the production of interleukin-2 by T helper c e l l s B Lymphocytes  and  (Asherson et a l . 1986).  Immunoglobulins  The second population of lymphocytes i s the B c e l l s . B c e l l s receive their name from the fact that they mature i n the bursa of Fabricius i n birds. For many years i t was unknown where B c e l l s matured  i n mammals as there was  no  obvious bursal equivalent. In recent years i t has become apparent that B c e l l s are processed i n the bone marrow (Cooper 1981). B c e l l s synthesize and secrete immunoglobulins. Immune responses mediated by immunoglobulins are termed humoral immune responses. Immunoglobulins are glycoproteins consisting of 4 protein chains covalently linked together (Figure 2). There are 2 heavy chains and 2 l i g h t chains i n each immunoglobulin molecule. The primary structure of both chains shows internal homology (Silverton et a l . 1977). They are made up of blocks of about 110 amino acids called domains (Silverton et a l . 1977). The l i g h t  chains consist of 2  domains while the heavy chains consist of 4 or 5 domains depending on the class of  immunoglobulin. The domains can be subdivided into variable and constant  domains. The variable domains are the regions of the immunoglobulin molecule that actually bind to antigen. The constant domains determine the class of the immunoglobulin. Digestive enzymes l i k e papain, w i l l cleave the immunoglobulin molecule  Figure 2. Structure of an IgG molecule (Based on Cooper et a l . 1984).  VL A N D V I : V A R I A B L E D O M A I N S Ci A N D C i : C O N S T A N T D O M A I N S  14 at the hinge region creating 3 fragments (Porter 1959). The two Fab fragments contain the variable regions of both the heavy and l i g h t chains 2). They r e t a i n their  (see Figure  a b i l i t y to bind antigen but lose the a b i l i t y  to bind  complement or bind to receptors on macrophages. The Fc portion of the immunoglobulin molecule w i l l not bind antigen but w i l l bind complement and bind to receptors on macrophages (Fc receptors). The secondary and t e r t i a r y structures of each domain are s i m i l a r . Each domain i s made up of two fi-pleated sheet structures connected by an intradomain d i s u l f i d e bond  (Silverton et a l . 1977). The cysteine residues that form the  d i s u l f i d e bond are conserved i n a l l immunoglobulin classes. In pigs, there are two types of l i g h t chains, lambda and kappa. There are 3 types of heavy chains u, x and a. The heavy chain type determines the class of the immunoglobulin molecule. Immunoglobulins containing u chains are called IgM,  x chains give IgG and a chains IgA. In many other species there are also  t and 5 heavy chains giving IgE and IgD respectively. . IgM i s the immunoglobulin synthesized f i r s t during B c e l l ontogeny (Kincaid 1981). It i s synthesized  and secreted during  a l l primary immune  responses.  Neonatal p i g l e t s do not normally encounter any antigens i n utero. Their immune responses are therefore primary immune responses and almost e n t i r e l y IgM (Allen and Porter 1977). IgM occurs i n two forms. IgM found bound to the membranes of B c e l l s i s a monomer while serum IgM i s a pentamer (see Figure 3) (Cooper et a l . 1984) with the f i v e IgM units arranged  r a d i a l l y with the antigen binding  s i t e s pointing outwards. The f i v e units of pentameric IgM are joined by protein called the J chain. IgM makes up about 5-10% of serum immunoglobulins. IgM i s the most e f f i c i e n t of the immunoglobulin classes at i n i t i a t i n g the complement cascade (Borsos and Rapp 1965) .  IgD  IgE  16  IgG,  the main immunoglobulin found i n the serum, makes up 75-85% of the  t o t a l serum immunoglobulins. It i s synthesized during a secondary immune response. IgG i s a monomer i n both the membrane bound and secreted forms. The x chain constant region has 3 domains compared to 4 for the u chain. In pigs two isotypes of IgG have been i d e n t i f i e d : IgGi and IgG2 sistant to papain  (Pery 1973). IgGi i s re-  digestion and does not f i x complement. IgG2 i s cleaved by  papain and i s e f f e c t i v e at f i x i n g complement. A t h i r d isotype of porcine IgG has been tentatively been i d e n t i f i e d using monoclonal antibodies but has not been characterized (Pescovitz et a l . 1985.). IgA  i s the main  immunoglobulin  class found  i n secretions  and on body  surfaces. It occurs as a monomer i n the membrane bound form and a dimer i n t h e secreted form (see Figure 8). The two monomers are joined by the J chain as f o r IgM.  In addition IgA synthesized i n the mammary gland i s covalently linked t o  a glycoprotein c a l l e d the secretory component. The secretory component increases the resistance of IgA to proteolytic enzymes i n the small intestine (Tomasi and Calvanico 1968). In pigs IgA i s i n high concentration i n sow's milk and i n the small intestine  (Allen and Porter 1977) and i s therefore of great importance  to the survival of the neonatal Only these  piglet.  3 classes of immunoglobulins have been characterized i n pigs.  IgE and IgD also exist i n other mammals including mice, rats and man,. IgE i s responsible f o r immediate h y p e r s e n s i t i v i t y reactions i n allergy. IgE binds t o Fc receptors on mast c e l l s . The binding of certain types of antigens causes the mast c e l l allergy occurs antigen  to release vasoactive molecules that produce the symptoms of  (Cooper et a l . 1984). IgD i s found only i n a membrane bound form. I t on the membrane of unprimed B c e l l s (Fu et a l . 1975).  and i s a membrane receptor f o r  17  The ontogeny of murine B c e l l s i s shown i n F i g u r e 4.  Stem c e l l s  d i f f e r e n t i a t e i n t o pre-B c e l l s . I n i t i a l l y the pre-B c e l l s do not e x p r e s s membrane-bound  immunoglobulins ( L a n d r e t h et a l . 1981). The f i r s t event i n the  p r o d u c t i o n of immunoglobulins i s the rearrangement of the 4 genes t h a t code f o r  F i g u r e 4. The ontogeny of B c e l l s  (Based on Cooper et a l . 1984).  18  the u c h a i n . They are the V, D, J and C genes  (Ravetch et a l . 1 9 8 1 ) (see F i g u r e  5). The V, D and J genes code f o r the v a r i a b l e domain of the heavy  chain.  There are many forms of each of these genes. During m a t u r a t i o n of the lymphocyte the genes undergo t r a n s l o c a t i o n so that one V, D and J gene l i e together all  the i n t e r v e n i n g genes having been e x c i s e d .  V, D and J genes leads  The combining of the d i f f e r e n t  to a l a r g e number of p o s s i b l e amino a c i d sequences i n  t h e * v a r i a b l e r e g i o n of the immunoglobulin molecule. In a d d i t i o n , t h i s l o c a t i o n i s extremely prone to e r r o r s so that more v a r i a b i l i t y  Figure 5. The g e n e t i c b a s i s of a n t i b o d y p r o d u c t i o n  Light  Vi  v2  V  Jl  J  i s generated.  Heavy Chain  2  C  Germ l i n e  I 1 1  DNA  Vi  v2  Jl  3  J  J  D  V  Variable  Di D  2  IgG  Lymphocyte RNA  Constant  V  V  Lymphocyte DNA  Variable  trans-  (Based on T i z a r d 1 9 8 6 b ) .  Chain  3  with  G  M  2  G  M  E  E  A  A  Production J  D  G  Constant  Protein  Complete antibody  19 About 9,600 d i f f e r e n t gene arrangements of the heavy chain genes are possible i n mice  (Max 1984). Another 800 possible arrangements are possible f o r the  l i g h t chains giving nearly 8,000,000 d i f f e r e n t s p e c i f i c i t i e s of antibody i n mice. There are C genes for a l l of the different classes of immunoglobulins. I n i t i a l l y only the u gene i s expressed and u chains appear i n the cytosol of the pre-B c e l l . A similar process occurs for the rearrangement of the lambda and kappa genes. The kappa gene i s rearranged f i r s t  (Korsmeyer 1981). If the  rearrangement i s successful then the lambda gene i s not rearranged. If i t i s unsuccessful the lambda gene i s rearranged. Only one of the lambda or kappa chains i s produced by any given B c e l l . Once the l i g h t chain has been r e a r ranged, IgM i s produced by the B c e l l and expressed on i t s membrane. IgD i s also produced and occurs on the membrane of the mature, v i r g i n B c e l l  (Fu et  a l . 1975). When mature, v i r g i n B c e l l s are activated by T helper c e l l s , they begin to divide and d i f f e r e n t i a t e into two types of c e l l s  (Cooper et a l . 1984). Plasma  c e l l s lose their surface immunoglobulin and begin to produce secretory type IgM. This primary response takes 10-14 days to develop. It i s the only type of response the neonatal p i g l e t i s capable of mounting. A small number of the dividing c e l l s d i f f e r e n t i a t e into memory B c e l l s . These c e l l s are long l i v e d . The memory c e l l s also undergo a switch i n the type of immunoglobulin they produce. They w i l l have the same variable regions as they started with but now they may produce IgG or IgA depending on type of antigen, the location of the immune response and other poorly understood factors. The next time the antigen that triggered the response i s met, the number of B c e l l s able to produce antibodies to i t w i l l be greatly expanded and the response w i l l be IgG or IgA. This i s termed a secondary response.  20  B c e l l s are the last link i n the immune response. The macrophages ingest, process and present antigen bound to class II MHC molecules. An antigen s p e c i f i c T helper c e l l binds to the antigen-class II complex causing the release of interleukin-1. The T c e l l clone i s then activated. I t i n turn activates antigen s p e c i f i c B c e l l s by c e l l - c e l l interactions and the release of activating factors. The B c e l l  begins to divide and form plasma  cells.  The plasma  cells  produce IgM for a primary response and IgA or IgG for a secondary response. B c e l l s also d i f f e r e n t i a t e into memory c e l l s to enhance the immune response i f the same antigen i s encountered again i n the future. When the foreign antigen has been eliminated, suppressor T c e l l s stop the immune response by acting upon T helper c e l l s . Complement The complement cascade i s a group of about 20 proteins found i n the bloodstream of a l l vertebrates. When the precursors of the complement cascade are activated, other complement components are activated sequentially i n a cascade. The blood-clotting system i s another example of a b i o l o g i c a l cascade system. Complement i s not part of the s p e c i f i c immune system, but interacts closely with many components of i t . The main functions of the complement cascade are: (1) production of inflammation; (2) opsonization of antigens for phagocytosis and (3) c y t o t o x i c i t y towards bacteria (Brown et a l . 1984). Cytotoxicity against bacteria i s the most obvious function of complement and can be activated by two pathways. The c l a s s i c a l pathway i s i n i t i a t e d by the binding of antibody to antigen (see Figure 6). IgM i s more e f f e c t i v e at i n i t i a t i n g the c l a s s i c a l pathway than IgG (Borsos et a l . 1981). This i s because two immunoglobulin molecules must be bound to antigen side by side. The pentameric structure of IgM makes this far more l i k e l y than for monomeric IgG. IgA  21 IgA w i l l not i n i t i a t e the complement cascade and may  block i n i t i a t i o n by IgG  ( G r i f i s s 1975). The binding of IgM or IgG to antigen changes the 3 dimensional structure of the Fc portion of the molecule. This allows the CI  Figure 6. The c l a s s i c a l ,  alternate and terminal pathways of the complement  cascade (Based on Tizard 1986c).  Antigen  component c f ccj.plarr.ent t o b i n d t o the immunoglobulin m o l e c u l e s . T h i s a c t i v a t e s CI which c l e a v e s two o t h e r complement p r o t e i n s . C4 i s c o n v e r t e d i s converted  t o C4a and C2  t o C2b. These p r o t e i n s then combine t o form C4a2b which c l e a v e s  C3 t o C3a and C3b ( T i z a r d 1986c) . The t e r m i n a l complement  pathway i s i n i t i a t e d  by C3b (see F i g u r e  6) . C3b  c l e a v e s C5 i n t o C5a and C5b. C5b then combines w i t h C6, C7 and C3 t o form a complex. T h i s complex serves  as a seed t o p o l y m e r i z e  Twelve t o f i f t e e n C9 molecules structure  then  inserts  immunoglobulin molecules terial  itself  the l a s t  component C9.  j o i n t o g e t h e r t o form a t u b u l a r s t r u c t u r e . T h i s into  the membrane of the b a c t e r i a t h a t the  were bound t o , l e a d i n g t o osmetic  l y s i s of the bac-  cell.  Complement can a l s o be a c t i v a t e d i n t h e absence of immunoglobulins by the a l t e r n a t e pathway (see F i g u r e 6 ) . The C3 molecule  w i l l spontaneously  breakdown  and i n i t i a t e t h i s pathway. T h i s c o u l d cause c o n s i d e r a b l e damage t o host t i s s u e s so i t i s t i g h t l y r e g u l a t e d by f a c t o r H which i n h i b i t s the a l t e r n a t e pathway. F a c t o r H i s p r e s e n t  i n high  c o n c e n t r a t i o n s on the s u r f a c e s of host  c e l l s . I t i s bound by s i a l i c a c i d which a l s o occurs i n h i g h c o n c e n t r a t i o n s or. the s u r f a c e s of host  cells  ( K a t z a t c h k i n e et a l 1979). B a c t e r i a however have  low c o n c e n t r a t i o n s of s i a l i c a c i d on t h e i r s u r f a c e s and t h e r e f o r e lew concent r a t i o n s of f a c t o r H. The a l t e r n a t e pathway can then proceed,  l e a d i n g t c the  l y s i s of the b a c t e r i a . Some components  of complement  have  other  functions  i n a d d i t i o n to t h e  a c t i v a t i o n of the cascade. C3b b i n d s t o b a c t e r i a l a n t i g e n s and f u n c t i o n s as an opsonin  f o r macrophages and n e u t r o p h i l s . I t a l s o promotes the p r o d u c t i o n of  immunoglobulins by B c e l l s . C5a promotes the chemotaxis c f p h a g o c y t i c  cells.  23 C3a and C5a provoke the i n f l a m m a t o r y repsonse by c a u s i n g mast c s l l degrar.ulatior. (Brown et a l . 1934) . D e f i c i e n c i e s i n complement l e a d t o r e c u r r e n t b a c t e r i a l i n f e c t i o n s  (Tizard  1936c). Complement i s an i m p o r t a n t e f f e c t o r mechanism of the immune system. I t i s a l s o i m p o r t a n t i n i t s own r i g h t as a component of n o n - s p e c i f i c host d e f e n s e .  The Ontogeny of the P i g l e t ' s Immune System The immune system of the p i g l e t d e v e l o p s i n a s e r i e s of w e l l d e f i n e d s t e p s d u r i n g the 115 day g e s t a t i o n p e r i o d begin  erythrcpoiesis  and  (see Table 3 ) . Stem c e l l s i n the y o l k sac  l y m p h o p o i e s i s on  days  15-16. The  thymus b e g i n s to  develop around day 21 ( S t e r z l and S i l v e r s t e i n 1967) . Stem c e l l s from the y o l k sac c o l o n i s e the thymus cn Day 23 and t h i s begins the p r o c e s s of T lymphocyte maturation. The p e r c e n t a g e of f e t a l p i g lymphocytes w i t h s u r f a c e immunoglobulins i s lew p r i o r to 70 days of g e s t a t i o n . The number of I g  +  c e l l s i n c r e a s e s r a p i d l y from  70 to 30 days. A s i m i l a r p a t t e r n i s seen i n serum immunoglobulins. On day of g e s t a t i o n , the c i r c u l a t i n g i n c r e a s e s to. 12.1 ug  rnL  -1  l e v e l s of IgG are 2.5  1  this  (Franz et a l . 1982). These v a l u e s are e x t r e m e l y lew  compared t o a d u l t serum IgG v a l u e s of 10-15 mg predominant  ug mLr . By day SO  50  immunoglobulin i n the serum and  rnL  -1  (Jonnson 1973). IgG i s the  amniotic f l u i d  while  IgM  i s in  h i g h e s t c o n c e n t r a t i o n i n the f e t a l l i v e r , i n t e s t i n e , s p l e e n and thymus (Franz et a l . 1932). The p e r i o d from 72 t o 83 days seems t o be the p e r i o d d u r i n g which the f e t a l p i g l e t d e v e l o p s immunocompetence (Solomon 1971) . B e f o r e t h i s p e r i o d t h e r e are few p e r i p h e r a l T c e l l s and the i n j e c t i o n of a l l o g e n e i c lymphoid c e l l s does not provoke an immune response. From 72 t o S3 days  ges-  24  tation,  the number o f p e r i p h e r a l T c e l l s  gestation,  injection  of a l l o g e n e i c  r e s p o n s e . The f e t a l p i g l e t  i n c r e a s e s m a s s i v e l y . At 80 days of  lymphoid  cells  provokes  a strong  i s a l s o a b l e t o mount an immune response t o sheep  e r y t h r o c y t e s (Solomon 1971) on day 80 of g e s t a t i o n . Once the p i g l e t to a g i v e n a n t i g e n , i t s response appears t o be a d u l t - l i k e humoral and c e l l u l a r  immune  immune components (Solomon  can respond  w i t h r e s p e c t t o both  1971) .  T a b l e 3. Ontogeny of the immune system o f the f e t a l  piglet.  Gestation Dav  Event  Reference  21  Epithelial  22  S p l e e n rudiment  28  Lymphocytes i n thymus  Mendel et a l . 1977  38  Lymphoblasts i n b l o o d  Solomon 1971  48  L y m p h o c y t o p o i e s i s i n Spleen  Mendel et a l . 1977  50  Peyer's patches  Chapman et a l . 1974  50  IgM, IgG and IgA found i n serum  Franz et a l 1982 .  52  L y m p h o c y t o p o i e s i s i n lymph nodes  Mendel et a l . 1977  74  Onset o f t r a n s p l a n t a t i o n  Solomon 1971  77  Lymphopoiesis i n Peyer's patches  80  S e n s i t i z a t i o n t o a l l o g e n e i c lymphoid  80  A n t i b o d y response t o Sheep e r y t h r o c y t e s  thymus rudiment  Mendel e t a l . 1977  appears  Mendel e t a l . 1977  rudiment  immunity  Solomon 1971 cells  Solomon 1971 Solomon 1971  25 Host Defenses i n the Neonatal Piglet The immune system of t h e n e o n a t a l p i g l e t i s capable of r e s p o n d i n g t o a n t i g e n s but i s unprimed. The p r i m a r y immune response o c c u r s t o o s l o w l y t o p r o v i d e much p r o t e c t i o n a g a i n s t pathogens t h a t t h e p i g l e t encounters on l e a v i n g the u t e r u s . The p i g l e t  must t h e r e f o r e r e l y  on p h a g o c y t i c  cells  such  as n e u t r o p h i l s and  macrophages and on a n t i b a c t e r i a l f a c t o r s such as complement, l a c t o f e r r i n and lysozyme. N e u t r o p h i l s occur i n g r e a t e r numbers i n the n e o n a t a l p i g l e t than i n a d u l t s (Sellwood e t a l . 1986). The n e u t r o p h i l s of t h e neonate a r e as e f f e c t i v e at k i l l i n g E. c o l i as those from a d u l t s but because the neonate l a c k s immunoglobu l i n s t o a c t as o p s o n i n s , n e u t r o p h i l b i n d i n g t o b a c t e r i a i s i m p a i r e d . C o l o s t r a l and m i l k immunoglobulins p r o v i d e a source of opsonins  and enhance k i l l i n g  n e u t r o p h i l s i n c a l v e s (Renshaw e t a l . 1976). The presence  of s p e c i f i c  by  immuno-  g l o b u l i n s i n the i n t e s t i n e a l s o has an e f f e c t on the e m i g r a t i o n of n e u t r o p h i l s i n t o the i n t e s t i n a l lumen (Sellwood et a l . 1986). For n e u t r o p h i l e m i g r a t i o n to take p l a c e , t h e r e must be c h e m o t a c t i c s i g n a l s from damaged t i s s u e and s p e c i f i c immunoglobulin  must be p r e s e n t i n the lumen of the g u t . I n c o l o s t r u m d e p r i v e d  p i g l e t s , and p i g l e t s r e c e i v i n g non-immune c o l o s t r u m t h e r e i s no e m i g r a t i o n of n e u t r o p h i l s i n response  t o a c h a l l e n g e t o E. c o l i .  Complement a c t i v i t y i s a p p r o x i m a t e l y one h a l f of a d u l t l e v e l s i n n e o n a t a l piglets  ( R i c e and Ecuyer  1963). Complement components a r e absorbed  from c o l o -  strum d u r i n g the f i r s t day of l i f e and i n c r e a s e the p i g l e t s complement a c t i v i t y (Day e t a l . 1969) . Complement a c t i v i t y reaches a d u l t l e v e l s by 2-4 weeks a f t e r birth. B c e l l numbers a r e about one t h i r d of the a d u l t l e v e l s i n the n e o n a t a l p i g l e t (Banks 1981). Serum immunoglobulin  c o n c e n t r a t i o n s a r e more d i f f i c u l t t o d i s c e r n  26 because  of  the presence  of  passively  absorbed  and  actively  synthesized  immunoglobulins i n naturally reared p i g l e t s . In p i g l e t s a r t i f i c i a l l y reared on bovine colostrum  the amount  of a c t i v e l y  synthesized  immunoglobulin  can be  determined. Serum IgM increases r a p i d l y during the f i r s t week after b i r t h and remains the predominant serum immunoglobulin u n t i l approximately (Klobassa  et a l . 1981). Serum IgG increases  slowly  until  21 days of age  day 21. It then  increases r a p i d l y and becomes the predominant immunoglobulin i n the serum. The number of B c e l l s i n the i n t e s t i n a l mucosa of the neonatal  piglet i s  extremely low. The colonization of the i n t e s t i n a l mucosa by B c e l l s begins i n the duodenum and the number of B c e l l s i s higher i n the duodenum than i n other portions of the gut throughout development (Allen and Porter 1977). IgM bearing c e l l s are the f i r s t to appear. IgA bearing c e l l s are i n the minority u n t i l 4 weeks of age. By 12 weeks of age, 90% of the B c e l l s of the i n t e s t i n a l mucosa bear surface IgA. The  T cell  function of neonatal  piglets i s different  ways. T c e l l numbers i n the peripheral blood neonatal  from adults i n two  and i n the small intestine of  p i g l e t s are low (Cepica and Derbyshire  1984a; Chu et a l . 1979) and  the r e l a t i v e numbers of the d i f f e r e n t T c e l l populations  are different from  adult pigs. Peripheral blood T c e l l s of neonatal p i g l e t s have a suppressive effect on the d i f f e r e n t i a t i o n of B c e l l s into immunoglobulin secreting c e l l s (Suganuma et a l . 1986). At 6 weeks of age the suppressive and B c e l l l e v e l s increase to approximately  effect  disappears  one half of adult l e v e l s . No s i g -  n i f i c a n t T helper c e l l function i s present at 6 weeks of age. There are also fewer cytotoxic T c e l l s i n the peripheral blood and small intestine of newborns (Cepica and Derbyshire  1984a; 1984b). T c e l l s , from newborn p i g l e t s show no  cytotoxic a c t i v i t y against transmissible g a s t r o e n t e r i t i s (TGE) virus infected  27 cells.  Low  lymphocyte numbers, direct  suppression and  lack of effector  and  helper T c e l l s a l l account for the s u s c e p t i b i l i t y of neonatal p i g l e t s to TGE and other v i r a l The  age  infections.  of weaning has  a significant  effect  on  both  cell-mediated and  antibody responses i n the p i g l e t . Weaning prior to 5 weeks of age resulted i n impaired c e l l mediated immunity (Blecha et a l . 1983). Weaning at 4 days of age followed by a r t i f i c i a l rearing  on immunoglobulin-free  milk replacer resulted  in decreased antibody responses to sheep red blood c e l l s or Salmonella antigen (Haye and  Kornegay 1979). The  factors  i n sow's milk responsible for these  effects have not been determined. In  summary,  the  neonatal  piglet  i s extremely  v i r t u a l l y no c i r c u l a t i n g immunoglobulins.  immunodeficient.  It  has  It does have high numbers of neu-  trophils and macrophages but the lack of immunoglobulins to act as  opsonins  decreases their a b i l i t y to phagocytize foreign p a r t i c l e s . Low levels of serum complement  increase the s u s c e p t i b i l i t y to b a c t e r i a l  infections.  Lymphocyte  numbers are low. B c e l l and T effector c e l l populations are reduced while T suppressor  cell  a c t i v i t y i s increased. For s u r v i v a l ,  e n t i r e l y dependent on sow's milk and immune system.  the p i g l e t  colostrum to augment and  i s almost  regulate i t s  28 PROTECTIVE FACTORS IN SOW'S COLOSTRUM AND MILK Sow's milk and  colostrum provide n u t r i t i o n and  immune protection to the  neonatal p i g l e t . P i g l e t s are born with very low energy reserves. Glycogen stores are about 65 Kcal while f a t stores add another  100 Kcal  (Aumaitre  and Seve  1978). Without an exogenous source of energy soon after b i r t h , p i g l e t s rapidly become hypoglycemic,  hypothermic  and d i e . Piglets also have limited stores of  protein, vitamins A, C and B complex and i r o n . Since n u t r i t i o n a l d e f i c i e n c i e s increase host s u s c e p t i b i l i t y to disease (Beisel 1984), i t i s extremely important to provide a balanced diet to the p i g l e t immediately following b i r t h . Early work with the a r t i f i c i a l rearing of p i g l e t s concentrated on the composition of sow's milk and developing a substitute (Braude et a l . 1947)  (see Table 4).  Table 4 . Approximate chemical analysis of sow's colostrum and milk  (Aumaitre  and Seve 1978).  omponent  c  g/100  mL of:  Colostrum  Dry Matter  23.0  Fat  Milk 18.3  4.7  6.6  13.8  5.3  Lactose  3.6  5.5  Ash  0.7  0.8  Protein (N x 6.38)  Energy content (Kcal 100 mL ) -1  140  107  29 The  first  attempts  to  rear  colostrum  deprived  p i g l e t s i n non-sterile  environments using these sow milk replacers resulted i n 100% mortality (Bustad et a l . 1947). Experiments using hysterectomy derived p i g l e t s reared  i n com-  p l e t e l y germ free environments were more successful and s u r v i v a l rates of from 70 to 95% were obtained a l I960;). The  (Young and Underdahl 1953;  expense and  Young et a l . 1955;  Betts et  complexity of rearing germ free p i g l e t s made i t  useless as a management tool to be used by producers. Obviously n u t r i t i v e properties of sow's milk  and  colostrum  more than the  are important  in  ensuring  p i g l e t s u r v i v a l . The main problem i s i n looking at sow's milk and colostrum  in  the same manner as other feeds. Milk i s not an amorphous l i q u i d . It i s a highly structured and complex system of selected proteins, l i p i d s , carbohydrates and other nutrients. Sow  milk replacers must replace the functional properties of  these components as well as their n u t r i t i o n a l properties. As i n the case of host defense mechanisms, the protective components of milk and colostrum  can be divided into non-specific and s p e c i f i c elements. The non-  s p e c i f i c protective components of milk and colostrum  can be further subdivided  into d i f f e r e n t functional groups. Nutrient  such as the  protein l a c t o f e r r i n and forms that  vitamin B12  make them unavailable  binders  iron-binding  binding proteins sequester nutrients i n for the  growth of  microorganisms  in  the  i n t e s t i n a l t r a c t . Complement components can destroy bacteria n o n - s p e c i f i c a l l y or  i n concert  directly  with s p e c i f i c  antibody. Milk  enzymes either attack  (lysozyme) or produce a n t i b a c t e r i a l products  (lactoperoxidase).  whole range of non-immunoglobulin proteins that mimic enterocyte for bacteria and viruses are present  A  receptors  i n milk. Bacteria and viruses that bind  to these soluble receptor analogues are prevented from binding to the cytes.  bacteria  entero-  30  In addition to these non-specific protective components, sow's milk and colostrum  also contain viable lymphocytes, macrophages, neutrophils  and im-  munoglobulins. These provide s p e c i f i c immune protection systemically and l o c a l l y in the i n t e s t i n a l t r a c t . Viable C e l l s Lymphocytes, macrophages and neutrophils are normal constituents of sow's milk and colostrum. These c e l l s are not random c o l l e c t i o n s of c e l l s from the sow's own immune system. They are selected populations  of c e l l s derived from  a compartment of the immune system known as the secretory immune system. The c e l l s are s p e c i f i c f o r enteropathogenic organisms and food antigens  that the  sow has encountered previously. Since the p i g l e t s are being reared i n the same environment as the sow, i t i s l i k e l y that the p i g l e t w i l l encounter the same bacteria, viruses and food The  secretory  antigens.  immune system i s the portion of the immune system that i s  concerned with immunity on the mucosal surfaces of the body. This includes the mucosal e p i t h e l i a of the g a s t r o i n t e s t i n a l tract, the mammary gland, the lungs, s a l i v a r y glands, uterus  and skin. Two things are t y p i c a l of secretory immune  responses: (1) c e l l s involved i n an immune response at one mucosal s i t e migrate to other mucosal s i t e s and provide IgA  i s synthesized  during  immune protection there and (2) secretory  secretory  immune responses. The portion  of the  secretory immune system that d i r e c t l y affects the s u r v i v a l of p i g l e t s i s the gut-mammary axis of immunity. Gut-associated  lyphoid tissue (GALT) consists of a covering of s p e c i a l i z e d  e p i t h e l i a l c e l l s c a l l e d M c e l l s overlying lymphoid tissue. The M c e l l s take up intact macromolecules from the lumen of the gut v i a pinocytosis and transport them  to the lyphocytes  and macrophages i n the underlying  lymphoid  tissue  (Bockman and Cooper 1973). In human studies i t has been found that small amounts of intact food antigens are transported from GALT to the mammary gland and into the milk  (Hemmings 1980). This exposes even exclusively breastfed infants to  food antigens. The B c e l l population of GALT consists of IgA and IgM bearing c e l l s . Antigen stimulation of GALT leads to the activation of only IgA secreting plasma c e l l s . No IgG secreting B c e l l s are produced. This effect i s caused by a population of "switch" T c e l l s i n GALT (Kawanishi et a l . 1983). These T c e l l s cause undifferentiated IgM bearing B c e l l s to d i f f e r e n t i a t e into IgA secreting c e l l s only. T c e l l s that regulate the synthesis of IgA by committed B c e l l s also e x i s t . These T c e l l s have Fc receptors f o r the a chain of IgA. They produce soluble help and suppression factors that bind to the surface IgA on B c e l l s  (Kiyono  et a l . 1985; Hoover and Lynch 1983). These T c e l l s regulate the synthesis of immunoglobulin by IgA secreting B c e l l s but have no effect upon IgG synthesis. Upon stimulation by T c e l l s , B c e l l s present i n GALT migrate to the mesent e r i c lymph nodes, the blood, l i v e r and spleen (McWilliams  et a l . 1975).  They  then home back to the gut or other mucosal s i t e s including the mammary gland (Phillips-Quagliata  et a l . 1983).  In a study  with  pregnant  women,  oral  immunization with nonpathogenic E. c o l i resulted i n the appearance of s p e c i f i c B c e l l s i n the colostrum of the volunteers (Goldblum et a l . 1975). In another human study,  the transfer of cell-mediated immunity to colostrum  after  oral  immunization was demonstrated (Parmely et a l . 1976). Bohl et a l . (1972), demonstrated that production of IgA s p e c i f i c for TGE virus i n the colostrum of sows depended on prior enteric i n f e c t i o n with the v i r u s . Presumably, migration of s p e c i f i c lymphocytes from the GALT to the mammary lymphoid tissue was r e sponsible for this mechanism.  The population of IgA secreting B c e l l s i n the sow's mammary gland increases markedly 1 to 2 weeks prior to farrowing (Brown et a l . 1975). One study found that i n mice this  increase i n c e l l s  around  p a r t u r i t i o n was under hormonal  control (Lamm et a l . 1977). V i r g i n female mice given injections of estrogen, progesterone and p r o l a c t i n had greatly increased numbers of IgA secreting B cells  i n their  mammary tissues. Treatment  with  testosterone decreased the  numbers of these c e l l s i n the mammary gland. Total c e l l counts i n sow's colostrum averaged 1 x 10 on the day of farrowing 7  and decreased to 1 x 10 by 10 days after farrowing (Evans et a l . 1982). The 6  d i f f e r e n t i a l c e l l counts from that study are shown i n Table 5. Sow's milk and colostrum d i f f e r s  from human and bovine milks i n that neutrophils are more  numerous than macrophages (Evans et a l . 1982).  Table 5. D:i f f e r e n t i a l c e l l counts for the mammary secretions of sows i n which b a c t e r i a l i n f e c t i o n was absent  (Evans et a l . 1982) •  Days post partum  % of t o t a l c e l l v i e l d neutrophils  macrophages  lvmphocvtes  epithelials  eosinophils  <1  71.7  1.3  26.4  0.4  0.2  3  55.4  15.0  22.8  6.1  0.7  10  39.2  14.6  13.7  31.4  1.1  15  51.3  5.5  11.2  31.3  0.7  20  32.1  0.0  0.0  67.9  0.0  33  The  lymphocytes  that appear i n milk and  colostrum  are derived from  the  secretory immune system and are d i f f e r e n t from lymphocytes found i n serum. Most of  the  B  cells  are  IgA  secreting  cells  (Parmely  and  Beer  1977) . A high  percentage of these B c e l l s are s p e c i f i c for enteric microorganisms. The T c e l l populations of milk and colostrum also show different r e a c t i v i t i e s to antigens than peripheral blood T c e l l s . The role that these c e l l s play i n protection of the p i g l e t s against enteric i n f e c t i o n has not been well explored. Colostral macrophages protect suckling rat pups against Klebsiella-induced necrotizing  enterocolitis  (Pitt  et a l .  1974). In v i t r o studies have shown that neutrophils from human milk can phagocytize and  kill  E. c o l i  (Robinson  et a l . 1978). Various studies have also  reported the transfer of c e l l mediated immunity v i a lymphocytes present i n milk and colostrum. Head and Beer (1979) found that c o l o s t r a l lymphocytes i n rat's milk could be absorbed d i r e c t l y into the bloodstream of suckling r a t s . Rat pups that were cross fostered to MHC incompatible mothers developed graft-versus host disease. Resistance to tumors can be transferred v i a milk i n mice (Head and Beer 1979). When mice susceptible to a Leydig c e l l  tumor were suckled by  their  natural mothers they had no resistance to challenge with tumor c e l l s . When the susceptible mice were suckled by resistant mothers they survived a challenge with tumor c e l l s . Since these types of experiments have not been performed with pigs, there i s no information on whether the piglet benefits from ingesting the c e l l s present i n sow's milk and colostrum. Immunoglobulins Most investigations of the protective effect of sow's colostrum and milk have concentrated on their immunoglobulin content. There are several reasons  34 for  t h i s . Immunoglobulins make up a larger percentage of the t o t a l solids i n  colostrum and milk than other protective factors so they are the most obvious factor to start with. They are simple to i s o l a t e and purify so that investigating their properties i s r e l a t i v e l y easy. They are available i n large quanti t i e s from sources such as abattoir blood, bovine milk and whey. This makes i t feasible to perform feeding experiments purified  using a r t i f i c i a l diets f o r t i f i e d with  immunoglobulins.  The immunoglobulin content of sow's colostrum during the f i r s t  day  after  p a r t u r i t i o n i s d i f f e r e n t from that found subsequently i n the milk (Table 6 ) .  Table 6. Ig levels in serum, colostrum, milk and i n t e s t i n a l juice of pigs (Bourne 1973).  Immunoglobulin concentration mg mL=-L IgG Sow Serum  IcrA  IcM  IgArlgG  24.3  2.4  2.9  0.10  61.8  9.7  3.2  0.16  11.8  3.8  1.3  0.32  Milk (2 d)  8.2  2.7  1.8  0.33  Milk (3-7 d)  1.9  3.4  1.2  1.79  Milk (8-35 d)  1.4  3.0  0.9  2.14  Colostrum  (0 hr)  Milk (24 hr)  I n t e s t i n a l Juice  0.2  2.6  Trace  13.0  In pigs, IgG i s present i n high concentrations i n colostrum while IgA predominates i n milk a few days after p a r t u r i t i o n . This pattern i s not followed  35 by a l l species however. In primates and rodents, IgA i s the p r i n c i p a l immunoglobulin i n both milk and colostrum. In ruminants,  IgG predominates i n both  milk and colostrum. A l l the IgG and 40% of the IgA i n the colostrum during the f i r s t after  parturition  are transferred  from  transferred d i r e c t l y into the colostrum  the blood  stream  few days  of the sow and  (Bourne and Curtis 1973). In cows,  s p e c i f i c receptors on the e p i t h e l i a l c e l l s of the mammary gland transport IgG from the bloodstream for  to the milk  (Brandon et a l . 1971). Selective receptors  immunoglobulin i n the mammary glands of sows may also exist (Franek et a l .  1975). After p a r t u r i t i o n , the transfer of immunoglobulins from the blood to the milk i s dramatically reduced. Three days after p a r t u r i t i o n , 90% of the IgA and 70% of the IgG i n sow's milk comes from l o c a l production by B c e l l s i n the mammary gland (Bourne and Curtis 1973). This pattern of IgG i n the colostrum and IgA i n the milk appears  to be  adapted to providing optimal immune protection to the p i g l e t . The immunoglobulins found directly  i n the colostrum are ingested by neonatal p i g l e t s and absorbed  into  the blood  stream  v i a pinocytosis. These immunoglobulins are  therefore responsible for providing systemic immunity i n the p i g l e t . Since the majority of the immunoglobulins present i n the colostrum are derived from the serum of the sow, they are i d e a l l y selected for this purpose. In addition IgG has a longer b i o l o g i c a l half l i f e  (14 days) than either IgA (2.5 days) or IgM  (5 days) (Bourne and Curtis 1973). IgG therefore provides much longer systemic protection than IgA or IgM. Since immunoglobulins are only absorbed  into the  blood stream during a short period after b i r t h , i t i s important that they should l a s t as long as possible. Secretory IgA that i s absorbed from colostrum has a  36 half l i f e of less than 24 hours (Bourne 1973). This i s not due to degradation of the slgA but rather i t s transport to mucosal surfaces where i t provides l o c a l immune protection (Bradley et a l . 1976). Serum immunoglobulin concentrations of conventionally reared p i g l e t s are shown i n Figure 7.  Figure 7 . IgG, IgA and IgM concentrations i n sera of naturally reared p i g l e t s (n=24) (based on data from Klobassa et a l . 1981).  Days  37  Absorption of macromolecules from the i n t e s t i n a l place i n two  distinct  steps  (Lecce  macromolecules by pinocytosis. The  1973). The  first  tract  by p i g l e t s  takes  i s i n t e r n a l i z a t i o n of  second i s transport of the  internalized  macromolecules across the enterocyte and into the blood stream. In rodents, a wide variety of macromolecules are internalized v i a pinocytosis but only IgG i s transported to the blood stream. This s e l e c t i v i t y does not exist i n p i g l e t s . They w i l l i n t e r n a l i z e a wide range of macromolecules including dextrans polyvinylpyrrolidone (Lecce et a l . 1961)  and  but IgG i s p r e f e r e n t i a l l y transported  into the blood stream i n the presence of other macromolecules (Leary and Lecce 1979). When porcine IgG or bovine albumin alone was  fed to p i g l e t s , they were  both absorbed at the same low l e v e l . When fed together, however, the absorption of IgG increased s i g n i f i c a n t l y while the bovine albumin was  absorbed  at the  o r i g i n a l low l e v e l . Leary and Lecce (1979) proposed that s p e c i f i c receptors on enterocytes bind IgG. Non-immunoglobulin macromolecules increase absorption of IgG  by  molecules  non-specifically  stimulating  pinocytosis. The  non-immunoglobulin  are internalized n o n - s p e c i f i c a l l y and are therefore absorbed  efficiently.  This  model has  not  been  proven.  macromolecules d i r e c t l y into the blood stream  Cessation  of  less  absorption  of  (closure) usually occurs between  24 and 48 hours after b i r t h . The ingestion of nutrients by the piglet seems to be the inducer of closure (Patt 1977). Starvation of p i g l e t s prolongs the period before closure (Lecce 1973).  After closure occurs, milk provides protection  against infections i n the i n t e s t i n a l tract of the p i g l e t . Sow's milk i s r i c h in secretory IgA. The properties of this immunoglobulin are i d e a l l y suited to providing l o c a l immune protection. The structure of human IgA i s shown i n Figure 8. The monomeric subunits  38 Figure 8. The covalent structure of human monomeric, dimeric and secretory IgA (Based on Underdown and Schiff 1986).  IgA lonoier  Secretory coiponent  Kacosal transport receptor l l t t t U t U l i O t t <<HMIimtt<UtMHH<| MIM|ltllMM>M«M«WltlltMUMUM«r  proteolytically l a b i l e segient  39  of slgA have the a b i l i t y to polymerize. The heavy chain of the IgA monomer has a C terminal extension  containing an extra cysteine  (Koshland  1985). These  cysteine residues allow the l i n k i n g of 2 or more IgA monomers v i a the J chain. Dimeric IgA i s the most common form but trimers, tetramers  and pentamers are  also formed  i s e s s e n t i a l for  (Radl  et a l . 1973). This polymerization  step  binding of the IgA to the mucosal transport receptor. IgA i s secreted into the lymphoid  tissue where i t i s synthesized.  To provide  protection on mucosal  surfaces i t must then be a c t i v e l y transported through the mucosal e p i t h e l i a and released. The transport of IgA from blood to b i l e i n perfused l i v e r s has been used to study the mechanism of this transport system (Fisher et a l . 1979). Secretory component i s synthesized i n the i n t e r i o r of the c e l l . It consists of the polypeptide found associated with free slgA plus an extra segment (Mostov et a l . 1979). This extra segment inserts i n the c e l l membrane and anchors the secretory component on the surface of the c e l l . IgA then binds to the membrane bound secretory component (Weicker and Underdown 1975). The entire complex then migrates across the c e l l where the membrane anchoring  segment i s cleaved o f f  and the slgA i s secreted (Solari and Kraehenbuhl 1984). This transport system i s c o n s t i t u i t i v e (Mullock et a l . 1980) and secretory component i s synthesized and moves across the e p i t h e l i a l c e l l s whether IgA i s present for transport or not. The energy expenditure  f o r this i s enormous. T h i r t y micrograms of secr-  etory component per gram of tissue must be synthesized every hour to maintain the transport system (Underdown and Schiff 1986). Such a high expenditure of energy indicates the importance of slgA i n the protection of mucosal surfaces. Secretory IgA must survive degradation  i n the digestive tract i f i t i s to  perform i t s protective function. The hinge regions of IgG and IgM molecules i n  40 particular  are  susceptible to degradation  by  digestive and  bacterial  teinases. The hinge region of IgA i s resistant to proteolysis and  pro-  therefore  better adapted to performing i t s protective functions i n the i n t e s t i n a l tract of the piglet than IgG or IgM The  (Plaut 1983).  protective functions of slgA are a l l related to i t s a b i l i t y  i f i c a l l y bind to antigen. Secretory IgA i n ingested milk causes the  to spec-  aggregation  of microorganisms i n the digestive tract of the p i g l e t . The dimeric structure of slgA increases i t s a b i l i t y to agglutinate p a r t i c l e s compared to IgG but i t i s i n f e r i o r to pentameric IgM i n this regard  (Ishizaka et a l . 1965). Underdown  and Schiff (1986) suggested that the structure of slgA i s a compromise between the agglutinating properties of IgM  and  the tissue permeability  of IgG.  In  addition to agglutination, slgA binds with cysteine residues i n the mucous coat on  i n t e s t i n a l epithelium.  This provides  a long  l a s t i n g coating of  specific  antibody on the mucosal surface of the small i n t e s t i n e . This coating of slgA i n h i b i t s adherence of microorganisms to enterocytes  (Walker and  Isselbacher  1974) . IgG does not form this protective coating. Microorganisms that do manage to adhere to the s p e c i f i c receptors on enterocytes  are prevented from being  internalized by slgA. IgG does not prevent i n t e r n a l i z a t i o n (Underdown and Schiff 1986). The  protective functions of IgA do not extend to k i l l i n g microorganisms.  Rather i t functions i n immobilizing and preventing adherence and  internaliza-  t i o n . Non-specific protective factors such as iron-binding proteins, lysozyme, lactoperoxidase and complement contribute to the k i l l i n g of microorganisms. In addition to binding microorganisms, slgA can also bind and neutralize toxins produced by pathogenic bacteria (Dobrescu and Huygelen 1976; Pierce and Reynolds 1975) .  41  Immunoglobulin F o r t i f i e d Milk Replacers The i n i t i a l f a i l u r e s i n rearing colostrum deprived p i g l e t s led to attempts to  simulate the immune properties of sow's colostrum and milk by  fortifying  milk replacers with immunoglobulins. Obviously the model for doing this i s the natural immunoglobulin content of colostrum and milk. The problem i s obtaining a r i c h source of porcine slgA. The only p r a c t i c a l source of porcine immunoglobulins i s pig abattoir blood which i s r i c h i n IgG and poor i n slgA (see Table 6). F o r t i f i c a t i o n of milk replacers with porcine blood immunoglobulins results i n a milk replacer immunoglobulin p r o f i l e similar to sow's colostrum but lacking the slgA found i n sow's milk. In vivo experiments using porcine blood immunoglobulins show that this lack of slgA does not have a detrimental effect  on  •survival (Owen and B e l l 1964; Scoot et a l . 1972; McCallum et a l . 1977). Ammonium sulphate fractionated porcine blood immunoglobulins fed at a l e v e l of 10 g Kg body weight  -1  on Day  1 and  2 g Kg body weight  -1  on Days 2-10  gave survival  rates of 70% (McCallum 1977). When immunoglobulins were given o r a l l y only during the f i r s t 24 hours of l i f e , the s u r v i v a l rates were very low (Owen et a l . 1961) . Porcine blood IgG provides adequate l o c a l protection i n the i n t e s t i n a l tract of  the p i g l e t . Large scale production of immunoglobulins from porcine blood i s possible  using a combination  of different  p u r i f i c a t i o n procedures.  fractionation i s the most common method i n use. E l l i o t  Ammonium sulphate  et a l . (1987) described  a continuous process for the large scale p u r i f i c a t i o n of immunoglobulin from blood. Continuous desludging centrifuges are used to remove the c e l l u l a r  and  f i b r i n components of blood. Saturated ammonium sulphate i s added to serum to give 45% saturation. The immunoglobulin containing p r e c i p i t a t e i s collected by  42  decanting centrifugation and ammonium sulphate i s removed by e l e c t r o d i a l y s i s or u l t r a f i l t r a t i o n . The remaining water i s removed by spray drying. This f r a c tionation fraction  method has several changes  phase  disadvantages. The immunoglobulin  making  resolubilization  necessary. Also,  containing extensive  processing using u l t r a f i l t r a t i o n or e l e c t r o d i a l y s i s i s required to remove the ammonium sulphate. A more recent method of fractionating serum immunoglobulins was described by Lee et a l . (1988). This method uses polyphosphates to fractionate  serum  immunoglobulins. Only 1.04% polyphosphate i s required and the immunoglobulin containing f r a c t i o n does not change phase. This method i s more readily adapted to continuous production of immunoglobulins than ammonium sulphate fractionation. In  addition to porcine blood, bovine blood, milk, whey and colostrum are  potential sources of immunoglobulins  f o r the a r t i f i c i a l  rearing of p i g l e t s .  Senft and Klobassa (1971) obtained survival rates of 92% feeding bovine colostrum to 232 p i g l e t s i n a non-isolated environment. The feeding of ammonium sulphate  fractionated  bovine blood immunoglobulins  to a r t i f i c i a l l y  p i g l e t s resulted i n 100% survival for a group of 8 p i g l e t s  reared  (McCallum 1977). A  direct comparison of bovine and porcine serum immunoglobulins on piglet performance has not been made however. One  thing that a l l i n vivo experiments have i n common i s that they use  immunoglobulins  as the sole protective component i n the milk replacer. As  mentioned previously immunoglobulins do not d i r e c t l y k i l l bacteria. Other nonspecific  factors  i n milk which  act i n concert  with  responsible for bacteriostasis and the direct k i l l i n g  immunoglobulins, are  of bacteria.  43 Lactoferrin Lactoferrin i s an iron-binding single chain glycoprotein unique to mammals. It shares close homology with t r a n s f e r r i n and ovotransferrin but d i f f e r s i n d i s t r i b u t i o n i n the body and i n function. Transferrin occurs mainly i n serum as a transport protein for i o n i c i r o n . Lactoferrin occurs i n milk, seminal f l u i d , tears and on body surfaces i n general. Its d i s t r i b u t i o n i s similar to IgA and this r e f l e c t s i t s functions as an important part of the immune response. Lactoferrin binds ionic iron, making i t unavailable to bacteria and thus preventing their growth. Lactoferrin i s the predominant  iron-binding protein i n  the colostrum and milk of commercially important a g r i c u l t u r a l species. For this reason there i s interest i n i t s use as a feed additive for milk replacers and starter d i e t s . Lactoferrin was 1960)  first  isolated by Sorensen and Sorensen i n 1939  (Groves,  as a red protein from milk. P o l i s and Shmukler (1953) obtained this red  protein i n an impure form and were able to characterize i t as containing i r o n . The most common method of p u r i f i c a t i o n presently i n use was hanssen  developed by Jo-  (1969) (Figure 9) .  Lactoferrin tends to form complexes with other molecules such as spermatozoal surface  components, DNA,  complexes  with other  serum  albumin  lactoferrin  and  molecules  fi-lactoglobulin. i n the presence  It also  forms  of C a  (Bez-  ++  korovainy, 1980). This makes i t d i f f i c u l t to prepare i n a pure monomeric form. For  this reason, estimates of the molecular weight of l a c t o f e r r i n vary from 77-  93 Kilodaltons (Groves, 1960; Castellino et a l . 1970; Weiner and Szuchet, 1975). Weiner and Szuchet  (1975) reported the molecular weight of bovine l a c t o f e r r i n  as 93 ± 2 Kilodaltons. This l a t t e r value i s probably the most r e l i a b l e because of the conditions under which i t was obtained. The protein was shown  44  Figure 9. The preparation or bovine l a c t o f e r r i n from milk (Based on Johanssen 1969).  Milk Centrifuge 8000 x g for 40 min Fat discarded Skim milk pH 7.0 i n 0.05 M K H 2 P O 4 / K 2 H P O 4 buffer Add 6 g CM-Sephadex C50 per L of milk S t i r 2 hours  Supernatant discarded  Wash CM-Sephadex C50 with deionized water Apply to column Elute with linear NaCl gradient 0.2-0.5 M i n pH 7.0 i n 0.05 M K H 2 P O 4 / K 2 H P O 4 buffer Lactoferrin-free — fractions discarded Lactoferrin enriched f r a c t i o n Gel f i l t r a t i o n with Sephadex G-200 Lactoferrin-free — fractions discarded Lactoferrin  to be homogeneous and the same molecular weight was obtained by both Svedberg and high speed sedimentation techniques. The p i of l a c t o f e r r i n was measured i n the same study as 8.0 ± 0.2. Lactoferrin  exists  i n two forms,  iron free and iron saturated. The forms  have quite d i f f e r e n t properties. Iron-saturated l a c t o f e r r i n i s more resistant to denaturation than iron-free l a c t o f e r r i n ( Baer et a l . 1979). P u r i f i e d i r o n free  l a c t o f e r r i n was denatured  at temperatures  as low as 45 °C while  iron-  saturated l a c t o f e r r i n remained stable up to 60 °C. They also found that the  45 a b i l i t y of iron-saturated l a c t o f e r r i n to bind iron was  p a r t i a l l y independent  of the degree of denaturation. That i s , the iron binding s i t e remained able to bind iron after other portions of the protein had been denatured. The effect of low temperature storage on l a c t o f e r r i n was studied by Goldsmith et a l . (1983). Storage of human milk samples at -20 °C for 4 weeks decreased the iron binding capacity of the milk l a c t o f e r r i n by The  30%.  primary structure of bovine l a c t o f e r r i n has been only p a r t i a l l y elu-  cidated. More work has been done on human l a c t o f e r r i n and w i l l be discussed here. Human l a c t o f e r r i n shares homology with t r a n s f e r r i n and hen ovotransferrin. In addition, l a c t o f e r r i n shows internal homology. The the N-terminal half  of the l a c t o f e r r i n molecule (residues 1-336) show marked  homology with those of the C-terminal half (Brock,  1985). This homology probably  the N-terminal  (residues 337-679) of l a c t o f e r r i n  arose as a result of gene duplication  during evolution. Each homology region has designated  amino acid residues of  one  iron binding s i t e which are  s i t e and the C-terminal  s i t e . The  two  carbohydrate  chains on the l a c t o f e r r i n molecule are located on the C-terminal region and are not involved i n iron binding (MacGillvray et a l . 1977). Lactoferrin  w i l l bind iron at a pH as low as 2.0 compared to 4.6 for trans-  f e r r i n (Van Snick et a l . 1973). This may r e f l e c t the fact that l a c t o f e r r i n must bind iron i n the acid environment of the stomach. The binding of two atoms of iron to l a c t o f e r r i n may HeLf + 2 F e  3+  + 2 HC0 - <-> 3  be represented  as:  F e L f ( H C 0 ) 2 +6H 2  3  +  The binding of a metal ion requires the simultaneous binding of an anion usually carbonate oxalate and  (Bates  and  Schlabach,  1973). Other anions  n i t r i l o t r i a c e t a t e can replace bicarbonate  such as EDTA,  (Schlabach  1975). Arginine i s the amino acid residue that binds the anion.  and  Bates,  46 The a b i l i t y of l a c t o f e r r i n to bind iron i s reduced by the presence of c i t r a t e (Reiter et a l . 1975). Citrate w i l l remove iron from l a c t o f e r r i n and the iron c i t r a t e i s a c t i v e l y taken up by bacteria (Rosenberg and Young, 1974). Iron has an octahedral ligand f i e l d and so requires 6 ligands to bind i t e f f e c t i v e l y . The carbonate anion i s the f i r s t of these ligands.  The other  amino acid residues involved i n the iron-binding s i t e have been studied i n several ways. Teuwisson et a l . (1972) used hydrogen ion t i t r a t i o n of l a c t o f e r r i n in i t s iron-free and iron-saturated forms to study the problem. Between pH 7 and 10,  the binding of one  Fe  ion by l a c t o f e r r i n liberates 3 protons.  3 +  possible ligands are t h i o l , phenol guanidinium, ammonium and  tryptophan.  The The  o p t i c a l spectrum or Fe2Lf resembles iron complexes with phenol so i t i s probable that tyrosine i s a ligand. The presence of tyrosine as a ligand i n the. iron binding s i t e was  confirmed  by c i r c u l a r dichroism  proton magnetic resonance (Woodworth et a l . 1970) spectroscopy  (CD)  (Mazurier et a l . 1976),  and u l t r a v i o l e t difference  (Krysteva et a l . 1976) .  Electron paramagnetic resonance spectra of lactoferrin-copper chelates are similar to those of copper-nitrogen  compounds (Aasa et a l . 1963)  at pH 7.5. At  this pH, imadazole nitrogen i s the only group that would remain unprotonated. This indicates that h i s t i d i n e i s present modification of h i s t i d i n e with  at the iron binding s i t e . Chemical  diethylpyrocarbonate  showed there i s only 1  h i s t i d i n e residue for the 2 iron atoms bound i n bovine l a c t o f e r r i n  (Krysteva  et a l . 1975). This suggest the iron binding s i t e s are d i f f e r e n t from each other. Tryptophan may also be a ligand since CD spectra show bands due to r e s t r i c t e d rotation  of  tryptophan  change when iron  i s bound. There i s evidence  that  suggests that although the conformation of tryptophan residues do change during iron  binding,  they  play no  role  as a ligand. Sulphenylation  of  tryptophan  47 residues of l a c t o f e r r i n did not change i t s a b i l i t y to bind iron (Ford-Hutchinson and Perkins 1972). Precise information on the nature of the residues involved i n binding iron in l a c t o f e r r i n w i l l have to wait u n t i l the complete primary structure of l a c t o f e r r i n has been determined and the iron-binding residues i d e n t i f i e d within the structure. There i s some controversy over whether the two iron-binding s i t e s are  equiv-  alent. Warner and Weber (1953) found strong cooperativity between the s i t e s i n conalbumin. That i s the binding of iron at one s i t e increased the a f f i n i t y for iron at the remaining s i t e . Aasa et a l . (1963), found no difference i n the i r o n binding  s i t e s i n t r a n s f e r r i n using  electron paramagnetic resonance spectra.  Mazurier et a l . (1976), using the same technique, found that the s i t e s were s i g n i f i c a n t l y d i f f e r e n t . Luk  (1971) showed that t r a n s f e r r i n w i l l bind only  ion of the large lanthanide ions Nd Tb  or H o . More recently  3+  3+  the  3+  or P r  3 +  but 2 of the smaller  iron-binding  constants  one  lanthanides  for each s i t e  was  measured by equilibrium d i a l y s i s . The results showed that the C terminal s i t e has a higher a f f i n i t y for iron than the N terminal s i t e  (Aisen et a l . 1978).  This conclusively demonstrated the non-equivalence of the s i t e s . Iron plays a central role i n the b a t t l e between host and b a c t e r i a l pathogens. That iron i s the nutrient that occupies this role i s not surprising when i t s properties are examined. The chemistry of iron i n aqueous systems i s dominated by i t s low s o l u b i l i t y . Under physiological conditions iron occurs i n the f e r r i c ( F e ) form which forms insoluble polymeric hydroxides. These compounds have 3+  a  solubility  product  of  lCr  3 7  (Weinberg,  s o l u b i l i t y free iron causes the formation  1974).  In  addition  to  its  low  of peroxides and hydroxyl r a d i c a l s .  For these reasons free iron i s rare i n l i v i n g systems and i s inevitably bound  48 by proteins or heme. This makes i t r e l a t i v e l y easy for the mammalian hosts to l i m i t iron a v a i l a b i l i t y to Transferrin  bacteria.  i s the primary iron transport protein i n the plasma. The binding  constant of t r a n s f e r r i n for iron i s about 10  36  (Aasa et a l . 1963). This means  that the concentration of free iron in the plasma i s about 10bacteria require iron at a concentration of about 10concentration of iron i s about 10  8  times too low  10  M,  18  M. Since most  (Kochan, 1977)  this  to support b a c t e r i a l growth.  On body surfaces, i n the mammary gland and i n milk, l a c t o f e r r i n has the same effect as t r a n s f e r r i n . It has bacteria  36  for iron. Many  are unable to grow i n the presence of iron-free, l a c t o f e r r i n .  In v i t r o studies of the i n h i b i t s the the  a binding constant of about 10  antibacterial  effects of l a c t o f e r r i n show that i t  growth of a wide variety of bacteria.  s u s c e p t i b i l i t y of various species of mastitis  Rainard  (1986a), compared  causing bacteria  to  lacto-  f e r r i n . A l l 35 strains of E. c o l i were susceptible to i n h i b i t i o n by l a c t o f e r r i n . Four of 10 strains of Staphylococcus aureus showed resistance to l a c t o f e r r i n induced bacteriostasis. A l l strains of Streptococcus aqalactiae and Str. uberis were unaffected by the presence of l a c t o f e r r i n . Rainard (1986a) observed that the iron requirements of the l a t t e r two  organisms are low. This gives them re-  sistance to i n h i b i t i o n by l a c t o f e r r i n .  Many other in v i t r o studies showed that  l a c t o f e r r i n i n h i b i t s the growth of E. c o l i (Rainard, 1986b; Reiter et a l . 1975; Samson et a l . 1979;  Bullen et a l . 1972).  For l a c t o f e r r i n to prevent the growth of E. c o l i i n the small intestine the suckling  animal i t must reach the small intestine i n t a c t . Lactoferrin  resistant to digestion  of is  by trypsin and chymotrypsin but not by pepsin (Brock et  a l . 1976). Lactoferrin isolated from the feces of breast fed infants was able to bind iron (Spik et a l . 1978) . Lactoferrin's  a b i l i t y to bind iron at  still pH's  49 as low  as 2.0  (Van  Snick et a l . 1973)  means that i t can bind iron in the  acid  environment of the stomach. Bovine milk contains 4-8 mM of c i t r a t e . Experiments using bovine milk showed no  bacteriostatic  dialysis  (Reiter  effect  due  to  l a c t o f e r r i n unless c i t r a t e was  removed  by  et a l . 1975). Citrate i s absorbed i n the upper duodenum i n  calves (Reiter, 1978). Bicarbonate i s also released i n the duodenum. This gives a low c i t r a t e , high bicarbonate environment, ideal conditions for  iron-binding  by l a c t o f e r r i n . There are f e r r i n . Two suckling  few  i n vivo studies on  the  a n t i - b a c t e r i a l properties of  lacto-  i n vivo models have been used, the bovine mammary gland and  guinea pig. Lactoferrin  the  concentrations in bovine mammary secretions  are shown i n Table 7. Seventeen l a c t a t i n g cows had  one  quarter infused  with a pathogenic s t r a i n  of E. c o l i . Thirteen out of 17 quarters became infected  (Reiter, 1985a). During  the dry period however, none of 14 quarters became infected. Two  dry quarters  infused with both iron and bacteria became infected. Clearly the high l a c t o f e r r i n levels i n the dry secretion of the mammary gland provides protection b a c t e r i a l i n f e c t i o n s . The  lower levels found during l a c t a t i o n along with  from the  high c i t r a t e levels mean a greater s u s c e p t i b i l i t y to i n f e c t i o n . Bullen et a l . (1972) studied the  effect of l a c t o f e r r i n on  the i n t e s t i n a l  f l o r a of suckling guinea pigs. Guinea pigs fed hematin as a source of iron  had  10,000 times the numbers of E. c o l i than guinea pigs receiving no hematin  had.  For  most virulent strains of E.  coli  this i n h i b i t i o n i s temporary. This i s  because many these strains are capable of obtaining iron via another  50  Table  7 . Concentration  of l a c t o f e r r i n  i n various secretions of the bovine  mammary gland (Smith and Schanbacher, 1977).  Type of mammary  Lactoferrin  secretion  (mg mhzA.)  Colostrum  1-5  Normal Milk  0.1-0.35  Early Involution 30 days of Involution C l i n i c a l Mastitis  1-8 20-30 1-8  mechanism. Bacteria can obtain iron from the environment i n two ways (Bullen et a l . 1978). The f i r s t i s by simple d i f f u s i o n of free iron or iron compounds through  the c e l l  membrane. This i s prevented  i n the presence  of iron-free  t r a n s f e r r i n or l a c t o f e r r i n . The second mechanism involves the synthesis by bacteria of iron chelators known as siderophores (Weinberg, 1984). In low iron environments many bacteria produce iron-chelators known as siderophores and membrane bound siderophore receptors. The a b i l i t y of bacteria to synthesize siderophores  and their  1973). Siderophores  receptors i s a recognized virulence factor  (Rogers,  are synthesized by many bacteria and secreted into the  tissues of the host. They have iron binding constants of up to 10' and are able 2  to  remove iron  from t r a n s f e r r i n  or l a c t o f e r r i n . The siderophore  receptors  transport the iron-siderophore complex into the b a c t e r i a l c e l l where enzymes remove the i r o n . To combat t h i s , mammalian hosts further l i m i t the a v a i l a b i l i t y of iron i n  51  the body. Leukocytic endogenous mediator-endogenous pyrogen (LEM-EP) i s secreted by leukocytes at the onset of a b a c t e r i a l i n f e c t i o n  (Powanda and Beisel, 1982;  Merriman et a l . 1977). This compound has three main effects on iron metabolism. The f i r s t i s a s h i f t of iron from the plasma to storage forms i n the l i v e r and spleen. Neutrophils secrete iron-free l a c t o f e r r i n at the s i t e of the i n f e c t i o n . The l a c t o f e r r i n removes iron from t r a n s f e r r i n . Macrophages then remove the ironsaturated  lactoferrin  from  the c i r c u l a t i o n .  (Van Snick  et a l . 1974). The  macrophages transport the iron to the l i v e r where i t i s sequestered i n storage forms. LEM-EP also causes a r i s e i n body temperature. The synthesis of siderophores i s inhibited by temperatures  greater than 37 °C. The r i s e i n body temperature  that occurs during infections has the effect of suppressing synthesis of mic r o b i a l siderophores iron  decreases  (Kluger and Rothenberg, 1979). Intestinal  during  infection  (Beresford  et a l . 1971).  absorption of LEM-EP  may be  responsible for this effect by increasing the production of f e r r i t i n i n mucosal c e l l s . When the mucosal c e l l s are shed this iron i s l o s t . (Sherman, 1984). In summary l a c t o f e r r i n has two physiological roles. The f i r s t i s to deprive bacteria of iron on body surfaces, the mammary gland and i n milk and colostrum. The second i s to scavenge iron from the body and return i t to the storage forms during b a c t e r i a l  infections.  The l a c t o f e r r i n content of sow's milk was measured i n two studies. Masson and Heremans (1971) found that sow's milk contained 20-200 jig rnL of lacto-1  f e r r i n and 20-200 ug rnL of t r a n s f e r r i n . E l l i o t et a l . (1984) measured the -1  l e v e l of l a c t o f e r r i n i n sow's milk throughout ranged from 1100-1300 pg mL dropped to 300 pg mL  -1  -1  a 21 day l a c t a t i o n . The levels  for the f i r s t three days after farrowing. This  on day 7 and 100 ug mL  -1  on day 21. They noted that the  52 drop i n l a c t o f e r r i n matched the r i s e i n immunocompetence of the p i g l e t . Lactoferrin iron  i s well  absorbed by piglets  (Fransson et a l . 1983).  3 9  Fe  labelled l a c t o f e r r i n was compared to FeSC-4  as a source of iron to p i g l e t s .  The  as well as the FeSC-4. However,  39  l a c t o f e r r i n iron was absorbed at least  l a c t o f e r r i n iron has the advantage of not being available to many species of b a c t e r i a l pathogens. Synthetic Iron  Chelators  In spite of l a c t o f e r r i n ' s desirable a n t i b a c t e r i a l properties, i t i s far too expensive to use as an additive to sow milk replacers at the present time. The same i s true of other iron-binding proteins such as t r a n s f e r r i n and conalbumin. A l l of these proteins have r e l a t i v e l y high molecular weights. Approximately 830 g of l a c t o f e r r i n are required to bind 1 g of i r o n . Normally, l a c t o f e r r i n i s only about one t h i r d saturated with iron (Bezkorovainy 1980). To attain this l e v e l of saturation, 2,490 g of l a c t o f e r r i n per g of iron are required. In addition to t h i s , commercial milk replacers contain higher concentrations  of iron than  sow's milk even when no supplemental iron i s added. This i s due to contamination during processing  and the iron content of the water used to reconstitute the  milk replacer. The iron content of sow's milk i s 1 ng rnL iron  content  of the reconstituted  described herein was 1.6 ug mL . _1  milk  replacer  used  -1  (N.R.C. 1978). The  i n the experiments  The amount of l a c t o f e r r i n required to bind  this amount of iron at 33% saturation i s approximately 4.0 mg mL . _1  about 13 times the concentration lactation  of l a c t o f e r r i n i n sow's milk  This i s  on Day 7 of  ( E l l i o t et a l . 1984). L a c t o f e r r i n has no enzymic properties and i t s  only b i o l o g i c a l role i s as an iron chelator (Brock 1985). It may therefore be possible to replace l a c t o f e r r i n with synthetic iron chelators. A number of synthetic iron chelators have been investigated for use i n the  53 treatment of i r o n overload i n humans (Grady and Jacobs 1981). Two compounds i n p a r t i c u l a r appear promising. N,N'-ethylenebis-[2-(o-hydroxyphenyl)]-glycine (EHPG) was f i r s t synthesized by Frost et a l . (1958). This compound i s also referred to as ethylene diamine-di-orthohydroxyphenyl a c e t i c acid (EDDA) and t h i s name w i l l be used here. Another i r o n chelator i s zyl)-ethylenediamine d i a c e t i c acid  N,N*-bis(o-hydroxyben-  (HBED). I t was f i r s t synthesized by  L'Eplattenier et a l . (1967). The structures of EDDA and HBED are shown i n Figure 10. Compared to l a c t o f e r r i n they have low molecular weights.  Figure 10. The molecular structure of EDDA and HBED (L'Eplattenier et a l . 1967;  Frost et a l . 1958).  HBED  -o—CO I  H H OC—OI /CHxCH*. | I  H  Ferric  "oocc;  CH,COO  _  H  EDDA  Ferric  HBED  54  EDDA has a molecular weight of 360.2 D and HBED has a molecular weight of 388.5 D. Both compounds are sexedentate ligands  (L'Epplatenier et a l . 1967). This  means that one molecule of either EDDA or HBED i s required to bind one atom of i r o n . Compared to the 830 g of l a c t o f e r r i n required to bind 1 g of iron, only 6.44 g of EDDA or 6.96 g of HBED are needed. EDDA and HBED are extremely s p e c i f i c for Fe of HBED for F e  3 +  is 10 1 8  3  3+  (see Table 8). The a f f i n i t y  times stronger than for the next most strongly bound  metal ion C u . In comparison the s t a b i l i t y constant for l a c t o f e r r i n 2 +  bound iron i s approximately 1 0  36  (Aasa et a l 1963) . HBED binds the f e r r i c ion  more t i g h t l y than l a c t o f e r r i n and EDDA, more weakly. The toxic effects of EDDA and HBED on animals have been studied. S t i f e l and Vetter (1967) drenched lambs with about 50 mg Kg body weight-1 day-1 of EDDA. This dose caused anorexia, awkward gait and increased urination. Two of 6 lambs receiving EDDA died. Postmortem toxicity  examinations revealed hepatic and pulmonary  and inflammation of the abomasal  i n j e c t i o n of 200 mg EDDA Kg body weight  -1  and i n t e s t i n a l  mucosa. A single  was not toxic to rats (Hershko et a l .  1984a). EDDA i s not well absorbed when administered o r a l l y to rats (Hershko et al 1984b). Poor absorption from the digestive tract would minimize systemic toxic e f f e c t s . HBED i s less toxic than EDDA. The LDso of HBED for rats was 800 mg Kg body weight-1 (Grady and Jacobs 1981). HBED i s also poorly absorbed from oral doses given to rats (Hershko et a l . 1986b). Miles and Khimji (1975) used EDDA as an indicator for synthesis of siderophores by bacteria. They found that 0.1 mg m l 10  4  Klebsiella  - 1  EDDA completely inhibited 7 x  spp.. Bacteria capable of synthesizing siderophores were not  55 i n h i b i t e d by EDDA however. No studies on the a n t i b a c t e r i a l properties of HBED Table 8 . Comparison of s t a b i l i t y constants of HBED and EDDA chelates (L'Eplattenier  et a l . 1967).  HBED  EDDA  LLO£_K!1ML  Ion  Mg  2+  Ca + 2  Log  K^ML  5  Log  10.51  8.00  9.29  7.20  2.09  K  2.51  Cu  2+  21.38  23.90  -2.52  Zn  2+  18.37  16.80  1.57  39.68  33^9  15.77  FejLi-  I  K  " M L  =  .. . [  H L  . . 3  THTTLT  where [ML] = the concentration of the metal-ligand complex [M] = the concentration of the free metal [L] = the concentration of the free ligand  have been published.  These two iron  chelators are promising  candidates as  replacements for l a c t o f e r r i n i n sow milk replacers. Vitamin B12 Binding Protein Like iron, vitamin B12 i s t i g h t l y bound by a s p e c i f i c milk protein (Gregory and Holdsworth 1955). Sow's milk i s p a r t i c u l a r l y r i c h i n B12 binding protein. Its B12 binding capacity i s 245 ng mL human milk and 0.5 ng mL  -1  -1  compared to 80 ng mL  -1  for  f o r cow's milk. The d i s t r i b u t i o n of B12 binding  protein i n the body i s similar to that of slgA and l a c t o f e r r i n . . It occurs i n tears, s a l i v a and gastric juice (Reiter 1985a). In v i t r o studies have shown that B12 binding protein i s able to i n h i b i t the  uptake of vitamin B12 by a variety of bacteria including E. c o l i  (Ford 1974).  The a n t i b a c t e r i a l properties of B12 binding protein have not been studied. B12 binding protein also increases the p i g l e t ' s absorption of vitamin B12 from the diet p a r t i c u l a r l y i n the f i r s t the f i r s t two weeks of l i f e (Trugo et a l . 1985). During this period the piglet does not synthesize adequate i n t r i n s i c factor and cannot therefore absorb vitamin B12. to  B12  binding protein however. Bi2  The piglet can absorb the vitamin bound binding protein deserves further i n v e s -  t i g a t i o n i n l i g h t of i t s r e l a t i v e l y high concentration i n sow's milk and i t s unknown a n t i b a c t e r i a l properties. Lvsozyme Lysozyme i s an enzyme found widely i n external secretions such as  tears,  s a l i v a , gastric secretions and milk. The concentration of lysozyme i n the milks of different species varies considerably. In human milk, i t occurs at a l e v e l of 400 mg mL  -1  while i n bovine milk i t occurs at a l e v e l of 180 ug mL  -1  (Jenness  1981). Lysozyme has  several b i o l o g i c a l  functions. The  first  involves i t s  enzymic a c t i v i t y . Lysozyme hydrolyzes the 1-4 6 linkage between N-acetylglucosamine and N-acetylmuramic acid i n the peptidoglycan layer of bacteria. This leads  to  lysis  of  the  bacteria and  prevents  the  outgrowth of  spores  and  vegetative c e l l s  (Wasserfall and Teuber 1979). Lysozyme's a b i l i t y to p r e v e n t  spore  may  outgrowth  account  for  the  low  numbers  or  complete  absence  of  C l o s t r i d i a i n the feces of breast fed infants. The products of the hydrolysis of peptidoglycan  by  lysozyme also serve  component i n Freund's adjuvant isoglutamine the  was  a protective function. The  found to be  active  N-acetylmuramyl-L-alanyl-D-  (Genco et a l . 1983). This compound i s similar to the products of  hydrolysis of  peptidoglycan  by  lysozyme. Adjuvants  stimulate  antibody  57 responses and  activate macrophages. Studies have shown that the products  hydrolysis of b a c t e r i a l c e l l walls by lysozyme also have an adjuvant  of  effect.  The l e v e l of slgA i n the feces of infants increased when lysozyme was included in  the  diet  (Lodinova  and  hydrolysis of b a c t e r i a l  cell  Jouja  1977). Feeding  the  products  of  lysozyme  walls to mice resulted i n increased levels of  salivary slgA i n mice (Morisaki et a l . 1983). IgM  and complement i n milk interact with lysozyme to increase i t s enzymic  activity can  (Reiter 1985b). Gram negative bacteria have an outer membrane which  prevent  lysozyme from penetrating  to the peptidoglycan  layer. IgM  and  complement produce lesions through the outer membrane of gram negative bacteria thus allowing lysozyme to attack i t s substrate (Reiter 1985b). Although lysozyme w i l l that this occurs  lyse bacteria i n v i t r o there i s s t i l l  no  evidence  i n vivo. Its low concentration i n sow's milk and  colostrum  indicates that i t i s probably of l i t t l e importance as a protective mechanism in naturally suckled p i g l e t s . It may however interact with other components in milk and increase the overall l e v e l of protection. Its enzymic properties and the immunostimulatory effects of i t s hydrolysis products  deserve further i n -  vestigation. The Lactoperoxidase  System  The lactoperoxidase system has not been studied i n sow's milk so the f o l lowing discussion refers to work done with bovine and human milks. The  lacto-  peroxidase system consists of the enzyme lactoperoxidase, H2O2 and SCN -  Lacto-  -  peroxidase and H2O2 form a complex which catalyzes the oxidation of SCN  -  +  NH^  to CO,,,  2-  , and S0  bacteria.  4  One  . The intermediary products of this reaction are i n h i b i t o r y to of  these  products  i s OSCN-  (Thomas et  a l . 1981). It has  a  chaotropic effect on the inner membrane of bacteria (Reiter et a l . 1976). Gram  58 negative,  catalase p o s i t i v e organisms such as E.  coli  require  an exogenous  source of H2O2 to be i n h i b i t e d or k i l l e d by the lactoperoxidase system (Carlsson 1980) . Gram p o s i t i v e , catalase negative  organisms excrete  be s e l f i n h i b i t o r y under aerobic conditions An  i n vivo t r i a l with calves was  s u f f i c i e n t H2O2 to  (Reiter 1985b).  conducted by Reiter and  Calves were fed raw milk or milk heat treated to destroy the system. When the calves were challenged  with oral E. c o l i ,  Harnulv  (1982) .  lactoperoxidase the calves  that  received the heat treated milk showed no reduction i n b a c t e r i a l numbers while the calves receiving raw  milk had  a 95% reduction  i n b a c t e r i a l numbers. The  addition of a magnesium peroxide to the diet as a s o l i d source of H2O2 resulted in a reduction of 99.99% i n b a c t e r i a l numbers. Several other (Waterhouse and Mullen 1980)  i n vivo studies  have shown that supplementing the natural l a c t o -  peroxidase system i n bovine milk by adding a source of H2O2 and SCN-  decreased  diarrhea and increased weight gains i n calves. A milk replacer for calves containing the activated lactoperoxidase  system i s available i n Sweden (Reiter  1985b). Experimental work on lactoperoxidase  supplemented milk replacers for  p i g l e t s i s p r a c t i c a l and desirable. Glycoconiuqate and Oligosaccharide Receptor Analogues Many pathogenic  bacteria and  b a c t e r i a l toxins  attach  to  the  intestinal  epithelium v i a glycoprotein, g l y c o l i p i d and oligosaccharide receptors. (Holmgren et a l . 1983) . Analogues of these receptors are also present  i n milk. In v i t r o  studies have shown that these molecules w i l l i n h i b i t hemagglutination of red blood c e l l s by E^. c o l i  (Holmgren et a l . 1983). Hemagglutination of red blood  c e l l s mimics adherence of bacteria to i n t e s t i n a l c e l l walls. In addition, the E. c o l i K88  adhesin which i s s p e c i f i c for pigs was  found to bind s p e c i f i c a l l y  to a receptor analogue on fat globule membranes from sow's milk but not cow  or  human milk (Reiter 1985b). These receptor analogues may protect suckling animals from b a c t e r i a l and v i r a l infections by i n h i b i t i n g attachment to the i n t e s t i n a l epithelium. In vivo studies on the receptor analogues have yet to be done. Sow's milk and colostrum nursing  piglet  contain a large number of factors that protect the  from b a c t e r i a l and v i r a l  i n f e c t i o n . These factors interact  extensively and i t i s probable that the protective value of the whole i s greater than the sum of the i n d i v i d u a l factors alone. The p r a c t i c a l application of our knowledge of these protective systems does not require that they a l l be replaced so that a milk replacer i s a perfect copy of maternal milk. Porcine serum immunoglobulins and bovine colostrum used as immunoglobulin sources for colostrum  have been successfully  deprived  p i g l e t s . Bovine serum  immunoglobulins have not been investigated however. A direct comparison of bovine and porcine serum immunoglobulins would show whether bovine serum i s a large potential source of immunoglobulins for sow milk replacers. The synthetic iron chelators EDDA and HBED have an a f f i n i t y  f o r iron similar to that of  l a c t o f e r r i n . They may provide an inexpensive way of mimicking the a n t i b a c t e r i a l properties of l a c t o f e r r i n i n sow milk replacers. Viable c e l l s i n sow's milk may enhance the p i g l e t ' s immune system. While the c e l l s selected, s p e c i a l i z e d population,  peripheral blood  i n sow's milk  leukocytes  are a  may provide a  suitable replacement just as serum immunoglobulins can replace c o l o s t r a l immunoglobulins. The  following experiments w i l l investigate i ) bovine versus porcine serum  immunoglobulins, i i ) the synthetic iron chelators EDDA and HBED as replacements for l a c t o f e r r i n and i i i ) peripheral blood leukocytes lostral  leukocytes.  as replacements f o r c o -  60 EXPERIMENT 1 Introduction Immunoglobulins from many sources have been used to provide passive immunity to colostrum deprived p i g l e t s with varying r e s u l t s . Porcine immunoglobulins from unfractionated serum provides adequate immune protection but i t s feeding i s impractical due to p a l a t a b i l i t y  problems  and r e l a t i v e l y  low immunoglobulin  content (Scoot et a l . 1972). Ammonium sulphate f r a c t i o n a t i o n of immunoglobulin from serum provides a cheap simple method of concentrating serum immunoglobulin. Immunoglobulins  from bovine sources have also been used as sources of passive  immunity for p i g l e t s . There i s interest i n bovine immunoglobulins f o r several reasons:(1) the a v a i l a b i l i t y of products l i k e bovine plasma, colostrum and acid whey; and 2) the possible use of single immunoglobulin source for both piglet and calf milk replacers. The objective of Experiment 1 was to compare immunoglobulins isolated from bovine and porcine serum as sources of passive immunity to colostrum deprived piglets.  The immunoglobulins  used  i n this  experiment  were prepared using  polyphosphate f r a c t i o n a t i o n . This was the f i r s t time immunoglobulins prepared by this method were used as a source of passive immunity for p i g l e t s . Materials and Methods Experimental Design There were 3 treatments i n which p i g l e t s received diets as shown i n Table 9 with 3 outcome groups of 11,11 and 12 p i g l e t s based on the a v a i l a b i l i t y of l i t t e r s . The term outcome group refers to a group of pigs that started the experiment at the same time.  61  Table 9 . Experimental protocol for studying the administration of immunoglobulins to p i g l e t s (Experiment  Treatment  Age of P i g l e t s (d)  Name Control Bovine  1  1  2 to 14  15 to 28  No Ig  No Ig  No Ig  20 BIgG  Porcine  4 BIgG  20 PIqG  A l l values are i n mg  1  1).  mL  No Ig  4 mg PlaG  No Ig  -1  These l e v e l s were based on r e s u l t s by McCallum (1977) who mg  mL  of PIgG on day  -1  1 and  5.33  mg  mL-  1  on days 2-20  found that  provided adequate  protection for colostrum deprived p i g l e t s . Levels of 13.3 mg mL 2.7  mg mL  -1  on days 2-20  provided inadequate  26.7  -1  on day 1 and  protection. The l e v e l s i n this  study were set between these two values to see i f an intermediate value would provide adequate immunity. Results were analyzed  using the General  Linear Models procedure  of  SAS  ( S t a t i s t i c a l Analysis System Institute Inc. 1985). The following least squares model was used to analyze the data i n Experiment 1.  where  Yu  = u + Ti + Gj + TiGj + E i j  Yu  = dependent variable  u  = o v e r a l l mean  Ti  = effect of the i t h treatment  Gj  = effect of the j t h outcome group  62 TiGj = i n t e r a c t i o n of the i t h treatment with the j t h outcome group Eij  = residual error for each sample  The i n t e r a c t i o n TiGj was not s i g n i f i c a n t for any of the measures so i t was added to the error term to increase precision and the r e s u l t s were recalculated. Analysis of covariance was performed on average d a i l y gains using i n i t i a l weight as covariate. Survival was analyzed by assigning 1 to p i g l e t s that survived the experiment and 0 to those that died. Differences between treatment means were analyzed using orthogonal contrasts. Preparation of Porcine and Bovine  Immunoglobulins  Citrated porcine whole blood and bovine plasma were obtained from Intercontinental Packers Ltd. of Vancouver, B.C..  Throughout the entire procedure  a l l materials were kept at 4 °C to minimize b a c t e r i a l growth. The porcine blood was allowed to s e t t l e overnight and the plasma was siphoned off the top. F i b r i n was precipitated by the addition of calcium carbonate and removed by c e n t r i fugation. Sera were fractionated using sodium polyphosphate "glass", a mixture of NaiaPiaCMo-NaaoPiaOog taining 114.4 g L MO) and 84.85 g L  _ 1  _ 1  (Lee et a l . 1988). One hundred mL of a solution con-  of sodium polyphosphate glass (Sigma Chemical Col, S t . l o u i s , NaCl were added to each L of plasma with constant s t i r r i n g .  After pH adjustment to 3.95 with 3M HCl, the mixture was s t i r r e d for 10 minutes and allowed to e q u i l i b r a t e overnight. The mixture was then centrifuged at 3000 g for 5 minutes and the supernatant retained. The immunoglobulin i n the supernatant was  concentrated using a P e l l i c o n u l t r a f i l t r a t i o n  system  (Millipore  Corporation, Bedford, MA) with a 100,000 nominal molecular weight l i m i t f i l t e r . The concentrated f r a c t i o n was l y o p h i l i z e d and stored at -18 °C i n sealed p l a s t i c containers.  63 Assay of Administered and Serum  Immunoglobulins  Quantitative analyses of bovine IgG and porcine IgG were done using the double antibody sandwich enzyme linked immunosorbent assay (ELISA) described by V o l l e r et a l . (1976). Antibodies were obtained from Sigma Chemical Co., St. Louis MO..  Antibodies to bovine IgG w i l l also cross react with porcine IgG. For  the bovine IgG assay, the cross reacting antibodies were removed by passing anti-bovine IgG anti-bodies over a column containing porcine serum proteins cross linked to agarose. Any cross reacting antibodies are bound i n the column. Likewise for the porcine IgG assay anti-porcine IgG antibodies were passed over a column containing bovine serum proteins cross linked to agarose. IgA and IgM were not assayed. Animal Management P i g l e t s were removed from sows immediately after b i r t h and placed i n boxes. A l l p i g l e t s over 800 g with no obvious anatomical or physiological defects (splay legs, a t r e s i a a n i i etc.) were used. When s u f f i c i e n t p i g l e t s were born for the outcome group they were randomly placed i n i n d i v i d u a l cages measuring 46 x 92 cm. The treatments were then assigned to the cages using a random number generator. The cages allowed contact between adjacently housed p i g l e t s . The room containing the cages was maintained at 32 °C. The cages were sanitized using high pressure washing and a quaternary ammonia disinfectant before the p i g l e t s were placed i n them. Milk Replacer The basal diet was a commercial, non-medicated,  a l l milk protein sow milk  replacer (Van Waters and Rogers Ltd., Vancouver B.C.). The milk replacer contained 21% crude protein, 31% f a t , and NRC requirement levels of a l l vitamins and minerals except i r o n . For this experiment 55 mg of iron as FeCl3 was added  64 for each Kg of milk replacer. The exact formulation of the diet i s proprietary information. A commercial milk replacer was used because this i s the type of diet that would be used i n any commercial applications of immunoglobulin f o r t i f i e d milk replacer. Results obtained with a p u r i f i e d laboratory diet might be less applicable to commercial applications. The dry diet was mixed with water to provide a 20% solids milk replacer. Freeze dried immunoglobulins were mixed with the dry milk replacer to provide the appropriate concentration. Feeding Regimen P i g l e t s were nipple fed by hand for the f i r s t 72 hours. They were fed hourly for  the f i r s t 6 hours and thereafter every 3 hours. After the period of hand  feeding, the p i g l e t s were fed from a nursing bottle located i n the cage and f i l l e d hourly by a p e r i s t a l t i c pump up to day 14. From day 15 to 28 they were fed  u n f o r t i f i e d milk replacer twice a day from p i g l e t waterers equipped  with  metal nipples. On day 1 p i g l e t s were fed ad l i b . On days 2 to 28 they were fed 375 mL K g  -1  body weight d a y  -1  to a maximum of 1,500 mL d a y . The p i g l e t s were -1  weighed every other day and the feed intake was adjusted accordingly. This l e v e l of  intake i s the same used by McCallum  (1977) and provides less feed than  naturally reared p i g l e t s receive from a sow. Diarrhea Scores Diarrhea was estimated using a scale described by Nocek et a l . (1984): 1) normal, no f l u i d 2) soft, mostly s o l i d 3) runny, mostly  fluid  4) watery, a l l f l u i d 5) watery, blood A l l observations were done by the same observer.  65 Protein Analyses Protein was determined using the macro Kjeldahl method. Ash Content The ash content of the bovine and porcine immunoglobulin  concentrates was  measured by heating the samples at 550 °C and determining the resultant change in weight. Blood Samples Blood samples were taken from the anterior vena cava at b i r t h and on days 1, 4, 7, 14, 21 and 28. The blood was collected i n 100 uL heparinized c a p i l l a r y tubes. The tubes were sealed with C r i t o s e a l and centrifuged for 15 minutes i n a Canlab International H i c r o c a p i l l a r y Centrifuge (Model MB) and read on a Canlab reader (Model (CR). The plasma samples were then stored at -18 °C. Post-Mortem Examinations Post-mortem examinations were performed within 24 hours of death at the P r o v i n c i a l Veterinary Pathology Laboratory (Abbotsford, B.C.) on a l l p i g l e t s that died during the experiment. B a c t e r i a l cultures of organisms found i n the i n t e s t i n e , lung, blood, spleen and kidneys were taken routinely. . Results Bovine and Porcine  Immunoglobulins  The y i e l d of freeze dried product was about 800-900 g f o r 120 L of porcine blood and 3100 g for 120 L of bovine plasma. The analysis of the freeze dried product i s shown i n Table 10. Gel electrophoresis was performed on porcine and bovine serum, and on both the p r e c i p i t a t e and supernatant fractions from the polyphosphate fractionation (see Figures 11 and 12). Survival Only 2 of 11 p i g l e t s i n the control group survived the entire 28 day period  66 (see Table 11). The mean survival f o r the control group was s i g n i f i c a n t l y lower than for the bovine or porcine immunoglobulin treatments. The difference i n surv i v a l between the porcine and bovine treatments was not s i g n i f i c a n t . There was a trend to higher survival i n the porcine group however. The apparent cause of death i n the f i r s t and second outcome groups was E. c o l i septicemia. Of the 5 piglets  that died i n the t h i r d outcome group only two died of E± c o l i sep-  ticemia. Both of these were controls. The other control p i g l e t that died i n this outcome group died of salmonellosis. One bovine treatment p i g l e t died i n the t h i r d outcome group. The pathogens isolated  from i t s tissues were E;. c o l i ,  K l e b s i e l l a spp., hemolytic Staphylococcus spp. and Pseudomonas spp.. The cause of death of the porcine treatment p i g l e t that died was Streptococcus suis type II. Diarrhea The  piglets  fed porcine or bovine immunoglobulins  did not d i f f e r  sig-  n i f i c a n t l y during any period (see Table 12). The controls had s i g n i f i c a n t l y more diarrhea during weeks 1 and 2 than the bovine or porcine immunoglobulin fed groups. Average Daily Gains and Piglet Weights Average d a i l y gains and p i g l e t weights were both analyzed. This was done because groups with small but non-significant differences i n average d a i l y gains can d i f f e r s i g n i f i c a n t l y i n weight at the end of the experiment. The controls had s i g n i f i c a n t l y lower average d a i l y gains during weeks 1,2 and 3 than the bovine  and porcine  immunoglobulin  fed piglets  (see Table 13). The bovine  immunoglobulin fed p i g l e t s had s i g n i f i c a n t l y lower average d a i l y gain than those that received porcine immunoglobulins during week 1. There were no treatment differences for b i r t h weights (see Table 14). The  67 controls had s i g n i f i c a n t l y lower mean weights on a l l days and had a mean weight of only 3,130  g at 28 days of age. The bovine immunoglobulin fed p i g l e t s had  s i g n i f i c a n t l y lower weights than the porcine immunoglobulin fed p i g l e t s on day 7. On days 14, 21 and 28, however, there were no s i g n i f i c a n t differences among outcome groups. Plasma  Immunoglobulins  Controls had no measurable plasma PIgG u n t i l 7 days of age (2.3 mg (see Table 15 and Figure 13). This rose rapidly to 15.3 mg mL 16.4 mg mL  -1  -1  mL ) -1  on day 21 and  on day 28.  The porcine immunoglobulin fed group showed a rapid r i s e i n plasma PIgG l e v e l s to 19.7 mg mL mg m l  -1  -1  at day 1. The l e v e l dropped to a minimum value of 8.3  on day 21. The controls had s i g n i f i c a n t l y higher plasma PIgG l e v e l s on  days 21 and 28 than the porcine group. The plasma of the bovine immunoglobulin fed p i g l e t s was analyzed for both BIgG and PIgG. On day 1, the plasma levels of BIgG of these p i g l e t s were s i g n i f i c a n t l y less than PIgG levels i n the porcine immunoglobulin fed p i g l e t s . The l e v e l of PIgG i n the bovine immunoglobulin treatment p i g l e t s was not measurable u n t i l day 4. It rose quickly to a mean of 13.1 mg m l  - 1  on day 21. The plasma  PIgG of the bovine immunoglobulin fed group was not s i g n i f i c a n t l y d i f f e r e n t from the other groups on days 21 and 28. The l e v e l of BIgG had declined to 0.2 mL-  1  mg  by day 28.  Discussion Survival of the control group i s an indication of the l e v e l of environmental pathogens.  Varley  et  a l . (1986)  achieved survival  rates  of  55-98% for  immunoglobulin deprived p i g l e t s i n extremely clean environmental conditions. The pigs were reared i n isolated cages with f i l t e r e d a i r . Under conditions more  68 t y p i c a l of commercial units, McCallum et a l . (1977) obtained s u r v i v a l rates of 0-15% . The s u r v i v a l rate of 22% i n the present experiment suggests a high l e v e l of  environmental pathogens. A negative control group has the disadvantage of acting as a reservoir of  contamination. Fecal cross contamination presumably allowed the spread of large numbers of pathogenic organisms from the controls to adjacent p i g l e t s . When McCallum et a l . (1977) deleted controls from one t r i a l , the survival rates and average d a i l y gains of the p i g l e t s increased markedly. There were no s i g n i f i c a n t differences i n survival between the three outcome groups but the cause of death changed as the experiment progressed. The apparent cause of death f o r p i g l e t s i n the f i r s t outcome group was Ej. c o l i  infection.  Piglets i n the second r e p l i c a t e died at an e a r l i e r age but the agent remained E. c o l i . In the t h i r d outcome group deaths were at about the same age as i n the second. The organisms responsible included Streptococcus suis, K l e b s i e l l a spp., Pseudomonas spp., hemolytic Staphylococcus spp. and Salmonella spp.. This pattern suggests f i r s t  a b u i l d up of numbers of Ej. c o l i  and then an  increase i n other less common environmental pathogens. An a l l i n - a l l out regimen with thorough s a n i t i z i n g would probably reduce the b u i l d up of disease i n the experimental room. Sampling blood from the vena cava i s s t r e s s f u l to the p i g l e t s and stress can decrease disease resistance. However, this method of blood sampling  probably  did not contribute to the low survival rate of the control p i g l e t s . In a similar type of experiment  (Scoot et a l . 1972) where no blood samples were taken,  control p i g l e t s had a survival rate of 0%. Diarrhea scores for the control group averaged 3.1 during the f i r s t week of l i f e . Control p i g l e t s a l l had diarrhea scores of 4 before death. In contrast  69 the  bovine immunoglobulin fed p i g l e t s that died had diarrhea scores of only 2  or  3. The mean score f o r this group was the same as f o r the porcine immuno-  globulin fed p i g l e t s . This indicates that although BIgG provides poor systemic protection i t provides adequate l o c a l protection i n the lumen of the small intestine. The growth rates of the bovine and porcine fed p i g l e t s d i f f e r e d s i g n i f i c a n t l y only during the f i r s t week. On weeks 2, 3 and 4 the average d a i l y gains of the bovine immunoglobulin fed group were not s i g n i f i c a n t l y d i f f e r e n t than the group that received porcine immunoglobulins. There was no compensatory gain and the weights of the bovine immunoglobulin fed group stayed about 400 g less than those that received porcine immunoglobulins from day 7 to 28. The growth rates of p i g l e t s i n this t r i a l were similar to those reported by McCallum  et a l . (1977) but lower than those f o r naturally reared  piglets.  Increasing the l e v e l of intake would probably improve the gains. Braude et a l . , (1970) obtained much higher average d a i l y gains with higher intakes. They also noted higher mortality and incidence of diarrhea at these l e v e l s . The plasma PIgG levels of p i g l e t s  that died on the control and porcine  immunoglobulin treatments were markedly lower than those of survivors. The one porcine immunoglobulin fed p i g l e t that died had a plasma PIgG l e v e l of only 9.0 mg mL  -1  on day 1. This was the lowest l e v e l of any p i g l e t on this treatment.  The two surviving controls were the only ones to have measurable plasma PIgG by day 7 ( 4.1 and 2.6 mg m L ) . A l l other controls were either dead or had -1  plasma PIgG l e v e l s less than 0.1 mg mL  -1  at 7 days of age. There was no apparent  relationship between the t o t a l plasma IgG and survival i n the bovine group. The plasma PIgG levels of the porcine immunoglobulin fed group were lower than published reports f o r naturally reared p i g l e t s . Klobassa et a l . (1981)  70 measured PIgG l e v e l s i n naturally suckled p i g l e t s and found that they peaked at 40.2 mg mL . -1  This i s more than double the 20.8 mg mL  i n the present study.  -1  This i s because sow colostrum provided a much higher l e v e l of immunoglobulins than  i n this  immediately Klobassa  experiment. The  average IgG  following p a r t u r i t i o n was  found  et a l . 1091). This decreased  concentration of sow's colostrum to be 95.6  to 14.2  mg  mL  mg mL -1  -1  at  24  i n one  study  hours  after  p a r t u r i t i o n . While i t i s possible to increase the l e v e l of IgG during the day 1, there may be no advantage i n doing so. In fact, there may be a disadvantage. Henry and Jerne (1968) found that passively administered IgG i n h i b i t s the active synthesis of IgG and IgM of the same s p e c i f i c i t y . In the present study, less IgG may have provided less i n h i b i t i o n to the development of active immunity by the p i g l e t . The naturally reared p i g l e t s studied by Klobassa et a l . (1981) had a plasma IgG l e v e l of only 6.7 mg m l  -1  on day 28. This did not increase u n t i l  after 5 weeks. The plasma IgG l e v e l of the porcine group began increasing after 3 weeks of age and reached 9.5 mg mL  -1  on day 28. The optimum l e v e l of pass-  i v e l y administered immunoglobulin would give adequate protection from disease and minimize i n h i b i t i o n of the development of active immunity. All  PIgG i n the plasma of the bovine immunoglobulin fed p i g l e t s was  syn-  thesized by the p i g l e t . Measurable levels of PIgG occurred by 7 days of  age.  It i s possible to approximate the PIgG synthesized by the porcine fed p i g l e t s using the half l i f e of 14 days for PIgG i n the bloodstream 1973).  (Curtis and Bourne  The calculated values show that no s i g n i f i c a n t synthesis took place  u n t i l after 21 days of age. Precocious development of active immunity by the bovine immunoglobulin fed group could be due to the low i n i t i a l l e v e l of plasma BIgG. It could also be that BIgG i s not as i n h i b i t o r y to active immunoglobulin synthesis. Henry and Jerne  (1968), found that rabbit IgG inhibited synthesis  71 of Ig of the same s p e c i f i c i t y i n mice. Cross-species i n h i b i t i o n may also take place between bovine and porcine IgG. The confounding effect of the low levels of BIgG i n the bovine group makes this impossible to determine i n the present study. The synthesis of PIgG by the control p i g l e t s was even more rapid than by the bovine immunoglobulin fed group. The difference was not s i g n i f i c a n t however. In  this case there was no passively administered immunoglobulin so there was  no i n h i b i t i o n to the development of active immunity. The means for the controls on days 14,21 and 28, are based on only two p i g l e t s however. The absorption of BIgG by the bovine immunoglobulin fed group was much lower compared to PIgG absorption by the porcine immunoglobulin fed group. Leary and Lecce  (1979)  found  that  the p i g l e t  selectively  absorbs  PIgG  when other  macromolecules are present i n the lumen of the i n t e s t i n e . Their proposed mechanism involves the presence of s p e c i f i c PIgG receptors on the surface of enterocytes  which  bind  PIgG s e l e c t i v e l y .  The non-immunoglobulin  macromolecules  stimulate pinocytosis but they are not bound by s p e c i f i c receptors and hence not  absorbed as e f f i c i e n t l y . The low absorption of BIgG i n this study may have  been due to the i n a b i l i t y of these receptors to bind and transport BIgG. In conclusion, bovine serum immunoglobulins are not well absorbed from the diet when fed to colostrum deprived p i g l e t s during the f i r s t day of l i f e . Bovine serum immunoglobulins cannot therefore replace porcine serum  immunoglobulins  on day 1. The function of dietary immunoglobulins after day 1 does not depend on absorption however. The similar levels of diarrhea seen on days 2-14 i n both bovine and porcine immunoglobulin fed p i g l e t s may indicate that either source of immunoglobulin w i l l provide adequate immune protection i n the small intestine after the f i r s t day of l i f e .  Table 10. Composition of freeze dried bovine and porcine immunoglobulin concentrates . 1  Component  Bovine Iq  Porcine Iq  74.5  77.9  IgG (%)  53.5  37.0  Ash (%)  12.4  10.1  Iron (uq/q)  29.8  17.6  Crude P r o t e i n  2  (%)  1  Analyses were performed on pooled samples prepared for Experiment 1.  2  Analyses are on a dry matter basis.  Table 11. The effect of bovine or porcine iinroioglobulins on the survival of colostrum deprived piglets (Experiment 1).  Number Surviving at day Birth  7  14  21  N  N  N  N  N  Control  11  5  3  2  2a 19.4 11.9  Bovine Ig  11  10  8  8  8  72.2 11.9  Porcine Ig  12  12  11  11  11  91.6 11.3  Treatment  28 %  N = the number of surviving piglets. a The contrast Control vs Bovine Ig and Porcine Ig is significant (P < 0.05).  Table 12. The effect of bovine or porcine imnuncglobulins on the average weekly diarrhea scores of colostrum deprived piglets (Experiment 1).  week 1 Treatment  X  2 SE  X  3 SE  4  X  SE  X  SE  Control  3.1a 0.05  2.4a 0.16  1.5  0.15  1.1  0.11  Bovine Ig  1.0  0.10  1.1  0.08  1.2  0.07  1.2  0.06  Porcine Ig  1.1  0.04  1.1  0.07  1.1  0.06  1.0  0.00  a The contrast Control vs Bovine Ig and Porcine Ig is significant (P < 0.05).  SE  74  Table 13. The effect of bovine or porcine immunoglofcilins on the average daily gains of colostrum deprived piglets (Experiment 1).  Average Daily Gains (g dayl) week 1  week 2  Treatment  X  Control  Oa  12  90b  24  150  16  9  140  12  Bovine Ig Porcine Ig  SE  150  •  week 3  week 4  X  SE  X  SE  X  SE  50a  33  60a  43  153  24  190  21  200  12  200  17  190  9  a The contrast Control vs Bovine Ig and Porcine Ig is significant (P < 0.05). b The contrast Bovine Ig vs Porcine Ig is significant (P < 0.05).  Table 14. The effect of bovine or porcine imminoglobulins on the body weights of colostrum deprived piglets (Experiment 1). Body Weights (g) Birth Treatment  day 7  day 14  day 28  X  SE  Control  1,290  58  1,280a 171  1,650a 342  2,060a 540  3,130  614  Bovine Ig  1,210  62  1,860b  81  2,900b 164  4,200b 258  5,580  293  Porcine Ig  1.240  55  2.320  66  3.290  4.670  5.980  236  X  SE  X  day 21  SE  132  X  SE  208  a The contrast Control vs Bovine Ig and Porcine Ig is significant (P < 0.05). b The contrast Bovine Ig vs Porcine Ig is significant (P < 0.05).  X  SE  75  Table 15. The effect of bovine or porcine immunoglobulins on the plasma immunoglobulin concentrations of colostrum deprived piglets (Experiment 1). Age of Piglets (days) 0 Treatment  X  1 SE  4  7  X  SE  X  SE  -  <0.1a  -  Control PIgG  <o.i  -  <0.1a  Bovine PIgG Bovine BIgG  <0.1 <0.1  -  <0.1 _ 4.0 0.5  'Bovine Total  <o.i  -  Porcine PIgG  <0.1  -  X  14 SE  X  SE  21 X  SE  28 X  SE  2.3a 2.6  6.8 2.6  15.3a 3.7  16.4a. 3.7  <0.1 _ 5.9 0.4  1.1 1.3 5.5 0.5  4.3 1.3 2.7 0.4  12.0 1.9 0.4 0.2  13.7 1.9 0.2 0.1  4.0b 0.5  5.9b 0.4  6.6b 1.3  7.0b 1.4  12.4 2.0  13.9 1.9  20.8 1.1  18.8 0.9  14.1 1.1  11.0 1.1  8.3 1.6  9.5 1.6  'For the Bovine Ig treatment, the total of PIgG and BIgG was compared to the other treatment means. a The contrast Control vs Bovine Ig and Porcine Ig is significant (P < 0.05). b The contrast Bovine Ig vs Porcine Ig is significant (P < 0.05).  Figure 11. Gel electrophoresis of polyphosphate fractions from porcine serum. (1) porcine serum; (2) polyphosphate supernatant f r a c t i o n ; (3) polyphosphate precipitate f r a c t i o n . A = Albumen; Ig =  A  Ig  Immunoglobulin.  A  Ig  Figure 12. Gel electrophoresis of polyphosphate fractions from bovine serum. (1) bovine serum; (2) polyphosphate supernatant f r a c t i o n ; (3) polyphosphate precipitate f r a c t i o n . A = Albumen; Ig = Immunoglobulin.  A  Ig  78 Figure 13. The effect of bovine or porcine immunoglobulins on the plasma immunoglobulin concentrations of colostrum deprived piglets (Experiment 1).  Day  79 EXPERIMENT 2 Introduction Lactoferrin i s an iron-binding protein found i n sow's milk and colostrum. It  i n h i b i t s the growth of bacteria i n the i n t e s t i n a l tract of the suckling  p i g l e t . Supplementation  of milk replacers with l a c t o f e r r i n may improve per-  formance of p i g l e t s reared on a r t i f i c i a l d i e t s . This use of l a c t o f e r r i n may be unfeasible however. There are several problems with using l a c t o f e r r i n as feed additive to p i g l e t milk replacers. F i r s t l y , l a c t o f e r r i n has a high molecular weight (93,000 daltons). This means that about 833 g of l a c t o f e r r i n are required to  bind  1 g of i r o n .  Secondly,  the iron  content  of most  commercial  milk  replacers i s r e l a t i v e l y high even i f no iron i s added. Contamination of milk replacers with iron during processing boosts the l e v e l of iron many times that of sow's milk. These two factors taken together mean that a very large amount of  lactoferrin  would be required to bind the iron  i n commercial sow milk  replacers. L a c t o f e r r i n i s uneconomical at the present time. Lactoferrin's a n t i - b a c t e r i a l effect i s due only to i t s a b i l i t y to bind i r o n . Other synthetic iron chelators may be able to replace l a c t o f e r r i n i n a r t i f i c i a l d i e t s . Two such compounds are ethylene diamine-di-orthohydroxyphenyl acetic acid (EDDA) and N,N'-Bis(o-hydroxybenzyl)-ethylenediamine  d i a c e t i c acid (HBED). The  objective of this experiment was to compare i n v i t r o , the a n t i b a c t e r i a l effect of  l a c t o f e r r i n , EDDA and HBED, with and without added  immunoglobulins.  Materials and Methods Experimental Design The treatments used are shown i n Table 16. The results were analyzed  80 Table 16. Experimental protocol for testing the effect of l a c t o f e r r i n , EDDA and HBED on the growth rate of E^_ c o l i 0 157 K88 (Experiment 2).  PIgG Chelator  0 mg/mL  Control  4 mg/mL  0.87 ug/mL F e  10.0 mg/mL l a c t o f e r r i n  0.94 mg/mL Fe  A  11.9%  12.9%  0.1 mg/mL EDDA  5.6%  6.0%  0.1 mg/mL HBED  6.1%  6.6%  A  The Control  values represent the amount of iron i n the growth medium. The  values f o r l a c t o f e r r i n , EDDA and HBED represent the percent saturation of the chelators with i r o n .  using the General Linear Models procedure of SAS ( S t a t i s t i c a l Analysis System Institute Inc. 1985) using the following least squares model. Y i j t = u + Ci + Pj + CiPj + E i j where  Y i j t = Log(CFU) mL" at time t 1  u  = the o v e r a l l mean  Ci  = the effect of the i  Pj  = the e f f e c t of the j  t  chelator  h  t  h  l e v e l of PIgG  CiPj = the interaction between the i  t  h  chelator and the  jth l e v e l of PIgG Eij  = the residual error f o r each sample  The Logio of the colony-forming units mL  -1  (Log(CFU) mL ) was the dependent -1  variable and represents the number of l i v i n g bacteria or clumps of bacteria i n  81 the growth medium. Treatment means were compared using the  Ryan-Einot-Gabriel-  Welsch multiple F test ( S t a t i s t i c a l Analysis System I n s t i t u t e Inc. 1985). Preparation of L a c t o f e r r i n Lactoferrin was separated from bovine colostrum using a method described by Law and Reiter (1977). Colostrum (7.5 L) was adjusted to pH 7.0 by the addition of K H C O 3 . Ammonium ferrous sulphate (8.6 g) was added to saturate the l a c t o f e r r i n with  i r o n . Dry  CM-Sephadex (90 g)  (C-50, Pharmacia Fine Chemicals  Uppsala, Sweden) prepared i n gel form i n 0.02 7.0)  was  added to the colostrum  Sephadex was  allowed  to s e t t l e  AB,  M potassium phosphate buffer (pH  and the mixture s t i r r e d for 2 hours. The  CM-  and  was  the supernatant was  followed by 3 washings of the Sephadex  decanted. This  with d i s t i l l e d water. The Sephadex was  then applied to a column at room temperature and eluted with 0.02M phosphate buffer containing  0.2M  NaCl  (pH  7.0)  until  no protein was  washings, based on absorbance at 280 nm.  The Sephadex was  0.02M phosphate buffer  NaCl  containing  0.5M  (pH  7.0)  detected  i n the  then eluted with a  and  the  lactoferrin  containing f r a c t i o n c o l l e c t e d . Iron was removed from the l a c t o f e r r i n by d i a l y s i s against trisodium c i t r a t e overnight followed by d i a l y s i s against deionized water for 24 hours at 4 °C. The l a c t o f e r r i n was  then freeze dried and stored at -18  °C. The unsaturated  iron binding capacity of the l a c t o f e r r i n was measured using  a method described by Bullen et a l . (1972). A 1% solution of l a c t o f e r r i n prepared i n phosphate buffered s a l i n e (0.01M phosphate, 0.15M  NaCl, pH  was  7.4).  The absorbance of the l a c t o f e r r i n at 470 nm was measured during t i t r a t i o n with 1.0 mM f e r r i c n i t r i l o t r i a c e t a t e (pH 7.4). The unsaturated iron binding capacity was  calculated from a plot of absorbance vs the amount of iron added. The  unsaturated  iron binding capacity was  found to be 1 g iron per 1370  g of l a c -  82 t o f e r r i n . The t h e o r e t i c a l value for the unsaturated iron binding capacity for iron free l a c t o f e r r i n , using a molecular weight of 93,000 daltons (Weiner and Szuchet 1975), i s 1 g iron per 833 g of l a c t o f e r r i n . Synthetic Iron Chelators EDDA was purchased from the Sigma Chemical Co. (St Louis, MO) i n 90% pure form and p u r i f i e d using the method of Rogers (1973). HBED was obtained as a g i f t from  the Strem Chemical Co  (Newbury, MA)  i n a pure iron  free form. Both  chelators were assayed for iron content prior to the experiment. Bacteria E. c o l i 0 157 K88 was obtained from the B r i t i s h Columbia Veterinary Pathology Laboratory.  It i s a known pathogen for pigs. Cultures were stored on  Trypticase Soy Agar (Difco Laboratories Inc., Detroit MI) slants at 4 °C and transferred monthly. I n h i b i t i o n Assays Trypticase  Soy broth,  lactoferrin,  EDDA, HBED and immunoglobulins were  s t e r i l i z e d by passage through a 0.22 um membrane f i l t e r (Millipore Corporation, Bedford MA).The iron content of the trypticase soy broth and the additives was measured. The l i q u i d trypticase soy broth contained 0.87 ug mL PIgG contained 0.07 mg mL  -1  -1  of i r o n . The  of iron at the concentration i t was used. Hemoglobin  was not present at a detectable l e v e l i n the PIgG solution. None of the other additives contained detectable levels of i r o n . E;. c o l i 0 157 K88 were grown overnight i n Trypticase Soy broth at 37 °C. The culture was then diluted to about 5 x 10  s  colony-forming units (CFU) mL . -1  One  mL of this culture was added to duplicate tubes containing 4 mL of trypticase soy broth and appropriate amounts of iron chelator and immunoglobulins for each treatment. The tubes were then incubated at 37 °C. Counts of viable bacteria  83 were done at 0, 2, 4, 6 and 12 hours on trypticase soy agar plates using a S p i r a l Plater  (Model CU, S p i r a l System Instruments Inc., Bethesda, MD.). The  plates were incubated 24 hours at 37 °C before counting. Iron Analyses Samples were ashed at 500 °C and dissolved i n 4M HC1. The sample was then analyzed using an atomic absorption spectrophotometer. Results There were no s i g n i f i c a n t hours  treatment differences i n the Log(CFU) mL  (see Table 17 and Figure 14). The overall  (1.02 x 10  9  mean Log(CFU) mL  -1  -1  at 0  was 5.01  CFU m i r ) . 1  The PIgG treatment had s i g n i f i c a n t l y fewer Log(CFU) m l  - 1  than the control  treatment at 2,4 and 6 hours. At 12 hours there was no s i g n i f i c a n t between the two treatments. The HBED treatment was not s i g n i f i c a n t l y  difference different  from the control treatment at any sampling time. Likewise, the HBED-PIgG treatment was not s i g n i f i c a n t l y different  from the PIgG treatment.  The l a c t o f e r r i n treatment was not s i g n i f i c a n t l y different  from the control  treatment at 2 hour incubation. At 4, 6 and 12 hours incubation however i t had s i g n i f i c a n t l y fewer Log(CFU) m l  - 1  than the control treatment. The l a c t o f e r r i n  treatment had s i g n i f i c a n t l y more Log(CFU) m l  - 1  at 2 hours than the PIgG treat-  ment. At 4 and 6 hours incubation, there were no s i g n i f i c a n t differences between the two treatments. At 12 hours the l a c t o f e r r i n treatment had s i g n i f i c a n t l y less Log(CFU) m l The  - 1  than the PIgG treatment.  lactoferrin-PIgG  and  the l a c t o f e r r i n  treatments  had  significantly  different Log(CFU) ml" only after 6 hours incubation. At that time l a c t o f e r r i n 1  PIgG had fewer Log(CFU) m l also had s i g n i f i c a n t l y  - 1  than the l a c t o f e r r i n treatment. Lactoferrin-PIgG  fewer Log(CFU) ml"  1  than the PIgG treatment after  6  84 hours. This may demonstrate synergism between l a c t o f e r r i n and PIgG. The results for EDDA were similar to the results for l a c t o f e r r i n . The  EDDA treatment had s i g n i f i c a n t l y fewer  Log(CFU) m l  - 1  than the control  treatment at 4, 6 and 12 hours. The EDDA treatment also had s i g n i f i c a n t l y fewer Log(CFU) m l PlgG  - 1  than the PIgG treatment at 6 and 12 hours. The EDDA and the EDDA-  treatments  were  incubation. At that Log(CFU) m l  - 1  significantly time  different  the EDDA-PIgG  only  treatment  after  4 and 6  hours  had s i g n i f i c a n t l y  fewer  than the EDDA treatment. The EDDA-PIgG treatment also had s i g -  n i f i c a n t l y fewer Log(CFU) m l  - 1  after 4 and 6 hours than the PIgG treatment. Once  again this may demonstrate synergism at these times. Discussion Sows milk i s b a c t e r i o s t a t i c or bacteriocidal c o l i (Nagy et a l . 1976a). Immunoglobulins  for many porcine strains of E.  and l a c t o f e r r i n are major factors i n  this e f f e c t . There i s disagreement whether either alone i s s u f f i c i e n t to i n h i b i t b a c t e r i a l growth or i f i n h i b i t i o n requires both immunoglobulins and l a c t o f e r r i n . Wilson (1972) found that immunoglobulins  alone are b a c t e r i o s t a t i c . Whey from  bovine colostrum or milk caused bacteriostasis  of an E^ c o l i s t r a i n after cows  were immunized f o r that s t r a i n . In another study the b a c t e r i c i d a l  effect of  bovine c o l o s t r a l whey was inactivated  and Brock  by heating to 56 °C (Reiter  1975). This treatment did not affect immunoglobulins but did destroy complement. Immunoglobulins  with no complement caused a temporary i n h i b i t i o n of the growth  of Ej. c o l i s i m i l a r to that i n the present experiment. Other studies found that immunoglobulins alone did not affect b a c t e r i a l growth at a l l (Spik et a l . 1978, Stephens et a l . 1980). Rainard (1986a) compared the effects of bovine IgGi and l a c t o f e r r i n on the growth of E. c o l i and found bovine IgGi alone was b a c t e r i o s t a t i c . They then used  85 a sonic treatment that broke up chains and clusters of bacteria before performing a viable count. Under these conditions, the immunoglobulin had no effect on b a c t e r i a l growth. Rainard suggested that the effect of immunoglobulins i s due  to the  formation of microcolonies  by  agglutination  rather  than  true  i n h i b i t i o n of growth. The bacteria are s t i l l able to multiply however. As the density of immunoglobulins  on the surface of bacteria decreases the micro-  colonies break up and the apparent i n h i b i t i o n ends. Microscopic examination of E_j. c o l i grown with PIgG i n the present experiment did reveal clusters of bacteria after 6 hours incubation. While immunoglobulins may have no effect on actual numbers of bacteria, the formation of microcolonies of bacteria may be of benefit to the suckling p i g l e t . Immunoglobulins coat the microcolonies of bacteria. This could prevent bacteria from adhering to the small i n t e s t i n e and causing disease (Nagy et a l . 1976b). The  immunoglobulins  might protect the p i g l e t u n t i l the microcolonies break up. Apparent i n h i b i t i o n of growth would show how long the immunoglobulins coat the bacteria and protect the p i g l e t . In the present experiment, immunoglobulins slowed apparent b a c t e r i a l growth for 6 hours. At 12 hours the Log (CFU) m l  - 1  was  the same as for the  control treatment. Studies of l a c t o f e r r i n also show c o n f l i c t i n g r e s u l t s . Some experiments showed that l a c t o f e r r i n i s more e f f e c t i v e when immunoglobulins are also present (Rogers 1973; 1976, Spik et a l . 1978). Other studies show no additive effect between immunoglobulins and l a c t o f e r r i n .  (Reiter et a l . 1975,  1979, Rainard, 1986a). Interestingly, Spik et a l .  Samson et a l .  (1978) found that l a c t o f e r -  r i n was more i n h i b i t o r y i n milk deactivated at 100 °C than i n peptone water. They speculated that milk either contributes d i r e c t l y to l a c t o f e r r i n ' s a c t i v i t y or i n d i r e c t l y s t a b i l i z e s l a c t o f e r r i n ' s structure.  86 The conditions of the experiment have much to do with the results obtained. The  present  study  found  an additive  effect  between  immunoglobulins and  l a c t o f e r r i n only at 6 hours. Lactoferrin had no s i g n i f i c a n t effect on b a c t e r i a l growth during the f i r s t 2 hours. Mellencamp et a l . (1981) found bacteria can use stored iron for growth even i n the presence of l a c t o f e r r i n . Bacteria grown i n iron poor conditions lose  t h i s i n t e r n a l iron store.  cause immediate and complete bacteriostasis.  Lactoferrin  can then  Rainard (1986a) grew bacteria i n  an iron poor media. The effect of l a c t o f e r r i n alone was so great that i t may not have been possible to detect any added effect of immunoglobulins. In an iron r i c h environment l a c t o f e r r i n i s only e f f e c t i v e after stores are depleted. Since immunoglobulins are e f f e c t i v e  the iron  f o r only a limited  period, the length of time an additive effect can occur i s small. The sampling time and l e v e l of iron i n the environment determine whether an additive  effect  occurs. EDDA and l a c t o f e r r i n inhibited growth during the 6 to 12 hour period while PIgG did not. Once a l l of the available it  was no longer  effective  chelators have a longer  immunoglobulin was bound to bacteria,  at preventing  l a s t i n g effect  further  on b a c t e r i a l  bacterial  growth.  Iron  growth. This has s i g -  nificance for the feeding of immunoglobulin f o r t i f i e d milk replacers to animals. The  i n t e r v a l between feedings must be short to be sure of a steady supply of  unbound immunoglobulins. EDDA caused complete bacteriostasis  over a 12 hour period. As mentioned i n  the l i t e r a t u r e review, Miles and Khimji the  (1975) used EDDA as an indicator f o r  synthesis of siderophores by bacteria.  completely inhibited 7 x 10 K l e b s i e l l a spp.. 4  They found that 0.1 mg m l  - 1  EDDA  Bacteria capable of synthesizing  siderophores were not inhibited by EDDA. A synergistic effect between EDDA and  87  PIgG was apparent i n the present study at 4 and 6 hours. The effect was not apparent at 12 hours but by t h i s time supply of available PIgG may have been exhausted. EDDA was able to completely i n h i b i t the growth of the test organism with or without PIgG. Lactoferrin was not as e f f e c t i v e i n i n h i b i t i n g the growth of the test organism. EDDA i s a small molecule compared to l a c t o f e r r i n . I t may be able to d i f f u s e the  into the b a c t e r i a l c e l l affecting  iron metabolism within  c e l l . This may account f o r i t s superior i n h i b i t i o n of b a c t e r i a l  growth  compared to l a c t o f e r r i n . HBED had no effect on b a c t e r i a l growth. Since i t has a stronger a f f i n i t y for iron than either l a c t o f e r r i n or EDDA t h i s i s surprising. No information on why HBED was i n e f f e c t i v e at preventing b a c t e r i a l growth was available i n the l i t e r a t u r e or from the manufacturer. From these results i t seems clear that EDDA can mimic l a c t o f e r r i n ' s a n t i b a c t e r i a l effect i n v i t r o f o r t h i s s t r a i n of E^ c o l i . HBED d i d not have any effect at a l l on b a c t e r i a l growth. Depending on the performance of EDDA i n vivo, i t may be an e f f e c t i v e a n t i b a c t e r i a l agent i n the diets of a r t i f i c i a l l y reared piglets.  88 Table 17. The effect of lactoferrin, EDDA and HBED, with and without porcine immunoglobulins, on the Mean Log(CFU) mL" of EL coli 0 157 K88 (Experiment 2). 1  Treatment  Chelator  PIgG  (mg/ml)  (mg/ml)  Time: (Hours) 0  2  4  6  12  0 4  5.01 5.00  5.12a 4.93bc  6.09a 5.51bc  7.03a 6.26bc  8.54a 8.59a  Lactoferrin 10 Lactoferrin Lactoferrin-PIgG 10 Lactoferrin  0 4  4.99 5.01  5.13a 5.74b 5.04abc 5.56bc  6.41b 6.03de  7.95b 7.93b  EDDA EDDA-PIgG  0.1 EDDA . 0.1 EDDA  0 4  5.04 5.01  5.04abc 5.41c 4.93c 5.07d  5.87e 5.18f  5.02c 4.84c  HBED HBED-PIgG  0.1 HBED 0.1 HBED  0 4  5.01 5.01  5.08ab 6.06a 5.02abc 5.50bc  7.10a 6.17cd  8.80a 8.53a  0.017  0.028  0.039  0.054  Control PIgG  SE  0 0  0.051  Means in the same column bearing the same letter are not significantly different (P < 0.05).  89 Figure 14. The effect of l a c t o f e r r i n , EDDA and HBED, with and without porcine immunoglobulins, on the Mean Log(CFU) mL  -1  (Experiment 2). T H E EFFECT OF LACTOFERRIN ON T H E GROWTH OF E. coli O 157 K88  | 3  T H E EFFECT OF EDDA ON T H E GROWTH OF E. coli 0157 K88  |  of E^. c o l i 0 157 K88  90 EXPERIMENT 3 Introduction In Experiment 2 the a n t i b a c t e r i a l effects of l a c t o f e r r i n , EDDA and HBED were tested i n an i n v i t r o system. Lactoferrin inhibited the growth of E^ c o l i 0 157 K88 over a 12 hour period. The large quantities of l a c t o f e r r i n required to bind a l l the iron present in commercial milk replacers makes i t unsuitable as a feed additive. The synthetic iron chelators EDDA and HBED have low molecular weights and are inexpensive. This makes them candidates to replace l a c t o f e r r i n in milk replacers. In Experiment 2, EDDA i n h i b i t e d the growth of E^. c o l i 0 157 K88 over a 12 hour period while HBED had no i n h i b i t o r y e f f e c t . The objective  of the  present experiment was to test the effects of adding EDDA or HBED to milk r e placers fed to colostrum deprived p i g l e t s . Materials and Methods Experimental Design The  treatments used i n Experiment 3 are shown i n Table 18. The experiment  was a 2 x 3 f a c t o r i a l layout with 2 levels of immunoglobulins and 3 levels of chelators.  The p i g l e t s were reared i n three outcome groups of 15,15 and 14  p i g l e t s . The outcome groups were started 28 days apart and an " a l l i n - a l l out" regimen was practiced. A l l p i g l e t s received the same diet on day 1. This was done to ensure that a l l p i g l e t s had approximately the same l e v e l of systemic PIgG. Any treatment differences  would thus be due to the effect of PIgG and iron chelators i n the  small i n t e s t i n e .  91  Table 18. Experimental protocol for testing the effect of EDDA and HBED on the performance of a r t i f i c i a l l y reared p i g l e t s (Experiment 3 ) . A  PIgG Treatment  dav 1  Chelator  dav 2-14  dav 2--14  POO  20  0  0  PPO  20  4  0  POE  20  0  0.1 EDDA  PPE  20  4  0.1 EDDA  POH  20  0  0.1 HBED  PPH  20  4  0.1 HBED  8  A  A l l values are i n mg mL .  8  The f i r s t l e t t e r of the treatment name indicates the type of immunoglobulin  -1  received on day 1, the second  letter  indicates  the type of  immunoglobulin  received on day 2-14 (P = porcine, 0 = none) the t h i r d l e t t e r indicates the type of chelator i n the diet on days 2-14 (E = EDDA, H = HBED, 0 = none).  The results were analyzed using the General Linear Models procedure of SAS ( S t a t i s t i c a l Analysis System I n s t i t u t e Inc. 1985) using the following squares model. Yijk = U + Ci + Pj + Gn + CiPj + CiGk +PjGk + CiPjGk + Eijk Where Yijk = the dependent variable u  = the o v e r a l l mean  least  92 Ci  = the e f f e c t of the i  Pj  = the e f f e c t of the j  Gk  = the e f f e c t of the k  t  chelator  h  t  h  l e v e l of PIgG outcome group  th  C Pj = the interaction between the i J  t  h  t  h  chelator and the j  t  h  l e v e l of  PIgG CiGk = the interaction between the i  chelator and the k  outcome  th  group PjGk = the i n t e r a c t i o n between the j  t  h  l e v e l of PIgG and the  k  th  outcome group CiPjGk = the i n t e r a c t i o n between the i and the k  th  t  h  chelator, the j  t  h  l e v e l of PIgG  outcome group  Eijk = the residual error for each sample Non-significant  interactions were added to the error term and  the results  recalculated. Differences between means were analyzed by orthogonal contrasts. Animal Management P i g l e t s were removed from the sow immediately after b i r t h and fed as i n Experiment 1 with the one difference that the p i g l e t s were nipple fed by hand for 48 hours instead of 72 hours. The  p i g l e t s adjusted  e a s i l y to the automated  feeder after 48 hours so the extra night of hand feeding was eliminated. Piglets were fed hourly for the f i r s t 6 hours then every 3 hours u n t i l 48 hours. From that point on the p i g l e t s were treated exactly as i n Experiment 1. P i g l e t s were injected with 100 mg of iron dextran on day 4 before blood samples were taken. Castration was Iron  performed on day  10.  Chelators  EDDA and  HBED were added as  concentrated  solutions  to the  liquid  milk  replacer before feeding. The f i n a l concentration of both iron chelators i n the  93 milk replacer was 0.1 mg mL . The iron content of the dry milk replacer was -1  6.1 pg g  - 1  on an as fed basis. The iron content of the water used to mix the  milk replacer was 0.5 pg mL . This gave a iron content of 1.6 ug mL -1  -1  of the  l i q u i d milk replacer as fed. The EDDA i n the p i g l e t diets was 10.3% saturated with i r o n . The HBED was 11.1% saturated with i r o n . Blood Samples Blood samples were taken from the o r b i t a l sinus with a IVt inch, 20 gauge needle. The blood was collected into heparinized 4 mL vacutainer tubes. One mL of blood was c o l l e c t e d at b i r t h and on day 1. Three mL of blood were collected at  a l l other sampling times. Two microhematocrit tubes were f i l l e d with blood  to determine packed c e l l volume. Another aliquot of blood was used to determine hemoglobin. The remainder plasma was c o l l e c t e d .  was centrifuged at 500 x g f o r 15 minutes and the  The plasma was frozen u n t i l the end of the experiment  when a l l other analyses were performed. Packed C e l l Volume Microhematocrits were done on each blood sample on the day of c o l l e c t i o n . Canlab heparinized microhematocrit tubes were f i l l e d with blood and sealed with C r i t o s e a l . The tubes were then centrifuged for 15 minutes i n a Canlab Internat i o n a l Microcapillary Centrifuge (Model MB) and read on a Canlab reader (Model CR). Hemoglobin Blood hemoglobin was measured by the cyanomethemoglobin method (Shoen and Solomon 1962). Duplicate analyses were done on the day of c o l l e c t i o n . Plasma Iron and Total Iron Binding Capacity Plasma iron and t o t a l iron binding capacity (TIBC) were measured on a Technicon autoanalyzer  (Model AA I I ) . Plasma iron was measured using  Technicon  94 method No. SE4-0025FL4. The procedure i s based on that of Giovaniello et a l . (1967) and Stookey  (1970). TIBC was measured by saturating the plasma trans-  f e r r i n with iron and removing the excess iron with s o l i d magnesium carbonate (Fielding 1980). The plasma was centrifuged and the supernatant was assayed for i t s iron content i n the autoanalyzer. Results Survival One p i g l e t on the POO treatment died (see Table 19). The apparent cause of death was  coli  septicemia. A l l other p i g l e t s  that  died  received  diets  containing HBED. This included 3 of 8 p i g l e t s on the POH diet and 2 of 7 p i g l e t s on the PPH d i e t . One piglet on the POH treatment died of E_j. c o l i septicemia. This piglet had an extremely high concentration of iron i n i t s l i v e r g-i compared to 175-472 ug g  - 1  (1073 ug  for other p i g l e t s from this experiment). Iron  t o x i c i t y was also given as a possible cause of death. The cause of death of the other two p i g l e t s was reported as chronic dermatitis with marked hyperkeratosis, parakeratosis and acanthosis. There were no v i s i b l e lesions of the l i v e r , brain or kidney of these p i g l e t s and no s p e c i f i c evidence of t o x i c i t y . The apparent cause of death of the two p i g l e t s on the PPH diet that died was E. c o l i septicemia. One p i g l e t also had non-hemolytic Staphylococcus aureus isolated from i t s spleen. Piglets receiving HBED had a s i g n i f i c a n t l y lower survival rate than those receiving either EDDA or no chelator. The survival rate for the POH treatment was s i g n i f i c a n t l y lower than the PPO, POE, PPE and PPH treatments. Diarrhea Significant differences i n the l e v e l of diarrhea occurred only i n the f i r s t week (see Table 20). Piglets receiving PIgG during days 2-14 treatments had less  95 diarrhea than those that did not. POE treatment p i g l e t s had a higher l e v e l of diarrhea than the combination of PPO, PPE and PPH. Average Daily Gains and Piglet Weights There were no s i g n i f i c a n t treatment differences i n the b i r t h weights of the piglets  (see Table 22). The Chelator x PIgG i n t e r a c t i o n was s i g n i f i c a n t f o r  average d a i l y gains during weeks 2,3 and 4. Because of this only  comparisons  between treatment means were analyzed (see Table 21). The interaction was caused by the high average d a i l y gains of the POE treatment compared to the POO and POH treatments. P i g l e t s that received only EDDA during days 2-14 gained as well as p i g l e t s on the PPO, PPE and PPH treatments. The r e s u l t s were similar for mean p i g l e t weights. The Chelator x PIgG i n t e r action was s i g n i f i c a n t on days 21 and 28. The i n t e r a c t i o n was caused by the high weights of the POE treatment p i g l e t s r e l a t i v e to the POO and POH treatments. The only s i g n i f i c a n t treatment differences occured on day 28. The p i g l e t s on the POH treatment weighed s i g n i f i c a n t l y less than the p i g l e t s on the POE, PPO, PPE and PPH treatments. Piglets that received only EDDA during days 2-14 weighed as much as p i g l e t s on the PPO, PPE and PPH treatments. Plasma Iron and Total Iron Binding Capacity The v a r i a b i l i t y for TIBC was high and no s i g n i f i c a n t differences were found for any sampling time (see Table 23). Blood samples on day 4 were taken 2 to 4 hours after  iron injections were given to the p i g l e t s . There was a rapid  increase i n TIBC following this i n j e c t i o n . TIBC values ranged from 269-302 ug dLr  1  on day 1 and 574-666 pg d l r on day 4. 1  Plasma iron was not measured at b i r t h or on day 1 (see Table 23). There were s i g n i f i c a n t differences i n plasma iron on day 14. P i g l e t s that received no PIgG on day 2-14 had higher plasma iron concentrations than those that did receive  96 PIgG. The contrast POE vs PPO, PPE and PPH was also  s i g n i f i c a n t . The POE  treatment had a higher plasma iron l e v e l than the other 3 treatments. Day 4 levels of plasma iron were markedly higher than the levels on day 7 due to the iron dextran i n j e c t i o n . Packed C e l l Volume and Hemoglobin Table 24 shows the packed c e l l volume and hemoglobin concentrations. On day 14 the packed c e l l volume of the p i g l e t s receiving no chelator was s i g n i f i c a n t l y higher than the combination of the EDDA and HBED treatment groups. There were no s i g n i f i c a n t contrasts for hemoglobin on any sampling day. Plasma PIgG Table 25 and Figure 15 show the plasma PIgG l e v e l s . There were no s i g n i f i c a n t differences  between any of the means for which comparisons were made.  Discussion The p i g l e t s i n t h i s experiment received 37.5 mg Kg body weight-1 day-1 of EDDA with no apparent i l l e f f e c t s . Lambs receiving 1  50 mg Kg body weight  -1  day  showed d e f i n i t e signs of t o x i c i t y and 2 of 6 died ( S t i f e l and Vetter 1967) .  EDDA i s not well absorbed when administered o r a l l y to rats  (Hershko et a l .  1984b). This may also be the case for p i g l e t s . Poor absorption from the digestive tract would minimize systemic toxic  effects.  HBED i s less toxic than EDDA. The LDso of HBED for rats was 800 mg Kg body weight-1 (Grady and Jacobs 1981). HBED was also poorly absorbed from oral doses given to rats  (Hershko et a l . 1986b). The 37.5 mg Kg body weight-1 day-1 fed  i n the present study should have caused no problems. The survival of piglets receiving  HBED was surprisingly  low however.  The severe dermatitis that affected the 2 piglets receiving the POH diet may have been caused by the HBED. The other p i g l e t that died on this treatment  97 had an unusually high l e v e l of iron i n i t s l i v e r but there were no overt signs of  t o x i c i t y except the skin l e s i o n s . The piglets on the PPH diet also had a  lowered survival rate. They d i d not display any skin lesions but did show a greater s u s c e p t i b i l i t y to E. c o l i . HBED, while not d i r e c t l y toxic, may i n t e r f e r e with the metabolism of iron and other metal ions i n the p i g l e t . Survival of the POO, PPO, POE and PPE treatment p i g l e t s was similar to the rates experienced i n Experiment 1 for the porcine immunoglobulin treatment. The survival of p i g l e t s on POO and POE diets was not adversely affected by the absence of PIgG after day 1. The " a l l i n - a l l out" regimen used i n this experiment allowed for a thorough cleaning of the experimental room among outcome groups of p i g l e t s . This prevented any build up of disease and meant that the l e v e l of environmental contamination was low as each outcome group began the experiment. The lack of a negative control group may also have contributed to the  cleanliness  of the environment.  If a negative control group  had been  included i n this experiment there may have been a higher l e v e l of disease and mortality. The diarrhea seen i n this experiment was d i f f e r e n t from Experiment 1. The highest score observed was a 3 for one of the POH p i g l e t s . A l l other p i g l e t s had a maximum score of 2. This low l e v e l diarrhea was widespread however, and most p i g l e t s had a score of 2 at some point during the experiment. The " a l l i n a l l out" regimen and the lack of a control group probably contributed to the low l e v e l of diarrhea seen i n the present experiment. EDDA and HBED had no effect on diarrhea during the present experiment. In Experiment 2 i t was found that EDDA inhibited the growth of E;_ c o l i 0 157 K88 but this d i d not translate into lower incidence of diarrhea. Not a l l d i arrhea i s caused by bacteria however. EDDA would probably have l i t t l e  effect  98 on diarrhea caused by viruses. PIgG had a s i g n i f i c a n t effect on the diarrhea scores during the f i r s t week of the experiment. PIgG would be e f f e c t i v e against viruses and t h i s may explain why i t was able to control diarrhea when EDDA could not. During the second week, diarrhea scores were the same whether the piglets received PIgG on days 2-14 or not. During the f i r s t week a l l p i g l e t s received the same diet on day 1. The effect of  this was to minimize treatment differences  i n average d a i l y weight gain  during the f i r s t week. During week 2, the b e n e f i c i a l effect of feeding immunoglobulins i n the diet after day 1 was manifest i n the increased average d a i l y weight gains of the PPO, PPE and PPH treatments compared to the POO and the POH d i e t s . Piglets on the POE diet also had higher average d a i l y weight gains than the POO and POH treatment p i g l e t s . POH piglets had a mean weight of only 5,090 g on day 28. POO p i g l e t s also had a r e l a t i v e l y low mean weight of 5,750 g. POE p i g l e t s weighed 6,240 g on average, as high as for any other treatment. There was no synergistic effect when EDDA and PIgG were fed together. The PPE treatment had weight gains and p i g l e t weights almost i d e n t i c a l to the POE treatment except during week 1. During that period the POE treatment piglets had lower average d a i l y gains than those on the PPE treatment. These findings  are similar to those i n Experiment 2 but are not,  however,  d i r e c t l y comparable. In Experiment 2, b a c t e r i a l i n h i b i t i o n was measured i n a closed system. The numbers of bacteria, the nutrients available, immunoglobulins and  iron chelators were fixed. In Experiment 3, these components were steadily  entering and leaving  the system, the system being the g a s t r o i n t e s t i n a l  tract  of the p i g l e t . EDDA appears to have an effect as b e n e f i c i a l as that of PIgG i n the small intestine of the p i g l e t .  The average d a i l y gains of the POH treatment p i g l e t s were worse than for POO treatment p i g l e t s . During weeks 1 and 2 the average d a i l y gains for POH were comparable to the other treatments. During weeks 3 and 4 t h i s treatment produced a marked depression i n rate of weight gain. PPH p i g l e t s  d i d not show this  depression i n weight gains during weeks 3 and 4. The absence of PIgG coupled with a possible t o x i c i t y from HBED may have been a greater challenge than the POH piglets  could withstand. This could have l e d to the lower survival and  weight gains. The PPO treatment and the porcine immunoglobulin treatment from Experiment 1 are i d e n t i c a l treatments. Average d a i l y gains of the p i g l e t s i n the present experiment were lower i n the f i r s t week than i n experiment 1 (90.8 vs 153.4 g day-1). This difference disappeared i n subsequent weeks and the 28 day p i g l e t weights were s l i g h t l y higher i n for Experiment 3 than Experiment 1. The t o t a l iron binding capacity  (TIBC) for the p i g l e t s measured agrees  with other studies (Furugouri, 1971;1972;1973). The TIBC was low at b i r t h and remained low u n t i l after  the iron dextran i n j e c t i o n . After  the iron dextran  i n j e c t i o n , the TIBC rose about 400 pg d L r i n the few hours. None of the con1  trasts were s i g n i f i c a n t for TIBC on any day. EDDA and HBED did have s i g n i f i c a n t effects on plasma i r o n . EDDA and HBED p i g l e t s had higher plasma iron levels on day 14 than those that received no chelator. This may have been due to the EDDA and HBED i n the blood. Any c i r culating EDDA or HBED would almost c e r t a i n l y be bound to iron and elevate plasma i r o n . A small quantity of the c i r c u l a t i n g chelators could affect plasma iron. The elevated plasma iron would not affect TIBC. Furugouri (1971) found that TIBC was independent of the plasma iron concentration. PIgG also affected plasma iron on day 14. Piglets receiving PIgG on days  100 2-14 had lower plasma iron concentrations than those that received no PIgG on days 2-14. This i s opposite of what would be expected. The PIgG i n the diet contained some iron so that p i g l e t s receiving PIgG on days 2-14 would obtain more dietary iron than those that received no PIgG. One might also expect an equal or higher i n f e c t i o n rate i n p i g l e t s not receiving PIgG on days 2-14.  Infection  causes plasma iron to decrease (Van Snick et a l . 1974). Piglets not  receiving  PIgG should therefore have an equal or lower plasma iron concentration. Plasma iron was not measured during the f i r s t two days after b i r t h . After the i n j e c t i o n of iron dextran on day 4, plasma iron was about 400 ug d l r for 1  a l l treatments. The l e v e l decreased to less than 200 on day 7. The concentration on day 21 was about the same as the plasma iron levels found i n other studies Furugouri (1971). The contrast No  chelator vs EDDA and HBED" was  packed c e l l volume. The difference  s i g n i f i c a n t on day 14 for  disappeared by day 21. EDDA and HBED may  i n t e r f e r e with the synthesis of red blood c e l l s but the effect was  small and  transitory. The packed c e l l volumes of p i g l e t s i n t h i s experiment were similar to those found i n other studies for the f i r s t 2 weeks (Zimmerman et a l . 1959, M i l l e r et a l . 1961, Kay et a l . 1980). For weeks 3 and 4 the values were 5-7% lower than those reported for naturally reared p i g l e t s . None of the contrasts were s i g n i f i c a n t for hemoglobin  concentration on any  day. The values found i n this experiment were similar to those found by M i l l e r et a l . (1961) but 2-3 g d l r higher than those reported by Kay et a l . (1980) 1  throughout the experimental period. Plasma PIgG l e v e l s were similar for a l l treatments. This was not unusual considering a l l p i g l e t s received the same diet on day 1. The difference PIgG ranged from 19.6 to 25.5 mg mL-1  i n plasma  on day 1. This range was much smaller on  101 day  4 (18.2-21.8). Most treatment means were at a minimum on day 21. The d i -  fference between the means on day 21 and 28 was not s i g n i f i c a n t . The values were similar to those i n Experiment 1 for the Porcine treatment group. In conclusion, HBED has no potential colostrum  deprived  as a l a c t o f e r r i n  piglets.  substitute  I t had no a n t i b a c t e r i a l  i n milk  replacers for  effect  i n v i t r o and  decreased survival and growth rates when fed to p i g l e t s . EDDA on the other hand appears to have promise as an additive to sow milk replacers. It has s i g n i f i c a n t antibacterial  properties i n v i t r o and did not have any negative effects on  p i g l e t performance when included i n the d i e t .  102  Table 19. The effect of EDDA or HBED, with or without porcine immunoglobulins on the survival of colostrum deprived piglets (Experiment 3). Number Surviving at day 28  Birth  7  14  21  Treatment  N  N  N  N  N  POO PPO  8 7  8 7  8 7  7 7  7a 88 7a 100  12.0 12.9  POE PPE  7 7  7 7  7 7  7 7  7d 100 7d 100  12.9 12.9  POH PPH  8 7  7 7  7 6  6 6  5b 5  12.0 13.1  %  62 70  SE  N = the number of surviving piglets. a The contrast POO and PPO vs POE, PPE, POH and PPH is significant (P < 0.05) b The contrast POH vs PPO, POE, PPE and PPH is significant (P < 0.05) d The contrast POE and PPE vs POH and PPH is significant (P < 0.05)  103 Table 20. The effect of EDDA or HBED, with or without porcine inrrmmoglobulins on the average weekly diarrhea scores of colostrum deprived piglets (Experiment 3). week Treatment  1  2  3  4_  POO PPO  1.6e 1.2  1.2 1.1  1.3 1.1  1.1 1.1  POE PPE  1.7ce 1.3  1.3 1.2  1.4 1.3  1.0 1.1  POH PPH  1.5e LJ2  1.2 h2  1.2 1.1  1.0 1.0  c The contrast POE vs PPO, PPE and PPH is significant (P < 0.05) e The contrast POO, POE and POH vs PPO, PPE and PPH is significant (P < 0.05)  104  Table 21. The effect of EDDA or HBED, with or without porcine innunoglobulins on the average daily gains of colostrum deprived piglets (Experiment 3).  Average Daily Gains (g day^-)  X  SE  X  SE  12 12  220 230  21 21  190 210  20 20  160 150  12 12  240 240  21 21  230 220  20 20  150 160  14 14  180 220  25 25  120b 200  23 24  X  SE  X  POO PPO  80 90  10 10  150 170  POE PPE  80 110  10 10  POH PPH  90 100  12 12  Treatment  week 4  week 3  week 2  week 1  SE '  b The contrast POH vs PPO, POE, PPE and PPH is significant (P < 0.05)  105  Table 22. The effect of EDDA or HBED, with or without porcine inrauncglobulins on the body weights of colostrum deprived piglets (Experiment 3).  Body Weights (q) Birth Treatment  dav • 7  dav 14  dav 21  dav 28  X  SE  X  SE  X  SE  X  SE  POO PPO  1,310 1,250  69 74  1,830 1,900  70 70  2,860 3,110  119 119  4,430 4,750  207 207  5,750 289 6,230 287  POE PPE  1,220 1,360  74 74  1,820 2,020  70 71  2,950 3,050  119 121  4,630 4,720  207 211  6,240 289 6,240 294  POH PPH  1,270 1,290  69 74  1,880 1,990  82 84  2,950 3,110  140 143  4,250 4,660  244 249  5,090 341 6,060 347  Contrasts b  -  -  -  -  The contrast POH vs PPO, POE, PPE and PPH is significant (P < 0.05)  X  b  SE  106 Table 23. The effect of EDM or HBED, with or without porcine immunoglobulins on the plasma iron and total iron binding capacity (TIBC) of colostrum deprived piglets (Experiment 3). Age of Piglets (days) 13 Treatment  X  SE  1 X  SE  4  7  X  SE  X  SE  14 X  21 SE  X  28 SE  X  SE  Plasma Iron (ug dlr ) 1  POO PPO  na na  na na  440 439  24 26  173 182  15 17  138ae 12 114a 13  97 95  9 9  69 69  3 4  POE PPE  na na  na na  428 403  29 22  156 192  19 14  174ce 15 132 11  93 101  11 8  65 73  4 3  POH PPH  na na  na na  441 392  26 26  188 181  17 17  153e 134  87 92  9 9  70 73  4 4  13 13  TIBC (ug dlr ) 1  POO PPO  249 23 269 25  294 281  22 24  666 654  32 35  526 541  61 68  406 411  53 59  548 535  22 24  588 570  24 26  POE PPE  291 21 268 23  302 273  20 22  643 598  29 32  484 455  57 61  442 434  50 53  544 562  21 22  570 543  22 24  POH PPH  231 26 263 26  269 291  25 25  574 587  37 37  423 504  71 71  494 442  62 62  548 569  26 26  555 654  27 27  a The contrast POO and PPO vs POE, PPE, POH and PPH is significant (P < 0.05) c The contrast POE vs PPO, PPE and PPH is significant (P < 0.05) e The contrast POO, POE and POH vs PPO, PPE and PPH is significant (P < 0.05)  107 Table 24. The effect of EDDA or HBED, with or without porcine inraunoglobulins on the packed cell volumes and hemoglobin concentrations of colostrum deprived piglets (Experiment 3).  Age of Piglets (days) 0 Treatment  X  1 SE  X  4 SE  X  7 SE  X  14 S  E  X  S  21 E  X  S  28 E  X  S  E  Packed Cell Volume (%) POO PPO  34.3 2.5 32.5 2.5  29.2 3.7 24.2 3.7  30.2 3.1 27.7 3.1  31.0 2.0 28.8 2.0  32.0a 1.7 31.3a 1.7  32.5 2.7 31.1 2.7  30.0 2.5 28.8 2.5  POE PPE  33.4 1.9 31.2 2.1  26.5 2.9 23.9 3.2  25.7 2.4 24.4 2.7  27.7 1.6 28.0 1.7  28.0 1.3 29.2 1.5  30.4 2.1 33.0 2.4  29.7 1.9 31.4 2.2  POH PPH  29.4 2.5 35.3 2.5  26.1 3.7 26.5 3.7  24.5 3.1 28.8 3.1  28.8 2.0 27.0 2.0  29.5 1.7 29.3 1.7  29.3 2.7 31.8 2.7  29.4 2.2 28.6 2.5  BBCGLCBIH (g d l / ' )  POO PPO  13.6 1.4 11.8 1.6  12.0 1.1 10.4 1.3  11.0 1.1 10.7 1.3  11.0 0.8 10.6 1.0  11.6 0.8 12.6 0.9  11.7 1.1 11.3 1.3  12.1 1.1 11.0 1.3  POE PPE  12.1 1.3 13.2 1.3  10.5 1.0 10.3 1.0  10.2 1.0 8.8 1.0  9.3 0.7 9.2 0.7  10.6 0.7 11.9 0.7  11.4 1.0 12.1 1.0  10.7 1.0 11.0 1.0  POH PPH  11.7 1.7 11.8 1.7  10.0 1.3 10.3 1.3  8.8 1.3 11.4 1.3  9.9 1.0 10.0 1.0  11.9 0.9 11.3 0.8  11.1 1.3 11.4 1.1  11.6 1.3 9.5 1.1  a The contrast POO and PPO vs POE, PPE, POH and PPH is significant (P < 0.05)  108 Table 25. The effect of EDDA or HBED, with or without porcine immunoglobulins on the plasma immunoglobulin concentrations of colostrum deprived piglets (Experiment 3) . 1  Age of Piglets (days) 0 Treatment  1  X  SE  POO PPO  <1.3 <1.3  -  19.6 3.0 20.3 3.0  21.6 21.4  2.4 2.4  POE PPE  <1.3 <1.3  -  25.5 3.0 19.6 3.0  POH PPH  <1.3 <1.3  -  20.8 22.3  1  X  4  SE  3.0 3.3  X  SE  7  28  SE  X  SE  X  SE  17.0 2.4 14.0 2.4  9.5 8.6  1.6 1.6  9.0 6.9  1.1 1.1  7.8 8.2  1.5 1.5  21.9 2.4 18.2 2.4  15.8 2.4 13.6 2.4  7.6 1.6 8.3 1.6  7.2 1.1 7.4 1.1  8.5 7.2  1.5 1.5  18.5 21.8  15.9 2.4 15.8 2.6  8.6 1.1 7.7 1.2  8.7 7.3  1.5 1.6  There were no significant contrasts (P < 0.05).  SE  21  X  2.4 2.7  X  14  10.6 10.1  1.6 1.7  Figure 15. The effect of EDDA or HBED, with or without  porcine  immunoglobulins on the plasma immunoglobulin concentrations of colostrumdeprived p i g l e t s (Experiment 3). For s t a t i s t i c a l analysis see Table 25.  110 EXPERIMENT 4 Introduction Providing passive immunity to a r t i f i c i a l l y reared p i g l e t s can be divided into two stages. The c o l o s t r a l stage occurs during approximately the f i r s t 24 hours of  life.  During  t h i s time immunoglobulins and other protective factors are  absorbed d i r e c t l y into the blood stream of the p i g l e t . This provides systemic immunity and regulates the immune system of the p i g l e t . The milk stage s t a r t s after the c o l o s t r a l stage ends and l a s t s u n t i l the p i g l e t i s weaned from the protective factors i n milk. These factors prevent enteric infections by preventing b a c t e r i a l growth and adhesion within the i n t e s t i n a l t r a c t . Experiment  1 demonstrated that during the c o l o s t r a l stage, porcine immuno-  globulins are required. Plasma IgG concentrations averaged 20.8 mg mL  -1  on day  1 when PIgG was fed. When BIgG was fed plasma IgG l e v e l s averaged 4.0 mg mL  -1  on day 1. During the f i r s t week of l i f e , p i g l e t s receiving PIgG gained weight at  a greater rate than those that received BIgG. There was also a trend to  higher survival among p i g l e t s receiving PIgG instead of BIgG. During the milk stage, the l e v e l of diarrhea i n p i g l e t s receiving BIgG was not s i g n i f i c a n t l y different from those fed PIgG. This may indicate that BIgG can replace PIgG during the milk stage. In Experiment  3, the addition of EDDA to milk replacers  during the milk stage had a b e n e f i c i a l effect on p i g l e t growth. The objective of Experiment  4 was to compare the effects of adding BIgG,  PIgG and/or EDDA to milk replacers to see which factor or combination of factors provided the best passive protection to the p i g l e t during the milk stage. Materials and Methods The treatments used i n Experiment  4 are shown i n Table 26.  111 Table 26. Experimental protocol f o r studying the administration of EDDA with porcine or bovine immunoglobulins  (Experiment 4 ) .  dav 1 Treatment Control  la  dav 2-•14 EDDA  Icr  dav 15--28  EDDA  Ig  EDDA  0  0  0  0  0  0  BBO  25 BIgG  0  5 BIgG  0  0  0  POO  25 PIgG  0  0  0  0  0  POE  25 PIgG  0.1  0  0.1  0  0  PBO  25 PIgG  0  5 BIgG  0  0  0  PBE  25 PIgG  0.1  5 BIgG  0.1  0  0  PPO  25 PIgG  0  5 PIgG  0  0  0  PPE  25 PlaG  0.1  5 PIgG  0.1  0  0  A l l values are i n mg mL  -1  The concentration of immunoglobulins i n the diets was increased from 20 mg mL  -  1  on day 1 and 4 mg mL  -1  on days 2-14 to 25 mg mL  closer to the 26.7 and 13.3 mg mL  -1  -1  and 5 mg mL . These are -1  l e v e l s recommended by McCallum  (1977). The  levels were increased for 2 reasons. F i r s t l y , to see i f any increase i n growth rate occurred at these higher l e v e l s . Secondly, to see what effect the increased dietary immunoglobulins on day 1 would have on the l e v e l of plasma immunoglobulins. In Experiment 3, plasma immunoglobulin levels actually increased from day 1 to day 4 for some treatments. Leary and Lecce (1979) proposed that immunoglobulin s p e c i f i c receptors on enterocytes are responsible for the selective absorption of PIgG from the diet into the bloodstream. It was hypothesized that 20 mg mL-  1 1 2  1  of PIgG i n the diet was  inadequate to saturate these receptors. This meant  that the absorption of PIgG continued after 24 hours thus giving higher plasma PIgG values on day 4. By increasing the l e v e l of PIgG to 25 mg mL  -1  i t was  hoped that the s p e c i f i c immunoglobulin receptors would be saturated within the f i r s t 24 hours of l i f e and gut closure would occur e a r l i e r . The p i g l e t s were raised i n 5 outcome groups. The outcome groups were farrowed less than 4 weeks apart so that newborn p i g l e t s entered the experimental room with the p i g l e t s from the previous outcome group s t i l l present i n the room. The r e s u l t s were analyzed using the General Linear models procedure of SAS using the following least squares model. Yu Where  Ytj  = u + Ti + Gj + TiGj + E i j = the dependent variable  u  = the o v e r a l l mean  Ti  = the effect of the i  Gj  = the effect of the j  t  treatment  h  t  h  outcome group  TiGj = the interaction between the i j Eij  t h  t  h  treatment  and  outcome group  = the residual error for each sample  Non-significant interactions were added to the error term and the results recalculated. Differences between the means were analyzed using orthogonal contrasts. All  other experimental methods and  periment  procedures  were as described for Ex-  3.  Results Survival The  survival  of  the Controls was  0 out  of 9  (see Table  27}.  This  was  113 s i g n i f i c a n t l y lower than for any other treatment. A l l p i g l e t s were dead before day 7. The cause of death for 8 of the 9 was septicemia. The major organism responsible was  E^. c o l i which was  i s o l a t e d from the tissues of every Control  p i g l e t that died, a hemolytic Staphylococcus spp. , fi hemolytic Streptococcus spp., K l e b s i e l l a spp. and Actinobacillus suis were also isolated Control  piglets.  Staphylococcus  spp.  and  Streptococcus spp.  from some  were found i n  Controls i n every outcome group. Actinobacillus suis was found i n the fourth outcome group  and K l e b s i e l l a  spp. was  isolated from both Controls from the  f i f t h outcome group. The remaining p i g l e t died of bronchopneumonia caused by E. c o l i . Four of 10 BBO treatment p i g l e t s died. This was s i g n i f i c a n t l y more than for any other treatment except the Controls. Bronchial pneumonia caused by E^. c o l i was the cause of death for the 2 p i g l e t s that died before 14 days of age. The two p i g l e t s that died during week 3 died of Streptococcus suis II i n f e c t i o n . There were no s i g n i f i c a n t differences i n survival rates between any of the treatments that received PIgG on day 1. Streptococcus suis II i n f e c t i o n was responsible for the death of the p i g l e t s on the PBO and the PBE treatments. One Porcine treatment  piglet  died. The  cause was  omphalitis (Kidney infection)  caused by E^. c o l i . Diarrhea The Control treatment had an average weekly diarrhea score of 3.4 during the first  week (see Table 28). This was  s i g n i f i c a n t l y higher than for any other  treatment. There were no diarrhea scores for weeks 2-4 since a l l Controls were dead after the f i r s t week. The type of immunoglobulins  fed during week 1 had a s i g n i f i c a n t effect on  the amount of diarrhea seen. P i g l e t s that received no immunoglobulins  during  114 week 1 had more diarrhea than those that received BIgG or PIgG. Piglets receiving BIgG i n week 1 had s i g n i f i c a n t l y more diarrhea than those receiving PIgG. The PPO treatment p i g l e t s had s i g n i f i c a n t l y less diarrhea than those on the BBO  treatment.  EDDA had no s i g n i f i c a n t e f f e c t on diarrhea during week 1. During week 2, p i g l e t s receiving no immunoglobulins  had more diarrhea than  p i g l e t s receiving PIgG or BIgG. No other effects were s i g n i f i c a n t during weeks 2 to 4. There was an increase i n diarrhea from week 2 to week 3 for groups receiving BIgG or PIgG during weeks 1 and 2. Average Daily Gains and P i g l e t Weights Tables 29 and 30 show average d a i l y gains and p i g l e t weights respectively. The p i g l e t weights for days 7, 14, 21 and 28 and a l l average d a i l y gains for the Control p i g l e t s were non-estimable. None of the comparisons  between treatments  for average  daily  gain were  s i g n i f i c a n t during week 1. During week 2 the p i g l e t s receiving EDDA had lower average d a i l y gains than those not receiving EDDA. Also during week 2, p i g l e t s that received no immunoglobulins  had lower average d a i l y gains than those that  received either BIgG or PIgG. The BBO treatment p i g l e t s had lower average d a i l y gains during week 3 than the p i g l e t s that received PIgG on day 1. On days 21 and 28, mean p i g l e t weights for the BBO treatment were lower than for p i g l e t s that received PIgG on day 1. P i g l e t s that received EDDA i n the diet had lower mean weights than those not receiving EDDA on days 14, 21 and 28. Also on days 14, 21 and 28 p i g l e t s that received no immunoglobulins  on days 2-14 had  lower weights than p i g l e t s that received either BIgG or PIgG on days Plasma Iron and Total Iron Binding  2-14.  Capacity  Table 31 shows the plasma iron and TIBC of the p i g l e t s . The Controls  had  115 s i g n i f i c a n t l y lower plasma iron and TIBC on day 4 than for a l l other treatments. The BBO treatment p i g l e t s had s i g n i f i c a n t l y lower plasma iron levels on days 7  and  14  than  a l l other  treatments.  The  treatments receiving  EDDA had  s i g n i f i c a n t l y higher plasma iron l e v e l s on day 7. On days 14 and 21, plasma iron was higher i n p i g l e t s that received no immunoglobulins on days 2-14 than in p i g l e t s that received either BIgG or PIgG. The same was true for TIBC on day 1. Packed C e l l Volume and Hemoglobin The Controls had a higher packed c e l l volume on day 4 than any other treatment (see Table 32). The only other s i g n i f i c a n t effect on packed c e l l  volume  and hemoglobin was due to EDDA. P i g l e t s that were fed EDDA had s i g n i f i c a n t l y lower packed c e l l volumes on days 1, 7 and 14. They also had s i g n i f i c a n t l y lower hemoglobin l e v e l s on days 1 and 14 (see Table 32). Plasma Immunoglobulins Table 33 and Figure 16 show the plasma immunoglobulin l e v e l s . PIgG and BIgG were measured for the BBO  treatment p i g l e t s . PIgG only was measured for a l l  other treatments. There were no s i g n i f i c a n t differences i n PIgG levels for any of the treatments receiving PIgG during the f i r s t 24 hours of l i f e . None of the Control  piglets  had  measurable  levels  of PIgG  during  the course  of the  experiment. The t o t a l of PIgG and BIgG concentrations i n the plasma of the BBO treatment p i g l e t s was compared to the PIgG levels i n the other treatments. The BBO p i g l e t s had s i g n i f i c a n t l y lower t o t a l IgG than the other treatments on days 1,4,7 and 14. Plasma PIgG was measurable on day 7 and rose to 10.1 mg mL  -1  by  day 21. Discussion The s u r v i v a l of the Control group was 0%. Furthermore a l l p i g l e t s died  116 before 7 days of age. Common environmental pathogens such as  c o l i . Strepto-  coccus spp. and Staphylococcus spp. were present throughout the experiment. Other less common pathogens such as K l e b s i e l l a spp. and Actinobacillus suis became apparent i n l a t e r outcome groups. Most Controls died of septicemia caused by one or more of the above pathogens. The pattern here i s similar to that found in Experiment 1. Experiment 3 had no Control treatment so that no comparisons are  possible. The BBO treatment p i g l e t s had a survival rate of 60%. This i s s l i g h t l y lower  than for the bovine immunoglobulin fed group i n Experiment 1 even though the levels of BIgG were higher i n the present experiment. The lower survival rate of both the Control and BBO p i g l e t s may indicate a higher l e v e l of contagion than i n Experiment 1. The room was i n continuous use i n this experiment for over 4 months compared to 2 months for Experiment 1. In Experiment 3 an " a l l i n - a l l out"  regimen was practiced. The l e v e l of environmental pathogens may have been  lower but with no Control group comparisons are d i f f i c u l t . The BBO p i g l e t s died of d i f f e r e n t causes than the Controls. E_j_ c o l i Bronchopneumonia was the cause of death of two p i g l e t s which died on days 7 and 14. Streptococcus suis II septicemia was the cause of death of the other two BBO p i g l e t s . They died on days 16 and 20. The BBO p i g l e t s appeared able to fend off  E^ c o l i septicemia successfully but they were susceptible to subsequent  infections by other organisms. The p i g l e t s that received PIgG on day 1 had a very low mortality rate. Only 3 of 60 p i g l e t s died. Streptococcus suis II was responsible for the deaths of the  one PBO and one PBE p i g l e t that died during week 3. The one PPO piglet that  died on day 2 of L  c o l i omphalitis weighed only 800 g at b i r t h and had a day  1 plasma PIgG l e v e l of only 8.5 mg mL . Mortality rates i n Experiment 3 were -1  117  similar  to those i n the present experiment  f o r analogous treatments. Both  experiments had POO, PPO, POE and PPE treatments. The mortality rate for those four treatments was 1 out of 29 i n Experiment 3 and 1 out of 40 i n the present experiment. The Controls i n the present experiment had a week 1 diarrhea score of 3.4 compared to 3.1 for Controls i n Experiment 1. The BBO p i g l e t s had week 1 diarrhea scores of 1.5 compared to 1.0 f o r the analogous Bovine Ig treatment i n Experiment 1. This also points to a higher l e v e l of environmental pathogens i n the present experiment. The addition of EDDA to BIgG or PIgG d i d not produce a marked decrease i n the l e v e l of diarrhea during week 1. In the absence of immunoglobulins however, EDDA was able to decrease the l e v e l of diarrhea during the f i r s t week of l i f e . During week 1, the POO treatment p i g l e t s had a diarrhea score of 1.7 compared to 1.4 for the POE p i g l e t s . This effect was not observed i n Experiment 3. Porcine immunoglobulins  f o r two weeks after b i r t h produced lower levels of  diarrhea than either BIgG or No immunoglobulins.  This i s similar to the result  found i n Experiment 3 where after day 14, the diarrhea scores went up for the PBO, PBE, PPO and PPE treatments. The p i g l e t s on these treatments were weaned from dietary immunoglobulins on day 14. This may have caused the increase i n diarrhea. The increase i n was moderate however. Most p i g l e t s experienced a few days of a diarrhea score of 2 and then returned to normal. The average d a i l y weight gains f o r the BBO p i g l e t s were low u n t i l week 4. The difference was only s i g n i f i c a n t during week 3 but the effect of 3 weeks of low average d a i l y gains gave the BBO treatment p i g l e t s the lowest day 28 weight of any treatment group. This i s d i f f e r e n t from the results f o r Experiment 1 where the bovine immunoglobulin fed p i g l e t s had depressed average daily gains  118 only during week 1. The reason for t h i s may be an increased l e v e l of environmental pathogens i n this experiment. EDDA i n t h i s experiment resulted i n decreased average d a i l y gains, and p i g l e t weights. This i s i n direct contrast to the results of Experiment 3 where EDDA had a positive difference  effect  on p i g l e t  weights and average d a i l y  gains. The only  between Experiment 3 and 4 i s that p i g l e t s received EDDA on day 1  in Experiment 4. EDDA i s not well absorbed from oral doses by rats (Hershko et a l . 1984b). This may also be the case for piglets after gut closure. Before gut closure takes place, EDDA may be absorbed d i r e c t l y into the blood stream along with  immunoglobulins.  increases i t s toxic  Extra  absorption  of EDDA on the f i r s t  day of l i f e  e f f e c t s . EDDA should not be fed u n t i l after gut closure  takes place. In Experiment 1 and the present experiment i t was noted that on day 1, PIgG i s not replaceable by BIgG. The treatments receiving  no immunoglobulins (POO  and POE) during days 2-14 had i n f e r i o r average d a i l y gains. The p i g l e t weights for these two treatments were low on days 14,21 and 28. There i s a s i g n i f i c a n t benefit to continued feeding of immunoglobulins after day 1. The day 28 weights for the PBO and PPO treatments were not s i g n i f i c a n t l y d i f f e r e n t . Both of these treatments received PIgG on day 1 but the PBO p i g l e t s got BIgG on days 2-14 while the PPO p i g l e t s received PIgG.  After day 1, BIgG and PIgG are equally  e f f e c t i v e i n promoting piglet growth. This may be due to an overlap i n the a n t i gens expressed by bovine and porcine enteric pathogens. This would lead to an overlap i n the immunoglobulin s p e c i f i c i t i e s found i n bovine and porcine sera and give the results seen here. Depending on the cost of producing PIgG and BIgG, either may be used to f o r t i f y p i g l e t diets after day 1. The Controls had a plasma iron concentration about 100 pg dLr lower than 1  119 the other treatments on day 4. B a c t e r i a l i n f e c t i o n causes a decrease i n plasma iron  (Cartwright et a l . 1946). Since the Controls experienced more b a c t e r i a l  infections  than any other group, i t i s not surprising  that their plasma iron  levels were lower. The piglets received 100 mg of iron dextran on day 4. Six of the 9 Controls died on days 4 or 5. Iron increases the a b i l i t y of bacteria tissues  to grow i n host  (Weinberg 1984). The iron dextran may have hastened the death of the  Controls by increasing  the a v a i l a b i l i t y of iron to bacteria.  Knight et a l .  (1984) found that 100 mg of iron dextran did not compromise the p i g l e t ' s a b i l i t y to l i m i t the a v a i l a b i l i t y of iron to bacteria. The experiment involved healthy 2 week o l d p i g l e t s , however. The Control piglets i n t h i s experiment may not have been able to synthesize enough l a c t o f e r r i n and t r a n s f e r r i n to chelate the injected  iron and sequester i t i n the l i v e r . Evidence that this was the case  can be seen i n the day 4 TIBC of the Controls. It was s i g n i f i c a n t l y lower than for any other group suggesting an i n a b i l i t y to rapidly synthesize iron-binding proteins. The plasma iron of the BBO treatment piglets was low on days 7 and 14. This treatment also suffered  from a high l e v e l of b a c t e r i a l i n f e c t i o n s . Presumably  t h i s was the cause of the low plasma iron concentrations i n these p i g l e t s . The  p i g l e t s receiving  similar to the result  EDDA had high plasma iron levels on day 7. This i s  i n Experiment 3 where EDDA and HBED had higher plasma  iron levels on day 14. This may be caused by EDDA bound iron present i n the plasma. The  piglets receiving  No Ig had higher plasma iron levels than those r e -  ceiving BIgG or PIgG on days 14 and 21. The same r e s u l t occurred i n Experiment 3 on day 14. It i s d i f f i c u l t to envision the mechanism that causes this e f f e c t .  120  Packed c e l l volumes and hematocrits for the Controls were high on day 4. This i s possibly due to dehydration. Most Controls had severe diarrhea on day 4. This may have caused dehydration severe enough to elevate these blood measures. Piglets receiving EDDA had depressed packed c e l l volumes  on days 1,7 and  14. They also had depressed hemoglobin values on days 1 and 14. This effect was present i n Experiment 3 to a lesser extent. Packed c e l l volumes were low only on day 14 i n that experiment and hemoglobin was not affected. The larger effect in the present experiment may be due to the increased absorbtion of EDDA on day 1. The effect on packed c e l l volume and hematocrit disappeared when EDDA was removed from the d i e t . In  Experiment  4 the l e v e l  increased to 25 mg mL  -1  of PIgG and BIgG included  on day 1 and 5 mg mL  -1  i n the diets was  on days 2-14. This resulted i n  an increase i n plasma PIgG concentrations compared to those i n the previous experiments. Day 1 plasma PIgG values ranged from 19.6 to 25.5 mg mL  -1  in  Experiments 1 and 3. In the present experiment, values ranged from 24.6 to 29.5 mg mL . This increase i n plasma PIgG i s nearly proportional to the increase -1  in dietary PIgG. The day 4 plasma PIgG levels were lower than the day 1 levels for  a l l treatments. This suggests that gut closure occurred within the f i r s t  24 hours after b i r t h . Experiment 1 concluded that the best l e v e l of PIgG on day 1 would provide adequate immune protection and minimize the i n h i b i t i o n of active immunity. The c r i t e r i a for "adequate" immunity are s u r v i v a l , diarrhea and weight gain. Comparing analogous treatments from Experiments 1,3 and 4 ( i . e .  PIgG on day 1,  PIgG on days 2-14) i t can be seen that survival did not change when the l e v e l of PIgG i n the diet was increased. Diarrhea, however, may have decreased s l i g h t ly i n the present experiment. Diarrhea i n the f i r s t two experiments ranged from  121  1.1 to 1.2 during the f i r s t for  the f i r s t  two weeks. In the present experiment i t was 1.0  two weeks. The l e v e l of environmental pathogens may have been  higher i n the present experiment than i n Experiments 1 and 3. In Experiment 1, the  room was i n continuous use for a shorter period of time than i n the present  experiment. In Experiment  3, an " a l l  in-all  out" regimen was used. In the  present experiment, the room was i n continuous use f o r a longer period and an " a l l i n - a l l out" regimen was not used. The Controls had 100% mortality. In spite of  this,  the weight gains of the p i g l e t s i n this experiment were s l i g h t l y  higher than i n Experiments 1 and 3. Day 28 p i g l e t weights for Experiments 1 and 3 averaged 5,980 and 6,060 g respectively. The average day 28 weights i n the present experiment were 6,350 g. The birthweights of pigs i n the 3 experiments were v i r t u a l l y the same. The increased l e v e l of dietary PIgG resulted i n an increase of about 300 g liveweight over 28 days. Based on a l l these c r i t e r i a , the  l e v e l of PIgG fed i n the current experiment i s better than the lower l e v e l  used i n the previous experiments. The plasma PIgG remained higher than i n the previous experiment throughout the  28 day period of the experiment. By day 28 the difference was n e g l i g i b l e .  The previous experiments ranged-from  7.2 to 9.5 mg mL  -1  on day 28. I n t h e  present experiment values ranged from 8.7 to 10.1 mg mL . -1  The BBO treatment p i g l e t s also received higher levels of BIgG on day 1 than the  analogous treatment i n Experiment 1. In Experiment 1 the day 1 plasma BIgG  was 4.0 mg mL . -1  In the present experiment the day 1 value was 5.7 mg  mL . -1  Increasing the l e v e l of BIgG i n the diet resulted i n a nearly proportional increase i n plasma BIgG. The plasma BIgG was s t i l l poorly absorbed compared to PIgG. I t also provided inadequate immune protection. The active synthesis of PIgG by the BBO treatment p i g l e t s was measurable by  122 day 7. This i s similar to the result obtained i n Experiment 1. The day 21 and 28 PIgG values for the BBO treatment p i g l e t s were s l i g h t l y lower than the levels found i n Experiment 1 but not markedly so. In conclusion, EDDA should not be fed u n t i l after gut closure takes place. EDDA had no deleterious effect on p i g l e t growth when fed on days 2-14 but when fed on days 1-14 s i g n i f i c a n t reductions i n p i g l e t growth rates occurred. There i s some question whether EDDA should be fed to colostrum deprived piglets at a l l . Immunoglobulins alone supported p i g l e t growth rates that were as good as EDDA alone  or EDDA with porcine immunoglobulins. This coupled  with EDDA's  potential t o x i c i t y may make i t unsuitable as an additive to sow milk replacers. Bovine immunoglobulins are not well absorbed from the diet during the f i r s t day of l i f e and porcine immunoglobulins must be fed to ensure adequate passive systemic immunity. After day 1, however, either bovine or porcine immunoglobul i n s may be used to provide l o c a l immune protection i n the i n t e s t i n a l t r a c t .  123 Table 27. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the survival of colostrum deprived piglets (Experiment 4). Number Surviving at day Birth  7  14  21  N  N  N  N  N  %  Control BBO  9 10  0 9  0 8  0 6  0a 6b  0 60  9.8 9.2  POO POE  10 10  10 10  10 10  10 10  10 10  100 100  9.3 9.4  PBO PBE  10 10  9 10  9 9  9 9  9 9  90 93  9.3 9.4  PPO PPE  10 10  9 10  9 10  9 10  9 10  88 100  9.8 9.2  Treatment  281 SE  N = the number of surviving piglets a The contrast "Controls vs a l l other treatments" is significant (P < 0.05) b The contrast "BBO vs POO, POE, PBO, PBE, PPO and PPE" is significant (P < 0.05)  124 Table 2 8 . The effect of EDDA, with and without bovine or porcine iinmunoglobulins, on the average weekly diarrhea scores of colostrum deprived piglets (Experiment 4).  week Treatment  3  4  Non-est 1.2  Non-est 1.4  Non-est 1.2  1.7d 1.4d  1.4d 1.4d  1.3 1.4  1.1 1.1  PBO PBE  1.3e 1.3ef  1.1 1.1  1.3 1.3  1.1 1.1  PPO PPE  1.0 1.1  1.0 1.1  1.2 1.3  1.1 1.1  1  2  Control BBO  3.4a 1.5  POO POE  1  1  Non-est = non-estimable  a The contrast "Controls vs a l l other treatments" is significant (P < 0.05) d The contrast "POO and POE vs PBO, PBE, PPO and PPE" is significant (P < 0.05) e The contrast 'TB0 and PBE vs PPO and PPE" is significant (P < 0.05) f The contrast "PBO vs PPO" is significant (P < 0.05)  125  Table 29. The effect of EDDA, with and without bovine or porcine iimnunoglobulins, on the average  daily gains of colostrum deprived piglets (Experiment 4).  Average Daily Gains (q day!) week 1 Treatment Control BBO  X  week 2  SE  Non-est 90 9 1  X  SE  week 3 X  SE  Non-est 100 13  Non-est 120b 19  week 4 X  SE  Non-est 200 16  POO POE  80 100  7 7  llOd 90cd  11 11  200 200  15 15  190 210  13 12  PBO PBE  100 80  8 8  160 110c  11 11  240 220  16 16  230 210  13 13  PPO PPE  110 80  8 8  180 120c  12 11  17 240 190 —•• 16  210 220  14 13  1  Non-est = non-estimable  b The contrast "BBO vs POO, POE, PBO, PBE, PPO and PPE" is significant (P < 0.05) c The contrast "POO, PBO and PPO vs POE, PBE and PPE" is significant (P < 0.05) d The contrast "POO and POE vs PBO, PBE, PPO and PPE" is significant (P < 0.05)  126 Table 30. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the body weights of colostrum deprived piglets (Experiment 4). Body Weights (g) Birth Treatment  dav 7 SE  X  SE  day 28  dav 21  X  SE  Control BBO  1,160 1,180  71 67  Non-est 1,810 65 1  Non-est 2,520 130  Non-est 3,370b 202  Non-est 4,750b 267  POO POE  1,060 1,210  67 67  1,730 1,850  52 51  2,500d 104 2,480cd 102  3,910d 161 3,900cd 157  5,260d 213 5,390cd 207  PBO PBE  1,090 1,270  67 68  1,910 1,750  54 54  2,990 108 2,520c 109  4,640 167 4,040c 168  6,240 221 5,510c 222  PPO PPE  1,260 1,160  71 67  1,970 1,770  58 53  3,230 116 2.610c 106  4,890 180 3,950c 165  6,350 237 5,470c 218  1  X  dav 14 X  X  SE  Non-est = non-estimable  b The contrast "BBO vs POO, POE, PBO, PBE, PPO and PPE" is significant (P < 0.05) c The contrast "POO, PBO and PPO vs POE, PBE and PPE" is significant (P < 0.05) d The contrast "POO and POE vs PBO, PBE, PPO and PPE" is significant (P < 0.05)  SE  Table 31. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the plasma iron and total iron binding capacity (TIBC) of colostrum deprived piglets (Experiment 4). Day 7 Treatment  XSE  XSE  XSE  XSE  14  21  XSE  XSE  28 X  SE  Plasma Iron (ug dlr ) 1  Control BBO  na na  na na  298a 27 389 15  Non-est 150b 20  Non-est 134b 16  Non-est 108 13  Non-est 90 5  POO POE  na na  na na  401 15 413 15  215 15 254c 15  194d 12 190d 12  112d 10 109d 10  94 86  4 4  PBO PBE  na na  na na  402 15 434 15  182 16 223c 16  154 12 160 13  109 11 98 11  84 84  4 5  PPO PPE  na na  na na  401 16 421 15  197 17 212c 15  161 14 170 12  107 11 110 10  90 92  5 4  Control BBO  246 14 233 13  289 18 294 17  381a 30 502 19  Non-est 461 21  Non-est 487 20  Non-est 544 18  Non-est 579 12  POO POE  267 13 259 13  318d 17 323d 17  493 19 494 19  461 16 495 16  520 15 502 15  545 14 549 14  577 10 566 10  PBO PBE  246 13 253 13  263 17 275 17  494 19 526 19  469 17 450 17  496 16 491 16  567 15 550 15  572 10 585 10  PPO PPE  242 13 250 13  296 17 299 17  508 20 503 19  485 18 455 17  508 17 502 15  577 15 537 14  586 11 574 10  1  TIBC (ug dlr ) 1  1  Non-est = non-estimable  a The contrast "Controls vs a l l other treatments" is significant (P < 0.05) b The contrast "BBO vs POO, POE, PBO, PBE, PPO and PPE" is significant (P < 0.05) c The contrast "POO, PBO and PPO vs POE, PBE and PPE" is significant (P < 0.05) d The contrast "POO and POE vs PBO, PBE, PPO and PPE" is significant (P < 0.05)  128 Table 32. The effect of EDDA, with and without bovine or porcine iimiunoglcibulins, on the packed cell volumes and hemoglobin concentrations of colostrum deprived piglets (Experiment 4).  Day 0  Treatment  X  4  1 SE  X  SE  X  7  SE  X  14  SE  X  28  21  SE  X  SE  SE  X  Packed Cell Volume (%) Control  32.7 1.7  30.2  1.6  33.5a 2.3  Non-est  Non-est  Non-est  Non-est  BBO  31.4 1.6  26.0  1.5  23.7 1.4  23.3  31.4  30.3  1.3  28.9 1.3  POO POE  34.3 1.6 31.9 1.6  28.9 1.5 27.4c 1.5  26.6 1.4 25.5 1.4  24.7 1.2 23.8c 1.2  31.4 1.1 26.1c 1.1  30.6 1.0 30.8 1.0  29.2 1.0 29.6 1.0  PBO PBE  32.6 1.6 32.9 1.6  30.6 1.5 27.9c 1.5  27.6 1.4 25.9 1.4  26.5 1.3 23.6c 1.3  31.1 1.2 28.2c 1.2  31.4 1.1 31.4 1.1  27.8 1.1 29.2 1.1  PPO PPE  33.6 1.6 31.7 1.6  30.4 1.5 26.0c 1.5  27.4 1.5 25.2 1.4  28.4 1.4 24.3c 1.2  33.3 1.2 27.3c 1.1  32.4 1.1 30.1 1.0  29.4  Non-est  Non-est  Non-est  Non-est  1  1.5  1.4  1.1 28.2 1.1  HQBGLOBIN (g dLr ) 1  Control  13.1 0.9  11.7  0.7  13.3  2.1  BBO  12.0 0.8  10.3  0.6  9.6  1.3  9.3  0.6  12.6  0.7  11.4 0.6  11.0 1.6  POO POE  13.5 0.8 12.7 0.8  11.7 0.6 10.7c 0.6  10.2 1.3 10.2 1.3  9.1 9.1  0.5 0.5  10.9 0.6 10.1c 0.6  11.9 0.4 11.5 0.4  10.6 1.3  PBO PBE  13.4 0.8 13.2 0.8  12.2 0.6 11.0c 0.6  10.6 1.3 10.7 1.3  9.7 8.8  0.5 0.5  12.3 0.6 10.6c 0.6  11.9 0.5 11.6 0.5  11.1 1.4 11.2 1.4  PPO PPE  13.9 0.8 11.8 0.8  11.9 0.6 10.4c 0.6  10.0 1.4 9.9 1.3  10.4 9.0  0.5 0.5  12.7 0.6 10.8c 0.6  12.2 0.5 11.7 0.5  11.6 1.4 10.5 1.4  1  Non-est = non-estimable  » The contrast "Controls vs a l l other treatments" is significant (P < 0.05) c The contrast "POO, PBO and PPO vs POE, PBE and PPE" is significant (P < 0.05)  10.4  1.4  129  Table 33. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the plasma iinmunoglobulin concentrations of colostrum deprived piglets (Experiment 4).  day 0  1  4  7  X  SE  X  SE  X  SE  Control  <1.3  -  <1.3a  -  <1.3a  -  BBO BIgG PIgG  <0.5 <1.3  -  5.7 <1.3  BBO Total IgG  <1.3  -  POO POE  <1.3 <1.3  -  26.0 24.6  2.8 2.9  22.8 22.5  2.4 2.5  19.4 17.9  1.9 2.0  13.5 12.7  PBO PBE  <1.3 <1.3  -  29.5 27.2  2.9 2.9  27.6 25.0  2.5 2.5  23.8 18.0  2.0 2.1  PPO' PPE  <1.3 <1.3  -  28.2 27.1  3.1 2.9  24.4 24.2  2.6 2.5  18.7 19.4  2.2 2.0  Treatment  1  0.1  -  5.7b 4.4  5.2 <1.3  0.1  -  5.2b 3.7  X  SE  X  SE  Non-est  Non-est  3.5 3.7  1.9 5.2  1  0.1 2.4  7.2b 3.1  2!3  21  14  SE  X  Non-est  X  SE  Non-est  -  0.1 1.8  0.7 10.1  0.1 1.3  <0.5 10.6  1.0  7.1b 2.3  10.8  1.5  10.6  1.2  1.4 1.5  10.8 9.9  1.0 1.0  8.7  8.7  0.8  15.3 12.4  1.5 1.5  12.2 10.5  1.0 1.0  10.1 9.7  0.8 0.8  14.2 15.1  1.6 1.5  10.0 11.1  1.1 1.0  9.5 9.3  0.8 0.8  Non-est = non-estimable  a The contrast "Controls vs a l l other treatments" is significant (P < 0.05) b The contrast "BBO vs POO, POE, PBO, PBE, PPO and PPE" is significant (P < 0.05)  0.7  130  Figure 16. The effect of EDDA, with and without bovine or porcine immunoglobulins, on the plasma immunoglobulin concentrations of colostrum deprived piglets analysis see Table 33.  Day  (Experiment  4). For  statistical  131  Experiment 5 Introduction EDDA has been shown to have s i g n i f i c a n t effects excretion  i n rats.  In experiments  hematocrit, hemoglobin, plasma iron  on iron metabolism and  3 and 4 iron  status  and t o t a l  binding  iron  was measured by capacity. These  measures do not give a detailed picture of iron metabolism. They also do not give any information on the effect that EDDA has on the excretion of iron i n the feces  and urine. The use of radioactive isotopes  metabolism  of iron  i s a well  established  technique  of iron  to study the  (Cavill,  1986).  This  objective of this experiment was to examine the effect that oral administration of EDDA had on iron metabolism and excretion i n p i g l e t s using [ F e ] . 5 9  Materials and Methods Experimental Design • Piglets  were randomly assigned  received 1.5 L d a y  -1  to one of two treatments. A l l p i g l e t s  of the same milk replacer used i n Experiments 1,3 and 4.  The Control group received milk replacer only. The EDDA group received 37.5 mg EDDA Kg body weight analyzed  -1  day  1  mixed with the milk  replacer. The results were  using the General Linear Models procedure of SAS.  Animal Management Eight p i g l e t s that had completed Experiment 4 were used. They were randomly assigned  to the two treatments. The p i g l e t s were fed their assigned  diets for  3 days before the i n j e c t i o n of [ F e ] . Piglets were weighed d a i l y and their EDDA S 9  intake  was adjusted  accordingly.  The piglets  were 28 days of age at the  beginning of the experiment and weighed between 5,550 and 7,250 g. The piglets were housed i n d i v i d u a l l y  i n stainless  steel metabolism cages measuring 1 m  square. The cages allowed separate c o l l e c t i o n of urine and feces.  132  Preparation of Radioiron Labelled Plasma The plasma was l a b e l l e d with [ F e ] C l 3 (Amersham Canada Ltd. Oakville Ont.) 3 9  using the procedure described by Cook and Finch (1980). Ten mL of blood were collected from a pig i n the same l i t t e r , but not i n the same experiment. blood was  The  centrifuged and the plasma removed into a s t e r i l e tube. A solution  of [ F e ] C l S 9  (3-20 mCi mg"  1  3  Fe) was prepared i n .01 N HC1 with s u f f i c i e n t  3.8%  sodium c i t r a t e to give a molar r a t i o i n excess of 20:1. The solution was added dropwise to the plasma with constant s t i r r i n g to give a f i n a l concentration of about  6 uCi mL  of plasma.  -1  The plasma was  held at room temperature  for at  least 30 minutes before being injected into the p i g l e t s . Injection of Radioiron and Sampling  regimen  The anterior vena cava of each p i g l e t was  catheterized and the catheter  was used for i n j e c t i o n of [ F e ] and for taking a l l blood samples. Before 59  i n j e c t i o n , blood samples were taken for hematocrit ation.  Standards  injecting  the  were prepared  [ F e ] . The 5 9  from  the  labelled  p i g l e t s were injected  and serum iron determinserum immediately  with about  l a b e l l e d plasma measured accurately to the nearest mg.  1000  mg  before of the  The catheter was  then  flushed with s t e r i l e saline to ensure that a l l of the l a b e l l e d plasma entered the bloodstream. Blood samples were collected at 15,30,45,60 and 120  minutes  after the i n j e c t i o n to measure plasma iron disappearance and plasma iron turnover i n the p i g l e t s . Samples were also taken at 24,72,120 and 144 hours to measure the incorporation of [ F e ] into the red blood c e l l s . Urine and feces 39  were collected twice d a i l y . After 6 days the p i g l e t s were s a c r i f i c e d . Blood samples were taken and the l i v e r s and spleens were removed, weighed and samples taken for counting. Samples were counted using a Packard Auto-Gamma counter (Model 500c).  133 Calculations A l l calculations were made using formulas given by Cook and Finch (1980) . The injected [ F e ] was calculated using the following formula: S9  Standard [ F e ] (CPM/mL) x weight of dose x 100 S9  Injected [ F e ] (CPM) = 39  Weight of standard  The plasma r a d i o a c t i v i t y at time 0 was calculated by taking the regression of the  logarithm of plasma r a d i o a c t i v i t y (CPM) against time. The y intercept was  used as the CPM at time 0. The Plasma Iron Disappearance Rate (T%) was calculated as the time required to. reduce the Time 0 plasma r a d i o a c t i v i t y by h a l f . Plasma volume was calculated using the following formula:  Injected [ F e ] (CPM) 39  Plasma volume .(ml)  Time 0 plasma  [ 3 F e ] (CPM ml" ) 9  1  Plasma iron turnover (PIT) rate was calculated using the following formula:  Plasma iron (ug 100 mL ) x (100 - Hematocrit) -1  PIT (mgLblood-i d a y ) = 1  fJ4 x 100  Red c e l l u t i l i z a t i o n of [ F e ] was calculated as: 39  Red c e l l Red c e l l u t i l i z a t i o n (%) =  [ F e ] (CPM mL" ) x red c e l l mass x 100 39  1  Injected L FeJ aa  activity  where  Plasma volume (mL) x 0.9 x Hematocrit Red c e l l mass (mL) =  100 - (0.9 x Hematocrit)  134 The d i s t r i b u t i o n of [ F e ] i n urine, feces, l i v e r and spleen was calculated as: s9  Sample [ F e ] (CPM) x sample weight x 100 =—,—,—_— - . _ — „ _ Injected [ F e ] a c t i v i t y 39  Sample d i s t r i b u t i o n (%) =  r  aw  59  Results The only s i g n i f i c a n t treatment differences occurred for VA and PIT (see Table 34). The Controls had a s i g n i f i c a n t l y lower T% than the EDDA group (75.5 vs 186.3 minutes). The Controls also had a s i g n i f i c a n t l y greater PIT than the EDDA group (0.90 vs 0.34 mg K g  -1  d a y ) . Figure 17 shows the plasma iron disap- 1  pearance curve. There was an i n i t i a l rapid incorporation of [ F e ] into the red c e l l s (See 39  Figure 18) . After 24 hours a s i g n i f i c a n t l y higher percentage of the o r i g i n a l dose of [ F e ] was incorporated into the red blood c e l l s of the Controls. This 39  difference remained s i g n i f i c a n t throughout the experiment. [ F e ] incorporation 59  reached a plateau on day 5 for the EDDA group but not for the Controls. The f e c a l and urinary excretion of [ F e ] was s i g n i f i c a n t l y higher for the 39  EDDA treatment  (See Figure 19). Excretion of [ F e ] i n the feces by the EDDA 39  group occurred at a f a i r l y constant rate throughout the 6 days o f the experiment. Urinary excretion occurred mainly i n the f i r s t 24 hours after i n j e c t i o n and then leveled o f f . Table 35 shows the excretion and d i s t r i b u t i o n of [ F e ] at the end o f the 39  experiment. The Controls excreted only 0.6% of the injected [ F e ] i n the feces 39  and 0.7% i n the urine while the EDDA group excreted 4.0% i n the feces and 6.3% in the urine. Both differences were s i g n i f i c a n t (p > 0.05). The [ F e ] incorpor59  ation into red blood c e l l s was s i g n i f i c a n t l y lowered by EDDA. The amount o f [ F e ] i n the spleen and l i v e r was not affected by treatment. The residual 39  135  [ F e ] l e f t unaccounted f o r was not 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 59  treatments. The feces, urine, red blood c e l l s , l i v e r and spleen accounted for a l l but 1.9% of the injected [ F e ] i n the Controls. The residual was 6.7% for 59  the  EDDA group.  Discussion Each p i g l e t consumed a l l the milk replacer i t was given. The average d a i l y gains of p i g l e t s i n the two treatments were not s i g n i f i c a n t l y different but there were large variations i n p i g l e t gains for the EDDA treatment. One p i g l e t on this treatment gained only 33 g d a y  -1  while another gained 183 g d a y . In -1  Experiment 4, EDDA caused an o v e r a l l decrease i n average d a i l y gains but here also the r e s u l t s were extremely variable. During the 14 day period i n which EDDA was fed i n Experiment 4, the average d a i l y gains ranged from 42 to 130 g d a y . In Experiment 3, during the same period gains ranged from 71 to 200 g -1  d a y . Only 2 p i g l e t s that received EDDA i n Experiment 3 showed marked decreases -1  in average d a i l y gains. The method of feeding EDDA was different i n the present experiment than in Experiments 3 and 4. The dose was 37.5 mg K g  -1  i n a l l 3 experiments but i n  Experiments 3 and 4 the concentration of EDDA i n the milk replacer was a constant 0.1 mg m l  - 1  . In the present experiment the amount of milk replacer fed  was a constant 1500 mL d a y  -1  and the concentration of EDDA varied with the  weight of the p i g l e t . This may have increased the amount of EDDA absorbed from the  diet i n the present experiment. EDDA had extremely variable e f f e c t s on piglet performance. This could be  due to d i f f e r i n g a b i l i t i e s to absorb of EDDA from the diet or differences i n the a b i l i t y of the p i g l e t s to detoxify the compound. EDDA i s also toxic to sheep ( S t i f e l and Vetter 1967). A dose of 50 mg mL  -1  caused weight loss and death i n  136  lambs. Plasma and red c e l l volumes were similar to those reported i n other studies. Talbot and Swenson (1970) found 4 week old p i g l e t s averaged about 75 mL Kg  -1  plasma volume and 25 mL K g  -1  red c e l l volume. Jensen et a l (1956) found  growing pigs had plasma volumes ranging from 33.9-60.3 mL K g  -1  and red c e l l  volumes from 20-44.9 mL K g . - 1  Hematocrits were s l i g h t l y lower than those expected for naturally reared p i g l e t s receiving  100 mg iron dextran i n j e c t i o n s . Talbot and Swenson (1970)  found that 4 week old p i g l e t s that received 100 mg of iron dextran had hematoc r i t s averaging 39.1. The values for p i g l e t s receiving no iron dextran averaged 20.8. The p i g l e t s used i n this t r i a l received an iron deficient milk replacer as their sole  source of i r o n . This probably caused the lowered  hematocrits.  Plasma iron was similar to those found i n other studies (Furugouri 1971;  Jensen  et a l . 1956). Furugouri (1974) reported T% values ranging from 23 minutes for newborns to 30 minutes for 10 day old p i g l e t s . Studies with growing pigs found values ranging from 43-100 minutes (Jensen et a l . 1956; Furugouri et a l . 1974; H r i s t i c et a l . 1970). The mean T& of 75.5 minutes for the Controls agrees well with these other studies. The EDDA p i g l e t s had a mean T% of 186.3 minutes. EDDA also affected plasma iron turnover (PIT). The Controls had a mean PIT of 0.90 mg Kg1  d a y . Other studies found values ranging from 0.4-2.0 mg K g -1  -1  day  1  (Jensen  et a l . 1956; Furugouri et a l . 1974; H r i s t i c et a l . 1970). The EDDA treatment had a mean PIT of 0.34 mg K g  -1  day . 1  EDDA has an apparently profound effect on the flow of iron i n the plasma. This apparent effect may not be r e a l . One possible interpretation of the results i s that EDDA bound iron i s a pool separate from t r a n s f e r r i n bound i r o n . When  137 the i n j e c t i o n of [ F e ] labelled t r a n s f e r r i n enters the blood stream the [ F e ] 39  39  i s free to exchange with the EDDA pool. This means that bloodstream longer  [ F e ] remains i n the 39  than i t would with no EDDA present.  Analysis  of plasma  samples counts both the t r a n s f e r r i n and EDDA pools of [ F e ] . This may lead to 3 9  an apparent decrease i n PIT where none e x i s t s . To test t h i s hypothesis transf e r r i n and EDDA bound [ F e ] would have to be analyzed 39  separately.  During the f i r s t 24 hours after the i n j e c t i o n of [ F e ] , the Controls had 3 9  a greater rate of [ F e ] incorporation into the red blood c e l l s than piglets 39  receiving EDDA. After 24 hours, however, the red blood c e l l [ F e ] incorporation 39  curves were nearly p a r a l l e l . The difference i n [ F e ] incorporation between the 39  Controls  and the EDDA group i s almost e n t i r e l y  explained  by the increased  excretion of iron i n the feces and urine. Other studies reported red blood  cell  incorporation rates of from 72-100% (Jensen et a l . 1956; Braude et a l . 1962). The Controls f e l l within this range. EDDA caused an increase i n the excretion of iron i n the feces and urine. The only other studies of the effect of EDDA on iron excretion involved rats. An intramuscular  i n j e c t i o n of EDDA was used rather than the oral administration  used i n the present experiment. (Hershko et a l . 1984a; 1984b). EDDA caused s i g n i f i c a n t excretion of iron i n both urine and feces i n these studies. Fecal excretion was f a r greater than urinary excretion however. In the present the reverse i s true. Excretion i n the feces depends on the [ F e ] g 39  and  _ 1  study  of feces  the t o t a l amount of feces produced. The feces showed a high a c t i v i t y per  gram but the t o t a l quantity of feces produced was small. The piglets, were fed only milk replacer. Since  the d i g e s t i b i l i t y of the milk replacer was nearly  100% this made for a small amount of fecal material and a lower excretion of [ Fe]. 3 9  138 In conclusion, EDDA has s i g n i f i c a n t effects on iron metabolism. The most important i s the increase i n urinary and fecal excretion of i r o n . The increase in excretion leads to a decrease i n the synthesis of red blood c e l l s and hemoglobin. This could be overcome by increasing the amount of supplemental iron dextran given to p i g l e t s . Of greater significance are EDDA's toxic e f f e c t s . A s i g n i f i c a n t number of p i g l e t s had reduced weight gains when EDDA was fed at 37.5  mg Kg body w e i g h t . These toxic properties make i t unsuitable -1  additive to p i g l e t milk replacers.  as an  139 Table 34. The effect of EDDA on individual piglets on the day of P'Fe] injection (Experiment 5).  Piglet  Average Weight Gain (g/dav) (g)  Plasma Volume (mL)  Red Cell Volume Hematocrit (mL) (%)  Plasma Iron (ua/dL)  T< (min)  PIT (mg/kg/dav)  Control Treatment 105 205 206 306  6,400 7,250 5,550 5,700  158 116 183 233  53.8 67.4 81.1 62.3  19.8 21.6 31.4 22.8  30.0 27.0 31.0 29.5  X SE  6,225 351  172 31  66.2 6.6  23.9 2.0  29.5 1.5  80 90 110 90 93 4.8  63.7 77.6 92.0 68.6  0.87 0.85 0.83 1.03  75.5a 11.0  0.90a 0.04  EDDA Treatment 102 201 203 303  6,400 6,650 6,750 5,400  75 166 183 33  57.5 58.3 48.7 82.8  23.8 28.6 24.3 27.4  32.5 36.5 37.0 29.0  X SE  6,300 351  114 31  61.8 6.6  26.0 2.0  33.8 1.3  90 100 95 90 94 4.8  a The treatments means are significantly different (p > 0.05).  198.2 215.4 183.3 148.2  0.31 0.29 0.33 0.43  186.3 11.0  0.34 0.04  140 Table 35. The effect of EDDA on excretion and distribution of P'Fe], (Experiment 5). 1  Treatment  Feces  Urine  X  X  SE  SE  Control  0.6a 0.8  0.7a 0.2  EDDA  4.0 0.8  6.3 0.2  1  Red Cells X  SE  86.5a 1.8 68.1 1.8  Liver X  SE  8.0 2.0 13.4 2.0  Spleen X  SE  2.3 0.3 1.5 0.3  Residual I  SE  1.9 3.2 6.7 3.2  Values are in percent of injected ["Fe] activity. The values for feces and urine  are from total collection during the entire experiment. The values for liver and spleen represent the total activity of the organ and includes the blood present within the organ. a The treatments means are significantly different (p > 0.05).  141  FIGURE 17. The e f f e c t of EDDA on plasma i r o n d i s a p p e a r a n c e  M'nies  (Experiment 5 ) .  142  FIGURE 18. The effect of EDDA on the incorporation of [ F e ] into red blood 39  cells  (Experiment 5).  Days  143 FIGURE 19. The effect of EDDA on urinary and fecal excretion of [ F e ] 59  (Experiment 5).  Feces  1  2  3  5  +  8  Day  Urine  1  2  3  4  5  8  144 EXPERIMENT 6 Introduction In  a l l previous experiments i n this series, the concentration of plasma  immunoglobulins was used as the only measure of p i g l e t immunity. In this experiment i t was decided to examine the effect a r t i f i c i a l rearing on cell-mediated  immunity.  Colostrum and milk contain viable leukocytes i n addition to immunoglobu l i n s . In mice and rats lymphocytes are absorbed from the milk into the bloodstream of the suckling animals (Parmely and Beer 1977; Head and Beer 1979). These  cells  can transfer  cell  mediated  immunity  from resistant  mothers  to  suckling mice. C o l o s t r a l lymphocytes may also be absorbed into the blood stream of the suckling p i g l e t and contribute to i t s immune status. Colostrum deprived p i g l e t s do not receive viable lymphocytes and may have decreased c e l l mediated immunity compared to sow reared p i g l e t s . Ideally viable lymphocytes  from sow  colostrum should be fed to colostrum deprived  piglets  during the f i r s t day of l i f e . The population of lymphocytes found i n colostrum i s d i f f e r e n t than the one found i n the peripheral blood (Parmely and Beer 1977) . A high proportion of c o l o s t r a l lymphocytes respond to enteric organisms and food antigens. Sow colostrum however, i s d i f f i c u l t  to obtain. For this reason  i t was decided to investigate whether feeding peripheral blood lymphocytes to colostrum deprived p i g l e t s could increase c e l l mediated immunity. The colostrum deprived p i g l e t s were also compared to l i t t e r mates that remained with the sow. Materials and Methods Experimental Design The treatments used are shown i n Table 36.  145 Table 36.  Experimental protocol used to study the effect of sow or a r t i f i c i a l  rearing on colostrum deprived p i g l e t s  Treatment  (Experiment 6).  dav 2-14  dav 1  dav 15-28  Sow reared  Sow's Colostrum  Sow's Milk  Artificial  25 mg mL  5 mg mL  -1  PIgG  0 mg mL  -1  PIgG  5 mg mL  -1  PIgG  0 mg mL  -1  PIgG  -1  PIgG  Sow's Milk  No leukocytes Leukocyte  25 mg mL 5 x 10  8  -1  PIgG  leukocytes  The p i g l e t s i n this experiment were reared i n four outcome groups. Outcome groups 1, 2 and 3 consisted of 6 p i g l e t s with 2 p i g l e t s assigned to the each treatment. Outcome group 4 consisted of 12 p i g l e t s with 4 p i g l e t s assigned to each treatment. Piglets that were not used i n the experiment  were l e f t  with  the sow. There was a minimum of 6 and a maximum of 8 p i g l e t s remaining with the sows f o r each outcome group. The outcome groups were a l l 1 week apart so that an " a l l - i n a l l out" regimen was not used. Piglets i n a l l outcome groups started the experiment  with other p i g l e t s  already present i n the room. The results were analyzed using the following least squares model. Yu where  = u + Ti + Gj + TiGj + E i j  Y u = the dependent variable u  = the o v e r a l l mean  Ti  = the effect of the i  t  h  Gj  = the effect of the j  t  h  treatment outcome group  146  TiGj = the i n t e r a c t i o n between the i  t  h  treatment and the j  t  h  outcome group Eij = the residual error for each sample Non-significant interactions were added to the error term and the results recalculated. Treatment differences were measured using orthogonal contrasts. Leukocytes Leukocytes were separated from porcine abattoir blood. The method employed does not remove monocytes so the c e l l preparations are referred to as leukocytes. About 1 L of blood was collected from 1 pig at Intercontinental Packers Ltd.  (Vancouver B.C.)  using sodium EDTA as an anticoagulant. The  blood  was  allowed to s e t t l e at room temperature for 2 hours. At this time the leukocytes formed a thick layer on top of the red blood c e l l s . The leukocytes were c o l lected into a s t e r i l e flask using a water pump. The collected c e l l s were mixed with 5 volumes of 0.83%  ammonium chloride to lyse any contaminating red blood  c e l l s . The leukocytes were harvested by centrifugation at 200 x g for 20 minutes and washed 3 times with Roswell Park Memorial Institute medium. The  v i a b i l i t y .of c e l l s  Trypan Blue dye exclusion. The  isolated by  containing  10%  fetal  calf  this procedure  v i a b i l i t y was  parations. Individual doses of 5 x 10 serum,  8  50  (RPMI) 1640 culture were measured by  greater than 95% for a l l pre-  c e l l s were made up i n 15 mL of RPMI-1640 IU  mL  -1  penicillin  and  50  ug  mL  -1  streptomycin. The doses were kept at 4 °C u n t i l use. A l l doses were used within 24 hours of preparation. Doses were slowly warmed to 32 °C before being given to p i g l e t s . In vivo Cell-Mediated Immunity Cell-mediated immunity was measured as the intradermal response to the T c e l l mitogen phytohemagglutinin  (PHA)  (Blecha et a l . 1983). A solution  (0.1  147  mL) containing 250 ug mL  -1  PHA was injected into the medial aspect of one flank.  The other flank was injected with s t e r i l e s a l i n e . Double skin f o l d thicknesses of each flank were measured at 24 and 48 hours after i n j e c t i o n using a constant tension micrometer. The increase i n  thickness i n the PHA flank minus the  increase i n thickness i n the Control flank was used as the measure of i n vivo cell-mediated immunity. Animal Management P i g l e t s were removed from the sow at b i r t h and placed i n cages i n the experimental room used i n previous experiments. The p i g l e t s were then randomly assigned to treatments. The p i g l e t s on the leukocyte treatment 10  8  received 5 x  leukocytes v i a intubation. The leukocytes were given i n 15 mL RPMI-1640  culture media supplemented with 10% f e t a l c a l f serum, 50 IU mL and 50 pg mL  -1  -1  streptomycin. The sow reared p i g l e t s and a r t i f i c i a l  penicillin treatment  p i g l e t s were intubated and given 15 mL RPMI-1640 containing 10% f e t a l serum, 50 IU mL ets  -1  p e n i c i l l i n and 50 pg mL  -1  calf  streptomycin. The sow reared p i g l -  were then returned to the sow where they remained for the duration of the  experiment. The a r t i f i c i a l and leukocyte treatment p i g l e t s were started on PIgG f o r t i f i e d milk replacer and were reared i d e n t i c a l l y to p i g l e t s i n Experiment 4. A l l p i g l e t s were given 300,000 IU of p e n i c i l l i n on days 2, 4 and 6. This i s a standard treatment that the a r t i f i c i a l  for a l l sow reared p i g l e t s at UBC and i t was decided  and leukocyte treatment  p i g l e t s should receive the same  treatment. Blood samples were taken from the suborbital sinus at b i r t h and on days 1,7,14,21 and 28. The blood was analyzed for PIgG. P i g l e t weights were taken every.other day and the feed intake of the a r t i f i c i a l and leukocyte  treatments  148 adjusted accordingly- Diarrhea scores were not kept i n t h i s experiment  because  of the d i f f i c u l t y of observing the feces of sow reared p i g l e t s . Results Survival One sow reared p i g l e t was overlain by a sow (see Table 37). Aside from that there were no disease problems or diarrhea among the sow reared p i g l e t s . A l l of  the a r t i f i c i a l  experimental  and  leukocyte  treatment  piglets  survived  the 28 day  period. There was a major disease problem among the l a s t  two  outcome groups however. Two piglets on the a r t i f i c i a l treatment and one piglet on the leukocyte treatment were euthanized at the end of the experiment. These p i g l e t s were both from outcome group 4. They had severe diarrhea, weight loss and were unable to stand at the end of the experiment. Post-mortem examinations found the cause was septicemia due to Streptococcus suis I I . Several piglets in outcome group 2 on the leukocyte treatment showed inflammation of the j o i n t s . These piglets did improve enough to be returned to the herd at the end of the experiment. Weight Gains and Piglet Weights Average d a i l y gains were not s i g n i f i c a n t l y different for any treatment (see Table 38). There was a trend towards higher average d a i l y gains for the sow reared p i g l e t s , especially during weeks 2 and 3. A l l treatments showed a depression i n average d a i l y gains during week 4. Mean p i g l e t weights for the sow reared piglets were s i g n i f i c a n t l y higher on days 14, 21 and 28 (see Table 39). At the end of the experiment  the sow  reared piglets had a mean weight of 7,360g compared to 6,310g for the leukocyte treatment p i g l e t s and 5,940 g for the a r t i f i c i a l Plasma Immunoglobulin Concentrations  treatment  piglets.  149 Tables 40 and Figure 20 show the plasma PIgG concentrations. Plasma PIgG of the sow for  reared p i g l e t s was  a l l days. The sow  s i g n i f i c a n t l y higher than the other treatments  reared p i g l e t s had a plasma PIgG l e v e l of 44.1  on day 1 compared to 26.9  and  27.1  mg mL  -1  for the a r t i f i c i a l  and  mg  mL  -1  leukocyte  treatments respectively. Intradermal The  Response to  intradermal  PHA  response to PHA  i s shown i n Table  41.  The  sow  reared  p i g l e t s had a s i g n i f i c a n t l y greater increase i n flank thickness at 24 and hours than either of the other treatments. and outcome group was  48  The i n t e r a c t i o n between treatment  s i g n i f i c a n t at 24 hours. Table 42 shows the treatment x  outcome group means at 24 hours. There was a wide v a r i a t i o n between outcome groups i n the increase i n flank thickness  due  to PHA.  The  sow  reared  piglets  had  a significantly  greater  response to PHA i n outcome groups 3 and 4 only. The leukocyte treatment p i g l e t s had a s i g n i f i c a n t l y greater response to PHA  than the a r t i f i c i a l  treatment i n  outcome groups 1 and 4. There were no s i g n i f i c a n t treatment differences at 24 hours for p i g l e t s i n outcome group 2. Discussion One of the advantages of a r t i f i c i a l rearing i s that i t eliminates traumatic i n j u r i e s to the p i g l e t by the sow.  One  sow  reared p i g l e t was  crushed  i n this  experiment. The crushing and trampling of p i g l e t s by the sow accounts for from 18-52% of a l l pre-weaning mortality (Braude et a l . 1954; Fraser 1966; Fahmy and Bernard 1971). Eliminating deaths due to trauma could save 1-2 weaned p i g l e t s per sow per year. All  the  artificial  and  leukocyte  treatment p i g l e t s  survived. However,  three p i g l e t s were too weak to return to the herd due to Streptococcus suis II  150  i n f e c t i o n . Streptococcus suis  II i n f e c t i o n might  have also been responsible  for the decrease i n weight gains for a l l treatments during week 4. The a r t i f i c i a l treatment i n the present experiment and the PPO in Experiment  treatment  4 were the same i n a l l respects. The weight gains are similar  for weeks 1, 2 and 3 i n both  experiments. During week 4 however, the p i g l e t s  i n the present experiment gained only 123.9 g d a y . The p i g l e t s i n Experiment -1  4 gained 209.5 g d a y . The same i s true of analogous treatments i n Experiments -1  3 and 1. There was no decrease i n average d a i l y gains during week 4. A possible reason for this was a high l e v e l of environmental contamination during the present experiment. At one point a l l p i g l e t s on the a r t i f i c i a l and leukocyte treatments were present i n the experimental room at the same time. This may  have allowed a build up of environmental contagion greater than i n  previous experiments. The weights of the sow reared p i g l e t s were higher than those of the other two treatments from day 14 on. This could be due to several things. The feed intake of the a r t i f i c i a l  and leukocyte treatment p i g l e t s was  than the feed intake of the sow reared p i g l e t s  probably lower  (Braude et a l . 1970; Pettigrew  et a l . 1985). Without actually measuring the feed intake of the sow reared pigl e t s , i t i s impossible to know how their feed intakes compared to the a r t i f i c i a l and leukocyte treatment p i g l e t s . Braude et a l . (1970) was able to increase the rate of gain of a r t i f i c i a l l y reared p i g l e t s by increasing the l e v e l of feeding. At a l e v e l of 100 g milk solids Kg body weight 1  1  -1  day  -1  p i g l e t s gained 326 g day-  . In Experiments 1, 3, 4 and 6, p i g l e t s fed 75 mg milk s o l i d s Kg body weight day  -1  gained approximately 170-185 g day  -1  -  over the 28 day experimental period  (See Table 43). Lewis et a l . (1978) examined the effect that milk y i e l d had on p i g l e t  151 weight gains. They found that sow milk y i e l d and percent milk s o l i d s accounted for only 44% of the variation i n p i g l e t weight gains. Environmental, genetic and immunological factors also affect p i g l e t gains. Since the p i g l e t s used were l i t t e r mates, environmental and immunological factors must be responsible f o r most of the remaining v a r i a t i o n . There i s no way to quantify the effect of the d i f f e r i n g environments on the p i g l e t s i n the present experiment. Plasma PIgG and intradermal response to PHA p a r t i a l l y measured the immunological d i f f e r ences. The day 1 plasma IgG of the sow reared p i g l e t s averaged mean of 44.1 mg mL . This i s similar to values of 40.2 mg mL  -1  found by Klobassa et a l . (1981)  and higher than the range of 18.7-39.0 mg mL  reported by Curtis and Bourne  -1  -1  (1973). The plasma PIgG concentrations of the sow reared p i g l e t s were 14.4 mg mL  -1  on day 28. This i s very high compared to the 6.7 mg mL  -1  found by Klobassa  et a l . (1981) on the same day. The reason f o r this discrepancy may be due to the ELISA used to measure the plasma PIgG. The antibody to PIgG used i n a l l of the experiments had s p e c i f i c i t i e s f o r the heavy and l i g h t chains of the PIgG molecule. Since PIgM and PIgA have the same l i g h t chains as PIgG there i s some cross r e a c t i v i t y present. The other studies c i t e d measured PIgG, PIgM and PIgA and would have used antibodies that did not cross react with d i f f e r e n t isotypes. In spite of t h i s , the comparison among treatments i n this experiment were v a l i d because the same assay was used for a l l measurements. The a r t i f i c i a l  and leukocyte treatment p i g l e t s d i d not d i f f e r i n plasma  PIgG at any time during the experiment. Both treatments had plasma PIgG concentrations similar to those found i n p i g l e t s on analogous diets i n Experiment 4. These  values were s i g n i f i c a n t l y lower  than  f o r the sow reared p i g l e t s  throughout the experiment. The higher l e v e l of PIgG i n the serum of the sow  152  reared p i g l e t s may have contributed to their increased weight gains. The sow reared p i g l e t s received their immunoglobulins from colostrum and milk while the other two treatments received blood derived  immunoglobulins.  Sow colostrum contains primarily IgG (Porter and Chidlow 1979). The s p e c i f i c i t i e s of c o l o s t r a l immunoglobulins are similar to those found i n the blood. On day  1 therefore, both sow reared  and a r t i f i c i a l l y  reared p i g l e t s  received  immunoglobulins that were similar i n nature. Sow milk contains mainly IgA and a high proportion of milk immunoglobulins are s p e c i f i c f o r enteropathogenic organisms encountered by the sow. On days 2-14, the sow reared p i g l e t s received mainly IgA that was highly s p e c i f i c f o r enteropathogenic organisms. The a r t i ficially  reared  piglets  received  immunoglobulins  that  were mainly IgG and  unselected for s p e c i f i c i t i e s against enteropathogens. In Experiments 3 and 4 i t was noted that p i g l e t s that received PIgG on day 1 a l l had comparable survival rates. The presence or absence of immunoglobulins i n the diet on days 2-14 had no effect on p i g l e t s u r v i v a l . One hypothesis i s that 20 mg mL  -1  of blood derived PIgG on day 1 ensures the survival  of colostrum deprived p i g l e t s . Dietary immunoglobulins after day 1 increase average d a i l y gains. In the present experiment, the sow reared and a r t i f i c i a l l y reared p i g l e t s both received immunoglobulins of a similar type on day 1. Survival was the same for  both groups. On days  s p e c i f i c for enteric  2-28 the sow reared p i g l e t s  organisms. The immunoglobulins  received  IgA highly  fed to the a r t i f i c i a l l y  reared p i g l e t s were the same as on day 1. Furthermore, immunoglobulins were only fed on days 2-14. The average d a i l y gains of the sow reared p i g l e t s were superior to the gains of the a r t i f i c i a l l y reared p i g l e t s . This effect may be due to the hypothesis given above. The day 1 immunoglobulins ensured the survival  153 of the p i g l e t s . After day 1, the immunoglobulins i n sow milk provided superior protection from enteric i n f e c t i o n and higher average d a i l y gains. The intradermal response to PHA i s a good measure of cell-mediated immunity in pigs, c a t t l e and chickens (Blecha et a l . 1983; Haggard et a l . 1980; Regnier and Kelley 1981) . The sow reared p i g l e t s had a more prolonged response to PHA than the a r t i f i c i a l greater  flank  or leukocyte treatment p i g l e t s . At 48 hours they had a  thickness  than either  the sow  reared  or leukocyte treatment  p i g l e t s . The s i t u a t i o n at 24 hours i s less clear because of the interaction between outcome group and treatment. There was  a large v a r i a t i o n i n the PHA  response of the sow reared p i g l e t s i n d i f f e r e n t outcome groups. For the sow reared p i g l e t s , this v a r i a t i o n was correlated with the parity of the sows used in the experiment. The sow for outcome group 1 was a second parity sow and the increase i n flank thickness at 24 hours was 2.2 mm.  The sows for outcome groups  3, 4 and 5 were a l l fourth parity sows. They averaged 3.2, 4.7 and 4.7 mm.  The  l e v e l of immunity provided i n the milk of the younger sow may have been less than for the older sows. There was  a trend to lessened response to PHA  from Outcome group 1 to  Outcome group 4 i n the a r t i f i c i a l and leukocyte treatments. Outcome groups 3 and 4 of these treatments had more disease problems than Outcome groups 1 and 2. Infection suppresses cell-mediated immunity response i n outcome groups 3 and 4 may  (Beisel 1984). The lower  PHA  have been caused by the increase i n  infection. As with immunoglobulins, the population of lymphocytes i n milk i s different from the population found i n the bloodstream (Parmely and Beer 1977). A high proportion of them respond to enteric organisms and food antigens encountered by the mother. Feeding peripheral  blood leukocytes would  c e r t a i n l y have a  154 different effect than feeding lymphocytes found i n milk. In the present experiment, there were s i g n i f i c a n t improvements i n response to PHA i n leukocyte treatment p i g l e t s i n outcome groups 1 and 4. There was no response i n outcome groups 2 and 3 however. This may be due to the Major Histocompatibility Complex (MHC) compatibility between the donor and recipient of the leukocytes. Normally a p i g l e t would ingest lymphocytes from i t s mother. The p i g l e t s own lymphocytes would share at least half of the MHC antigens  expressed  by these ingested lymphocytes. The leukocyte treatment p i g l e t s received lymphocytes from an unrelated donor. The donor and recipient may have none or many MHC  antigens  i n common. Many functions  r e s t r i c t e d . The closeness  of c e l l  mediated  of the match between MHC types  immunity  are MHC  of the donor and  recipient may determine i f intradermal response to PHA i s increased or not. This has some relevance  to the practice of cross f o s t e r i n g p i g l e t s at  b i r t h . Head and Beer (1979) found that rats that suckled their mothers before being fostered to a MHC incompatible mother developed normal immune Rats moved to MHC incompatible mothers at b i r t h showed decreased  responses.  responsiveness  to alloantigens and 38% died of graft-versus host disease. A similar experiment using pigs would be of considerable i n t e r e s t . In conclusion, sow reared p i g l e t s gained weight faster, had higher levels of plasma PIgG and 2 of 4 l i t t e r s , had greater intradermal responses to PHA compared to the a r t i f i c i a l treatment p i g l e t s . Feeding  peripheral blood  leuk-  ocytes i n addition to immunoglobulins caused s i g n i f i c a n t increases i n i n t r a dermal PHA responses i n 2 of 4 l i t t e r s  but this did not have any effect on  average d a i l y gains. Incompatibility between the MHC of the donor.leukocytes and  the p i g l e t s that received them may be responsible f o r the inconsistent  results.  155  Table 37. The effect of sow or artificial rearing on piglet survival (Experiment 6).  day Birth  7  14  21  Treatment  N  N  N  N  N  Sow Reared  10  9  9  9  9  Artificial  10  10  10  10  10  100  6  Leukocyte  10  10  10  10  10  100  6  1  There were no significant contrasts between treatment means (P < 0.05).  28  1  %  SE  91  6  156  Table 38. The effect of sow or artificial rearing on piglet average daily gains (Experiment 6).  Average Daily Gains (g dayl) week 1 Treatment  X  week 2  week 3  week 4  SE  X  SE  X  SE  X  SE  18.2  260.4  23.6  260.4  23.0  191.9  24.2  Sow Reared  158.4  Artificial  140.3  19.6  196.0  25.4  207.8  24.7  123.9 11.6  Leukocyte  132.8  18.2  211.8  23.6  219.8  23.0  155.8  1  1  9.4  There were no significant contrasts between treatment means (P < 0.05).  Table 39. The effect of sow or artificial rearing on mean piglet weights (g) (Experiment 6),  Birth  dav 7  dav 14  Treatment  X  SE  X  SE  Sow Reared  1,240  46  2,370  127  4,200a 222  Artificial  1,340  49  2,250  137  3,620  Leukocyte  1,330  47  2.190  128  3,680  X  SE  dav 21 X  day 28 X  SE  6,020a 310  7,360  434  239  5,070  334  5,940  468  223  5,210  311  6,310  435  SE  a The contrast "Sow reared vs Artificial and Leukocyte"is significant (P < 0.05).  Table 40. The effect of sow or artificial rearing on plasma PIgG levels (mg mL ) (Experiment 6). -1  day 0 Treatment  _1  XSE  7  14  21  XSE  XSE  XSE  XSE  28 XSE  Sow Reared  <0.1  -  44.1a 1.8  36.4a 1.6  27.2a 1.6  18.9a 1.3  14.4a 0.7  Artificial  <0.1  -  26.8 1.9  19.8 1.7  13.0 1.6  9.3 1.2  8.8 0.7  Leukocyte  <0.1  -  27.0 1.8  20.8 1.6  15.2 1.5  9.9 1.2  8.4 0.6  a The contrast "Sow reared vs Artificial and Leukocyte"is significant (P < 0.05).  158 Table 41. The effect of sow or artificial rearing on piglet intradermal response to PHA at 3 weeks of age (Experiment 6).  Increase in Flank Thickness 1  24 Hrs Treatment  1  X  SE  48 Hrs X  SE  Sow Reared  3.7a 0.3  2.8a 0.3  Artificial  2.2  0.3  1.6  0.3  Leukocyte  2.5  0.3  1.5  0.3  Increase in Flank thickness =(PHA flank thickness at 24 or 48 hours - preinjection  thickness) - (Saline flank thickness at 24 or 48 hours - preinjection thickness). a The contrast "Sow reared vs Artificial and Leukocyte"is significant (P < 0.05).  159 Table 42. The effect of sow or artificial rearing on piglet intradermal response to PHA at 24 1  hours for treatment x outcome group interaction (Experiment 6).  Outcome group 1 Treatment  X  SE  2 X  4  3 SE  X  SE  X  SE  Sow Reared  2.2 0.3  3.2 0.3  4.7a 0.3  4.7a 0.3  Artificial  2.5b 0.3  2.9 0.4  2.1 0.3  1.5b 0.2  Leukocyte  3.2 0.3  2.9 0.3  2.0 0.3  2.1 0.2  1  Increase in Flank thickness =(PHA flank thickness at 24 hours - preinjection  thickness) - (Saline flank thickness at 24 hours - preinjection thickness). a The contrast "Sow reared vs Artificial and Leukocyte"is significant (P < 0.05). b The contrast "Artificial vs Leukocyte" is significant (P < 0.05).  160 FIGURE 2 0 . The effect of sow or a r t i f i c i a l rearing on p i g l e t plasma PIgG (Experiment 6). For s t a t i s t i c a l analysis see Table 40.  Day  161 GENERAL DISCUSSION P i g l e t s require porcine immunoglobulins  on day 1 f o r adequate  passive  systemic immunity. P i g l e t s fed porcine immunoglobulins had plasma IgG levels many times higher than p i g l e t s fed bovine immunoglobulins. They also had i n creased survival rates and higher average d a i l y gains (See Table 43). A f t e r day 1, the addition of immunoglobulins to the diet results i n higher average d a i l y gains but does not increase s u r v i v a l (See Table 43). In Experiment 4, i t was shown that bovine and porcine immunoglobulins  are equally e f f e c t i v e at  increasing average d a i l y gains when fed on days 2-14. The important consideration w i l l be the r e l a t i v e costs of porcine and bovine immunoglobulins. HBED has no potential as an additive to milk replacers for a r t i f i c i a l l y reared p i g l e t s . I t had no i n v i t r o a n t i - E ^ c o l i a c t i v i t y . When added to p i g l e t diets i t lower survival rates and decreased average d a i l y gains. EDDA inhibited the  growth of E^ c o l i i n an i n v i t r o test. When added to p i g l e t diets on days  2-14 i n Experiment 3, i t increased p i g l e t average d a i l y gains as much as porcine immunoglobulin. However, when added to diets on day 1-14 i n Experiment 4, i t caused a decrease i n average d a i l y gains (See Table 43). This may be due to increased absorption of EDDA on day 1 before gut closure takes place.  EDDA  increased the excretion of iron i n the urine and feces thus reducing the iron available for the synthesis of hemoglobin. Packed c e l l volumes and hemoglobin concentrations were low i n p i g l e t s fed EDDA i n Experiment 4. Since EDDA was not  superior to porcine immunoglobulins when fed on days 2-14 EDDA i s not r e -  commended as an additive to p i g l e t milk replacers. The c e l l  mediated immunity  of a r t i f i c i a l l y reared p i g l e t s was impaired  compared to sow reared piglets i n 2 of 4 l i t t e r s . The feeding of leukocytes to a r t i f i c i a l l y reared piglets s i g n i f i c a n t l y increased c e l l mediated immunity i n  162  2 of 4 l i t t e r s . The  major h i s t o c o m p a t i b i l i t y complex types of the donor and  recipients of the leukocytes may be responsible for the uneven r e s u l t s . Future experiments should measure the MHC the uneven r e s u l t s ,  types of the pigs involved. In addition to  increases i n the intradermal  responses to PHA  were not  correlated with increases i n average d a i l y gain. C l e a r l y , more work needs to be done i n this area. The questions that need to be addressed are: 1) the normal levels  of  cell  mediated immunity  in piglets;  mediated immunity i s transferred from sow  2)  the  to p i g l e t ;  extent  to which  cell  3) the effect of  cell  mediated immunity on piglet survival and average d a i l y gains and 4) the effect of MHC  compatability i n the transfer of c e l l mediated immunity via dietary  leukocytes. The s u r v i v a l of the p i g l e t s that received porcine immunoglobulins on day 1 and  excluding p i g l e t s that received HBED was  116  out  of 121  p i g l e t s that were free from defects and weighed over 800  or 95%.  Only  g were used so the  results are not comparable to s u r v i v a l i n commercial herds. Ten n a t u r a l l y reared p i g l e t s i n Experiment 6 had  a survival  rate of 9 out  p i g l e t s were chosen using the same c r i t e r i a  of 10 p i g l e t s . These  as for the a r t i f i c i a l l y  reared  p i g l e t s . The one sow reared p i g l e t that died i n Experiment 6 was crushed by the sow.  The major advantage of a r t i f i c i a l rearing i s the reduction of  i n j u r i e s caused by the sow.  traumatic  This factor alone accounts for up to one half of  pre-weaning mortality (Fahmy and Bernard 1971). While there are many factors i n sow's milk and colostrum  that protect piglets against enteric infections,  immunoglobulins alone provide an adequate l e v e l of protection. The rearing of p i g l e t s using milk replacers f o r t i f i e d with 25 mg mL 1 and 5 mg mL  -1  of either PIgG or BIgG on days 2-14  natural rearing.  -1  artificial PIgG on day  i s a viable a l t e r n a t i v e to  163  Table 43. The average daily gains and survival of piglets in Experiments 1, 3, 4 and 6.  Experiment  n  Ig day 1 (mg/mL)  1  Ig day 2 i[mg/mL)  Chelator (mg/mL)  Survival Mean Daily Gain  1  (%)  (a/d;  1  11  0  0  0  19  66  4  9  0  0  0  0  -  1  11  20 BIgG  4 BIgG  0  72  156  4  10  25 BIgG  5 BIgG  0  60  128  4  10  25 PIgG  5 BIgG  0  90  184  4  10  25 PIgG  5 BIgG  93  151  1  12  20 PIgG  4 PIgG  0  92  169  3  8  20 PIgG  0 PIgG  0  88  159  3  7  20 PIgG  4 PIgG  0  100  178  3  7  20 PIgG  0 PIgG  0.1 EDDA  100  179  3  7  20 PIgG  4 PIgG  0.1 EDDA  100  174  3  8  20 PIgG  0 PIgG  0.1 HBED  62  136  3  7  20 PIgG  4 PIgG  0.1 HBED  70  170  4  10  25 PIgG  0 PIgG  100  150  4  10  25 PIgG  0 PIgG  100  149  4  10  25 PIgG  5 PIgG  88  181  4  10  25 PIgG  5 PIgG  100  154  6  10  25 PIgG  5 PIgG  0  100  164  6  10  25 PIgG  5 PIgG  0  100  178  6  10  Sow Colostrum and Milk  91  219  0.1 EDDA  0 0.1 EDDA 0 0.1 EDDA  Mean daily gain is for the entire 28 days of the experiments.  164 CONCLUSIONS 1) Bovine serum  immunoglobulins  are poorly absorbed from the diet  into the  blood stream i n the f i r s t 24 hours after b i r t h and porcine serum immunoglobulins are required during this period i n order to ensure adequate passive systemic immunity. 2) Bovine and porcine  immunoglobulins  are equally  effective  at increasing  average d a i l y gains on days 2-14. 3) The c e l l mediated immunity of a r t i f i c i a l l y reared piglets may be impaired compared to sow reared p i g l e t s . The feeding of leukocytes to a r t i f i c i a l l y reared piglets s i g n i f i c a n t l y increased c e l l mediated immunity i n 2 out of 4 l i t t e r s . It had no effect on survival or average d a i l y gains however. 4) The best average d a i l y gains of any treatment group of a r t i f i c i a l l y reared piglets  i n these experiments (184 g d  - 1  ) , were unacceptably low compared to  those of sow reared p i g l e t s . Further studies on the i d e a l plane of feeding are required. 5) HBED has no potential  as an additive to milk replacers for a r t i f i c i a l l y  reared p i g l e t s . 6) EDDA i s not recommended as an additive to p i g l e t milk replacers due to i t s potential t o x i c i t y , i t s effects on iron metabolism and the fact that i t i s not demonstrably superior to immunoglobulins.  165  LITERATURE CITED  Aasa R. Malmstrom B.G. Saltman P. and Vanngard J . . 1963. 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