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Ala}! .Izvbtoulrr‘.» . l3}. #5; .4... niri 9111.."th (.10"! ~ B) t. (I A («1.53.5.1 \ . . . . \‘ 114.. .IHX ’Aur )‘t\s\l.‘|‘x\l Ania-thziltltiglll HLIMIUI. LIX; fill . 111‘; . A. . . . .Illki if . - 6%.“); . #24:. ilsybliioltofcl E I I HAT“... v .««.‘.s .«L.L.L4 \« lint! . .. lliiuxllz 4. . . (39321213111111. . 5:3)... \iit. x... n LIBRARY Michigan Sta re Uniwrsity , Wm; This is to certify that the thesis entitled IMMUNIZATION OF THE RAT AGAINST TAENIA TAEN | AEFORMI 5 presented by JOSEPH MAI NA AYUYA has been accepted towards fulfillment of the requirements for iI/I 8 degree in J I] C [(061017 0/, \/ Arc/\(Cm ' . 0‘ Major professor Dmfl aorta? U O~7639 IMMUNIZATION OF THE RAT AGAINST TAENIA TAENIAEFORMIS BY Joseph M. Ayuya A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1978 Joseph M. Ayuya the resistance stimulated by orally administered saline soluble antigens, whether the adjuvant was given together with the antigen or separately in the musculature. Both antigens were shown to confer a systemic resistance when given per 05. Low levels of homocytotropic antibody were detected in rats immunized intramuscularly with either antigen using B. pertussis or aluminum hydroxide as adjuvants. None was detectable in those rats immunized orally or intramuscularly using Freund's complete adjuvant with either antigen. Passive transfer of resistance was not observed in recipients of serum from rats that had been vaccinated orally or intramuscularly with either antigen employing B. pertussis as an adjuvant. Dedicated to my People and the Struggle ii ACKNOWLEDGEMENTS I wish to express my sincerest appreciation to Dr. J. F. Williams who welcomed, advised, encouraged and guided me throughout the course of my studies and preparation of this thesis. His optimism, insistence and persuasion were positively catalytic even when problems seemed insurmountable. W. Leid, S. B. The advice offered and time spent by Drs. R. Singh and R. J. Moon as members of my guidance committee is gratefully appreciated. For their companionship, helpful criticism and encouraging discussions, I am greatly indebted to Drs. Antony J. Musoke, Roger W. Cook, Bruce Hammerberg, Mr. John Picone, Mr. Stephen Hustead, Miss Anne Zajac, Miss Martha Calkins, Miss Marla Signs and other student members who have been and still are with Dr. Williams. The technical assistance rendered by Miss Alma Shearer and Miss Sharon Garland was indeed invaluable, and is sincerely acknowledged. I am also obliged to the University of Nairobi and U.S.A.I.D. for their joint scholarship offered to me, to the Department of Microbiology and Public Health and to the Veterinary College, whose accomodating welcome enabled me to pursue and complete my iii M.S. program in Michigan State University. To my friends and folks, including my parents, wife and children; - God bless you all for your moral support, prayers and inexhaustible patience. iv TABLE OF CONTENTS INTRODUCTION . LITERATURE REVIEW Life cycles and medical and veterinary importance of Taeniid cestodes Experimental Cysticercosis and immunity . General characteristics in animal infections Immune mechanisms in Jaenia taentaeformts infection . Protective humeral response . Other aspects of host— —parasite interactions Intestinal lymphoid tissue and the immune response. Gut- associated lymphoid tissue . . . Immunoglobulins in intestinal secretion Intestinal immune response to antigens and infection Viral Bacterial Helminth infections Immune exclusion by the gut and orally induced systemic tolerance . Adjuvants and helminth infections REFERENCES . ARTICLE — THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH ZWENIA YMEWJAEFORMLS. VII. IMMUNIZATION BY ORAL AND PARENTERAL ADMINISTRATION OF ANTIGENS . . . . Page 10 14 I4 16 18 19 19 21 24 26 30 45 Table LIST OF TABLES Immunization of rats against T. taeniaeformis with IVP and SSA using FCA Immunization of rats against T. taeniaefbrmis with IVP and SSA using B. pertussis as adjuvant. Immunization of rats against T. taeniaefbrmis with SSA and IVP. Effect of B. pertussis when given with antigen by the intramuscular or intraperitoneal route. Intramuscular immunization of rats against T. taeniaejbrmis with SSA and IVP. Effects of FCA, BP and Al(OI-I)3 as adjuvants Oral immunization of rats against T. taeniaeformis with SSA and IVP Intramuscular immunization of rats against T. taeniaejbrmis with SSA and IVP, and pass1ve transfer of serum to recipients Oral immunization of rats against T. taeniaeformis with SSA and IVP, and passive transfer of serum to recipients vi Page 53 54 S6 S7 64 66 LIST OF FIGURES Figure Page 1. Reaginic antibody titres in the serum of rats immunized with SSA and IVP using B. pertussis or Al(OH)3 as adjuvants . . . . . . . . . . . . . 59 vii cur imp the fai lik ime' gat' stu of des met ant pro ces dea 36C Introduction Cysticercoses affecting man and domesticated animals are currently increasing on a global scale and their socio—economic impact cannot be taken lightly. In the absence of suitable chemotherapeutic agents, and in the face of difficulties and failures with other control measures, more and more attention is being focussed on the exploration of immunological alternatives to control these infections. Laboratory models of cestodiasis like T. taeniaejbrmis in the rat are a vital part of these exper- imental immunological investigations. The hope that information gathered from such models would be relevant and contribute to studies on eventual immunization against the important cysticercoses of livestock is now being realized to some degree. The work described herein centers on vaccination of rats against T. taeniaefermis using two types of antigens derived from the metacestode. The effects of routes of administration of the antigens and the adjuvants employed are investigated. The literature review has therefore been organized to provide, in the first part, a background of information on taeniid cestodes of medical and veterinary importance. The second part deals with experimental cysticercoses and immunity. In this section, the general characteristics of acquired resistance det: The: cel reS] prm ant: the dis use IN) to cestode infections in animals are described, followed by a detailed account of the immune mechanisms in T. taeniaefbrmis. These include protective humoral responses, and other humoral and cellular aspectscnihost-parasite interactions. In the third part, the intestinal lymphoid tissue and its associated immune responses are examined. The tOpics dealt with are immunoglobulins produced in the intestinal secretions; the immune response to antigens and infections; the phenomenon of immune exclusion by the gut, and orally-induced systemic tolerance. Lastly, a discussion is presented of the nature of adjuvants and their use in protective immunization against helminth diseases. class of bot famil) where: the i] in th. excel relat on ma Ansar of th impor of ti Provi a bri Shed Ofe eggs LITERATURE REVIEW The phylum Platyhelminthes contains many members in the class Cestoda, order Cyclophyllidae, Family Taeniidae which are of both medical and veterinary importance. Adult tapeworms in this family parasitize the intestinal tract of their definitive hosts, whereas the metacestodes, or larval forms, occur in various tissues of the intermediate hosts. Man and domestic animals are commonly involved in these cycles, but several of the taeniids of rodents serve as excellent models for laboratory investigations on host—parasite relationships in cestodiasis. In recent reviews of the literature on natural and experimental taeniiasis, Leid (1973),Musoke (1975), Ansari (1975) and Hustead (1976) have provided comprehensive accounts of the biology, immunology, epidemiology and clinical and economic importance of the major taeniid parasitoses. An exhaustive account of these aspects will not be attempted here, but for the purpose of providing background to the specific areas addressed in this thesis a brief overview is apprOpriate. Life cycles and medical and veterinary importance of Taeniid cestodes Adult taeniid cestodes in the gut of the definitive host shed terminal proglottids, or segments, which contain large numbers of eggs, within each of which is an embryo. Both segments and eggs pass out with the faeces into the environment, and the interm eggs. or one mucosa or lym these which defini The la the gr 1 host t import as a 1 but tI leadh ovis . Cysti econo In su devel for T (dog 0f no but t brain intermediate host usually becomes infected by ingesting dispersed eggs. Through the action of gastrointestinal fluids the embryos, or oncospheres, are liberated and actively penetrate the intestinal mucosa. They reach their sites of predilection via the blood or lymph, and by migrating through intervening tissues. At these sites they differentiate into sessile cystic larval forms which must survive in the tissues until consumed by the carnivorous definitive host in order for the life cycle to be completed. The larval forms develop into adult ribbon—like tapeworms in the gut in about 6 weeks. It is the growth and development of larvae in intermediate host tissues which leads to the public health and veterinary importance of taeniids. For example, Teenia saginata occurs as a relatively benign adult tapeworm in the intestine of man, but the cystic larvae (cysticerci) occur in the muscles of cattle, leading to disfiguring lesions throughout the carcasses. Taenia ovis lives as an adult in the gut of domestic dogs, but its cysticerci invade the muscles of sheep leading to considerable economic and animal protein loss through carcass condemnation. In some instances the picture is complicated by the accidental development of larvae in the tissues of man, and such is the case for T. sOZium (man - pig cycle) and Eehinoeoeeus granulosus (dog - sheep cycle). There the infection of human tissues is of no significance in terms of continuation of the life cycle, but the growth of cystic masses in vital organs in humans (e.g. brain, eye, spinal cord) has major clinical consequences. On a global basis, the prevalence of the cysticercosis- hydatic the pa: have e: econom. clearl certai determ Taenii on the availa long-l in tre metace chemo1 infee metac infec acqui with artif a dis enhar dist] rene\ EXpej to h hydatidosis complex of diseases has shown an upward trend over the past few decades. Both developed and developing countries have experienced this trend, though the behavioral and socio- economic factors which have contributed to the increases are clearly different in these distinct environments. However, certain biological characteristics of the taeniids are important determinants of the success of this group under all circumstances. Taeniid eggs are known to survive for months to years, depending on the environment, and there are no practical chemical disinfectants available which will kill them. Adult worms themselves are long-lived and prolific, and although certain drugs are useful in treatment, none of these is entirely satisfactory. The metacestodes often survive in tissues for years and no satisfactory chemotherapeutic agents are presently at hand for treatment of infected animals or humans. Polyembryonic multiplication of metacestodes occurs in some species and this also enhances the infection potential of taeniids. Finally, although a marked acquired resistance develops to superinfection in hosts infected with metacestodes, there is, as yet, no practical approach to artificial immunization as a preventive measure. The realization that tapeworm infections are occurring with a disturbing frequency, and that their propagation may even be enhanced by the advent of modern hygienic sewage treatment and distribution measures (Pawlowski and Schultz,1972), has led to renewed interest in research on these parasites in recent years. Experimentally, some new chemical anthelmintics have been shown to have lethal effects on tapeworm larvae (Salazar et al,1972; Campbe 1974), tissue advanc cestod host-p promis in thi Experi Genera cysti< actiVI trans his Cl 1932, demon T. pi the r fecti Nemet is no Urquh (Swea and c and ( Campbell and Blair,1974; Heath and Chevis,1974; Thienpont et a1, 1974), but a great deal of work needs to be done on toxicology, tissue residues and effective dose levels. Likewise, important advances have been made in our understanding of immunology of cestodes, but much remains to be determined. The exploration of host-parasite relationships in laboratory animals is one of the promising avenues which has been pursued in the work reported in this thesis. Experimental Cysticercosis and Immunity General characteristics in animal infections It has been established conclusively that resistance to cysticercosis may occur in the intermediate host as a result of active infection or artificial immunization, and that passive transfer can be achieved with serum or colostrum. Miller and his coworkers (Miller, 1931, 1932, 1935; Miller and Gardiner, 1932, 1934; Miller and Kerr, 1932; and Kerr, 1935) convincingly demonstrated these characteristics for T. taeniaefermis and TC pisijbrmis. Campbell (1936, 1938a and b) confirmed most of the results of Miller's earlier work, and resistance to superin- fection against T. pisifbrmis was confirmed by Solomon (1934), Nemeth (1970), and Heath (1973b and c). Acquired resistance is now known to occur to T. saginata (Penfold et a1, 1936; Urquhart, 1958; Soulsby, 1963; Froyd, 1964), T. hydatigena (Sweatman, 1957), T. Ovis (Gemmell, 1962a) and T. soZium (Herbert and Oberg, 1974). Passive transfer of resistance with serum and colostrum has been achieved against T. hydatigena and T. evis (Blunde Arundel In other w workers of viat product General antigei viable sheep and E. of ces (1955) of tae 1970; a new Antige shown chalh showe in me CaVit reSis Heath with OHCOE (Blundell—Hassell et a1, 1968; Gemmell et a1, 1969; Rickard and Arundel, 1974). In early work on artificial immunization, Miller and most other workers used crudely ground-up worm homogenates. Subsequent workers have focussed their attention on parenteral inoculation of viable eggs and/or activated embryos, and excretory-secretory products released during the in vitre culture of metacestodes. Generally water-in—oil adjuvants have been used as vehicles for antigens in these studies. Gemmell (1964, 1965, 1966), using viable eggs and activated oncospheres, successfully immunized sheep against challenge infection with T. hydatigena, T. Ovis and E. granulesus. Following studies on the hatching and activation of cestode ova by Silverman (1954),and Silverman and Maneely (1955), great improvements have been made on the in vitro culture of taeniid metacestodes. These improvements (Heath and Smyth, 1970; Heath and Eldson-Dew, 1971; Heath, 1971, 1973a) opened a new avenue to artificial immunization against cysticercosis. Antigens produced in Uitre by oncospheres of T. ovis were first shown to be effective when used in vaccinating lambs against challenge infection by Rickard and Bell (1971a). They also showed that larvae of T. taeniaefbrmis and T. evis contained in membrane diffusion chambers and implanted into peritoneal cavities of rats and lambs, respectively, conferred significant resistance against challenge infection (Rickard and Bell, 1971b). Heath (1976) succeeded in immunizing rabbits against T. pisifermis with in vitre products. Vaccination of calves with activated oncospheres of T. saginata also produced a highly significant resist; Wikerh: of Rid Freund A carrie emu provid ”. tae Chroma T. pit fracti on Se} charm and e metac activ molec purit less re5p( indi( Will resistance against challenge infection (Wikerhauser et a1, 1971; Wikerhauser et a1, 1974). Their success was followed by that of Rickard and Adolph (1976) in which in vitro products plus Freund's adjuvant were used to immunize calves against T. saginata. Attempts to characterize protective antigens have not been carried out to any great extent to date. Campbell (1936, 1939) found that proteinaceous fractions of ground-up worm material provided protective immunity in vaccination trials against T. taeniaeformis, whereas polysaccharide fractions were ineffective. Chromatographic fractionation of crude in Uitre products of T. pisiformis (Rickard and Katiyar, 1976) failed to yield any fraction that produced protective immunity. Using gel—filtration on Sephadex 6-200, Kwa and Liew (1977) claim to have purified and characterized protective antigens from crude worm homogenates and excretory-secretory products derived from T. taeniaefbrmis metacestodes maintained in uitro. They found that the protective activity in both types of antigens resided in a sample with a molecular weight of approximately 140,000, but the criteria of purity and the assessment of immunity in their experiments are less than satisfactory. Immune mechanisms in Taenia taeniaefermis inIEction Protective humeral response Advances made recently in research in the immunological response of the rat and mouse to T. taeniaefermis serve as important indicators for investigations on other cysticercoses. Leid and Williams (1974a) have confirmed the earlier observations of Miller and Ga] that s< taeniae infect: that tl Subsem izatio were a parasi passiv was ma confer rats. immmc rats. not or remair evidei may a( (Musol (1975. IgGl aCtiv aCtiv mouse Provo and Gardiner (1932, 1934) and Campbell (1938a and b) by showing that serum taken 14, 21 and 28 days after infection with T. taeniaefOPmis passively protected recipient rats against challenge infection. They fractionated 28 day - immune serum and found that the protective capacity resided in the IgGZa immunoglobulins. Subsequent work (Leid and Williams, 1974b) led to the character- ization of antibodies in the IgE immunoglobulin class which were also shown to be stimulated during infection with this parasite. These reaginic antibodies appeared at a time when passive transfer of resistance with serum from infected rats was maximal. It was shown that fractions enriched for reagin conferred a highly significant degree of resistance on recipient rats. However, these reagin—enriched fractions contained other immunoglobulins which by themselves, passively protected recipient rats. The exact role that IgE may play in protective immunity not only against cysticercosis but in other helminthic infections remains a matter of controversy (Murray, 1972). Some recent evidence suggests that in cysticercosis IgE-mediated reactions may accelerate humoral antibody attack on challenge organisms (Musoke et al, 1978). In mice infected with T. taeniaefermis, Musoke and Williams (1975a) showed that fractions of immune serum containing both IgG1 and IgGZ, but not those containing only IgGZ, had protective activity. Hence it appeared that, unlike in the rat where this activity localized in IgGZa, it probably resided in IgGl in the mouse. Intraperitoneally implanted cysticerci in the rat also provoked a high degree of resistance to oral challenge with eggs absok< serum < lg61 a in rat those serum F that a in the It was antibo attain ibilit Colost a sigr Ghsol when 1 coloy These may h andn other immm with OVera 10 (Musoke and Williams, 1976), but the protective activity in immune serum obtained from such rats was found to reside mainly in the IgGl and IgM antibody classes. Cysticerci implanted intraperitoneally in rats with concurrent hepatic infections were killed whereas those implanted in uninfected animals survived even when immune serum was given simultaneously. Further studies by Musoke and Williams (1975b) demonstrated that as infection in the rats progressed, the protective activity in their serum was extended to other immunoglobulin fractions. It was also found that hepatic cysticerci were susceptible to antibody up to five days post infection, but thereafter parasites attained insusceptibility both in vivo and in Uitro. The suscept- ibility prior to the fifth day was shown to be complement—dependent. Colostral antibodies from infected rats were also shown to play a significant role in conferring passive resistance to young rats (Musoke et al, 1975). Immune colostral IgA conferred resistance when fed or when injected into the intestinal lumen, while immune colostral lgGl and lg62 were effective when given parenterally. These observations as a whole indicate that the protective mechanism may involve distinct antibody types, depending upon the duration and nature of the exposure of the rat to parasite antigens. Other aspects of'hosi-parasite interactions It has become increasingly clear that many components of the immune mechanism may be involved in the response to infections with T. taeniaefbrmis although the role of these reactions in the overall host-parasite relationship remains to be clarified. Ansari primar) starth the fi eosino week p cells with e respon serum after mmbm T. tae IgE-m: J larva the a integ taken antig the E was 1 into antfl the in ti COnt 11 Ansari and Williams (1976) found that during the course of a primary infection in the rat, there is a peripheral eosinophilia starting in the second week post infection which peaks before the fifth week and thereafter declines. There was a marked eosinophilic infiltration in the liver tissue during the 2-5 week post infection period with a wide zone of eosinophilic cells surrounding the developing larvae. When challenged orally with eggs, infected rats also showed a brisk secondary eosinophilic response within a week. Rats given intravenous doses of immune serum similarly had a secondary eosinophilia within a few hours after an oral challenge with eggs (Ansari et al, 1976). Mast cell numbers also increase significantly in the tissues of rats with T. taeniaefbrmis and their increased numbers may be important in IgE-mediated reactions to challenge (Cook and Williams, 1978). Hustead and Williams (1977a) showed that T. taeniaefvrmis larvae are capable of absorbing a variety of proteins, and that the absorbed macromolecules retain their structural and functional integrity following transport. Host serum immunoglobulins were taken up both in vitro and in vivo, and were shown to retain both antigen binding capacity and biologic functions associated with the Fc regions. By employing radiolabelled macromolecules it was further shown that the rate of transportation of these molecules into the metacestode was increased by the presence of both antibodies and complement (Hustead and Williams, 1977b). In vitro the larvae were shown to deplete functional complement levels in the surrounding medium, thus restoring the normal permeability control. Fu showed complen produc1 both tl anaphy activi The an other Kassis these larvae sulfat protei and ht immun shown larva get t Trans been 1972] gati< eVid BOI‘U Stru 12 Further investigations by Hammerberg and Williams (1978a) showed clearly that parasite larval products interacted with complement in vitra and in viva. Cyst fluids and in vitra products from T. taeniaefarmis larvae consumed complement by both the alternate and classical pathways, cleaved C3 and generated anaphylatoxin in vitra, depressed circulating hemolytic complement activity and caused alterations in vascular permeability in viva. The anticomplementary substances were shown to be present in other cestodes too (Hammerberg et al, 1976, 1977; Herd, 1976; Kassis and Tanner, 1976). Physico-chemical characterization of these complement-interacting factors from T. taeniaafarmis larvae (Hammerberg and Williams, 1978), suggested that they are sulfated polysaccharides or proteoglycans composed of carbohydrate, protein, sulphate and hexosamine, free of sialic and uronic acids and heterogeneous in net negative charge but resistant to proteolysis. The occurrence of intact and biologically functional host immunoglobulins, including those of the IgG class that have been shown to be protective, in the cyst fluids of T. taeniaefarmis larvae leaves several questions unanswered. How do these molecules get there and what is their function once in the cystic fluids? Transportation of macromolecules across the cestode membrane has been suggested to be by simple diffusion (Varela-Diaz and Coltorti, 1972) possibly through tegumental pores. Ultrastructural investi- gations on a variety of taeniid metacestodes have not revealed evidence of pores that might serve this function (Morseth, 1966; Bortoletti and Ferretti, 1971, 1973; Lascano et al, 1975). Ultra- structural and functional analogies have been drawn between the tegume mammal by Bra which epithe of mac and re (Picor serve sourcs benef direc the s aspec while that antib furth the n antit aroui consl Therr afte: Pred anap 13 tegument of larval cestodes and intestinal epithelium of neonatal mammals (Beguin, 1966; Slais, 1966). Pinocytosis has been postulated by Brambell (1966) and Rodewald (1973) to be the process by which proteins are specifically taken up by the neonate intestinal epithelial cells. This process may account for the translocation of macromolecules across the tegument of taeniid metacestodes, and recent evidence suggests that this is the case in T. taeniaefarmis (Picone, 1977). Whether host macromolecules in the cyst fluids serve in osmoregularity control (Dixon et al, 1973), provide a source of nutrients for the parasite or are there for some mutual benefit to host and parasite is not clear. There is presently no direct evidence to support any prOposal. From the foregoing it is possible to construct a picture of the sequence of events which follow infection in the rat. Some aspects of this picture have been established experimentally while others are susceptible to future investigation. It appears that the parasites rapidly stimulate a complement-dependent humoral antibody protective mechanism which protects the host against further challenge. During and after oncospheral reorganization, the metacestode evolves a mechanism of elaborating and releasing anticomplement factors. In viva their effects are exerted locally around the cyst by a non—immunological triggering of complement consumption through both the classical and alternate pathways. There is no marked inflammatory process around the metacestodes after the first several weeks of infection when eosinophils predominate. It is possible that the host enzymes deactivate the anaphylatoxins locally leading to reduced inflammatory activity. There taenia releas direct inflam net re reject rded resp01 at thi cells enhan of in may 0 trans has b event which theSe 14 There is histochemical evidence of release of proteases by T. taeniaefarmis in situ (Lewert and Lee, 1957). Other factors released by the parasites may serve to affect, directly or in— directly, the differentiation, infiltration, and function of inflammatory cells such as eosinophils and mast cells. The net result is prolonged survival of the larvae, without local rejection, in hosts which can mount an immediate and highly effective rejection of new challenge organisms. The character of the antigens which provoke this protective response remains to be seen. The response itself probably occurs at the level of the gut at least in part. IgE—sensitized mast cells living in the gut wall may accelerate the process, or enhance access of complement and protective antibody to the site of invasion. It seems possible that local gut-related immune reactions may occur which are not detectable in experiments on parenteral transfer of systemic antibody or cells. This aspect of cysticercosis has been little studied, but in recent years the nature of immune events in the intestine has been characterized in many ways which are relevant to this discussion of taeniiasis, and some of these are reviewed below. Intestinal lymphoid tissue and the immune response Gut-associated lymphaid tissue The gastro-intestinal epithelium is exposed to a wide variety of potential antigens. These include dietary and food contaminant antigens, microorganisms and drugs. It is not surprising therefore that p other mammal of org made U P are th Ultras contai 1974). that 1 are 01 is un] strat a r01 cells IgA, lgD—c 1972) tissu Struc deper the ( TOWa] Whid 15 that portions of the gut wall are well endowed with lymphoid and other elements capable of immunological response. In most of the mammalian species studied, these elements have a similar pattern of organization in the intestinal wall, the major portion being made up by the so-called “Gut Associated Lymphoid Tissue” (GALT). From 10 to 15 percent of cells within the intestinal epithelium are thelio—lymphocytes, a term introduced by Fichtelius (1968). Ultrastructurally these cells resemble lymphocytes, but they contain granular inclusions reminiscent of mast cells (Bienenstock, 1974). Their origin is unclear. Bienenstock (1974) suggests that they are derived from mast cells, and Guy-Grand, et al (1974) are of the opinion that they are of T cell origin. Their function is unknown, although their resemblance to lymphocytes and their strategic position in the epithelium suggest that they may have a role to play in the local immune response. In the lamina propria below the epithelium are numerous cells including lymphocytes, plasma cells, macrophages, eosinophils and mast cells. About 80 percent of the plasma cells contain IgA, 15 percent IgM and 3 percent IgG. IgE, and in some species, IgD—containing plasma cells are present in lesser numbers (Jones, 1972). Within the lamina propria are found organized lymphoid tissues, the ”Peyer's patches”. They are composed of three structural elements; the domes, the follicles and the thymus— dependent areas (Waksman, 1973). The follicles are found below the domes and the thymus—dependent areas in between the follicles. Towards the lumen the domes are covered by a specialized epithelium which has pinocytotic capabilities (Bockman and Cooper, 1973). Current immune pinocyt directl prolife These c hence i 'home' secret: lflmmgwg S are de perhap IgG, 1 can be by the which human (Roiti dimerf heldl one fl a Spe in th PTOdu 16 Currently it is suspected that Peyer's patches function in the immune response after antigenic material enters through the pinocytotic cells overlying the dome. The antigens then interact directly or indirectly with B lymphocytes, inducing them to proliferate and triggering them into terminal differentiation. These cells make their way into the draining lymphatics and hence into the blood stream, from whence they preferentially 'home' to the intestinal lamina propria and mature into antibody- secreting plasma cells (Bienenstock, 1974; Cooper et al, 1974). Immunagiaaalina in intestinal secretions Some of the immunoglobulins produced by these plasma cells are detectable in secretions, though others cannot be found, perhaps because of their susceptibility to proteolytic degradation. IgG, IgD, and IgE fall into this category. IgM on the other hand, can be present in active form in secretions and may be protected by the addition of secretory polypeptide chains, similar to IgA, which is the most abundant immunoglobulin in the lumen. In the human lgA forms about 13 percent of total serum immunoglobulins (Roitt, 1974). It occurs in a variety of forms — monomeric, dimeric or higher polymers. The polymers including the dimer are held together by a J (joining) chain. The dimer which is the main one found in the intestinal lumen is thought to be secreted by a special mechanism (Heremans, 1974). The monomers are assembled in the lamina propria plasma cells in which the J chain, also produced in these cells, is used to join them into a dimer. The dimer then passes into the epithelial cells by an as yet undetei dimer v the se< 1974;] releasr It has resist When 1 secret lgA (B l of imm also 1 IgG an Whethe as to envirc T Precir generz destrr cells may P react intes (Fret and H 17 undetermined means (Threadgold, 1967; Allen et al, 1973). The dimer with J chain is then combined with yet another molecule, the secretory component, formed in the epithelial cells (Brandtzaeg, 1974; Roger and Lamm, 1974). This secretory IgA (S—IgA) is then released into the intestinal lumen by an unknown mechanism. It has been suggested that the secretory component confers resistance to proteolysis on S-lgA (Tomasi and Grey, 1972). When IgA is deficient, lgM producing cells may increase and secretory IgM in the lumen may compensate for the lack of secretory IgA (Brandtzaeg et al, 1968). Immunization or infection leads to an increased synthesis of immunoglobulins at the local level. Intestinal stimulation also leads to the development of circulating antibodies (mainly IgG and IgM), and these may then enter the intestinal lumen. Whether their origin is local or circulatory, the question remains as to the protective function of antibodies in the intestinal environment. Through properties such as neutralization, agglutination, precipitation or opsonization, (Nossal and Ada, 1971) antibodies generally serve to identify or recognize antigens for subsequent destruction by effector systems which may involve phagocytic cells, complement cascade, etc. It has been suggested that IgA may play a rather different protective role in the gut lumen by reacting with bacteria and preventing their adherence onto the intestinal mucosa, and hence interfering with successful colonization (Freter, 1972; Fubara and Freter, 1973; Gibbons, 1974). Walker and Hong (1973) conducted studies with soluble antigens and found that i homolc that t withir hypoti the 11 degra< l secrm and L: and e pathw: lmen in pr and h and m but a of by seem Intes \ ferri and e resul ce11s 18 that in orally immunized animals, subsequent administration of homologous antigen resulted in reduced uptake. It was later shown that this reduction was due to enhanced degradation of antigen within the intestinal lumen (Walker et al, 1974). An additional hypothesis, then, is that IgA combines with soluble antigen in the lumen and prevents its uptake, leading to enhanced enzymatic degradation. Although complement components may occur in the intestinal secretions, complement fixation does not occur in the lumen (Mestecky and Lawton, 1974). IgA antibodies do not ordinarily fix complement, and effector mechanisms involving either the classical or alternate pathways are not considered likely to be important in the intestinal lumen. It has, however, been shown that secretory IgA is bacteriocidal in presence of complement and lysozyme (Adinolfi et al, 1966; Hill and Porter, 1974). Secretory IgA also agglutinates efficiently and may opsonize antigen for phagocytosis (Bienenstock, 1974), but agglutinated or neutralized antigen would be quickly disposed of by peristalsis or proteolysis, so that phagocytosis does not seem to be an essential part in the intestinal immune system. Intestinal immune respanses ta antigens and infectians Crabbe, et a1 (1969) showed that immunization of mice with ferritin orally resulted in IgA producing cells in the intestine and exclusively IgA antibodies in the serum. Parenteral immunization resulted in IgM and IgG antibodies in the serum and IgA producing cells in the intestine. Rothberg, et a1 (1973) suggestedtflufi oral immunization of rabbits with bovine serum ablumin (BSA) was follow of sys lympho sugges lympho of abs immune viral, Viral ( after in thi entiri polio mucos prodt Gast newb subs Bac 19 followed by local antibody production and a gradual sensitization of systemic lymphoid tissue by the dissemination of sensitized lymphocytes. In later studies (Rothberg et al, 1974) they further suggested that, as well as antigen-reactive cells, systemic lymphoid tissue could be sensitized by immunogenic concentrations of absorbed antigens. There is now plenty of evidence that local immune mechanisms operate in the intestine in response to some viral, bacterial and helminth infections. Viral Studies on poliovirus (Ogra et a1, 1974) showed that only after oral immunization did significant antibody titers occur in the nasal and intestinal secretions. This antibody was entirely IgA and was considered to protect by preventing the poliovirus from crossing the nasopharyngeal and gastrointestinal mucosae into the circulation. Parenteral immunization, in contrast, produced predominantly IgG with some IgM and IgA, but in the serum. Recent experiments with an attenuated strain of Transmissible Gastroenteritis Virus have indicated thatit is possible to immunize newborn pigs orally with the strain and confer resistance to a subsequent challenge with a virulent strain (Puruuchi et a1, 1976). Bacterial Extensive work has been carried out on the immune response to cholera in both laboratory animals and humans. Freter (1956) was able to confer protection against fatal enteric cholera in guinea pigs by active oral immunization using heat killed Vibria chalerae. Passive immunization was achieved by oral dosing with antibody from vaccinated donors. In human volunteers (Freter and Gan induced tract . systemi V. ch01 given 1 1972a z (coproz also 0~ Fubara mmmi (1974) in pas infect antib: of th icall clarf (197 afte fra< 20 and Gangaraso, 1963) a heat killed oral V. chalerae vaccine induced formation of locally produced coproantibody in the intestinal tract. Oral immunization resulted in productioncfi?both local and systemic antibodies in humans given an apathogenic strain of V. chalerae (Sanyal and Mukerjee, 1969), and in humans and rabbits given lysates of L-forms of V. chalerae (Agarwal and Ganguly, 1972a and b). The predominant locally produced antibodies (coproantibodies) were of the IgA class, although IgM and IgG also occurred (Fubara and Freter, 1972a and b; Shimamura, 1972). Fubara and Freter (1972a, 1973) have demonstrated protective immunity to cholera with secretory IgA, and Pierce and Reynolds (1974) showed that humoral IgG antitoxin conferred protection in passive immunization. To be effective against the cholera infection protective antibodies must be antitoxic as well as antibacterial to prevent the toxin effect and reduce colonization of the gut by the vibrios. Exactly what roles locally and system— ically produced antibodies play after oral exposure remains to be clarified (Tomas and Grey, 1972). Recently Agarwal and Sundararaj (1977) have provided evidence that cell-mediated immunity occurs after oral immunization of rabbits with Ribonucleic Acid—Protein fractions of V. chaZerae. Escherichia caZi antigens as dietary additives for oral immunization of pigs (Porter et al, 1973) have been shown to improve the performance of piglets. Further detailed studies by Porter, et a1 (1974) showed that the antibody activity in the locally produced secretions in the intestine of piglets was predominantly associated with IgA, although IgM and IgG were also detect secret most 1 single did no lack c admini nmmm HeZmir gastrr manif claim capac Only expul exten BXpul Worms and 1 Upon Dinet Rothl agai Shee 21 detectable. Lysozyme, but not complement, was present in the secretion leading to the suggestion that bacteriostasis was the most likely antibacterial mechanism attributable to the IgA. A single dose of antigen followed by a second one 3 to 4 weeks later did not result in increased antibody secretion, suggesting a lack of memory in the secretory immune system. Repeated antigen administration for up to 3 weeks was necessary to induce maximal numbers of antibody producing cells in the lamina propria. HeZminth Infectians It has been convincingly demonstrated that many parasitic gastrointestinal helminths evoke immune responses which are manifested in the gut of their hosts. These responses have been claimed to result in effects such as reducing the reproductive capacity of worms, and/or stunting and/or expulsion of parasites. Only a brief mention of some examples will be made here. Among the nematodes, the immune mechanism leading to the expulsion of Nippastrangylus arasiZiensis has been the most extensively examined. It is now generally accepted that this expulsion involves three components of the immune response. The worms are first damaged by antibodies (Jones and Ogilvie, 1971) and they become susceptible to an expulsive step which is dependent upon both lymphoid and myeloid factors (Keller and Keist, 1972; Dineen et al, 1973; Dineen and Kelly, 1973; Kelly et a1, 1973). Rothwell and Merritt (1974) have demonstrated IgA and IgM antibodies against Trichastrangylus caZubrifarmis in serum of infected sheep, and thymus—dependent lymphocytes were shown to play a role in the resistance of guinea pigs to this nematode (Dineen and Adams, T. cot and So shown et al, in the wherea et al, serum T. Spl IgM, 1 but 01 et al in th cesti dog a Herd done antit and l he 31 IgA. tegm argu, 22 Adams, 1971). Both humoral and cell-mediated responses against T. calubrifarmis have since been conclusively demonstrated (Dobson and Soulsby, 1974) and histamine and 5-hydroxytryptamine have been shown to participate in its expulsion in the guinea pig (Rothwell et a1, 1974). IgA and IgG antibodies have been demonstrated in the gut extracts of rabbits infected with TrichineZZa spiralis, whereas the serum contained IgM antibodies in addition (Crandall et a1, 1967; Crandall and Crandall, 1972). The gut IgG and the serum IgG and IgM antibodies were shown to be specific for T. spiralis. In mice infected with Hemataspiraides dubius, IgM, IgA and antibodies of the IgG class occurred in the serum but only IgG antibodies were detected in the intestine (Crandall et al, 1974). Among the cestodes, immune mechanisms have been implicated in the resistance of birds to secondary infections with HaiZZietina cesticittus (Gray, 1973) and in the successful immunization of the dog against H. granulasus (Turner et a1, 1933; Gemmell, 1962b; Herd and Chappel, 1975). Most studies on adult cestodes have been done on Hymenalepis species, particularly H. nana, H. diminuta and H. micrastama. In his recent studies Befus (1975) has demonstrated antibody responses to experimental infections with H. diminuta and H. micrastama in the mouse. By immunofluorescence techniques, he showed that the teguments of these worms are covered with IgA, IgG, IgG2 and IgM. C3 was also found to be fixed to the tegument of H. diminuta. Correlating a number of factors he argues that these immunoglobulins are in fact, specific antibodies. These factors include the time of appearance of the immunoglobulins and the on the ' bodies the wor represe through Ti larvae nemator epithei and h0( and b; of the most 1 penetr 3 to 4 1969a) level, immunr wall, in ral (lass been Baner T- ta infec tiSSu and the intensity of the infection, the rate of their accumulation on the worms and the surface area they cover. Even if the anti— bodies were specific their role in the immune events harmful to the worms remains to be clarified. The possibility that they represent nutrient material in the process of being taken in through the tegument cannot be discounted. There is no direct evidence of immune responses to cestode larvae occurring at the gut level. Unlike the lumen dwelling nematodes and cestodes these larvae rapidly penetrate the intestinal epithelium by the combined action of their penetrating glands and hooks (Silverman and Maneely, 1955; Banerjee and Singh, 1969a and b; Heath, 1971). The time taken to complete the penetration of the intestinal wall and to reach the site of predilection will most likely vary with each taeniid larva. For T. taeniaefarmis penetration of the villi is complete within 15 minutes, and in 3 to 4 hours the parasites reach the liver (Banerjee and Singh, 1969a). If any mechanism is to affect these larvae at the gut level, be it specific or non-specific, immunological or non- immunological, it has to operate in the gut lumen or in the gut wall. Gut level immunity to taeniid larvae was proposed to occur in rabbits immunized against T. pisifarmis by Leonard and Leonard (1933) but substantive evidence in support of this has not yet been developed. A gut phase of resistance was considered by Banerjee and Singh (1969c) to be present in rats infected with T. taeniaefarmis. The oncospheres given subsequent to primary infection showed very reduced migration through the intestinal tissues. mere. h testin respon Parrot respon in rat they w lymphc antib< feces fed r to an and m with chall react clea1 feedf show< with and‘ indu 1974 of t 24 Immune exclusian_bi the gut and Orally induced systemic taZerance Initial presentation of some antigens through the gastroin— testinal tract has been observed to alter significantly the response of the organism to a subsequent exposure. Thomas and Parrot (1974) showed that the ability to mount a humoral antibody response to bovine serum albumin (BSA) was significantly lowered in rats which had been fed BSA previously. In these experiments, they were unable to demonstrate anti-BSA producing cells in any lymphoid tissue, including that of the lamina propria, and no antibody activity could be detected in the intestinal juice or feces. Recently, David (1977) showed that rats which had been fed ragweed or horse serum developed specific unresponsiveness to anaphylactic sensitization. They did not form IgE antibodies and maintained a normotensive response to intravenous challenge with antigen. Prefeeding Ovalbulmin followed by homologous intragastric challenge in mice resulted in a lowered amount of ovalbumin reaching the circulation, and there was no accompanying increased clearance (Swarbrick et a1, 1977). It was concluded that antigen feeding induces immune exclusion by the gut. These mice also showed a reduced antibody response when subsequently challenged with ovalbumin parenterally. The phenomena of immune exclusion and orally induced systemic tolerance seem to be simultaneously induced. Both mechanisms are antigen specific (Thomas and Parrott, 1974; David, 1977). Administration of small but frequent doses of the antigen leads to a more complete state of unresponsiveness than f in his in ind 2 mont an app preven IgA ar them i of sys and P2 they : toler with of th at mo and t the f by fe Where Showc Proti Perk anti; Elna 25 than feeding of large amounts at longer intervals. David (1977), in his experiments, suggests that age is also an important factor in induction of tolerance. Termination of antigen feeding for 2 months restored the ability to become anaphylactically sensitized. To explain immune exclusion Walker, et a1 (1974) have proposed an appealing hypothesis where IgA antibodies combine with antigens preventing their uptake. It has further been suggested that such IgA antibodies blanket normal flora and food antigens protecting them from other immune responses. On the other hand the induction of systemic tolerance has been difficult to explain. Thomas and Parrott (1974) have suggested two explanations: firstly they suggest that antigens may be formed of both immunogens and tolerogens which are separated from each other by the gut, with the immunogens producing a local immune response in the wall of the gut andthe tolerogens being absorbed to produce tolerance at more distant sites. Secondly, they feel that both immunogens and tolerogens may be absorbed, but that the liver sequesters the former, allowing the latter to exert their effect. Pathophysiological disturbances have also been observed by feeding soya protein in calves and piglets (Barrett et a1, 1977). Whereas there was no evidence of tolerance, intestinal biopsies showed that there were morphological disturbances following soya protein ingestion. In orally sensitized animals inhibition of peristaltic flow occurred in Thiry—Vella loops into which soya antigen was perfused. It is clear from the above discussion that the response of an animal locally and systemically to antigens presented via the gut d6 The s; (Bern: all a some 1 feedh aller immun the g antig facto Adjuv to ar work mate] and< t0 h Spec most adeq non- diff and hard the 26 gut depends on many factors which need further investigation. The species of the animal and its age, the nature of the antigen (Bernstein and Ovary, 1968) and the period of exposure to antigen all seem to be important. Further research will undoubtedly reveal some more factors. David (1977) suggested that the potential of feeding antigen as a prophylactic measure in the management of some allergic diseases should not be ignored. The potential for immunization against infectious micro— and macro-organisms via the gastrointestinal route is even greater. It appears that each antigen/animal relationship merits special considerations of factors that will favor a desired response. Adjuvants and helminth infections Adjuvants are substances which potentiate the immune response to antigens, and they have been used in much of the experimental work on artificial immunization of animals against helminths with materials extracted from or secreted by parasites. The selection and route of administration of adjuvants for these studies appear to have been arrived at empirically, rather than being based on specific characteristics which are known and desirable. For the most part, Preund’s complete adjuvant has been used, often without adequate controls for the effects of water—in—oil emulsions on non—specific host resistance mechanisms. Consequently, it is difficult to interpret the results of such vaccination trials, and in the event that significant protection was produced it is hard to determine what the effect can be attributed to, or what the contribution of the adjuvant was. activ miner World of ad adjuv trans immur molec speci as t} are hydr comp pert form and the both lynm rest Heal mat] 19h Wor 27 There are many substances which are known to have adjuvant activity, including microbial products, nucleic acids, certain minerals, emulsions and even vitamins (Jolles and Paraf, 1973; World Health Organization Technical Report, 1975). Two levels of adjuvant activity may be recognized. At the antigen level the adjuvant may induce conformational changes at specific sites, transform a non or poorly immunogenic molecule into a strongly immunogenic one and lower the catabolic rate of the antigenic molecule. At the host level the activity is directed at both Specific immunocompetent cells and non-specific mechanisms, such as those regulating protein synthesis and cell multiplication. Among the most widely used adjuvants for experimental work are aluminum compounds (including aluminum phosphate, aluminum hydroxide and aluminum oxide), Freund's incomplete and Freund's complete adjuvant which are water—in-oil emulsions, and Bordetella pertussis. The aluminum compounds and the Freund's adjuvants form a slow-releasing repository of antigen at the injection site. Antibody-producing plasmacytes form in the draining lymph nodes and around the local granuloma as it develops. In addition to the adjuvants acting as vehicles for antigens, macrophages ingest both antigen and adjuvant material and help disseminate them to lymphatic tissues further away, elevating the overall immune response (Freund, 1953, 1956; White 1967; Allison, 1973; World Health Organization Technical Report, 1975). B. pertussis activates macrophages and is a strong lymphocytosis inducer (Dresser et al, 1970; Maillard and Bloom, 1972; Allison, 1973; Finger, 1974; World Health Organization Technical Report, 1975). Light and electr of emu associ It is are di (or 11 in mo: et al is in isola also adju\ cause are' agai beer wer fro electron microscopic studies have shown that the effectiveness of emulsions as immunological adjuvants depends on the anatomical associations formed by their components (Dvorak and Dvorak, 1974). It is also now evident that the adjuvant effects of microorganisms are due to their subcellular or even molecular components. Wax D (or its derivatives) is thought to be one of the active portions in most mycobacteria (Freund, 1953, 1956; White, 1967). Mota, et al (1974) showed that the adjuvant activity of B. pertussis is in a lipOpolysaccharide fraction. A histamine-sensitizing factor isolated from B. pertussis(Lehrer et a1, 1974, 1975, 1976) has also been found to possess adjuvant activity. In the clinical practice of active immunization against infection adjuvants have found their greatest use in vaccines for diseases caused by microorganisms, especially bacteria and Viruses. There are presently no vaccines involving adjuvants for active immunization against helminth infections, although some promising findings have been reported. Silverman,et a1 (1962), using aluminum hydroxide, were able to enhance the protective ability of in vitro antigens from Dictyoeaulas uiuiparua and Triehostrongylus eolubriformis in guinea pigs and from Strongyloides papillosus in rabbits. Freund‘s adjuvant was successfully used in similar vaccinations against As-aria Zumbrieoides and A. swam in the guinea pig (Soulsby, 1957, 1963). Immunization against T. outs with in vitro products and Freund's complete adjuvant has been achieved in lambs (Rickard and Bell, 1971; Rickard et al, 1976) and against T. saginata in calves (Rickard and Adolph, 1976). However, no vaccination experi there operat immuni somati adjuve (1974: adjuv taeni obtai more in en again immur experiments have been done in which adjuvant was not used, and there is no indication of the site at which the protective response operates. Kwa and Liew (1977) employed Freund's adjuvant in immunization against T. taenidejbrmia in the rat using both somatic and excretory-secretory antigens, but the effect of the adjuvant in their experiments is not clear. Varela—Diaz, et a1 (1974) and Musoke and Williams (1976) have shown that Freund's adjuvant alone may significantly reduce the survival of implanted taeniid metacestodes in rodents, and similar results have been obtained in schistosomiasis (Capron et a1, 1969). Clearly, a more detailed examination is necessary of the role of adjuvants in enhancing the develOpment of protective immune mechanisms against helminths, and especially taeniid cestodes, where artificial immunization has great practical potential. REFERENCES Adinolf Agarwal Agarwal Agarwal Allen, Alliso Ansari Ansarj AHSarj Bauer; REFERENCES Adinolfi, M., Glynn, A. A., Lindsay, M. and Milne, C. M. 1966. Serological prOperties of 7A antibodies to Escherichia coli present in human colostrum. Immunology 10:517-26. Agarwal, S. C. and Ganguly, N. K. 19723. Experimental oral immunization with L. forms of Vibrio cholerae. Inf. immunity 5:31-34. Agarwal, S. C. and Ganguly, N. K. 1972b. Oral immunization with L. forms of Vibrio cholerae in Human Volunteers. Inf. and Immunity 6:17-20. Agarwal, S. C. and Sundararaj. 1977. Cell mediated immunity after oral immunization with Ribonucleic acid protein fractions of Vibrio cholerae L-form lysates. Inf. Immunity 16:527-530. Allen, W. D., Smith, C. C. and Porter, P. 1973. Localization of intracellular immunoglobulin A in porcine intestinal mucosa using c zyme—labelled antibody. Immunology 25:55—70. Allison, A. C. 1973. Effects of adjuvants on different cell types and their interactions in immune response. p. 72-99. In: Immunopotentiation. Ciba Foundation Symposium 18 (new series). Elsevier-Excerpta Medica—North Holland. Ansari, A. 1975. Experimental studies of eosinophilia in rats infected with Taenia Laeniaejbrmis. M.S. degree thesis; Michigan State University, East Lansing, Michigan. Ansari, A. and Williams, J. F. 1976. The eosinophilic response of the rat to infection with Yaenia taeniaefbrmis. J. Parasitol. 62:728—730. Ansari, A., Williams, J. F. and Musoke, A. J. 1976. Antibody— mediated secondary eosinophilic response to Taenia taeniaefbrm— is in the rat. J. Parasitol. 62:737-740. Banerjee, D. and Singh, K. S. 1969a. Studies on Cystieereus jaseiolaris I.Studies on the early stages of infection in cysticercosis in rats. Ind. J. Animal Sci. 39:149-154. 30 Baner Bauer Barra Befus Bern: Bien Blun Bock Bort 31 Banerjee, D. and Singh, K. S. 1969b. Studies on Cysticereus faseiolaris II.Histochemical studies on Taenia taeniaefbrmis, changes in rats' intestine and oncosphere during penetration. Ind. J. Animal Sci. 39:155-163. Banerjee, D. and Singh, K. S. 1969c. Studies on Cysticereus fascia- Zaris IV.Immunity to cysticereus faseiolaris in rats. Ind. J. Animal Sci. 39:250-253. Barratt, M. E. J., Strachau, P. M. and Porter, P. 1977. Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets. p. 13-14. In: The British Society for Immunology 1977 Autumn Meeting (Abstracts). London. Befus, A. D. 1975. Intestinal immune response of mice to the tapeworms figmenoiepis iiminutu and H. mierostoma. Doctor of Philosophy degree thesis; Department of Zoology, University of Glasgow. Beguin, F. 1966. Etude au microscope electronique de la cuticle et de ses structure anoiiree che quelques cestodes. Essai d'histOIOgie comparie. Z. Zellforsch, mik rosk Anat. 72:30-76. Bernstein, I. D. and Ovary, Z. 1968. Absorption of antigens from the gastrointestinal tract. Int. Arch. Allergy and Appl. Immunol. 33:521-527. Bienenstock, J. 1974. The physiology of the local immune response and the gastrointestinal tract. p. 197-207. In: Progress in Immunology II.vol. 4. (L. Brent and J. Holborow, eds.) Amsterdam, Oxford; North—Holland Publishing Co. Blundell, S. K., Gemmell, M. A. and MacNamara, F. M. 1968. Immunological responses of the mammalian host against tapeworm injections. VI.Demonstration of humoral immunity in sheep induced by the activated embryos of T. hydatigena and T. ouis. Exp. Parasitol. 23:79-82. Bockman, D. E. and Cooper, M. D. 1973. Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix and Peyer‘s patches. An electron microscopic study. Am. J. Anat. 136:455—478. Bortoletti, L. and Ferretti, G. 1971. Observations on the ultrastructure of the tegument in the larval forms of Hydatigera (Taenia) Caeniaefbrmis and considerations on the development of the cyclophyllidean cestode larvae. Riv. Parassitol. 32:249-271. Bortolet Brambell Brandtz: Brandtz Campbel Campbel Campbej Campb e Campbe Capror Cook, C00pe 32 Bortoletti, L. and Ferretti, G. 1973. Investigation on larval forms of Echinococcus granulosus with electron microscope. Riv. Parassitol. XXXIV(2), 89—110. Brambell, F. W. R. 1966. The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet Nov. 1087-1093. Brandtzaeg, P. 1974. Mucosal and glandular distribution of immunoglobulin components: differential localization of free and bound SC in secretory epithelial cells. J. Immunol. 112:1553-1559. Brandtzaeg, P., Fjellanger, I. and Gjeruldsen, S. T. 1968. Immunoglobulin M: local synthesis and selective secretion in patients with immunoglobulin A deficiency. Science 160:789—791. Campbell, D. H. 1936. Active immunization of albino rats with protein fractions from Taenia taeniaefbrmis and its larval form Cysticercus fasciolaris. Am. J. Hyg. 23:104—113. Campbell, D. H. 1938a. The specific protective property of serum from rats infected with Cysticercus crassieoZZis. J. Immunol. 35:195-204. Campbell, D. H. 1938b. The specific absorbability of protective antibodies against Cysticercus crassicoZZis in rats and C. pisifbrmis in rabbits from infected and artificially immunized animals. J. of Immunol. 35:465-476. Campbell, D. H. 1939. A polysaccharide fraction from Cystieercus crassicolis and its role in immunity. J. Inf. Dis. 65:12—15. Campbell, W. C. and Blair, L. S. 1974. Treatment of the cystic stage of Taenia crassiceps and Echinococcus multilocularis in laboratory animals. J. Parasitol. 60:1053—1054. Capron, A. and Lesoin,M. A. 1969. Role de protection du BCG dans la Schistosomiase experimentale a S. mansoni. C. R. Hebd. Seanc. Acad. Sci. Paris. Ser. D. 269:2110—2112. Cook, R. W. and Williams, J. F. 1978. Gastrointestinal changes in Taenia taeniaefbrmis infection in the rat. J. Comp. Path. (submitted for publication). Cooper, M. D., Kincade, P. W., Bockman, D. E. and Lawton, A. R. 1974. Origin, distribution and differentiation of IgA producing cells. p. 13—22. In: The Immunoglobulin A System. (J. Mestecky and A. R. Lawton, eds.). Plenum Press; New York; London. Crabbe, P. b( g< w: Crandall, t i I Crandall, i Crandall, i 1 David, F Dineen, Dineen, Dineen, Dixon, Dobson DreSsg Crabbe, Crandall, R. B., Cebra, J. J. and Crandall, C. A. 1967. The rela— Crandall, R. B. and Crandall, C. A. 1972. TrichineZZa spiralis: Crandall, R. 8., Crandall, C. A. and Franco, J. A. 1974. David, F. M. 1977. Prevention of homocytotropic antibody formation Dineen, Dineen, Dineen, 33 P. A., Bazin, H., Eyssen, H. and Heremans, J. F. 1969. Anti- bodies of the IgA type in intestinal plasma cells of germ free mice after oral or parenteral immunization with ferritin. J. Exp. Med. 130: 723-744. tive proportions of IgG, IgA and IgM containing cells in rabbit tissues during experimental trichinosis. Immunology 12:147-158. immunologic response to infection in mice. Exp. Parasitol. 31:378-398. Helegineosomoides polygyrus (=Nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice. Exp. Parasitol. 315:275-287. and anaphylactic sensitization by prefeeding antigen. J. Allergy and Clin. Immunol. 60:180-187. J. K. and Adams, D. B. 1971. The role of the recirculating thymus-dependent lymphocyte in resistance to Trichostrongylus colubrifbrmis in the guinea pig. Immunology 20:109-113. J. K. and Kelly, J. D. 1973. Expulsion of Nippostrongylus brasiZiensis from the intestine of rats: the role of a cellular component derived from bone morrow. Int. Arch. Allergy and Appl. Immunol. 45:759-766. J. K., Ogilvie, B. M. and Kelly, J. D. 1973. Expulsion of Nippostrongylus brasiliensis from the intestine of rats, collaboration between humoral and cellular components of the immune response. Immunology 24:467-475. Dixon, S. N., Gibbons, R., Parker, J. and Sellwood, R. 1973. Dobson, Characterization of glycoprotein in the cyst fluid of Cysticercus tenuieollis from the goat. Int. J. Parasitol. 3:419—424. C. and Soulsby, E. J. L. 1974. Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriforms: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions and in vitro lymphocyte trans- formation during primary infections. Exp. Parasitol. 35:16-34. Dresser, D. W., Wortis, H. H. and Anderson, H. R. 1970. The effect of pertussis vaccine on the immune response of mice to sheep red blood cells. Clin. Exp. Immunol. 7:817-831. Dvorak, A. C( t( Fichteliu: 1« Finger, H I e Freter, R P J Freter, R M 1 Freter, F < Freund, ; ( Freund, . Froyd, c Fubara, Fubara: Fubara Furuu Gemmg 34 Dvorak, A. M. and Dvorak, H. F. 1974. Structure of Freund's Complete and Incomplete Adjuvants. Relation of adjuvanticity to structure. Immunology 27:99-114. Fichtelius, K. E. 1968. The gut epithelium - a first level lymphoid organ? Exp. Cell Research 49:87-104. Finger, H. 1974. Bordetella pertussis as adjuvant. In: The Immune System and Infectious Disease (E. Nefer and F. Milgrom, eds.). Basel, New York, S. Karger. Freter, R. 1956. Coproantibody and bacterial antagonism as protective factors in experimental enteric cholera. J. Exp. Med. 1041419-426. Freter, R. 1972. Parameters affecting association of vibrios with the intestinal surface in experimental cholera. Inf. Immun. 6:134-141. Freter, R. and Gangarosa, J. 1963. Oral immunization and production of coproantibody in human volunteers. J. Immunol. 91:724—729. Freund, J. 1953. The effect of paraffin oil and Mycobacteria on antibody formation and sensitization. Am. J. Clin. Pathol. 21:645-656. Freund, J. 1956. The mode of action of immunologic adjuvants. Advanc. Tuberc. Res. 7:130—148. Froyd, G. 1964. The effect of post infection serum on the infectability of calves with Taenia eggs. Brit. Vet. J. 120:162-166. Fubara, E. S. and Freter, R. 1972a. Source and protective function of coproantibodies in intestinal disease. Am. J. Clin. Nutrition 25:1357—1363. Fubara, E. S. and Freter, R. 1972b. Availability of locally synthesized and systemic antibodies in the intestine. Inf. and Immunity 62965—981. Fubara, E. S. and Freter, R. 1973. Protection against enteric bacterial infection by secretory IgA antibodies. J. Immunol. 1112395-403. Furuuchi, S., Shimizu, Y. and Kumagai, T. 1976. Vaccination of newborn pigs with an attenuated strain of transmissible gastroenteritis virus. Am. J. Vet. Res. 37:1401—1404. Gemmell, M. A. 1962a. Natural and acquired immunity factors inhibiting penetration of some hexacanth embryos through the intestinal barrier. Nature 194:701-702. Gemmell, i c Gemmell, Gemmell, I Gemmell, Gemmell, Gibbons, Gray, Jl GUY-Gral Hammerb Hammerb Hammerb 35 Gemmell, M. A. 1962b. Natural and acquired immunity factors interfering with development during the rapid growth phase of Echinococcus granulosus in dogs. Immunology 5:496-503. Gemmell, M. A. 1964. Immunological Responses of the Mammalian Host Against Tapeworm Infections. 1. Species specificity of Hexacanth embryos in protecting sheep against Taenia hydatigena. Immunology 71489-499. Gemmell, M. A. 1965. Immunological Responses of the Mammalian Host Against Tapeworm Infections. III. Species specificity of Hexacanth embryos in protecting sheep against T. ovis. Immunology 8:281-290. Gemmell, M. A. 1966. Immunological Responses of the Mammalian Host Against Tapeworm Infections. IV. Species specificity of Hexacanth embryos in protecting sheep against Echinococcus granulosus. Immunology 11:325-335. Gemmell, M. A., Blundell—Hassell, S. K. and MacNamara, F. N. 1969. Immunological responses of the mammalian host against tapeworm infections. IV. The transfer via colostrum of immunity to Taenia hydatigena. Exp. Parasitol. 26:52-56. Gibbons, R. J. 1974. Bacterial adherence to mucosal surfaces and its inhibition by secretory antibodies. p. 315-325. In: The Immunoglobulin A System. (J. Mestecky and A. R. Law- ton, eds.). New York, London, Plenum Press. Gray, J. S. 1973. Studies on host resistance to secondary infections of Raillietina cesticillus. Parasitology 67:375-382. Guy—Grand, D., Griscelli, L. and Vassalli, P. 1976. The gut associated lymphoid system: nature and properties of large dividing cells. European J. Immunol. 4:435-443. Hammerberg, B. and Williams, J. F. 1978a. Interaction between Taenia taeniaefbrmis and the complement system. J. Immunol. in Press. Hammerberg, B. and Williams, J. F. 1978b. Physico—chemical characterization of complement—interacting factors from Taenia taeniaefbrmis. J. Immunol. in Press. Hammerberg, B., Musoke, A. J., Hustead, S. T. and Williams, J. F. 1976. Anticomplementary substances associated with Taenia taeniaefbrmis larvae. In: Pathophzsiology of Helminth Infections. (E. J. Soulsby, ed.). London—— and New York, Academic Press. Hammerbe Heath, E Heath, [ Heath, 1 Heath, 1 Heath, Heath, Heath, Heath, HErbert Herd, } Herd, 36 Hammerberg, B., Musoke, A. J. and Williams, J. F. 1977. Activation of complement by hydatid cyst fluid of Echinococcus granulosus. J. Parasitol. 63:327—331. Heath, D. D. 1971. The migration of oncospheres of Taenia pisifbrmis, T. serialis, and Echinococcus granulosus within the intermediate host. Int. J. Parasitol. 1:145-152. Heath, D. D. 1973a. An improved technique for the in vitro culture of Taeniid larvae. Int. J. Parasitol. 3:481-484. Heath, D. D. 1973b. Resistance to Taenia pisifbrmis larvae in rabbits. I. Examination of the antigenically protective phase of larval development. Int. J. Parasitol. 3:485-489. Heath, D. D. 1973c. Resistance to Taenia pisifbrmis larvae in rabbits. II. Temporal relationships and the development phase affected. Int. J. Parasitol. 3:491-498. Heath, D. D. 1976. Resistance to Taenia pisifbrmis larvae in rabbits: Immunization against infection using non—living antigens from in vitro culture. Int. J. Parasitol. 6:19-24. Heath, D. D. and Elsdon-Dew, R. 1971. The in vitro culture of Taenia saginata and Taenia taeniafbrmis larvae from the oncosphere, with observation on the roles of serum for in vitro culture of larval cestodes. Int. J. Parasitol. 2:119-130. Heath, D. D. and Chevis, R. A. F. 1974. Mebendazole and hydatid cysts. Lancet 7874:218—219. Heath, D. D. and Smyth, J. D. 1970. In vitro cultivation of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisifbrmis and T. serialis from oncosphere to cystic larva. Parasitology 61:329-343. Herbert, I. V. and Oberg, C. 1974. Cysticercus in pigs due to infection with Taenia soZium, Linnaeus 1758. In: Parasitic Zoonoses: Clinical and Experimental Studies. (E. J. L. Soulsby, ed.). Academic Press, New York, San Francisco, London. Herd, R. P. 1976. The cestocidal effect of complement in normal and immune sera in vitro. Parasitology 72:325-330. Herd, R. P., Chappel, R. J. and Biddell, D. 1975. Immunization of dogs against Echinococcus granulosus using worm secretory antigen. Int. J. Parasitol. 5:395-399. Heremans Hill, I. Hustead Hustead Hustead Jolles, Jones, Jones, Kassis, Keller, Kelly, Kerr, 37 Heremans, J. F. 1974. The IgA system in connection with local Hill, I. and systemic immunity. p. 3—11. In: The Immunoglobulins A System. (J. Mestecky and A. R. Lawton, eds.). New York, London, Plenum Press. R. and Porter, P. 1974. Studies of bactericidal activity to Escherichia coli of porcine serum and colostral immuno— globulins and the role of lysozyme with secretory IgA. Immunology 25:1239—1250. Hustead, S. T. 1976. Permeability studies on taeniid metacestodes. M.S. degree thesis; Michigan State University, East Lansing, Michigan. Hustead, S. T. and Williams, J. F. 1977a. Permeability studies of taeniid metacestodes. I. Uptake of proteins by larval stages of Taenia taeniaefbrmis, Taenia crassiceps and Echinococcus granulosus. J. Parasitol. 63:314-321. Hustead, S. T. and Williams, J. F. 1977b. Permeability studies Jolles, of taeniid metacestodes. II. Antibody mediated effects on membrane permeability in larvae of Taenia taeniaefbrmis and Taenia crassiceps. J. Parasitol. 63:322-326. P. and Paraf, A. 1973. Chemical and Biological Basis of Adjuvants In Molecular Biology, Biochemistry and Biophysics 13. (A. Kleinzeller, G. F. Springer and H. G. Wiltman, eds.). Springer—Verlag, New York-Heidelberg Berlin. Jones, E. A. 1972. Immunoglobulins and the gut. Gut 13:825—835. Jones, V. E. and Ogilvie, B. M. 1971. Protective immunity to Kassis, Keller, Nippostrongylus brasiliensis: the sequence of events which expels worms from the rat intestine. Immunology 20:549-561. R. and Tanner, C. E. 1976. The role of complement in hydatid disease: in vitro studies. Int. J. Parasitol. 6:25-36. R. and Keist, R. 1972. Protective immunity to Nippostrongylus brasiliensis in the rat: central role of the lymphocyte in worm expulsion. Immunology 22:767-773. Kelly, J. D., Dineen, J. K. and Love, R. J. 1973. Expulsion of Nippostrongylus brasiliensis from the intestine of rats: evidence for a third component in the rejection mechanism. Int. Arch. Allergy Appl. Immunol. 45:767—779. Kerr, K. B. 1935. Immunity against a cestode parasite, Cysticercus pieifbrmis. Am. J. Hyg. 22:169-182. Kwa, B. E Lascano, Lehrer, Lehrer, Lehrer, Leid, R Leid, R Leid, E Leonar( Lewert M81113 Mestec Miller 38 Kwa, B. H. and Liew, F. Y. 1977. Immunity in Taeniasis - Cysticerco- sis. I. Vaccination against Thenia taeniaefbrmis in rats using purified antigens. J. Exp. Med. 146:118—131. Lascano, E. F., Coltorti, E. A. and Varela-Diaz, v. M. 1975. Fine structure of the germinal membrane of Echinoeoceus granulosus cysts. J. Parasitol. 61:853-860. Lehrer, S. B., Tan, E. M. and Vaughan, J. H. 1974. Extraction and partial purification of the histamine - sensitizing factor of Bordetella pertussis. J. Immunol. 113:18—26. Lehrer, S. B., Vaughan, J. H. and Tan, E. M. 1975. Immunologic and biochemical properties of the histamine sensitizing factor from Bordetella pertussis. J. Immunol. 114:34—39. Lehrer, S. B., Vaughan, J. H. and Tan, E. M. 1976. Enhancement of reaginic and hemagglutinating antibody production by an extract of Bordetella pertussis containing histamine- sensitizing factor. J. Immunol. 116:178-183. Leid, R. W. 1973. Immunological events associated with Taenia taeniaefbrmis infection in the laboratory rat. Ph.D. dissertation; Michigan State University, East Lansing, Michigan. Leid, R. W. and Williams, J. F. 1974a. Immunological responses of the rat to infection with Taenia taeniaefbrmis. I. Immunoglobulin classes involved in passive transfer of resistance. Immunology 27:195—208. Leid, R. W. and Williams, J. F. 1974b. Immunological responses of the rat to infection with Taenia taeniaeformis. II. Characterization of reaginic antibody and an allergen associated with the larval stage. Immunology 27:209—225. Leonard, A. B. and Leonard, A. E. 1941. The intestinal phase of the resistance of rabbits to the larvae of Taenia pisifbrmis. J. Parasitol. 27:375-378. Lewert, R. M. and Lee, C. L. 1957. The collagenase—like enzymes of skin-penetrating helminths. Am. J. of Trop. Med. Hygiene 6:473-479. Maillard, J. and Bloom, B. R. 1972. Immunological adjuvants and the mechanism of cell cooperation. J. Exp. Med. 136:185-190. Mestecky, J. and Lawton, A. R. 1974. The Immunoglobulin A System. New York, London, Plenum Press. Miller, H. M., Jr. 1931. The production of artificial immunity in the albino rat to a metazoan parasite. J. of Preventive Med. 5:429-452. Miller, H a A Miller, 1 1 Miller,l 1 Miller, Miller, Morseth, Mota, I Murray, Musoke, Musoke MUSoke MUSoke 39 Miller, H. M., Jr. 1935. Transmission to offspring of immunity against infection with a metazoan (cestode) parasite. Am. J. Hyg. 21:456-461. Miller, H. M., Jr. and Gardiner, M. L. 1932. Passive immunity to infection with metazoan parasite, Cysticercus fhsciolaris, in the albino rat. J. Prev. Med. 6:479-496. Miller, H. M., Jr. and Gardiner, M. L. 1934. Further studies on passive immunity to a metazoan parasite, Cysticereus fuseiolaris. Am. J. Hyg. 20:424-431. Miller, H. M., Jr. and Kerr, K. B. 1932. Attempts to immunize rabbits against a larval cestode, Cysticercus pisifbrmis. Proc. Soc. Exper. Biol. Med. 29:670-671. Miller, H. M., Jr. and Massie, E. 1932. Persistence of acquired immunity to Cysticercus faseiolaris after removal of the worms. J. Prev. Med. 6:31—36. Morseth, D. 1966. The fine structure of the tegument of adult Echinocoecus granulosus, Taenia hydfitigena and Taenia pisifbrmis. J. Parasitol. 54:15-27. Mota, I., Perini, A. and Trindade, V. S. 1974. The mechanism of the adjuvant effect of Bordetella pertussis: the substance responsible for the selective enhancement of IgE antibody production. Int. Arch. Allergy 471:425—432. Murray, M. 1972. Immediate hypersensitivity effector mechanism. 11. In vivo reactions. p. 155 In: Immunity £9_Animal Parasites. (E. J. Soulsby, ed.). Academic Press, New York. Musoke, A. J. 1975. Immunologic mechanism in resistance to experimental infection with Taenia taeniaefbrmis. Ph.D. dissertation; Michigan State University, East Lansing, Michigan. Musoke, A. J. and Williams, J. F. 1975a. Immunoglobulins associated with passive transfer of resistance to Taenia taeniaeformis in the mouse. Immunology 28:97—102. Musoke, A. J. and Williams, J. F. 1975b. Immunological response of the rat to infection with Taenia taeniaeformis. V. Sequence of appearance of protective immunoglobulins and the mechanism of action of 7SY2a antibodies. Immunology 29:855—865. Musoke, A. J. and Williams, J. F. 1976. Immunological response of the rat to infection with Taenia taeniaeformis: protective antibody response to implanted parasites. Int. J. Parasitol. 62265—269. Musoke, Musoke, Nemeth, Nossal, Ogra, P Pawlow: Penfol Picone Pierce Forte] Porte Musoke, Musoke, Nemeth, Nossal, Ogra, P. Pawlowski, Z. Penfold, Picone, Pierce, Porter, Porter, 40 A. J., Williams, J. F. and Leid, R. W. 1978. The immunologi- cal response of the rat to infection with T. taeniaefbrmis. VI. The role of immediate hypersensitivity in resistance. Immunology in Press. A. J., Williams, J. F., Leid, R. W. and Williams, C. S. F. 1975. The immunological response of the rat to infection with Taenia taeniaefbrmis. IV. Immunoglobulins involved in passive transfer of resistance from mother to offspring. Immunology 29:845-853. 1. 1970. Immunological study of rabbit cysticercus. II. Transfer of immunity to Cysticercus pisifbrmis (Block, 1780) with parenterally administered immune serum or lymphoid cells. Acta Veterinaria Academiae Scientiarum Hungaricae 20:69-79. G. S. V. and Ada, G. L. 1971. Antigens, Lymphoid Cells and the Immune Response. New York, London, Academic Press. L., Wallace, R. B., Umana, A., Ozga, S. S., Grant, D. K. and Moray, A. 1974. Implications of secretory immune system in viral infections. p. 271—282. In: The Immuno— globulin_A System. (J. Mestecky and A. R. Lawton, eds.). New York, London, Plenum Press. and Schultz, M. 1972. Taeniiasis and cysticercosis (Taenia saginata). Advances in Parasitol. 10:269—344. J. W., Penfold, B. H. and Phillips, M. 1936. Acquired active immunity in the Ox to Cysticercus bovis. Med. J. Australia, Vol. 1, 417-423. J. 197 . Electron microscopy of post-oncospheral development of Taenia taeniaefbrmis. (Personal communication). N. F. and Reynolds, H. Y. 1974. Immunity to experimental cholera. 1. Protective effect of humoral IgA antitoxin demonstrated by passive immunization. J. Immunol. 113:1017- 1023. P., Kenworthy, R., Holme, D. and Horsfield, S. 1973. Escherichia coli antigens as dietary additives for oral immunization of pigs: Trials with pig creep feeds. Vet. Record 92:630-636. P., Kenworthy, R., Noakes, D. E. and Allen, W. D. 1974. Intestinal antibody secretion in the young pig in response to oral immunization with Escherichia coli. Immunology 27:841-852. Rickard, Rickard, Rickard, Rickard Rickard Rodewal Roitt, Roger, Rothbe Rothbe ROthwe 41 Rickard, M. D. and Arundel, J. H. 1974. Passive protection of lambs against infection with Taenia ovis via colostrum. Aust. Vet. J. 50:22-24. Rickard, M. D. and Bell, K. J. 19713. Successful vaccination of lambs against infection with Taenia ovis using antigens produced during in vitro cultivation of the larval stages. Res. Vet. Sci. 12:401-402. Rickard, M. D. and Bell, K. J. 1971b. Immunity produced against Taenia ovis and T. taeniaefbrmis infections in lambs and in rats following in vitro growth of their larvae in filtration membrane diffusion chambers. J. Parasitol. 57:571-575. Rickard, M. D. and Adolph, A. J. 1976. Vaccination of calves against Taenia saginata infection using a ”parasite—free” vaccine. Vet. Parasitol. 1:389-392. Rickard, M. D. and Katiyar, J. C. 1976. Partial purification of antigens collected during in vitro cultivation of the larval stages of Taenia pisifbrmis. Parasitology 72:269-279. Rodewald, R. 1973. Intestinal transport of antibodies in the newborn rat. J. Cell Biol. 58:189—211. Roitt, I. 1974. Essential Immunology. Oxford, London, Edinburgh, Melbourne, Blackwall Scientific Publications. Roger, M. E. and Lamm, M. E. 1974. Localization of free and bound secretory component in human intestinal epithelial cells. J. Exp. Med. 139:629—642. Rothberg, R. M., Kraft, S. C. and Michalek, S. M. 1973. Systemic immunity after local antigenic stimulation of lymphoid tissue of the gastrointestinal tract. J. Immunol. 111:1906—1913. Rothberg, R. M., Kraft, S. C. and Michalek, S. M. 1974. The establishment of systemic immunity following antigenic stimulation of the lymphoid tissue of the gastrointestinal mucosa. p. 473-478. In: The Immunoglobulin A System. (J. Mestecky and A. R. Lawton, eds.). New York, London, Plenum Press. Rothwell, T. L. W. and Merritt, G. C. 1974. Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubrifbrmis infected sheep. Int. J. Parasitol. 4:63-71. Rothwell Salazar, Sanyal, Shimamun Silverm: Silverm Silvern Slais, Solom( Souls Souls Soul 42 Rothwell, T. L. W., Prichard, R. K. and Love, R. J. 1974. Studies on the role of histamine and S-hydroxytryptamine in immunity against the nematode Trichostrongylus colubri- fbrmis. I. In vivo and in vitro effects of the amines. Int. Arch. of Allergy and Appl. Immunol. 46:1-13. Salazar, M., Gonzalez, D. and Vega, M. V. 1972. Ensayo de tratamiento de la cisticercosis con metrifonato. Rev. Invest. Solud. Publica 32:1-7. Sanyal, S. C. and Mukerjee, S. 1969. Live oral cholera vaccine: Report of trial on human volunteer subjects. Bull. W.H.O. 40:503-511. Shimamura, T. 1972. Immune response in germ—free mice orally immunized with Vibrio cholerae. Keio J. Med. 21:113-126. Silverman, P. H. 1954. Studies on the biology of some tapeworms of the genus Taenia. I. Factors affecting hatching and activation of Taeniid ova and some criteria of their viability. Ann. Trop. Med. Parasit. 48:207-215. Silverman, P. H. and Maneely, R. B. 1955. Studies on the biology of some tapeworms of the genus Taenia. III. The role of the secreting gland of the hexacath embryo in the penetration of the intestinal mucosa of the intermediate host and some of its histochemical reactions. Ann. Trop. Med. Parasitol. 49:326-330. Silverman, P. H., Poynter, D. and Podger, K. R. 1962. Studies on larval antigens derived by cultivation of some parasitic nematodes in simple media. Protection tests in laboratory animals. J. Parasitol. 48:562-572. Slais, C. 1966. Beitrug zur morphogenese des Cysticercus ceZZuZosae and C. bovis. Folia parasitol. (Prah) 13:73-92. Solomon, G. 1934. Some points in the early development of Cysticercus pisifbrmis. J. Helminthol. 12:197-204. Soulsby, E. J. L. 1957. Immunization against Ascaris Zumbricoides in the guinea pig. Nature 179:783—784. Soulsby, E. J. L. 1963a. "Immunological unresponsiveness to helminth infections in mammals.” Proceedings 17th Int. Vet.Congress, 1962. Vol. 1, 761—767. Hanover. Soulsby, E. J. L. 1963b. The nature and origin of the functional antigens in helminth infections. Annals of the New York Academy of Sciences 113:492—509. Swarbric Sweatman Thienpon Thomas, Threadg Tomasi, Turner, Urquha] Varela, Varela Walker Walks] 43 Swarbrick, E. T., Stokes, C. R. and Soothhill, J. F. 1977. The absorption of antigen after oral immunization and the induction of systemic tolerance. In: p. 13 British Society for Immunology 1977 Autumn meeting (Abstracts) in London. Sweatman, G. K. 1957. Acquired Immunity in Lambs Infected with Ibenia hydatigena. Canad. J. of Comp. Med. 21:65—70. Thienpont, D., Vanpary, O. and Heremans, L. 1974. Anthelmintic activity of mebendazole against Cysticercus fhsciolaris. J. Parasitol. 60:1052-1053. Thomas, H. C. and Parrot, M. V. 1974. The induction of tolerance to a soluble protein antigen by oral administration. Immunology 27:631-639. Threadgold, L. T. 1967. The Ultrastructure of the Animal Cell. Pergamon Press. Tomasi, T. B. and Grey, H. M. 1972. Structure and function of Immunoglobulin A. p. 81-213. In: Progress in Allergy - Vol. 16 (P. Kallos, B. H. Walhman and A. deWeck, eds.). Basel, S. Karger. Turner, E. L., Berbarian, D. A. and Dennis, E. W. 1933. Successful artificial immunization of dogs against Taenia echinococcus. Proc. Soc. Exp. Biol. New York 30:618-619. Urquhart, G. M. 1958. The production of experimental cysticercosis in calves in Kenya. Bull. of Epizootic Diseases of Arica 6:385-393. Varela—Diaz, V. M. and Coltorti, E. A. 1972. Further evidence of the presence of host immunoglobulins in hydatid cyst membranes. J. Parasitol. 59:484-488. Varela—Diaz, v. M., Williams, J. F., Coltorti, E. A. and Williams, C. S. F. 1974. Survival of cysts of Eehinococcus granulosus after transplantation into homologous and heterologous host. J. Parasitol. 60:608-612. Walker, W. A. and Hong, R. 1973. Immunology of the gastrointestinal tract: Part I. J. Pediatrics 83:517-530. Walker, W. A., Isselbadrer, K. J. and Bloch, K. J. 1974. The role of immunization in controlling antigen uptake from the small intestine. p. 295-303. In: The Immunoglobulin A System. (J. Mestecky and A. R. Lawton:_3ds.). New York, London, Plenum Press. Wak sman , White , R Wikerhai Wikerha1 World H 44 Waksman, B. H. 1973. The homing pattern of thymus-derived lymphocytes in calf and neonatal mouse Peyer's patches. J. Immunol. 111:878-884. White, R. G. 1967. Characterization of Mycobacterial components of adjuvant mixtures. p. 49-58. In: International Symposium on adjuvants of immunity. Symposia series in Immunobiological Standardization, No. 6. S. Karger Basel (Switzerland) New York. Wikerhauser, T., Zukovic, M. and Dzakula, N. 1971. Taenia saginata and T. hydatigena: Intramuscular vaccination of calves with oncospheres, Exp. Parasitol. 30:36—40. Wikerhauser, T., Zukovic, M., Dzakula, N., Marau, R. 1974. Immunization of calves against the infection with Taenia saginata and subcutaneous vaccination with the homologous oncospheres and eggs. p. 195—197. In: Parasitic Zoonosis. (E. J. L. Soulsby, ed.). Academic Press, New York. World Health Organization. 1975. Technical Report series, WHO. Immunoglocial adjuvants: report of a WHO scientific group. Geneva, World Health Organization 1975. ARTICLE THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMIS VII. IMMUNIZATION BY ORAL AND PARENTERAL ADMINISTRATION OF ANTIGENS. The immunological response of the rat to infection with Taenia taeniaefbrmis VII. Immunization by oral and parenteral administration of antigens J. M. Ayuya and J. F. Williams Department of Microbiology and Public Health Michigan State University East Lansing, MI Rat antigens signific intrapel effectii require< were in« hydroxi‘ of anti adjuvan Reagini of rats but nor of reag Sf protec admini; but 51 advant extend antibo but tk Challe CUSSe( Rats immunized with in vitro (IVP) products and saline soluble antigens (SSA) derived from Taenia taeniaefbrmis were found to be significantly protected against challenge infection. Oral and intraperitoneal administration of antigen solutions alone was effective in stimulating resistance. However, adjuvants were required for successful immunization when single doses of antigens were inoculated intramuscularly. Bordetella pertussis and aluminum hydroxide were able to improve markedly the protective effects of antigens given parenterally by either route, but Freund's complete adjuvant (FCA) was not effective as an adjuvant in this system. Reaginic antibodies to parasite antigens were detected in the serum of rats vaccinated with IVP or SSA and B. pertussis or A1(OH)3, but none were detected in those given FCA. The possible role of reaginic antibodies in immunity to T. taeniaefbrmis is discussed. Single doses of antigens given orally produced significant protection. Increasing the number of daily doses of antigen administered orally enhanced the degree of protection to a limited but significant extent. However, there did not appear to be any advantage to giving large doses (>1 mg protein) of antigen, or extending the immunizing schedule over several weeks. Reaginic antibodies were not detected in the serum of rats immunized orally, but these animals were resistant to both oral and intravenous challenge infection with parasites. These observations are dis- cussed in relation to the phenomena of immune exclusion of antigen 45 by the g with res Sel ineffec1 in quani donors ‘ and B.‘ to infe to our from ra transfe The rec to ass< of ant' are em per os 46 by the gut, and gastrointestinally induced systemic tolerance with respect to IgE production. Serum from rats immunized by all routes was found to be ineffective in conferring resistance upon recipients when given in quantities up to 1.5 ml per rat. Furthermore, serum from donors vaccinated intramuscularly with saline soluble antigens and B. pertussis increased the susceptibility of recipient rats to infection with T. taeniaefbrmis. This is in sharp contrast to our previous experiences in which we have shown that serum from rats with an active infection is highly effective in passive transfer. Possible reasons for these observations are discussed. The requirements for adequately controlled immunization procedures to assess the contributory effects of adjuvant type and the route of antigen inoculation in immunizing against taeniid infections are emphasized in the discussion. The potential for vaccination per 05 against other helminthiases is also raised. It immunize with inc (Miller, obtainec 1932; K( artific been no: which w diffusi 1968; W However larvae for she in cult and the is curr De 0f prot Which t model I istics1 of a p( We rep( route ( 0f 1mm] INTRODUCTION It has been known for many years that rats can be successfully immunized against challenge infections of Taenia taeniaefbrmis with inoculations of homogenates or extracts of dead metacestodes (Miller, 1931; Campbell, 1936). Comparable results have been obtained with the rabbit-T. pisifbrmis system (Miller and Kerr, 1932; Kerr, 1935; Heath, 1976), but in domesticated ruminants artificial immunization schemes for cysticercosis have generally been most promising when animals were exposed to living parasites, which were either inoculated at abnormal sites or implanted in diffusion chambers (Gemmell, 1964, 1965a,b, 1966; Gemmell et a1, 1968; Wikerhauser et al, 1971, 1974; Rickard and Bell, 1971a). However, recent advances in the in vitro cultivation of cestode larvae have lead to the development of an experimental vaccine for sheep which consists of taeniid parasite antigens collected in culture media (Rickard and Adolph, 1976; Rickard et al, 1976), and the potential for application of this procedure in cattle is currently under field test (Rickard, personal communication). Despite these successes very little is known about the nature of protective immunogens of taeniid helminths or the responses which they provoke in vaccinated animals. The rat—T. taeniaefbrmis model provides an excellent opportunity for study of these character- istics, and Kwa and Liew (1977) have described the partial purification of a potent immunizing factor in metacestodes of this species. We report here our observations on the influence of antigen source, route of administration and adjuvant selection on the efficacy of immunization against T. taeniaefbrmis. Protective antigens 47 were de and the when t1 an effe results animals immune Expery F expert Sparta given killed from h water were I BME (( 1000 1 0f pol mediLu water 48 were detected in parasite extracts and in in vitro culture products, and these produced highly significant immunity to challenge, even when they were administered orally. Bordetella pertussis was an effective adjuvant in potentiating the immune response, and the results suggest that the mechanism of resistance in vaccinated animals is different from that which develops in rats which become immune following active infection. MATERIALS AND METHODS Experimental Animals Female 21 day old Spartan [Spb(SD)BR] rats were used for the experiments, except where specified. They were purchased from Spartan Research Animals, Haslett, Michigan. The animals were given proprietary brand food and water ad Zibitum. Preparation of Antigens i) In Vitro Products (IVP) Rats infected with T. taeniaefbrmis for over 3 months were killed with C02 vapour. The metacestodes were carefully dissected from hepatic cysts and washed three times each in triple distilled water and sterile physiological saline. Fifty strobilocerci were placed in culture vials each containing 150 mls of Hank's BME (Grand Island Biological Co., Grand Island, New York) with 1000 ug/ml of streptomycin, 1000 U/ml of penicillin, 500 U/ml of polymixin B and incubated at 37°C. After 24 hrs the culture medium was collected, dialysed for 48 hrs against triple distilled water and then for 12 hrs against phosphate buffered saline (PBS). The med. or by d When co dialyse then de and kep ii) S2 M: in PBS for 12 2 1/2 conten Protei T by the modifi was a the i Michi used contz anti, 49 The medium was then concentrated by either negative vacuum dialysis, or by dialysis against polyethylene glycol (Carbowax, Union Carbide). When concentration was by the latter method, the medium was further dialysed against PBS for 48 hrs. The protein content per ml was then determined. These In Vitro Products (IVP) were then frozen and kept at -20°C until used. ii) Saline Soluble Antigens (SSA) Mature strobilocerci obtained as described above, were ground in PBS in a glass mortar. The ground material was left to stir for 12 hrs at 4°C after which it was centrifuged at 50,000g for 2 1/2 hours. The supernatant was carefully withdrawn, its protein content per ml determined and then stored at -20°C until used. Protein Determination The protein concentration of the IVP and the SSA was determined by the Folin—Ciocalteau reaction (Williams and Chase, 1968), a modification of the method of Lowry,et al (1951). Adjuvants Freund's complete adjuvant (FCA, Difco, Detroit, Michigan) was emulsified with an equal amount of SSA or IVP when used in the intramuscular immunizations. Where B. pertussis (BP) vaccine cells (kindly supplied by the Michigan Department of Public Health, Lansing, Michigan) were used as an adjuvant, a suspension was prepared such that 1 m1 contained 1 x 1010 organisms and 1 mg of the appropriate parasite antigen. W PCP (1974a). and 0.1 appropr: the rat to be t antigen Evans b Any rea was cla include tested. the po: T. tae G at 21 a dose as des three disser CYSts each . 50 Homologousgpassive cutaneous anaphylaxis (PCA) PCA tests were carried out as described by Leid and Williams (1974a). The backs of retired female breeder rats were shaved and 0.1 ml quantities of undiluted or doubling dilutions of appropriate serum were injected intradermally. 72 hours later the rats were challenged intravenously (IV) with the antigen to be tested. Each rat received 1 ml containing 1 mg of the antigen, after the latter had been mixed in a 1:1 ration with Evans blue. Reactions were read 15 to 30 minutes after challenge. Any reaction showing intense blueing of more than 3 mm in diameter was classified as positive. Negative and positive sera were included in each recipient and two rats were used for each sample tested. The negative serum was obtained from normal rats, and the positive one from rats that had been harbouring an active T. taeniaefbrmis infection for at least five weeks. General Experimental Procedure Groups of rats were vaccinated in various ways (see results) at 21 days of age. Three weeks later they were challenged with a dose of 250 eggs obtained from T. taeniaefbrmis and counted as described by Leid and Williams (1974a). After an additional three weeks the rats were killed with CO2 vapour, their livers dissected out of the abdominal cavities and the number of hepatic cysts was counted: Further procedural details are described with each experiment. Analyti 11 where) by eid Scheffi varian and th an unl two or 0.05 a The tw respec a sing the st was t1 intrm Each in a divid these day c the a five 51 Analytical Assessment of Resistance The number of cysts per liver was transformed into ./§IET§Z where y = number of cysts. This transformed data was then analyzed by either the Bonferroni t procedure (Miller, 1966) or the modified Scheffe's method (Gill, 1977). The former was used when the variance of all groups in a single experiment was homogeneous, and the latter when it was heterogeneous. These methods permitted an unlimited number of comparisons or contrasts to be made between two or more groups. For both types of analyses, P values of only 0.05 and 0.01 can be determined from tables currently available. The two values were considered significant, and highly significant respectively. The data given in the tables are the results of a single experiment in each case, but unless otherwise indicated the statistical significance of the differences between groups was the same in replicate experiments. RESULTS In the preliminary experiments groups of six rats were immunized intramuscularly (IM), per 05 (PO) and intraperitoneally (IP). Each rat received not more than one mg of antigenic protein material in a ml of fluid. Intramuscular vaccination was achieved by dividing the dose of antigen into four equal portions, and injecting these into the muscles of each of the four limbs on the first day of each experiment. For intraperitoneal and oral vaccination, the antigen dose was given in a series of equal daily portions for five days. Oral and intraperitoneal vaccination with IVP conferred signifi and C; immnu resists effect intrap< H: SSA in Table and PB A, E a in the parasi thisx of th when immm not 5 (Tabl 52 significant resistance against subsequent challenge (Groups B and C; P < 0.05 and P < 0.01, respectively — Table la). Unexpectedly, immunization with IVP in FCA (group A) did not confer any significant resistance as compared to the untreated control (group D). No effect on resistance was shown with intramuscular FCA alone, or intraperitoneal PBS (groups of E and F). Highly significant resistance resulted from immunization with SSA in both oral and intraperitoneal routes (groups B and C; P < 0.01, Table 1b). Vaccination with SSA in FCA, FCA alone intramuscularly, and PBS intraperitoneally did not result in any resistance (Groups A, E and F - Table lb). In both experiments (a and b) the rats in the FCA control groups (E) had a higher average number of parasites than did the untreated control groups (D), although this was not statistically significant in either case. In a subsequent experiment, intramuscular administration of the antigens conferred no significant resistance, although when combined with B. pertussis they stimulated a highly protective immunity (groups A, P < 0.01 Table 2a,2b). The groups given B. pertussis alone intramuscularly and BME orally (E and C) were not significantly different from the untreated control group (D) (Table 2a). Table Group Treat Mean paras and r Conti and P Va] Grom Trea Mean para Cont and P Va 53 Table l. Immunization of rats against T. taeniaefbrmis with IVP and SSA using FCA a.) IVP Group A Group B Group C Group D Group E Group F Group and Treatment IM+FCA PO IP Untreated FCA IP-PBS control Mean No. of 33.5 3.7 1.3 32.8 50.5 41.8 parasites (16—63) (0-13) (0-4) (17-60) (16-64) (27-75) and range ( ) Contrasts and A vs D, NS B vs D, C vs D, E vs D, NS P Values P<0.05 P<0.01 b.) SSA Group A Group B Group C Group D Group E Group F Group and Treatment IM+FCA PO IP Untreated control FCA IP—PBS Mean No. of 24.5 3.8 2.0 35.5 49.7 35.8 parasites (5-53) (2-7) (1-4) (26—50) (24—70) (21-58) and range ( ) Contrasts and A vs D, NS 8 vs D, C vs D, E vs D, F vs D, P Values P<0.01 P<0.0l NS NS NS = Not Significant \l‘l‘l‘l‘lllllillllill‘ .m MEHWS H SHHB WVENNKMUHRMDQ .& Hmcfldwm mumh MO EOHHMNHCSEEH .N ®HQNH Pfid>5n©d mm WHWMSDRMQ 54 ucaouencwam uoz u mz once uoz u 92* monam> a Ho.ovm mo.o a Ho.ovm was mz .Q m> m o m> u .o m> m an w> < mummhucou m U emcee can flee-ONc msm-mfic flav-vma nmm-mc mmm-ma Am-0a mouamanaa m.om w.H~ Dz o.mm n.mH w.e~ m.m mo .02 new: mZm Hosucoo psoauwonh om 2H mm wouwmhwcz aH om mm+EH can QDOHO o msowo a macho m msouu o msoso u macho m mzoaw < @3090 m Ho.0va mo.ovd Ho.ovm was .a m> u .o m> m .a m> < mpmwsucou A V swamp was flas-oma awe-amc Ams-wmv ham-ssc Am-oa an-ec flN-oc mouamauma n.mm n.wm m.nm N.om n.H n.HH o.H mo .02 sec: mZm Hopucou psoaumohh oa 2H am sausages: aH oa am+2H use macho o macho a macaw m macho o macaw u macho m mzoso < @3090 a>H h.m ucm>sflem mm mwmmzuamm .m wcflm: H :uflz wessokmcwzmeu .& pmcfiewm mums mo compmNflcssaH .N oases give with high comp tect adju sign alon as m pTOU (Tab: resi C an( FCA 1 effe< B an< infe( culat Vacci vaccj antil antig effec 55 B. pertussis was also found to be effective as an adjuvant when given by the intraperitoneal route (Table 3). Whether in combination with B. pertussis or not, single doses of both antigens stimulated highly significant resistance (groups A,B,D, and E, P < 0.01), as compared to the control rats (group G). However, the levels of pro- tection conferred when the antigens were combined with B. pertussis adjuvant and given intraperitoneally (groups B and E) were themselves significantly different (P < 0.01) from those obtained with antigen alone (groups A and D). Intramuscular immunization with B. pertussis as an adjuvant (groups C and F) also conferred high levels of protection (P < 0.01). Aluminum hydroxide [Al(OH)3] was almost as effective as B. per— tussis as an adjuvant, when given intramuscularly using both antigens (Table 4). Significant (P < 0.05) and highly significant (P < 0.01) resistance was obtained with the SSA and the IVP respectively (groups C and F), compared to the untreated controls (group H). As before FCA was ineffective (groups A and D), whereas B. pertussis was highly effective in enhancing the protective ability of the antigens (groups B and E; P < 0.01). It was also found that rats which developed resistance to challenge infection (groups B,C,E, and F - Table 4) had detectable levels of cir- culating homocytotropic antibody beginning at the second week post— vaccination. Antibodies detectable by PCA were present 21 days post- vaccination at which time the rats were challenged. The titres of the antibody were depicted in figure 1. The development of resistance following oral administration of antigen was further investigated in order to establish the most effective dosing regimen. .OHSOR HMOCOHHHOQGHHCH .HO HNHSUWDEMHHEH OSH Am EOMHHCN Sufi: C®>HM SOS; MFWWSMRDQ .m MO Poowmm .m>H UCG m m2 .w m> m medac> a Ho.ovm Ho.ovm Ho.ovm Ho.ovm Ho.ova Ho.ovm cam w m> a .Q m> m w m> o w m> w .< m> m .w m> < mummsucow A w emcee cam Amm-flmw ha-cw Am-vc hm-aw mN-Ow flofi-mw mouamauam N.nN N.o 0.0 w.m o.N m.H o.w mo .02 ado: Hcpuccw ccumcsucw am+a>Hqu mm+a>HlmH m>HuaH am+9 comaucm guflz cc>flw ccgz mwmmzpsmm .m mo uccwmm .d>H was H USN m m2 .w m> m Ho.ov.m mo.ovm mo.ovm .m w> m .< m> w .< m> m mcsam> m Ho.ovm Ho.ovm mo.ovm Ho.ovm mz was .2 m> m .2 m> m mz .2 m> o .2 m> w .2 m> m .2 m> < mummnuccw m w swamp can flosm-mnw flasa-wsw Am-ow ha-ow Asma-emw Acm-mmw flos-mw sem-om wepfimauam N.vm m.wm m.N N.o o.nn w.~v m.HN AH.Nm mo .02 sec: Hoeucow Hohuccw sausages: mflzowa< Mmzow~<+a>m am+a>H H mnzowa<+sflea mm mflzowm< use am .H cam > m .P d ”m. O m. Lac: V . u .m we. 3 iv w mu .w m we. mfcj<¢2m am dam mic:<.