iMMUNIZATSON 0F PORCWE FETUSES 5N UTERD WETH ESCHERIGHiA COL! 0149: K91(BB:'K88(L} AND ”RESPONSE OF SROTO-BEOTIC PIGS TO CHALLENGE ' AT BIRTH Thesis for the Qegree of M. S. MiCHEGAN STAE UNIVERSZTY CAROL A. YROMPSON 1976 - “JP”- , ‘ Q' " imam. of r _ * IIOAE 8-30“ . mom 1 mun, q ABSTRACT IMMUNIZATION OF PORCINE FETUSES IN UTERO WITH ESCHERICHIA COLI Ol49:K9l(B);K88(L) AND RESPONSE OF GNOTOBIOTIC PIGS TO CHALLENGE AT BIRTH BY Carol A. Thompson The immunologic response of fetal pigs to in utero injection of Escherichia coli Ol49:K9l(B);K88(L) and the response of neonates to challenge at birth were studied in 34 gnotobiotic pigs. A laparotomy was performed at 98 days of gestation. Escherichia coli bacterin (5 x 109 organisms) was injected in utero into either the amniotic fluid or the muscles of the hind limb of 14 fetuses. The remaining fetuses were either injected in utero with saline in the same manner or were uninjected. After delivery by hysterotomy, viable E. coli organisms were given orally to 23 of the gnotobiotic pigs (chal- lenged). The remaining 11 gnotobiotic pigs were given saline orally (controls). Eighteen of the 23 challenged pigs (78%) died following exposure. The in utero injection of either bacterin or saline did not appear to alter the susceptibility of the neonate at birth to viable E. coli of the same serotype. The clinical signs and gross and microscopic lesions were consistent with those associated with colibacillosis. Carol A. Thompson Antibody to E. coli antigen was not detected by using the bac- terial agglutination and passive hemagglutination tests in the serum of the pigs at birth. However, antibody to E. coli antigen was detected in the serum of 2 challenged pigs at 11 days of age. The results indicate that with the present techniques, porcine neonates previously injected in utero with E. coli antigen are not protected when challenged with live organisms of the same strain, nor are detectable antibodies against E. coli produced by fetal pigs. However, antibodies to E. coli antigen are formed by neonatal pigs before 11 days of age. Thus, one can assume that pigs are immuno- competent to E. coli antigen sometime between 98 days of gestation and 11 days of age. IMMUNIZATION OF PORCINE FETUSES IN UTERO WITH ESCHERICHIA COLI Ol49:K9l(B);K88(L) AND RESPONSE OF GNOTOBIOTIC PIGS TO CHALLENGE AT BIRTH BY Carol A. Thompson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1976 To my Mother and Father ii ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to her major professor, Dr. G. L. Waxler, for his assistance and guidance throughout the course of this research. Gratitude is also extended to members oftmn:academic committee, [Ma S. D. Sleight and Dr. R. F. Langham of the Department of Pathology and Dr. E. Sanders of the Department of Microbiology and Public Health, for their invaluable assistance. Thanks are also extended to Dr. G. H. Conner and Dr. T. Riebold of the Department of Large Animal Surgery and Medicine for their assistance during portions of this research. Sincere appreciation is extended to the Upjohn Corporation, Kalamazoo, Michigan, for the financial support given to the author during her two years at Michigan State University. Special thanks are extended to members of the technical staff of the Department of Pathology, particularly Mr. John Allen and Mr. James Southern, Animal Caretakers, Veterinary Research Farm, and Mrs. Shirley Howard and Mrs. Helen Davidson, Medical Technolo- gists, Department of Pathology. Finally, the author wishes to thank Dr. A. W. Dade and Dr. E. S. Carlisle for their encouragement and support. iii TABLE OF CONTENTS INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . OBJECTI VES O O I O I O O O O O I O O O O O O I O I O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . General Immunology . . . . . . . . . . . . . . . . . . . . Immunization of the Fetus. . . . . . . . . . . . . . . . . Colibacillosis in Neonatal Pigs. . . . . . . . . . . . . . Pathogenesis. . . . . . . . . . . . . . . . . . . . Clinical Signs. . . . . . . . . . . . . . . . . . . Gross and Microscopic Findings. . . . . . . . . . Passive Immunization of the Neonate Against Colibacillosis Gnotobiotic Pigs . . . . . . . . . . . . . . . . . . . . . Methods in Immunology. . . . . . . . . . . . . . . . . . . Direct Bacterial Agglutination Test . . . . . . . . Passive Hemagglutination Test . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . RESULTS Animals. . . . . . . . . . . . . . . . . . . . . . . . . . Preparation of Bacterin. . . . . . . . . . . . . . . . . . Fetal Injection. . . . . . . . . . . . . . . . . . . . . . Preparation of Gnotobiotic Equipment . . . . . . . . . . . Preparation of Live Challenge Organisms. . . . . . . . . . Gnotobiotic Procedures . . . . . . . . . . . . . . . . . . Specimen Collection. . . . . . . . . . . . . . . . . . . . Serum Samples . . . . . . . . . . . . . . . . . . . Bacteriologic Samples . . . . . . . . . . . . . . . Tissue Samples. . . . . . . . . . . . . . . . . . . Immunologic Procedures . . . . . . . . . . . . . . . . . . Direct Bacterial Agglutination Test . . . . . . . . Passive Hemagglutination Tests. . . . . . . . . . . Surgical Procedures. . . . . . . . . . . . . . . . . . . . Laparotomy Procedures . . . . . . . . . . . . . . . Hysterotomy Procedures. . . . . . . . . . . . . . . Clinical Response of Pigs to Challenge . . . . . . . . . . Direct Bacterial Agglutination and Passive Hemagglu- tination Tests . . . . . . . . . . . . . . . . . . . . . iv Page 21 21 21 23 24 26 26 27 27 27 28 29 29 29 31 31 31 31 33 36 Gross Findings . . . . . . . . . . . . . . . . . Histopathologic Findings . . . . . . . . . . . Mandibular and Mesenteric Lymph Nodes and Thymus. Ileum . . . . . . . . . . . . . . Liver, Spleen, and Kidney . . . . . . . . Bacteriologic Findings . . . . . . . . . . . . . DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . Clinical Responses of Gnotobiotic Pigs to Challenge at Birth 0 O O O O O O O O O O O O O O O O O O Histopathologic and Gross Findings . . . . . . . Immunologic Response of Fetal Pigs to Injection. SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . VITA. . . . . . . . . . . . . . . . . . . . . . . . . . Page 36 39 39 44 49 49 53 53 56 57 6O 62 69 LIST OF TABLES Table Page 1 Numbers of fetuses, solutions injected, routes of injection and identification of fetuses injected in utero O O O I O O O O O O O O O O I I O O O O O O O O O 24 2 Results of laparotomy and hysterotomy and fetal injection. I O O O O I O O O O O O O O O O I O O O O O O O 32 3 Responses of neonatal pigs to oral exposure to E. coli Ol49:K9l(B);K88(L) following in utero injection of bacterin or saline. . . . . . . . . . . . . . 34 4 Direct bacterial agglutination and passive hemag- glutination titers of serum from pigs injected in utero with E. coli bacterin (Group 1) or saline (Group 2) or uninjected (Group 3). . . . . . . . . . . . . 37 vi Figure lA,lB 2A,ZB 4A,4B 5A,5B LIST OF FIGURES Photomicrographs of the mesenteric lymph node of pig No. 14, previously injected in utero with E. coli bacterin, challenged with viable E. coli at birth, and euthanatized at 3 days of age. . . Photomicrographs of the mesenteric lymph node of pig No. 31, previously injected in utero with E. coli bacterin, challenged with viable E. coli at birth, and euthanatized at 18 days of age . . Photomicrograph of the ileum of pig No. 15, pre- viously injected in utero with E. coli bacterin, challenged at birth with viable E. coli and dying at 2 days of age . . . . . . . . . . . . . . . . Photomicrographs of the ileum of pig No. 32, pre- viously injected in utero with E. coli bacterin, given saline orally at birth and euthanatized at 11 days of age . . . . . . . . . . . . . . . . . Photomicrographs of the ileum of pig No. 28, pre- viously injected in utero with saline, challenged at birth with viable E. coli and euthanatized at 11 days of age. . . . . . . . . . . . . . . . . . . vii Page 41 43 46 48 INTRODUCTION Active immunization of the fetus in utero may be an important means of protecting the neonate against infectious diseases, such as colibacillosis, which are difficult to control using conventional methods. Enteric colibacillosis, caused by enteropathogenic strains of Escherichia coli, affects a variety of domestic animals and is a major cause of death of newborn pigs. Numerous studies on active immunization of the bovine fetus in utero for protection of the neonate against colibacillosis have been conducted in recent years. The results of these studies indicate that prenatal immunization may be a means of preventing this disease. The purpose of the present research was to determine whether previous exposure of the porcine fetus to E. coli antigen in utero would protect the pig against challenge with the live organism at birth. OBJECTIVES The objectives of this research were: 1. To determine whether porcine fetuses could be immunized with a killed inoculum of Escherichia coli (E. coli) Ol49:K9l(B)- K88(L) by in utero injection into either the amniotic fluid or muscles of the thigh. 2. To determine whether porcine neonates previously injected in utero with E. coli bacterin were protected from oral challenge with the live organism of the same strain. 3. To compare the clinical responses and gross and microscopic lesions in porcine neonates given either viable E. coli organisms or saline orally. 4. To quantitate the immune responses of pigs by direct bac- terial agglutination test. 5. To gain experience in research investigative techniques. th‘fmu deri Vg LITERATURE REVIEW General Immunology The immune system is important to the survival of man and animals in an environment containing large numbers of potentially harmful infectious agents. This system enables the animal to produce either specific immunoglobulins, or cells, or both, which will react specifically with foreign substances that enter the body. Lympho— cytes are an intrinsic part of the immune system and the principal cells involved in immune reactions. The peripheral lymphoid tissues of mammals and birds contain 2 different types of lymphocytes which originate from 2 central lymphoid organs (Raff, 1973). The thymus (T) lymphocytes are derived from the thymus and are responsible for the cell-mediated immunity. The bursa (B) lymphocytes, which are responsible for humoral mediated immunity, are derived from the bursa of Fabricius in birds and the equivalent of the bursa in mammals (Raff, 1973). Lymphocytes are produced by the thymus and bursa independently of antigenic stimuli (Raff, 1973). Lymphocytes first appear in the thymus of the developing fetus. They are derived from hemopoietic stem cells which migrated to the thymus from the fetal liver. In the adult, these stem cells are derived from the bone marrow (Raff, 1973). Once in the thymus, the stem cells proliferate and differentiate into thymus lymphocytes 4 (Raff, 1973). Some of the lymphocytes in the thymus migrate to peripheral lymphoid organs to establish the T lymphocyte population (Weissman, 1967). In mammals, the B cell precursors or stem cells arise from the bone marrow and, in some unknown tissue, differentiate into B lymphocytes (Eisen, 1974b). These stem cells may undergo dif- ferentiation in the gut-associated lymphoid tissues such as tonsils or appendix (Eisen, 1974b; Raff, 1973). The most widely accepted theory concerning the ability of lympho— cytes to recognize specific antigens is the clonal selection theory. The clonal selection theory suggests that ...some time in the ontogeny and independently of anti- gen, individual lymphocytes (or clones of lymphocytes) become committed to responding to one, or a relatively small number of antigens; they express the commitment through antigen-specific receptors on their surface. (Raff, 1973) When antigen enters the body it combines with those B or T lymphocytes which possess the corresponding receptor, thereby stimu- lating these cells to proliferate and differentiate into blast cells. When the B lymphocytes are stimulated by antigen, some of the result- ing blast cells become plasma cells. These cells secrete antibody into the circulating blood (Raff, 1973). The remaining blast cells revert back to B lymphocytes and circulate in the blood and lymph as memory cells to await future contact with the antigen (Eisen, 1974b; Raff, 1973). When T lymphocytes are stimulated by antigen, the resulting blast cells secrete specific lymphokinase, which is important in the cell-mediated response (Raff, 1973). 5 Immunization of the Fetus The discovery that the mammalian fetus is capable of an active immune response to antigenic stimuli has led to the development of many new techniques and experimental models to aid in the understand- ing of the various aspects of immunogenesis (Sterzl and Silverstein, 1967). There is one decided advantage in studying the immune response of the fetus to antigenic stimuli in utero. The mammalian placenta protects the developing fetus from a variety of organisms in the environment of the mother (Sterzl and Silverstein, 1967). Therefore, the immune system of the fetus has not been challenged by antigen. Thus, one could be reasonably certain that the immunological response of the fetus to experimentally injected antigen would be caused only by those antigens injected (Sterzl and Silverstein, 1967). Also, in certain animals such as the cow, horse, sheep, and pig there are no maternal antibodies present in the circulation of the fetus to inter- fere with the immune response of the fetus to the antigen (Soloman, 1971; Sterzl and Silverstein, 1967). Fennestad and Borg-Petersen (1957) were among the first investi- gators to study active antibody formation by the fetus in utero. A laparotomy was performed in each of 2 cows at 261 and 264 days of gestation. A live suspension of Leptospira saxkoebing was injected into the placentome of each cow. Antibodies against this antigen were found in the serum of the newborn. Silverstein et a1. (1963) injected fetal lambs in utero with bacteriophage ¢Xl74, horse ferritin, ovalbumin, Salmonella typhosa, diphtheria toxoid, and viable Bacillus Calmette—Guerin (BCG) at varying times during the gestation period. These fetal lambs were able to produce specific antibody to the first 6 3 antigens as early as the 66th to 70th day of the lSO-day gestation period. However, antibodies were not formed to Salmonella typhosa, diphtheria toxoid or BCG at any time during fetal or early neonatal life. The immune response of fetal dogs has also been studied (Jacoby et a1., 1969). Fetal dogs were able to produce antibody to bacteriophage ¢Xl74 and ovine erythrocytes at 40 and 48 days of ges- tation, respectively. Antibodies were not produced in these fetuses to bovine serum albumin at any time during gestation. These experi- ments using the fetal lamb and dog indicate that the immunocompetence of the fetus to various antigens does not develop simultaneously but sequentially. The reasons for this are not clear. One possible explanation for the sequential occurrence of antibody is that the clonal lymphocytic precursors may develop or mature at a different rate (Silverstein et a1., 1963). Also, whether the lymphocyte is stimulated by its antigen depends on the nature of the antigen and complex interaction with other lymphocytes and macrophages (Raff, 1973). Fetal pigs in utero have been found to be immunocompetent to such antigens as bacteriophage ¢Xl74 (Hajek et a1., 1969), sheep red blood cells (Schultz et a1., 1971), swine erysipelas adsorbed bacterin (Wellmann and Reblin, 1972), and parvovirus strain 3060 (Bourne, 1974). To this investigator's knowledge, there are no reports in the literature on the immune response of the fetal pig injected in utero with Escherichia coli antigen. Conner et a1. (1973) found that ovine and bovine neonates pre- viously given E. coli antigen by in utero injection into the amniotic fluid were protected when challenged at birth with viable organisms C) V h (1 r9 L). Q 7 of the same strain. However, detectable antibody to E. coli was not found in the serum of some of these neonates at birth. These inves- tigators postulated that E. coli antigen stimulated the intestines to produce antibody locally, thereby protecting the calves and lambs to challenge at birth. Further studies by Wamukoya and Conner (1976) demonstrated that antibodies are produced locally in the intestines of fetal calves following injection of E. coli antigen into the amniotic fluid. The vaccination of the bovine fetus with E. coli antigen may be a means to protect the neonate against colibacillosis (Conner et a1., 1973; Gay, 1975; Olson and Waxler, 1976). Colibacillosis in Neonatal Pigs Enteric colibacillosis is caused by enteropathogenic strains of E. coli, a gram-negative, nonspore-forming rod belonging to the family Enterobacteriaceae. Enteric colibacillosis is known to occur in pigs, calves, and lambs and is a major cause of losses in the newborn of these species (Blood and Hnerseon, 1968). Poultry and man are also affected (Sojka, l965a). Enterotoxic colibacillosis, entertic E. coli infection, E. coli diarrhea, and cholera-like E. coli infection are synonymous with enteric colibacillosis (Moon, 1974). Jensen, in 1893, was the first to observe the association of E. coli with diarrhea in calves (white scours). The same investi- gator, in 1899, suggested that diarrhea in piglets, like white scours in calves, was associated with E. coli infection (Sojka, l965b,c). However, since E. coli is a normal inhabitant of the intestinal tract of healthy pigs, there was considerable controversy concerning the importance of this organism in causing colibacillosis (Rutter, 1975; Sojka, l965a). The controversy continued until serological 8 procedures were developed for the classification of E. coli organisms (Kauffmann, 1947). It then became clear that only a few serotypes of E. coli are pathogenic. The Kauffmann-Knipschildt-Vahlne serological scheme for the classification of E. coli is based on the identification of the domi- nant antigens of the organism, namely the somatic (O), capsular (K) and flagellar (H) antigens (Kauffmann, 1947). The O antigens are heat stable lipopolysaccharide complexes which form part of the cell wall. The K antigens are composed of polysaccharides and form an envelope or capsule around the cell wall. These antigens prevent agglutination of a live suspension of homologous 0 antigen with its antisera. The K antigens are thermolabile, being inactivated by heating at 100 C or 121 C. The K antigens are subdivided into L, A and B types according to differences in heat stability and anti- genicity. The H antigens are thermolabile, are inactivated at 100 C, and appear to be protein in nature. Approximately 150 O, 90 K and 50 H serotypes have been identified (Nielsen et a1., 1968). Only a few of these serotypes are enteropathogenic (Moon et a1., 1966; ¢rskov et a1., 1964; Smith and Halls, 1967a). Pathogenesis In recent years much work has been done to aid in the under- standing of the pathogenesis of enteric colibacillosis. However, more detailed information is needed for complete understanding of the disease. Currently, the most acceptable pathogenesis involves ...a susceptible pig infected with an enteropathogenic strain of E. coli which has the capacity to proliferate in the proximal small intestine and to produce and 9 release enterotoxins in adequate amounts to cause alteration in the normal fluid and electrolyte trans- port functions of the intestines, with resultant diarrhea, dehydration, and death. (Kohler, 1972) Enteropathogenic strains of E. coli are widely distributed in the environment and can be found wherever pigs are raised (Kohler, 1972; Nielsen et a1., 1968; Rutter, 1975). Heavily populated, poorly ven- tilated farrowing houses and poor management practices greatly increase the numbers of enteropathogenic E. coli organisms in the environment of susceptible pigs (Kohler, 1972; Nielsen et a1., 1968). The risk of infection increases with the added numbers of enteropathogens in the environment (Kohler, 1972). The susceptible pig is infected by ingestion of the enteropatho- genic E. coli (Rutter, 1975). Many of the E. coli organisms are destroyed by the low pH of the stomach contents. However, in the newborn pig the gastric pH is high during the first 48 hours of life. Smith and Jones (1963) examined the pH of the stomach contents of healthy pigs during the first 24 hours after birth and obtained values of 5.5, 5.1, and 4.3 at 6, 12, and 24 hours, respectively. They also found the number of E. coli in the intestines of l-day-old pigs to be quite high. These results indicated that the high gastric pH allowed the organisms to proliferate in the stomach and that these organisms continued to proliferate after passing into the small intestine. This factor may predispose the neonatal pig to infection by the enteropathogenic organisms (Nielsen et a1., 1968; Smith and Jones, 1963). Other factors may be responsible for the ability of the entero- pathogenic strains of E. coli to proliferate in the anterior part of 10 the small intestine. Intestinal motility is one factor. Chyme, and the bacteria it contains, are continually moved down the intestinal tract by peristaltic movement. Any factor which decreases or prevents peristalsis will increase the number of organisms in the lumen (Drees and Waxler, 1970; Kohler, 1973; Nielsen et a1., 1968). Nutritional factors may also be important as suggested by the occurrence of enteric colibacillosis after weaning (Steven, 1963a; Svendsen et a1., 1974). The ingestion of colostrum and the continuous supply of antibodies in the milk of the sow greatly enhance the pig's resistance to colibacillosis (Kohler et a1., 1975; Porter et al., 1970a; Svendsen and Wilson, 1971). The ability of enteropathogenic strains of E. coli to proliferate in the small intestine is not unique to these strains. Nonentero- pathogenic strains proliferate at the same rate and achieve numbers equal to or greater than those of enteropathogenic strains (Kohler and Bohl, 1966b; Smith and Halls, 1967a). However, enteropathogenic strains of E. coli have the ability to adhere to and colonize the intestinal epithelial cells in great numbers (Bertschinger et al., 1972a,b). Nonenteropathogenic strains are mainly observed in the intestinal lumen (Bertschinger et a1., 1972a,b). The factors which allow enteropathogenic strains to adhere to the intestinal epithelial cells are not clear. It has been suggested that K88 antigens may play a role in this process (Jones and Rutter, 1972; Smith and Linggood, 1971). A transmissible plasmid controls the production of K88 antigens of strains E. coli (¢rskov and ¢rskov, 1966; Smith and Linggood, 1971). 11 Jones and Rutter (1972) conducted a study in which both conven- tional and germfree pigs were given orally either enteropathogenic strains of E. coli which possessed K88 antigen (K88-positive strains) or a strain which lacked K88 antigen (K88-negative strain). These investigators found the K88-positive strain adhered to and colonized the intestinal epithelial surface of the conventional pigs, while K88-negative strain did not. Also, the mortality rate and severity of diarrhea were much greater in those pigs given the K88-positive strain.) There was no observable difference between the K88-positive and K88-negative strains' ability to colonize the small intestines of gnotobiotic pigs or in the ability of these strains to cause diarrhea. These researchers suggested that the reduced motility of the germfree gut may have been responsible for the colonization of the intestinal epithelial cells by the K88-negative strain. Also, many E. coli enteropathogens which lack K88 antigen colonize the small intestine of pigs (Moon, 1974). More studies are needed to determine the mechanisms of how K88-negative strains colonize the epithelial surface of the small intestine (Moon, 1974). Although proliferation and colonization of the enteropathogenic strains of E. coli in the anterior part of the small intestine are important factors in the development of colibacillosis, they alone are not suf- ficient to cause the fluid accumulation that occurs in the intestines of infected pigs (Jones and Rutter, 1972). De and Chatterje (1953) and De et a1. (1956) discovered that ligated intestinal loops of rabbits became distended with fluid when injected with Vibrio cholerae and E. coli organisms isolated from human patients with cholera. Smith and Halls (1967b) found that 12 bacteria-free fluid (enterotoxin) prepared from cultures of E. coli isolated from pigs with diarrhea, when injected into ligated intes- tinal loops of the same species, caused fluid accumulation. Fluid accumulation was not observed in those loops inoculated with bacteria- free fluid from E. coli strains which did not cause diarrhea. These investigators postulated that proliferating E. coli organisms in the anterior part of the small intestine of the intact host produce enterotoxins and that these enterotoxins induce the diarrhea which occurs in colibacillosis. Other investigators have provided strong evidence that diarrhea seen in this disease is probably enterotoxin induced (Smith and Gyles, 1970; Smith and Linggood, 1971). Two similar enterotoxins have been isolated from enteropatho- genic strains of E. coli: heat labile (LT) and heat stable (ST) (Smith and Gyles, 1970; Smith and Halls, 1967b). The LT enterotoxin's ability to cause fluid accumulation in ligated intestinal loops of pigs is greatly reduced when heated at 121 C for 30 minutes and almost completely inactivated when heated at the same temperature for 2 hours (Smith and Halls, 1967b). AntiSera prepared against the living organisms neutralizes the dilating effect of this enterotoxin (Smith and Gyles, 1970). The ST enterotoxin is not antigenic or affected by antisera prepared against live E. coli organisms (Smith and Gyles, 1970; Smith and Halls, 1967b). A close relationship exists between K88 antigens on a strain of E. coli and the ability of the strain to produce enterotoxins (Smith and Gyles, 1970). Both K88 antigen and enterotoxins (ENT) are produced by separate trans- ferrable plasmids (Smith and Gyles, 1970; Smith and Linggood, 1971). However, K88 plasmid can be removed from a strain without affecting 13 the production of LT or ST enterotoxins by the strain. The reverse is also true: a strain to which the ENT plasmid is transported does not acquire the ability to produce K88 antigens (Smith and Gyles, 1970). There is probably no difference between enterotoxins produced by different strains of E. coli in the same species, but there are differences in those enterotoxins produced in different species (Smith and Halls, 1967b). Enterotoxins produced by the proliferating E. coli enteropatho- gens induce 1055 of fluid across an intact mucosa (Moon, 1974; Moon et a1., 1971). The enterotoxins of V. cholerae have a similar effect on the intestines of people affected with cholera (Carpenter, 1972). Moon et a1. (1971) compared the responses of ligated intestinal loops of rabbits and pigs to the injection of enterotoxins from V. cholerae and E. coli. No differences were observed in the gross and microscopic response of the intestinal loops to enterotoxins from these 2 organisms. The composition of the fluid secreted by the intestine in response to enterotoxins from E. coli and V. cholerae was also similar. The fluid in comparison to serum was , , ++ ++ , , + - - + low in protein, Ca , and Mg and high in Na , Cl , HCO , and K . 3 These investigators concluded that the response to V. cholerae and E. coli enterotoxins was similar. Carpenter (1972) reported similar findings in people with cholera. In most species, the anterior part of the small intestine appears to be more sensitive to the effects of enterotoxins than does the posterior part (Moon, 1974). Carpenter and Greenough (1968) reported that the rate of secretion of fluid in response to 14 V. cholerae enterotoxins in the duodenum and ileum of the dog was essentially the same and that the lesser net fluid output of the ileum was due to the greater capacity of the ileum to absorb the fluid. The cells responsible for the secretion of fluids and the exact mechanisms involved in response to E. coli enterotoxins are not known. Nielsen et a1. (1968) postulated that the crypts of Lieberkuhn secrete fluid in response to injury of the mucosa by enterotoxins. However, there is some evidence that V. cholerae enterotoxins stimu- late the intracellular enzyme adenylate cyclase, which catalyzes the conversion of adenosine triphosphate (ATP) into 3',5'-cyclic adenosine monophosphate (cAMP), which in turn regulates the secretion by the small intestine exposed to this toxin (Lehninger, 1975; Moon, 1974). The increase of cAMP inhibits the active transport of sodium from the lumen of the small intestine into epithelial cells and stimulates the active secretion of chloride. This results in a net secretion of electrolytes and water (Lehninger, 1975; Moon, 1974). Escherichia coli enterotoxins may have the same mechanism of action as those of V. cholerae (Evans et a1., 1972). Further studies are needed as to the specific mechanism of action of E. coli enterotoxins in causing the fluid loss observed in enteric colibacillosis. The final step in the pathogenesis of enteric colibacillosis is the clinical response of the affected pig. The loss of fluids into the intestinal lumen results in diarrhea and dehydration with eventual acidosis and hemoconcentration. Death usually occurs if the pig is unable to correct the dehydration and electrolyte losses (Kohler, 1972). 15 Clinical Signs Enteric colibacillosis usually affects pigs of 3 definite age groups: 1) neonatal pigs 1 to 4 days of age, 2) piglets approxi- mately 3 weeks of age, and 3) pigs 10 to 12 weeks of age (Stevens, l963a,b). The clinical signs of colibacillosis in baby pigs usually appear 12 hours after birth. At this time several pigs may be found dead or moribund without showing evidence of diarrhea. Others may appear listless and Show varying degrees of diarrhea (Moon, 1969; Stevens, 1963a). The pigs will usually nurse until they are too weak to stand (Moon, 1969). The same clinical signs have been observed in conventional and germfree pigs experimentally infected with entero- pathogenic strains of E. coli (Christie and Waxler, 1973; Drees and Waxler, 1970; Meyer and Simon, 1972; Miniats and Gyles, 1972). Some 3-week-old pigs develop sudden diarrhea which may result in decreased growth rates of these pigs (Sojka, l965c). Death may occur in a few of the affected pigs (Sojka, l965c). Changes in management practices, such as the introduction of solid food to the diet of these pigs, may be responsible for the development of coli- bacillosis in this age group (Stevens, l963b). Postweaning enteritis is observed in pigs 2 weeks after weaning. These pigs exhibit a short period of diarrhea. The mortality rate in this age group is usually low (Stevens, l963a,b). Gross and Microscopic Findings The characteristic gross lesions observed in pigs affected with enteric colibacillosis are usually confined to the stomach and 16 intestinal tract. The stomach is distended with gas and coagulated milk (Christie and Waxler, 1973; Moon, 1969; Stevens, 1963a). The small intestine is dilated and flaccid and contains large quantities of fluid, clots of undigested milk and mucus (Christie and Waxler, 1973; Drees and Waxler, 1970; Kohler, 1967; Meyer and Simon, 1972; Moon, 1969; van Dreumel, 1972; Waxler et a1., 1971). Petechial to diffuse hemorrhage and congestion of the intestinal mucosa and engorgement of the mesenteric blood vessels have been reported (Christie and Waxler, 1973; Meyer and Simon, 1972; Miniats and Gyles, 1972; Svendsen et a1., 1974). The cecum and colon may also be dis- tended with gas. Gross lesions are not always observed in pigs affected with this disease (Smith and Jones, 1963). Histopathologic examination of tissues from pigs affected with colibacillosis has revealed such lesions as: severe inflammation and massive numbers of polymorphonuclear leukocytes in mesenteric lymph nodes (Miniats and Gyles, 1972);.vacuolation of villous epi- thelial cells (Miniats and Gyles, 1972); edema of the lamina propria (Christie and Waxler, 1973; Drees and Waxler, 1970; van Dreumel, 1972); and congestion of the submucosa of the small intestine (Svendsen et a1., 1974). Hemorrhage and surface necrosis with sloughing of the villi has also been reported (Christie and Waxler, 1973). However, microscopic lesions may not be seen in pigs affected with enteric colibacillosis (Kohler, 1967). Passive Immunization of the Neonate Against Colibacillosis The pig is virtually devoid of immunoglobulins at birth. Traces of immunoglobulins antigenically related to 196 have been detected in the serum of the pig at birth (Sterzl and Silverstein, 1967). 17 This immunoglobulin is probably produced by the fetus in utero since there is no transfer of immunoglobulins across the placenta in the pig (Kim et a1., 1966; Porter, 1969; Porter and Allen, 1972; Solomon, 1971; Sterzl et a1., 1966). Immunoglobulins and antibodies are acquired from the maternal colostrum by absorption from the small intestine within the first 24 to 36 hours of life (Porter, 1973; Porter and Allen, 1972; Wilson and Svendsen, 1972). After this time, no antibodies or immunoglobulins are absorbed by the newborn pig. The level of passively acquired immunoglobulins and antibodies to E. coli in sera of young pigs significantly declines after the first week of life (Porter, 1969; Wilson, 1972; Wilson and Svendsen, 1972). Actively acquired antibodies to E. coli are not detected in the sera of neonates until after the third week of life (Wilson and Svendsen, 1972). Thus, after the first week of life, the pig is par- ticularly vulnerable to pathogenic E. coli organisms and must rely on local antibodies present in the intestinal tract. Secretory IgA is the major immunoglobulin and source of anti- body to E. coli in the intestines of young pigs (Porter et a1., 1970a). The function of secretory IgA antibodies is protection of the mucosal surfaces from invasion by pathogenic viruses and bacteria (Tomasi, 1967; Williams and Gibbons, 1972).. Secretory IgA present in the intestines of young pigs is obtained mainly from the milk of the sow. Also, some secretory IgA is produced in the intestines of young pigs. Porter et al. (1970b) detected, by the use of immunoelectrophoresis, secretory IgA in the intestinal secretion of young pigs about 10 days of age. Antibodies of the IgA class are produced by immunocytes in the lamina propria of the small intestine and are then transported 18 through the epithelial cells of the mucosa, where the secretory piece is added (Porter and Allen, 1972; Tomasi, 1967). Recognition of the importance of E. coli antibody in the colostrum and milk of the sow for the passive protection of young pigs against invasion by enteric pathogens has resulted in several studies in active immunization of the sow with E. coli antigens. Svendsen and Wilson (1971) found that when colostrum from vaccinated sows was given orally to gnotobiotic pigs, there was some protection against experimentally produced colibacillosis. Protection against coli- bacillosis was not observed in gnotobiotic pigs given colostrum from nonvaccinated sows. Similar results have been obtained using experi- mentally infected conventional pigs (Kohler et a1., 1975; Rutter and Anderson, 1972). Kohler and Bohl (1966b) and Miniats et a1. (1970) studied the value of feeding antisera to experimentally infected gnotobiotic pigs. These investigators found that these pigs were protected from clinical signs of disease during the time that the antisera were being administered. When the antisera were withdrawn, the pigs developed clinical signs of disease. The antiersa may have inter- fered with the effects of enterotoxins on the intestinal tract (Kohler and Bohl, 1966b; Miniats et a1., 1970). Gnotobiotic Pigs In studies concerning the development of immunity, it is of primary importance that there is adequate control of antigenic stimuli (Sterzl and Silverstein, 1967). The porcine fetus in utero is usually in a sterile environment and does not come into contact with external l9 antigens until after birth. The number of antigens in the environ— ment of the newborn pig can be controlled when pigs are raised under gnotobiotic conditions. Also, pigs are virtually devoid of anti- bodies at birth since maternal antibodies do not cross the placenta in this species (Kim et a1., 1966; Porter, 1969; Sterzl et a1., 1966; Sterzl and Silverstein, 1967). Thus, one could be nearly certain that the antibody response of gnotobiotic or fetal pigs to the injection of antigen is caused by that antigen without interference of other antigens and antibodies (Kohler and Bohl, 1966b; Porter and Kenworthy, 1970; Sterzl and Silverstein, 1967). Gnotobiotic pigs are used in the study of certain infectious agents, such as E. coli. One can study the effects of specific serotypes and strains of E. coli without the interference of intes- tinal flora or pathogenic organisms or passively acquired antibodies (Sterzl and Silverstein, 1967; Waxler et a1., 1971). These factors cannot be controlled under field conditions (Waxler et a1., 1971). Methods in Immunology Direct Bacterial Agglutination Test Bacterial cells in suspension will usually clump when mixed with specific antiserum. According to the lattice theory for the precipitation of soluble antibody-antigen complexes, each antibody molecule is linked to more than one antigen molecule, and in turn each antigen molecule is linked to more than one antibody molecule. The aggregates so formed will become large enough to fall out of solution to form a visible precipitate. The principles are more or less the same for the agglutination reaction (Eisen, 1974a). 20 The bacterial agglutination test can be used to detect and roughly quantitate antibodies in sera when known cells are used (Eisen, 1974a). This test is more sensitive than the precipitin test but not as sensitive as the passive hemagglutination test. The bacterial agglutination test can detect approximately 0.1 ug/ml of antibody (Eisen, 1974a). The bacterial agglutination test has been used to detect and quantitate antibodies to E. coli in both porcine (Corley et a1., 1973) and bovine (Conner et a1., 1973) species. Passive Hemagglutination Test Soluble antigens can be detected by the agglutination reaction when these antigens are attached to the surface of erythrocytes. Erythrocytes will readily absorb polysaccharides and some proteins. This test is very sensitive and can be used to detect as little as 0.01 ug/ml of antibody (Eisen, 1974a). The passive hemagglutination test can be conducted using either the tube method (Sharpe, 1965; Svendsen and Wilson, 1971) or the microtitration method (Sever, 1962). The chief advantages of the microtechnique are the saving of reagents and the rapid performance of microdilution (Sever, 1962). The passive hemagglutination test has been used to detect antibodies against E. coli in the serum of pigs (Svendsen and Wilson, 1971). MATERIALS AND METHODS Animals Six gilts (Yorkshire, Hampshire, and mixed breeds), bred by natural service, were obtained as a source of gnotobiotic pigs.a The gilts were housed in a barn and were allowed access to a small pasture. Their diet consisted of gestation ration with water ad libitum. Food and water were withheld 24 hours before each surgery. Three Dutch Belted rabbits were used for the production of antisera to be used in the direct bacterial agglutination test and the passive hemagglutination test. Preparation of Bacterin Stock culturesb of E. coli 0149:K91(B);K88(L) were stored in the dark at room temperature in tightly sealed slant tubes containing trypticase soy agarC with no dextrose. Subcultures, on the same media, were prepared at regular intervals, incubated overnight at 37 C, sealed, and stored. aObtained from the Department of Animal Husbandry, Michigan State UniverSity. bObtained from Dr. H. W. Moon, National Animal Disease Center, Ames, Iowa 50010. CBBL, Division of Broquest, Cockeyville, Maryland. 21 22 Preliminary studies were performed to determine the maximum growth curve of the E. coli organisms. Sterile test tubes containing 7 ml of brain heart infusion (BHI) brothd were inoculated with 1 standard loopful of E. coli organisms taken from the stock culture and incubated at 37 C for a variable number of hours. The cultures were washed 2 times in sterile saline and resuspended in sterile saline to their original volume. Serial dilutions were made, and 1 m1 from each dilution tube was plated in trypticase soy agar and incubated at 37 C for 24 hours. Those plates containing less than 30 or greater than 300 colonies were discarded, and the remaining plates were counted and tabulated. The preliminary studies revealed the maximum growth to occur at 10 hours. Bacteria used for injection of the fetuses were prepared with slight modification according to Conner et a1. (1973). A culture of E. coli organisms was grown for 10 hours at 37 C in BHI broth, washed twice in sterile saline, seeded in bottles containing trypticase soy agar, and incubated at 37 C for 24 hours. The growth was washed off the agar medium with sterile saline and pooled. Formalin (0.4%) was added to the suspension after samples were taken for bacteriologic examination to determine purity and viable cell count. The formalin- ized cell suspension was incubated in a shaking water bath at 37 C for 18 to 24 hours. The formalinized bacterin was washed 3 times in sterile saline, resuspended in sterile saline to a concentration of 5 x 109 organisms/ml, and stored at 4 C in sterile, heat-sealed ampules. Merthiolate (0.02%, w/v) was added as a preservative. d . . . . . Difco Laboratories, Detr01t, Michigan. 23 Fetal Injection A right flank laparotomy was performed at 98 days of gestation. Anesthesia was produced in the first 3 gilts by epidural injection of 25 m1 of 2.5% procaine hydrochloridee into the lumbosacral space. A general anesthetic (Halothane 2%,f nitrous oxide, oxygen mixture) was given to the last 3 gilts. Five milliliters thiamylal sodium9 in a 10% solution was given intravenously as preanesthetic medica— tion. The fetuses, enclosed within the uterus, were brought through the surgical incision one at a time and inoculated through the uterine wall. Each fetus and the uterus at the site of the fetus were marked for later identification. Approximately 0.25 ml of sterile India ink was injected into the subcutaneous tissue of the fetus, and suture material was placed in the serosa of the uterus. During the experiment, injections were made into a total of 49 fetuses from 6 litters. The numbers of fetuses, solutions injected, routes of injection and identification of fetuses are summarized in Tfiflel. The gilts were allowed to recover from surgery. Their progress h'i and temperature were observed and antibiotics were given for several days following the laparotomy. Observations of food and eEpidural solution, Haver-Lockhart Labs, Kansas City, Missouri. fAyerst Laboratories, New York, New York. gSurital Sodium, Parke, Davis & Company, Ann Arbor, Michigan. hCombiotic, Charles Pfizer and Co., Inc., New York, New York. lLiquamycin (50 mg/cc), Pfizer, Inc., New York, New York. 24 Table 1. Numbers of fetuses, solutions injected, routes of injection and identification of fetuses injected in utero Numbers of fetuses injected in utero, the solution injected and routes of injection 9 Numbers of Bacterin (5 x 10 ) Saline Sites of India sutures placed AF* IM* AF IM ink injection in uterine wall 27 shoulder 1 18 hip region 2 3 dorsal midline 3 l dorsal midline l (lumbar region) (mattress) * AF = amniotic fluid; IM = intramuscularly. water consumption, as well as the general condition of the sow, were made throughout the experiment. Preparation of Gnotobiotic Equipment The rearing isolators were prepared for receipt of the gnoto- biotic pigs according to procedures outlined by Waxler et a1. (1966). Each of the 3 isolators was washed with a detergent solution, rinsed with clear water and dried using cheesecloth to prevent stains. J was used to remove persistent stains and marks and to Polykleen polish the isolators both inside and outside. The cages, floor mats, caps, bands, and other equipment were washed and dried. The plastic JSchwartz Chemical Company, Inc., New York, N.Y. 25 outer sleeve of the intake filter of each isolator was removed to inspect the filter for damage. The filters were sterilized, using dry heat, at 150 C for 3 hours. The exhaust valve apparatus of each isolator was checked for fluid level. The cages were inspected for cleanliness and sharp edges before they were autoclaved at 250 F for 30 minutes. The cages were then wrapped in brown paper and again autoclaved at 250 F for 30 minutes. After each isolator and gloves were carefully checked for holes, each isolator and the floor mats, bands, and inner cap within each isolator were sprayed with 2% peracetic acid solutionk with a wetting agentl added in a concentration of 0.1%. The cages were unwrapped, and 4 were placed in each isolator and sprayed with peracetic acid. The isolators were allowed to stand for 30 minutes before air flow was started through the filters. The surgical isolator was washed and sterilized using the same procedures as described above. The surgical instruments, towels, and umbilical clamps were placed in a stainless steel, filter- equipped sterilization cylinder and autoclaved at 250 F for 30 to 45 minutes. A vinyl sleeve, previously sprayed with peracetic acid, was attached to the cylinder and to the stainless steel ring in the wall of the isolator. The instruments and material within the cylinder were then passed directly into the isolators. Blood tubes, towels, needles, syringes, and other equipment which would be needed kFMC Corporation, Buffalo, New York. lNacconal NRSF, National Analine Division, Allied Chemical Corporation, New York, N.Y. 26 during the experiment were placed in a similarly constructed cylinder, autoclaved, and passed into one of the rearing units using the same technique as described above. Preparation of Live Challenge Organisms To prepare live challenge organisms, a culture of E. coli was grown for 10 hours at 37 C in a tube containing 7 ml of BHI broth. The challenge organisms were washed 2 times in sterile saline. Based upon previous growth of lO-hour cultures, sterile saline was added to the washed challenge organisms to give a concentration of approximately 3 x 106 organisms/ml. Challenge organisms were placed in sterile ampules, heat sealed, and used within a few hours. A viable count was obtained to determine the actual concentration of the challenge organisms. Gnotobiotic Procedures A total of 34 pigs from 3 litters was delivered by hysterotomy at 112 days of gestation and raised under gnotobiotic conditions according to techniques described by Waxler et al. (1966). Anesthesia was produced by epidural injection of 25 m1 of 2.5% procaine hydro- chloride at the lumbosacral space. Promazine hydrochloridem (0.5-1.0 mg/lb body weight) was given as a tranquilizing medication. Once the pigs were obtained, the sow was euthanatized. The pigs from each sow were separated into 3 rearing isolators. Each isolator contained pigs which received in utero injection of E. coli bacterin or saline or were uninjected. After blood and mSparing, Wyeth Laboratories, Inc., Philadelphia, Pennsylvania. 27 bacterial samples were taken, the pigs in 2 isolators from each litter were given orally 1 ml of live challenge organisms (2-7 x 106 organisms). The pigs in the remaining isolator from each litter were given 1 ml of sterile saline and kept as controls. Specimen Collection Serum Samples The gnotobiotic pigs were bled at birth and at the termination of the experiment at 10 or 18 days of age. Those pigs which appeared very weak before the end of the experiment were bled and then euthana- tized. Approximately 5 ml of blood was collected from the anterior vena cava into 10 ml plastic syringes, placed in test tubes and allowed to clot. The serum samples were obtained according to accepted procedures and frozen at -24 C until used. Bacteriologic Samples Bacteriologic procedures were performed according to Waxler et a1. (1966) with minor changes. Swabs were taken from the rectum of pigs and waste material in pans of the cages of each isolator at the beginning and end of the experiment. The specimens from each iSO1ator were streaked on blood agarn plates and incubated aerobi— cally and anaerobically at 25 C, 37 C and 55 C. Thioglycollate brothn was also inoculated and incubated aerobically at the same tempera- tures as above. In addition, PPLO brothn was inoculated and incu- bated at 25 C and 37 C. Every 3 days, 1 loopful of the bacterial nDifco Laboratories, Detroit, Michigan. 28 sample in PPLO broth was transferred into fresh medium and incubated at the same temperatures. On the 12th day, the bacterial sample in PPLO broth was transferred to PPLO agar and incubated at 25 C and 37 C for 1 month. Cecal material from pigs which died or were euthanatized was taken with sterile swabs, streaked onto trypticase soy agar, and incubated at 37 C for 24 hours, at which time the plates were inspected for growth. Tissue Samples Those pigs which did not die were euthanatized by intravenous injections of pentobarbital sodium0 or by electrocution. A muscle relaxant was given prior to electrocution. The pigs were placed in dorsal recumbency and a midline incision was made to expose the abdominal and thoracic organs. A general examination of the organs was made at that time. The cecum was opened with sterile instruments and its contents were sampled for bacteriologic examination. A small section of the ileum was taken starting at the ileocecal valve and progressing anteriorly. Samples of mesenteric lymph node adjacent to the ileum were also taken. Other tissue samples were taken from the liver, spleen, right kidney, thymus, and mandibular lymph nodes. Any other tissue which appeared abnormal was taken for histopatho- logic examination. The tissues were placed in 10% (v/v) formalin solution, processed according to standard procedures, cut at 6 microns, and stained with hematoxylin and eosin (Luna, 1968). oHaver-Lockhart Labs, Kansas City, Missouri. 29 Immunologic Procedures Direct Bacterial Agglutination Test The antibody levels of sera were determined with slight modifi- cation, according to procedures described by Conner et a1. (1973). Briefly, the 0 antigen was prepared by growing E.'coli cultures in BHI broth at 37 C for 10 hours. The culture was heated in a boiling water bath at 100 C for 1 hour and allowed to cool, and formalin (0.5%) was added. The 0 antigen was either used immediately without washing or stored at 4 C. The K antigen was prepared from cultures grown in BHI broth at 37 C for 10 hours. Formalin (0.5%) was added, and the antigen was used within 18 hours. Serial twofold dilutions of sera were prepared in 0.5 ml of saline with the beginning dilution of 1:2, and 0.5 m1 of antigen was added. Rabbit antiserum against E. coli was used for the posi- tive control. The O agglutination tubes were incubated at 50 C for 20 hours. The K agglutination tubes were incubated at 37 C for 2 hours, then at 4 C overnight, and placed at room temperature for 1 hour before results of the tests were read. Passive Hemagglutination Tests The passive hemagglutination test was performed on selected serum samples according to methods described by Shape (1965). A 48-hour growth, on trypticase soy agar, was harvested using 4 ml of saline. The bacterial cell suspension was heated in a boiling water bath at 100 C for 1 hour, allowed to cool, and centrifuged at 3000 rpm for 30 minutes. The resulting supernatant was stored at 4 C until used. 30 Hemolysins were removed from each serum sample (2 ml) by mixing the sample with 0.1 ml of washed packed sheep red blood cells (SRBC). The mixture was left at room temperature for 3 hours and then cen- trifuged, and additional SRBC (0.2 ml) were added to each serum sample. The serum samples were then stored overnight at 4 C. Washed SRBC (1 ml) were added to 5 ml of 0 antigen in 94 ml of phosphate buffered saline (PBS) and incubated at 22 C for 45 minutes. The coated SRBC were washed 3 times in PBS, and a 1% suspension of washed coated SRBC in PBS was prepared. Serial twofold dilutions of the serum in 0.025 ml amounts were made in PBS starting with a dilution of 1:2. Control tests included coated SRBC and PBS, uncoated SRBC and PBS, and uncoated SRBC and sera. In addition, rabbit antiserum against E. coli was used for a positive control. The plates were incubated at 22 C for 45 minutes and then at 4 C overnight. RESULTS Surgical Procedures The results of the surgical procedures and fetal injections are summarized in Table 2. Laparotomy Procedures The epidural procedure produced anesthesia which was adequate initially in that there was minimal reaction to the incision through the abdominal wall. However, the surgical procedure was time con- suming and, before it was completed, there was considerable strug- gling on the part of the sow. Subsequently, 2 of the 3 sows which were given the epidural anesthesia aborted following surgery. The remaining gilt had an uneventful surgical recovery. The general anesthesia administered to the last 3 gilts alleviated the problems encountered with the epidural anesthesia. These gilts had an uneventful recovery from surgery. HysterotomyiProcedures The epidural anesthesia was adequate for this procedure. Forty- five live and l mummified fetuses were delivered by hysterotomy at 112 days of gestation. Four pigs from Litter 4 were euthanatized. Two fetuses could not be identified by using the uterine and fetal markings. It was assumed that 1 pig was given saline by in utero injection into the amniotic fluid. However, since neither pig could 31 32 .nuuwn um mmfim omuomflcfl Iwcflamm gnu mo moo mufiucwofi no: oasou+++ nomuflumcmnusm mums H50m++ acmAMHEEsE was mam mco+ “musumeum mxmms m mums mafia one “unadumsemuucw u 2H «madam cauoflsfim u .m.¢ «ea «« « imcmauonmc OH oH Homucm>mcs In: m H v Hmumomm o +++ Amcmzuonmv NH NH Homucm>mc9 H m N m Hmumcwm m Amcmauonmv NH ha aswucm>mcd 1|: m III m Hmumcwm o i + Snowman umuww III OH whom 0H Uwuuono II; N II: v amusoflmm m wummusm umumm i: 3 $86 a 6388... l: m u-.. N. 333% m in I: 33:26:: I-.. m I- 4 3339.... a ucmEflummxm umuufla a“ uHHo mo mum>00mm 2H .m.< «azH «.m.¢ haououwmmH .0: CA poms mmfim mmmsuwm mo mcwamm swuwuumm you Mammcummcm uflwo mo .oc Hmuoa .0: Hobos Dump: cw omuowncfi momsuwm mo .02 mo mama coauownCH Houmm can hsououmumhz one MEouoummoH mo muasmmm .m wanna 33 be identified, they were considered to be uninjected controls. The remaining fetuses given either saline or E. coli bacterin in utero were identified by their fetal or uterine markings, or both. The 7 pigs of Litter 1 were found to be approximately 21 days premature at the time of the surgical procedure. These pigs died within 1 hour after birth. Clinical Response of Pigs to Challepge The clinical responses of the 23 neonatal pigs to challenge with viable E. coli are summarized in Table 3. Eighteen of the 23 chal- lenged pigs died. This was a mortality rate of approximately 78%. Fourteen (61%) of these pigs were either found dead or became mori- bund and were euthanatized within 48 hours after challenge. Clinical signs were observed in 12 (52%) of the challenged pigs and were those associated with colibacillosis, as evidenced by anorexia, depression, and generalized weakness. These signs were usually observed within 24 to 36 hours after challenge. Diarrhea was observed in 8 chal- lenged pigs and appeared within 24 to 36 hours after challenge, persisting in 7 of these pigs until death occurred at 2 to 3 days of age. Diarrhea was observed in the remaining pig (No. 28) for about 3 days, subsided, and was not again observed during the experiment.) Five challenged pigs survived until euthanatized at the termination of the experiment at 10 to 18 days of age. Clinical signs of colibacillosis were observed in l of these pigs (No. 28). Clinical signs associated with colibacillosis were not observed in the 11 control pigs. However, 8 of these pigs died before the termination of the experiment. 34 v I III III m m NN +OH I III m m m HN +m + oOH x mN.h III N m 0N +N + 00H x mN.h HEva N m 0H v I 00H x mN.> m N m wH +0H I o0H x mN.h +++AEHVm N m NH +N + 00H x mN.> III H m 0H N I 00H x mN.> ++HEHVm H m mH +m + 00H x mN.h m H m VH N II 00H x mN.n m H m MH N I III m m v NH m I III III m v HH v I III m m v 0H N I III III m v m H + 00H x mo.o III N v m N + oOH x mo.m III N v n N I 00H x mo.m m N v o N I oOH x mo.o m N v m N I oOH x 00.0 m H v v N + 00H x No.6 m H v m m + 00H x 00.0 m H v N N I 00H x mo.© III H v H memov shown no «uwm:mHHm:0 HmEmH:mmH0 «:OHuumfl:H .0: msoum .0: .0: mon m0 0o: uwumm wwwomHo HNOU .m mHQMH>o chub: :w mo 0Q>B Mo wouwHomH wouUHH mHm HmuH:HH0 mo m:mHm mmoo 00:0HHmnu Hmuo 0:HHmm no :Humuomn mo :OHuomm:H oumus :w m:H30HHom.HHmemxflmonxnmvHo HNOU .m CH magmomxm Hmuo ou mmHm Houm:00: mo mmm:0mmmm .m mHQMB 35 .0mm m0 m>m0 MH um o0m:0HHm:U < “SHAMHDUmSEmuu:H touumfl:H 0:HHmm HEHVm+++ xxHMMHsomsEmuu:H owuomw:H :Humuomn u HE ++ «MHmm:mnusm um mmHm m0 mm:+ “0mm mo mxmo m um 605:0HHMEU “mmwmme HMOH:HH0 u + .mmmmmHo HmoH:HH0 0: u I «a: «« “omuomn:H:o: u III .xHHmuHuOH:EMImuu:H 0:HH0m u m .aHHmoHuoH:EMImuu:H omuomn:H :Humuomn u m i +mH I III III m w on H I III m m 0 mm +HH I III m m 0 NM oH x Hm.N on x m.m +mH I 00H x mo.N m N m Hm mpv nummo um hTsimmvcmeHmEB HmEmH:mmuo h..:0H..H00.H:H .0: macho .0: .0: mmHm m0 00d “mum: mmmmme HHoo .m 0HQMH>V 0H0“: CH mo mama Ho HoumHOmH umuuHH mHm HMUHcHHu mo m:mHm muoo mmcmHHmso Hmuo H.©.u:00v m mHndB 36 Direct Bacterial Agglutination and Passive Hemagglutination Tests The results of the direct bacterial agglutination test for pig sera with K and O antigens, as well as the passive hemagglutination test with O antigens, are summarized in Table 4. All the pigs had negative 0 and K agglutinin titers at birth. The sera of pigs No. 28 and 31 had 0 agglutinin titers at 11 days of age. The reciprocal of the titers was 16 and 4, respectively, With the bacterial agglu- tination test and equal to or greater than 256 with the passive hemagglutination test. The serum of pig No. 31 was tested again at 18 days of age using the bacterial agglutination test. The O agglutinin titer had not changed. Only the serum of pig No. 28 had K agglutinin titer at 11 days of age. Gross Findings The changes observed in most of the pigs given live challenge organisms orally were confined to the organs of the digestive system and were those associated with colibacillosis. The stomach, posterior 1/3 of the jejunum, cecum, and spiral colon were distended with gas. The stomach was filled with undigested coagulated milk. The intes- tine of some pigs also contained an abundant yellowish ingesta. Other gross lesions were observed in only a few pigs. These lesions con- sisted of hemorrhagic enteritis and colitis (pig No. 15), hyperemia of the stomach and jejunum (pig No. 18), and hyperemia of the jejunum, ileum and spiral colon (pigs No. 25 and 26). Also, pig No. 25 had hemorrhagic areas involving the mucosa of the fundus of the stomach. Partial atelectasis of the apical lobe of the right lung was observed 37 Table 4. Direct bacterial agglutination and passive hemagglutination titers* of serum from pigs injected in utero with E. coli bacterin (Group 1) or saline (Group 2) or uninjected (Group 3) Group Pig Age at time sample 0149 antigens K antigens no. no. taken (days) Directf Passive++ Direct 1 2 0** _ NT*** _ 4 0 - - — 5 O - NT - 10 0 - NT - l4 0 - NT - 3 - NT - 15 0 - - - 18 0 — — - 4 - NT - 19 O - NT - 2 - NT - 23 0 - NT - 26 0 - - - 27 0 - NT - 29 0 - - - 11 - NT - 18 - NT - 31 0 - - - ll 4 256+++ - 18 4 NT - 32 O - NT - 11 - NT - 2 3 0 NT - 6 0 - NT - 12 0 - NT - 13 0 - NT - 17 0 - NT - 10 - NT - 21 0 - NT - 10 - NT - 28 0 - - - 11 16 256+++ 4 33 0 - NT - 38 Table 4 (cont'd.) Group Pig Age at time sample 0149 antigens K antigens no. no. taken (days) Direct Passive Direct 3 l 0 - NT - 7 O - NT - 8 0 — NT - 9 O - NT - ll 0 - NT - 16 O - NT - 2 - NT - 20 0 - NT — 22 0 - NT — 24 0 - NT - 25 0 - - - 3O 0 - NT - 11 - NT — 34 0 — NT - * Titer expressed as reciprocal of the highest dilution showing agglutination. ** 0 = at birth. *** NT = not tested. Direct bacterial agglutination. T . . . Pass1ve hemagglutination. +++Titer was equal to or greater than 256. 39 in pig No. 7. No gross changes were observed in challenged pigs that survived 10 days or more after challenge. The stomach and entire intestinal tract of 2 of the pigs given sterile saline orally at birth were distended with gas. Organisms were not isolated from the cecal contents of these pigs. It is possible that the gas entered the stomach and intestine of these pigs with food ingested or during the process of dying, or both. Also, the gas may have been produced by organisms not detected with the bacteriologic procedures used. One control pig (No. 23) died when fetal membranes lodged in the larynx and upper 1/3 of the trachea. The entire right lung of pig No. 33 was collapsed. No other gross changes were observed in control pigs at necropsy. Histopathologic Findings Sections from mandibular and mesenteric lymph nodes, thymus, ileum, liver, spleen, and kidney were evaluated. Mandibular and Mesenteric Lymph Nodes and Thymus Evaluations were made of the mandibular and mesenteric lymph nodes of all but 4 experimental pigs. There were no significant morphologic differences observed between these 2 lymph nodes. The lymph nodes taken from most of the pigs had only poor demarcation between cortex and medulla. Follicular development of lymphoid tissue was observed in 11 pigs (Figure 1A, 13). Germinal centers were observed in mandibular lymph nodes taken from pigs No. 21 and 28 and mesenteric lymph nodes of pigs No. 17, 21, 28, 29 and 31 (Figure 2A, 23). All of these pigs, with the exception of pig No. 40 Figure 1A, 1B. Photomicrographs of the mesenteric lymph node of pig No. 14, previously injected in utero with E. coli bacterin, challenged with viable E. coli at birth, and euthana- tized at 3 days of age. Notice the poor demarcation between the cortex and medulla, the absence of lymphoid follicles and germinal centers, and the relatively small numbers of lymphocytes in the cortical area. H&E stain; x 50 (A) and x 125 (B). 41 Figure l 42 Figure 2A, 28. Photomicrographs of the mesenteric lymph node of pig No. 31, previously injected in utero with E. coli bacterin, challenged with viable E. coli at birth, and euthana- tized at 18 days of age. Antibodies were detected in the serum of this pig at 11 and 18 days of age. Notice the lymphoid follicles with germinal centers and numerous lymphocytes in the cortical areas. H&E stain; x 50 (A) and x 125 (B). 43 Figure 2 44 21, were given orally the live challenge organisms and survived 10 days or more postchallenge. Pig No. 21 was given saline at birth and survived until the termination of the experiment at 10 days of age. Numerous eosinOphils and neutrophils were observed in the medullary regions of many of the lymph nodes examined. Massive hemorrhage was observed in the mesenteric lymph node of pig No. 15. No differences in the appearance of the mesenteric or mandibular lymph nodes could be attributed to in utero injection of E. coli bacterin. However, the development of germinal centers in pigs No. 17, 28, 29 and 31 may have been due to the administration of viable E. coli organisms at birth. The thymuses of 33 pigs were evaluated. The thymus of all these pigs was well developed with distinct cortical and medullary regions. Hassall's corpuscles were observed in the medulla. 11:11.12 Morphologic changes were observed in the ileum of 11 challenged pigs. These changes consisted of sloughing and erosion of the epi- thelium of the villi. Extensive hemorrhage and sloughing of villous epithelium were observed in sections of the ileum of pig No. 15 (Figure 3). The same lesions were observed to a lesser extent in pig No. 13. Vacuolation of villous epithelial cells and numerous goblet cells were observed in all sections examined. However, in most instances vacuoles were more numerous in epithelial cells of the ileum taken from the control pigs that survived until the termi- nation of the experiment (pigs No. 21, 22, 32, and 34) (Figure 4). Lesions were not observed in sections of ileum of 4 of the 5 challenged 45 Figure 3. Photomicrograph of the ileum of pig No. 15, previously injected in utero with E. coli bacterin, challenged at birth with viable E. coli and dying at 2 days of age. Notice the extenSive hemorrhage with sloughing of the villous epithelial cells and the edema and congestion of blood vessels in the submucosa. H&E stain; x 50. 46 Figure 3 47 Figure 4A, 4B. Photomicrographs of the ileum of pig No. 32, previously injected in utero with E. coli bacterin, given saline orally at birth and euthanatized at 11 days of age. Notice extensive vacuolation of the villous epithelial cells and the long fingerlike projections of the villi. H&E stain; x 50 (A) and x 125 (B). 48 I ' ', was; 49 pigs that survived until 10 or 18 days of age. Slight erosions of villous epithelium were observed in sections taken from challenged pig No. 28. Also, the least number of vacuolated epithelial cells were observed in the ileum of this pig (Figure 5). Neutrophils were observed among the epithelial cells of the villi, around the crypts, and in the lamina prOpria of some of the sections evaluated. Lesions were not observed in sections of the ileum taken from control pigs. Liver, Spleen, and Kidney Vacuolation of the hepatic cells and numerous foci of extra- medullary hematopoiesis were observed in all sections examined. The splenic lymphoid nodules of all the experimental pigs were generally quite small and lacked germinal centers. Changes observed in sections of the kidney were cloudy swelling and hydropic degeneration of the cortical tubular epithelial cells. No differences were observed in the liver, spleen, or kidney of these pigs which could be attributed to the in utero injection of E. coli antigen or to viable E. coli organisms given to pigs at birth. Bacteriologic Findings No growth was obtained from the isolators at the beginning of any of the experiments. Bacteriologic cultures indicated growth of E. coli in the isolators housing challenged pigs. Also, gram- positive cocci were isolated from fecal samples collected from iso- lator 2 that housed Group 2 of Litter 6. No growth was obtained from isolators housing the control pigs. 50 Figure 5A, SB. Photomicrographs of the ileum of pig N0. 28, previously injected in utero with saline, challenged at birth with viable E. coli and euthanatized at 11 days of age. Notice the relatively small numbers of vacuolated epithelial cells when compared to the ileum of pig No. 32. H&E stain; x 50 (A) and x 125 (B). 51 Figure 5 52 Cecal contents from each pig were bacteriologically evaluated. Heavy to moderate growth of E. coli was isolated from 17 of the challenged pigs. Slight growth of E. coli was isolated from chal- lenged pigs No. 19, 29, 30 and 31. Bacteria were not isolated from challenged pigs No. 17 and 18, nor from any of the control pigs. Gram-positive cocci were isolated from the cecal contents of pigs No. 30 and 31 at 18 days of age. These organisms were present in the isolator housing these pigs, and probably entered the isolator between the 10th and 18th day of the experiment, since these organisms were not isolated from the cecal contents of pig No. 29 at 10 days of age. DISCUSSION The results of the direct bacterial agglutination and passive hemagglutination tests indicate that antibody against E. coli was not produced in those pigs previously injected in utero with the E. coli Also, the in utero injection of either the bacterin or saline did not appear to alter the susceptibility of the neonate to challenge at birth. The responses of these pigs to challenge suggest that local antibodies against E. coli were probably not produced by the intestines. Clinical Responses of Gnotobiotic Pigs to Challenge at Birth There was one primary factor which may have influenced clinical response of challenged pigs. The viable bacterial count of the chal— lenge dose administered to gnotobiotic pigs at birth in Litters 4, 5 and 6 ranged from 2 x 106 to 7.25 x 106 (Table 3). However, the pigs in Group 2 of Litters 5 and 6 probably received a much smaller number of viable organisms. Clinical signs of disease were observed in pigs No. 19 and 20, but only a few E. coli organisms were isolated from the cecal contents of these pigs. Escherichia coli organisms were not isolated from the cecal contents of pigs No. 17 and 18, nor from the cages and waste pans of the isolator at the termination of the experiment. Peracetic acid used to sterilize the ampules cone taining the viable organisms may have entered when the ampule was 53 54 opened, killing many of the bacteria present. The reason that only pigs No. 19 and 20 appeared to receive the challenge organisms is not known. One could postulate that these 2 pigs may have been the first pigs in the isolator challenged and that some of the organisms were still viable at that time. However, this explanation does not account for the inability to recover E. coli organisms from the waste pans or cages of the isolator housing these pigs. One would have thought that the remaining organisms in the isolator would have multiplied. The bacteriologic samples may have been taken from the cage and waste pans used by one of the pigs that had no E. coli organisms in the cecal contents. Also, peracetic acid may have entered the tube containing the bacteriologic sample while the tube was being paSsed out of the isolator. Escherichia coli organisms were not isolated from the ampule containing the viable cell suspension used to infect the pigs in Group 2 of Litter 6 at birth. After all the pigs in Group 2 of Litter 6 were reinfected at 5 days of age and 2 pigs (Nos. 30 and 31) again at 13 days of age, only a few E. coli organisms could be isolated from the cecal contents and from the cages and waste pans of the isolator. Peracetic acid may have entered the ampule killing many of the E. coli organisms. Also, the pigs may have been more resistant to infection at 13 days of age. Another possible explana- tion for the difficulties encountered in challenging these pigs is that the organism may have been losing its virulence. Another factor that may have influenced clinical responses of the challenged pigs was the number of pigs in Litter 4. There were 17 pigs in this litter. These pigs were very small and weak at birth. 55 How much of an effect this had on the clinical responses of these pigs to challenge is not known. However, there appeared to be no difference in the clinical responses of these pigs when compared to clinical responses of pigs that received the intended dose of challenge organism in Litters 5 and 6. The weakness of the pigs in Litter 4 at birth may have been a factor in the death of all the control pigs of this litter. The clinical signs of disease observed in challenged pigs were consistent with the observation reported by other investigators (Christie and Waxler, 1973; Drees and Waxler, 1970; Meyer and Simon, 1972; Miniats and Gyles, 1972). Organisms of the strain of E. coli used in this experiment seem to be quite virulent, as evidenced by the death of 14 of the challenged pigs within 48 hours after chal- lenge. Also, many of these pigs died peracutely before the appearance of diarrhea. The survival of pigs No. 17, 29, 30 and 31 is diffi- cult to interpret since the challenge doses given to these pigs were probably much too low. The results from challenge pig No. 28 indi- cate that this pig was able to survive the injurious effects of the organisms upon the intestines. Antibodies against E. coli were detected in this pig at 10 days of age. The antibodies present in the serum probably had little effect on the organisms present in the intestinal lumen of this pig (Porter, 1973). It is possible that local antibodies were produced by cells in the intestines of this pig which enabled the pig to survive infection (Porter and Allen, 1972; Tomasi, 1967; Wamukoya and Conner, 1976). However, the investigation of the development of a local immune response by this pig is beyond the scope of the present research. 56 Histopathologic and Gross Findings The poor development of the mesenteric and mandibular lymph nodes and lymphoid structures of the spleen of all pigs that died before 5 days of age appeared to be independent of the in utero injection of either bacterin or saline. This is a further indica- tion that the in utero injection of the bacterin did not cause an immune response in these pigs, since in the absence of antigenic stimuli the lymphoid tissues of fetal and germfree pigs mature slowly (Kruml et a1., 1970; Sterzl and Silverstein, 1967). It is possible that pigs given live challenge organisms orally at birth died before the E. coli antigen could stimulate maturation of the lymphoid tissue and subsequent production of antibodies. The maturation of the mesenteric and mandibular lymph nodes of pigs that survived until the termination of the experiment appeared to be dependent on the administration of challenge organisms, age and individual variations. The challenge organisms probably stimu- lated development of lymphoid tissues of pigs No. 17, 21, 29 and 31 but not pig No. 30. Age seemed to be a factor in the developnent of lymphoid tissues of pig No. 21, since this pig was given saline at birth (Waxler and Drees, 1973). The significance of the sloughing of villous epithelial cells of the ileum was difficult to interpret due to the autolytic changes observed (Cross and Kohler, 1969). The extensive vacuolation observed in the ileum of pigs No. 17, 21, 22, 32, and 34 was probably due to slow replacement of absorptive epithelium that occurs in the germfree ileum (Moon et a1., 1973). The least number of vacuolated epithelial cells were observed in the ileum of pig No. 28. The 57 presence of E. coli organisms probably caused the epithelial cells to turn over at a faster rate with the eventual loss of much of the vacuolated epithelial cells (Moon, 1972). The gross lesions observed in the challenged pigs were con- sistent with those of colibacillosis (Christie and Waxler, 1973; Drees and Waxler, 1970; Meyer and Simon, 1972; Waxler et a1., 1971). Immunologic Response of Fetal Pigs to Injection As previously stated, the results of the direct bacterial agglutination test and passive hemagglutination test and the clinical response of the neonate to challenge indicate that antibody against E. coli was not produced in those pigs previously injected in utero with the E. coli bacterin. The failure of these fetuses to produce antibody to E. coli bacterin could stem from several possibilities: (l) the immunologic unresponsiveness of the fetal pigs to E. coli; (2) the dose of the antigen; (3) the routes of injection; and (4) the sensitivity of the immunologic tests employed. Previous studies of fetal immunization have shown that immune competence to antigen develops sequentially rather than simultaneously (Jacoby et a1., 1969; Silverstein et a1., 1963). According to the clonal selection hypothesis, when antigen enters the body it binds to those lymphocytes which already have receptors for the antigen on their surface. The interaction of antigen with its receptor initiates the activation of these cells to produce antibody (Eisen, 1974b; Raff, 1973). It has been postulated that clonal lymphocytic precursors may develop or mature at different rates (Sterzl and Silverstein, 1967). If this is true, then perhaps the clonal lymphocytic 58 precursors for E. coli antigen had not developed in these fetal pigs when the antigen was injected at 98 days of gestation. However, if all the lymphocytic precursors developed and matured at the same rate, then perhaps enzymes needed to catabolize E. coli antigen before it is presented to the specific lymphocytes may be absent in fetal pigs. A second consideration is the dose of the antigen administered to fetal pigs in utero. The reaction of lymphocytes to antigen depends largely on the concentration and nature of the antigen. It is not known whether the number of organisms given to the fetuses by in utero injection into either the amniotic fluid or muscles of the thigh caused high dose tolerance. Germfree piglets have been found to have small numbers of immunocompetent cells (Sterzl and Silverstein, 1967). If the same can be said of the fetal pig, then a high concentration of antigen would be required to make contact with the few cells. However, E. coli is a complex antigen having repeating identical determinants and is poorly catabolized (Raff, 1973). This antigen chiefly stimulates B cells. More antigen is needed to stimulate B cells than T cells, but B cells may be paralyzed by very high concentrations of antigen. Bovine fetuses have been stimulated to produce antibody to E. coli antigen by.in utero injection of E. coli antigen into the amniotic fluid (Conner et a1., 1973) and muscles of the body (Gay, 1975). Recent studies by Wamukoya and Conner (1976), with the bovine fetus, have shown that injection offlE. coli antigen into the amniotic fluid probably offers the best protection of the neonate against colibacillosis. The swallowing of the amniotic fluid containing 59 antigen stimulated the intestine to produce antibodies locally. Thus, one could speculate that the in utero injection of antigen into either the amniotic fluid or muscles of the hind limb of porcine fetuses would have caused a response similar to that of bovine fetuses if the fetal pigs were immunocompetent to E. coli antigen at the time of injection. The final consideration is the immunologic test used. The bacterial agglutination test is not as sensitive in detecting anti- body as the passive hemagglutination test (Eisen, 1974a). This was shown by the marked differences in the O agglutinin titer of pigs No. 28 and 31 when determined by the 2 methods. It is possible that the bacterial agglutination test failed to detect antibody in the sera of pigs taken at birth. However, this possibility is not likely since antibodies were not detected in sera of pigs at this age when the passive hemagglutination test was used. This investi- gator is aware that the antibody binding property of the KB antigen of the E. coli organisms was not inactivated by heating at 100 C for 1 hour. Thus the KB antigen was present along with the 0 antigen in the supernate used in the passive hemagglutination test. It is possible that part of the O agglutinin titer of these 2 pigs was attributed to the presence of KB antigen. The neonatal pig is able to produce antibodies to E. coli as indicated by the detection of antibody in sera of pigs No. 28 and 31 at 10 days of age. Since these pigs were in constant contact with the E. coli organisms, it is difficult to postulate at what time between birth and 11 days of age these antibodies were produced. SUMMARY AND CONCLUSIONS The immunologic response of fetal pigs to.in utero injection of E. coli 0149:K9l(B);K88(L) and the response of neonates to challenge at birth were studied in 34 gnotobiotic pigs. A laparotomy was performed at 98 days of gestation. Escherichia coli bacterin (5 x 109 organisms) was injected in utero into either the amniotic fluid or the muscles of the hind limb of 14 fetuses. The remaining fetuses were either injected in utero with saline in the same manner or were uninjected. After delivery by hysterotomy procedure, viable E. coli organisms were given orally to 23 of the gnotobiotic pigs (challenged). The reamining 11 were given saline orally (controls). Eighteen of the 23 challenged pigs (78%) died following exposure. The in utero injection of either bacterin or saline did not appear to alter the susceptibility of the neonate at birth to viable E- COli of the same serotype. The clinical signs, gross and microscopic lesions were consistent with those associated with colibacillosis. Antibody to E. coli antigen was not detected using the bacterial agglutination and passive hemagglutination tests in the serum of the pigs at birth. However, antibody to the E. coli antigen was detected in the serum of 2 challenged pigs at 11 days of age. The results indicate that, with the present techniques, porcine neonates previously injected in utero with E. coli antigen are not protected when challenged with live organisms of the same strain, nor 6O 61 are detectable antibodies against E. coli produced by fetal pigs. However, antibodies to E. coli antigen are formed by neonatal pigs before 10 days of age. Thus, one can assume that pigs are immuno- competent to E. coli antigen sometime between 98 days of gestation and 11 days of age. BIBLIOGRAPHY BIBLIOGRAPHY Bertschinger, H. U., Moon, H. W., and Whipp, S. 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Hyg. I Abt. Orig. A222, (1972): 374-395. Williams, R. C., and Gibbons, R. J.: Inhibition of bacterial adher- ence by secretory immunoglobulin A: A mechanism of antigen disposal. Sci., 177, (1972): 697-699. Wilson, M. R.: Role of immunity in the control of neonatal coli- bacillosis in pigs. J. Am. Vet. Med. Assoc., 160, (1972): 585-590. Wilson, M. R., and Svendsen, J.: Immunity to Escherichia coli in pigs: Serum gamma globulin levels, indirect hemagglutinating antibody titer and bactericidal activity against Escherichia coli in pigs up to five weeks of age. Can. J. Comp. Med., 6, (1972): 38-43. VITA The author was born in Chicago, Illinois, on November 16, 1949. Her primary and secondary education was completed in Chicago. She fulfilled her preveterinary medicine requirements at Kennedy- King Junior College in Chicago, and entered Tuskegee Institute College of Veterinary Medicine in 1970. She received the degree of Doctor of Veterinary Medicine in 1974. In September of the same year the author entered Michigan State University to begin the Master of Science degree program in the Department of Pathology. The author is a member of the American Veterinary Medical Association and the Phi Zeta Honorary Society of Veterinary Medicine. 69