Date llllllllllllllllllllllllllllllgllllll This is to certify that the thesis entitled INTESTINAL IMMUNE RESPONSE OF THE BOVINE FETUS TO IN UTERO VACCINATION WITH ATTENUATED CALF DIARRHEAL CORONAVIRUS presented by Thomas P . Mullaney has been accepted towards fulfillment of the requirements for M. S . degree in Pathology Z i 5% g. Major professor May 10; 19 79 0-7 839 LIBRARY Michigan $3” UM” MAGIC 2 APR} {5.1999 OVERDUE FINES ARE 25¢ PER DAY _ PER ITEM Return to book drop to remove this checkout from your record. INTESTINAL IMMUNE RESPONSE OF THE BOVINE FETUS TO IN UTERO VACCINATION WITH ATTENUATED CALF DIARRHEAL CORONAVIRUS BY Thomas P. Mullaney A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1979 ABSTRACT INTESTINAL IMMUNE RESPONSE OF THE BOVINE FETUS TO IN UTERO VACCINATION WITH ATTENUATED CALF DIARRHEAL CORONAVIRUS BY Thomas P. Mullaney Bovine fetuses were vaccinated during the last 7 weeks of gestation by inoculation of cell-culture-attenuated calf diarrheal coronavirus (7 fetuses) or sterile physiological saline (3 fetuses) into the amniotic fluid. The calves were delivered by cesarean section at normal parturition time and maintained in a closed gnotobiotic environment. Methyl green-pyronine stained sections of ileum and lymph node from 1-day-old calves contained appreciable numbers of plasma cells while only a small number of plasma cells were present in sections from control calves. Frozen sections of ileum and lymph node were stained with mono-specific anti-bovine IgG, IgM and IgA. Positively stained plasma cells were numerous in tissue sections from vaccinated calves. There were approximately equal numbers of IgM and IgA staining cells observed and these cell types predominated, especially in the lamina propria. Smaller numbers of IgG staining cells were also observed. There were no IgA staining cells and a very small number of IgG and IgM staining cells in tissue sections from control calves. Thomas P. Mullaney These results indicate the ability of the near term fetus to respond to an orally administered attenuated coronavirus by the production of IgA, IgM and IgG in intestinal and lymphoid tissues. ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. C. K. Whitehair for his guidance and assistance during this research. I am grateful to Dr. R. F. Langham and Dr. G. L. Waxler of the Department of Pathology for their advice and assistance during my training at Michigan State University. Sincere thanks to Dr. L. E. Newman of the Department of Large Animal Surgery and Medicine for his constant cooperation and help during this research. I also thank Dr. R. W. Leid of the Department of Pathology for his generous assistance and Dr. K. Ames of the Department of Large Animal Surgery and Medicine for his help with surgical procedures. The assistance of research technicians Barbara Goelling and Shirley Howard and research animal caretaker John Allen is also appreciated. ii TABLE OF CONTENTS INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O LITE%TURE REVIEW 0 O O O O O O O O O O O O O O O O O O O OBJECTI Escherichia coli Diarrhea. . . . . . . . . . . . . Rotavirus Diarrhea . . . . . . . . . . . . . . . . Calf Diarrheal Coronavirus . . . . . . . . . . . . Agents Other Than EPEC, Rotavirus and Coronavirus. General Immunology . . . . . . . . . . . . . . . . Bovine Immunoglobulins . . . . . . . . . . . . . . Ontogeny of the Bovine Immune Response . . . . . . Prenatal Immunization. . . . . . . . . . . . . . . Summary. . . . . . . . . . . . . . . . . . . . . . VES O O O C O O O O O O O O O O O O O O O O O O 0 0 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . RESULTS DISCUSS Experimental Animals . . . . . . . . . . . . . . . General Design . . . . . . . . . . . . . . . . . . Vaccine. . . . . . . . . . . . . . . . . . . . . . Vaccination Procedure. . . . . . . . . . . . . . . Gnotobiotic Equipment. . . . . . . . . . . . . . . Cesarean Section . . . . . . . . . . . . . . . . . Thiry-Vella Loop Surgery . . . . . . . . . . . . . Anti-Bovine Immunoglobulins. . . . . . . . . . . . Immunoelectrophoresis. . . . . . . . . . . . . . . Conjugation of Anti-IgA to Fluorescein Isothio— cyanate (FITC) . . . . . . . . . . . . . . . . . Cutting and Staining of Frozen Sections. . . . . . Histopathologic Technique. . . . . . . . . . . . . Immunofluorescent Microscopy . . . . . . . . . . . Plasma Cell Counts . . . . . . . . . . . . . . . . Light Microscopy . . . . . . . . . . . . . . . . . ION. O O I O O O O O O O O O O O O O O I O O O O O Interval Between Inoculation and Parturition . . . In utero Vaccination . . . . . . . . . . . . . . . iii Page 10 ll 13 l6 19 21 22 23 23 23 24 24 27 28 31 34 34 35 36 36 37 39 42 50 51 51 52 Page Immunofluorescent Localization of Immunoglobulins in Intestinal Tissue . . . . . . . . . . . . . . . . . 53 Future Research. 0 O O O O O O O O O O O O O O O O O O O 54 SUMMRY O O O O O O O O O O O O O O O O O O O I O 0 O O O O O O 5 5 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . 56 iv Table LIST OF TABLES Experimental procedure . . . . . . . . . . . . . . . . Interval between vaccination and birth . . . . . . . . Plasma cell counts in tissues stained with fluorescein— conjugated anti-IgM immunoglobulin . . . . . . . . . . Plasma cell counts in tissues stained with fluorescein- conjugated anti-IgG immunoglobulin . . . . . . . . . . Plasma cell counts in tissues stained with fluorescein- conjugated anti-IgA (Miles Laboratories) immunoglobulin. Plasma cell counts in tissues stained with fluorescein- conjugated anti-IgA immunoglobulin . . . . . . . . . . 43 44 45 46 Figure 10 11 12 LIST OF FIGURES The cow on the operating table . . . . . . . . . . The calf in the Primary calf isolator. . . . . . . The surgical isolator for intestinal 100p surgery. Removal of tissue biopsy specimens .rgical isolator . . . . . . . . . . . . . . . . - . . . . Photograph of the immunoelectrophoretic pattern of anti-IgG and anti-IgM. . . . . . . . . . . . . . . Photograph of the immunoelectrophoretic pattern of al’lti-IgA O O O I O I O O O O O I O O O O O O O C 0 Photograph of the immunoelectrophoretic pattern of anti-IgA obtained from Dr. Ward. . . . . . . . . . Photomicrograph of frozen section of ileum of calf number 804 stained with fluorescein-conjugated anti-IgM immunoglobulin. . . . . . . . . . . . . . Photomicrograph of frozen section of ileum of calf number 803 stained with fluorescein-conjugated anti-IgA iHlIUUDOQ'lObUlin. o o o o o o o o o o o o o Photomicrograph of frozen section of ileum of calf number 801 stained with fluorescein-conjugated anti-IgG immoglObUI in o o o o o o o o o o o I o o Photomicrograph of frozen section of ileum of calf number 803 stained with fluorescein-conjugated anti-19A imunOglObUIino o o o o o o o o o o o o o Photomicrograph of frozen section of ileum of calf number 711 stained with fluorescein-conjugated anti-IgM immunoglobulin. . . . . . . . . . . . . . vi Page 30 30 33 33 4O 4O 41 47 47 48 48 49 INT RODUCTI ON Neonatal diseases of calves are an important worldwide problem and cause great economic loss to the cattle industry. The mortality rate from conception to 2 months of age in the United States has been at least 10% for beef calves and 15-20% for dairy calves (National Academy of Sciences, 1968; Oxender et al., 1973; Speicher and Hepp, 1973). Much of the literature relating to neonatal diseases of calves emphasizes treatment and the use of antibiotics, electrolytes and fluid replacement. Treatments have not significantly influenced the mortality rate since surveys in Great Britain in 1936, 1948 and 1962 indicate little variation in mortality rate despite introduction of antibiotics between the second and third survey (Roy, 1970). California workers (Martin et al., 1975) state, "There has been no published evidence to indicate a decrease in the rate of calf mortality since the turn of the century." The treatment, control and prevention of neonatal mortality in calves is often difficult, frustrating and unrewarding because the cause cannot be determined quickly and accurately (Radostits et al., 1975). Neonatal diarrhea in calves occurs as the result of several infectious (Acres et al., 1977; Morin et al., 1976) and non-infectious processes (Smith, 1962). Specific etiologic diagnoses of infectious diarrheal diseases of calves are difficult because numerous agents 2 are involved, clinical signs and lesions are frequently non-specific and asymptomatic infections occur (Acres et al., 1977). There are also several host factors that predispose the calf to infection and clinical disease (Moon et al., 1978). One of the most important aspects in the control of enteric infections in calves is prevention. Since there is no transplacental transfer of maternal immunoglobulins in the cow, calves at birth are hypogammaglobulinemic. The value of colostrum in prevention of enteric disease has been emphasized for many years (Kaester and Sutton, 1948). Reports on efficacy of vaccination of the pregnant dam are conflicting (Gay, 1971) and poor results may be due to the difficulty of diagnosis and the many agents involved. Active immuni- zation of the newborn calf with coronaviral and rotaviral vaccines has been attempted; however, the efficacy of these vaccines in the field has been questioned (Acres and Radostits, 1976; Newman et al., 1973). Research in recent years has led to a better understanding of the bovine immune system. For many years protection against many pathogenic organisms was correlated with serum antibody levels. It is now evident that protection against infection by organisms that enter the body via the intestinal tract may depend on local immunity or may be mediated by a combination of local and systemic immune mechanisms. Porter, a widely recognized authority on intestinal secretory immunity of neonates, states, "There is every possibility that this [intestinal secre- tory immune system] could contribute to resistance against enteric infections during the first few weeks of life." (Porter et al., 1975) 3 Studies on the ontogeny of the humoral and cellular immune system of bovine fetuses indicate the capability to develop detectable immune responses to a variety of antigens. This informa- tion has provided the impetus to explore the possibility of injecting antigens into amniotic fluid of the near—term fetus to actively stimulate specific immune responses to agents frequently pathogenic in the first few weeks of life. LITERATURE REVIEW This literature review summarizes the literature on Escherichia coli, rotavirus and other agents involved in neonatal diarrhea of calves and concentrates on calf diarrheal coronavirus, bovine immunoglobulins, ontogeny of the immune response and in utero vaccination because these subjects relate more directly to this research. Escherichia coli Diarrhea Jensen in 1893 was the first to observe an association of E. coli with white scours in calves (Sojka, 1965). Research workers for many years were unable to assess the significance of E. coli isolates from calves with diarrhea because E. coli were found in the intestinal tract of normal calves. Laboratory techniques to differentiate enterOpathogenic E. coli (EPEC) from non-EPEC have been developed (Smith and Balls, 1967). These techniques have not only aided diag- nosis, but have also helped define the mechanisms by which E. coli causes diarrheal disease. Diagnosis of EPEC infection is based on demonstration of enterotoxins and ability of an E. coli isolate to proliferate to high numbers in the anterior small intestine (Smith and Halls, 1967). Demonstration of enterotoxins has required expensive and time-consuming in vivo assays (Moon et al., 1976; Myers et al., 1975), and these are rarely used routinely by diagnostic laboratories. In vitro assay methods for enterotoxins have been 5 reported recently (Evans and Evans, 1977; Greenberg et al., 1977), and these may be more suitable for routine diagnostic purposes. Other researchers have attempted to relate EPEC strains to particular somatic (O) antigens (Glantz, 1971; Fey, 1972). Clinical disease has recently been found to correlate more closely with presence of distinct protein capsular (K) antigens (Isaacson, 1977), and certain antigenic types are associated with particular host species. The K99 antigen has been demonstrated in 80-97% of enterotoxigenic E. coli (ETEC) and in 0-7% non-ETEC strains (Ellens et al., 1978). Immunofluorescent examination of ileum for K99 antigen is a rapid technique and may soon be a valuable aid to routine diagnosis of ETEC in calves (Moon et al., 1978). The lesions of EPEC induced diarrheal disease in calves have been described (Pearson et al., 1978). Severe stunting of villi occurred in the distal half to one-third of the small intestine. There were occasional areas of coagulative necrosis of epithelial cells resulting in exposure of small areas of lamina propria to the lumen. Neutrophils were present in those areas, both in the lamina propria and on the villous surface. The submucosa of the distal ileum was edematous and contained neutrophils, lymphocytes and plasma cells. The role of colostral immunoglobulins in immunity to EPEC and invasive E. coli has been studied in detail (Logan et al., 1974; Logan and Penhale, 1971). There have been numerous attempts to increase passive resistance by vaccination of the pregnant dam, but results are conflicting (Gay, 1971; Acres and Radostits, 1976). Recently there has been interest in the use of bacterial pili as vaccines. The K99 antigen of E. coli is a plasmid mediated surface 6 protein with fimbriae (pili) and adhesive properties which act as colonization factors facilitating adhesion to intestinal mucosa. It is reasonable to expect that prevention of intestinal colonization will prevent E. coli induced diarrheal disease (Isaacson et al., 1978). The K99 antigen has been purified (Isaacson, 1977) and used as an experimental vaccine in sows (Isaacson et al., 1978) and cows (Acres et al., 1978) with promising results. Rotavirus Diarrhea Researchers have suspected for many years that viruses play a role in neonatal enteric infections (Light and Hodes, 1943; Amstutz, 1965). Rotaviral study commenced with the isolation of a reovirus— like agent from calves at the University of Nebraska (Mebus et al., 1969). This virus was subsequently characterized as a member of the Reoviridae family of viruses with a proposed genus Rotavirus (Flewett et al., 1974; Mebus, 1976; Flewett and Woode, 1978). Diarrhea induced by a rotavirus has been experimentally repro— duced in gnotobiotic calves (Mebus et al., 1969). The clinical signs, lesions and pathogenesis of the disease have been described (Mebus et al., 1971a; Mebus, 1976). Diagnosis is based on detection of viral antigen by fluorescent antibody staining of distal ileum and spiral colon and by electron microscopic examination of feces for virions having a rotavirus morphology in association with clinical signs and lesions (Mebus et al., 1971b; Mebus, 1976). Rotaviruses are not host specific; they seem to be almost ubiquitous in nature and infect a wide range of mammals (Woode and Crouch, 1978). A cell-culture—attenuated rotavirus vaccine is available, but its efficacy in the field has been questioned (Acres 7 and Radostits, 1976). Woode (1978) states, "From the data available on antigenic and cross pro— tection studies it is unlikely that a [rotavirus] vaccine prepared from one serotype will protect against all serotypes." Calf Diarrheal Coronavirus Coronaviruses were pr0posed as a new taxonomic group of viruses in 1968 to account for structural differences between avian infec- tious bronchitis virus and myxovirus. The name coronavirus was proposed to describe the petal shaped projections which resemble the solar corona. Coronaviruses are widely distributed in nature and infect man, calves, mice, pigs, rats, dogs, chickens, turkeys and possibly foals (Sharpee et al., 1976). Coronaviruses were first isolated from calves with diarrhea in 1971 during a field experiment with an attenuated reovirus-like (rotavirus) vaccine (Mebus et al., 1972). The vaccine was effective in preventing diarrhea caused by rotavirus, but diarrhea appeared in several herds when the vaccinated calves were 5 to 21 days old. Electron microscopy of diarrheal feces from these calves, and experimentally inoculated calves, revealed a coronavirus-like agent (Mebus et al., 1972; Stair et al., 1972). The virus was characterized and classified as a coronavirus based on the following properties: 1) characteristic surface structure, 2) size, 3) replication within cytoplasmic vesicles, 4) ribonucleic acid content, 5) presence of an essential lipid envelOpe, and 6) low particle density (Sharpee et al., 1976). The clinical signs and lesions in gnotobiotic calves infected with calf diarrheal coronavirus have been described (Mebus et al., 1973a; Mebus, 1976). Fifteen to eighteen hours after inoculation 8 with 10 ml of feces from a diarrheic gnotobiotic calf, calves were depressed and anorectic and had strings of saliva hanging from the mouth. The incubation period before onset of diarrhea varied from 19 to 24 hours. Feces were watery and yellow with volume depending on the amount of milk fed. Infected calves continued to have diarrhea for 5 to 6 days; some calves became moribund or died 48 to 62 hours following onset of diarrhea. Mortality varied from less than 1% to over 50% (Mebus et al., 1973a). Lesions of calf diarrheal coronavirus infection occur in small and large intestine. In gnotobiotic calves, virus infected mainly villous epithelium of upper, middle and lower small intestine and superficial and crypt colonic epithelium causing lysis of infected cells. There was absence of infection in upper and middle small intestine of colostrum fed calves. At onset of diarrhea villous epithelial cells were infected but morphologically normal. Forty- eight to ninety-six hours after onset of diarrhea villous atrophy was present, some adjacent villi were fused and villous epithelium was composed of low cuboidal to squamous cell types. Colonic ridges were atrophied and covered by cuboidal epithelium. Scattered colonic crypts were dilated and lined by squamous to cuboidal epi- thelium, and dead cells were present in the lumen (Mebus et al., 1973a). Based on scanning electron, light and immunofluorescent micro- scopy, the following pathogenesis for calf diarrheal coronavirus infection was suggested (Mebus et al., 1975). It was proposed that diarrhea initially results from decreased intestinal absorption due to a redirection of epithelial cell function from absorption to viral production. The continuing diarrhea in calves results from reduced 9 absorptive capacity of intestine due to 1) immature villous epi- thelium, 2) reduced surface area because of small intestinal villous and colonic ridge atrOphy and 3) continuing infection of intestinal epithelium (Mebus et al., 1975). During investigations with rotavirus in experimental calves, it was observed that calves which recovered from virulent viral infec- tion did not develop diarrhea when reinoculated with virulent virus. It was postulated that oral administration of an attenuated virus would protect the calf against virulent infection (Mebus et al., 1976). The calf diarrheal coronavirus was attenuated by consecutive passage in a fetal bovine kidney cell line (Mebus et al., 1973b). Twenty gnotobiotic calves orally vaccinated with attenuated calf diarrheal coronavirus and given virulent virus 96 hours later remained clinically normal during post—vaccination and post-challenge observation periods, indicating the vaccine was safe and efficacious. Nine non-vaccinated control calves developed severe diarrhea when given virulent virus and 3 died (Mebus et al., 1976). The mechanism of protection following vaccination was determined using isolated intestinal loops (Thiry—Vella loops) prepared in the lower ileum of two one—day-old colostrum-deprived calves and one two—day-old colostrum-fed calf. When calves were 4 days old, 4 ml of coronavirus vaccine was introduced into the cranial end of the loop. Four days post-inoculation virus was present in loop fluid and no detectable neutralizing antibody was present in loop fluid or serum. During the period of viral shedding no interferon was detected in the loop fluid. Six to seven days post-inoculation there was no detectable virus, but there was neutralizing antibody 10 in loop fluid. It was proposed that early protection resulted from a viral interference phenomenon with later resistance due to anti— body present in the intestine. This antibody was primarily IgM and IgA (Mebus et al., 1976). The replication of the coronavirus in the intestinal loop of the colostrum-fed calf which had a high serum neutralizing antibody titer confirmed results in a previous report which indicated that calves with circulating coronavirus antibody were not protected against calf diarrheal coronavirus infection (Mebus et al., 1973a; Mebus et al., 1976). Agents Other Than EPEC, Rotavirus and Coronavirus Cattle of all ages can be infected with Salmonella sp., but calves between the ages of 2 to 6 weeks seem to be most susceptible to acute and fatal infections (Hughes et al., 1971; Moore et al., 1962). The most common clinical signs are fever, anorexia and fetid diarrhea. Chlamydia sp. have also been implicated in neonatal diarrhea of calves (Eugster and Storz, 1971). Viruses involved in the syndrome include infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, bovine adenoviruses, bovine parvoviruses, bovine syncytial virus, rhinoviruses, enteroviruses and caliciviruses. A coccidium of the genus Cryptosporidium is frequently associated with neonatal diarrhea in calves (Moon et al., 1978) and in one study was estimated to be similar in economic impact to rotaviral infection (House, 1978). Mixed infections are frequently diagnosed in calves with diarrhea (Moon et al., 1978; Morin et al., 1978). 11 General Immunology Immunity represents one of the most important means by which an individual maintains its well being in an environment. Individuals are continually exposed to a wide variety of potentially harmful substances and microorganisms. Resistance of an immunologically competent host to foreign antigens may be due to specific immune mechanisms or non-specific mechanisms, or both. Non-specific natural factors include epithelial surfaces, perspiration, saliva, tears, intestinal secretions and phagocytic cells. Phagocytic cells include macrophages such as blood monocytes, connective tissue histiocytes, Kupffer cells of the liver, microglial cells of nervous tissue, reticulum cells of lymphoreticular tissue, and granulocytes (neutrophils, eosinophils and basophils). Non-specific processes protect against most harmful substances and microorganisms. When non-specific processes are unable to combat foreign antigens, specific immune processes are activated. The specific immune system consists of cell mediated and humoral components, also referred to as thymic and bone marrow systems, respectively (Claman and Chaperon, 1969). The lymphore— ticular system harbors most of the cells (lymphocytes and macrOphages) that are necessary for development of an immune response. Stem cells of lymphocytes are in the liver during fetal development and begin to seed bone marrow during the last third of gestation and post- natal life. Stem cells give rise to lymphocytes, some of which migrate via the blood vascular system to the thymus. While in the thymus these cells acquire new physical and biological character- istics. This is accomplished upon exposure of stem cells to the thymic hormones thymosin and thymopoietin (Eisen, Immunobiology, 12 1974). After leaving the thymus, cells known as thymic lymphocytes (T cells) migrate to the lymph nodes and spleen. T cells tend to localize in the paracortical areas of lymph nodes and in the adven- titial sheath of branches of the splenic artery (Osburn, 1973). Cells continually circulate by leaving lymph nodes via lymphatics and the thoracic duct and return to lymphoid tissue from blood by traversing post-capillary venules. Thymic lymphocytes, which constitute approximately 80% of lymphocytes observed in circulating blood, are the principal elements involved in cellular immunity (Dumonde and Mairi, 1971). Lymphocytes leaving bone marrow may go directly to lymphoid tissue and localize in the cortex and medullary cords of lymph nodes, primary and germinal centers of lymph nodes, and white pulp of the spleen. These cells, known as bone marrow lymphocytes (B cells), eventually become plasma cells which produce immunoglobulins with specific antibody activity (Osburn, 1973). The 2 populations of lymphocytes (T cells and B cells), macro- phages, and antigen provide the principal factors necessary for immunization to occur (Osburn, 1973). When an antigen enters the body it binds to phagocytic type cells (macrophages) and if specific antibody is present it facilitates adherence of antigen to the phagocytic cells by a process called opsonization (Eisen, 1974). The antigen is engulfed by the macrophages and usually broken down by intracellular lysosomal enzymes. Subsequently, most antigens become closely associated with the cytOplasmic membranes of the macrophages (Unane and Askonas, 1968). In this position the trapped antigen is in a position to come in contact with T cells. Once appropriate T cells with specific receptor sites on their cytOplasmic 13 membrane for antigen come in contact with Specific antigen, a series of events occurs, some of which are not completely understood (Feldman and Basten, 1972). The T cell, after contact with antigen, may 1) undergo blastogenesis, 2) release lymphokines, or 3) process antigen which interacts with specific immunoglobulin on the surface of B cells (Claman and Chaperon, 1969). The biologic effect of lymphokines is quite diverse and includes mitogenic factor, macrophage-activating factor, lymphocytotoxic factor and chemotactic factor (Dumonde and Mairi, 1971). In addi- tion, the lymphokines appear to amplify the B cell response associated with some antigens (Claman and Chaperon, 1969). The principal func- tion of B cells is to secrete specific antibodies when apprOpriately stimulated by complementary antigen. Not all antigens require macrophage-T cell-B cell interaction to produce an immune response. Certain antigens which appear in nature as polymerized substances can directly activate IgM bearing B cells that recognize those antigenic determinants. These antigens include a protein with repeating determinants found on certain bacterial flagellae and lipopolysaccharide (endotoxin) of some gram negative bacteria. They are called T independent antigens. Bovine Immunoglobulins Immunoglobulins are a family of high molecular weight proteins that share common physicochemical characteristics and antigenic determinants. These proteins occur in the serum and other body fluids of animals and possess Y or slow 8 electrophoretic mobility. Immunoglobulins include all molecules with antibody activity as well as other chemically related normal or pathological proteins. The 14 family of immunoglobulin molecules has a related structure. All immunoglobulins appear to be either monomers or polymers of a 4-chain molecule consisting of 2 light polypeptide chains and 2 heavy poly- peptide chains. Bovine immunoglobulins have been identified which are analogous in their physicochemical and antigenic characteristics to human immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA). Two subclasses of IgG exist. These are designated IgG1 and IgG2, and they have immunochemical and biological differences (Porter, 1973a). It has been suggested that the cow has an immunoglobulin similar to human immunoglobulin E (IgE) (Schultz, 1973). The bulk of immunoglobulins occurring in serum and lacteal secretions of the cow are of the IgG class. Immunoglobulin G1 is selectively transported by the udder from the circulation to the lacteal secretions by an unknown mechanism (Butler, 1969). It is, therefore, the predominant immunoglobulin in bovine colostrum and milk and post-colostral calf serum (Pierce and Feinstein, 1965). In adult serum, IgG and IgG occur in relatively equal amounts (Duncan 1 2 et al., 1972). Within the IgG class complement fixation and homolo- gous skin sensitization are principally properties of the IgGl immunoglobulins (Porter, 1973a). Molecules of the IgG class have a sedimentation coefficient of approximately 78 and contain 2 to 4% carbohydrate. The molecule consists of 2 heavy (H) chains and 2 light (L) chains symmetrically arranged into a monomeric unit. The monomer has a molecular weight of 150,000 Daltons. Bovine IgM occurs in serum, colostrum, milk and other secretions (Butler, 1969, 1973). Serum levels of IgM seem to be generally higher than in man and account for 10-20% of total serum immunoglobulin. 15 Evidence suggests partial concentration of IgM occurs in bovine colostrum, but it is present only as a trace constituent in most other secretions (Butler, 1973). Immunoglobulin M is important in the primary immune response,in complement fixation and as an agglu- tinating antibody in the serum (Butler, 1969). Immunoglobulin M consists of a pentamer of 5 subunits linked together by a polypeptide or J chain. Each subunit of the IgM pentamer is in the shape of a single IgG monomer. The sedimentation coefficient of IgM is 195 and the molecular weight 900,000 Daltons. Considerable variation exists among the reported values for IgA in bovine serum, but all suggest a value significantly lower than that present in normal human serum (Butler, 1973). Secretory IgA is the predominant immunoglobulin present outside the vascular system in all exocrine secretions of cattle studied except lacteal secretions (Butler et al., 1972; Mach and Pahud, 1971). Immunoglobulin A can exist in a series of polymeric forms which are held together by a polypeptide J chain. Monomeric IgA is abundant in human serum, but in bovine serum IgA occurs predominantly as a dimer (Mach and Pahud, 1971; Duncan et al., 1972). Secretory IgA is in the dimer form plus an additional non-immunoglobulin called secretory component (Duncan et al., 1972). It has been demonstrated by immunofluorescent studies of intes- tinal mucosa that secretory component is associated with crypt epithelium and intestinal mucins (Allen and Porter, 1973). Secretory component is not present in the plasma cells of the submucosa which stain specifically for IgA. Synthesis and secretion of secretory component proceeds separately and independently from IgA. The secreted IgA complex is formed at some point in epithelium while l6 immunoglobulin is being transported from plasma cells in the lamina propria to the external surface. The biological function of secre— tory component appears to be protection of immunoglobulin against enzyme degradation and to bind immunoglobulin in surface mucus (Porter, 1973b). The main responsibility for antibody defense of the external surface is apparently borne by IgA. A homocytotropic antibody which is apparently the bovine homolog of IgE has been detected recently (Butler, 1973). This IgE-like protein was concentrated in colostrum. The IgE proteins play an important role in allergic reactions. Little definitive work has been done on the immediate form of hypersensitivity in cattle. One specific condition, milk allergy, a naturally occuring syndrome in cattle, has been described as a model of a disease produced pre- dominantly by hypersensitivity of the immediate type (Campbell, 1970). Ontogeny of the Bovine Immune Response Studies on lymphoid tissue development suggest that the bovine fetus is in many respects comparable to those of other species (Schultz, 1973). Lymphocytes were first recognized in the thymus at 42 days of gestation (Schultz et al., 1970). Hassall's corpuscles were first observed in a 65 day fetus, and they were regularly present in the thymus after that age (Schultz et al., 1973). The weight of the thymus in relation to total body weight (thymic index) was reported for fetuses from 40 days to birth. The thymic index reached a maximum value in fetuses at 205 to 220 days and decreased beyond that age (Schultz et al., 1973). There was a sharp reduction in the thymic index near birth which may have resulted from elevation of steroids that trigger parturition (Eberhart and Patt, 1971). 17 Lymphocytes were first recognized in bone marrow at 55 days, but were difficult to recognize at all ages of fetal life due to their paucity and the numbers of other hemopoietic cells (Schultz et al., 1973). Lymphocytes were recognized in the peripheral blood at 45 days, approximately the same age at which the thymus appeared. Lymphocytes were the only leukocytes in the peripheral blood until 120 days, when granulocytes were first observed (Schultz et al., 1971). 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 (Sattar et al., 1967). Lympho— cytes were observed in lymph nodes and gut associated lymphoid tissue later in development, and by 175 days of gestation lymphoid cells were recognized in most peripheral lymphoid tissue. Premature lymphoid tissue development occurred in fetuses stimulated overtly with numerous antigens (Schultz, 1973). Fetal and neonatal studies of development of immunoglobulins in animals not overtly stimulated with antigens have focused on intracytoplasmic immunoglobulin detected by immunofluorescence (Schultz et al., 1973; Porter et al., 1972) and serum immunoglobulins detected by immunoelectrophoresis and radial immunodiffusion (Schultz et al., 1971). Using immunofluorescent techniques, cells containing IgM were observed in the spleen of a 59 day old fetus (Schultz et al., 1973). In the same study IgG containing cells were first recog- nized in a 145 day old fetus. Numerous lymphoid organs contained these cells but the spleen was the organ which most frequently had immunoglobulin containing cells. Serum IgM and IgG were detected by radial immunodiffusion at 130 to 135 days. Immunoglobulin A has 18 not yet been conclusively identified in the bovine fetus (Schultz, 1973). A wide variety of microbial and non-microbial antigens have been studied in the bovine fetus. Immunoglobulin levels in fetuses infected with viruses, bacteria and chlamydia have been reported (Braun et al., 1973; Osburn and Hoskins, 1971; Sawyer et al., 1973). In most instances levels of 196 and/or IgM were elevated in infected animals when compared to control animals. Viral infection in the fetus can lead to measurable or no measurable immunologic response with death or survival of the fetus (Schultz, 1973). Fetuses become immunologically competent to BVD, IBR and PI-3 viruses at 90 to 120 days (Kahrs et al., 1970; Kendrick, 1973; Dunne et al., 1973). Immunologic competence to Leptospira antigens, Brucella antigens, Anaplasma antigens and ferritin also develOped at approximately 100 days (Schultz, 1973). Few studies have been reported on cell mediated immunity in the bovine fetus. In a study on the occurrence of phytohemagglutinin (PHA) 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). Phytohemag— glutinin responsive cells began to decline at approximately 250 days of gestation with minimal responsiveness near parturition. The suppression of cell mediated immunity may result from a rise in corticosteroids required to initiate parturition (Osburn et al., 1972). Hemolytic complement has been found in the fetus at 60 to 70 days of age (Gerwurz et al., 1968). Total complement levels 19 increased with increasing age, but birth values were well below adult values. Prenatal Immunization It was thought for many years that fetuses of most species were not immunologically competent. That theory was refuted when Silverstein et a1. (1963) reported that the ovine fetus responded to several types of antigen at various times during fetal life. Subse- quently, numerous researchers utilized laparotomy for intracardiac, intramuscular and intra—amniotic fetal vaccination (Gay, 1971, 1975; Richardson et al., 1968; Richardson and Conner, 1972; Cegnar et al., 1975). Richardson et a1. (1968) found that the ovine fetus synthe- sized high levels of anti-Brucella agglutinins after stimulation late in gestation. The response was slow with low levels of anti— body in ovine fetuses vaccinated during midgestation (Richardson et al., 1971). However, the initial stimulus had a priming effect because a second dose of antigen administered in utero or at birth resulted in a secondary response with antibody levels as high as those attained in adult sheep given secondary stimulation. The feasibility of using the oral route to stimulate the immune response was investigated in the ovine fetus by injecting Brucella antigen into the amniotic fluid at 19 and 50 days before birth (Richardson and Conner, 1972). The results indicated that vaccina- tion by the oral route stimulated immunity in fetal life. Prenatal vaccination with E. coli antigen deposited in amniotic fluid pro- tected newborn lambs and calves eXposed to oral challenge of E. coli capable of causing death of non—vaccinated neonates (Conner et al., 1973). Protection against challenge was independent of humoral 20 agglutinins because humoral E. coli agglutinin was present in some, but not all, of the prenatally immunized lambs and calves at birth. Gay (1975) inoculated E. coli antigen into bovine fetuses in utero and found that vaccination with a single serotype of E. coli resulted in heterogenetic protection against neonatal colisepticemia. Non-surgical techniques have been described for in utero vaccina- tion of the bovine fetus (Conner and Carter, 1975; Gay, 1975). Fetuses were vaccinated by inoculation of antigen into amniotic fluid by needle puncture through the intact flank of the dam. Results, using Evans blue dye, confirmed that injection was made into the amniotic cavity by this method, and examination of the fetuses showed dye present in the intestinal contents (Gay, 1975). This technique of prenatal immunization by the oral route has been used to demon- strate that calves develop serum neutralizing antibodies against calf diarrheal rotavirus following vaccination with a cell culture attenuated rotavirus (Conner and Carter, 1975). Other researchers have similarly used E. coli antigens in utero and found calves were protected against challenge with homologous E. coli serotypes (Wamukoya and Conner, 1976; Olson and Waxler, 1976, 1977). Prenatal vaccination of bovine fetuses by the oral route is an effective means of stimulating immunity to enteric pathogens fre- quently encountered during the first few days of life. However, the frequency of premature birth, stillbirth, and abortion following intra-amniotic vaccination precludes recommendation of this technique in widespread field trials (Gay, 1975; Conner et al., 1977; Hamid et al., 1977). 21 Summary Diarrhea in newborn calves is an important disease and many infectious agents are involved in the syndrome. Research in recent years has focused more on prevention. Numerous vaccines are cur- rently being evaluated against EPEC infection. Attenuated corona- viral and rotaviral vaccines are now available for field use. There is a paucity of knowledge about responses to these viral vaccines in terms of specific immunoglobulin classes. This research investi- gated the immune response of gnotobiotic calves to in utero vaccina- tion with attenuated coronavirus vaccine. The response was measured by determining the classes of antibody (IgG, IgM, IgA) produced in the ileum and ileal lymph nodes of l-day-old calves. OBJECTIVES The objectives of this research were: 1. To determine the types of antibody (IgM, IgG, IgA) produced in the ileum and associated lymph nodes of gnotobiotic calves following in utero vaccination with a cell-culture-attenuated coronavirus. 2. To determine by histOpathologic examination whether increased numbers of immunocytes were present in the ileum and lymph nodes of vaccinates. 22 MATERIALS AND METHODS Expermental Animals Fifteen pregnant Holstein cows bred to Holstein bulls were used in this research. Calving dates were estimated by rectal palpation of cows at frequent intervals. The research was initiated in fall 1977 and completed in spring 1978. Cows were maintained on pasture with an open-sided barn for protection from inclement weather. During late fall and throughout winter they were fed legume hay free choice and ground corn. Trace mineral salt and dicalcium phosphate were also available free choice. The cows were moved to the Veterinary Clinical Center at the time of in utero vaccination and maintained there until cesarean section surgery. Feed was withheld for 12 to 24 hours and water for 8 to 12 hours prior to cesarean section. General Design This research was carried out in cooperation with Newman (1978). The in utero vaccination and cesarean sections were done jointly and tissue biopsies were obtained during isolated loop intestinal surgery on l-day-old gnotobiotic calves. Gut loop washings were obtained each day for 5 days, and the calves' immunity was then challenged with virulent coronavirus (Newman, 1978). 23 24 Ten of fifteen cows in this study were subjected to in utero vaccination of the fetus with modified life calf diarrheal corona- virus, 4 were inoculated in utero with sterile 0.85% saline solution and one remained unvaccinated. Experimental procedures were completed on 11 calves from these 15 cows. Seven calves received modified live virus, 3 received saline solution, and one was unvaccinated (Table 1). Vaccine The experimental modified live coronavirus vaccine used in this research was the only vaccine agent available and had been used extensively in other trials and experiments (Mebus, 1976). The vaccine, serial number x-S, propagated on bovine kidney diploid cell line BK4, was obtained from Norden Laboratories, Lincoln, Nebraska. Characterization, propagation and attenuation of calf diarrheal coronavirus has been described (Mebus et al., 1973b; Sharpee et al., 1976). The lyophilized vaccine was refrigerated until it was recon- stituted with diluent immediately prior to use. This experimental 6.1 vaccine contained approximately 10 TCID50 (median tissue culture infective dose) per test dose (Norden Laboratories, Lincoln, NB). Vaccination Procedure The technique used for in utero vaccination was a non-surgical method developed by Conner at Michigan State University and described in detail by Olson (1975). The cows were vaccinated when it was estimated by rectal palpation that parturition would take place in 21 days. In preparation for vaccination the cow was restrained in a cattle chute, the tail tied and the position of the fetus located by abdominal ballottement of the right flank. If fetal ballottement was not possible, the point of vaccination was located 5 cm dorsal 25 Table 1. Experimental procedure 11 pregnant Holstein cows f l j 3 inoculated in l non-inoculated 7 vaccinated in utero with saline control utero with attenu— ated coronavirus cesarean sections on all cows using germfree procedures to obtain gnotobiotic calves Thiry-Vella loop surgery in l-day-old calves biopsy of ileum and ileal lymph node preservation of tissues frozen at -70 C fixed in 10% for immunofluores- formalin for his- cent examination topathologic examination 26 and 25 to 30 cm anterior to the fold of the flank. The area was clipped, scrubbeda 6 times and swabbed 6 times with a surgical solution.b A 25 gauge 1 cm needle attached to a 3 ml syringe was used to infiltrate the skin at the point of vaccination with 2.5% procaine hydrochloride. An 18 gauge 4 cm needle attached to a 15 ml syringe filled with 2.5% procaine hydrochloride was used to anesthetize the abdominal wall and peritoneum. The area was again scrubbed 6 times with surgical scruba and 6 times with surgical solution.b Two persons who had surgically scrubbed and donned sterile gloves were required for this vaccination procedure in order to guarantee aseptic technique. A small skin incision was made over the anesthetized area, and a 12 gauge 5 cm needle was inserted through muscles and peritoneum. This needle acted as a cannula into which a 16 gauge 30 cm needle was inserted and gently pushed through the uterine wall until the tip struck the fetus. The fetus usually kicked when pricked by the needle and in some instances continued vigorous activity throughout the procedure. Location of the needle tip was determined by aspira- tion with an attached 10 ml syringe. Amniotic fluid is normally clear, colorless and mucoid. If the needle tip was not within the amniotic cavity, it was likely that clear, watery, amber-colored allantoic fluid would be withdrawn into the syringe. It was then necessary to redirect the needle and reassess needle tip location. The syringe containing vaccine was attached to the needle and vaccine aBetadine Surgical Scrub, Purdue Frederick Co., Norwalk, CT. bBetadine Solution, Purdue Frederick Co., Norwalk, CT. 27 injected into the amniotic cavity. The needles were removed follow- ing vaccination and a topical dressingC applied. Amniotic fluid was cultured to determine if bacteria were present at time of vaccination. All cows were given intramuscular injections of vitamin A and Dd (5 m1) and selenium—vitamin Be (5 ml) at the time of in utero inoculation because previously 3 calves had been lost with lesions of selenium deficiency (Newman, 1978). The cows were examined each day following in utero vaccination for signs of impending parturition. Cesarean sections were performed when udder engorgement and ligament relaxation indicated parturition was imminent. Gnotobiotic Equipment The isolator system used was a modification of the closed system described by Trexler (1968), adapted to swine by Waxler et a1. (1966), and to goats by Oxender et a1. (1971). Four flexible plastic film isolators each fitted with 2 pairs of shoulder length rubber gloves were used: 1) a surgical isolator which was attached to the cow and into which the calf was delivered at the time of cesarean section, 2) a transport isolator in which the newborn calf was transported from the Veterinary Clinical Center to the research barn, 3) a primary calf isolator in which the calf was maintained for the duration of the research, and 4) a calf surgical isolator in which Thiry—Vella loop surgery was performed on the 1-day-old calf. CTopazone, Eaton Veterinary Laboratories, Norwich, NY. dVitamin A and D Injectable, Pfizer Inc., New York, NY. eBO-SE Injection, Burns-Biotec Laboratories, Oakland, CA. 28 One isolator could be attached to another with a sterile lock formed by metal rings built into the wall of each isolator. The rings were attached with adhesive tape and covered at both ends with removable vinyl caps. Thus the calf and other materials could be passed from one isolator to another without exposure to the outside environment. The isolators were cleaned and checked for leaks by the use of freon and a freon leak detector (Trexler and Reynolds, 1957; Trexler, 1961). Surgical instruments and other supplies were wrapped in polyethylene, heat sealed, sterilized with ethylene oxide gas and placed in the isolators. The isolator and its contents were sterilized with an aerosol of 2% peracetic acidf and 0.1% wetting 9 Thirty minutes later, filtered air was passed through the agent. isolators, and the exhaust air passed through a liquid—filled trap which prevented back flow of air into the sterile environment. Cesarean Section The cow was restrained in stocks, washed and prepared for surgery. The hair on the left side from the midline dorsally to the subcutaneous abdominal vein ventrally and from the 8th rib to the external angle of the ileum was clipped and shaved. The surgical site was scrubbeda 6 times, defatted 6 times with ether and swabbed 6 times with a surgical solution.b The area was anesthetized by , L and L using 20 m1 of 2.5% paravertebral nerve block of T 2 3 13' L1 procaine hydrochlorideh at each site (Wright, 1971). Ten milliliters f . . . . . . FMC Corporation, Industrial Chemical DiViSion, Buffalo, NY. gNacconol NRSF, National Analine Division, Allied Chemical Corp., New York, NY. hEpidural, Haver Lockhart Co., Kansas City, MO. 29 of 2.5% procaine hydrochlorideh was also administered epidurally immediately before the cow was tied in right lateral recumbency on an operating table (Figure 1). A partially inflated inner tube was placed under the right shoulder of the cow to prevent radial paralysis. The grounding plate for the electro-surgical unit was placed under the neck of the cow. The surgical area was again scrubbeda 3 times, swabbed with ether 3 times, swabbed 6 times with a surgical solutionh and 6 times with 70% alcohol. The bottom of the surgical isolator was cleaned with alcohol and covered with a sheet of plastic. An aerosol of 2.0% peracetic acidf with 0.1% wetting agent9 was sprayed between the isolator and the plastic sheet at least 30 minutes prior to the time of surgery. The sheet of plastic was removed and the isolator bottom dried with a sterile towel at the same time that preparation of the surgical site was complete. The isolator was attached to the surgical site with a sterile aerosol adhesive1 and tied to the cow with cord around the cow at 4 locations. The bottom of the isolator was incised with a cautery tip.j The skin and subcutaneous tissue were incised with an electro— surgical pencilk to prevent contamination which might be caused by bacteria in hair follicles or glands of the skin. Towels were attached to the edges of the incised skin with towel clips to prevent the surgeon's gloves from coming into direct contact with skin. 1Vi-Drape Adhesive, Parke-Davis and Company Medical-Surgical Division, Detroit, MI. 3National Electric Instrument Co., Inc., Long Island, NY. kSolid-State Electrosurgery Model SSE2 with Isobloc and Cautery Pencil Lectro Switch Model Number E2502, Valley Lab, Boulder, CO. 9., . ' ’ 30 Figure 1. The cow on the operating table. In the background (A) surgical isolator, (B) transport isolator. Figure 2. The calf in the primary calf isolator. 31 Abdominal muscles and peritoneum were bluntly dissected or incised with scissors. Part of the uterus containing a foot of the calf was exteriorized and incised. Obstetrical chains were placed over the forefeet and the calf pulled from the uterus into the transport isolator, which was then sealed and separated from the surgical isolator. The surgical isolator was removed from the cow and a sterile drape placed over the surgical site. The uterus was closed using No. l chromic catgut and replaced in the abdominal cavity. The peritoneum and muscle layers were closed separately with continuous sutures. The skin incision was closed with non-absorbent suture material1 using a continuous lockstitch pattern. The calf was dried with towels in the transport isolator and given intramuscularly 2-ml injections of vitamins A and D,d selenium- vitamin E,e iron dextran complexm and B-vitamins.n The calf was transferred to a primary calf isolator (Figure 2) at the research barn and 3-6 hours later was allowed to nurse 1 liter of autoclaved (250 F for 35 minutes) whole milk. Thiry-Vella Loop Surgery A Thiry-Vella fistula (Markowitz et al., 1964) is an isolated segment of small intestine with an intact blood and nerve supply. The 2 ends of the isolated intestine are brought out through the skin surface as fistulous openings and the cut ends are anastomosed. 1 . . . Vetafil, Haver—Lockhart Laboratories, Kansas City, MO. mNonemic (Iron Dextran Injection), Burns-Biotec Laboratories, Omaha, NB. nVi-B Complex Injectable, W. A. Butler Co., Columbus, OH. 32 The l-day-old calf was anesthetized with 1 ml of a 10% experi- mental anesthetic agent.0 The calf was transferred to a surgical isolator (Figure 3) and placed in left lateral recumbency. The surgical site halfway between the external angle of the ileum and the last rib was clipped and draped with 4 linen towels and a surgi— cal drape. A dorsoventral incision approximately 7 cm long was made, muscle incised, peritoneum incised, and the cecum located and exteriorized. Color coded Allis tissue forceps were clamped over the ileum 30 cm anterior to the ileocecal valve. Ileum was severed between the forceps. A 3-cm segment of ileum was removed together with part of an associated lymph node. A second division was made in the ileum 150 cm anterior to the first. The isolated ends of the ileum were brought to the surface of the skin and the remaining ends anastomosed by the end-to-end method. The 2 ends of the isolated intestine were positioned with the distal end at the dorsal commissure of the incision and the anterior end at the ventral commissure. The wall of the ileum was sutured to peritoneum, muscle layers, and skin at 4 locations with interrupted mattress sutures. These calves were utilized in other research (Newman, 1978). Segments of ileum and lymph node were obtained, immediately wrapped in gauze, and removed through a small port in the wall of the isolator (Figure 4). When the tissues were placed in the port, a rubber stopper was inserted into the port from the inside of the isolator to maintain a sterile environment. oC1-744, Parke-Davis and Company, Detroit, MI. 33 Figure 3. The surgical isolator for intestinal loop surgery. Figure 4. Removal of tissue biopsy specimens from surgical isolator. 34 The segments of ileum and lymph node were cut into thin slices, placed on cork discs to which a few drops of embedding mediump had been added, and frozen on acetone and dry ice. The tissues were wrapped in foil, marked and stored at -70 C. Tissue sections were also fixed in 10% buffered formalin for histopathologic examination. Anti-Bovine Immunoglobulins Fluorescein isothiocyanate (FITC) conjugated anti-bovine IgG, IgM and IgA prepared in rabbits were purchased.q Specificity was checked by immunoelectrophoresis. No immunoprecipitin line was visible on immunoelectrophoresis of the original sample of anti- bovine IgA, so additional material was purchased and results obtained with this antiglobulin verified with unconjugated anti-bovine IgAr obtained from another source. Immunoelectrophoresis Immunoelectrophoresis was carried out according to the micro- method described by Scheidegger (1955). Barbital sodiumS buffer (0.1M, pH 8.0) was prepared as described by Leid and Williams (1974) and used in the buffer chamber and to make agar gel.t Two percent agar gel was prepared by adding barbital buffer to dried agar and boiling the mixture until all agar had melted. Merthiolate was added to a concentration of 1% as a preservative. Six microscope slides pO.C.T. Compound, Lab-Tek Products, Naperville, IL. qResearch Products, Miles Laboratories, Inc., Elkhart, IN. rKindly supplied by Dr. A. C. Ward, University of Idaho, Moscow, ID. sScientific products, 17150 Southfield Road, Allen Park, MI. t . . . Noble Agar, Difco Laboratories, DetrOit, MI. 35 were placed in an electrophoresis frame. Melted agar was poured into the frame and allowed to harden and wells and troughs cut. Antigens (B.W.S. [bovine whole serum], bovine saliva and colostrum) were placed in the wells with capillary pipettes, and electrophoresis was performed in a buffer filled chamber with a current of 12 amperes per frame for 60 minutes. Antiglobulins were placed in the troughs, and the frame was incubated in a moist chamber at room temperature for 24 to 48 hours. Slides were washed in 2% sodium chloride and distilled water for 48 hours with 2 changes of washing solution. Slides were covered with moist paper strips and dried at room tempera- ture. Slides were stainedu for 10 minutes and excess stain removed with 3 changes of 2% glacial acetic acid. Conjugation of Anti-IgA to Fluorescein Isothiocyanate (FITC) Anti-IgA (obtained from Dr. Ward) was prepared in rabbits, pre— cipitated by saturated ammonium sulfate and lyophilized. It was reconstituted with phosphate buffered saline to a concentration of 10 mg protein per ml of solution. The conjugation was performed as described by The and Feltkamp (1970). Fluorescein isothiocyanatev was dissolved in NaHPO4 (l ml/mg FITC). One milliliter of FITC solution per 100 mg protein was added to anti-IgA solution while stirring at room temperature. The reaction was continued for 60 minutes, and the pH was continually adjusted to 9.5 by adding 0.1 M NaHPO4 solution. The conjugate was placed on a Sephadex G-25 coarse uAniline Blue Black, Matheson, Coleman and Bell, Norwood, NJ. vSigma Chemical Co., St. Louis, MO. 36 column,w equilibrated and eluted with phosphate buffered saline, and the unconjugated FITC removed. Cutting and Staining_of Frozen Sections Frozen sections of ileum and lymph node were cut at a thickness of 4 u on a cryostat.x Sections were incubated overnight at 37 C on glass slides and fixed in acetone for 10 minutes. Fixed tissues were stained with conjugated antisera, incubated for 30 minutes at 37 C, rinsed and washed in 2 changes of phosphate buffered saline for 60 minutes while stirring gently. The tissues were mounted in buffered glycerol and examined under a fluorescent microscope. Normal bovine IgG, IgM and IgAy were added to their respective conjugated anti- sera, incubated at 37 C for 30 minutes and tissues stained as previously described as controls. Histopathologic Technique Tissues fixed in 10% formalin were embedded in paraffin and stained with hematoxylin and eosin. A methyl green—pyronine stain was used to detect plasma cells. wPharmacia Fine Chemicals, Piscataway, NJ. xInternational Equipment Co., Needham Heights, MA. yResearch Products, Miles Laboratories, Inc., Elkhart, IN. RESULTS Ten of fifteen cows utilized in this research were vaccinated in utero with a cell-culture-attenuated coronavirus. One cow aborted an emphysematous fetus 3 days post-vaccination. Four cows had live premature calves on days 9 and 10 post-vaccination. Five cows were utilized as controls. Four were inoculated in utero with sterile 0.85% saline and l was not inoculated. All 5 cows maintained normal pregnancy until delivery by cesarean section. The length of time from in utero vaccination to parturition is recorded in Table 2. BiOpsy specimens of ileum and lymph node were obtained from 7 calves which had been vaccinated in utero with attenuated corona- virus. Six biopsy specimens were obtained during the course of Thiry-Vella loop surgery on l-day-old calves. Calf number 803 died soon after cesarean section and a segment of ileum and lymph node was obtained immediately and preserved as described for other tissues. Tissues were also obtained from four l-day-old control calves during Thiry-Vella loop surgery. Biopsy specimens were not obtained from 3 vaccinated and 1 control calf. Two cows, numbers 715 and 709, calved during a snowstorm and gnotobiotic cesarean sections could not be performed. One cow, number 810, aborted an emphysematous fetus, and tissues were not collected from calf number 805 because there was inadequate time to prepare for gnotobiotic surgery. 37 38 Table 2. Interval between vaccination and birth Calf Number Number of Days Non-Inoculated Control 712 normal calf --— Saline Inoculated Controls 704 normal calf 49 710 normal calf 54 715 normal calf 14 806 normal calf 27 Vaccinates 705 live premature calf 9 706 normal calf 14 709 live premature calf 9 711 live premature calf 9 714 live premature calf 10 801 normal calf 36 803 died 1 hour after birth 14 804 normal calf 24 805 normal calf 7 810 abortion, fetus emphysematous 3 39 Immunofluorescent Microscopy Antiglobulins obtained from Miles Laboratories were checked for monospecificity using immunoelectrophoresis. Samples of anti-IgG and anti-IgM were reacted with whole bovine serum (Figure 5). Single precipitin lines were produced with anti-IgG corresponding to the electrophoretic mobility of IgG immunoglobulin and anti-IgM correspond— ing to the electrophoretic mobility of IgM immunoglobulin. The two samples of anti-IgA were reacted with whole bovine serum, bovine colostrum and saliva. The Miles antiserum produced a single precipi- tin 1ine corresponding to the electrophoretic mobility of IgA immuno- globulin. However, the line of precipitation was not clear and was difficult to photograph (Figure 6). The anti-IgA obtained from Dr. Ward had a precipitin line corresponding to the electrophoretic mobility of IgA, and a second unidentified precipitin line (Figure 7). Two sections of each tissue were stained with anti-bovine IgM, anti—bovine IgG and anti-bovine IgA. Normal bovine IgM, IgG and IgA were incubated for 30 minutes at 37 C with their respective anti- globulins and 1 section of each tissue was stained to rule out the possibility of a non-specific reaction. In addition, 1 tissue section was first flooded with unconjugated anti-IgA for 30 minutes and then stained with conjugated anti-IgA.) The optimal dilution for conju- gated anti-IgM and anti-IgG was 1:32 and for anti-IgA was 1:4. Each stained section was examined by fluorescent microscopy and the total number of positive staining plasma cells in 20 consecutive micro- scopic fields was determined under magnification of 250. 40 Figure 5. Photograph of the immunoelectrophoretic pattern of anti-IgG and anti—IgM. Anti-IgG (G), anti— IgM (M), bovine whole serum (B). Notice the immuno- precipitate bands IgG (9), IgM (m). Figure 6. Photograph of the immunoelectrophoretic pattern of anti-IgA. Anti-IgA (A), bovine whole serum (B), bovine colostrum (C), bovine saliva (5). Notice the immunoprecipitate band IgA (a). Anti-IgA obtained from Miles Laboratories. 41 Figure 7. Photograph of the immunoelectrophoretic pattern of anti-IgA obtained from Dr. Ward. Anti-IgA (A). Notice the immunoprecipitate band IgA (a). Uniden— tified immunoprecipitate band (b). 42 Plasma Cell Counts The results of staining tissue sections with conjugated anti- IgM and anti—IgG are recorded in Tables 3 and 4, respectively. Immunoglobulin A plasma cell counts were repeated twice using 2 different sources of anti-IgA, and the results are recorded in Tables 5 and 6. Tissues from calves in non-inoculated and saline- inoculated control groups had no evidence of fluorescent plasma cells except for a small number of IgM-staining cells in lymp hode of calf number 806 and IgG-staining cells in lamina propria of calf number 710. Large numbers of positively stained plasma cells were observed in lamina propria of ileum of coronavirus vaccinated calves. These cells were predominantly IgM (Figure 8) and IgA (Figure 9) containing cells. Smaller but appreciable numbers of IgG—containing cells were also observed (Figure 10). In 1 calf, number 801, small numbers of all 3 types of plasma cells were observed and cells con— taining IgG were predominant. In sections stained with anti-IgA immunoglobulin there was positive staining of apical cytoplasm of crypt epithelial cells (Figure 11). Counting of positively stained cells was difficult in the lamina propria of all sections because of non-specific fluorescence caused by numerous eosinophils (Figure 12). Positively stained plasma cells were not observed in Peyer‘s patches stained with anti-IgA immunoglobulin. Small numbers of positive cells were observed in Peyer's patches of 3 calves stained with anti-IgM and of 4 calves stained with anti-IgG immunoglobulin. Most vaccinated calves (6 of 7) had appreciable numbers of positively stained cells in lymph nodes. All 3 classes of 43 Table 3. Plasma cell counts in tissues stained with fluorescein- conjugated anti-IgM immunoglobulin Lamina Propria Peyer's Lymph Calf Number of Ileum Patches Node Non-Inoculated Control 712 o -—-a o Saline Inoculated Controls 704 0 O O 710 ' O O O 806 0 O O Vaccinates 705 57b --- 36 706 13 --- 20 711 25 O 18 714 19 O O 801 10 2 12 803 270 19 41 804 44 21 39 a . . . Peyer's patches not present in section of ileum. bNumber of positively stained plasma cells in 20 consecutive high power microsc0pic fields. 44 Table 4. Plasma cell counts in tissues stained with fluorescein- conjugated anti-IgG immunoglobulin Lamina Propria Peyer's Lymph Calf Number of Ileum Patches Node Non-Inoculated Control 712 0 --_.a 0 Saline Inoculated Controls 704 0 0 0 710 6 0 0 806 0 0 0 Vaccinates 705 43b --- 32 706 11 --- 26 711 17 8 38 714 ll 0 0 801 28 10 17 803 64 7 22 804 37 28 20 aPeyer's patches not present in section of ileum. bNumber of positively stained plasma cells in 20 consecutive high power microscopic fields. 45 Table 5. Plasma cell counts in tissues stained with fluorescein- conjugated anti-IgA (Miles Laboratories) immunoglobulin Lamina Propria Peyer's Lymph Calf Number of Ileum Patches Node Non-Inoculated Control 712 o ---a o Saline Inoculated Controls 704 0 0 0 710 0 0 0 806 0 0 0 Vaccinates 705 95b --- 39 706 20 -—- 12 711 12 0 5 714 7 0 0 801 10 0 22 803 88 0 42 804 56 0 0 a . . . Peyer's patches not present in section of ileum. bNumber of positively stained plasma cells in 20 consecutive high power microscopic fields. 46 Table 6. Plasma cell counts in tissues stained with fluorescein- conjugated anti—IgAa immunoglobulin Lamina Propria Peyer's Lymph Calf Number of Ileum Patches Node Non-Inoculated Control 712 0 -—-b 0 Saline Inoculated Controls 704 0 0 0 710 0 0 0 806 0 0 0 Vaccinates 705 78c --- 55 706 15 --- 10 711 14 0 0 714 4 0 0 801 7 0 10 803 75 0 30 804 47 0 8 aKindly supplied by Dr. A. S. Ward, University of Idaho, Moscow, ID. b . . . Peyer‘s patches not present in section of ileum. CNumber of positively stained plasma cells in 20 consecutive high power microscopic fields. 47 Figure 8. Photomicrograph of frozen section of ileum of calf number 804 stained with fluorescein- conjugated anti-IgM immunoglobulin. Notice positively stained plasma cells (arrows). Figure 9. Photomicrograph of frozen section of ileum of calf number 803 stained with fluorescein- conjugated anti—IgA immunoglobulin. Notice positively stained plasma cells (arrows). 48 Figure 10. Photomicrograph of frozen section of ileum of calf number 801 stained with fluorescein- conjugated anti-IgG immunoglobulin. Notice positively stained plasma cells (arrows). Figure 11. Photomicrograph of frozen section of ileum of calf number 803 stained with fluorescein- conjugated anti—IgA immunoglobulin. Notice positively stained plasma cell (a) and positive staining of the apical cytoplasm of crypt epithelial cells (b). 49 Figure 12. Photomicrograph of frozen section of ileum of calf number 711 stained with fluorescein— conjugated anti-IgM immunoglobulin. Notice non- specific fluorescence caused by eosinophils (arrows). 50 immunoglobulins were present, and no consistently predominant anti- body class was observed. Light Microscopy Differences were not observed between vaccinates and controls in hematoxylin and eosin (H&E) stained sections of ileum and lymph node. Sections of ileum were similar to those previously described by Mebus and co-workers (1975) in gnotobiotic calves. Lamina prOpria of all sections of ileum contained numerous eosinophils, and it was impossible to determine whether increased numbers of plasma cells were present in sections from vaccinated calves. In sections stained with methyl green-pyronine, appreciable numbers of plasma cells were observed in lamina propria of coronavirus vaccinated calves. Very few plasma cells were observed in sections from control calves. DISCUSSION The literature has little information on the types of immune responses induced by oral vaccination of calves with attenuated coronavirus. This research demonstrated a wide spectrum of immune responses in the gnotobiotic calf following in utero vaccination with attenuated coronavirus. The calves were challenged with viru- lent coronavirus and all vaccinated calves remained clinically normal during the post-challenge observation period. Control calves developed diarrhea 19 to 22 hours post-challenge (Newman, 1978). In addition, Newman characterized the classes of antibody in the intestine and found IgA predominated in intestinal secretions. Immunofluorescent examination demonstrated IgM, IgA and IgG in the immunocytes of the ileum and associated lymphoid tissue of vaccinated calves. The demonstration of protection of calves at birth by in utero vaccination with attenuated coronavirus has important implica- tions in terms of reducing calf mortality. This procedure also circumvents the suppressive effects of colostral antibodies on coronavirus vaccines given to calves immediately after birth. Interval Between Inoculation and Parturition Breeding dates were not available for the cows used in this experiment. The approximate age of the fetus was determined by rectal palpation of the pregnant cow and the time between in utero inoculation and parturition varied between 7 and 54 days (Table 2). 51 52 This time variation probably affected the immunological response of the calves. In future research of this kind, cows with known breed- ing dates should be used. In utero Vaccination Abortion and premature birth of calves has also been encountered by other researchers following in utero vaccination with E. coli antigens (Gay, 1975; Conner et al., 1973; Hamid et al., 1977). Gay (1975) considered the possibility that abortions might be due to effects of E. coli endotoxin. The dose of antigen used may also be important, because Conner et a1. (1973) observed that abortion occurred in ovine fetuses injected intra-amniotically with 1012 killed E. coli. Abortion did not occur when fewer organisms were used. The occurrence of abortion and premature birth places strict limitation on field use of fetal vaccination. The factors present in the coronavirus vaccine used in this experiment which caused abortion and premature birth have not been identified. Newman (l978)‘observed that premature delivery 9 or 10 days following in utero vaccination may be related to onset of antibody production, other immune responses or altered fetal corticoid levels. Also considered were further attenuation of modified live virus vaccines and vaccine dose reductions to avoid vaccine-induced abortion and premature birth. Further research on the feasibility of in utero vaccination at approximately 7 months of gestation is necessary (Newman, 1979). There is a paucity of knowledge about responses to vaccines in general. Porter (1973a) observed that workers in applied research 53 will no longer be satisfied to use simple serologic methods in their evaluation of vaccines. More sophisticated techniques need to be adOpted to assess the spectrum of responses in terms of immunocytes and specific immunoglobulin Classes. In utero vaccination may be an excellent method for this type of vaccine evaluation in the future. Immunofluorescent Localization of Immunoglobulins in Intestinal Tissue Plasma cell counts in lamina propria of ileum of coronavirus vaccinated calves indicated approximately equal numbers of Ing and IgA-producing cells. Intestinal immunoglobulin content in loop washings was determined in 5 vaccinated calves and IgA levels con- sistently predominated (Newman, 1978). There are at least 2 explana- tions for this occurrence. Porter (1973b) found an abundance of IgM-producing cells in the intestinal tissues of young pigs, but only 10% of the immunoglobulin in the secretions was IgM. Immuno- globulin A predominated in the intestinal secretions. It seems probable that IgA is preferentially transported by the intestinal epithelium. Alternatively, if IgM and IgA were secreted in equal amounts, the finding of low levels of IgM in the secretions could be explained by IgM being more susceptible to enzyme degradation than secretory IgA. It has been suggested that secretory component blocks sites on IgA which are subject to enzyme action (Tomasi, 1967). It seems, therefore, that the main responsibility for anti- body defense of the intestinal surface is borne by IgA. The finding of relatively large numbers of IgM containing cells in the lamina propria is still significant. Their presence sub- stantiates the theory of Porter (1973b) that IgM probably acts in 54 concert with IgA, partly by secretion, but mainly by providing a second line of defense in the lamina propria. Very large numbers of all three immunoglobulin—producing cell types were observed in tissues from calf number 803. This is interesting because calf number 803 was delivered by cesarean section when rectal palpation indicated the calf was a full-term fetus. However, ligament relaxation had not occurred in the dam at the time of cesarean section. The high cell counts might be related to the fact that this calf was not subjected to the immunosuppressive effects of the glucocorticoid surge which usually initiates parturi- tion in the cow. Future Research The suggestion by Newman (1978) that a smaller dose of antigen be used earlier in gestation to prevent vaccine-induced abortions is particularly relevant when one considers the mechanism by which the specific immune system protects against most enteric infections induced by viruses. Antibodies must be present in the intestine to provide protection (Snodgrass and Wells, 1978). Further research is necessary to determine if a small dose of viral antigen will "prime" the immune system. If so, a second oral vaccination immedi- ately after the calf is born would probably induce coproantibody formation before the age (5 days) at which most calves become susceptible to virus-induced diarrhea. SUMMARY Bovine fetuses were vaccinated during the last 7 weeks of gesta— tion by inoculation of cell-culture-attenuated calf diarrheal coronavirus (7 fetuses) or sterile physiological saline (3 fetuses) into the amniotic fluid. The calves were delivered by cesarean section at normal parturition time and maintained in a closed gnoto- biotic environment. Methyl green-pyronine stained sections of ileum and lymph node from l—day-old calves contained appreciable numbers of plasma cells while only a small number of plasma cells were present in sections from control calves. Frozen sections of ileum and lymph node were stained with monospecific anti-bovine IgG, IgM and IgA. Positively stained plasma cells were numerous in tissue sections from vaccinated calves. 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