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Artificial rearing of low birth-weight pigs Whiting, Richard John 1982-03-31

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ARTIFICIAL REARING OF LOW BIRTH-WEIGHT PIGS by RICHARD JOHN WHITING Sc. Ag., The University of British Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 © Richard John Whiting, 1982 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of A/"Wy*»( ^ <X~*ir~L*t The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Four experiments were conducted to evaluate a crude immunoglobulin preparation fractionated from abattoir porcine serum with ammonium sulphate. The preparation was used as an additive to milk replacer for colostrum deprived, low birth-weight pigs reared in a non-isolated environment. Six pigs per treatment were used in experiments 1 and 3. Eight pigs per treatment were used in experi ments 2 and 4. In the first experiment, members of the negative control group did not survive and mortality of the other three groups which received some immunity was high. In the second experiment, the negative control group was eliminated from the trial, so those receiv ing only colostrum for 12 hours died, but the two groups receiving immunoglobulin treatment showed improved survival (63, 50%). In the third experiment, higher levels of immunoglobulin (15g./kg. body weight, initially followed by 5g./kg/day) did not show a significant effect on survival in comparison with the previous levels of lOg./kg. to 2g./kg.. However, rate of gain in body weight was significantly higher in the high dose level of immunoglobulin. In the fourth experiment the pigs were maintained on immunoglobulin for 10, 15, and 21 days and it was found that 21 day treatment eliminated deaths. The highest rate of gain was achieved with those on 21 day treatment in experiment 4. However, these rates of gain were considerably below those achieved with Digs on the sow. The causes of mortality were predominately E. coli scours, septicaemia due to E. coli and other bacteria, pneumonia, and 1n the \n ii two experiments, Salmonellosis. The prevention of death in experiment 4 by the immunoglobulin extract, indicated the success of the preparation against the Salmonella species encountered. iii TABLE OF CONTENTS Page 1. INTRODUCTION 1 2. LITERATURE REVIEW 2 2.1 The Mammalian Immune System 2 2.1.1 The Development of Immunocytes 2 2.1.2 The Intestinal Secretory Immune System .... 4 2.1.3 Antibody 5 The Structure of Antibodies 5 2.1.4 Complement 10 2.1.5 Antibody Reactions in the Gut Associated Lymphoid Tissue (GALT) 13 2.2 Prenatal Development of the Porcine Immune System . 16 2.3 Postnatal Development of the Porcine Immune System . 18 2.4 The "Runt" Phenomenon . 23 2.5 Mechanisms of Action of Bacteria in the Intestine. . 26 2.6 Diarrhea in Piglets 28 2.7 Non-Specific Gastro-Intestinal Immune Mechanisms . . 30 2.7.1 The Gastric Trap 32.7.2 Intestinal Motility 0 2.7.3 Intestinal Microflora 31 2.7.4 Non-Immunological Factors in the Secretions of the G. I. Tract 32 2.8 Humoral and Cellular Immunity in Porcine Mammary Secretions 4 2.9 Preweaning Mortality 36 2.9.1 Causes of Preweaning Mortality 36 iv 2.9.2 Effect of Low Birth-Weight on Survival ... 38 3. EXPERIMENTAL 42 3.1 Introduction3.2 Experiment I 44 3.2.1 Objective3.2.2 Materials and Methods 44 Experimental Animals 44 Preparation of Porcine Immunoglobulins 44 Dietary Treatments 44 Feeding Regimen . 45 3.2.3 Measurments and Observations 45 3.2.4 Results and Discussion 46 3.3 Experiment II 49 3.3.1 Objective3.3.2 Materials and Methods 49 Experimental Animals 49 Dietary Treatments 49 Feeding Regimen 50 3.3.3 Measurments and Observations 50 3.3.4 Results and Discussion 51 3.4 Experiment III 54 3.4.1 Objective3.4.2 Materials and Methods 54 Experimental Animals 54 Dietary Treatments 54 v Feeding Regimen 55 3.4.3 Measurments and Observations 55 3.4.4 Results and Discussion 56 3.5 Experiment IV 64 3.5.1 Objective3.5.2 Materials and Methods 64 Experimental Animals 64 Dietary Treatments 64 Feeding Regimen 5 3.5.3 Measurments and Observations . . . 65 3.5.4 Results and Discussion 63.6 General Discussion 70 3.7 Conclusions 5 4. BIBLIOGRAPHY 77 vi LIST OF TABLES Table Page 2.1 Immunoglobulin Structure 9 2.2 Mechanisms of Antibody Anti-Bacterial Activity 14 2.3 Prenatal Development of the Porcine Immune System ... 17 2.4 A Summary of Major Causes Contributing to Death in Baby Pigs 32.5 Effect of Birth Weight on Mortality 39 3.1a Experiment I: Experimental Animals 47 3.1b Experiment I: Results 43.2a Experiment II: Experimental Animals 52 3.2b Experiment II: Results 53.3a Experiment III: Experimental Animals 57 3.3b Experiment III: Results 58 3.4a Experiment IV: Experimental Animals 66 3.4b Experiment IV: Results 6vii LIST OF FIGURES Figure Page 2.1 The IgG Antibody Molecule: a Schematic Presentation of Structure 6 2.2 Activation Pathways and Physiological Functions of Complement Components 12 2.3 Serum Immunoglobulin Profiles of the Neonatal Pig. . . 20 3.1a Growth in Experiment III, Replicate 1 60 3.1b Growth in Experiment III, Replicate 2 61 3.2 Scouring Frequency 63 3.3 Cumulative Weight Gain: Experiment IV 68 3.4 Comparative Growth Curves 71 vi i i ACKNOWLEDGMENTS I would like to gratefully acknowledge the wonderful patience and understanding of my project supervisor, Dr.B.D. Owen who provided strong support during some difficult periods. I should also like to thank Dr. R.M. Beames for his assistance toward completion of the study. Dr. R. Peterson provided assistance with statistical analyses. I am indebted to the people at B.C. Veterinary Laboratory in Abbotsford for carrying out autopsies and bacteriological assess ments. Finally, I would like to express thanks to the three summer students, Brigitte Sonendrucher, Larry Nault, and Don Roberts, who helped during odd hours of the night and day to carry out the experiments. ix 1 1. INTRODUCTION Several studies have been done on the artificial rearing of neonatal pigs using supplemental immunoglobulins added to the diet (Owen, Bell, and Williams, 1961; Scoot, 1972; McCallum, 1977; Lodge and Elliot, 1979). The experiments reported herein were attempts to raise low birthweight pigs, including so-called runts, in a farm environment using previously studied levels and periods of serum-derived immuno globulin supplementation. Below a birthweight of 900 to 1000 grams, pig mortality during the first week of life dramatically increases (Pomeroy, 1960; Sharpe, 1966; Lefce, 1971; Fahmy and Bernard, 1971; Bereskin et al., 1973; English and Smith, 1975). Previous workers have found that, given an environment removed from competition and trauma, low birth weight pigs can be artificially reared with low mortality and more satisfactory growth rates (Lecce, 1971; England, 1974). .However, these earlier studies utilized a semi-isolated environment and sterile food. The current project involved attempts to raise these low birthweight pigs in a non-isolated environment using added immunoglobulins to eliminate the need for an isolated environment. 2 2. LITERATURE REVIEW 2.1 The Mammalian Immune System 2.1.1 The Development of Immunocytes The major effector cells in immunity are the lymphocytes, of which there are two classes. There are T lymphocytes where "T" refers to their derivation in the thymus gland and there are B lymphocytes where the "B" refers to the major differentiation region in the bird called the Bursa of Fabricus (Cooper and Lawton, 1974). The bursa has no analogous region yet demonstrated in the mammal although similar development of B cells here seems to occur in the fetal liver or bone marrow. (Cooper & Lawton 1974). These lymphocytes are derived from a common stem cell for hemopoiesis from which other circulating cells are derived. Thus, lymphocytes, monocytes, poly morphonuclear leucocytes, macrophage? megakaryocytes (from which platelets are derived), mast cells, eosinophils and erythrocytes are all derived from the same stem cell. All, except erythrocytes, are involved in the immune system. The primordial stem cell first develops in the blood islands of the embryonic yolk sac and then migrates to the liver of the fetus. Later in gestation and during adult life these stem cells are found in the bone marrow, having migrated from the fetal liver, and they continue the very active pro cess of hemopoiesis throughout the life of the individual (Hood e_t a_K, 1978). In the hemopoietic tissues a subtle and as yet undetermined differentiation occurs giving rise to cells which will individually home to a specific tissue microenvironment. Here further differentiation and a commitment occurs which means that the cell is set on a uni-directional path of development. T cell precursors become committed in the microenvironment of the thymus under the control of thymus hormone; B cells are committed in the fetal liver, bone marrow and, possibly Peyer's patches (Bienstock, 1979); macrophages and polymorphonuclear leucocytes in the bone marrow and spleen; and mast cells in the bone marrow (Hood et aj_., 1978). The lymphocytes develop within these specific tissues in an antigen independent differentiation until they are capable of responding to antigens. At this stage both B and T cells carry antigen receptor molecules on their membranes. The B cell receptor molecule is an immunoglobulin. The T cell receptor molecule is as yet unidentified but does not seem to be immunoglobulin (Benaceraf and Unanue, 1979). The B cell immunoglobulin is first IgM but later bears the isotype to be produced by the end cells. (Hood et al., 1978). If these lymphocytes bind a cognate antigen to these surface receptors they become activated; they enlarge with the formation of polysomes, microtubules and macromolecules in a process called blast transformation (Hood et al., 1978). These activated cells proliferate and continue to differentiate. Thus clones of T cells are formed containing both effector cells and memory T cells. Similarly, clones of effector and memory B cells are formed. The memory cells remain in circulation and provide a continuously reactive clone of cells which respond in a secondary immune response upon later antigen stimulation. The major effector cells are directly involved in antibody production as with T helper and T suppressor and plasma cells (B end cells) or in cell mediated immunity as with T killer and T - delayed hypersensitivity cells. (Hood et al., 1978). The function of the lymphoid system is to provide maximal contact of any antigen with its repertoire of antigen specific lymphocytes. Foreign material is phagocytized by macrophages which present antigen from 4 the foreign material to the T cells. These T cells (T helpers) are then activated and stimulate maximal antibody production by the plasma cells. Any antigen which enters tissue is gathered into the lymphatic system by the lymph fluid and carried to a nearby filtering lymph node or the spleen to stimulate antibody production. Antigens which enter the upper respiratory tract or gastro-intestinal tract encounter local lymph nodes as well as specialized organs: tonsils, adenoids, Peyer's patches, and the appendix. Blood borne antigens are dealt with mainly by the spleen. (Hood et al., 1978), 2.1.2 The Intestinal Secretory Immune System A major area of contact with antigenic material in the environ ment is at the mucosal lining of the gastro-intestinal system. Consequent to the development of gut microflora, the lamina of the gut becomes infiltrated with immunocytes producing predominately IgM and IgA which are transported across the gut epithilium into the lumen (Porter et al_., 1976). Initially in the development of a young animal IgM producing cells predominate but later there is a greater proliferation of IgA cells (Allen and Porter, 1973). The intestinal tract is especially adapted for production of antibody to the lumenally occurring antigens of bacteria, viruses, and other food borne materials. The mucosa overlying the Peyer's patches appears to be adapted to the uptake of large molecules and even intact microorganisms (McClelland, 1979). In this way precursor lymphocytes are sensitized in the Peyer's patches and are released to circulate and home into the intestinal lamina propria where they develop into IgA secreting plasma cells (Pierce and Cowans, 1975; Hasland et al_., 1976). The development of a systemic antibody production due to the gastro-intestinal immunization route may also occur from IgG or IgM forming 5 cells which are seeded by Peyer's patches in extra-intestinal lymphoid tissues. Also there is the possibility of antigen reaching lymphocytes in the spleen or peripheral lymph nodes from the gut (Craig and Cebra, 1971). Serum IgA also originates from gastrointestinal secretion (Tomasi, 1976). Mammary secretions of antibody are found to be sensitized to intestinal antigens (Chidlow and Porter, 1979). This is the gut-to mammary axis referred to by Parmely et aK, (1976). IgA secreting lympho cytes, possibly originating in Peyer's patches and travelling through the mesenteric lymph nodes into circulation are trapped by active mammary tissue under the influence of mammotropic hormones (Lamm et al_., 1978). 2.1.3 Antibody Antibody is produced by plasma cells which are end B cells (Nossal, 1976). The antigen is held by the immunoglobulin receptor in the cell membrane and is processed by internalization or endocytosis. The binding of antigen by B cells is essential for the development of production of antibody (Benaceraf and Unanue, 1979). The antibody is of five isotypes: M, G, A, D, or E. M, G, or A are responsible for humoral and secretory defense while IgD is presently only known as a membrane bound receptor on many lymphocytes, and IgE is concerned with the allergic response (Benaceraf and Unanue, 1979). The Structure of Antibodies (figure 2.1) Antibodies belong to a class of proteins called immuno globulins. The basic unit of structure is a complex of four polypeptides, two identical "light" (low molecular weight) chains and two identical "heavy" (high molecular weight) chains linked together by disulphide 6 L Choin Hypervoriable Regions CH3 Figure 2.1. The IgG Antibody Molecule: a' Schematic Presentation of Structure (Benaceraf and Unanue, 1979) 7 bonds (Hood et.al., 1978). The five classes of antibodies are called isotypes and their different structures are determined by their heavy chains. Thus, IgG has gamma heavy chains, IgM has mu, IgA has alpha, IgD has delta, and IgE has epsilon heavy chains. All isotypes may have one of two types of light chain, either kappa or 1 ambda. The heavy chains have three constant regions and one variable region (at the amino-terminal end) while the light chains have one constant with one variable region. The variable regions are further composed of framework regions, and hypervariable regions which are the least homogenous regions of the immunoglobulin (Beneceraf and Unanue, 1979). The variable regions of the heavy and light chains form a cleft which is the specific binding site of the antibody for an antigen (Searhart et al_., 1981). The binding site is precisely complementary to the structure of the antigen and binds to the antigen by weak electrostatic attraction, hydrogen bonding, and Van der Waal forces (Bach, 1978). The synthesis of these immunoglobulins is controlled by gene clusters (Hood et a_K, 1978). For each heavy or light chain a diverse cluster of V (variable) region genes of unknown number is linked with a cluster of C (constant) region genes on one chromosome. Thus, heavy chain clusters of V and C regions may be on one chromosome, a kappa C region with a cluster of V regions on another, and a lambda C region with a cluster of V regions on another. A single V region on the chromosome for the heavy chain can be associated with two or more C region genes during the different iation of antibody producing cells (Hood et.al., 1978) resulting in identical idiotypes (i.e. identical variable regions) among different isotypes. Further work by Tonegawa et a]_., (1978) found the presence of J (joining) 8 loci on light chain chromosomes, and both J and D (diversity) loci on heavy chain chromosomes. Diversity of antibody production to deal with the many possible antigens in the environment, in theory, results from two possible mechanisms (Hood et al_., (1978). The first is diversity within the same germ line (i.e. diversity of V region genes). Thus, a combination from the selection of variable light chain genes with variable heavy chain genes produces a multiple of them in antibody clefts. In combination with J and D loci there is a further multiplication of diversity (Gearhart et al_., 1981). The second mechanism of antibody diversity could result from somatic mutation or recombination which could occur during an individual's immune development. The consequence of these mechanisms is a very diverse repertoire of antibody specificities which, in the presence of antigen stimulation, leads to a continuously circulating array of antigen specific lymphocytes ready to pro liferate under heavier antigen stimulation. The newborn is inexperienced in the environment which is constantly presenting diverse antigens and consequent ly has an undeveloped repertoire of lymphocytes to respond. The antibody classes differ in gross formation ( Table 2.1) IgG is a monomer of two heavy and two lights chains of immunoglobulin. IgM is a pentamer which is held together by a joining (J) chain with disulphide bonds (Tomasi, 1976). IgM may also be associated in disulphide or non-covalent bonds with another protein, the secretory component (Brandtzaeg & Baklien, 1977) at least in human IgM. IgA may exist as a monomer, or as a dimer with a J chain as in serum. It may also exist as secretory IgA (S-IgA) which is a dimer with J chain and secretory compoent (SC). The binding of SC in IgM is weaker (more non-covalent bonding) than in S-IgA (Brandtzaeg & Baklien, 1977). IgD Immunoglobulin Light Chain Type Heavy Chain Type Other Components Structure 10 and IgE both exist as monomers. Both J chain and Secretory Component are polypeptides (Tomasi, 1976). According to Brandtzaeg & Baklien (1977) J chain is synthesized in the plasma cell with immunoglobulin where it polymerizes the IgA or IgM. SC is produced by serous type epithilial cells and acts as a specific receptor for the immunoglobulin. Covalent and non-covalent interaction between SC and immunoglobulin are completed during the passage of the complex through the epithilial cells and, conjugated IgA (or IgM) with SC, plus free SC is transported into the lumen by exocytosis (Benaceraf and Unanue, 1979). Since S-IgA is the predominate immunoglobulin in all body fluids outside the vascular system (with the exception of colostrum which contains mostly IgG) (Porter, 1976), this model probably applies to all exosecretions of secretory IgA. SC conjugated IgA is resistant to proteolysis by gastro-intestinal enzymes (Tomasi and Calvanico, 1968). SC is also thought to facilitate binding of the immunoglobulin to the mucosa coat of the intestinal epithilium (Porter et al_., 1972). 2.1.4 Complement The term complement refers to a complex group of enzymes in normal blood serum (Mayer, 1973). Thus, the complement system consists of 17 plasma proteins (Benaceraf and Unanue, 1979) which comprise a significant portion of the serum globulin fraction (Hood et aj_., 1978; Mayer, 1973). These proteins fall into two functional groups. One group is the classical com ponents which are symbolized with a capital C and a number, one to nine. The other group is the components of the alternate pathway which are denoted with a capital letter and are B, D, and P (properdin). Many of these proteins are cleaved during complement reactions and cleavage fragments are suffixed with \ 11 lower case letters, for example, C3a and C3b, with the suffix b denoting the larger of the two fragments (Beneceraf and Unanue, 1979). The functions of complement are several-fold. Completion of the complement sequence on an attacked cell leads to cytolysis. The cleavage product of C3, C3B, binds to microorganism cell surfaces producing immune adherence which facilitates phagocytosis (Mayer, 1973; Benaceraf and Unanue, 1979). C3a and C5a cleavage fragments are anaphylotoxins which are noted for causing lethal bronchospasm in guinea pigs and wheal and flare reactions in human skin by degranulating mast cells (Benaceraf and Unanue, 1979). Anaphylotoxins are active in other mammals also, causing acute inflammation (Hood et al_., 1978). C5a and free C5b-6-7 complex are chemo-tactic factors which attract polymorphonuclear leucocytes (PMN), which are phagocytes, to a site of complement activity (Hood et al_., 1978). The sequence of the complement cascade is most simply demonstrated in figure 2.2. Essentially, there is an effector pathway (C5 to C9) which "punches" a hole in the affected cell and can be activated by either the classical sequence involving Cl, C2, C4 and calcium or the alternate sequence involving B, D, P and magnesium. The classical sequence requires specific antibody which must be either IgM or IgG. A single molecule of IgM bound to an antigen on a cell membrane will initiate the classical sequence whereas two adjacent IgG molecules are necessary. Since antibody is scattered over the cell surface quite randomly, the probability of two IgG molecules occupying adjacent sites is quite small and therefore the frequency with which IgG activates the classical sequence is low (Mayer, 1973). The alternate pathway is a non-specific mechanism of immune defense (Mayer, 1977) since it doesn't require antibody for initiation. It is important in defense against 12 activation antigen-antibody complex (classical pathway) physiological ^ functions endotoxin (alternative pathway) properdin factors B and D immune adherence and phagocytosis , CS freeCSb.6,7 C7 complex CB OB | holes in membrane (lysis) | Acute inflammation blood-vessel dilation transudation PMN chemotaxls phagccytosii lysosomal activation necrosis repair regeneration Figure 2.2 Activation Pathways and Physiological Functions of Complement Components (Hood et al_., 1978) 13 gram-negative bacteria that inhabit the gut. Lipopolysaccharide from the cell walls of these organisms (endotoxins) combine directly with the serum factor, properdin (Hood et al_., 1978). There is indication that S-IgA will also act with lysozyme to activate complement fixation by the alternate pathway (Hill and Porter, 1974). Both the classical activation sequence and the alternate pathway lead to cleavage of C3 which initiates the effector sequence. 2.1.5 Antibody Reactions in the Gut Associated Lymphoid Tissue (GALT) Table 2.2 lists most of the known activities of antibody both systemically and externally as "secretory antibody". Within the gut associated lymphoid tissue (GALT) there are essentially two lines of antibody defense as interpreted by Tomasi (1976). The first line of defense involves reactions within the intestinal lumen and the second line of defense involves reactions in the tissues of the GALT allied to systemic immunity. The first line of defense is mainly carried out by S-IgA (as copra antibodies). Miler et aK (1975), using ligated porcine intestinal segments and live E. coli cultures as challenge injections, found that local protective effects of immunoglobulins IgG, IgM, and IgA were different, the minimum effective concentrations being 0.5, 0.05, andO.005 mg/ml respectively for IgG, M, and A. Miler et al_. (.1975) further stated that IgA does not possess enterotoxin neutralizing activity and that the mechanisms of action might be due to inhibition of absorption of bacteria or by blocking of binding of enterotoxin to receptors on epithelial cells. The efficacy of IgA was further demonstrated by Steele et al_. (1974). They found that of the immuno globulins, S-IgA was most effective in reducing mortality in rabbits infected with live cholera organisms. 14 Table 2.2 Mechanisms of Antibody Anti-bacterial Activity Class of Antibody Activity IgG Toxin neutralization Agglutination1 IgM Opsonization1 Bacteriolysis by complement fixation Inhibition of bacterial adherence* Toxin neutralization1 Agglutination* OpsonizationJ i Bacteriolysis by complement fixation IgA Anti-toxin activity* Agglutination M Inhibition of bacterial motility Bacteriostasis5 Inhibition of bacterial adherence* Complement fixing by the alternate pathway * Binding to mucin of mucosa* Inhibition of bacterial enzymes Inhibition of foreign antigen uptake J 1. Benaceraf and Unanue, 1979 2. Tomasi, 1976 3. Brandenburg and Wilson, 1974 4. McClelland, 1979 5. Porter, et al_., 1976 15 The second line of defense, or systemic immunity, depends largely on IgG and IgM. The activities of IgG and IgM are quite different. Due to its pentameric form, IgM has more active antibody binding sites than IgG (Bach, 1978). Thus, the activity of IgM is considered to be greater in antibody reactions. Rowley and Turner (1966) found the 8 IgM molecules/bacterium were necessary as opsonins whereas 2200 IgG molecules/ bacterium were necessary. Brandenbourg and Wilson (1974) stated that IgM was 500 to 1000 times more effective as an opsonin than IgG. They also stated that IgM was much more effective in complement fixation (See Section 2.1.4). Steele et al_., (1974) developed data supporting the greater activity of IgM using rabbit Ig against V. cholerae. Porter et al^, (1977) say that the dominant immunoglobulins found in the lamina propria are IgM and IgA. Since IgM may form only a small part of the secreted Ig, it may form a strong second line of defense in tissues behind the epithilium (Allen and Porter, 1973) where it can carry out complement fixation, opsonization agglutination, and toxin neutralization against pathogenic invaders. 16 2.2 Prenatal Development of the Porcine Immune System (see Table 2.3) Working with the fetal lamb and fetal mouse it was found that development of competence in the mammalian fetus occurs in a controlled, step-wise fashion. "A hierarchy of antigens exists to which the fetus develops competence at different stages of gestation" (Silverstein, 1977). The immunologic competence maturation is independent of lymphoid develop ment. The fetus before having organized lymphoid tissue is able to respond to some antigens presumably utilizing lymphoid cells in the fetal liver. Once it is competent to a certain antigen, the fetus is able to produce comparable antibody titres to an adult, and forms them in the usual sequence of immunoglobulin classes (IgM followed by IgG) (Silverstein, 1977). How ever, in contradiction, there may be some restrictions to fetal response. The level of antigen needed for stimulation may be higher (Sterzl, 1963), or there may be fewer stem cells, or there may be clonal restriction in the initial response (Klinman and Press, 1975). 17 Table 2.3 Prenatal Development of the Porcine Immune System Gestational Time of Occurrence Event Day 28 Day 32 Day 38 Day 40 Day 51 Before 65 days Day 77 After 70 - 80 days After 80 days At birth Lymphoid cells are detected in the region of the thymus' The lymph nodes begin forming without germinal centres (unless the animal is infected/ , Lymphoid cells are detected in the liver and blood Components of the complement system are present * Number of lymphocytes gradually begin to increase in the spleen1 Less than 1% of the lymphocytes have surface immunoglobulin^ Histologically, the thymus is identical to development at birth' In the lamina propria of the terminal ileum lymphocytes occur in follicular aggregations' Approximately 10% of blood lymphocytes have surface immunoglobulin indicating development of immunocompetence* A more generalized follicular lymphoid structure is developing with larger populations of lympho cytes. Immune responses can be shown against several antigens: formalized Salmonella* , sheep red blood cells* and phage6 . Transplantation immunity, to allogeneic cells was induced after 80 days ; if injected at 60 days, allogeneic cells induce tolerance to skin grafts.7 Peyer's patches are present' 1. Kruml et al_., 1970 2. Pestana et al_., 1965 3. Day et al_., 1969 4. Binns, 1973 5. Binns and Symons, 1973 6. Tlaskalova et al_., 1970 7. Binns, 1967 18 2.3 Postnatal Development of the Porcine Immune System In germ-free neonatal piglets, no immunoglobulin is found with the exception of a small "half molecule" of IgG type which has been shown to consist of one heavy and one light chain and is not antigen responsive (Prokesova et al_., 1970). This fragment has been theorized as a released, membrane-associated immune receptor molecule from lymphocytes (Porter, 1979). Due to an epithiliochorial placenta, the pig fetus is prevented from receiving antibody from the dam's circulation. The reason for this is unclear, because, although in the early embryonic stages as many as six histologically distinct layers separate the two bloodstreams, in later pregnancy the embryonic capillaries develop more in sub-epithilial positions and invade the embryonic trophoblast so that maternal and fetal bloods are in close proximity (Marrable, 1971). Whatever the situation of the circulatory supply, no maternal antibodies (or immeasurable quantities) pass (Kim et al_., 1967). The placenta is also thought to block antigen stimulation (Prokesova et al_., 1970). Immunization of a newborn piglet with a large dose of antigen results in a relatively rapid onset of antibody formation in the blood. Antibodies are produced within 36 hours as IgM, and after 6 days both IgM and IgG which were antigen responsive are found (Prokesova et al_., 1970). However, if certain antigens (e.g. Hog Cholera Virus) are used, injection in neonates induces tolerance to that antigen. (Weide et aj_., 1962). This neonatal induction of tolerance to certain antigens implies an immaturity of development and is commonly demonstrated in mice (Beneceraf and Unanue, 1979). After birth, lymphocytes are antigenically stimulated at the 19 Peyer's patches and/or the mesenteric lymph nodes to precommitment for IgA production; they then enter the circulation and home mainly to the lamina propria of the small intestine. Thus, a response of the intestinal secretory immune system requires antigenic stimulation. In the duodenum, Brown and Bourne (1976) found considerable numbers of cells staining for each of the three classes of immnuglobulin after the first week. The presence of IgM cells was greater than IgA cells for the first 3 weeks; IgA exceeded IgM cells after 3 weeks during which time IgG cells were present in considerable but smaller numbers. After about 1 month, the plasma cell population of the ducdenum was like that of an adult. Humoral immunity also begins with the presence of synthesized IgM first during the end of the first week; IgA is found in the serum after 7-12 days; IgG is found in the serum after 14 days (Bourne and Curtis, 1973), The immune mechanism of an adult can be contrasted with that of a neonate. The adult has been primed, during its antigen experience, to a great diversity of antigens from bacterial flora, and this response may be cross-reactive to an even larger array of invaders. Thus, a response which appears primary in adults may really be a secondary and more rapidly occurring response, whereas in the young or germ-free animal a primary response is truly primary; (Solomon, 1971). It is puzzling why a piglet which is immunocompetent at birth should be so susceptible to infection particularly when deprived of maternal antibody. But this may be answered that because of antigenic virginity, there may be a reasonable delay in response. Essentially a race occurs between the multiplication of antigen stimulated cells and pathogens (Solomon, 1971). 20 21 The general development of the immune system after birth is a continuous maturation due to stimulation by natural antigens. This may be observed in the rapid enlargement of lymphoid organs such as the spleen and the slower development of lymph nodes with age (Solomon, 1971). These structures do not develop nearly as fast in germ-free animals. In the alimentary tract of germ-free pigs there is virtually no development of lymphoid tissues by 31 days (Kenworthy, 1970). Contrasting results have been found with regard to the effect of colostrum antibody in the newborn pig. Segre and Myers (1964) in their trials found that piglets seemed to respond better to antigen-antibody complexes where the antibody is supplied by colostrum. Adult pigs respond just as well to antigen alone which, according to these workers, is pre sumably because there is sufficient natural antibody present in adults. However, Sterzl, et al_., (1965) found that colostrum tended to inhibit the active production of antibody. Later results tend not to resolve this contradiction except that low levels of antibody will sometimes enhance an immunological response whereas high levels of antibody will cause a poor response (Solomon, 1970). The later case may be understood either to be a case of removal of antigenic stimulation by the antibody (Dixon et al., 1967) or a case of negative feedback. Low levels of antibody may facilitate phagocytosis by macrophages and enhance antigen presentation to T-helper cells (Kim et al_., 1966). Sera from pre-colostral, neonatal, germ-free piglets is bactericidal for rough strains of E. coli, and this activity can be correlated with the presence of complement (Sterzl et al_., 1964). This is a mechanism which can function non-specifically against gram negative 22 bacteria without the presence of antibody and could be effective against any gut flora which become invasive. 23 2.4 The "Runt" Phenomenon As birthweight decreases, mortality increases (Carroll et aJL, 1962; Sharpe, 1966; Fahmy and Bernard, 1971). Smaller pigs often require manual assistance to suckle and additional warmth to survive. Failure to suckle can lead to hypoglycaemia and death (Newland et al^., 1952); lack of warmth can lead to hypothermia and death. Exceptionally small pigs at birth are termed "runts" and are distinguished from normal pigs by both their light weight and distinctive physical characteristics. Cooper et al_.» (1970) defined runts as being the lightest 5% of the total population at birth. Perry and Rowel 1 (1969) took a runt to have a prenatal weight of two-thirds of the average for its uterine horn. Physically, runts are small, thin, and have a disproportionately larger and more domed head than normal progeny. The physical characteristics of a runt are the result of intra-uterine growth retardation which is not the result of fetal anomoly or maternal illness (Cooper et al_., 1978). The runting phenomenon may be a result of intrinsic fetal growth factors or be secondary to placental function (Cooper et al_., 1978) but the cause is not clear. When five or fewer feti are in each horn of the uterus, the position of the embryo has no effect on size. But, as the number increases, those at either end tend to have an increasing advantage over those in the middle of the horn, and those at the ovarian end have a greater advantage than those at the cervical end (Perry and Rowel 1 , 1969). Marrable (1971) suggested that even with small litter sizes, the positional effect will be operative later in 24 pregnancy. Waldorf et al_., (1957) suggested that the uterine arteries provided higher blood pressure at terminal sites but, although this may be relevant in other multiparous mammals, the porcine vascular system is such that supply to the cervix and uterine body is augmented by a branch from the urogenital artery (Marrable, 1971). Several studies have been done about prenatal appearance of runting. Pomeroy (1960) found significant runting appearance at 74 days of gestation but not at 51 days. Perry and Rowel 1 (1969) found feti of less than two-thirds the weight for the average of the uterine horn at 31 to 49 days of gestation and identified these as runts appearing(y>Cooper et al_., (1978) found runts at gestational ages of 44, 53, 56 and 75 days of gestation. Obviously these data do not pin-point a conclusive stage of gestation at which fetal growth retardation occurs. However, since 99 per cent of fetal growth (by weight) occurs after day 40 of gestation (Marrable, 1971), the occurrence of runting this early suggests that intra-uterine retardation may be determined before it becomes recognizable and may occur throughout fetal development in some cases. Widdowson (1971) compared the development of organs in the runt with that of a fetus taken at an equal size and with that of a litter-mate of normal size. Two outstanding differences were noted: firstly, muscle development, as measured by quadriceps size was significantly less in the runt than in either the fetus of equal weight or the normal litter-mate; secondly, the livers of the fetus and runt were comparable in size yet very much smaller than the liver of the normal littermate with the added characteristic of a very low carbohydrate content in the runt. Heart, spleen and stomach of the runt were intermediate in weight between those 25 of the fetus and normal littermate. The brain of the runt was nearer to normal weight than any other part of the body. Generally, Widdowson (1971) found that the decrease in organ size of the runt was due to both hypoplasia (decreased cell size) and hypotrophy (decreased cell number). Widdowson (1971) went on to compare the growths of a runt and a normal littermate for three years and found that the runt was 60 kg. lighter at the end of this term. Since there were no restrictions in diet or as a result of environmental effects after weaning, it may indicate that intra uterine growth retardation may provide a permanent set-back. 26 2.5 Mechanisms of Action of Bacteria in the Intestine Within the intestine there are three major ways that bacteria may associate with the intestinal mucosa to produce disease. Firstly, they can attach to the mucosa without penetration and induce disease by multi-3 plying and producing exotoxin . This occurs in E. coli enteritis of the small intestine and is caused by enterotoxin producing E. coli. Secondly, bacteria can attach to and penetrate the mucosa but not the subepithilial tissues and induce disease by damaging epithilial cells and also produce exotoxin. This can occur in E. coli enteritis of the large intestine and is caused by invasive strains of E. coli. Thirdly, bacteria can attach to, and penetrate the mucosa to reach the subepithelial tissues, multiply in the submucosa, and spread systematically also growing intracellular^ in phagocytes. This mode of action occurs with Salmonella enteritis caused by S. typhi. (McClelland, 1979). A major phenomenon involved in bacterial invasion of epi thelial surfaces is bacterial adherence. Since peristalsis and flushing with secretions tends to remove bacteria and maintain a continuously lower level of growth and prevent invaders from proliferating, attachment to the gut epithelium by adherence is a major mechanism affecting the ability of a pathogen to proliferate. Best known in this connection is E. coli with its varying adhesion fimbriae. The most studied is the K88 antigen which has non-flagellar filamentous surface pilli carried on the genome of a plasmid. (Smith and Linggood, 1971). Other adherence factors of E. coli in the neonatal porcine intestine are the antigens K99, strain 987 type, and type 1 common pilli of which each has a different receptor site on the intestinal epithelium (Dupond and Pickering, 1980). All of these surface 27 structures are antigenic and elicit antibody responses where the antibody acts as an anti-adherence factor possibly by masking the adherence site of the bacterium. In reference to the K88 antigen, the porcine receptor site for it is controlled by a dominant gene and therefore animals which are double recessive are immune to K88 strains of bacteria unless other factors are present determining virulence. (Rutter, 1975; Rutter et al_., 1976). The mechanism of enterotoxins is not fully understood. However two enterotoxins have been shown to be produced by strains of E. coli patho genic for swine: one is antigenic and heat labile (LT) and the other is non-antigenic and heat stable (ST) (Dupont and Pickering 1980). Both toxins are transmitted by a plasmid and both may be produced by the same strain of E. coli. It has been suggested that the non-antigenic ST was important in the diarrhea of swine (Gyles, 1971). 28 2.6 Diarrhea in Piglets In a normal, healthy intestinal mucosa, salts and water are absorbed by an energy requiring mechanism. The driving force of water absorption is an active transport of electrolytes, particularly sodium and chlorine. Acute enteritis,which causes diarrhea in newborn animals^produced watery stools from fecal fluid primarily originating in the small intestine (Tennant and Hornbuckle, 1980). The rapid de hydration causes hemoconcentration and along with the movement of ions, leads to metabolic acidosis caused by renal failure to excrete hydrogen ions and by increased production of organic acids resulting from decreased tissue oxygenation. Hyperkalemia (hiigh potassium) also results as does hypoglycemia due to decreased gluconeogenesis and increased anaerobic glycolysis (Tennant and Hornbuckle, 1980). In the specific case of the enterotoxic mechanism of E. coli, the toxin (LT only) acts by binding to receptors on the mucosal cell membranes to activate adenyl cyclase which converts ATP to cyclic AMP. The cAMP acts as a second messenger to influence permeability of the membrane. The net result is that sodium absorption is blocked and chlorine is secreted. The chlorine ion pulls water and cations with it into the gut lumen to maintain osmotic equilibrium. This is the cause of fluid loss (Dupont and Pickering, 1980). E. coli heat stable toxin (ST) does not activate adenyl cyclase however, as has been discovered recently, in mice and rabbits, its action may be mediated through the build-up of guanyl cyclase which produces cGMP (Hughes et al_., 1978). In the case of Salmonella infection, similar dehydration and hydrogen ion and electrolyte disturbances are the result. Salmonella 29 is an invasive organism and penetrates the mucosa and proliferates in the gut associated lymphoid tissue (Dupont and Pickering, 1980). Its effect can occur throughout the intestine although it usually invades the lower gut mucosa more readily. Using the rat, Powell et al_., (1971) showed diarrhea increased especially the ileal secretion of water, sodium and chlorine and potassium. They found an increased hydrostatic pressure in the lamina propria, perhaps caused by venous or lymphatic obstruction, with resultant increased membrane permeability. 30 2.7 Non-Specific Gastro-intestinal Immune Mechanisms There is a series of mechanisms which operate in defense against pathogenic bacteria and in some cases, viruses and parasites. 2.7.1 The Gastric Trap A gastric trap is provided by the HCl which is anti-bacterial and is highly effective against almost all bacteria. However, if the gastric mucosa is not functional, as, possibly, in the neonate, or if the challenge dose is large enough, or if bacteria are not reached by the acidity of the stomach, bacteria may pass this trap to proliferate later in the gastro-intestinal tract (Giannella et al_., 1971). In neonates, the work on gastric acid secretion seems quite incomplete and contradictory. Cranwell et al_., (1968) found that piglets in a clean environment had acid secretion at one week of age, while those raised in a conventional environment didn't secrete until 30 days of age. Later, Cranwell and Titchen (1974) discovered that secretion could begin from 2 days of age using a surgically separated pouch from the stomach and that the secretion was significant. Hill (1970) stated that acid secretion was very minimal during the first 2 weeks. Jones (1972) referred to work which stated that HCl secretion does not begin until 20 days of age of milk fed or 14 days if given cereals. The likely explanation of discrepancies is that the piglet has a competent gastric mucosa at least within a few days of birth, but environmental or nutritional factors control its development (Cranwell and Titchen, 1974). 2.7.2 Intestinal Motility The rate of passage of chyme will determine to a large extent whether local propagation will occur. There is evidence that the time of 31 contact between the epithilial lining of the gut and an invasive pathogen such as a Salmonella strain of bacteria determines the development of disease (Sprinz, 1969). It has been shown by the use of a ganglion blocking drug which slowed the rate of peristalsis in rats that there was a substantial increase in the number of coliforms in the small intestine. (Dixon and Paulley, 1963). Diarrhea is associated with decreased motility (Christiansen et al., 1972) and gut stasis (Rutter, 1975; White et.al., 1972; Kohler, 1972). Related to intestinal motility is the level of secretion of large amounts of sterile fluid by the duodenum which washes out bacteria (Nielson e_t aj_-, 1968). 2.7.3 Intestinal Microflora In the developing piglet raised conventionally, while gastric activity is low, bacteria are ingested from the environment and success fully adapted strains proliferate. Probably, the establishment of a healthy and natural microflora occurs here and evolves as the diet changes from milk to solid food. In a healthy young pig the ecology of the gut maintains a homeostasis which is further ensconced in a more mature animal. In the nursing piglet lactobacilli proliferate producing lactic acid which in its acidity inhibits other bacteria. Later, the resident bacteria create antibacterial catabolites including lactic acid and short chain volatile fatty acids to lower the pH. In addition, the resident flora dominate the demand for space and nutrients to prevent other forms from gaining access to grow (Freter and Abrams, 1972; Freter, 1974). Working with continuous flow cultures, Ozawa and Freter (1964) found that resident stains used nearly all energy sources and any invader strain couldn't proliferate unless a source not used by the residents was supplied. 32 Another factor of the environment of the lumen is that it has a low redox potential which allows only certain strains of bacteria to survive (McClelland, 1979). A further factor influencing bacterial ecology in the gut is that some bacteria can produce specific antimicrobials of limited spectrum which appear to influence the ability of a particular strain to survive (Branche et aK, 1963). In treating with antibiotics against an infection (e.g. Salmonella) the magnitude of the challenge dose may be reduced and the infection may be prolonged by reducing the total intestinal flora and allowing the infective strain to proliferate (Aserkoff and Bennett, 1969). The stability of the microflora suggests that immunity is operative against the effects of invasive or mucosal associated bacteria and that low grade pathogens (i.e. the intestinal flora) either are restricted to the gut lumen where immunological inter actions are prevented or display a low degree of antigenicity. (Shedlofsky and Freter, 1974). 2.7.4 Non-Immunological Factors in the Secretions of the G.I. Tract Several non-specific inhibitors of bacterial growth are present in the duodenal secretions and in milk of the nursing individual. Bile salts are metabolized by the resident bacteria and may form anti metabolites to certain bacteria (McClelland, 1979). Lactoferrin and lysozyme are both present in intestinal secretions and milk and, with secretory IgA, are bactericidal (Hill and Porter, 1974; Knopf et al., 1971) . Lactoferrin, which is commonly present in milk chelates iron which restricts bacterial growth (Orson and Reiter, 1981; Bullen et al., 1972) . Complement may be involved in reactions in or near the mucosa. The alternate or properdin pathway of complement activation is accepted 33 as important in the defense against gram negative bacteria of the gut (Hood et al., 1978). This is supported by the discovery that some complement components are synthesized by the intestinal mucosa (Lai A Fat et ai., 1976). 34 2.8 Humoral and Cellular Immunity in Porcine Mammary Secretions Colostrum is the fluid secreted by the mammary gland during the first 24 hours (Karlsson, 1966) and milk is secreted during the remainder of lactation. IgG is the primary immunoglobulin component of serum and colostrum; IgA is the primary component of milk; and IgM is a minor component of both. In the first 24 hours IgG decreases five fold and within the first week it decreases thirty-fold. Thus it drops from forming 80% of total colostral immunoglobulin to 25% of total milk immunoglobulin (Curtis and Bourne, 1971). IgA only decreases three-fold during the first week and hence emerges as the major immunoglobulin in sow milk, accounting for 50 to 60% of milk immunoglobulin (Curtis and Bourne, 1971). Colostrum functions mainly in protection against systemic infection because the immunoglobulins it contains are absorbed through the gut mucosa into the circulation. This occurs in a period of time during which the piglet intestine absorbs macromolecules intact before gut closure after 24 to 36 hours (Payne and Marsh, 1962). Milk provides copra-antibodies which function within the intestinal lumen during the pre-weaning period (Wilson, 1974; Brandtzaeg, 1973). 125 Bourne and Curtis (1973) using I labelled immunoglobulin determined the proportions of serum derived and locally produced immuno globulin in mammary secretions. They found that all colostral IgG came from the serum and that 60% of colostral IgA was synthesized by the mammary gland (a small portion of total colostral immunoglobulins), and, conse quently, considered that colostrum was a serum transudate (Bourne, 1973). They found that milk, on the other hand, was a true mammary secretion since 35 more than 90% of the IgA and IgM and 70% of IgG was produced locally. In conjunction with immunoglobulins, porcine milk contains a trypsin inhibitor which occurs firstly at high levels and falls to a very low level by the fifth day (Laskowski et al_., 1957). This probably functions to prevent degradation of colostral immunoglobulins before they can be absorbed in the intestine (Scoot, 1972). Although no information exists for porcine colostrum, human colostrum shows a relatively high level of T lymphocytes and macrophages (Parmely and Beer, 1977). Parmely and Beer suggested that it was possible, but not proven, that these cells could survive in the gastro-intestinal tract. They further suggested that the mammary tissue may "package" specific cellular components in the colostrum to contribute to immuno-competency in the neonate and that this cell mediated immunity (CMI) from the mammary gland depended upon inductive events at distant mucosal surface (bronchial associated and gut associated lymphoid tissues). 36 2.9 Preweaning Mortality In a survey of the literature from 1937 to 1976 McCallum (1977) found that levels of preweaning mortality have consistently ranged between 18 and 30 per cent of pigs born (including stillbirths). Two studies (Fahmy and Bernard, 1971; English and Smith, 1974) recorded that 50 per cent of the total preweaning mortality occurred before 3 days of age. Pomeroy (1960) noted that 70.2 per cent of total preweaning mortality occurred before 3 days of age, and Jones (1972) recorded 63.1 per cent. Thus, any attempt to reduce mortality must affect the earliest days of post natal existence. 2.9.1 Causes of Preweaning Mortality The causes were fairly consistent among the authors cited and are summarized in Table 2.3 These are trauma, starvation, general weakness, disease, congenital abnormalities, and other causes. Trauma involves crushing or trampling of pigs by the sow. Some of the data may reflect the effect of other primary causes predisposing trauma such as malnutrition, general weakness, or chilling. Starvation, a major primary cause of death, could result from two different contributing factors. The first is agalactia of the sow which, in the studies of English and Smith (1974) contributed to 27 per cent of the starvation. The other factor, contributing 73 per cent, is severe competition within litters due either to supernumary pigs in relation to available teats or to dis parity in birthweights which results in unequal competitive advantage for heavier pigs (English and Smith, 1974). The common result of starvation is hypoglycaemia (Sharpe, 1966; Edwards, 1972) which may be aggravated by chilling (Curtis, 1974). General weakness, which contributes to high Table 2.4 A Summary of Major Causes Contributing to Death in Baby Pigs (per cent contribution) Reference Trauma Starvation Relative Disease Congenital Other Weakness Abnormality Sharpe (1966) 21.2 17.1 9.6 9.6 2.1 40.4 Fahmy and Bernard 19.2 — 26.9 17.5 14.2 22.2 (1971) English and Smith 18.2 42.8 14.9 — 12.3 11.8 (1974) Rodeffer et al., 30.9 17.6 14.7 18.2 — 13.1 (1975F 38 losses, is most clearly associated with parturition aberrations of which the most common result is prenatal anoxia or hypoxia (English and Smith, 1974). "Parturition aberrations" include premature umbilical cord rupture and a prolonged farrowing interval between births (Rodeffer e_t aJL. 1975) Disease, as a cause of death, includes different diseases depending on the study. Sharpe (1966) listed the major causes as gastroenteritis and septicaemia. Fahmy and Bernard (1971) listed scours and pneumonia as major disease factors. Rodeffer et al_., (1975) listed transmissable gastroenteritis (T.G.E.), other diarrheas, and pneumonia. Congenital abnormalities varied among the studies. English and Smith (1974) listed the major traits including atresi ani, cardiac abnormality, congenital splay-leg, cleft palate, and hypoplasia kidney. 2.9.2 Effect of Low Birthweight on Survival "The effect of birth weight on survival is essentially linear within a wide range of values" (Bereskin et al_.» 1973). Thus, low birth weight is a major contributing factor to reducing survival (Sharpe, 1966;; Fahmy and Bernard, 1971; Bereskin_et al_., 1973). Table 2.4 lists mortality values found for low birthweight pigs by several researchers. These high mortality rates may vary with strain of pig or environment which, in turn, may include temperature and milking ability of the sow (Pomeroy, 1960). These high mortality rates reflect a greater susceptibility to the major causes of death. There was a higher incidence of trauma in low birthweight pigs (Sharpe, 1966); a markedly higher incidence of starvation (Sharpe, 1966; English and Smith, 1974); and low birthweight had some effect on the incidence of septicaemia (Sharpe, 1966). Also, low birth weight pigs are much more susceptible to chilling. Newland et al_., (1952) Table 2.5 Effect of Birth Weight on Mortality Reference Weight Range Per Cent Mortality Pomeroy (1960) <900g 83.0 (before 3 days) Sharpe (1966) <800g 82.1 Fahmy and Bernard <910g 60.0 (1971) ( English and Smith <907g 74.6 (1974) VO 40 found that the drop in body temperature after chilling was inversely related to body weight. Pomeroy (1960) cited three factors contributing to higher mortality in low birth weight pigs: 1. they may be less vigorous and active, and less competitive at the teats during nursing. 2. they have a larger surface area in relation to body weight. Therefore there is a relatively greater heat loss per unit of body weight. 3. they may be physiologically immature. 41 Foot-Notes 1. Immunocytes include all cells of the immune system. 2. The failure of the immune system, as a result of previous contact with antigen, to respond to the same antigen, although capable of responding to tohers. Tolerance is best established by neonatal injection of an antigen (Benaceraf and Unanue, 1979). 3. An exotoxin is a soluble poisonous substance passing into the medium during growth of a microorganism. An endotoxin is a poisonous substance present in bacteria but separable from the cell body only on its disintegration. An enterotoxin is a toxin of bacteria produced within and affecting the intestine. (Websters 7th New Collegiate dictionary, 1965) 42 3. Experimental 3.1 Introduction Colostrum-deprived pigs have been successfully reared in a non-isolated environment since 1961 (Owen et al_., 1961). After work by Owen and Bell (1964), Scoot (1972), and McCallum (1977), McCallum (1977) concluded that it was possible "to raise colostrum-deprived neonatal pigs in an ordinary swine barn environment and achieve survival rates comparable to those presently tolerated under natural conditions" using abattoir serum-derived immunoglobulins in milk replacers. Both Scoot (1972) and McCallum (1977) concluded that oral administration of 10 grams per kilogram of body weight of immunoglobulins on the first day followed by 2 grams per kilogram on succeeding days could confer adequate passive immunity on colostrum-deprived pigs. Scoot (1972) found that administration of immunoglobulins over 21 days increased survival compared to 10 day treatment (from 75 per cent to 88 per cent) and McCallum (1977) observed a sharp increase in mortality following removal of immunoglobulin after 10 days compared to those continued on treatment for 20 days. The purpose of experiments reported herein was to evaluate the effect of orally administered immunoglobulins on colostrum-deprived pigs of low birthweight. Experiments of Lodge and Elliot (1979), using the artificial rearing technique of McCallum et al., (1977), found that 21 day weight, 56 day weight and age at 90kg. were highly correlated with birthweight, but that low birthweight was less inhibiting under artificial rearing than under natural rearing. Lodge and Elliot (1979) also found that artificial rearing reduced mortality of low birthweight pigs. 43 The trials described herein were carried out in a barn environment on a commercial farm 1 in an environmentally controlled room adjacent to the farrowing rooms. A series of 4 experiments was carried out to determine effects of low birth weight on survival and growth. 44 3.2 Experiment I 3.2.1 Objective The objective of this experiment .was to assess the efficacy of abattoir derived porcine immunoglobulin extract from serum as a milk replacer additive for rearing low birthweight pigs. 3.2.2 Materials and Methods Experimental Animals Twenty-four low birth weight pigs weighing less than lOOOg of Yorkshire X Landrace breeding were allotted within a ten day period. Farrowings were attended and experimental pigs were removed from the sow at birth or 12 hours later. Pigs were weighed at birth, ear tagged, and individually penned in a nursery room. Pigs were randomly assigned to treatments. Cages were adjoining wire mesh 46cm x 25cm x 20cm in two tiers. Prior to the trial the room and cages were thoroughly cleaned and dis infected. Cages were washed periodically during the trial. Temperature in the nursery room was maintained between 30°C and 33°C. All pigs received iron dextran injections and had their needle teeth and tails clipped at two days of age. A second iron dextran injection was given at 10 days of age. Preparation of Porcine Immunoglobulins Immunoglobulins used in all trials were prepared by Canada Packers Ltd., Toronto, Ontario. The immunoglobulin -fraction derived at 40 per cent saturation with (NH4)2 SO^ was washed, dialyzed, mixed with condensed whole milk and spray dried (Owen, personal communication). Dietary Treatments In all treatments, pigs received a non-medicated commercial 2 milk replacer . Feed intake was adjusted to 7.5 per cent of their body 45 weight in air dry matter which was diluted 1:4 with water. For the first 3 days diets were formulated with a 10 per cent dextrose solution. Pigs were weighed every other day and feed allowances were adjusted accordingly. Immunoglobulin was mixed with the milk replacer and fed at levels set out in the following schedule: 1. No colostrum, milk replacer only. 2. Colostrum for 12 hours, followed by milk replacer only. 3. Colostrum for 12 hours, plus immunoglobulin at 2g/kg body weight/day for 9.5 days. 4. Immunoglobulin extract on first day (lOg/kg body weight), plus immunoglobulin (2g/kg body weight/day) for 9 days. Scouring animals were treated, as necessary, on an individual 3 pig basis with Furoxone . The trial was of 21 days duration. Feeding Regimen Since the pigs would not feed from shallow bowls immediately after birth, they were force fed with syringes until they would nipple feed. In later trials nipple feeding was successful from birth but during this trial some difficulty was encountered. At 10 days of age all pigs were switched to shallow bowls. Feeding was every 2 hours for the first 4 days, every 4 hours on days 5 to 8 and every 6 hours from days 9 to 21. Fresh water was made available from day 5 and creep feed was offered fresh daily after 10 days of age. 3.2.3 Measurements and Observations Pigs were weighed every 2 days. Mortality was recorded and post mortems were carried out on all dead pigs by the Provincial Veterinary Laboratory, Abbotsford, B.C. Infectious agents and causative factors were 46 identified whenever possible. Mortality was analyzed statistically by giving a survivor a zero value and a dead pig a 1 value. The analysis used was analysis of variance (ANOVA) with the design model being: Y^j = p + T- + e^j where T.. = Treatment Effects y = Population Mean £.. = Experimental Error Rate of gain was also analyzed with this model. The mean birth-weights of survivors and dead pigs were compared with a "t" test (Choi, 1978). 3.2.4 Results and Discussion Mortality was high (Table 3.1.b) with all pigs in the negative control group dying before 10 days of age. Those which received colostrum plus supplemental immunoglobulin extract for 10 days showed the highest survival. The differences between treatments were not significant (P 0.05). The mean birth-weight of the surviviors was significantly higher than that of the dead pigs (P 0.01). This difference suggests that higher birth-weight pigs had an advantage in this trial. The cause of death was predominantly aspiration pneumonia complicated by E. coli septicaemia. The pneumonia was invariably caused by the presence of foreign material in the bronchi and lungs introduced while force feeding with syringes. A major characteristic of dead pigs, regardless of treatment, was a flaccid, dilated intestine or distended stomach. The cause of this state could have been over-feeding and could have involved E. coli enteritis. All pigs were diarrhetic befor death. Most pigs which died showed a marked lack of depot fat indicating no energetic reserves for survival, and 47 Table 3.1a Experiment 1: Experimental Animals Treatment Number in Treatment Mean Birth-Weight Group t S.D. No colostrum; no immuno- 6 765 ± 157 g globulin Colostrum; no immuno- 6 854 + 121 g globulin Colostrum + immunoglobulin 6 843 + 190 g (2g/kg/day) Immunoglobulin (lOg/kg/day 6 752 + 190 g to 2g/kg/day) Table 3.1b Experiment 1: Results Treatment % Survival % Survival Rate of Gain to Day 10 to Day 21 ( q/day ) No colostrum; no immuno- 0 globulin Colostrum; no immuno globulin 33 33 46.5 + 24.2 Colostrum + immunoglobulin 50 50 42.1 + 23.5 (2g/kg/day) Immunoglobulin (lOg/kg/day 16 16 7.5 (1 pig)-to 2g/kg/day) 48 dehydration, no doubt a result of the diarrhea. A higher level of disease carrying microbes in the environ ment might have been present due to the reservoir of infection carried by the negative control animals which were all ill from the beginning of the trial. Sanitation and high moisture levels were difficult to control by washing the cages. The resulting moist, humid, and non-sterile conditions may have been ideal for the growth of microbes. It was assumed that a 12 hour nursing period was adequate for attaining sufficient immunoglobulin levels in the plasma. This is supported by work of Scoot (1972) and Carlson and Lecce (1973). 49 3.3 Experiment II 3.3.1 Objective The objective of this experiment was to further assess the efficacy of abbatoir derived porcine serum immunoglobulin extract as a milk replacer additive for rearing low birth weight pigs. To reduce the pool of infection, negative controls receiving no immunoglobulins (artificial or colostrum derived) were not included in this trial. 3.3.2 Materials and Methods Experimental Animals Twenty-four low birth weight pigs weighing less than lOOOg of Yorkshire X Landrace breeding were allotted within a ten day period. Farrowings were attended and experimental animals were removed at birth or 12 hours later. Pigs were weighed at birth, ear tagged, and individually penned in a nursery room. Pigs were randomly assigned to treatments. Cages were adjoining wire mesh 46cm x 25cm x 20cm in two tiers. Prior to the trial the room and cages were thoroughly cleaned and disinfected. Cages were left unwashed throughout the trial period. Temperature in the nursery room was maintained between 30°C and 33°C. All pigs received iron dextran injections and had their needle teeth and tails clipped at two days of age. A second iron dextran injection was given at 10 days of age. Dietary Treatments 2 Pigs received a non-medicated commercial milk replacer . Feed intake was reduced from the level in experiment I to 6 per cent of body weight in air dry matter which was diluted 1 : 4 with water. For the first 3 days diets were formulated with a 10 per cent dextrose solution. Pigs were weighed every other day and feed allowances were adjusted accord-50 ingly. Immunoglobulin extract was mixed with the milk replacer and fed at levels set out in the following schedule of treatments: 1. Colostrum for 12 hours, followed by milk replacer only. 2. Colostrum for 12 hours, followed by immunoglobulin at 2g/kg body weight/day for 9.5 days. 3. Immunoglobulin (lOg/kg body weight) on the first day followed by immunoglobulin at 2g/kg body weight/day for 9.5 days. Scouring animals were treated, as necessary, on an 4 individual pig basis with Anistat , a broad spectrum antibiotic. The trial was of 21 days duration. Feeding Regimen Pigs were nipple fed dor one week then changed to bowl feeding for the remainder of the trial. Feeding was every 2 hours for the first 4 days, every 4 hours on days 5 to 8, and every 6 hours from days 9 to 21. Fresh water was available to them from day 5 and creep feed was offered fresh daily after 10 days of age. 3.3.3 Measurements and Observations Pigs were weighed every 2 days. Mortality was recorded and post mortems were conducted on all dead pigs by the Provincial Veterinary Laboratory, Abbotsford, B.C.. Infectious agents and causative factors were identified whenever possible. Mortality and rate of gain were analysed as in Experiment 1, using analysis of variance. The mean birth weights of survivors and dead pigs were compared with a "t" test (Choi, 1978). 51 3.3.4 Results and Discussion Mortality (Table 3.2.b) decreased from the level achieved in Experiment 1. This may be attributed to improved technique, elimination of the negative control group (as a pool of infection), and improved environmental conditions. The group receiving only colostrum for 12 hours showed a mortality rate at 21 days significantly higher than the other two groups (P 0.05). There was no significant difference in mortality between thos receiving colostrum with immunoglobulin and those receiving immunoglobulin extract instead of colostrum. Since it was observed in Experiment 1 that feeding at 7.5 per cent of body weight led to a distended stomach and possible gastric stasis producing scours, the level of feeding was reduced to 6 per cent of body weight in air dry matter, and this problem was consequently reduced. White et al_., (1969) found in their trials, that scour was always preceded by gastric stasis. They found that a direct relationship existed between pH and growth of bacteria in the stomach. A lack of lactic acid producing organisms in the stomach of the achlorhydric pig , with resultant high pH in the stomach, might lead to rapid growth conditions for coliforms which could then pass to the intestine and cause a scour syndrome. Endotoxin release in the stomach by coliform bacteria could also lead to reduced gastric acid secretion exacerbating the conditions. There was no significant difference between birth weights of surviving versus dead pigs. 52 Table 3.2a Experiment 2; Experimental Animals Treatment Number in Treatment Mean Birth-weight Group ± S.D. Colostrum; no immuno- 8 677 ± 111 g globulin Colostrum + immunoglobulin 8 707 ± 170 g (2g/kg/day) Immunoglobulin (lOg/kg/day 8 736 ± 143 g to 2g/kg/day) Table 3.2b Experiment 2: Results Treatment % Survival % Survival Rate of Gain to Day 10 to Day 21 ( g/day ) Colostrum; no immuno- 37 0 globulin Colostrum + immunoglobulin 75 63^ 35.2 + 15.4 g (2g/kg/day) Immunoglobulin (lOg/kg/day 63 50b 39.8 ± 16.9 g to 2g/kg/day) (differing superscripts denote statistically different values at P= .05 ) 53 Causes of mortality were general septicaemia for 6 pigs probably due to coliform infection, definite coliform septicaemia for 3 pigs, and coliform enteritis with septicaemia for 7 pigs. All dead pigs showed characteristics of emaciation and dehydration which may be a result of the diarrhea which occurred in all animals which died. Rate of gain did not differ significantly between the two groups of surviving pigs (Table 3.2 b) and overall remained at a very low level. Consumption of creep feed and water supplied ad libitum could not be weighed accurately because of wastage. 54 3.4 Experiment III 3.4.1 Objective The objective of this experiment was to compare a lower level of immunoglobulin extract administration, (lOg/kg body weight for the first day followed by 2g/kg body weight/day for 9 days) with a hioher immunoglobulin level (15g/kg body weight for the first day followed by 5g/kg body weight/day). Treatment groups of pigs nursing colostrum for 12 hours instead of high initial doses of immuno globulin were also included for comparison. 3.4.2 Materials and Methods Experimental Animals Two consecutive replicates were carried out. To each replicate 24 low birthweight pigs of less than lOOOg of Yorkshire X Landrace breeding were allotted within a 10 day period. Farrowings were attended and experimental pigs were removed at birth or 12 hours later. Pigs were weighed at birth, ear tagged, and individually penned in a nursery room. Pigs were randomly assigned to treatments. Cages were adjoining wire mesh 46cm x 25cm x 20cm in two tiers. Prior to the trial the room and cages were thoroughly cleaned and disinfected. Cages were left unwashed throughout the trial period. Temperature in the nursery was maintained between 30°C and 33°C. All pigs received iron dextran injections and had their needle teeth and tails clipped at two days of age. A second iron dextran injection was given at 10 days of age. Dietary Treatments 2 Pigs received a non-medicated commercial milk replacer . Feed intake was set at 6% of body weight of air dry matter which was diluted 55 1 : 4 with water. For the first 3 days diets were formulated with a 10 per cent dextrose solution. Pigs were weighed every other day and fed accordingly. Immunoglobulin extract was mixed with the milk replacer and fed at levels set out in the following schedule of treatments: 1. 15g/kg bodyweight immunoglobulin on the first day followed by 5g/kg body weight/day for 9 days. 2. lOg/kg body weight immunoglobulin on the first day followed by 2g/kg body weight/day for 9 days. 3. 12 hours of colostrum nursing followed by 9.5 days of immunoglobulin at 5g/kg body weight/day. 4. 12 hours of colostrum nursing followed by 9.5 days of immunoglobulin at 2g/kg/day. 4 In replicate 1 the antibiotic Anistat was used. In 3 replicate 2 the antibacterial Furoxone was used. Feeding Regimen Pigs were nipple fed for one week then changed to bowl feeding for the remainder of the trial. Feeding was every 2 hours for the first 4 days, every 4 hours on days 5 to 8, and every 6 hours from days 9 to 21. Fresh water was available from day 5 and creep feed was offered fresh daily after 10 days of age. 3.4.3 Measurments and Observations Measurments and statistical analyses were the same as in the two previous experiments except that another dependant variable was included, frequency of scouring. The design was a randomized block design since two replicates were carried out. 56 3.4.4 Results and Discussion Mortality (Table 3.3..b) was similar to that in Experiment 2 where it was approximately 50 per cent in the treated groups. There was no significant difference between treatments for mortality. Thus, the results indicated no significant difference between those receiving colostrum and those receiving immunoglobulin extract, or between those receiving a higher level of immunoglobulin and thowe receiving a lower level. The assumption that 12 hours of nursing was adequate was based on the findings of Scoot (1972) and Carlson and Lecce (1973). This assumption relies on the pig receiving adequate colostrum during nursing. However, low birth weight pigs are frequently disadvantaged in this respect, not being as competitive or not being able to reach a teat. Thus, in some cases within this trial, individuals may not receive an adequate level of systemic passive immunity which may lead to susceptibility to infection affecting the mortality rate. As was the case, those receiving artificial immunoglobulin extract instead of colostrum, showed a high survival before day 10, but the difference was not significant. The difference between mean birth weights of survivors and dead pigs was not significant. It was observed that Table 3.3a Experiment 3: Experimental Animals 57 Replicate Treatment Number in Treat- Mean Birth-Weight ment Group ± S.D. Immunoglobulin (15g/kg 6 761 ± 166 g /day to 5g/kg/day) Immunoglobulin (lOg/kg 6 729 ± 227 g /day to 2g/kg/day) Colostrum + immuno- 6 835 + 99 g globulin (5g/kg/day) Colostrum + immuno- 6 746 ± 192 g globulin (2g/kg/day) 2 Immunoglobulin (15 to 6 814 ± 83 g 5 g/kg/day) Immunoglobulin (10 to 6 671 + 96 g 2 g/kg/day) Colostrum + immuno- 6 704 ± 218 g globulin (5g/kg/day) Colostrum + immuno- 6 globulin (2g/kg/day) 834 i 59 g Table 3.3b Experiment 3: Experimental Results Replicate Treatments % Survival % Survival Rate of Gain Scouring Frequency - to Day 10~~~ to Day 21 (g/day) days scouring/survivor) Mean Scouring • Frequency 1 15 g/kg for Day 1 then 50 46.3 + 4.0a 7 5 g/kg/day for 9 days 10 g/kg for Day 1 the 2 g/kg/day for 9 days Colostrum for 12 hrs. 5g/kg/day for 9.5 days Colostrum for 12 hrs.; 2g/kg/day for 9.5 days 15 g/kg for Day 1 then 50 55.7 + 3.5a 9 5 g/kg/day for 9 days 10 g/kg for Day.1 then 83 55.7 ± 3.5a 8.8  then 50 39.7 ± 1.2b 5.3  hrs.; 33 51.5 ± 0.7a 5 ; 66 55.3 + 8.8a 3.3 5.0 + 2.6C 2 g/kg/day for 9 days Colostrum for 12 hrs.; 17 59.0(1 only)" 15 5 g/kg/day for 9.5 days Colostrum for 12 hrs.; 50 60.0 ± 8.7a 7 8.9 + 3.5d 2 g/kg/day for 9.5 days Combined 15 g/kg for Day 1 then 84 50 51.0 ± 6.la 5 g/kg/day for 9 days 10 g/kg for Day 1 ther 2 g/kg/day for 9 days Colostrum for 12 hrs.; 5 g/kg/day for 9.5 days Colostrum for 12 hrs.; 2 g/kg/day for 9.5 days n 84 66 41.0 ± 6.5b ; 75 25 54.0 + 4.4a ; 75 58 57.3 ± 8.4a co 59 under500g birth weight there was no survival, however the number of observations was few. Cause of death during the first replicate, in 11 cases was Salmonellosis causing diarrhea and septicaemia. The actual serotype was identified as Group B Salmonella in 4 cases while in the remaining cases serotype was unidentified. In one case death was due to E. coli enteritis. During the second replicate, Salmonella were heavily implicated, but, due to the interference of antibiotics given during treatment, could not be isolated in all cases. Klebsiella was also isolated from two pigs. Rate of gain (Table 3.3.b) was increased over that found in Experiment II. Those receiving immunoglobulin at the lower level showed a significantly lower rate of gain in both replicates (P = 0.05) (Figures 3.1.a and 3.1.b). Since the pigs receiving 2g/kg body weight/ day of immunoglobulin after receiving colostrum showed a significantly higher (P = 0.05) growth rate than those which received artificial immuno globulin (lOg/kg body weight on the first day) instead of colostrum, the effect may be due to an insufficient initiating dose of lOg/kg body weight for passive systemic immunity. However, even if this treatment affected growth rate, it did not show any significant affect on mortality rate. Both Scoot (1972) and McCallum (1977) found that the higher dosage of immunoglobulins (15 and 5g/kg body weight/day) increased rate of gain slightly. Scoot (1972) indicates that blood globulin levels were not as high with artificially supplemented as with sow nursed pigs. Frequency of scouring of the survivors showed no significant differences between the scheduled treatments, however, the frequency of scouring was significantly different between the two replicates (Table Figure 3.1.a Growth in Experiment 3, Replicate 1 Fiqure 3.1.b Growth in Experiment 3, Rep 2000 1500 A -Colostrum + m - " + 2g/kg/day Ig 5g/kg/day Ig • -10 to 2 g/kg/day Ig O -15 to 5 g/kg/day Ig 1000 U If IB 20 62 3.3.b). The obvious reason for this difference seems to have been that different anti-scour drugs were used in each replicate suggesting that 4 the antibiotic Anistat was more effective in controlling scours than 3 the antibacterial Furoxone . In both replicates, the scouring frequency of survivors increased the highest level after removal of pigs from immunoglobulin supplementation at 10 days of age (Figure 3.2). This suggests that the pigs required further passive protection with immuno globulins beyond the tenth day. Figure 3.2. Scouring Frequency (per cent of survivors scourinq) 100 A Experiment 3; Replicate 1 A Experiment 3; Replicate 2 ° Experiment 4 A A A O A A 2 _L_ A A A A A 8 JL A A L_ o o J 1 L_ A A O A J I 1 3 5 Days on Trial 11 13 15 17 19 21 64 3.5 Experiment IV 3.5.1 Objective The objective of this experiment was to compare periods of immunoglobulin feeding. The periods selected for comparison were 10, 15 and 21 days on immunoglobulin extract at a level of lOg/kg body weight for the first day followed by 2g/kg body weight/day for 9 days. 3.5.2 Materials and Methods Experimental Animals Twenty-four low birthweight pigs of less than lOOOg of Yorkshire X Landrace breeding were allotted within a ten day period. Farrowings were attended and experimental pigs were removed at birth. Pigs were weighed at birth, ear tagged, and individually penned in a nursery room. Pigs were randomly assigned to treatments. Cages were adjoining wire mesh 46cm x 25cm x 20cm in two tiers. Prior to the trial the room and cages were thoroughly cleaned and disinfected. Cages were left unwashed throughout the trial period. Temperature in the nursery room was maintained between 30°C and 33°C. All pigs received iron dextran injections and had their needle teeth and tails clipped at two days of age. A second iron dextran injection was given at 10 days of age. Dietary Treatments 2 Pigs received a non-medicated commercial milk replacer . Feed intake was adjusted to 6 per cent of body weight in air dry matter which was diluted 1 : 4 with water. For the first 3 days diets were formulated with a 10 per cent dextrose solution. Pigs were weighed every other day and feed allowances were adjusted accordingly. Immunoglobulin was mixed with the milk replacer and fed at one level: lOg/kg body weight 65 on the first day followed by 2g/kg body weight/day for succeeding days. Three periods of immunoglobulin feeding were applied: 10 days, 15 days, and 21 days. Scouring was treated, as necessary, on an individual pig 3 basis, with Furoxone . Feeding Regimen Pigs were nipple-fed throughout the 21 day trial period. Creep feed was offered from day 10 but water was offered periodically, only, by bottle, to prevent spillage. Feeding was every 2 hours for the first 4 days, every 4 hours on days 5 to 8 and every 6 hours from days 9 to 21. 3.5.3 Measurements and Observations Pigs were weighed every 2 days. Mortality was recorded and post mortems were conducted on all dead pigs by the Provincial Veterinary Laboratory, Abbotsford, B.C. Infectious agents and causative factors were identified whenever possible. Mortality, rate of gain, and frequency of scouring (for survivors) were statistically analysed with analysis of variance using the same model as in Experiment 1. Also the mean birth weights of ; survivors and dead pigs were compared with a "t" test. 3.5.4 Results and Discussion Mortality rate (Table 3.4.b) improved compared to previous 66 Table 3.4a Experiment 4: Experimental Animals Treatment Number in Treatment Mean Birth-weight Group t S.D. 10 days on immunoglobulin 8 674 + 148 g. 15 days on immunoglobulin 8 708 + 111 g. 21 days on immunoglobulin 8 749 + 116 g. Table 3.4b Experiment 4: Results Treatment % Survival % Survival Rate of Gain Frequency of  to Day 10 to Day 21 (g/day) Scouring (Days scouring/ survivor) 10 days 63 50a 40.8 + 23.2cd 1.0 15 days 100 8Rab 37.4 + 14.5C 1.1 21 days 100 100b 60.3 i 10.6d 0.3 67 trials. Mortality was significantly higher (P = 0.05) on the 10 day treatment compared to the 21 day treatment. However, 3 out of 4 which died in the 10 day treatment group did so while receiving immunoglobulin before 10 days of age. Therefore, the result is spurious. The effect of prolonged feeding of immunoglobulin definitely reduced mortality by maintaining better health and preventing scours in those pigs receiving immunoglobulin after 10 days. Death was due to bacterial infection causing diarrhea in 4 cases and pneumonia in 1 case. Salmonella was isolated from 4 of the 5 dead and presumed present also in the fifth. Thus the infection causing death was diagnosed as Salmonellosis in all cases. There was no significant difference between mean birth weights of survivors and dead Pigs. Rate of gain (Table 3.4.b and Figure 3.3) was highest in the group receiving immunoglobulins for 21 days, but only significantly higher (P = 0.05) than the group receiving immunoglobulins for 15 days. The 21 day group had the highest mean rate of gain of all groups in the four experiments carried out. Frequency of scouring (Table 3.4.b) was very markedly reduced during this trial compared to Experiment III where it was also recorded. The group receiving immunoglobulin for 21 days showed no diarrhea after 4 days of age and the other two groups showed a much reduced frequency between 13 and 20 days of age (Figure 3.2). Since bowl feeding was eliminated and nipple-feeding was continued throughout, chances of spreading contamination in the feed were likely markedly reduced. Water provision was limited to periodic bottle feeding since it couldn't be provided ad libitum without spilling leading to a wet Figure 3.3. Cumulative Weight Gain. Experiment 4 1400 f 1200 1000 800 600 400 200 A - 10 days on Immunoglobulin O - 15 days on Immunoglobulin • - 21 days on Immunoglobulin 11 13 15 Days of Age 69 environment which seemed to be deterimental to the pigs from experience in the preceding trials. 70 3.6 General Discussion The approach of this study was two-fold. Firstly, arti ficial rearing was seen as a mean for increasing survival of low birth weight pigs. Secondly, this artificial rearing technique with immuno globulin extract was applied in a non-isolated environment. Thus, the results can be discussed from both perspectives. An important observation in these trials was a lower rate of gain than previously found by McCallum (1977) using a very similar diet, and by other workers (Figure 3.4). The rate was highest with the 21 day treatment in Experiment IV where scouring was markedly reduced but was still well below rates found with sow reared pigs of low birth weight within the same herd (60g/day vs 125g/day). Two outstanding reasons for this are evident. Firstly, the pigs on trial may have been restricted in their food intake too much for optimum growth. Considering the diet to be adequate, the main contributing factor could be too infrequent feeding since their intake at each feeding was limited by their capacity. However, without automatic feeding, more frequent feeding would be difficult. Secondly, the lower rate of gain may have been due to their low birth weights. Low birthweight seems to have a definite effect on rate of gain. However, Lodge and Elliot (1979) indicated that a lower birth weight was less inhibiting on growth under artificial rearing than under natural rearing. Widdowson (1971), in a trial, showed that runt pigs never reach the potential of large litter mates. One must distinguish between low birthweight pigs which are normal anatomically , but small, and true runts. Not all pigs under lOOOg are necessarily runts. The Figure 3.4 Comparative Growth Curves A -A -o -: Perry and Lecce (1968); artificial rearing » " " " ; sow rearing Siers et al. (1977); artificial rearing ; sow rearing O - This trial: Experiment IV (21 days on Ig) 10 12 14 16 18 20 22 Days of Age 72 manifestation of runt characteristics is largely indicated by size, however pigs above 600g don't seem to show any runting appearance. True runts seem underdeveloped, possibly due to prenatal undernourishment. Their physical appearance of very small size and disproportionately large domed heads suggests this. Small, but non-runt pigs may not be as disadvantaged as true runts when provided adequate nutrition and hence may show adequate compensatory growth to make up for low birthweight. Indeed, in all but the first trial, birthweight did not affect survival. However, all those below 500g at birth, died. It is possible that the survival of very small true runts (e.g. below 500g birthweight) requires more extraordinary attention to hygiene and environment. During sampling of the sow-reared low birthweight pigs, variability on growth rate was found to be high (coefficient of variation = 30.4 per cent). This is understandable if the operative factors deter mining growth at this pre-weaning age are considered. Such factors include: 1. Nursing success which is determined by aggressiveness, milk production of the sow, and access to a productive teat. 2. Freedom from scours or other disease maladies. 3. Genetic potential of the individual. Since low birthweight pigs are less competitive during nursing and are more susceptible to chilling (as explained by the relationship between weight, surface area, and heat loss (Monteith and Mount, 1974)), artificial rearing in a controlled environment would eliminate many of the stress ful factors and allow a more equitable situation. Runts may be even 73 further disadvantaged for survival because of a low reserve of carbohydrate in the liver and muscles at birth for energy (Widdowson, 1971). The high survival levels in Experiment IV provide support for the advantage of artificial rearing of low birthweight pigs. The factors contributing to the efficacy of the immuno globulin extract can be analysed. This extract, which is a precipitate "salted out" with a 40 per cent (NH4)2 SO^ solution, contains all of the gamma-globulin and much of the beta-globulin fractions of serum (Owen, 1961). Like colostrum, which is considered a serum transudate (Bourne, 1973), it would contain over 80 per cent IgG, less IgA then colostrum (8 vs 13 per cent), and more IgM than colostrum (10 vs 4 per cent) (Bourne, 1973). Also, unlike colostrum, serum derived immungolobulin contains no secretory IgA (S-IgA). One could presume that the efficacy of the extract was predom inately due to IgG. However, the presence of IgM and non-secretory IgA cannot be ignored. Miler et al_., (1975) found that the minimum effective concentrations for local protective effect in ligated loop tests with pigs were 0.5, 0.05, and 0.005 mg/ml of IgG, IgM, and IgA respectively. There fore, even lower levels of IgM and IgA may have a significant protective effect. IgG itself, has been found to be of significant protection against enteric colibacillosis (Brandenburg and Wilson, 1972). How IgG functions within the gut lumen is open to question but it has antitoxin and agglu tinating properties (Beneceraf and Unanue, 1979). Although it may not be able to adhere to the gut lining as S-IgA does, agglutination of bacteria would faciliate their peristaltic removal. Finally, the efficacy of this extract against Salmonella bacteria is of considerable interest. To date, this technique has been 74 used to prevent mortality due to E. coli enteritis (Owen e_t aj_., 1961; Scoot, 1972; McCallum, 1977; and Kenelley et_ al_., 1979). Since it protects against Salmonella, as shown in the present study, many of the herds from which the blood for immunoglobulin extraction was taken must have antigenic quantities of Salmonella infesting them. The contribution of these herds must have been great enough to provide a sufficiently high antibody titre against Salmonella to be preventive in these experiments. 75 Conclusions 1. The artificial rearing technique with artificial immunoglobulins used in these trials will save low birthweight pigs if weaned at birth. However, growth rate is restrained, possibly by restrictive feeding or by their low birth weight. 2. The level of immunoglobulin of 10 g/kg./body weight on the first day followed by 2 g/kg/day on succeeding days seems to be effective as a dosing level. 3. Longer periods of immunoglobulin administration may be necessary in a non-isolated environment. Whether this is a result of low birthweight is not known. 4. This immunoglobulin extract used was effective protectio against the Salmonella bacteria encountered in these trials. Certain types of Salmonella may be present at endemic levels in many swine herds. 5. 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