.~~. "—an :- U' w. n m. o. . s u u . mm b 9%. I A .. n5 .¢ 1...: L. .58 4.. a nu... “3!; 7‘ .d J.” ( :1." I": y i. A>Ina '. :21. . 7 . . . .MJM ...v. ”.1” .. ... 34.. 7 3; . at; .. A.» ,. .. 4.4:. .. t . .. v This is to certify that the thesis entitled IgA Immunoglobulin Response to Eacherichia coli in Prenatal and Postnatal Calves presented by Behzad Yamini has been accepted towards fulfillment of the requirements for Ph . D . degree in Pathology ma fléfi‘ Major professor Date January 26, 1976 0-7639 n' " “" ' WT: ABSTRACT IgA IMMUNOGLOBULIN RESPONSE TO ESCHERICHIA COLI IN PRENATAL AND POSTNATAL CALVES BY Behzad Yamini This study emphasized the chronologic appearance of immuno- globulin A (IgA)-containing plasma cells and their distribution and numbers in the intestinal tract, spleen and mesenteric lymph nodes in beef calves vaccinated in utero with Escherichia 0011 (E. coli) bacterin. The immunologic and pathologic effects of revaccination at birth and of challenge with E. coli were also studied. The 17 principal calves were vaccinated in utero by a non- surgical procedure with E. coli 026:K60:NM bacterin (5.0 x 1010 organisms) being inoculated into the amniotic fluid during the last 6 weeks of gestation. Nine control calves were given an intrauterine inoculation of saline. The calves were deprived of colostrum after birth and were divided into 5 principal and 5 control groups. Calves in 2 out of 5 principal groups were given a single in utero injection of bacterin, 1 group was killed at birth, and 1 group was given E. coli at birth and killed 2 to 4 days after challenge. Calves in the other 3 principal groups were revaccinated orally at birth after a previous single in utero injection of bacterin. The calves in l of Behzad Yamini the revaccinated groups were killed at 5 to 7 days of age. Calves in 1 group were given E. coli at 3 days of age and were killed at 3 to 5 days after challenge. Calves in the last group were killed at 14 days of age. Calves in l of the 5 groups of controls were killed at birth, 1 group of calves at 9 days and another at 14 days of age. Control calves in a fourth group were challenged with E. coli at birth and killed at 1.5 to 4 days of age. Control calves in the last group were given E. coli at 3 days of age and killed at 4 days after challenge. The challenge doses were 1.5 x 1010, 1.0 x 1011 or 1.5 x 1011 E. coli organisms. Only 1 calf from the principal group and l calf from the con- trol group had clinical signs of colibacillosis after challenge with the intermediate or high challenge dose, respectively. The most prominent histologic lesions in these 2 calves were congestion and edema of the small intestine and infiltration of bacteria into the brush border of the villi. There was serologic evidence to suggest that 2 of the control calves had consumed colostrum. These 2 calves failed to have clinical signs of colibacillosis after the challenge dose and acquired colostral antibody was the most probable cause of the resistance. Gel filtration and immunoelectrophoresis in agar-gel were used to purify the 19A in samples of bovine colostrum. The purified IgA was injected into guinea pigs. Serum from these guinea pigs was used as the source of the anti-IgA for the studies utilizing immuno- fluorescence and immunoelectrophoresis. The serum tube agglutination and serum passive hemagglutination assays (PHA) titers to 026 antigen at necropsy were higher than at Behzad Yamini birth from calves in principal groups. The PHA titers of intestinal washings were very low or negative. By IEP, intestinal washings were positive with anti-IgA immunoglobulin in 4 principal calves and 2 control calves, but these results did not correlate with the PHA titers. Studies using immunofluorescence indicated that there were appreciable numbers of IgA-producing plasma cells in in utero vac- cinated calves at birth. These cells became more numerous in orally vaccinated or challenged calves. The lower jejunum and ileum and related lymph nodes had more IgA-producing cells than any of the other tissues examined. Furthermore, in revaccinated and challenged calves the spleen was especially active in the formation of IgA- containing plasma cells. The duodenum had more limited activity. The results indicate that the entire small intestine, the draining lymph nodes, and the spleen were involved in IgA formation in these young calves. The control calves had no indication of fluorescent plasma cells before 9 days of age. This further demonstrates the importance of age in IgA immunoglobulin production. The immuno- electrophoretograms of serum samples of principal and control calves at birth and necropsy were negative for IgA immunoglobulin. IgA IMMUNOGLOBULIN RESPONSE TO ESCHERICHIA COLI IN PRENATAL AND POSTNATAL CALVES BY Behzad Yamini A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1976 Dedicated to Love and Caring ii ACKNOWLEDGEMENTS I wish to express my gratitude and appreciation to Dr. S. D. Sleight and Dr. G. L. Waxler from the Department of Pathology for their guidance and direction during this research. I am grateful to Dr. G. H. Conner of the Department of Large Animal Surgery and Medicine, and to Dr. J. F. Williams of the Department of Microbiology and Public Health for their assistance. My thanks is also extended to Dr. R. w. Leader, Chairman of the Department of Pathology, for advice and for reading the manuscript. Particular appreciation is extended to Dr. R. F. Langham of the Department of Pathology for his generous assistance and advice throughout my training at Michigan State University. I also thank Drs. Morrill, Whitehair, Sanger, Keahey, Trapp and Dade from the Department of Pathology for the training which I received in practical aspects of pathology. Sincere thanks is extended to the other members of the Depart- ment of Pathology, especially to Ms. Joann Finn, Mrs. Mae Sunderlin and Mrs. Frances Whipple for their help. My gratitude and appreciation are also extended to Dr. M. Kaveh, General Director of Razi State Institute, Tehran, Iran, and Dr. H. A. Neshat, the Dean of the School of Veterinary Medicine, University of Tehran, Iran. iii Sincere thanks to the members of the Department of Pathology, School of Veterinary Medicine, University of Tehran, and the Department of Pathology, Razi State Institute. During this research, I was supported through a senior graduate assistantship from Michigan State University and by Razi State Institute, Iran, and the University of Tehran, Iran. iv INTRODUCTION. OBJECTIVES. TABLE OF CONTENTS REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . . . . . General Immunology . . . . . . . . . . . . . . . . . . Ontogenic Development of Immune Response in Calf . . . Lymphoid Tissue Development . . . . . . . . . . Immunoglobulin Development. . . . . . . . . . . Cell Mediated Immunity (CMI). . . . . . . . . . Antibody to Microbial Agents. . . . . . . . . . Antibody to Nonmicrobial Antigens . . . . . . . Transmission of Immunity in the Bovine Species . . . . Transmission of Antibodies Before Birth . . . . Transmission of Antibodies After Birth. . . . . Transmission of Homologous y-Globulins After Birth. . . . . . . . . . . . . . . . . . . . Termination of the Transmission of Passive Immunity . . . . . . . . . . . . . . . . . . The Immune Globulin of Colostrum. . . . . . . . Transport of Globulin from the Intestine to the Circulation. . . . . . . . . . . . . . The Decline of Passive Immunity and Development of Active Immunity . . . . . . . . . . . . . IgA and Secretory Immunoglobulin . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . Structure of Serum IgA. . . . . . . . . . . . . Physicochemical Properties . . . . . . . Subunit Structure. . . . . . . . . . . . Secretory IgA . . . . . . . . . . . . . . . . . J Chain. . . . . . . . . . . . . . . . . Secretory Component (SC) . . . . . . . . Immunoglobulin Other Than IgA in Secretions . . Synthesis of Secretory Immunoglobulin . . . . . Ontogeny of the IgA System. . . . . . . . . . . Page l3 14 14 15 16 18 18 19 20 22 22 23 24 24 25 25 26 27 The Mechanism by Which Secretory IgA Operates . . IgA in Bovine Serum and in Other Secretions . . . The Bovine Intestinal Secretory Immune System . . Colibacillosis . . . . . . . . . . . . . . . . . . . . . Normal Bacterial Flora of the Calf Intestine and Its Aberrations in Cases of Colibacillosis . . Identification and Classification of the E. coli Group . . . . . . . . . . . . . . . . . . Epidemiology of Colibacillosis in Calves. . . . . Pathogenesis. . . . . . . . . . . . . . . . . . . The Effect of Metabolic Changes in the Patho- genesis of Neonatal Diarrhea . . . . . . . . . Treatment and Control . . . . . . . . . . . . . . Enteric Disease Other Than Colibacillosis. . . . . . . . Immunization Before Birth (Intrauterine Vaccination) . . MATERIAL AND METHODS. . . . . . . . . . . . . . . . . . . . . . Animals. . . . . . . . . . . . . . . . . . . . . . . . . cows 0 O O O O O O O O O O I O O O O O O O O O O 0 Laboratory Animals. . . . . . . . . . . . . . . . Antigens . . . . . . . . . . . . . . . . . . . . . . . . Bacterin (Vaccine). . . . . . . . . . . . . . . . Challenge Inoculum. . . . . . . . . . . . . . . . Fetal Inoculation. . . . . . . . . . . . . . . . . . . . Preservation and Collection of Tissues . . . . . . . . . Protein Analysis . . . . . . . . . . . . . . . . . . . . Immunologic Procedure. . . . . . . . . . . . . . . . . . Immunoelectrophoresis (IEP) . . . . . . . . . . . Double Immunodiffusion (Ouchterlony Method) . . . Direct Bacterial Agglutination Passive Hemag- glutination Assay (PHA) and Antiglobulin Augmentation . . . . . . . . . . . . . . . . Preparation of Anti~Whole Bovine Serum (AWBS) . . Separation of IgA from Colostrum. . . . . . . . . Preparation of Antisera to IgA. . . . . . . . . . Conjugation of Anti-IgA to Fluorescein Iso— thiocyanate (FITC) . . . . . . . . . . . . . . Frozen Section Procedure (Cutting, Staining). . . vi Page 28 29 3O 32 33 34 36 38 4O 41 42 42 45 45 45 47 47 47 48 48 49 50 50 50 52 52 52 53 55 56 56 RESULTS Bacteriologic Procedure. Histopathologic Technique. Photography. DISCUSSION. SUMMARY REFERENCES. VITA. vii Page 57 57 57 58 78 88 91 106 Table LIST OF TABLES Page Data related to in utero injections of bovine fetuses with E. coli 026:K60:NM bacterin (Groups 1-5) or saline (Groups 6-10) . . . . . . . . . . . . . . 61 Direct bacterial agglutination titers of serum and passive hemagglutination titers from serum and intestinal washings from neonatal calves previously injected in utero with E. coli 026:K60:NM bacterin (Groups 1-5) or saline (Groups 6-10) . . . . . . . . . . 70 Plasma cell counts in tissues stained with fluorescein conjugated anti-IgA immunoglobulin and from neonatal calves previously injected in utero with E. coli 026:K60:NM bacterin (Groups 1-5) or saline (Groups 6-10). . . . . . . . . . . . . . . . . . . . . viii Figure LIST OF FIGURES Experimental groups of calves and ages at reimmuniza— tion, challenge and euthanasia. The number of animals in each group is indicated in parentheses. Photograph of the immunoelectrophoretic pattern of monospecific anti—IgA produced in guinea pigs. Monospecific anti-IgA (A); semipurified IgA from colostrum (C); antiwhole bovine serum (B) produced in rabbit. Notice the immunoprecipitate bands IgA (a), IgM (m), IgGl (g) . . . . . . . . . . . . . . . . Photograph of the Ouchterlony assay (1) and immuno- electrophoretic pattern (2) of monospecific anti- bovine IgA produced in guinea pig. Monospecific anti-IgA (a); monospecific anti-IgA received from Cornell (b); semipurified IgA from colostrum (C). Notice that the antiglobulin is monospecific . . . . Photomicrograph of the lower jejunum of control calf 4657 (group 7) challenged at birth with E. coli 026:K60:NM and euthanatized at 3 days of age. Notice extensive edema beneath mucosal epithelium (1). Photomicrograph of mesenteric lymph node of control calf 1912 (group 7) challenged at birth with high dose of E. coli 026:K60:NM and euthanatized at 1.5 days of age. Notice extensive infiltration of neutro- phils (arrow) into the sinusoids . . . . . . . . . . . Photomicrograph of the villi of the ileum from con— trol calf 1912 (group 7) challenged at birth with a high dose of E. coli 026:K60:NM and euthanatized at 1.5 days of age. Notice edema (l), inflammatory cells (2), and accumulation of bacteria at brush border (3) . . . . . . . . . . . . . . . . . . . . Photomicrograph of the Spleen of principal calf 1214 (group 2) previously vaccinated in utero, revac- cinated orally at birth and challenged at 3 days of age with E. coli 026:K60:NM. Calf was euthanatized at 6 days of age. Notice plasma cells (arrow) . ix Page 46 59 6O 64 65 67 68 Figure Page Photomicrograph of frozen section from spleen or principal calf 1214 (group 2) previously vaccinated in utero, revaccinated orally at birth and challenged at 3 days of age with E. coli 026:K60:NM. Calf was euthanatized at 6 days of age. Stained with fluorescein conjugated anti-IgA immunoglobulin. Notice positively stained plasma cells (arrows). . . . . . . . . . . . . . 76 Photomicrograph of frozen section from ileum of principal calf 1214 (group 2). Stained with fluore- scein conugated anti-IgA immunoglobulin. Notice positively stained plasma cells (arrow). . . . . . . . . 77 INTRODUCTION Calf mortality due to neonatal diseases is a major problem and economic losses continue despite extensive efforts at preven- tion and control in most parts of the world, including the United States and Iran. The enteric form of colibacillosis and colisepti- cemia caused by Escherichia coli (E. coli) are probably the most common causes of death or stunted growth in young calves. Chemotherapy or vaccination of the dam or calf after birth with formalin-killed bacterins have been of little help so far. Poor sanitation and ventilation, overcrowding, inadequate feeding of colostrum and stress are important in increasing the suscepti- bility of calves to E. coli. Considerable work has been done in recent years on immunologic aspects of host defense mechanisms in disease. Studies on onto- genetic development of the fetal calf and its capacity to respond to antigenic stimulation may have provided the impetus for exploring the possibility of in utero vaccination. It is now very well documented that secretory immunoglobulins secreted by plasma cells in mucous membranes provide protection against pathogenic microorganisms. One of the secretory immuno- globulins (IgA) is thought to be important in the prevention of respiratory and gastrointestinal diseases in neonates. It is believed that the young calf receives the IgA from colostrum and l is not capable of producing secretory IgA at least during the first 10 to 14 days after birth. OBJECTIVES The objectives of this research were: 1. To obtain evidence as to the presence of IgA immunoglobulin in the intestinal tissue and washings of neonatal calves. 2. To study the response of calves to primary prenatal and secondary postnatal vaccination with E. coli. 3. To evaluate the resistance of vaccinated calves after exposure to pathogenic E. coli. REVIEW OF LITERATURE General Immunology One of the most important means by which an individual main- tains its well being in an environment is by immunity. A true appreciation of the complex nature of the immune system and the consequences of a deficiency in one or more parts of this system have become apparent within the last 10 years (Osburn et al., 1974). There is now good evidence that many diseases, including neoplasms, result from failure of the host's immune system to eliminate the invading organisms or cells. A similar situation exists in the developing fetus and neonate, and the deficiency may be the result of an immature immune system or of an acquired deficiency. It is now recognized that animals possess "multipotential" immune response mechanisms consisting of both a cell—mediated and a humoral component or the thymic and bone marrow system, respec- tively (Claman and Chaperon, 1969; Cooper et al., 1968). The lympho- reticular system harbors most of the cells (lymphocytes and macro- phages) that are necessary for immunization. The stem cells of lymphocytes are in the liver in fetal development and in the bone marrow during postnatal life. The stem cells give rise to lympho- cytes, some of which migrate via the blood vascular system to the thymus. In the thymus, these cells acquire new physical and bio- logical characteristics. After leaving the thymus, the cells, known 4 as thymic lymphocytes (T cells), migrate to lymph nodes and the spleen, where they localize in specified anatomic sites. The cells continually circulate by leaving the lymph nodes via lymphatics and the thoracic duct. They return to lymphoid tissue through the blood by traversing the postcapillary venules. Thymic lymphocytes, which constitute approximately 80% of the lymphocytes observed in circu- lating blood (Ritzmann et al., 1973), are the principal element involved in cellular immunity (Dumonde and Mairi, 1971). In addi- tion, lymphocytes leaving the bone marrow may go directly to lymphoid tissue, where they locate in the cortex and medullary cords of lymph nodes. These cells, which are known as bone marrow lymphocytes (B cells), eventually become plasma cells, which produce serum immunoglobulins with specific antibody activity (Claman and Chaperon, 1969; Cooper et al., 1968). One important function of T cells is to serve as bearers of cellular immunity. When the appropriate antigen interacts with the complementary receptor sites on the membrane of the T cells, the cell is stimulated to release lymphokines (Dumonde and Mairi, 1971). The biologic effects of the lymphokines are quite diverse and include mitogenic factor, macrophage—activating factor (MAF), migration- inhibition factor (MIF), lymphocytotoxic factor, and chemotactic factor (Dumonde and Mairi, 1971). In addition, lymphokines appear to amplify the B-cell response associated with some antigens. The principal function of the B cell is to secrete specific antibodies when appropriately stimulated by complementary antigens. Evidence suggests that before successful antibody production occurs there 6 must be an interaction between T cells, B cells and macrophages (Claman and Chaperon, 1969). Ontogenic Development of Immune Response in Calf Knowledge of the time at which immunologic competence develops in the fetus and neonate and the cells and organs concerned with that development are of significance in a basic understanding of the immune response (Sterzl and Silverstein, 1967). The under- standing of these basic concepts made it logical to attempt to control disease by intrauterine and intrafetal vaccination (Conner et al., 1973; Gay, 1971, 1975; Kendrick, 1973; Schultz et al., 1973). Ontogenetic development of immunologic function is largely dependent on two variables: species and length of gestation. The average gestation period for cattle is approxi- mately 280 days and generally only one fetus is present (Brambell, 1970). Lymphoid Tissue Development Although lymphoid precursor cells are presumably present in the yolk sac and fetal liver, lymphocytes were first recognized (Schultz et al., 1970) at 42 days of gestation in the developing lobules of the fetal thymus. Schultz et a1. (1973) first observed Hassell's corpuscles in a 65-day fetus and they were present in the thymus thereafter. Lymphocytes were first recognized in the bone marrow at 55 days. Lymphoid cells in bone marrow were difficult to recognize at all ages due to their relative paucity when compared to the other hemopoietic cells, primarily granulocytes and immature erythrocytes. Peripheral lymphoid tissues developed later than the thymus. The spleen was observed at 55 days; however, lymphoid cells were not recognized until 59 days of gestation. Hematopoiesis was reported to begin at approximately 60 days in the spleen. Red and white pulp was partially differentiated in the 80— to lOO—day fetus. The prescapular and prefemoral lymph nodes were recognized at 60 days of gestation. Popliteal lymph nodes were observed at 65 days, the supramammary node at 90 days, and the cervical, mediastinal and mesenteric nodes by 100 days. There were few lymphocytes present and cortical and medullary areas were not differentiated nor were germinal centers observed at early developmental stages. True lymphoid tissue was observed in the tonsil at approximately 175 days. Lymphoid development of the gastrointestinal tract, particularly the lower ileum, occurred at about 175 days with a few lymphocytes present in the lamina propria at approximately 150 days (Schultz et al., 1973). Lymphocytes were first recognized in the peripheral blood at 45 days, approximately the same age at which the lymphoid thymus appeared; and lymphocytes were the only leukocytes in the peripheral blood up to 120 days when granulocytes were observed (Schultz et al., 1971). A sharp elevation in total neutrophils was observed near parturition (Hubbert and Hollen, 1971; Schultz et al., 1971). Immunoglobulin Development At least 3 distinct immunoglobulin classes are present in cattle: IgG, IgM, and IgA (Butler, 1969; Butler et al., 1971; Mach and Pahud, 1971). There are 2 distinct subclasses of IgG: IgGl 8 and IgG (Butler et al., 1971). It is also suggested that the cow 2 has an immunoglobulin similar to human IgE (Hammer et al., 1971; Schultz et al., 1973). Employing the immunofluorescent technique, Schultz et a1. (1973) reported that cells containing IgM were present in the spleen of a 59—day-old fetus. In the same study IgG-containing cells were first recognized in a l45-day-old fetus. Numerous lymphoid organs contained these cells, but the spleen was the organ which most fre- quently had Ig-containing cells. Several reports are available on the occurrence of immuno- globulins in sera of bovine fetuses not overtly stimulated with antigens. Pierce (1955), using immunoelectrophoresis, reported that precolostral calf serum contained a low level of Ig. Schultz et a1. (1971) compared immunoelectrophoresis and qualitative radial immunodiffusion and reported that 13 of 95 fetuses of varying ages contained 19 by immunoelectrophoresis and 39 out of 95 samples con- tained Ig as determined by radial immunodiffusion in fetuses which had not received overt antigenic stimulation. Initial detection of IgM was made in serum at 130 days and IgG at 145 days. Schultz et a1. (1971) reported that 10 fetuses older than 220 days had IgM- and IgG-containing cells in their spleen but did not have IgA in the serum nor IgA-containing cells in the tissues. In a more recent study (Schultz, 1973), IgA was detected in the serum of 2 of 135 fetal and precolostral serum samples. The gas— trointestinal tract of the calf, which would be expected to contain large numbers of IgA-containing cells, had in addition to IgA- containing cells a large number of IgM-containing cells (Porter et 9 al., 1972). A similar observation was made by Schultz et al. (1971) in the lacrimal gland of late term fetuses (265 to 275 days). Cells with IgA were expected but only cells with IgM were present. This may suggest that IgM-containing cells occur early in development to be replaced later by cells which produce IgA. An intraclonal switch in the constant region of the heavy chain of IgM to IgA synthesis may occur similar to the switch mechanism proposed for IgM to 196 (Lawton et al., 1972). Cell Mediated Immunity (CMI) Few studies have appeared on CMI in the bovine fetus. In a study on the occurrence of phytohemagglutinin responsive cells in various lymphoid tissues of the bovine fetus, the youngest fetus examined had PHA responsive cells in its peripheral blood at 105 days of age (Schultz, 1973). Osburn (1972) found a decrease of PHA responsive cells beginning at approximately 250 days of gestation with a minimal responsiveness near parturition. The suppression of CMI presumably results from a rise in corticosteroids required to initiate parturition. Billingham and Lampkin (1957) studied skin graft rejection in newborn calves and demonstrated that a 264-day-old premature calf was able to reject skin allografts in a manner similar to older calves. Antibody to Microbial Agents One of the first viral infections in which neutralizing anti— body was detected in fetal or precolostral calf serum was bovine virus diarrhea (BVD). Ward et a1. (1969) found that 4 of 11 calves 10 from cows infected with BVD at 5 to 7.5 months of pregnancy had anti- body before suckling and that the other 7 maintained their antibody for 6 months before a decline in the titer, thus suggesting active immunity in these calves. Braun 9t al. (1973) reported on intrafetal inoculation with BVD virus at different stages of gestation. Virus isolation, IgM and IgG values and neutralizing antibody titers of fetal sera were determined. Virus was isolated from the fetus for 56 days after inoculation and for as long as 170 days from the amniotic fluid. The youngest fetus inoculated at 100 days of age had elevated IgG and IgM values as determined by radial immunodif- fusion and had a serum neutralizing titer to the virus at birth. Brown (1973) studied fetuses from 14 cows infected with BVD at 150 days of gestation. The fetuses were taken by hysterotomy from 4 days after infection to birth. The IgM values increased at 2.5 weeks after infection and were maximally elevated at 3 to 4 weeks, declining toward parturition. The IgG values increased at approxi- mately 3 weeks and were maximal at about 10 weeks after infection. With the exception of one fetus, IgG values remained low and IgA 2 was not observed except for the fetus with elevated IgG2. In a serologic survey of 100 aborted fetuses, Dunne et a1. (1973) reported that HI antibodies to PI-3 virus occurred in 53% of the fetuses. Antibodies were first observed during the 4th month of gestation. Fetuses younger than 4 months were negative for HI antibodies. Gibson (1971), using an attenuated strain of IBR virus, initially observed a serum neutralizing antibody titer in a 6- month-old fetus. Antibody titers to the virus were regularly 11 observed after that age. Fetuses inoculated in the 4th and 5th month with IBR virus did not develop neutralizing antibody. Kendrick (1973) reported on the intrafetal vaccination of 7 fetuses with inactivated IBR virus. All fetuses from 3 to 8 months of age had circulating antibody and/or antibody could be extracted from splenic cells 35 to 56 days after inoculation. The immune response of the bovine fetus to several bacteria has been examined. Fennestad and Borg-Peterson (1962) subjected 8 pregnant cows to laparotomy and inoculated them intraplacentally with Leptospira saxkoebing. Six of the fetuses produced agglutina- ting antibody to the bacteria when tested 32 to 62 days later. They concluded that the bovine fetus can produce antibody at less than 164 days of gestation and that antibody synthesis was accom- panied by lymphoid tissue development and the formation of plasma cells. Gibson (1971) studiedtfimaimmune response of fetuses, 4 to 9 months of age, to heat inactivated Brucella abortus strain 19 vaccine and a sonic extract of Br. abortus cells. Both the whole organism and extract were incorporated in complete Freund's adjuvant. TWO fetuses inoculated at 90 to 100 days produced antibody which was detected by the indirect hemagglutination test 57 days after inoculation. Osborn and Hoskins (1971) inoculated viable Vibrio fetus into the placental cavities of 11 cows at 122 to 245 days of gestation. Cows inoculated at 122 to 182 days of gestation aborted dead fetuses 5 to 7 days after inoculation. Cows infected after 182 days had increased IgM levels and specific antibody. 12 Gay (1971) inoculated E. coli intrafetally to determine if the immunized fetuses could survive challenge at birth with a nonrelated virulent strain. Twenty-one fetuses were inoculated at approxi- mately 245 days or older. Eight were inoculated intramuscularly with E. coli in Freund's incomplete adjuvant. Thirteen were inocu- lated intra-amniotically, 11 without adjuvant and 2 with Freund's incomplete adjuvant. Fetuses vaccinated by all of the above routes for 11 or more days survived challenge. However, those vaccinated for less than 11 days had colisepticemia and died in 30 to 96 hours. The problems most often encountered after intrafetal vaccination were premature births and stillborn fetuses. In addition to bacteria and viruses, bovine fetuses have been inoculated with other microbial agents, including: Chlamydia, Coxiella burnetti and Anaplasma marginale (Sawyer et al., 1973; Trueblood, 1971). Newborn calves were found to be immunologically competent to bacteriophage 0X-l74 (Schultz, 1970). Antibody to Nonmicrobial Antigens Bovine fetuses inoculated with ferritin and ovalbumin in com- plete Freund's adjuvant during the 4th month of gestation developed antibody to ferritin within approximately 30 days after inoculation. Antibody to ovalbumin was first observed when a 5-month-old fetus was inoculated and a sample was collected 56 days after inoculation (Gibson, 1971). Antibodies to sheep red blood cells were reported in 16 of 22 samples of precolostral sera (Rice and Duhamel, 1972). 13 Transmission of Immunity in the Bovine Species The bovine species has specialized regions of the uterine mucosa called caruncles where placental attachment occurs. There are 70 to 120 caruncles in the uterus of the cow, and they are circular or oval, free from glands and regularly arranged in rows. They are present at all times but enlarge during pregnancy. The fetal tropho- blast of the allantochorion, where it is in contact with a caruncle, thickens and gives rise to allantochorionic villi which grow into the caruncle, the uterine epithelium of which degenerates so that the fetal villi are lodged in crypts in the highly vascular subepi- thelial connective tissue. These tufts of villi are called cotyledons and with their corresponding caruncles form placentomes. Since the chorionic trophoblast is in direct contact with the uterine subepi- thelial connective tissue, there is one less layer of tissue, the uterine epithelium, intervening between the maternal and fetal circu- lations than in the epitheliochorial placenta of the mare and the sow (Steven, 1968). Transmission of Antibodies Before Birth The evidence suggests that there is no transmission of immunity before birth in the cow. McAlpine and Rettger (1925) found that calves before suckling are always negative for both agglutination and complement fixation tests for Brucella abortus, regardless of whether or not their mothers were positive reactors. Brown (1958) could detect no antibodies in the sera of calves from cows immune to rinderpest when samples were obtained before the calves had Suckled. Graves (1963) showed that cows vaccinated with the virus 14 of foot and mouth disease did not transmit neutralizing antibodies to their calves before suckling. Kulangara and Schechtman (1963) injected human serum albumin intravenously into cows near full term and, in another experiment, these authors also injected human serum albumin into the uterine lumen. Although the albumin was present in significant quantity in the maternal serum at parturition, it could not be detected in the serum of the calf. Transmission of Antibodies After Birth Orcutt and Howe (1922), McAlpine and Rettger (1925), and McDiarmid (1946) confirmed that antibodies to Brucella abortus appeared in calf serum within 2 hours of suckling, and titers increased up to 24 hours. Thereafter the concentration of antibody declined gradually over the next 6 months. Diphtheria and tetanus antitoxins and antibodies to Hemophilus pertussis, vaccinia, rabies, rinderpest, foot and mouth disease, Escherichia coli, Trichomonas fetus, and Salmonella sp. are all transmitted to calves by way of the colostrum (Brambell, 1970). Transmission of Homologous Y-Globulins After Birth Howe (1921, 1924) and Orcutt and Howe (1922) found that the amounts of euglobulin and of pseudoglobulin in the serum of the newborn calf were negligible before suckling. These amounts rose rapidly after suckling and reached peak values at about 1 day. SanClemente and Huddleson (1943) examined the sera of newborn calves before and after the ingestion of colostrum. They found that before ingestion of colostrum, the outstanding characteristic of the serum was the extremely high concentration of o—globulin and the nearly 15 complete absence of y-globulin. Within 4 hours of ingesting colostrum the y-globulin might account for about 15% of the total protein or, if the cow had been infected with Brucella, as much as 30 or 40%. Polson (1952) agreed that the calf at the time of birth has no Y- globulins in its serum, though they are present at high concentra- tions within a few hours after the first feeding of colostrum. There is considerable evidence that other soluble proteins, as well as serum proteins which reach the intestine of the newborn calf, are transmitted to the circulation indiscriminately (Pierce et al., 1964). Termination of the Transmission of Passive Immunity The gut of the calf transmits immunoglobulins from the lumen to the circulation for only a few hours after birth under normal circumstances. The transmission is rapid and intensive. The ruminants resemble the pig and horse in this respect and differ from rats and mice in which transmission continues almost throughout lactation. It is now well documented that the gut of the newborn calf loses its permeability to large molecules during the first 24 to 30 hours of life. Smith et al. (1964), after injecting bovine y-globulin into the amniotic fluid during the 6th, 7th, and 8th months of gestation, were unable to find any evidence of absorption by the fetal calf gut. Calves delivered 2 to 3 weeks prematurely and maintained on milk and aminosol-dextrose for 38 hours thereafter were unable to absorb y-globulin from colostrum. However, calves that received colostrum immediately after premature delivery absorbed y-globulin readily. 16 The Immune Globulin of Colostrum Since the gut of the newborn ruminant appears to transmit pro- teins in solution nonselectively, it follows that the normal young animal receives into its circulation all proteins that occur in colostrum. These include maternal serum proteins secreted by the mammary gland and other proteins which are synthesized in the gland. Transmission of immunity depends therefore on the particular components of the immune globulins which are transferred from the circulation to the colostrum and on any that may be locally synthesized by the lymphoid tissue of the mammary gland (Brambell, 1970). Crowther and Raistrick (1916) distinguished caseinogen, lactal- bumin and lactoglobulin in both colostrum and milk. They determined that the protein content of the mammary secretion declined rapidly with milking over the first few days. This decline was much greater in globulin than in albumin and was least in caseinogen. Smith (1946) showed that immune lactoglobulin is the predominant protein in bovine colostrum. Subsequently, he showed that the protein con- centration of colostrum within a few hours of parturition was as high as 15 to 26% or 2 to 3 times the concentration of protein in plasma. The immunoglobulins constituted 50 to 60% of the total colostral proteins and 85 to 90% of the total colostral whey pro- teins. Garner and Crawley (1958) found that 131I-labeled bovine y-globulin intravenously injected into a cow in late pregnancy was concentrated two- to three-fold by the mammary gland. This did not occur when the labeled globulin was injected into a cow which was 5 months pregnant. 17 There is no doubt that the concentration of immune globulins in the colostrum exceeds that in the serum, whereas the concentration in the milk is very low. Regmann (1920) observed that the antibody titer of the colostrum was higher than that of the serum in an immune cow, whereas the concentration in milk whey was only 1/40 to 1/80 of that in serum. The amount of antibody present in the colostrum at parturition approximately equaled the amount that had disappeared from the circulation during colostrum formation, indicating trans- mission. The cessation of v-globulin secretion by the udder during the early part of the dry period is reflected by the development of a hypergammaglobulinemia. The removal of y-globulin from the circulation by the udder, during colostrum formation, results in an antepartum hypogammaglobulinemia. There is clear evidence of the selective secretion and concen- tration in the colostrum, not only of the immunoglobulins in pre- ference to other serum proteins but also of some components of the immune globulins in preference to others (Smith, 1946a). Most of the immune globulin in colostrum of a normal animal is derived from the circulation, but there is also clear evidence indicating local synthesis of immunoglobulins (Lascelle, Outteridge and MacKenzie, 1966). There is no doubt that heterologous serum proteins in the maternal circulation can be transmitted to the colostrum. Dixon et al. (1961) found that heterologous v-globulins are concentrated in bovine colostrum to a degree similar to homologous y-globulin. Mid-I- lr..l‘h.‘ Hm 18 Transport of Globulin from the Intestine to the Circulation It has been shown by Comline, Roberts and Titchen (1951) that the absorption of colostral globulin in newborn calves takes place entirely in the small intestine and that none is absorbed from the abomasum or large intestine, when these have been isolated by liga- tures from the small intestine. Antibody globulin in colostral whey, introduced into the duodenum at 6 to 27 hours after birth, appears in the lymph after 1 to 2 hours. It was shown by cannula- tion of the intestinal lymphatic duct and the thoracic duct that the transport of the absorbed globulin to the circulation is entirely lymphatic and that none enters the circulation directly. There is evidence that the absence of proteolytic activity in the digestive tract of the newborn calf may facilitate the colostral proteins reaching the small intestine without degradation and, to this end, the colostral trypsin inhibitor may contribute. The absorption is effected by the epithelial cells of the jejunum and ileum. The Decline of Passive Immunitykand Development of Active Immunity The normal young calf that has taken colostrum and acquired its maximum concentration of maternal immune globulin by the second day enters upon a period when the decline in the passively acquired globulin overlaps the rise in autogenous y-globulin and tends to obscure it. Consequently, a point is reached sometime after birth when the y-globulin concentration is at a minimum and the loss of passively acquired y-globulin is equaled by the production of l9 autogenous y-globulin (Tennant et al., 1969). Smith (1948) reported that the globulin acquired from the colostrum decreased steadily in calves after 2 days of age, reaching about half its initial concentration by 20 days. Some globulins persisted for many months. Kerr and Robertson (1954), studying immunity to the Trichomonas fetus, found that adult animals, being immunized or suffering from the disease, had a "natural" antibody agglutinin to this organism at a titer of 1/48 to 1/96. This natural antibody was passively transmitted with colostrum to the calf. In the circulation of the calf it declined gradually and disappeared by the 17th to the 55th day. Autogenous antibody began to appear by the 35th to 60th day and was fully established by the 63rd to 113th day. IgA and Secretory Immunoglobulin As mentioned earlier, an individual possesses multipotential immune response mechanisms consisting of both cell-mediated and humoral components. The humoral or antibody component is further subdivided into an internal and an external secretory system (Bellanti, 1971; Butler, 1969). The internal secretory system is made up of lymph nodes, thymus, and spleen and produces circulating antibodies of principally the IgG and IgM types. The external secretory system consists of lymphoreticular cells located in sali- vary, lacrimal, and mammary glands and in the submucosa of the respiratory, gastrointestinal and genitourinary tracts. These cells produce antibodies, predominantly of the IgA type, which are elaborated in glandular and mucous membrane secretions (Bellanti, 20 1971; Butler et al., 1970; Mach and Pahud, 1969, 1971; Tomasi and Bienenstock, 1968). The available evidence thus far in man and animals indicates that the external secretory antibody system functions independently from the internal secretory system and is easily activated by the local application of antigen. The main value of this system seems to be the protection of external surfaces of superficial membranes which are not in contact with circulating antibodies. This is not the only protection available at the local site of infection. Cell- mediated immunity undoubtedly plays a significant, though as yet undefined, role. In the presence of inflammation there is transuda- tion or effusion of serum which may carry antibodies into mucous membranes. Additionally, such nonspecific factors as mucoprotein inhibitors and interferon provide protection to surface cells of superficial membranes (Todd, 1973). History The presence of a local immune system was proposed by Beseredka more than 45 years ago (Besredka, 1927). However, the theory that serum antibody "spilled over" into external secretions and thereby resulted in mucosal immunity was widely held until the last decade, despite the excellent work of Walsh and Cannon (1938). They used experimental animals to show that antibody in the nasal cavity was locally produced. Fazekas de St. Groth and Donnelley (1950) showed that antibody to influenza virus in mice was most efficiently stimu— lated by local application of antigen and that protection against influenza correlated better with the presence of antibody in 21 bronchial secretions than with the titer of antibody in serum. The "spill-over" concept, however, was finally put to rest with the demonstration of IgA (a relatively minor immunoglobulin in serum) as the predominant immunoglobulin class in external secretions (Hanson, 1961; Tomasi and Zigelbaum, 1963; Waldman and Ganguly, 1974). The first identification of IgA in normal human sera occurred during the early studies of Grabar and Williams (1953) using the technique of immunoelectrophoresis. They noted a fast migrating component which they originally designated 82 globulin and which was renamed 82A by Burtin et al. (1957) to distinguish it from another similarly migrating component of high molecular weight (82M) isolated by Kunkel et al. (1956). Particularly important was the work of Slater et al. (1955) on proteins isolated from the sera of patients with multiple myeloma. These workers demonstrated the existence of antigenically distinct although related groups of proteins in the slow and fast y—globulin regions on electrophoresis. The existence of the two classes of myeloma proteins and their counterparts in normal serum provided the first clear separation of the IgG and IgA classes of y-globulins. Heremans et al. (1959), using a zinc precipitation method, succeeded in iso- lating IgA from normal serum and first described some of its important physical and chemical properties. Heremans et al. (1959) also clearly showed the antigenic relationship of IgA to the y-globulins and thereby pointed out its potential importance in the immune response. 22 Structure of Serum IgA Physicochemical Properties. Many of the physicochemical proper- ties associated with IgA are shared with immunoglobulins of other classes, and there are relatively few distinctive features which serve to distinguish IgA from these other immunoglobulins. It is only when antigenic properties and relatively subtle chemical tech- niques such as amino acid sequence analysis are used that IgA emerges as a distinct immunoglobulin. Perhaps the most distinctive physical characteristic of IgA is its polydisperse nature with regard to molecular size. The monomeric IgA (abundant in human serum) has a sedimentation coefficient of approximately 6.3 8. However, a variety of polymers with increasing sedimentation coefficients up to 18 S are also found. This heterogeneity in the size of IgA is considerably greater than that found in any other immunoglobulin class, although monomer-polymer forms are also observed in IgM and occasionally in IgG (Capra and Kunkel, 1970; Klein et al., 1967; Smith et al., 1965). The IgA molecule is relatively acidic and, on boundary electro- phoresis, migrates between the B and 7 regions. In terms of its general chemical properties, IgA is quite similar to other immuno- globulins. It has a nitrogen content of 16.2% and its amino acid (composition is similar to that of the IgG heavy chain. Immunoglobulins irlgeneral can be quite clearly distinguished by their amino acid (Ramposition from nonimmunoglobulin proteins. There is a relatively leirge amount of carbohydrate in IgA compared to IgG. It contains '7‘to 8% carbohydrate compared to 2 to 3% present in IgG. This is 23 somewhat less, however, than IgM, 19D and IgE, all of which contain 12 to 13% carbohydrate (Davie and Osterland, 1968). As mentioned before, IgA can exist in a series of polymeric forms. In the bovine species polymeric IgA is the predominant form in serum. The sedimentation coefficients for these polymers are approximately 10 S, 13 S, 15 S and 17-18 S (Vaerman et al., 1965), and these coefficients represent the dimer, trimer, tetramer and pentamer, respectively, of the basic monomeric structure. Their molecular weights are 320,000, 480,000, 640,000 and 800,000, respectively. Monomeric IgA has a molecular weight of approximately 160,000 (Vaerman et al., 1965). The polymers of IgA are bonded to one another by disulfide bridges and reprsent the majority of the secretory IgA immunoglobulin in secretions (Tomasi and Bienenstock, 1968). Subunit Structure. The basic 4 polypeptide chain structure which has been established for IgG is also found in IgA. IgA con- sists of heavy (a) and light (A or K) chains. As is the case with other immunoglobulins, the heavy chains and light chains of IgA are held together by disulfide bonding as well as noncovalent interactions. In man there are 2 IgA subclasses: IgA and IgA 1 2 (Grey et al., 1968). Digestion of IgA with papain leads to the formation of a 3.5 S fragment similar to antigen-binding fragment (Fab). Pepsin digestion results in the production of 3.5 and 5 S fragments with L chain antigenic determinants presumably analogous to F(ab')2 and Fab' of IgG (Tomasi and Grey, 1972). However, thus far there has 24 been no convincing evidence that any proteolytic treatment of IgA results in the formation of a crystallizable fragment (Yakulis et al., 1969). Secretory IgA It now seems quite certain that the molecular weight of both the 11 S salivary and colostral IgA is approximately 385,000. Under appropriate conditions the secretory IgA molecule can be split in such a way as to yield a dimeric IgA molecule with a sedimentation coefficient of about 10 S. This dimeric molecule is free of secre- tory component (SC). It can be further split into a monomeric form by removing the joining chain (J) (Halpern and Koshland, 1970). In addition to predominant 11 8 IgA species, most external secretions which have been carefully studied also contain smaller amounts of 7 S as well as polymers of IgA with sedimentation coef- ficients greater than 11 S (Newcomb and DeVald, 1969). J Chain. Secretory IgA molecules contain an additional chain, unrelated to the secretory component. This component is referred to as the J (for joining) chain and has a molecular weight of 23,000 and an amino acid composition different from the L chain (Halpern and Koshland, 1970). It was found, following complete reduction, that the J chain was evident only in polymeric IgA and not in the 7 S monomer. The function of the J chain may be to pro— Vide linkage in polymeric immunoglobulins. The J chain from secre— tory IgA and IgM had similar electrophoretic mobility, molecular weight, amino acid composition and peptide map (Mestecky'et al., 1972). 25 Secretory Component (SC). Tomasi and Calvanico (1968) have shown that approximately 20% of the L chain and SC in the secretory molecule are bonded noncovalently while the remaining 80% bonds covalently and can be released only after reduction of disulfide bonds. The secretory piece does not appear to contain any immuno- globulin antigenic determinant. It has a high carbohydrate content, about 9.5%, and contains sialic acid. It is a single polypeptide chain with a molecular weight of 50,000 to 60,000, and its amino acids are different from those in the gamma A polypeptide chain. There are 4 moles of disulfide bound per mole of SC. Secretory gamma A globulins are highly resistant to proteolysis with enzymes such as trypsin, chymotrypsin, and pepsin, all of which will attack the serum immunoglobulins. It seems quite clear that the SC conveys a great measure of stability to this antibody molecule (Tomasi and Grey, 1972). Regardless of the question of resistance to proteolysis, some proteolysis of human colostral IgA occurs with both trypsin and pepsin and the product is primarily 5.0 S F(ab)2 and 3.5 S Fab (Tomasi and Calvanico, 1968). Immunoglobulin Other Than IgA in Secretions Immunoglobulins other than IgA, including IgG, IgM and IgE, have been detected in most external secretions in varying propor- tions in different fluids. There is evidence that the IgG present in many secretions is derived in large part from serum and lacks SC (Bienenstock and Tomasi, 1968). Small amounts of IgM can be 26 isolated from the secretions of individuals with a selective deficiency of IgA. There was SC associated with IgM in the serum of 3 patients and the duodenal fluid of 1 patient in a selective deficiency of IgA (Thompson, 1970). The binding studies suggested that IgM, although capable of binding SC in vitro, does so with less avidity than IgA (Thompson, 1970). Evidence has recently been presented that IgE is present in saliva, colostrum, nasal fluid, and urine of normal individuals and the concentration of this immunoglobulin is markedly increased in the respiratory fluids of patients with certain allergic disorders (Ishizaka et al., 1971; Salmon, 1970). The IgE present in secretions appears to have the same molecular size as serum IgE (200,000) and it lacks SC (Salmon, 1970). Synthesis of Secretory Immunoglobulin It now appears that immunoglobulins in secretions may reach mucosal surfaces by 2 routes: by transudation from serum or by local synthesis in plasma cells lying in intimate relationship to the mucous membrane or glandular epithelium (Tomasi and Grey, 1972). Abundant evidence is available that the majority of the predominant 11 S species of IgA is synthesized locally. Using fluorescent anti- body and in vitro culture techniques, local synthesis of IgA in the gastrointestinal tract has been demonstrated in many species (Nash et al., 1969; Bistany and Tomasi, 1970). Secretory component also appears to be locally synthesized but in a different cell type than IgA. Evidence indicates that SC is synthesized in epithelial cells of many secretory organs (Tourville et al., 1969). The bulk of 27 evidence suggests that IgA is synthesized primarily as a dimeric molecule in the secretory plasma cells. This is consistent with observations in certain species that the major type of circulating IgA is dimeric and originates in large part in secretory sites particularly in the lamina propria of the gastrointestinal tract (Tomasi and Grey, 1972). Good evidence is now available that small amounts of IgM (Tomasi and Bienenstock, 1968) and IgG (Brandtzaeg et al., 1970) and primarily IgE are synthesized locally in several secretory sites. Ontogeny of the IgA System In all species so far studied, including man, the serum of the unsuckled newborn is severely deficient in IgA. Very little IgA passes the placenta or yolk sac, even in those species, such as man, monkey, rabbit, guinea pig, mouse and dog, where the fetal IgG concentration approaches or exceeds that in the maternal circu- lation (Tomasi and Grey, 1972). In most studies serum IgA is reported to reach adult levels at a later age than either IgG or IgM. In a recent study of human sera (Uffelman et al., 1970), the IgA levels were approximately 25% of adult value by 6 months of age and reached 50% by the end of 4 years. In some cases IgA can be detected in saliva or in tears as early as 10 days of age in the apparent absence of serum IgA (McKay and Thom, 1969). In one study (Selner et al., 1968), 92% of normal infants had adult levels of IgA in their saliva by 28 days of age. The more rapid appearance and development of IgA in secretions than in serum could be related to a greater antigenic 28 stimulation initially occurring at mucous membranes (Tomasi and Grey, 1972). The Mechanism by Which Secretory IgA Operates The immune mechanism by which secretory antibody might operate in the complex environment of these biological fluids and mucous surfaces largely remains a matter of speculation (Waldman and Ganguly, 1974). Secretory IgA antibodies possess antiviral activity in the absence of complement (Dowdle et al., 1971). They may act as an initial immunologic barrier to the entry of invasive viral infec- tions such as polio, measles and rubella (Ganguly et al., 1973). However, when this mucosal immunity is overcome, dissemination depends on the systemic immune mechanisms. In some cases lack of mucosal antibodies might result in a "carrier" state even in the presence of systemic immunity (Ganguly et al., 1973). It is well established that IgA does not fix complement in the classical manner (Ishizaka et al., 1966). The antibacterial action of IgA is more controversial. Four mechanisms have been suggested so far in this matter. I. Adinolfi et al. (1966) have shown that secretory IgA anti- body against coliform organisms is capable of bacteriolysis in the presence of complement and lysozyme, especially in view of the fact that some mucosal secretions are rich sources of lysozyme. II. Secretory IgA antibody may exert antibacterial activity by the alternate pathway of complement fixation. Ellman et al. (1971) demonstrated in vitro that antigen-antibody complexes were 29 capable of fixing the late-acting components of complement in sera of the copper-deficient guinea pig. III. Wernet et al. (1971) purified colostral immunoglobulins from pigs and rabbits immunized against E. coli. They demonstrated that while IgA lacked activity in the direct bactericidal test, it was very active as an opsonin. IV. A fourth possible mechanism is the inhibition of absorp- tion and growth of bacteria on mucosal cells in the presence of secretory antibodies. That the adherence of bacteria to the mucosal cell wall may be an important feature in determining growth and pathogenicity has been suggested for various bacteria, including Streptococcus sp. (Group A), V. cholerae, Mycoplasma pneumoniae, E. coli, Neisseria gonorrhoeae, and Shigella sp. (Gibbons and Van Houte, 1971). IgA in Bovine Serum and in Other Secretions Although there is considerable variation among the reported values for IgA in bovine serum, all reports suggest a value sig- nificantly lower than that present in normal human serum (human 2.5 mg/ml, bovine 0.5 mg/ml) (Butler, 1973). Unlike IgA in man, but similar to IgA in most other species studied, IgA in bovine serum occurs predominantly as a dimer (Butler, 1971; Duncan et al., 1972; Mach and Pahud, 1971; Wang and Fudenberg, 1974). It seems reasonable that the IgA in bovine serum and that of most other species is produced outside of the central compartment of the humoral immune system. In contrast, the higher level of IgA in 30 human serum results from the predominance of 7 S IgA, presumably a product of the spleen and lymph nodes. Outside of the vascular system, secretory IgA (SIgA) is the predominant immunoglobulin present in all exocrine secretions of cattle studied except for lacteal secretions (Butler et al., 1972; Mach and Pahud, 1971; Porter, 1971). The failure of SIgA to pre- dominate in bovine colostrum as it does in the colostrum in man or rabbits is understandable in terms of the differences in the route of passive transfer of immunity to offspring among species (Butler et al., 1972). The SIgA in human or rabbit colostrum is typically not absorbed and presumably has an important role in the defense against infection directly in the lumen of the gut. In baby pigs, where absorption of maternal immunoglobulin all but ceases 3 to 4 days postpartum, IgA replaces IgG as the predominant lacteal immuno- globulin after that time. The failure of the IgA:IgG ratio in bovine colostrum to change in the same manner after closure suggests that IgG continues to have a primary function against pathogens in the gut or mammary gland (Butler, 1973). The Bovine Intestinal Secretory Immune System Curtain et al. (1971) suggested that IgG predominates in bovine mucous secretions and also in plasma cells locally situated in the external mucosa. Vaerman (1970) had earlier failed to detect many IgA cells in the lamina propria of bovine intestinal tissue. The initial results in studies by Porter and Noakes (1970) of bovine intestinal secretions obtained from fistulated calves were in complete contradiction to the above studies. The level of 31 IgA was comparable to those in the pig in which an intestinal secre- tory IgA system had been adequately defined (Allen and Porter, 1973; Porter et al., 1972). Furthermore, Mach and Pahud (1971) also found IgA to be a major immunoglobulin in washings from the epithelium of the bovine gastrointestinal tract. Immunochemical studies of intestinal secretions and immuno- fluorescent studies of intestinal tissues confirmed the presence of a secretory IgA system similartx>that described in other species (Porter et al., 1972). Free and bound secretory component and 11 S IgA were demonstrated in the secretions of fistulated calves and immunofluorescent localization of IgA and secretory component in the intestinal tissues was similar to that described in man (Tourville et al., 1969) and the pig (Allen and Porter, 1973; Porter and Allen, 1972). Secretory component was demonstrated only in crypt epithelial cells, being narrowly confined to the apical cytoplasm. The IgA was present in the apical cytoplasm of the epithelial cells in the lower region of the crypts, in the mucin covering the apical surfaces, and in numerous plasma cells in the underlying lamina propria. Nevertheless, IgM consistently exceeded the level of IgA and its localization in lymphoid cells of the lamina propria and crypt epithelial cells suggested that IgM, too, may normally play an important role in local intestinal defense in the calf (Porter and Noakes, 1970). The possibility is now emerging that IgM may be involved in the primary immune response of the external secretory immune system (Allen and Porter, 1973). Thus, in the internal system a switch to I96 synthesis takes place and this is the predominant immunoglobulin 32 in the blood. In the external secretory system a switch to IgA synthesis must take place. Colibacillosis The syndrome associated with colibacillosis has been divided into 3 forms: septicemic, enteric and enteric toxemic (Gay, 1965). The septicemic or colisepticemic form usually results in rapid death of the calf and is associated with an E. coli bacteremia. The clinical signs of colisepticemia are sudden anorexia, rapid dehydration and increased cardiac and respiratory rates. Although many strains of E. coli have been isolated from calves with coli- septicemia, the isolations from internal organs of a given case are usually of a single strain in a pure culture. The enteric form, often called "white scours", has the greatest incidence in the calf. The predominant clinical signs are mild to severe diarrhea, anorexia and dehydration. The feces have a pasty to watery con- sistency. Death may or may not occur depending upon the severity of the physiological derangements produced. The enteric toxemic form is a comparatively rare type of colibacillosis. It is charac- terized by sudden collapse, extreme prostration and death within 6 to 16 hours after clinical onset. This form is associated with a massive proliferation of a mucoid strain of E. coli with A type K antigens in the small intestine of the calf. There is no diarrhea or bacteremia (Gay, 1965, 1971; Fey, 1972). 33 Normal Bacterial Flora of the Calf Intestine and Its Aberrations in Cases of Colibacillosis Now it is evident that there are great variations in distribu- tion among various bacterial species in the intestine in the normal animal. These variations are influenced by such factors as age, diet, pH, oxidation-reduction potentials in the various portions of the intestine, and antagonisms and synergisms between and within the bacterial species involved (Goldman, 1924; Haenel, 1961; Johanson, 1949; Smith, 1961). Although Hagan (1917) and Williams et al. (1920) found evidence of in utero contamination of the calf intestine, it is evident that the main colonization of the intestine occurs after birth with the bacteria originating from the environment. In terms of total fecal flora, the maximal numbers are reached by 24 hours (Roberts et al., 1954; Smith, 1960, 1961). At this time, the flora is comprised mainly of E. coli, Streptococcus sp. and Clostridium perfringens, with Lactobacillus and Bacteroides sp. appearing on the second day. These various bacterial species persist in high numbers for periods of time which vary with the species and gradually decrease in number (Smith, 1961; Gay, 1965). There is inadequate information about the distribution of pathogenic strains of E. coli in the intestinal tract of calves with colienteritis. The main difficulty in estab- lishing E. coli as an enteric pathogen is its normal presence in the intestine and feces. The establishment of a new E. coli type as an enteric pathogen is an enormous task, but there is no doubt that additional types are potentially pathogenic. Once statistical and epidemiological evidence for pathogenicity of a given strain seems 34 to be acceptable, one still has to exercise great caution in branding any E. coli type as the cause of enteritis (Fey, 1972). In the studies by Carpenter and Woods (1924) and Smith and Orcutt (1925), the intestinal flora of healthy calves was compared with that of scouring calves. In the healthy calf, E. coli and other bacteria were prevalent only in the large intestine and in the distal portion of the small intestine. The medial and proximal portions of the small intestine were described as being virtually free of bacteria. In contrast to this, in calves dying of "scours" E. coli was present in large numbers in the upper small intestine. Smith (1963) examined the intestinal flora of calves. In contrast to the earlier studies, he found that E. coli was prevalent through- out the intestinal tract of the healthy calf. He also examined the intestinal flora of scouring calves and found no evidence of a pro- liferation of E. coli in the small intestine. He concluded that E. coli and other bacteria are not directly responsible for calf scours and calf diarrhea is noninfectious in many instances and may occur as a result of environmental factors. Identification and Classification of the E. coli Group The family Enterobacteriaceae is composed of gram-negative rods which may or may not be motile and attack glucose with the production of acid or acid and gas. Nitrates are usually reduced to nitrites. On the basis of further biochemical reactions, the family may be arranged into divisions and thus into groups, one of which is E. coli. These groups are not distinct but form dense populations within the family which have certain biochemical properties. Within the E. coli group, the individual strains are identified by serological methods. These serological types can be further divided or classified by biochemical characteristics, phage susceptibility and susceptibility to colicines (Smith and Crabb, 1956; Barry et al., 1962; Linton, 1960). In 1947, Kauffmann published an antigenic schema for E. coli and this has subsequently been expanded by other workers (Edwards and Ewing, 1962). There are three main antigens of E. coli which are used in its identification. These include the O or somatic antigens, the K antigens which occur in capsules or microcapsules, and the H or flagella antigens. The difficulty encountered by earlier workers in serotyping E. coli was apparently due to the K antigen which, when present, prevents the agglutination of the O or somatic antigen by the homologous sera. The K antigen can be removed by heat. However, there are differences in heat susceptibility of these K antigens, and it is on this basis that they are further subdivided into three groups. I. The L-type K antigens are completely destroyed by heat at 100 C for 1 hour, thus rendering the 0 antigen agglutinable in O antisera. They retain no antigenicity and lose their ability to combine with homologous K antisera. II. The B-type K antigens are also destroyed by heat at 100 C for 1 hour and lose their antigenicity. However, they retain their ability to bind or combine with their homologous antisera. III. A-type K antigens are not inactivated by heat at 100 C but require heat at 121 C for 2.5 hours before the 0 antigen becomes agglutinable. 36 ¢rskov et al. (1961) showed that there is some variation with certain K antigens and that certain E. coli strains may possess more than one type of K antigen. The O antigens are not single antigens, but they are composed of several antigenic components and, therefore, are called 0 group antigens. Different 0 groups may share some of these antigenic components. Hence, there are many cross reactions among 0 groups. As might be expected, these antigenic cross reactions are not restricted to within the E. coli group but also occur between it and other groups in the family Enterobacteriaceae (Ewing et al., 1956). Flagella, or H antigens, may or may not be present. All of these antigens are named numerically, the 0 group anti- gen being given first, followed by the K antigen and then the flagella antigen (Edwards and Ewing, 1962). Epidemiology of Colibacillosis in Calves Epidemiological studies have shown that many of the human sero- types are capable of spreading and actually causing epidemics of diarrhea in nurseries and hospitals (Harris et al., 1956). If ISVidence of this kind were available from epizootics of colibacillosis, it would provide convincing evidence of the pathogenicity of certain Strains of E. coli for calves. Specific serotypes of E. coli are associated with certain (iiJSeases in animals and man. The evidence of this association in CCfilibacillosis of calves has been presented (Glantz et al., 1959). 'Iqifié majority of serological studies have been on E. coli isolated from calves with the septicemic form of colibacillosis. It is now 37 evident that certain serotypes and 0 groups of E. coli are commonly associated with this syndrome. There have been few serological studies on E. coli associated with the enteric form of colibacillosis and the serotypes associated with these syndromes are largely unknown. This is partly because of difficulty in determining the etiologic significance of an isolation of E. coli from the intestine of a scouring calf. The clinical and bacteriological syndrome asso- ciated with the enteric-toxemic form of colibacillosis is sufficiently distinctive to allow its differentiation from other causes of death. However, there is at present no means by which the enteric form of colibacillosis can be differentiated from other causes of diarrhea in calves. Because of this no great significance can be placed on reports of the isolation of given serotypes of E. coli from the intestine of individual scouring calves (Gay, 1965). Unfortunately, the factors associated with virulence of a strain are unknown, and there is no in vitro test that will detect these strains. For example, the susceptibility to serum bactericidal activity (Muschel, 1960) or to phagocytin (Namioka and Marata, 1962) cannot be directly correlated with pathogenicity. Similarly, routine in vivo tests, such as pathogenicity for laboratory animals (Lindberg and Young, 1956; Sjostedt, 1946) or chick embryos (Hughes and Lovell, 1962), appear to be of little value. These tests do show, however, that the presence of K antigen is associated with a markedly increased virulence over the K negative form. An in vivo test was described which held some promise for the identification of enteropathogenic strains. This involved the injection of living cultures of E. coli into an isolated loop of 38 rabbit small intestine. The rabbits were killed 24 hours later, and the enteropathogenicity of the strains was assessed according to the severity of the reaction of the gut (Taylor et al., 1958, 1961). The enteropathogenicity of 6 strains of E. coli was evaluated via the ligated loop technique in 15 gnotobiotic swine 3 to 4 weeks old (Davidson and Waxler, 1975). The surest method of determining the pathogenicity of a sero- type of E. coli is to test, by experimental infection, its ability to reproduce diarrhea in calves (Gay, 1971). Pathogenesis As stated by Gay (1965), there has been little difficulty in experimentally reproducing colisepticemia in calves deprived of colostrum, providing they are challenged with the invasive strains of E. coli. Examination of calves dying of colisepticemia indicated that they were either agammaglobulinemic or markedly hypogammaglobu- linemic (Fey and Margadant, 1961). Fey (1962) postulated that this deficiency in y-globulins was a major factor in the incidence and pathogenesis of colisepticemia in calves. Smith (1963) has also observed that some calves thought to have been fed colostrum possessed very low levels of y-globulin, and variations have been found in the y-globulin levels of calves fed colostrum. The reason for such variation in the y—globulin content of various sera cannot be fully explained. Gay (1965) suggested that the enteropathogenic strains of E. coli must have an unknown mechanism for becoming established as a part of the intestinal flora and later cause an enteritis. The infective coli strain is either ingested and 39 established in the intestine or endogenous colibacteria ascend from the lower to the upper part of the intestine. For reasons unknown, they begin to proliferate massively, causing an enteritis. The consequence of inflammation is an increased permeability of the gut wall, which enables the colibacteria to spread within the whole body. Colisepticemia would then be the consequence of the preceding colienteritis (Fey, 1972). From bacteriological examination of cases of colisepticemia, it is evident that invasion need not necessarily occur from the intestinal flora but may occur via the navel or even from other areas such as the nasal or pharyngeal mucosa (Fey and Margadant, 1961). The factors associated with invasiveness in E. coli and the mechanisms by which they invade are as yet unknown. However, this does not account for many of the cases of colisepticemia in which the septicemic strain cannot be found as part of the intes- tinal flora (Fey, 1962). Experimental calves that have been fed colostrum and have absorbed y-globulins are resistant to septicemic invasion by E. coli. However, if colostrum is given too late the calf may die of colisepticemia despite absorbing y-globulins to normal or near- normal levels since the virulent colibacteria became established when the animal was unprotected (Fey, 1972). The factors associated with resistance to the septicemic form of colibacillosis in the colostrum-fed calf are unknown. Resistance does not appear to be associated with agglutinating antibody, since many healthy calves in the field are devoid of demonstrable agglutinins against E. coli strains associated with colibacillosis (Fey, 1972). 40 Calves fed colostrum are susceptible to the enteric form of colibacillosis; however, almost nothing is known of the pathogenesis of these infections (Gay, 1965). The Effect of Metabolic Changes in the Pathogenesis of Neonatal Diarrhea Diarrheic calves expel volumes of watery feces in excess of their fluid intake. Some calves die in a few days as a result of acidosis and dehydration. Packed cell volumes (PCV) may change remarkably because of hemoconcentration. The change in PCV may be less noticeable in prolonged cases because dehydration is not marked and the electrolyte/fluid balance remains almost normal. The effect of severe diarrhea on the plasma of the calf is to decrease the volume, usually to decrease the sodium and chloride concentration, to increase the urea concentration, and sometimes to increase the potassium concentration. The level of plasma proteins goes down because of lowered intake of food and due to losses through effusion of protein into the intestine. The hydrogen ion concentra- tion in plasma rises and the bicarbonate concentration decreases so that metabolic acidosis ensues. Spontaneous recovery can occur, whereupon the plasma parameters return to normal. Recovered calves may remain emaciated for a long time (Fisher, 1971). One of the most important predisposing factors in the patho- genesis of colibacillosis may be the failure of some colostrum-fed calves to acquire gamma globulin in their serum from the colostrum, thus resulting in persistent agammaglobulinemia or hypogammaglobulinemia. It is generally accepted that colibacillosis occurs in calves after exposure to a pathogenic strain of E. coli because the calves had 41 acquired no protective antibody in the colostrum against that sero- type (Smith, 1963; Gay et al., 1965; Fey, 1971). It has been sug- gested that colostrum should be fed to calves in the first 6 hours or less of life (Gay et al., 1965; Fisher, 1971) and that at least 2 kg be fed twice daily for 4 days (Klaus et al., 1969). Other factors which may influence the incidence of colibacillosis include those directly affecting the host, such as age, species, genetics, nutritional levels of the dam and calf, preexisting diseases and season. Included are management failures like improper planning, overcrowding of animals, poor sanitation and ventilation, labor shortages, and stresses of any kind (Reisinger, 1965; Barnum et al., 1967; Oxender et al., 1971). Treatment and Control Sulfonamides and antibiotic compounds, tetracycline, dihydro~ streptomycin, neomycin, nitrofurans and chloramphenicol have been used therapeutically and prophylactically. It was difficult to establish successful chemotherapeutic control in herds where the disease frequently occurred because of the emergence of multiple resistant strains of E. coli. Treatment should be based on prior drug sensitivity tests (Smith, 1958). Smith (1960) has shown that the efficiency of chemotherapeutic agents in eliminating sensitive strains of E. coli is matched by the extreme speed at which resistant strains may replace them during chemotherapy. Radostits (1965) has recommended fluid replacement therapy with balanced electrolytes and a source of energy (acetate or lactate) especially in prolonged diarrhea. 42 There are conflicting opinions as to the value of vaccination of the dam as a means of protection of calves against colibacillosis (Gay, 1971). The validity of passive immunization is also question- able. Commercial serotype-specific antisera would protect calves only against the homologous serotype of E. coli and would probably not be effective against most natural epizootics involving other serotypes (Gay, 1971). Vaccination of calves against colibacillosis with formalin-killed bacterins has not been successful (Gay, 1971). Enteric Disease Other Than Colibacillosis Young calves can also be affected by enteric diseases caused by certain viruses. The viral enteric pathogens that have been reported include infectious bovine rhinotracheitis virus (Amstutz, 1965), bovine viral diarrhea virus (Steck et al., 1971; Lambert et al., 1974), parainfluenza 3 virus (Steck et al., 1971), rhinoviruses, enteroviruses (Mayr et al., 1964), adenoviruses (Steck et al., 1971), a reovirus-like agent (White et al., 1970; Mebus et al., 1971, 1973) and a coronavirus-like agent (Mebus et al., 1973). These viruses were reported as the primary cause of viral enteritis. The age of the calf and the ingestion of protective colostrum are recognized as 2 factors important in determining if a virus will infect a calf. It has been suggested that a virus and E. coli may both be involved in some cases of diarrhea in young calves (Amstutz, 1965; Mebus et al., 1973). Immunization Before Birth (Intrauterine Vaccination) Gay (1971) was one of the first to attempt to vaccinate calf fetuses during laparotomy by using a single serotype of E. coli. 43 Calves responded and were protected by producing serotype specific antibody. Some calves also produced heterogenetic antibody to other E. coli strains. In a more recent work Gay (1975) reported that nonsurgical in utero vaccination with a single serotype of E. coli can result in hterogenetic protection against neonatal colisepticemia. Research at the College of Veterinary Mecicine at Michigan State University has shown that vaccination of the calf before birth is effective in preventing one form of diarrhea (scours). Intra- uterine vaccination of the bovine fetus can be accomplished without performing surgery on the cow. The procedure requires less than 10 minutes and, with continued use, could probably be performed in 5 minutes under field conditions. It therefore appears that vaccina- tion of the bovine fetus can become a practical, routine clinical procedure. When an E. coli antigen (bacterin) was deposited in fetal fluids 3 to 4 weeks prior to birth, the newborn possessed serum antibodies and they were also protected against an oral challenge dose of virulent E. coli which was the same organism as was in the antigen (Conner et al., 1973). Knowledge gained from this research is now being applied in several local dairy herds under farm conditions. Prenatal vaccina- tion is being performed in herds previously suffering losses from colibacillosis (diarrhea, septicemia). At this time there are not sufficient data from the field for a definitive evaluation, but immunization before birth appears to protect calves from colibacil- losis. The occurrence of premature birth in some calves vaccinated in utero is a problem requiring continued study. 44 While speculative at this juncture, it may be possible to immunize the bovine fetus (with a single intrauterine injection) against many of the bacterial and viral diseases that plague the cattle industry (Conner, 1975). MATE RI AL AND METHODS Animals % Thirty pregnant Hereford cows bred by Aberdeen Angus or Beef Friesian bulls were obtained from the Premier Corporation, Fowlerville, Michigan. The cows were fed brome-alfalfa hay twice daily and water ad libitum in a confined pole barn and were kept under 24-hour observation after birth of the first calf and for the duration of the calving period. Soon after natural delivery and before suckling the calves were transported to a calf barn and were kept in individual pens. The calves were divided into principal and control groups depending upon whether they had been given an intrauterine inoculation of E. coli 026:K60:NM bacterin or saline. They were further subdivided as shown in Figure l. The immunized calves were inoculated in utero with 5 x 1010 (1 m1) killed, washed E. coli organisms and were reimmunized at birth by oral inoculation of the same dose. Challege doses were 1.5 x 1010, 1.0 x 1011 or 1.5 x 1011 organisms (5, 30 or 50 ml, respectively) of live, unwashed E. coli organisms. The calves were bled from the jugular vein at birth and at death. They 45 46 .momosucoumm cfi oopmoflocfi ma macho some :H mHmEflcm mo Hones: one .mflmmcmnuso cam omcoaamso .cofiuMNACSEEflou um wood can mo>amo mo mmsoum Hmucoefinmmxm .H ousmflm Amy mxoos m we ooHHHM . H msouw Rev 1H5 mxmmz N mane m-o um ooHHflM um ooaafix _ msou x S u E as: nwsmwsmmmm um ooaflfix _ moouo moon m m 6 Adv msme a pm emsaflx s INS . m mo him meme m um um omaaax coco o Hamcu m msouw m macho Amy Amv _ mawo vra mxmo vnm um o fix e_HH um ewaaax and um emacmemgo Amy sauna lav genus nuuan on cause on msouu um emflaau um emflaax emmcmmflmno coflumuflcssshmm h m msouu m msouo v msouu _ _ _ _ oumuz cw one“: Cw Amsmflcmmuo oaoaxmv cfluouomo OGHme MO coauothH NNOU .m MO cowuumnCH monouo Houucoo onmmo qaezmszmmxm monouo Hmmflocflum 47 were fed 120 gm of a milk replacer dieta in 500 m1 of warm water twice daily. Laboratory Animals Eight male Hartley-White guinea pigs averaging 500 gm each and 10 male Dutch Belted rabbits averaging 2 kg were obtained through the Center for Laboratory Animal Resources, Michigan State University. Initial serum samples from the laboratory animals were negative for antibody activity against bovine serum by the Ouchterlony method (Campbell et al., 1970). Antigens Bacterin (Vaccine) For vaccination purposes, a culture of 026:K60:NM was grown overnight on trypticase soy agar slants. The growth was removed by washing with 0.85% NaCl solution and then seeded into bottles containing the same medium and incubated for 24 hours at 37 C (Conner et al., 1973). The growth was suspended in 0.85% NaCl solution and samples from pooled suspension were examined for purity by streaking on blood agar. A viable count was taken by following the method described by Norris and Ribbons (1969). The bacteria were killed by 0.4% formalin and incubated in a shaking water bath at 37 C for 24 hours. The cell suspension was standardized to 5 x 1010 cells/ml in 0.85% saline containing 0.02% merthiolate and was kept at 4 C and used within 5 to 20 days. a . . . . . MMPA Premium Calf Milk Replacer, Michigan Milk Producers Association, Detroit, MI. 48 Challenge Inoculum The E. coli inoculum used as a challenge dose for the newborn calves was prepared from the viable stock agar culture by inoculating trypticase soy broth and incubating 24 hours at 37 C. The viable count of challenge organisms in the broth after incubation was approximately 3 x log/m1. The preparation was kept at room tempera- ture in the dark and used within 48 hours (Conner, 1973). Fetal Inoculation The in utero vaccination procedure which was used in this work was a nonsurgical method developed by Conner at Michigan State University. During the procedure the cows were in a standing position. The fetus was located in the region of the right flank of the cow by palpation and ballottement within a triangular area formed by points which were approximately 25 cm anterior to the division between the front and rear quarters of the udder, the stifle joint and the distal aspect of the 10th rib. The site of inoculation was clipped and cleansed with a surgical scrub,b and a local anestheticC was infiltrated into the skin, muscles, and peri- toneum at the site where a needle puncture was made. The area was scrubbed and disinfected again. The operator, wearing sterile rubber gloves, inserted a sterile lZ-gauge 5 cm cannulated needle through the skin, muscle and peritoneum (it was easier to insert bBetadine scrub and solution, The Purdue Frederick Co., Norwalk, CO. cProcaine hydrochloride, 2.5%, Bio-Ceutic Lab, Inc., St. Joseph, MD. 49 the needle if a small incision was first made in the skin). A 16- gauge 30 cm needle was attached to a 10 ml empty syringe and was passed through the cannula and was gently pushed towards the wall of the uterus until the fetus was touched. The fetus usually reacted vigorously to the needle stimulus. At this time the amniotic fluid was aspirated to make sure that the amniotic cavity had been entered. When the end of the needle was in the amniotic cavity, the syringe was replaced with another one containing 1 ml of either E. coli bacterin (5 x 1010 organisms) or sterile 0.85% NaCl solution. The injection was made into the amniotic cavity. The needles were withdrawn and antibiotic powder was applied at the injection site. The samples of amniotic fluid were cultured for bacterial growth and later analyzed for biochemical properties. The cows were observed for several days after the in utero injection. Preservation and Collection of Tissues Specimens were collected at necropsy after the calves were killed by electrocution. Tissue samples from the liver, spleen, kidney and mesenteric lymph nodes were collected aseptically for bacteriological examination from certain animals. The small intes- tine was divided into 4 parts, duodenum, upper and lower jejunum and ileum, respectively. Each part was clamped at both ends and intestinal contents were mixed and washed carefully, by pipetting 20 ml of a chilled mixture of phosphate buffered saline (PBS) (0.01M phosphate, pH 7.2; 0.15M NaCl) and dithiothrietold (0.001M) d . . . DTT, Sigma Chemical Corp., St. Louis, MO. 50 into the lumen. The intestinal washings were collected in flasks and later were centrifuged at 10,000 g for 10 minutes at 4 C. The supernates were dialyzed in 6 liters of PBS at 4 C for 48 hours and then concentratede to 2 ml and stored at -70 C. Sections from the 4 segments of intestine and corresponding mesenteric lymph nodes and spleen were collected. The tissues were immediately put into 2-methylbutanef and placed in liquid nitrogen vapor for several minutes. The tissues were then placed into plastic bags, marked and stored at -70 C. The tissue sections were also collected from the same earlier mentioned areas and were fixed in formalin for histopathologic examination. Protein Analysis The protein content of the whole bovine serum and the anti-IgA sera was determined by a combination of 3 methods: the biuret procedure (Coles, 1974), the method of Lowry (Lowry et al., 1951) or by refractometry.g Immunologic Procedure Immunoelectrophoresis (IEP) Immunoelectrophoresis was performed following the micromethod described by Scheidegger (1955). Barbital sodiumh buffer (0.1M, ePolyethylene Glycol 20,000, Fisher Scientific Co., Fairlawn, NJ. f . . . Obtained from Eastman Organic Chemicals, Rochester, NY. gAmerican Optical Co., Buffalo, NY. hScientific Products, 17150 Southfield Road, Allen Park, MI. 51 pH 8.0) was prepared by the method described by Leid and Williams (1974) and was used in the buffer chamberi after allowing to stand for 24 hours to reach maximum electrolytic dissociation. Two percent agar gel was prepared by melting and boiling the Nobelj agar in 250 ml distilled water and then by adding 250 ml of the barbital sodium buffer. Merthiolate (1:10,000) was added to a concentration of 1% as a preservative. The mixture was dispensed into stoppered tubes in 14 ml volumes and was stored at 4 C. The electrophoresis frames were covered by 6 microscopic slides. Melted agar was poured on the slide covered frame and, after hardening, the wells and troughs were cut. Antigen was placed into the wells and electrophoresis was done in the buffer filled chamber. A current of 12 amperes per frame was used for 70 minutes. Afterwards, antiserum was placed into the troughs and the frame was incubated at room temperature for 24 to 48 hours in a moist chamber. To preserve the desirable slides, the slides were washed in 2% sodium chloride and distilled water for 48 hours with 2 changes of each to remove nonprecipitated proteins and excess salt (Grabar and Burtin, 1964). The slides were covered with moist paper strips and were dried at room temperature. The slides were stained with aniline blue blackk (Grabar and Burtin, 1964) for 10 minutes and destained with 2 changes of 2% glacial acetic acid. The slides were preserved by submerging into an acetic acid-glycerol- water mixture for 15 minutes, after which they were air dried and stored. 1Gelman Deluxe Electrophoresis Chamber. 3Difco Laboratories, Detroit, MI. k . . Aniline Blue Black, Matheson, Coleman and Bell, Norwood, NJ. 52 Double Immunodiffusion (Ouchterlony Method) The previously described agar-gel was used on microscopic slides. The wells were 3 mm in diameter and were spaced 5 or 6 mm apart, center to center. Incubation, drying and staining were the same as for IEP. Direct Bacterial Agglutination Passive Hemagglu- tination Assay (PHA) and Antiglobulin Augmentation Antibody levels were determined by tube agglutination and passive hemagglutination assay on serum samples and intestinal washings. The methods were those described by Conner et al. (1973) and Wilson and Svendson (1971), respectively. Sheep red blood cells were aseptically collected from a donor and stored for 1 week at 4 C in sterile Alsevier's solution (Campbell et al., 1970). Antiglobulin (IgA) augmentation of tube agglutination and PHA assay were carried out by the method described by Altemeier and Robbins (1966). The selected samples of serums and intestinal wash- ings were mixed with equal amounts of anti-IgA immunoglobulin. The mixtures were incubated for 1 hour at 37 C and then used for agglu- tination and PHA assay. This was conducted to detect the presence of IgA type immunoglobulin with E. coli 026 specificity (Williams, 1976). Preparation of Anti-Whole Bovine Serum (AWBS) Four rabbits were used to produce AWBS. Blood obtained from a normal cow was clotted at 25 C for 1 hour and then overnight at 4 C. The serum was separated and was diluted with 0.1M phosphate buffered saline (pH 7.3). Equal amounts of diluted serum and complete 53 Freund's adjuvant1 (6 m1 + 6 ml) were mixed and homogenized. Each rabbit was given 2 ml of homogenate with a total protein content of 2.15 mg. Two weeks later the rabbits were given a booster injection with the same amount of protein. The rabbits' serums were tested after 8 days and the animals that had serum with strong precipitin arcs on IEP were bled and the antisera were stored at -70 C (Williams and Chase, 1967). Separation of IgA from Colostrum To identify the presence of IgA immunoglobulin in the intestinal washings, serum and plasma cells of the lamina propria there was need for anti-IgA. Although bovine colostrum is not a rich source of IgA compared to other secretions, it is more easily available in large amounts. The first colostrum from Holstein-Friesian cows was used and IgA was partially purified by salting out, dialysis and gel filtration chromatography. Finally the product was purified by specific immunodiffusion and was injected into guinea pigs to pro- duce anti-IgA. One hundred milliliters of first colostrum was pooled from 3 Holstein-Friesian cows. Pat was removed from the colostrum by centrifugation for 60 minutes at 2500 x 9 after which the fat layer was removed with a spatula. The caseins were removed by lowering the pH to 4.6 with 0.1M HCl, incubating for 30 minutes, and centri- fuging for 30 minutes at 2500 x g. The supernate was dialyzed 6 hours against cold running tap water at 10 C and then 48 to 72 hours l Obtained from Difco Laboratories, Detroit, Michigan. 54 against 3 changes of distilled water at 4 C to effect precipitation of the euglobulins. The water soluble fraction was precipitated by slowly adding an equal volume of saturated ammonium sulfate while stirring. The stirring was continued for 3 hours at room tempera— ture and then the solution was centrifuged at 10,000 g for 30 minutes. The resuspended precipitate was considered the water soluble immuno— globulin fraction (WSIF). The supernatant, although still rich in immunoglobulins, is called the water soluble nonimmunoglobulin fraction (WSNF) and can be used for the preparation of free secre- tory component (Butler and Maxwell, 1972). The precipitate (WSIF) was dissolved in PBS to 1/3 the starting volume. The redissolved protein was reprecipitated with saturated ammonium sulfate and dissolved in starting buffer for column chroma— tography. The sample was then dialyzed in 4 liters of starting buffer for 48 hours at 4 C until complete removal of sulfate and was filteredm (0.45 Mm) and used for gel filtration chromatography (King, 1968). Sephadex G-200n was swelled and boiled in distilled water for 3 hours. The gel was cooled and resuspended in chilled Tris-HCl buffer0 (0.1M Trizma, pH 8.2) to which sodium azide (400 mg/l) had been added as preservative. The gel was degassed in vacuum for 3 hours and poured into a columnp (2.5 cm x 90 cm) at 4 C. After m . Metrical CrA-6, 25 mm, Gelman Instrument Co., Ann Arbor, MI. nSephadex G-200, Pharmacia Fine Chemicals, Piscataway, NJ. oSigma Technical Bulletin No. 106B, Sigma Chemical Co., St. Louis, MO. pGlenco Scientific Inc., Houston, TX. 55 packing, the flow rate was adjusted to 9 ml/hr and buffer was passed continuously. The sample (total volume did not exceed 1% of the total bed volume) was layered onto the surface of the gel and sample fractions were collected in 3 m1 volumes with a fraction collectorq and monitored at 280 um of ultraviolet light in a spectrophotometer.r Selected fractions were examined by IEP and were pooled and concen- trated by ultrafiltration in a vacuum (Craig, 1968) and stored at 4 C. Preparation of Antisera to IgA The eluates which were pooled from peak 1 by gel filtration were rich in IgA as determined by monospecific antiserum for IgAS but also contained by I96 I96 and IgM. The eluates were sub- 1' 2 jected to IEP with anti-IgAs and precipitin arcs were extensively washed as described previously and cut carefully out of agar gel. They were dissolved in PBS and emulsified with an equal volume of adjuvantt before injection into guinea pigs (Smith et al., 1964; Nansen et al., 1971). Four guinea pigs were used for production of antisera and each animal received 12 IgA precipitin arcs. Half the volume of material was injected intramuscularly into the thigh and the rest was injected subcutaneously at several sites in the posterior thoracic region. qulumetric Fractionator, Model V-10, Gilson Medical E1ect., Middleton, WI. r Model DB, Beckman Instruments Co., Fullerton, CA. sKindly supplied by Dr. R. D. Schultz, Cornell University, Ithaca, NY. t . . . Freund Complete Adjuvant, Difco Lab, DetrOit, MI. 56 Two weeks later the guinea pigs were given a booster injection with 6 precipitin bands each and exsanguinated 10 days later. Blood from guinea pigs with a strong monospecific response to IgA was collected. Antisera were divided into several tubes and stored at -70 C. Conjugation of Anti-IgA to Fluorescein Isothiogyanate (FITC) The anti-IgA which was collected from guinea pigs was precipi— tated by saturated (X3) ammonium sulfate to remove albumin. The conjugation of anti-IgA was performed by following the method described by The et al. (1970). Fluorescein isothiocyanateu (8 mg/g of protein) was dissolved in NaZHPO4 (1 ml/mg of FITC). The pH of the FITC solution was adjusted to 9.5 by adding 0.1M Na3PO4 (pH 12) solution. The protein was placed into a 25 m1 beaker and, as the FITC was added, the pH was constantly adjusted to 9.5. The conjugation was allowed to proceed for 1 hour at room temperature while gently stirring. The unconjugated FITC was removed by passing the mixture through Sephadex G25 gel.v Frozen Section Procedure (Cutting, Staining) Frozen sections of the tissues collected from the calves were cut on an International-Harris Cryostat. Sections cut at a thickness of 10 u were immediately fixed in chilled acetone for 15 minutes. The fixed tissues were stained with conjugated antibody for 30 minutes at room temperature, rinsed and washed in PBS for 15 minutes, u . . . . Obtained from Sigma Chemical Co., St. Louis, MO. v . . . . . Obtained from PharmaCia Fine Chemicals, Piscataway, NJ. 57 and mounted in buffered glycerin (1 part PBS and 9 parts glycerin) (Richardson et al., 1972). For controls the tissues were also stained with normal conjugated guinea pig sera or frozen sections were first flooded with unconjugated anti-IgA for 20 minutes and then were stained with conjugated IgA for 30 minutes. Bacteriologic Procedure Tissues and samples of amniotic fluid were cultured for bac- Incubation was at 37 C for terial organisms on blood agar plates. 24 hours. Isolates were identified by standard biochemical tests. Histopathologic Technique Tissues were fixed in 10% formalin-sodium acetate and routinely stained with hematoxylin and eosin. Periodic acid-Schiff and Giemsa stains were used for plasma cells. Gram's stain was used for bac- teria (Luna, 1968). Photography Unstained immunoprecipitin bands in agar-gel were photographed by the use of a multipurpose industrial view cameraw and black and white film.x wPolaroid Mp-3 Land Camera, Polaroid Corp., Cambridge, MA. xPolaroid Land Roll Film Type 47, Polaroid Corp., Cambridge, RESULTS The first step in this study was the preparation of purified anti-IgA immunoglobulin. The first peak, which was eluted by G-200 gel filtration chromatography, was rich in IgA but also contained IgM and IgG (Figure 2). The eluate was purified by IEP on agar-gel l as described before by using the semipurified IgA from the first peak and anti-IgA received from Cornell.a The anti-IgA which was finally produced in laboratory animals had monospecific activity corresponding to the electrophoretic mobility of IgA immunoglobulin. A single line was produced by IEP and by the Ouchterlony method when a semipurified eluate of colostrum was reacted with anti-IgA and antiwhole bovine serum (Figures 2 and 3). Two of the 30 calves which initially had been planned for this research died during parturition. One fetus was aborted 3 days after the intrauterine injection. A fourth calf was discarded because 2 attempts were needed for a successful intrauterine injection and the calf was born 2 days after the second injection. The results of the intrauterine injections, clinical responses and responses to challenge are shown (Table 1). None of the calves that were given an oral challenge dose of live E. coli died as a aKindly provided by Dr. A. D. Schultz, Cornell University, Ithaca, NY. 58 » figure 1. Photograph of the immunoelectro— ghoretic pattern of monospecific anti-IgA produced in guinea pigs. Monospecific anti-IgA (A): semi- purified IgA from colostrum (C); antiwhole bovine serum (B) yruduced in rabbit. Notice the immunopre— cigitato bands IQA (a), IgM (m), Iqu (g). (35' 21w 3 l‘imtoarag h of the Cuchterloriy ’3 say: (1) and immurmlL-ctro}Emretic pattern (_) of monospecific antibovino 1qu produced in guinea iii}. Non/Ha} egi tic .artti—IOA (a); m'nicsgwacific Li~Iv:i-. r1 velvei from Cornell (l ); szrr‘i; :zri {it ri ‘f'i “r 1* 2'?» 2:33- . ., ’fi 3 «7’ {'12} \- l. cuuan um I I 2 mo nmm mo ocaamm mama cuuan um I I m we czo om mcaamm oamm cuuao um I I 2 0m vnm em moaamm vmm o rowan um I I 2 no amm mm oauwuowo mmvm Logan um I I 2 we dzo om caumuomo mama rowan um I I m mm mum om aaumuomn omoa Logan um I I m mm amm oa cauouomn ommm m .amcqumom v I aaoa x m.a 2 we mmm mm oaumuomo vmma .amcqumOQ a I aaoa x m.a 2 Ne azo em caumuomn mmem .amnqumOQ m + aaoa x o.a 2 am mmm ma caumuomn mama .amnqumom m I oaoa x m.a m I New aa saumuomo oamm .amcqumom m I oaoa x m.a 2 mm van oa cauouomo mmva v n I I m we «42o vv cauouomo v m I I m mm mum aa oauouomo oaam m 1 6 .amnqumOQ a I aaoa x m.a a on mam he caumuomn omom .amcqumom m I aaoa x o.a m we vmm ma aauwuomn vama .amBUIumod m I oaoa x m.a z I own m caumuomn no .amnqumOE v I oaOa x m.a m I mom m caumuomn mmma m ea I I 2 on amm me cauouomo meow «a I I m ow mmm we oauouomo mmva a am>mov cocoaamno aaaoo .m xom anav amxmov UOauom sumac can GOauoomca .02 .0: wammcmnuoo ammo pound manda>v unmao3 :Oaumumou coauoonca oaoo: ma wamu msouo mo oEaB mcmam mmoo cocoa nuuam coosuon when mo omwe aaoacaao Iamno amuo aoan mmsouuv ocaaMm uo amIa masouuv aauouomn zzuooxnomo waoo .m spa: momsuow oca>on mo mcoauoonCH Chou: CH 0» UmuMaou upon .a magma 62 Ca mo>amo com nuuan um waoo .M No omoc mmcoaamco m co>am muw3 n msouw ca mo>amu omoo omcoaamso w co>am oumz v moouo Ca mo>amu ammo .Cauouomn zzuooxuomo Haoo .m sea: cuuao um ooumcaoom> mums m can m .a mmsouo ca mo>amu .wmm mo mxoo m on comcoaawoo muo3 m moouu .nuuao um waoo .m m0 .maQMaam>m uoc moon .1 .wmo mo mmmo m we mmz m msouo ca mo>aoo now cocoaamco ea I I m me I mv moaamm acmm oa m I I m I mom m moaaMm mwom m .amnqumOS a I aaoa x m.a m em omm mm mcaamm soma m .am;UIum0d m + aaoa x m.a 2 mm ohm om meaamm mama .awnqumod a I aaOa x o.a 2 mm mam mm acaamm mam .amnqumoha m I oaoa x m.a 2 mo omm ma ocaamm nmov n amxmov cocoaamno aaaoo .m xom Anav amxmov coaumm Susan can GOauooflCa .0: .oc mammcmnuso amuo umumm oanma>v unmao3 coaumpmow COauoonca Chou: CH mamo moouu mo oEaB woman omoo cocoa nuuam cmwzuon mama mo mama amoacaau Iamno ammo Apnoeaucoov a manna 63 result of infection. One newborn vaccinated calf (1377) and 1 new- born control calf (1912) had clinical signs after an intermediate 11 . ll . (1.0 x 10 ) and a high (1.5 x 10 ) challenge dose, respectively. The clinical signs included anorexia, depression, dehydration and generalized weakness. A mild diarrhea with mucous and blood-covered feces was present. The challenge dose of viable E. coli was either 1.5 x 1010 (5 ml), 1.0 x 1011 (30 ml) or 1.5 x 1011 (50 ml). The initial low challenge dose failed to produce clinical disease. The intermediate and high challenge doses produced clinical signs in l principal and 1 control calf. The pathogenicity of the 026:K60:NM strain of E. coli had been demonstrated previously in dairy calves (Conner, 1973). Two dairy calves were given the low challenge dose at birth. The calves died within 36 hours and had typical signs of colibacillosis. Bacteriologic examination from heart, liver, spleen and kidney tissues from calves 1377, 299 and 1912 showed light growth of non- hemolytic E. coli. The gross pathologic changes in those calves with clinical disease were edema and congestion of mesenteric lymph nodes, hyper- emic mesenteric tissue, petechial hemorrhage of the thymus and con— gestion of the spleen. The intestinal wall appeared thicker than normal and was hyperemic. Congestion and edema of the villi, sub- mucosa and mesenteric lymph nodes were the outstanding histopatho- logic lesions in calves with clinical disease. There was dilatation of central lacteals and infiltration of inflammatory cells into the mesenteric lymph node (Figures 4 and 5). Calf 1912 from the control group and calf 1377 from the principal group had an extensive 64 Figure 4. Photomicrograph of the lower jejunum of control calf 4657 (group 7) challenged at birth with E. coli 026:K60:NM and euthanatized at 3 days of age. Notice extensive edema beneath mucosal epithelium (l). H&E stain; X125. 65 Figure 5. Photomicrograph of mesenteric lymph node of control calf 1912 (group 7) chal— lenged at birth with high dose of E. coli 026: K60:NM and euthanatized at 1.5 days of age. Notice extensive infiltration of neutrophils (arrow) into the sinusoids. HGE stain; X312. 66 infiltration of bacteria in the lower jejunum and ileum. These bacteria had adhered to the surface of the intestinal villi (Figure 6). Calf 4657 (Table 1) from the control group, which was challenged with the low dose, had bacterial infiltration and some bacterial adhesions to the intestinal villi. The 2 dairy calves which were challenged with a low dose of E. coli at birth had extensive edema, congestion and hemorrhage of the intestine and lymph nodes with an extensive infiltration of neutrophils. There were areas of necrosis and bacterial colonies in the intestinal mucosa. There were numerous plasma cells in tissues of the vaccinated calves and these were detected by special staining with Giemsa and PAS. The spleen and mesenteric lymph nodes of calves from groups 2 and 4 contained many huge plasma cells (Figure 7). These cells were especially prominent in calf 2036, which was orally revaccinated and later challenged with the high challenge dose (Figure 7). Anti-IgA immunoglobulin was used for the qualitative IEP test on calf sera at birth and at necropsy and on intestinal washings from the duodenum, upper and lower jejunum and ileum. The IEP pro- files from serum samples were negative in all calves with the exception of control calves 299 and 1567. These 2 calves had posi- tive IgA bands in serum samples at birth and at necropsy. The initial IEP test from intestinal washings was positive only for the washings from the ileum of principal calves 1429, 2036, 4, 1534 and control calf 2085, and for lower jejunal washings from control calf 2201. Further attempts to demonstrate a positive IgA reaction in intestinal washings resulted in negative or very weak reactions. 67 Figure 6. Photomicrograph of the villi of the ileum from control calf 1912 (group 7) chal- lenged at birth with a high dose of E. coli O26: K60:NM and euthanatized at 1.5 days of age. Notice edema (l), inflammatory cells (2), and accumulation of bacteria at brush border (3). Gram's stain; X500. 68 Figure 7. Photomicrograph of the spleen of principal calf 1214 (group 2) previously vaccinated in utero, revaccinated orally at birth and chal- lenged at 3 days of age with E. coli 026:K60:NM. Calf was euthanatized at 6 days of age. Notice plasma cells (arrow). Giemsa stain; X500. 69 The Direct Bacterial Agglutination Test and Passive Hemagglutina— tion Assays (PHA) were conducted on calf serum samples at birth and at necropsy with 026 and K60 E. coli antigens. The PHA tests were done on intestinal washings from the duodenum, upper and lower jejunum and ileum with 026 antigen. The results are summarized in Table 2. The passive hemagglutination titers were higher than they with the direct agglutination titer and serum samples at necropsy had from 2- to 5-fold higher titers than the serum samples taken at birth. There was no K60 agglutinin activity as measured by either direct or PHA methods on selected samples. The PHA titers from intestinal washings were mostly negative or very low. When the tube agglutination or PHA tests were repeated, the titers were negative or lower than initial titers. There was no correlation between the IEP positive reaction of intestinal washings to anti- IgA, and the PHA titers when 026 antigen was used. The attempts to potentiate the direct agglutination or PHA titers by anti-IgA immunoglobulin were unsuccessful. The absorption of immunoglobulin from selected samples of serum and intestinal washings with anti—IgA failed to decrease the tube agglutination and PHA titers. The results of staining tissue sections with conjugated anti- body are shown in Table 3. Several sections were made of each frozen tissue. Five sections of each tissue were stained with conjugated anti-bovine IgA immunoglobulin. To rule out the possi- bility of a nonspecific reaction, 1 section of each tissue was stained with conjugated normal guinea pig serum and 1 section was first layered with unconjugated anti-IgA and then stained with 70 I I I I I I Nana I I I I I I SamN I I I I I I 4mm I I I I I I NmeN I I I I m I mama I I I I I I coma I I I I N I ommN I I I I mmN ma e I emma I N I I ma N I I mmeN I I I I N I I I mama I I I I I I I I oamN I I I I I I I I mmea v N mNa v a I e I I I I I I I I oaaN omom I vama no I I I mmma I I I Nvom I I I I ma I N I mmva l N I I V NCON N NN I ‘71‘ H ha n3 0 C Q «as «in .0G «I«o>ammmm o>ammmm uoouao mamu wmcacmmz aMCaumoucH Eduom cmmauca oNo .oc moouo aoan mmoouov ocaamm no amIa mmsouuv cauouooo 27uooxuomo waoo .m cuaz came: 2a omuoonca wam30a>oum mo>amo amumcooc Eoum mmcanmm3 amcaummuca can Edumm Eoum muouau QOaumcausammmEoc o>ammmm can Esuom mo «muouau COHuMCausammm amauouomn uoouao .N manna 71 .xao>auoommou .omm mo mxmo ea com o um ooaaax mums oa msouo ca mm>amo com m msouw Ca mm>amu .suuan um ooaaax oum3 m can m mmsouw ca mo>amu .omm mo mxmo m um oomcoaamno mumz m msouo ea mo>amo com .nuwao um waoo .M NO omoo wocoaamso w co>am ouo3 n moouw Ca mo>amo .nuu an um aaou .M NO mmOp mocwaamco m cm>am oum3 v msouo ca mo>amu .mmm mo mwmo m on mp3 m moouo Ca mo>amo you once moccaamco amuo .cHumuomn Haoo .m nuaz suwan um covenaoow> mums m pom N .a mmsouofifiamo>amo .Edmaa u H .Escshom uo30a n ma .Edcsmofl Momma u no .Edcoooso Q .xmmouooc um maQEMm Edumm n c unuuan um mamEMm Eoumm o «as «a .waou .m mo comaucm omo Spas coumoo Ommm mo COaDMCauoammmEoL moazogm c0au5aa© uwmcman mnu mo amuoumaomu ecu mm owmmmumxm mum mumuaa k. I I I I I I I I aONN I I I I I I I I mNON I I I I ma I I I Noma I I I I m I I I mama I I I I ma N N I mmN I I I I I I I I emov oa H he as a a n «In «In .0: «I«o>ammmm o>ammwm uomuao mamo mmmanmoz aaoaumoucH Esumm comauca oNo .oc moouu xemscaucooc N magma 72 I I I I I I I I No hana I I I I I I I I om omam I I I I I I I I em emm 0 ma am I ma ma ma I I mm mmem ma mm I am ma ma I I om mama I ea I oa I I I I om omma I I I I I I I I oa ommm m om mm om mm ea ma Om mm mm emma em oe oa ae ma mm I an em mmem oa am oa ma I I I mm ma puma ma mm I oa I I I ma aa oamm I ON I I I I I ma oa mmea e om ao mma mm mm mm om mo ea e oa mm mm cm I I I mm aa oaam m mm moa oe mm mm ae ma mmm he omom ow mm om oma I 0a Om 0am ma eama em mo am me mm ON I oma m no am mm mm cm I I I om m mmma N mm mm m ma ma aa I mm me meow mm 0a oa mm ma ON I om ee mmea a zaH H zana ma zans ha a cooamm gouan can coauoonca .o: .0: «maamu «Emmam mo Nonssz coozuon wwwo Mano msouo aoan mmsouov ocaaMm Ho amIa mmsouwv cauouomn zzuooxuomo waoo .m nua3 camps Cw oouoohca wamooa>oum mo>amo amumcooc Eoum pom CaasQOamocsEEa mmHIaucw ooummoncoo saooonOSam nuaz cesawum moSmmau ca mucooo aamo manuam .m magma .>ao>auoommou .omm no name ea one m on ooaaax mums oa com 0 monouw Ca mo>amo .nuuan up coaaax mums m can m mmsouo ca mo>amo .omo No name m on ommcmaamno mumz m msouo Ca mo>amo com sumac um «Moo .m we omoo mmcoaamco m cm>am ones 5 macaw ca mo>amu .nuuao um aaoo .m «o mmoo omcoaawco m co>ao ouw3 e moose Ca mo>amo .006 m0 m>mc m an mp3 m moouu Ga mo>amo now omcwaamgo ammo .cauouomo Haoo .m QuaB cuuan um conceaoom> mums m can m .a mmsouw ca mo>amo .oooc nmswa Edoaa u zaH .Edoaa u H .oooc nmexa Escsnoh uo30a n zana .Escshmm umBOa u ha .mp0: £QE>a Edcsflwn nomad u zqns .Edsoflwn momma u no .Edcooooo n o .aommxv moaoam oamoom Iouoae o>auooomcoo om ca ooucsoo ouo3 £0a£3 maaoo mammam mo nomads amuou opp oumoaoca mousmam .1 73 ma mm I ON I m I em me aomm oa I ma I I I I I mm m mmom m I I I I I I I I mm noma w I I I I I I I I om mama I I I I I I I I mm mom I I I I I I I I ma nmoe n qu H zaaq ha zqno as o cmmamm nonan 62m coauomaca .oc .oc «maamu meMam mo Honesz cmo3uwn m>mo mamu moouo Aemscaocooc m manna 74 conjugated anti-IgA. The optimal dilution for conjugated antiserum was 1:4. Each stained section was observed by fluorescence microscopy and the total number of plasma cells in 20 consecutive microscopic fields was determined under (x250) magnification. Calves 334, 2816 and 1717 in control group 6 (Table l), which were killed at birth, had no evidence of fluorescent plasma cells. Calves in control group 7 (4657, 299 and 1912), which were chal— lenged at birth, and 1 control calf from group 8 (1567), which was challenged at 3 days of age and killed at 7 days of age, had no indication of fluorescent plasma cells. One calf in each control group (group 9, calf 2085; group 10, calf 2201), which were killed at 9 and 14 days of age, respectively, had appreciable numbers of fluorescent plasma cells in the spleen and ileum (Table 3). The calves in principal group 5 (Table l) were vaccinated in utero and killed at birth. The time of vaccination ranged from 10 to 28 days before parturition. Calf 2290, which was vaccinated 10 days before birth, had no evidence of fluorescent plasma cells. The other 3 calves in this group had variable numbers of fluorescent plasma cells in the jejunum and ileum, but none had any plasma cells in the spleen or duodenum (Table 3). The calves in principal group 4 (Table l) were vaccinated in utero, challenged at birth, and were killed at 2 to 4 days of age. Calves 1459 and 2510, which had been vaccinated 10 to 11 days (Table 1) before birth, had small numbers of fluorescent plasma cells in the spleen and ileum. Calves 1377, 2459 and 1534, which had been vaccinated in utero 18, 34 and 28 days before birth, had higher numbers of cells in the spleen and cells in other regions of 75 the intestine. Calves 2116 and 4 from group 3 were vaccinated 11 and 44 days before birth, revaccinated orally at birth, and were killed at 5 and 7 days of age, respectively. Calf 4 had higher numbers of fluorescent plasma cells, and the cells were present in all tissues examined (Table 3). Calves 1322, 67, 1214 and 2036 from group 2 were vaccinated in utero at times ranging from 8 to 47 days (Table 1) before birth. They were revaccinated orally at birth, were challenged at 3 days of age and were killed at 4, 5, 3 and 4 days after challenge. Calves 1214 and 2036 of this group had the highest numbers of fluorescent plasma cells in the experiment (Table 3; Figures 8 and 9). Calves 1429 and 2042 in group 1 were vaccinated 44 or 49 days before birth, revaccinated at birth and were killed at 14 days of age. Both calves had appreciable numbers of fluorescent plasma cells in all tissues examined, with the exception of the duodenum. 76 Figure 8. Photomicrograph of frozen section from spleen or principal calf 1214 (group 2) pre- viously vaccinated in utero, revaccinated orally at birth and challenged at 3 days of age with E. coli 026:K60:NM. Calf was euthanatized at 6 days of age. Stained with fluorescein conjugated anti- IgA immunoglobulin. Notice positively stained plasma cells (arrows). X400. 77 Figure 9. Photomicrograph of frozen section from ileum of principal calf 1214 (group 2). Stained with fluorescein conjugated anti-IgA Notice positively stained plasma cells (arrow). X250. immunoglobulin. DISCUSSION Some portions of this research were done jointly with Olson (1975). These include the basic designing of the experiment, the in utero injection of the cows, the handling of the calves, and bacteriologic procedures. Olson was interested mainly in evaluation of the IgM and IgG immunoglobulins in the intestine. Wamukoya (1975) used immunofluorescent techniques to detect IgM- and IgG-containing plasma cells in calves in groups 4, 5, 6 and 7 (Figure 1). Several problems arose during the course of the experiment which made it necessary to alter the design. These problems and alterations increased the number of variables, decreased the numbers of calves in certain groups, and resulted in data which in many instances were difficult if not impossible to analyze statistically. Four of the 30 calves were lost because of problems with injections, abortions or stillbirths. Failure of the primary challenge dose to produce clinical disease was the principal factor which made it necessary to vary the experimental design. The challenge dose used initially had earlier been shown to be effective in the production of clinical disease in dairy calves (Conner, 1973; Gay, 1971). We orally inoculated 2 dairy calves with the dosage used by Conner and were successful in producing clinical signs of colibacillosis. In our experiments with beef calves we were 78 79 unable to produce the clinical disease in either the principals or the controls by giving the dosage used in dairy calves. Further— more, by increasing the dosage we were able to produce clinical signs in 1 principal calf and 1 control calf (Table 1). These results indicate that beef calves may be more resistant to coli- bacillosis than dairy calves. Most of the cows which were used in this experiment were naturally bred on pasture. This made a precise determination of the time of gestation impossible. As a result the time between the in utero inoculation and parturition varied from 5 to 62 days (Table 1). This time variation between inoculation of bacterin and birth definitely affected the immunological response in the calves. Although there were no meaningful differences in the clinical manifestations among calves in the various groups, there were dif- ferences in the histologic changes in the intestinal tract. Of particular interest were the accumulations of large numbers of bacteria on the brush border of the intestinal villi of unvaccinated calves given a challenge dose of E. coli at birth. There also were intestinal edema and congestion in these calves. Since similar changes were not seen in vaccinated calves given the same challenge dose, it is likely that immunoglobulins or a combination of immuno- globulin and other factors provided a local protective mechanism in the intestine. These factors may prevent bacterial attachment to the villi and, if the bacteria cannot become attached, they are moved down the tract and are unable to cause disease. Waldman and Ganguly (1974) suggested that a possible mechanism for the inhibition of absorption and growth of bacteria on mucosal cells is the presence 80 of secretory antibodies. The ability to adhere to the mucosal cell wall may be an important feature in determining growth and patho- genicity for various bacteria. Taylor et al. (1958) observed that when the more virulent strains of E. coli were inoculated into ligated intestinal loops of rabbits, they adhered in a compact layer over the epithelium and covered the villi. The less virulent strains grew freely in the lumen. They suggested that the role of secretory antibodies might be to interfere with adherence of patho- gens, with or without involvement of other nonspecific factors in this complex environment. It is difficult to prepare IgA which is as pure as IgM and IgG preparations, particularly when biological integrity is a strong consideration (Butler, 1972). The method which was used in this experiment for final purification of IgA, the immunoprecipitation of antigen by monospecific antiglobulin in agar-gel, was the simplest and most direct way to purify IgA from semipurified material. Possibly the use of isoelectric focusing or the use of immunoabsor- bents may be methods of choice for final purification. Plasma cells were numerous in tissues from calves in vaccinated groups and were especially numerous in calves in groups 2 and 4. These calves had been given challenge doses of E. coli. Special staining with PAS and Giemsa revealed many plasma cells in the spleen of these calves. Examination of splenic tissue from these calves by the use of fluorescent microscopy also revealed many plasma cells. Apparently the challenge dose of E. coli induced a secondary response which potentiated the conversion of lymphocytes to plasma cells. 81 Fey (1972) stated that colisepticemia is a disease of agamma- globulinemic or markedly hypogammaglobulinemic calves. Agamma- globulinemic calves are extremely susceptible to both experimentally induced and naturally occurring colisepticemia. There is a marked decrease in susceptibility in calves whose serum immune globulin values approach 400 to 500 mg of IgG/100 ml, and normogammaglobulinemic calves are resistant. The gut of the newborn calf transmits protein in solution nonselectively, and the normal newborn animal receives into its circulation all proteins that occur in colostrum (Brambell, 1970). There was considerable evidence which strongly suggests that 2 of the control calves (299 and 1567) may have suckled after birth. Both of these calves were resistant to the challenge dose, and it is presumed that acquired colostral immunoglobulins were responsible for the resistance. Evidence to support this presumption consisted of the presence of strong IgA bands in the IEP profile of serum samples taken shortly after birth and at necropsy. Olson (1975) also presented data which indicated that calf 299 may have suckled. He reported that both of these control calves had a serum immuno- globulin pattern on IEP which was identical to adult serum. All control calves' sera had negative 026 agglutinin titers at birth, with the exception of these 2 control calves. It is difficult to explain why one of the vaccinated calves had clinical signs of colibacillosis after being given the challenge dose. In utero vaccination had taken place 18 days before birth, and IgA-positive plasma cells were in the tissues examined. Wamukoya (1975) found IgM- and IgG-positive plasma cells in tissues from this 82 calf. Evidently the calf was not adequately stimulated by intra— uterine vaccination and was not protected from the challenge dose. Most of the plasma cells could have appeared because of a rapid response to the challenge dose. It was difficult to interpret the results of tube agglutina- tion and PHA tests on serum samples and intestinal washings. Olson (1975) reported that the initial titers were much higher than their duplicates which were done later. Also in the present experiment duplicate tests of the samples resulted in negative titers or much lower titers than the initial titers. The lowering of titers was more pronounced in tests on intestinal washings. Apparently the IgA and IgM immunoglobulins are very sensitive to thawing and freezing (Williams, 1976). This sensitivity may explain the failures in attempts to potentiate the tube agglutination and PHA titers by anti-IgA immunoglobulin or to decrease the titers by absorbing the serum with anti-IgA. The IEP-positive reactions of intestinal washings could not be correlated with the PHA titers of the same washings, but the reac- tions of intestinal washings did correlate well with the fluorescent plasma cell counts in tissue sections of the ileum. Improved handling of serum samples and intestinal washings may be essential in order for future workers to obtain more meaningful results. In the species so far studied, including man, the serum of the unsuckled newborn is severely deficient in IgA (Tomasi and Grey, 1972). In most studies serum IgA is reported to reach adult levels at a later age than either IgG or IgM (Uffelman et al., 1970). The IEP tests of serum samples at birth and necropsy were negative for 83 IgA immunoglobulin in all calves with the exception of serum from the 2 calves (299 and 1567) which apparently consumed colostrum. These results indicate that 1- to l4-day-old calves do not have detectable IgA immunoglobulin in serum if they have not been fed colostrum. There are various ways that one can interpret the results of counting fluorescent plasma cells (Table 3). The procedure provides a means to monitor the appearance and to quantitate IgA-containing plasma cells which have appeared as a result of in utero vaccination and subsequent revaccination and challenge. The results also may be a direct measurement of naturally appearing IgA-containing plasma cells without apparent antigenic stimulation. It is also likely that some of the IgA-containing plasma cells seen in the older calves could have appeared naturally and some appeared as a result of the various stimuli provided during the experiment. The first indication of the presence of IgA-containing plasma cells in controls was in calf 2085 of group 9, which was killed at 9 days of age. In this calf fluorescent plasma cells were present in the spleen and ileum. Control calf 2201 from group 10, which was killed at 14 days of age, had numerous IgA-containing cells in the spleen, upper and lower jejunum and ileum. These observations indicate that naturally appearing IgA-positive plasma cells appear as early as 9 days of age and initially appear in the ileum and spleen. Calves in control groups 6, 7 and 8 (Table 3) had no evidence of fluorescent plasma cells in any of the tissues examined. There also was no indication of IgG- and IgM-containing plasma cells in the tissues of calves from control groups 6 and 7, which were 84 either killed at birth or challenged at birth and killed at 3 or 4 days of age (Wamukoya, 1975). Of considerable interest were the calves in group 5, which were injected in utero and killed at birth (Table l), in order to measure the primary response of calves to a single injection of bacterin. Generally the greater the time between intrauterine vac- cination and birth, the greater the number of fluorescent plasma cells. A wider distribution of plasma cells in the various tissues also occurred when the time was greater. Calf 2290, in which intra- uterine vaccination had occurred 10 days before birth, had no visible IgA-containing plasma cells. Calf 1690, which had received an intrauterine injection 20 days before birth, had some fluorescent plasma cells in the ileum and lower jejunum. The 2 other calves in this group, which were injected 20 and 28 days before birth, had numerous IgA-containing plasma cells in the jejunum, ileum and related lymph nodes (Table 3). The calves in principal group 4 were vaccinated in utero and were given a challenge dose of E. coli at birth (Table l). Apparently, the challenge dose had the effect of inducing a secondary response. Calves 1459 and 2510 were vaccinated 10 and 11 days before birth and were killed 3 days after challenge. There were some IgA-containing plasma cells in the spleen, jejunum and ileum. The other calves in this group, which were vaccinated at times ranging 18 to 34 days before birth, had varying numbers of fluorescent plasma cells in the spleen, jejunum and ileum. One calf (1534) had numerous fluorescent plasma cells in the duodenum. 85 The 2 calves in group 3 were vaccinated in utero, revaccinated at birth, and killed at 5 or 7 days. The revaccination was apparently effective in inducing a secondary response since fluorescent plasma cells were seen in higher numbers and had a wider distribution. Calf 4, which was vaccinated 44 days before birth, had more fluorescent cells than calf 2116, which had been given an intra- uterine vaccination 11 days before birth. The calves in group 2 were vaccinated in utero at times ranging from 8 to 47 days, revaccinated at birth, given a challenge dose of E. coli at 3 days of age and killed at times ranging from 3 to 5 days after challenge. The calves in this group had the highest number of fluorescent plasma cells, and these cells had a wider distribution in the various tissues. These results indicate that a challenge with E. coli can further stimulate a calf which has been treated as described. In this group calves 1322 and 67, which had been given an intrauterine vaccination only 8 days before birth, had lower numbers of IgA-containing cells than the other 2 calves. This further emphasizes the importance of an adequate time between vaccination and birth. The calves in group 1 were vaccinated in utero 44 and 49 days before birth and revaccinated orally at birth. They were killed at 14 days of age (Table 1). These calves had numerous plasma cells :Ln all tissues observed. The spleen had the highest number, but tflnere were no fluorescent cells in the duodenum. The population of Illasma cells at 14 days of age may be a combination of naturally appearing cells and those resulting from the experimental stimulation. 86 In summary, there were appreciable numbers of IgA-producing plasma cells in in utero vaccinated calves at birth. These cells were more prominent in orally vaccinated or challenged calves. The lower jejunum and ileum and related lymph nodes had more IgA- producing cells than any of the other tissues examined. Furthermore, in revaccinated and challenged calves the spleen was especially active in the formation of IgA-containing plasma cells. The duodenum had more limited activity. The results indicate that the entire small intestine, the draining lymph node and the spleen were involved in IgA formation in these young calves. The control calves had no indication of fluorescent plasma cells before 9 days of age. This further demonstrates the importance of age in IgA immunoglobulin production. The immunoelectrophoretogram of serum samples of principal and control calves at birth and necropsy were negative for IgA immunoglobulin. The results of the hemolytic plaque assay (Olson, 1975) showed the importance of IgM in the early development of the local intes- tinal immune system. The principal target organs for antigenic stimulation were the intestinal and mesenteric lymph nodes. Immuno- electrophoretograms of serum from control calves tested at birth showed the presence of immunoprecipitate bonds resembling IgG1 and IgG and suggested that the calves were not agammaglobulinemic at 2 birth. The fluorescent antibody study using monospecific conjugated 133M and IgG (Wamukoya, 1975) showed no evidence of fluorescent Ellesma cells in control calves at birth or in calves challenged at lDirth and killed at 5 days of age. The principal calves vaccinated 87 in utero and calves vaccinated in utero and orally revaccinated had numerous fluorescent plasma cells, especially in the jejunum and ileum. The IgG-producing plasma cells were more prominent than the IgM-producing cells. SUMMARY This study emphasized the chronologic appearance of immuno- globulin A (IgA)-containing plasma cells and their distribution and numbers in the intestinal tract, spleen and mesenteric lymph nodes in beef calves vaccinated in utero with Escherichia coli (E. coli) bacterin. The immunologic and pathologic effects of revac- cination at birth and of challenge with E. coli were also studied. The 17 principal calves were vaccinated in utero by a non- surgical procedure with E. coli 026:K60:NM bacterin (5.0 x 1010 organisms) being inoculated into the amniotic fluid during the last 6 weeks of gestation. Nine control calves were given intrauterine inoculation of saline. The calves were deprived of colostrum after birth and were divided into 5 principal and 5 control groups. Calves in 2 out of 5 principal groups were given a single in utero injection of bacterin, 1 group was killed at birth, and 1 group was given E. coli at birth and killed 2 to 4 days after challenge. Calves in the other 3 principal groups were revaccinated orally at birth after a previous single in utero injection of bacterin. The calves in l of the revaccinated groups were killed at 5 to 7 days of age. Calves in one group were given E. coli at 3 days of age and were killed at 3 to 5 days after challenge. Calves in the last group were killed at 14 days of age. Calves in l of the 5 groups of control calves were killed at birth, 1 group of calves at 9 days 88 89 and another at 14 days of age. Control calves in a fourth group were challenged with E. coli at birth and killed at 1.5 to 4 days of age. Control calves in the last group were given E. coli at 3 days of age and killed at 4 days after challenge. The challenge doses were 1.5 x 1010, 1.0 x 1011 or 1.5 x 1011 E. coli organisms. Only 1 calf from the principal group and 1 calf from the control group had clinical signs of colibacillosis after challenge with the intermediate or high challenge dose, respectively. The most prominent histologic lesions in these 2 calves were congestion and edema of the small intestine and infiltration of bacteria into the brush border of the villi. There was serologic evidence to suggest that 2 of the control calves had consumed colostrum. These 2 calves failed to have clinical signs of colibacillosis after the challenge dose and acquired colostral antibody was the most probable cause of the resistance. Gel filtration and immunoelectrophoresis in agar-gel were used to purify the IgA in samples of bovine colostrum. The purified IgA was injected into guinea pigs. Serum from these guinea pigs was used as the source of the anti-IgA for the studies utilizing immunofluorescence and immunoelectrophoresis. The serum tube agglutination and serum passive hemagglutination assay (PHA) titers to 026 antigen at necropsy were higher than at birth from calves in principal groups. The PHA titers of intestinal washings were very low or negative. By IEP, intestinal washings were positive with anti—IgA immunoglobulin in 4 principal calves and 2 control calves, but these results did not correlate with the PHA titers. 90 Studies using immunofluorescence indicated that there were appreciable numbers of IgA-producing plasma cells in in utero vac- cinated calves at birth. These cells became more numerous in orally vaccinated or challenged calves. The lower jejunum and ileum and related lymph nodes had more IgA-producing cells than any of the other tissues examined. Furthermore, in revaccinated and challenged calves the spleen was especially active in the formation of IgA- containing plasma cells. The duodenum had more limited activity. The results indicate that the entire small intestine, the draining lymph nodes, and the spleen were involved in IgA formation in these young calves. The control calves had no indication of fluorescent plasma cells before 9 days of age. This further demonstrates the importance of age in IgA immunoglobulin produc- tion. The immunoelectrophoretograms of serum samples of principal and control calves at birth and necropsy were negative for IgA immunoglobulin. REFERENCES REFERENCES Adinolfi, M., Glynn, A. A., Lindsay, M., and Milne, C. M.: Sero— logical properties of VA antibodies to E. coli present in human colostrum. Immunology, 10, (1966): 517-526. Allen, W. D., and Porter, P.: Localization by immunofluorescence of "secretory component" in porcine intestinal mucosa. Immunology, 24, (1973): 363-373. Altemeier, W. A., Robbins, J. B., and Smith, R. T.: Quantitative studies of the immunoglobulin sequence in the response of the rabbit to somatic antigen. J. Exp. Med., 124, (1966): 443. Amstutz, H. E.: Occurrence and etiology of infectious calf diarrhea. In Symposium on Infectious Diarrhea in Calves. J.A.V.M.A., 147, (1965): 1360-1363. Barnum, D. A., Glantz, P. J., and Moon, H. W.: Colibacillosis. Ciba Veterinary Monograph Series - 2, Ciba Pharmaceutical Co., Summit, N.J., (1967). Barry, G. T., Abbot, V. D., Everhart, D. L., Leffler, R. J., and Mynant, E.: Relationship of serotype to colicine type in E. coli. Bacteriol. Proc., (1962), p. 51. Bellanti, J. A.: Immunology. W. B. Saunders Co., Philadelphia, Pa., (1971): 13-59. Besredka, A.: Local Immunization. Williams and Wilkins, Baltimore, Md., (1927): 181 p. Bienenstock, J., and Tomasi, T. B.: Secretory yA in normal urine. J. Clin. Invest., 47, (1968): 1162. Billingham, R. E., and Lampkin, G. H.: Further studies in tissue homotransplantation in cattle. J. Embryol. Exp. Morphol., 50, (1957): 351-357. Bistany, T. S., and Tomasi, T. B.: Serum and secretory immuno- globulins of the rat. Immunochemistry, 7, (1970): 453. Brambell, F. R.: The Transmission of Passive Immunity from Mother to Young. Amer. Elsevier Pub. Co., New York, (1970). 91 92 Brandtzaeg, P., Fjellanger, I., and Gjeruldsen, S. T.: Human secre- tory immunoglobulins. I. Salivary secretions from indi- viduals with normal or low levels of serum immunoglobulins. Scand. J. Haemat. (Suppl.), 12, (1970): 4. Braun, A. K., Osburn, B. I., and Kendrick, J. W.: The immunological response of the bovine fetus to bovine viral diarrhea virus. Am. J. Vet. Res., 34, (1973): 1127—1132. Brown, R. D.: Rinderpest immunity in calves. I. The acquisition and persistence of maternally derived antibody. J. Hyg. Camb., 56, (1958): 427-434. Brown, T. T.: Pathogenetic studies of BVD infection in the bovine fetus. Ph.D. thesis, New York State Veterinary College, Cornell University, (1973). Burtin, P., Hartmann, L., Heremans, J., Scheidigger, J. J., Westerdorp- Boerma, F., and Wiemer, R.: Etudes immunochimiques et immuno- electrophoretiques des macroglobulinemies. Rev. Franc. et Clin. Biol., 2, (1957): 5. Butler, J. E.: Bovine immunoglobulins: A review. J. Dairy Sci., 52, (1969): 1895-1909. Butler, J. E.: Physicochemical and immunochemical studies of bovine IgA and glycoprotein-a. Biochem. Biophys. Acta, 251, (1971): 435. Butler, J. E.: Synthesis and distribution of immunoglobulins. J. .V.M.A., 163(7), (1973): 795-800. Butler, J. B., Groves, M. L., and Coulson, E. J.: The identification of a secretory immunoglobulin in the cow that is antigeni- cally related to glycoprotein-a. Fed. Proc., 29, (1970): 642 (Abstract). iButler, J. E., and Maxwell, C. F.: Preparation of bovine immuno- globulins and free secretory component and their specific antisera. J. Dairy Sci., 55(22), (1972): 151-164. IButler, J. E., Maxwell, C. F., Pierce, C. S., Hylton, M. B., Asofsky, R., and Kiddy, C. A.: Studies on the relative synthesis and distribution of IgA and IgG in various tissue and body fluids of the cow. J. Immunol., 109, (1972): 38. Butler, J. E., Winter, A. J., and Wagner, G. G.: Symposium: Bovine immune system. J. Dairy Sci., 54, (1971): 1309-1340. C3ampbe11, D. H., Garvey, J. S., Cremerin, B., and Subsdrof, D. H.: Methods in Immunology, 2nd ed. W. A. Benjamin, New York, (1970). 93 Capra, J. D., and Kunkel, B. G.: Aggregation of gamma G3 proteins. Relevance to the hyperviscosity syndrome. J. Clin. Invest., 49, (1970): 610. Carpenter, C. M., and Woods, B. A.: The distribution of the aerogenes group of bacteria in the alimentary tract of calves. Cornell Vet., 14, (1924): 218-225. Claman, H., and Chaperon, E.: Immunological complementation between thymus and marrow cells - A model for the two cell theory of immunocompetence. Transplant. Rev., 1, (1969): 92-113. Coles, E. H.: Veterinary Clinical Pathology, 2nd ed. W. B. Saunders Co., Philadelphia, Pa. (1974): 558-559, 575-576. Comline, R. S., Roberts, H. B., and Titchen, D. A.: Route of absorption of colostrum globulin in the newborn animal. Nature (London), 167, (1951): 561-562. Conner, G. H.: Personal communication, (1975). Conner, H. G., Richardson, M., and Carter, G. R.: Prenatal immuniza- tion and protection of the newborn: Ovine and bovine fetuses vaccinated with E. coli antigen by the oral route and exposed to challenge inoculum at birth. Am. J. Vet. Res., 34(6), (1973): 737. Cooper, M., Peyer, D., Peterson, R., Gabrielsen, A., and Good, R.: The two-component concept of the lymphoid system. In Immuno- logic Deficiency Disease in Man, Birth Defects. Original Article Series, Vol. 4, Ed. by D. Bergsma and R. Good. National Foundation, New York, N.Y., (1968). Craig, L. C.: Dialysis and ultrafiltration. In Methods in Immunology and Immunochemistry, II. Edited by C. A. Williams and M. W. Chase. Academic Press, New York, (1968): 119-133. Crowther, C., and Raistrick, H.: A comparative study of the proteins of the colostrum and milk of the cow and their relations to serum proteins. Biochem. J., 10, (1916): 434-452. (Zurtain, C. C., Clark, B. L., and Duffy, J. H.: The origins of the immunoglobulins in the mucous secretion of cattle. Clin. Exptl. Immunol., 8, (1971): 335-344. IDavidson, J. P., and Waxler, G. L.: Enteric colibacillosis: Evalua- tion of strains of Escherichia coli utilizing the ligated loop technique in gnotobiotic swine. Am. J. Vet. Res., 36, (1975): 765-770. IDavie, J. M., and Osterland, C. R.: Chemical characterization of glycopeptides from human gamma M globulins. J. Exp. Med., 128, (1968): 699. 94 Dixon, F. J., Weigle, W. 0., and Vazquez, J. J.: Metabolism and mammary secretion of serum proteins in the cow. Lab. Invest., 10, (1961): 216-237. Dowdle, w. R., Coleman, M. T., Schoenbaum, S. C., Mostow, S. R., Kaye, H. S., and Hierholzer, J. C.: Studies on inactivated influenza vaccines. National Institute of Child Health and Human Development, (1971): 113-127. Dumonde, D., and Mairi, R.: The clinical significance of mediators of cellular immunity. Clin. Allergy, (1971): 123-139. Duncan, J. R., Wilkie, B. N., Hiestand, F., and Winter, A. J.: The serum and secretory immunoglobulins of cattle. Characteri- zation and quantitation. J. Immunol., 108, (1972): 965. Dunne, H. W., Ajinkya, S. M., Bubash, G. R., and Grield, L. C. Jr.: Parainfluenza-3 and bovine enteroviruses as important factors in bovine abortion. Am. J. Vet. Res., 34, (1973): 1121-1126. Edwards, P. R., and Weing, W. H.: Identification of Enterobacteriaceae. Burgess Publishing Co., Minneapolis, (1962). Ellman, L., Green, I., and Frank, M. M.: Immune function in C4 deficient guinea pigs. Demonstration of an alternate pathway for activation of the complement sequence. Clin. Res., 19, (1971): 440. Ewing, W. H., Tatum, H. W., Davis, B. R., and Reavis, R. W.: Studies on the serology of the E. coli group. Communicable Disease Center, Atlanta, Ga., (1956). Fazekas, de St. Groth, S., and Donnelley, M.: Studies in experimental immunology of influenza. IV. The protective value of active immunization. Aust. J. Exp. Biol. Med. Sci., 28, (1950): 61-75. Fennested, K. L., and Borg—Peterson, C.: Antibody and plasma cell production by bovine fetuses infected with Leptospira sax- koebing. J. Infect. Dis., 110, (1962): 63-69. Fey, H.: Neuere untersuchungen fiber die colisepsis des Kalbes. Schweizer Arch. fur Tier., 104, (1962): 1-12. Fey, H.: Immunology of the newborn calf: Its relationship to coli- septicemia. In Neonatal Enteric Infections Caused by Escherichia coli. Ann. N.Y. Acad. Sci., 176, (1971): 49-63. Fey, H.: Colibacillosis in Calves. Verlag Hans Huber, Bern, Switzerland, (1972). Fey, H., and Margadant, A.: Hypogammaglobulinamie bei der colisepsis des Kalbes. Pathol. Microbiol., 24, (1961): 970-976. 95 Fisher, W. E.: Hydrogen ion and electrolyte disturbances in the neonatal calf diarrhea. Ann. N.Y. Acad. Sci., 176, (1971): 223. Ganguly, R., Ogra, P. L., Regas, S., and Waldman, R. H.: Rubella immunization of volunteers via the respiratory tract. Infec. Immun., 8, (1973): 497-502. Garner, R. J., and Crawley, W.: Further observations on the maternal transference of antibodies in the bovine. J. Comp. Path. Ther., 68, (1958): 112-114. Gay, C. C.: Escherichia coli and neonatal disease of calves. Bact. Rev., 29, (1965): 75-101. Gay, C. C.: Problems of immunization in the control of Escherichia coli infection. In Perspectives in the Control of Neonatal Enteric Disease, IV. Ann. N.Y. Acad. Sci., 176, (1971): 336-349. Gay, C. C.: In utero immunization of calves against colisepticemia. J.A.V.M.A., 36, (1975): 625-630. Gay, C. C., Anderson, N., Fisher, E. W., and McEwan, A. D.: Gamma- globulin levels and neonatal mortality in market calves. Vet. Rec., 77, (1965): 148-149. Gibbons, R. J., and Van Houte, J.: Selective bacterial adherence to oral epithelial surfaces and its role as an ecological determinant. Infec. Immun., 3, (1971): 567-573. Gibson, C. D.: The immune repsonse of the bovine fetus. Ph.D. Thesis, U. of Minnesota, (1971). Gill, T. J., III: Methods for detecting antibody. Immunochem., 7, (1970): 997-999. Glantz, P. J., Dunne, H. W., Heist, C. B., and Hakanson, J. F.: Bacteriological and serological studies of E. coli serotypes associated with calf scours. Penn. State Univ. Agr. Exp. Sta. Bull., 645, (1959): 22. Goldman, A.: Studies in intestinal bacteriology. J. Infect. Dis., 34, (1924): 459-501. Grabar, P., and Bortin, P.: Immunoelectrophoretic Analysis. Elsevier Publishing Co., Amsterdam, (1964). Grabar, P., and Williams, C. A., Jr.: Methode permettant l'etude conjugee des proprietes electrophosetique et immunochimiques d'un melange de proteines. Application au serum sanguin. Biochim. Biophys. Acta, 10, (1953): 193. 96 Graves, J. H.: Transfer of neutralizing antibody by colostrum to calves born of foot and mouth disease vaccinated dams. J. Immun., 91, (1963): 251-256. Grey, H. M., Abel, C. A., Yount, W. J., and Kunkel, H. G.: A sub- class of human yA-globulins (YA2) which looks the disulfide bonds linking heavy and light chains. J. Exp. Med., 128, (1968): 1223. Haenel, H.: Some rules in the ecology of the intestinal microflora of man. J. Appl. Bacteriol., 24, (1961): 242-251. Hagan, W. A.: The etiology and mode of infection of white scours. Cornell Vet., 7, (1917): 263-283. Halpern, M. S., and Koshland, M. E.: Novel subunit in secretory IgA. Nature (London), 288, (1970): 1276. Hammer, D. K., Kiekofen, B., and Schmid, T.: Detection of homocyto- tropic antibody associated with a unique immunoglobulin class in the bovine species. Europ. J. Immunol., 1, (1971): 249- 257. Hanson, L. A.: Comparative immunological studies of the immuno- globulins of human milk and blood serum. Int. Arch. Allergy Appl. Immunol., 18, (1961): 241-267. Harris, A. H., Yankauer, A., Crosby Green, D., Caleman, M. B., and Pharseuf, M. Y.: Control of epidemic diarrhea of the newborn in hospital nurseries and pediatric wards. Ann. N.Y. Acad. Sci., 66, (1956): 118-128. Heremans, J. F., Heremans, M. T., and Schultz, H. E.: Isolation and description of a few properties of the B2A—globu1in of human serum. Clin. Chim. Acta, 4, (1959): 96. Howe, P. E.: An effect of the ingestion of colostrum upon the composition of the blood of new born calves. J. Biol. Chem., 49, (1921): 115-118. Hubbert, W. T., and Hollen, E. J.: Cellular blood elements in the developing fetus. Am. J. Vet. Res., 32, (1971): 1213-1219. Howe, P. E.: The relation between the ingestion of colostrum or blood serum and the appearance of globulin and albumin in the blood and urine of the new-born calf. J. Exp. Med., 39, (1924): 313-320. Hughes, B. A., and Lovell, R.: Virulence of E. coli for chick embryos. Intern. Cong. Microbiol. 8th, Montreal. Abstr. No. E338, l, (1962): 114. 97 Ishizaka, K., Ishizaka, T., Tada, T., and Newcomb, R. W.: Site of synthesis and function of 7E. In Kaufman Secretory Immuno- logic System. U.S. Govt. Printing Office, Washington, D.C., (1971): 71. Ishizaka, T., Ishizaka, K., Borsos, T., and Rapp, H. J.: C'l fixa- tion by human isoagglutinins: Fixation of C‘l by 7G and yM but not by yA antibody. J. Immunol., 97, (1966): 716-726. Johanson, K. R., and Sarles, W. B.: Some consideration of the bio- logical importance of intestinal microorganisms. Bacteriol. Rev., 13, (1949): 25-45. Kauffmann, F.: The serology of the coli group. J. Immunol., 57, (1947): 71-100. Kendrick, J. W.: The effects of IBR-IVP virus on the fetus. J.A.V.M.A., 163, (1973): 852-854. Kerr, W. R., and Robertson, M.: Passively and actively acquired antibodies for Trichomonas fetus in very young calves. J. Hyg., Camb., 52, (1954): 253-263. King, T. P.: Chromatographic separations of macromolecules on porous gels and cellulose ion exchangers. In Methods in Immunology and Immunochemistry, II. Ed. by C. A. Williams and M. W. Chase. Academic Press, New York, (1968): 135-162. Klaus, G. G. B., Bennetl, A., and Jones, E. W.: A quantitative study of the transfer of colostral immunoglobulins to the newborn calf. Immunology, 16, (1969): 293-299. Klein, F., Mattern, P., Radema, H., and Zwet, T. L.: Slow sedi- menting serum components reacting with anti IgM sera. Immunology (London), 13, (1967): 641. Kulangara, A. C., and Schechtman, A. M.: Do heterologous proteins pass from mother to fetus in cow, cat and guinea pig? Proc. Soc. Exp. Biol. Med., 112, (1963): 220-222. Kunkel, H. G., Smith, w. K., Joslin, F. G., Natvig, J. B., and Litwin, S. D.: Subgroups of yA immune globulins. Proc. Soc. Exp. Biol. Med., 122, (1955): 1247. Lambert, G., McClurkin, A. W., and Fernelius, A. L.: Bovine viral diarrhea in the neonatal calf. J.A.V.M.A., 164, (1974): 287-289. Lascelles, A. K., Outteridge, P. M., and Mackenzie, D. D.: Local production of antibody by the lactating mammary gland fol- lowing antigenic stimulation. Aust. J. Exp. Biol. Med. Sci., 44, (1966): 169-180. 98 Lawton, A. R., Belf, K., Royal, S. A., and Cooper, M. D.: Ontogeny of B-lymphocytes in the human fetus. Clin. Immunol. and Immunopath., l, (1972): 84-93. Leid, W. R., and Williams, J. E.: The immunological response of the rat to infection with Taenia taeniaeformis. I. Immuno- globulin classes involved in passive transfer of resistance. Immunology, 27, (1974): 195. Lindberg, R. B., and Young, V. M.: Observation on enteropathogenic E. coli. Ann. N.Y. Acad. Sci., 66, (1956): 100—107. Linton, K. B.: The colicine typing of coliform bacillae in the study of cross infection in urology. J. Clin. Path., 13, (1960): 168-172. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, (1951): 265-275. Luna, L. G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill Book Co., New York, (1968): 32-46. Mach, J. P., and Pahud, J. J.: Secretory IgA with secretory piece in bovine colostrum and saliva. Nature, 223, (1969): 952. Mach, J. P., and Pahud, J. J.: Secretory IgA, a major immunoglobulin in most bovine external secretions. J. Immunol., 106, (1971): 552—563. Mayr, A., Kalich, I., and Mehnert, B.: Kalber Krankheiten. Wiener Tier. Monat., 51, (1964): 74-92. McDiarmid, A.: The transference of agglutinins for Brucella abortus from cow to calf and their persistence in the calf's blood. Vet. Rec., 38, (1946): 146-149. McAlpine, J. G., and Rettger, L.‘F.: Serological studies on bovine infectious abortion. J. Immunol., 10, (1925): 811-828. McKay, B., and Thom, H.: Observations on neonatal tears. J. Pediat., 75, (1969): 1245. Mebus, C. A., Stair, E. L., Rhodes, M. B., Underdahl, N. R., and Twiehaus, M. J.: Calf diarrhea of viral etiology. Ann. Rech. Vet., 4, (1973): 71-78. Mebus, C. A., Stair, E. L., Underdahl, N. R., and Twiehaus, M. J.: Pathology of neonatal calf diarrhea induced by a Reo-like virus. Vet. Path., 8, (1971): 490-505. 99 Mestecky, J., Kulhavy, R., and Kraus, F. W.: Studies on human secretory immunoglobulin A. II. Subunit structure. J. Immunol., 108, (1972): 738. Muschel, L. H.: Bactericidal activity of normal serum against bacterial cultures. Proc. Soc. Exp. Biol. Med., 103, (1960): 632-636. Namioka, S., and Murata, M.: Studies on the pathogenicity of E. coli. Cornell Vet., 52, (1962): 289-296. Nansen, P., Flagstad, T., and Pedersen, K. B.: Preparation of anti- sera to bovine immunoglobulin classes by immunization with agar-gel precipitates. Acta Path. Micro. Scand., 179B, (1971): 459-465. Nash, D. A., Vaerman, J. P., Bazin, H., and Heremans, J. F.: Identification of IgA in rat serum and secretions. J. Immunol., 103, (1969): 145. Newcomb, R. W., and DeVald, B. L.: Protein concentrations in sputa from asthmatic children. J. Lab. Clin. Med., 73, (1969): 734. Norris, J. R., and Ribbons, D.: Methods in Microbiology, Vol. 1. Academic Press, (1969): 614-616. Olson, D. P.: Prenatal and postnatal immune responses of the beef- type calf to E. coli 026:K60:NM. Ph.D. thesis, Michigan State University, (1975). Orcutt, M. L., and Howe, P. E.: The relation between the accumula- tion of globulins and the appearance of agglutinins in the blood of newborn calves. J. Exp. Med., 36, (1922): 291-308. Orskow, I., Orskow, F., Sojka, w. J., and Leach, J. M.: Simultaneous occurrence of E. coli B and L antigens in strains from diseased swine. Influence of cultivation temperature. Two new E. coli K antigens K87 and K88. Acta Path. et Micro. Scand., 53, (1961): 404—422. Osburn, B. I.: Some practical aspects of immunity in the bovine animal. Bovine Practitioner, Nov., (1972): 13-16. Osburn, B., and Hoskins, R.: Infection with Vibrio fetus in the immunologically immature fetal calf. J. Infect. Dis., 123, (1971): 32-40. Osburn, B. I., Stabenfeldt, G. H., Ardans, A. A., Trees, C., and Sawyer, M.: Perinatal immunity in calves. J.A.V.M.A., 164, (1974): 295-298. 100 Oxender, W. D., Newman, L. B., and Morrow, A. D.: Factors influencing dairy calf mortality in Michigan. J.A.V.M.A., 162(6), (1971): 458. Pierce, A.: Electrophoretic and immunological studies on sera from calves from birth to weaning. I. Electrophoretic studies. J. Hyg., 53, (1955): 247-260. Pierce, A. E., Risdall, P. C., and Shaw, B.: Absorption of orally administered insulin by the newly born calf. J. Physiol., Lond., 171, (1964): 203-215. Polson, A.: Comparative electrophoretic studies of bovine and human colostrum in relation to neonatal immunity. Ondersteport J. Vet. Res., 25, (1952): 7-12. Porter, P.: Immunoglobulin IgA in bovine mammary secretions and serum. Biochim. Biophys. Acta, 236, (1971): 664. Porter, P., and Allen, W. D.: Classes of immunoglobulins related to immunity in the pig. J.A.V.M.A., 160, (1972): 517-518. Porter, P., and Noakes, D. E.: Immunoglobulin IgA in bovine serum and external secretions. Biochim. Biophys. Acta, 214, (1970): 107-116. Porter, P., Noakes, D. I., and Allen, W. D.: Intestinal secretion of immunoglobulins in the preruminant calf. Immunol., 23, (1972): 299-312. Radostits, O. M.: Clinical management of neonatal diarrhea in calves with special reference to pathogenesis and diagnosis. J.A.V.M.A., 147, (1965): 1367-1376. Reisinger, R. C.: Pathogenesis and prevention of infectious diarrhea (scours) of newborn calves. J.A.V.M.A., 147, (1965): 1377- 1386. Rice, C. E., and Duhamel, L.: A comparison of the complement, con- glutinin and natural anti-sheep red cell antibody titers of the serum of newborn and older calves. Canad. J. Comp. Med., 29, (1972): 109-115. Richardson, M., and Conner, G. H.: Prenatal immunization by the oral route: Stimulation of Brucella antibody in the fetal lamb. Infect. and Immunity, 5(4), (1972): 454. Ritzmann, S., Daniels, J., Sakai, H., and Beathard, G.: The lympho- cyte in immunobiology. Ann. Allergy, 31, (1973): 109-125. 101 Roberts, H. E., Worden, A. N., and Rees-Evans, E. T.: Observation on some effects of colostrum deprivation in the calf. J. Comp. Pathol. Therap., 64, (1954): 283-305. Salmon, 5. E.: IgE immunoglobulin in secretions. Clin. Res., 18, (1970): 135. SanClemente, C. L., and Huddleson, I. P.: Electrophoretic studies of the proteins of bovine serums with respect to Brucella. Michigan State College, Agricultural Experiment Station, Technical Bulletin 182, (1943): 3-44. Sawyer, M., Osburn, B. I., Knight, H. D., and Kendrick, J. W.: A quantitative serologic assay for diagnosing congenital infec- tion. Am. J. Vet. Res., 34, (1973): 1281-1284. Scheidigger, J.J.: Une micro-methode de 1'immunoe1ectrophorese. Int. Arch. Allergy, 6, (1955): 103-110. Schultz, R. D.: Comments on the immune response of the fetus and neonate. J.A.V.M.A., 163, (1973): 804-806. Schultz, R. D.: Ontogeny of the bovine immune response. Ph.D. thesis, Pennsylvania State University, University Park, Pa. (Dissertation Abstracts, Vol. XXXI, No. 9, 1970). Schultz, R. D., Confer, F., and Dunne, H. W.: Occurrence of blood cells and serum protein in bovine fetuses and calves. Canad. J. Comp. Med., 35, (1971): 93-98. Schultz, R. D., Dunne, H. W., and Heist, C. E.: Ontogeny of the bovine immune response. Fed. Proc., 29, (1970): 699. Schultz, R. D., Dunne, H. W., and Heist, C. E.: Ontogeny of the bovine immune response. J. Dairy Sci., 54, (1971): 1321- 1322. Schultz, R. D., Dunne, H. W., and Heist, C. E.: Ontogeny of the bovine immune response. Infect. Immunity, 7, (1973): 681- 691. Selner, J. C., Merrill, D. A., and Claman, H. N.: Salivary immuno- globulin and albumin and albumin development during the newborn period. J. Pediat., 72, (1968): 685. Sjostedt, S.: Pathogenicity of certain serological types of E. coli. Acta Pathol. Microbiol. Scand., Suppl. 63, (1946): 1-148. Slater, R. S., Ward, S. M., and Kunkel, H. G.: Immunological rela- tionships among the myeloma proteins. J. Exp. Med., 85, (1955): 101. Smith, Smith, Smith, Smith, Smith, Smith, Smith, Smith, Smith, Smith, Smith, Steck, Sterzl, 102 E., Kochwa, S., and Wasserman, L. R.: Aggregation of IgG in vivo. Am. J. Med., 39, (1965): 35. H., Gallop, R. C., and Tozer, B. T.: The production of spe- cific rabbit antibodies by injecting individual antigen- antibody complex separated from mixed antigens. Immunology, 7, (1964): 111-117. H. W.: Further observations on the effect of chemotherapy on the presence of drug-resistant Bacterium coli in the intestinal tract of calves. Vet. Rec., 70, (1958): 575-580. H. W.: The ecology of the intestinal bacteria of the calf with particular feference to E. coli. Vet. Rec., 72, (1960): 1178-1183. H. W.: The development of the bacterial flora of the feces of animals and man. The change that occurs during aging. J. Appl. Bacteriol., 24, (1961): 235-241. H. W.: Observations on the etiology of neonatal diarrhea in calves. J. Pathol. Bacteriol., 84, (1963): 147-168. H. W., and Crabb, W. E.: The typing of E. coli by bacterio- phage. Its application to the study of the E. coli population of the intestinal tract of healthy calves and of calves suf- fering from white scours. J. Gen. Microbiol., 15, (1956): 556-574. E. L.: The immune proteins of bovine colostrum and plasma. J. Biol. Chem., 164, (1946): 345-358. E. L.: The isolation and properties of the immune proteins of bovine milk and colostrum and their role in immunity: A review. J. Dairy Sci., 31, (1948): 127-138. T., and Orcutt, M. L.: The bacteriology of the intestinal tract of young calves with special reference to early diarrhea. J. Exp. Med., 41, (1925): 89-106. V. R., Reed, R. E., and Erwin, S. E.: Relation of physio- logical age to intestinal permeability in the bovine. J. Dairy Sci., 47, (1964): 923-924. F., Nicolet, F., and Schipper, E.: Aetiologische unter- suchungen uber virale und bakterielle infektionen in kalberund rindermastbetrieben. Berliner und Munchener Tier. Woch., 84, (1971): 21-24. J., and Silverstein, A.: Developmental aspects of immunity. Adv. Immunobiol. Academic Press, New York, 6, (1967): 337- 460. 103 Steven, D. H.: Placental vessels of the fetal lamb. J. Anat., 103, (1968): 522-539. Taylor, J., Maltby, M. P., and Payre, J. M.: Factors influencing the response of ligated rabbit gut segments to injected E. coli. J. Pathol. Bacteriol., 76, (1958): 491-499. Taylor, J., Wilkins, M. P., and Payne, J. M.: Relation of rabbit gut reaction to enteropathogenic E. coli. Brit. J. Exp. Pathol., 42, (1961): 43-52. Tennant, B., Harrold, D., Reina-Guerra, M., and Laben, R. C.: Neo- natal alterations in serum gamma globulin levels of Jersey and Holstein-Friesian calves. Am. J. Vet. Res., 30, (1969): 345-354. The, T. H., and Feltkamp, T. E. W.: Conjugation of fluorescein iso- thiocyanate to antibodies. I. Experiments of the conditions of conjugation. Immunology, 18(6), (1970): 865. The, T. H., and Feltkamp, T. E. W.: Conjugation of fluorescein iso- thiocyanate to antibodies. II. A reproducible method. Immunology, 18(6), (1970): 875. Thompson, R. A.: Secretory piece linked to IgM in individuals deficient in IgA. Nature (London), 226, (1970): 946. Todd, J. D.: Immune response to parenteral and intranasal vaccina- tions. J.A.V.M.A., 163(7). (1973): 807-809. Tomasi, T. B.: The gamma A globulins: First line of defense. In Immunobiology, ed. by R. A. Good and D. W. Fisher. Sinauer Associates, Stamford, Conn., (1971): 76-83. Tomasi, T. B., and Bienenstock, J.: Secretory immunoglobulins. Advances Immunol., 9, (1968): 1-96. Tomasi, T. B., and Calvanico, N.: Human secretory IgA. Fed. Proc., 27, (1968): 617. Tomasi, T. B., and Grey, H. M.: Structure and function of immuno- globulin A. Progr. Allergy, 16, (1972): 81-213. Tomasi, T. B., Jr., and Zigelbaum, S. D.: The selective occurrence of ylA globulins in certain body fluids. J. Clin. Invest., 43, (1963): 1552-1560. Tourville, D. R., Adler, R. H., Bienenstock, J., and Tomasi, T. B.: The human secretory immunoglobulin system. J. Exp. Med., 129, (1969): 411-423. Trueblood, M., Swift, B., and Bear, P.: Bovine fetal response to Anaplasma marginale. Am. J. Vet. Res., 32, (1971): 1089-1090. 104 Uffelman, J.A., Engelhard, W. E., and Jolliff, C. R.: Quantitation of immunoglobulins in normal children. Clin. Chim. Acta, 28, (1970): 185. Vaerman, J. P.: Studies on IgA immunoglobulins in man and animals. Thesis, University of Belgium, Louvain, Belgium, (1970). Vaerman, J. P., Fudenberg, H. H., Vaerman, C., and Mandy, W. J.: On the significance of the heterogeneity in molecular size of human serum gamma G globulin. Immunochemistry, 2, (1965): 263. Waldman, R. H., and Ganguly, R.: Immunity to infections on secre- tory surfaces. J. Infect. Dis., 130(4), (1974): 419-440. Wallenius, G., Trautman, R., Franklin, E. C., and Kunkel, H. G.: Heavy components of human serum. Fed. Proc., 15, (1956): 378. Walsh, T. E., and Cannon, P. R.: Immunization of the respiratory tract: A comparative study of the antibody content of the respiratory and other tissue following active-passive and regional immunization. J. Immunol., 35, (1938): 31-46. Wamukoya, J. P. 0.: Local immune responses following in utero vaccination of the bovine fetus with E. coli. M.S. Thesis, Michigan State University, (1975). Wang, A. C., and Fudenberg, H. H.: IgA and evolution of immuno- globulins. J. Immunogenetics, 1, (1974): 3-31. Ward, G. J., Roberts, S. J., McEntee, K., and Gillespie, J. H.: A study of experimentally induced BVD disease in pregnant cows and their progeny. Cornell Vet., 59, (1969): 525-538. Wernet, P., Breu, H., Knop, J., and Rowley, D.: Antibacterial action of specific IgA and transport IgM, IgA, and IgG from serum into the small intestine. J. Infect. Dis., 124, (1971): 223-226. White, R. G., Mebus, C. A., and Twiehaus, M. J.: Incidence of herds infected with a neonatal calf diarrhea virus. Vet. Med./Sm. An. Clin., 65, (1970): 487-489. Williams, C. A., and Chase, M. W.: Methods in Immunology and Immunochemistry, I. Academic Press, New York, N.Y., (1967): 1-385. Williams, J. P.: Personal communication, Michigan State University, (1976). 105 Williams, W. L., Hagan, W. A., and Copenter, C. M.: White or calf scours. J.A.V.M.A., 57, (1920): 124-146. Wilson, M. R., and Svendsen, J.: Immunity to Escherichia coli in pigs: Serologic response of sows given formalin-treated live E. coli vaccine. Am. J. Vet. Res., 32, (1971): 891-898. Yakulis, V., Costea, N., and Heller, P.: Cleavage of the Fe fragment of IgA. J. Immunol., 102, (1969): 488. VITA VITA The author was born in Tehran, Iran, on August 20, 1939. He graduated from the School of Veterinary Medicine, University of Tehran, in 1963. Following graduation from 1963 to 1965 he served in charge of clinic at Army in Varamin Station. The author began his work as research assistant in Razi State Institute in September 1965. For the following six years the author was engaged in research and diagnostic work in the Department of Microbiology and Pathology of Razi State Institute and co-authored several papers. In September 1971 he began his graduate studies in pathology at Michigan State University. From September 1971 through June 1973 he was supported by FAO of the United Nations postdoctoral fellowship. In June 1973 he received a Senior graduate assistantship from Michigan State University and started diagnostic work for prac- ticing veterinarians in the State of Michigan. The author completed the requirements for the Master of Science degree in pathology in December 1973. 106 RIES H “'aniimuijgiMingigmflmlmfgnm