IMMUNQLOGiCAL EVENTS ASSOCIATED A WETH TAEI‘HA TAEMAEFGRMIS iNFgC‘mNS iN, THE LABORATORY, RAT Dissertation for the Degree of Ph. D. MECHEGAN STATE UNWERSITY R. WES LEtD 1973 _ LIBRARY ' 1 Wed” This is to certify that the - thesis entitled IMMUNOLOGICAL EVENTS ASSOCIATED WITH TAE'IVIA TAE'NIAE- FORMIS INFECTIONS IN THE LAKJRATORY RAT presented by R. Wes Leid has been accepted towards fulfillment of the requirements for % degree in Microbiology nd Public Health .32 WW Major professor Date 7/9/71? ill: on; t 330x mum we uunAfijl am" | 45;“:- w. 1;" 1-5 $~~ ABSTRACT IMMUNOLOGICAL EVENTS ASSOCIATED WITH TAENIA TAEWLAEFORMLS INFECTIONS IN THE LABORATORY RAT By R. Wes Leid Serum from rats infected for 28 days with cysticerci of Taenia taeniaefbrmis was shown to protect recipient rats against homologous challenge. Furthermore, this activity resided in the globulin fraction of immune serum as determined by (NH4)ZSO precipitation. Gel filtra- 4 tion and anion exchange chromatography of immune globulin solutions resulted in a separation of rat globulins into yM, Y2a'+ YZb’ YZa‘+ Y2b + 7E and Y2a + YZb + Y1 containing fractions. Highly significant pro-A tection (P<0.001, 0.001 and 0.01, respectively) was associated only with the latter 3 fractions, the DEAE eluates corresponding to 0.01M phosphate, 0.05M phOSphate and 0.10M phosphate. Further chromatography gave rise to antibodies of a single well defined immunoglobulin class, 7SY2a’ which was able to confer highly significant protection (P<0.0l) when given alone. The protective capacity exhibited by the 0.01M, 0.05M and 0.10M eluates may have been due to the presence of 78y2a in all these fractions as it was a consistent feature detectable by double immunodiffusion and immunoelectrophoresis. The presence of the different immunoglobulin classes in each fraction was detected by mono— specific antiserum to each class. A variety of absorption techniques were used in attempts to reduce the effectiveness of immunoglobulin R. Wes Leid solutions. All were unsuccessful and may have resulted from a lack of effective antigens in extracts from cysticerci of T. taeniaefbrmis. Infection with cysticerci of T. taeniaefbrmis resulted in the appearance of skin fixing antibody or reagin during the third week of infection. Peak titers were reached during the fifth week and declined thereafter. The physico-chemical and biological characteristics of this reagin were consistent with those of the rat immunoglobulin, designated yE. In no instance was it possible to demonstrate a short term skin- fixing response due to 73y23 in protective serum from 28-day-old infections even though antibodies were present in this class. Nonethe- less, short-term sensitization was possible at 2-6 hours and this reactivity was determined by physico-chemical means to be due solely to reagin or 7E. An allergen implicated in the reagin response was purified to a single defined protein, which contained carbohydrate but no lipid, was negatively charged and was able to provoke passive cutaneous anaphylaxis (PCA) in sensitized rats in approximately 5 pg quantities. A monospe- cific antiserum raised to this allergen removed all PCA reactivity from larval extracts. However, allergenic activity was only slightly reduced by absorption of adult worm extracts with this same antiserum. It was totally ineffective in reducing the ability of in vitro culture concentrates of the cysticerci to provoke PCA. Thus the presence of two different allergens was indicated. The partial removal of aller- genic activity from adult worm extracts might indicate the presence of yet a third allergen. This is only the second instance in which a parasitic allergen has been so purified and the presence of more than one allergen shown to occur in helminthic extracts. R. Wes Leid This work has demonstrated the ability of antibodies in a single well defined immunoglobulin class, 7SY2a, to passively protect recipient rats. A pure allergen associated with the reaginic antibody response was isolated and partially characterized. IMMUNOLOGICAL EVENTS ASSOCIATED WITH TAEWIA TAENIAEFORMIS INFECTIONS IN THE LABORATORY RAT By I 5/. it” bf RZ'Wes Leid A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1973 Dedication This dissertation is dedicated to my wife Katie and my sons Rory and Jeffrey ii ACKNOWLEDGEMENTS I am extremely appreciative of my privilege in working with my mentor, Dr. J. F. Williams, during the course of this study. For his guidance and encouragement I am very grateful and will remember him as a teacher, colleague and friend throughout my career. I would also like to thank the following people for their valuable assistance during the course of this work: Ms. Anndy Whipple, Dr. David H. Bing, Dr. A. J. Musoke, A. Wayne Roberts, Dr. G. R. Carter, Dr. Kathy Morris and Ms. Sandy Spurlock. I would like to say thank you to my wife Katie for her help in the pursuit of my degree as without her moral and financial support this would not have been possible. Also I would extend my appreciation to the Department of Microbiology and Public Health for their financial assistance and the opportunity to study at Michigan State University. Finally I would like to thank my guidance committee for their encouragement throughout my tenure here: Dr. H. C. Miller, Dr. H. W. Cox, and Dr. R. A. Patrick, who replaced Dr. D. H. Bing upon the latter's move to Harvard. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . . . 3 General Biology, Epidemiology, Pathology and Economics of Taeniid Infections . . . . . . . . . . . . . 3 Taenia saginata. . . . . . . . . . . . . . . . . . . 3 Taenia solium. . . . . . . . . . . . . . . . . . . . 7 Taenia taeniaefbrmis . . . . . . . . . . . . . . . . 9 Rat Imuno logy O O O O O O O O O O O O O O O O O O O O O O O 14 Reagin O O O O O O O O O O O O O O O O O O O O O O O O O O O 16 REFERENCES 0 O O I O O O O O O O O I O O O O O O O O O O O O O O O 20 ARTICLE 1 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMLS. I. IMMUNOGLOBULIN CLASSES INVOLVED IN PASSIVE TRANSFER OF RESISTANCE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . 29 INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . 30 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . 31 Maintenance of the parasite. . . . . . . . . . . . . 31 Experimental animals . . . . . . . . . . . . . . . . 31 Passive transfer . . . . . . . . . . . . . . . . . . 32 Preparation of immune sera . . . . . . . . . . . . . 32 Immunoelectrophoresis (I.E.P.) and double immunodiffusion (D.I.D.) . . . . . . . . . . . . . 32 Measurement of protein concentration . . . . . . . . 33 Chromatography . . . . . . . . . . . . . . . . . . . 33 Preparation of antisera. . . . . . . . . . . . . . . 34 Metacestode saline extracts. . . . . . . . . . . . . 40 Absorption of immune globulins . . . . . . . . . . . 40 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . 41 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . 55 ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . . . 61 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . 62 iv Page ARTICLE 2 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENJA TAEWIAEFORMLS. II. CHARACTERISATION OF REAGINIC ANTIBODY AND AN ALLERGEN ASSOCIATED WITH THE LARVAL STAGE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . 68 INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . 69 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . 70 Maintenance of the parasite. . . . . . . . . . . . . 70 Experimental animals . . . . . . . . . . . . . . . . 70 Reaginic serum . . . . . . . . . . . Immunoelectrophoresis (I-E-P-) and double immunodiffusion (D.I.D.) . . . . . . . . . . . . . 71 Preparative electrophoresis. . . . . . . . . . . . . 71 Measurement of protein concentrations. . . . . . . . 72 Chromatography . . . . . . . . . . . . . . . . . . Homologous passive cutaneous anaphylaxis (PCA) . . . 73 Polyacrylamide gel electrophoresis (PAGE). . . . . . 74 Parasite extracts. . . . . . . . . . . . . . . . . . 74 In vitro maintenance of T. taeniaefbnmis . . . . . . 75 Preparation of antisera. . . . . . . . . . . . . . . 75 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . 76 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . 106 ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . . . 113 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . 113 LIST OF TABLES Table Page ARTICLE 1 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMLS. I. IMMUNOGLOBULIN CLASSES INVOLVED IN PASSIVE TRANSFER OF RESISTANCE l PASSIVE PROTECTIVE CAPACITY OF IMMUNE SERUM AND IMMUNO- GLOBULIN FRACTIONS IN RECIPIENT RATS CHALLENGED WITH 300 EGGS OF TAENIA TAENIAEFORMLS. . . . . . . . . . . . . . 48 2 PASSIVE PROTECTIVE CAPACITY OF IMMUNE SERUM AND IMMUNO- GLOBULIN FRACTIONS IN RECIPIENT RATS CHALLENGED WITH 600 EGGS OF T. TAENIAEFORMIS. . . . . . . . . . . . . . . . 52 ARTICLE 2 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMIS. II. CHARACTERISATION OF REAGINIC ANTIBODY AND AN ALLERGEN ASSOCIATED WITH THE LARVAL STAGE 1 OPTIMAL SENSITISATION OF RAT SKIN WITH REAGINIC ANTIBODIES TO TAE’NIA TAENIAEFORMIS. . . . . . . . . . . . . 81 2 SENSITIVITY OF RAT REAGIN TO T. TAEWIAETORMIS T0 HEAT TREATMENT AND REDUCTION AND ALKYLATION . . . . . . . . 83 3 CROSS REACTIVITY OF VARIOUS HELMINTH EXTRACTS . . . . . . . 105 vi LIST OF FIGURES Figure Page 1 Life cycle of Taenia saginata . . . . . . . . . . . . . . . 6 2 Life cycle of Taenia taeniaefbrmis. . . . . . . . . . . . . 12 ARTICLE 1 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAETURMIS. I. IMMUNOGLOBULIN CLASSES INVOLVED IN PASSIVE TRANSFER OF RESISTANCE l Immunoelectrophoretic analysis of normal rat serum (NRS) versus Fc specific guinea pig anti—7872 (l), guinea pig anti-7371 (2), sheep anti-YM (3) and sheep anti-yA (4). . . 37 2 Elution profile at 280 nm of the globulin fraction (50% (NH4)ZSO4) of immune rat serum after gel filtra- tion on Sephadex G-200. Fraction 1 was concentrated and tested for protective activity. Fraction 2 was further fractionated on DEAE-cellulose. . . . . . . . . . . 44 3 Elution profile at 280 nm of the 78y globulin fraction (50% (NH4)ZSO4) of immune rat serum fractionated on DEAE cellulose with phosphate buffers and 2M NaCl. All phosphate buffers were made 0.015M in NaCl. Fractions 1, 2 and 3 were tested for activity in passive transfer experiments. . . . . . . . . . . . . . . . 46 4 Elution profile at 280 nm of the 78y globulin fraction (50% (NH4)ZSO4) of immune rat serum fractionated on DEAE cellulose with phosphate buffers and 2M NaCl. All phosphate buffers were made 0.015M in NaCl. Fractions 1 and 2 were tested for activity in passive transfer experiments. . . . . . . . . . . . . . . . . . . . 51 5 Representative livers from groups of rats passively immunised with the DEAE fractions indicated. The animals were challenged per 08 with 600 eggs of T. taeniaefbrmis and sacrificed 21 days later. . . . . . . . . 54 6 Immunoelectrophoretic analysis of normal rat serum (bottom well) and fraction 1, 0.005M eluate (upper well) from DEAE cellulose fractionation (FIG. 4) versus rabbit anti-whole rat serum (aWRS). . . . . . . . . . . . . 57 vii Figure Page ARTICLE 2 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENLA TAEWIAETORMLS. II. CHARACTERISATION OF REAGINIC ANTIBODY AND AN ALLERGEN ASSOCIATED WITH THE LARVAL STAGE 1 An analysis of the pattern of appearance of skin sensi- tising antibody (or reagin) to infection with Taenia taeniaefbrmis in the rat. Serial bleedings were obtained at the times indicated and PCA tests done with the sample. A booster dose of 1,000 eggs was given 81 days after primary infection (arrowed) . . . . . . 78 2 Elution profile at 280 nm of rat serum obtained 35 days after infection with T. taeniaefbrmis and passed through Sephadex G-200. Fractions were pooled as indicated and those with pOsitive PCA reactivity are shown in black. . . . . . . . . . . . . . . . . . . . . . . 85 3 Elution profile at 280 nm of rat serum Obtained 35 days after infection with T. taeniaefomis and sub— jected to DEAE cellulose chromatography. All phosphate buffers were made 0.015M in NaCl. Fractions indicated were pooled and tested in PCA. Reactivity was limited to the blacked out areas. . . . . . . . . . . . . . . . . . 88 4 Elution profile at 280 nm of extract prepared from cysticerci of T. taeniaefbrmis and subjected to DEAE cellulose chromatography. All buffers were made 0.015M in NaCl. Fractions indicated were pooled and tested for their ability to provoke PCA in sensitised rats. Positive reactivity is indicated by blacked out areas . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 5 Elution profile at 280 nm of extract prepared from cysticerci of T. taeniaefbrmis and subjected to CM Sephadex chromatography. Fractions indicated were pooled and tested for their ability to provoke PCA in sensitised rats. Positive reactivity is indicated by blacked out areas. . . . . . . . . . . . . . . . . . . . 94 6 Elution profile of an extract prepared from cysticerci of T. taeniaefbrmis eXpressed as protein content of 1 cm fractions from Pevikon block electrophoresis. Fractions were pooled as indicated and tested for their ability to provoke PCA in sensitised rats. Reactivity was demonstrable in pools 4-10 although the most intense reactions were seen with 7-10. . . . . . . 96 7 Five percent polyacrylamide gel electrophoresis of pools l-lO from preparative electrophoresis of larval extract. Fractions were stained with a 1% solution of aniline blue black . . . . . . . . . . . . . . . . . . . 99 viii Figure Page Pool 9 from preparative block electrophoresis sub— jected to 5% polyacrylamide gel electrophoresis and stained for protein (1), carbohydrate (2) and lipid (3) . . 101 Immunoelectrophoretic analysis using sheep antiserum against an allergen from cysticerci of T. taeniae- fbrmis in the troughs. Antigens in the wells from top to bottom were as follows: adult extraCt, larval -extract, larval extratt and in vitro-culture concentrate o o I O o o o o o o o o o o o o o 10 3 ix INTRODUCTION Cysticercosis and hydatidosis are cyclo-zoonotic infections caused by members of the order Cyclophyllidea, Family Taeniidae. The cosmo- politan distribution of these parasites demonstrates that members of this parasite family are not limited by geographical boundaries and infection rates are high under a variety of socio-economic conditions. The larval or cysticercus stage of taeniid parasites is of greatest concern to man and domesticated food animals. Both the economic loss in protein from infected animals and the loss in human health and pro- duction have come to assume increased importance as other major world diseases are brought under control. These losses are of particular relevance in the developing countries of the world where reduction in total protein available for food consumption and lowered industrial output due to worker absence can be ill afforded. Since the cysticerci are not susceptible to any practical chemotherapy and indeed may only be treated by surgical excision if their location is amenable to surgery, immunological control of the cysticerci offers one avenue of therapy that should be pursued. The immunological events associated with specific acquired resistance to these helminths remain ill-defined and a further analysis of these phenomena in laboratory and food animals may permit specific prophylaxis and therapy. The rat Toenia taeniaefbnmis system has served as an eXperimental model system for investigations on cysticercosis with particular emphasis on the immunology of such an infection. The literature review 1 2 has been divided into three main headings to provide a background on clinical and experimental cysticercosis and rat immunology. The first area of concern is the general biology, epidemiology, pathology and economics of infection with taeniid parasites. Here the emphasis is on Thenia solium and T. saginata and their significance both to public health and veterinary medicine. The second section discusses the immunology of experimental infec- tions in domestic animals and the biology and immunology of T. taeniae- fbrmis infections in the rat. Taenia taeniaefbrmis is discussed in relation to the immunological reactions occurring during parasitism and how these may be comparable to those seen in the more costly large animal systems. The last topic covers rat immunology with a particular emphasis on reagin mediated immediate hypersensitivity reactions. Earlier workers had indicated that serum antibody was important in passive protection to T. taeniaefbrmis and this section discusses the nomenclature, biological and physico-chemical properties of the different immuno- globulin classes in the rat. The allergic response is a prominent feature of helminthiasis in general and of cysticercosis in particular and with the pathogenesis of taeniid infections resulting from an intense hypersensitivity reaction. Therefore, reagin mediated hyper— sensitivity has been reviewed at some length with respect to the type of antibody involved, the molecular characteristics of such a protein and the means by which clinical allergic disease is manifested. LITERATURE REVIEW General Biology, Epidemiology, Pathology and Economics of Taeniid Infections Cestodes of the genera Taenia and Echinococcus are members of the Family Taeniidae, Order cyclophyllidba, one of the two medically important orders of cestodes in both man and domesticated animals. Members of this family have a life cycle which includes a larval stage in an intermediate host with the adult worm in a definitive host. Taenia saginata Biology. The life cycle of Taenia saginata is shown in Figure l and is typical of taeniid parasites. Taenéa saginata, the beef tape- worm, resides as an adult in the small intestine of man with the larval stage present in the muscles of cattle. It is a cosmopolitan parasite with a more widespread distribution than that exhibited by T. solium. Man becomes infected by the consumption of raw or undercooked beef con- taining the cysticerci. Cattle become parasitized by the ingestion of eggs present on pastures contaminated by human feces. Epidemiology, Urquhart (1961) has suggested that dairy calves in East Africa may also become infected during the first few days of life by the native stockman when mucus is cleaned from the calves' mouths or when the fingers are used to induce the calf to drink from a pail. A small number of eggs underneath the fingernails would be capable of 3 4 producing the infections seen and it has been noted that approximately 30% of the dairy cattle in East Africa have cysticerci (Soulsby, 1965). Silverman and Griffiths (1955) have suggested that an indirect means of transmission may occur in Great Britain whereby sea birds pick up eggs as a result of feeding on raw sewage, with the subsequent passage of these eggs through the birds' digestive tracts. This provides for con- tamination of pastures where cattle are grazing. This mechanism would help explain infection of cattle in the absence of adult T. saginata in peOple on the farm or in the surrounding locality. Although T. saginata is present in most countries of the world, the incidence of both the larval stage and the adult are particularly high in Africa with East African states having the highest rates. Froyd (1960) has found 31.7% of the people in Kenya harboring the parasites. Urquhart (1961) has reviewed reports on infection levels in Africa and these figures reach levels of 75-86% of the cattle parasitized with cysticerci of T. saginata depending upon the locality. Economics. Presence of cysticerci in cattle represents a serious economic loss not only in terms of outright condemnation of the carcass but also in a reduction of the value of the meat containing small numbers of the larvae. In the case of carcasses with low numbers of the parasites, the meat must be kept frozen for several weeks to kill the cysticerci and as a result a lower price is obtained at marketing. Pathology and Control. Under normal conditions infection with larvae does not cause clinical disease in cattle. The adult worms bring about little pathology in the human host and can be successfully removed by various chemical agents. Prevention and control of the Figure 1. Life cycle of Taenia saginata. MA cysticercus 99 Figure l 7 disease could be effected should generally accepted personal hygienic and community health standards be followed. Taenia soZium Biology and Epidemiology. Thenia solium, the pork tapeworm, resides as an adult cestode in the small intestine of man with the pig serving as the usual intermediate host. Man becomes infected with the adult parasites by the consumption of raw or undercooked meat and may also become infected with the larval stage by the ingestion of infective eggs (Soulsby, 1965). The global intensity of this infection is not as high as that of T. saginata. Pathology. If the larval stage is present in man, serious complica— tions may be brought on as there is a marked tendency for the cysticerci to localize in the brain (Faust, Russell and Jung, 1970). In a study of cysticercosis in Mexico, 25 out of 100 cases diagnosed as human cerebral tumors were actually a result of infection with cysticerci of T. soZium (Mazzoti, 1944). Also, epileptiform attacks are frequently seen in human beings after death and degeneration of the cysticerci (Soulsby, 1965). Clinically these larvae when found in the brain have been referred to as cysticercus racemosus because of their tendency to expand within the cerebral cavities. The larval stage has also been found in the eye, musculature, heart, liver, lungs and abdominal cavity. The presence of the growing parasite provokes a local cellular reaction which includes infiltration with neutrophils, eosinophils, lymphocytes, plasma cells and sometimes giant cells. Fibrosis and necrosis follow this cellular picture with an eventual caseation and calcification of the cyst. These pathological findings are of tremendous importance should the larvae localize in the brain. Here the parasite may cause 8 little pathology while alive but upon death a great variety of neuro— logical symptoms may develop, which can lead to a rapid demise of the patient. Such parasitized individuals may show varied clinical signs, including epilepsy, epileptiform seizures, disordered behavior, transient pareses, intermittent obstructive hydrocephalus, dysequilibrium, meningo- encephalitis and failing vision (Faust, Russell and Jung, 1970). Economics. In addition to the pathology of the disease in humans, this infection in the intermediate host, swine, constitutes both an important financial loss and also a loss in available protein. In a 3-year study of cysticercosis in slaughter houses of 6 Latin American countries, Garrick (1967) showed that 2.13% of all hogs slaughtered were infected with cysticerci of T. solium, with a resultant loss of over $500,000 in condemned carcasses. Control and Treatment. The only known treatment for infection with the larval stage in man is surgical removal and, if the larvae have spread throughout the cerebral cavities, the prognosis is very grave. As with T. saginata, adult worms cause little pathology and can be removed by chemical means. Prevention of this disease revolves around the two basic concepts, personal hygiene and general sanitary measures (Faust, Russell and Jung, 1970). As long as people in endemic areas continue to eat raw or undercooked pork and do not dispose of human excreta according to accepted modern sanitary practices, the parasite and the disease which it causes will flourish. Immunology of Experimental Infections. Immunologically-mediated host defense reactions have been shown to develop in domestic animals against cysticerci of taeniid parasites. Naturally acquired resistance 9 has been demonstrated in sheep infected with T. hydbtigena (Gemmell, 1961, 1969a; Sweatman, Williams, Moriarty and Henshall, 1963), T. ovis (Gemmell, 1969a) and Ebhinococcus granulosus (Sweatman, 1957). Also cattle under field conditions are resistant to superinfection with T. saginata (Urquhart, 1961). Experimentally, sheep infected or arti- ficially immunized with T. hydhtigena. T. ovis or E. granulosus are resistant to homologous challenge infections (Gemmell, 1962, l964a,b, 1965a, 1966; Rickard and Bell, 1971a). Soulsby (1963) showed that cattle infected with T. saginata were resistant to superinfection by the same parasite. Furthermore, resistance to challenge infections with T. hydbtigena in sheep could be passively transferred to recipient animals by serum (Blundell, Gemmell and Macnamara, 1968) and also by colostrum (Gemmell, Blundell-Hassell and Macnamara, 1969). Taenia hydatigena embryos treated by physical or chemical means were capable of eliciting a strong resistance in sheep to challenge infection (Gemmell, 1969b). These embryos did not develop into mature metacestodes and, since killed eggs or activated embryos did not induce immunity (Gemmell, 1964b, 1969b), it seems that elaborated antigens of the parasite are responsible for induction of acquired resistance. In this regard the experiments of Rickard and Bell (1971b) are important. Their studies indicated that antigens excreted or secreted by growing larvae of T. ovis contained in membrane diffusion chambers and implanted into peritoneal cavities of sheep were responsible for protection to challenge infections. Taenia tqgniaeffirmis Biology. The laboratory animal model for cysticercosis of T. taeniaefbrmis in the rat has served in the study of the immunological 10 reactions to these tissue invading parasites. The life cycle of this tapeworm is shown in Figure 2. This particular parasite in the field is cycled through rodents (rats and mice) as intermediate hosts while the cat serves as the definitive host in a predator-prey relationship. The intermediate host becomes infected by the ingestion of eggs. The egg is composed of an outer layer of keratinized blocks with several membranes inside of the blocks and finally a 6-hooked or hexa- canth embryo within the last membrane (Morseth, 1965, 1966). The embryophoric blocks are passively digested away as the egg is carried through the stomach and into the small intestine. At the latter site the intestinal enzymes set in motion, through an as yet undetermined means, the activation of the larvae. The embryo initiates intense movement and tears the oncospheral membrane, moving to the villi of the small intestine (Silverman and Maneely, 1955; Banerjee and Singh, 1969a,b; Heath, 1971). Penetration of the villus takes place within 15 minutes (Banerjee and Singh, 1969a) and occurs through lysis of host tissues, destruction of the same tissue by the hooks of the embryos or a combination of both methods (Silverman and Maneely, 1955; Banerjee and Singh, 1969a,b; Heath, 1971). The embryo migrates until it reaches a venule which carries the larvae passively to the site of predilec- tion, a place that varies according to the species described (Heath, 1971). In the case of T. taeniaefbrmis the final site is the liver, through which the embryo migrates until such time as the host encap- sulates it with fibrous 'tissue' (Singh and Rao, 1967; Smyth and Heath, 1970). The larvae continue development until the parasite reaches a fully infective metacestode stage by 60 days after infection (Hutchison, 1958). It is termed at this time a bladder worm and 11 Figure 2. Life cycle of Taenia taeniaeformis. 12 CAT cysticercus egg RAT Figure 2 l3 morphologically shows several analogies to the mammalian embryo (Smyth, 1969). Upon oral consumption of this infective cysticercus by the defini- tive host the scolex evaginates from the bladder, attadhes to the small intestine, usually in the crypts and initiates new growth, finally developing into an adult cestode. Infective eggs and gravid proglottids are passed in the cat feces 45 days later and thus the cycle is main— tained. Similar cycles are perpetuated using other intermediate and definitive hosts for the remaining members of this family, Taeniidae. Immunology. Miller (1931a) demonstrated conclusively that the rat becomes resistant to superinfection with T. taeniaefbnmis. Further experimental work by Miller and his colleagues showed that resistance could be artificially induced, passively transferred with serum from infected rats and maternally transferred to young rats from females with experimental infections (Miller and Gardiner, 1932, 1934; Miller, 1931b, 1932, 1935). A more detailed examination of this work was accomplished by Campbell (1936, 1938a,b,c). He confirmed Miller's early observations on the success of artificial immunization in the T. taeniaefbrmis system and extended those findings by testing a variety of antigenic and immunogenic preparations. He demonstrated that serum obtained at increasing times post infection was capable of conferring greater resistance to challenge in recipients than serum taken early in the infection. His further studies on the passive transfer of resistance led him to postulate the possible existence of two distinct populations of antibodies capable of mediating resistance to challenge. He visualized two processes which he termed "early" and "late" immunity. The former 14 was characterized by its appearance early in the infection, ability to cause parasite death before liver establishment and susceptibility to removal by absorption with larval extracts. The "late" immunity involved serum antibodies that not only brought about destruction of larvae before establishment but also under appropriate conditions were able to result in death of parasites after their successful invasion of the liver. This late protective capacity was not for the most part reduced by any absorption procedures. Rickard and Bell (1971) showed that implantation into recipient rats of growing larvae of T. taeniaefbrmis, contained within membrane diffusion chambers, was capable of inducing a significant protection to challenge. Reduction in parasite numbers was observed with 1 week implants and complete resistance was demonstrated at 3 weeks. This would indicate that the living parasite is important in the induction of a protective response by the host. Murrell (1971) showed that larvae incubated in serum obtained 35 days after infection with T. taeniaefbrmis were unable to control efflux and influx of certain radiolabelled amino acids and sugars. Further- more, this reaction was shown to be in part complement dependent. The direct extrapolation from the rat T. taeniaefbrmis model to taeniid infections in domestic animals and man is not justified. None- theless, the knowledge gained from the rat model on the immunological events associated with T. taeniaefbrmis infections may serve to illumin- ate areas for fruitful exploration in the larger animals. Rat Immunology The physico-chemical and biological properties of immunoglobulins in general, and those of the rat in particular, have been studied 15 extensively over the past decade. In the rat at the present time, there are 6 classes of distinct immunoglobulins demonstrable: 7SY2a’ 7SY2b9 7SY1: YA: YE: and NM: Arnason, de Vaux St-Cyr and Relyveld (1964) demonstrated that the rat had at least 3 immunoglobulin classes as revealed by immunoelectro- phoresis: yG, yM and 1A. No attempt was made at this time to isolate the 7A protein. The yA molecules were so named because of their electro- phoretic migration. Banovitz and Ishizaka (1967) were able to show antigen binding in 5 precipitin arcs using radioimmunoelectrophoresis. The 1G class has been separated electrophoretically into 2 com- ponents (Nussenzweig and Binaghi, 1965) designated by 7872a and 7872b (Binaghi and Sarandon de Merlo, 1966). These 2 classes were able to be separated chromatographically on DEAE-cellulose using a low ionic strength initial buffer (Stechschulte, Austen and Bloch, 1967; Bloch, Morse and Austen, 1968). Both of the 78y2 antibody classes have agglu- tinating and hemolytic properties (Morse, Bloch and Austen, 1968). The 7Sy23 antibodies also have the capacity to fix for short periods of time in recipient rats for participation in passive cutaneous anaphy- laxis (PCA) reactions. They also prepare rat peritoneal cells for the antigen induced release of slow reacting substance of anaphylaxis, SRS-A (Stechschulte et aZ., 1967; Orange, Valentine and Austen, 1968; Morse et a1. , 1968). The rat immunoglobulin class originally designated yA by Arnason et al. (1964) and Binaghi and Sarandon de Merlo (1966) has now been shown to be a 7Sy1 (Jones, 1969), although it is not the biological EQUivalent of 7SY1 from mice and guinea pigs (Binaghi, 1971). This antibody class has been shown to have agglutinating activity. It also binds complement and is active in hemolytic assays (Jones, 1969; Morse 16 et aZ., 1968), although its 1ytic properties were not as pronounced as with the 7Sy2 antibodies. There has now been shown to be an immunoglobulin class designated yA which predominates in secretions and is prominent at mucosal sur- faces (Nash, Vaerman, Bazin and Heremans, 1969; Stechschulte and Austen, 1970; Bistany and Tomasi, 1970; Nash and Heremans, 1972). It is, however, present at very low levels in serum and all of its physico- chemical characteristics have been shown to be similar to yA molecules in other species. The 1M immunoglobulin class has been studied extensively by Binaghi and his colleagues (Binaghi and Oriol, 1968; Oriol, Binaghi and Coltorti, 1971). They have defined an antibody class which has 30 times more agglutinating activity than the 7S antibodies and is approxi- mately 300 times as effective in lysis of sensitized red cells. They have shown that the rat 7M antibody molecule has a valence of 10, which is similar to that now demonstrated experimentally for 1M antibodies of other species. Other workers (Van Breda Vriesman and Feldman, 1972) have shown that the half-life of electrophoretically slow yM is between 60-65 hours. Reagin Rat reaginic antibody, which appears in response to both immuniza- tion and infection (Binaghi and Benacerraf, 1964; Ogilvie, 1967) has now been defined by Austen and his co-workers (Stechschulte, Orange and Austen, 1970) to be the rat immunoglobulin yE. This antibody was similar to the immunoglobulin class designated yE in humans by Ishizaka and Ishizaka (1967). Orange, Stechschulte and Austen (1970) demonstrated that this yE antibody sensitized rat peritoneal cells for antigen l7 induced release of histamine and SRS—A. 7E antibody was also shown to be responsible for long-term skin fixation (Stechschulte at al., 1970). The optimal conditions for the in vitro release of histamine has been further studied by Bach, Bloch and Austen (l97la,b). A possible compe- tition for receptor sites on the peritoneal cells between the yE and 7872a antibodies has been demonstrated both in vitro and in viva (Bach et aZ., 1971b; Ohman and Bloch, 1972). Immediate hypersensitivity reactions have been shown to be mediated by skin sensitizing antibodies. In many animal species studied, there are two types of these skin fixing antibodies, one which fixes to target cells for short periods of time (7G) and another (1E) which sensitizes cells for an extended period (Binaghi, 1971; Bloch, 1968; Bloch and Ohman, 1971; Ishizaka, 1971). In humans, however, no short- term activity due to yG molecules has been conclusively demonstrated. Reagin or yE mediated hypersensitivity reactions appear to be one of the main causes of clinical allergic disease. It has been known for over 50 years that this immediate type hypersensitivity reaction could be passively transferred with serum. This fact indicated that a serum antibody was responsible for the immediate reactions rather than a cellular involvement of the delayed type as seen with a tuberculin skin reaction. As the separation of plasma proteins became more refined and the immunoglobulins of man and other animal species were determined, more intensive investigations on the nature of the skin sensitizing antibody were initiated. Early investigators (Ishizaka, Ishizaka and Hornbrook, 1963; Vaerman, Epstein, Fudenburg and Ishizaka, 1964) suggested that the immunoglobulin class yA might be the carrier of reaginic hypersensi- tivity. However, later it was shown through the use of an elaborate 18 physico-chemical separation procedure and a sensitive radioimmunoassay that reaginic activity was confined to a new immunoglobulin class, 7E (Ishizaka, Ishizaka and Hornbrook, l966a,b). Most of the previous work was confirmed when a myeloma protein was obtained which did not cross react with the known heavy chain specific antisera, but did show the presence of immunoglobulin light chains (Johansson, Bennich and Wide, 1968). The biological, physical and chemical properties of this immuno- globulin class were determined in a series of investigations by Johansson and his colleagues (Johansson and Bennich, 1967; Bennich, Ishizaka, Ishizaka, and Johansson, 1969; Johansson, Bennich and Berg, 1970; Bennich, 197la,b). The Ishizakas and their colleagues (Ishizaka and Ishizaka, 1968a,b, l969a,b; Ishizaka, Ishizaka and Lee, 1970a; Ishizaka, Tomioka and Ishizaka, 1970b; Ishizaka, Johansson and Bennich, 1969; Ishizaka and Ishizaka, 1971; and Ishizaka, 1971) also played a major role in investigations on yE. These studies showed a molecule with an approximately 88 sedimen- tation coefficient, a molecular weight of 190,000-200,000, a fast gamma electrophoretic ability, a susceptibility to heat and reduction and alkylation. This antibody also failed to cross the placenta and had a half-life at passively sensitized sites of 8.5-14 days as opposed to 2-3 days in the serum. The levels in normal individuals were shown to be around 100-300 ng/ml. This antibody lacked any complement fixing activity even when aggregated (Ishizaka, Soto and Ishizaka, 1972). Through inhibition studies, it has become evident that the yE antibody binds to target cells by the Fc portion of the immunoglobulin molecule (Stanworth, Humphrey, Bennich and Johansson, 1968; Ishizaka et al., 1970a). This fixation by the Fc portion of the immunoglobulin would allow binding of specific antigen by the active sites located on 19 the Fab region. The result would be a process whereby release of vaso- active components from the target cells would occur by secretory means. This is in contrast to the cellular destructive pathways as would be the case should 7G molecules be involved (Lichtenstein, 1972). The vasoactive mediators, primarily SRS-A and histamine, would be responsible for the clinical signs observed as these agents have profound effects on smooth muscles and vascular permeability (Lichtenstein, 1972). REFERENCE S REFERENCES Arnason, B., Vaux St-Cyr, C. and Relyveld, E. (1964). 'Role of the thymus in immune reactions in rats. IV. Immunoglobulins and antibody formation.’ Int. Arch. Allergy, 25, 206-224. Bach, M. K., Bloch, K. J. and Austen, K. F. (1971a). 'IgE and IgGa antibody-mediated release of histamine from rat peritoneal cells. 1. Optimum conditions for in vitro preparation of target cells with antibody and challenge with antigen.’ J. Exp. Med., 133, 752-771. Bach, M. K., Bloch, K. J. and Austen, K. F. (1971b). 'IgE and IgGa antibody-mediated release of histamine from rat peritoneal cells. II. Interaction of IgGa and IgE at the target cell.‘ J. Exp. Med., 133, 772-784. Banerjee, D. and Singh, K. S. (19693). 'Studies on cysticercus fasciolaris. I. 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Acquired resistance to the larval phase.‘ Aust. Vet. J., 45, 521-524. Gemmell, M. A. (l969b). 'Immunological responses of the mammalian host against tapeworm infections. X. Immunization of sheep against Taenia hydatigena and T. ovis with chemically or physi- cally treated embryos.‘ Exptl. Parasit., 26, 58-66. Gemmell, M. A., Blundell-Hasell, S. K. and Macnamara, F. N. (1969). 'Immunological responses of the mammalian host against tape- worm infections. IX. The transfer via colostrum of immunity to Taenia hydatigena.' Emptl. Parasit.. 26. 52-56- Heath, D. D. (1971). 'The migration of oncospheres of Taenia pisiformis, T. serialis and Echinococcus granulosus within the intermediate host.‘ Int. J. Parasitol., 1, 145-152. Hutchison, W. M. (1958). 'Studies on Hydatigera taeniaefbrmis. I. Growth of the larval stage.' J. Parasit., 44, 574-582. 23 Ishizaka, K. (1971). In Amos, B. (ed.). (1971). Progress in Immunology, p. 859-874. Academic Press, New York. Ishizaka, K. and Ishizaka, T. (1967). 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ImunOZo , 101’ 68-780 Ishizaka, K. and Ishizaka, T. (l969a). 'Physicochemical properties of human reaginic antibodies. VII. Effect of reduction and alkyla- tion on yE antibodies.' J. IMmunol., 102, 69-76. Ishizaka, K. and Ishizaka, T. (1969b). 'Immune mechanisms of reversed type reaginic hypersensitivity.‘ J. Immunol., 103, 588-595. Ishizaka, K. and Ishizaka, T. (1971). 'Mechanisms of reaginic hyper- sensitivity.‘ Clinical Allergy, 1, 9-24. Ishizaka, T., Ishizaka, K., Johansson, 8. G. O. and Bennich, H. (1969). 'Histamine release from human leukocytes by anti-yE antibodies.' J. Immunol., 102, 884-892.. Ishizaka, K., Ishizaka, T. and Lee, E. H. (1970a). 'Biologic function of the fragments of E myeloma protein.‘ Immunochemistry, 7, Ishizaka, K., Tomioka, H. and Ishizaka, T. (1970b). 'Mechanisms of passive sensitisation. I. Presence of IgE and IgG molecules on human 1eukocytes.‘ J. Immunol., 105, 1459-1467. Ishizaka, T., Sota, C. S. and Ishizaka, K. (1972). 'Characteristics of complement fixation by aggregated IgE.' J. Immunol., 109, 1290-1295. 24' Johansson, S. G. O. andiBennich, H. (1967). 'Immunological studies of an atypical (myeloma) immunoglobulin.‘ Immunology, 13, 381-394. Johansson, S. G. 0., Bennich, H. and Berg, T. (1970). In Miescher, F. A. (ed.). (1970). Immunopathology VI International Symposium, pg. 172-180. Schwabe and Co., Basel. Johansson, S. G. 0., Bennich, H. and Wide, L. (1968). 'A new class of immunoglobulin in human serum.‘ Immunology, 14, 265-272. Jones, V. E. (1969). 'Rat 7S immunoglobulins: Characterization of Y2 and 71-Anti-Hapten antibodies.' Immunology, 16, 589-599. Lichtenstein, L. M. (1972). 'Allergy.' In Bach, F. H. and Good, R. A. (ed.). (1972). Clinical Immunobiology, Vol. 1, p. 243-269. Academic Press, New York. Mazzotti, C. (1944). 'Datos sobre la cisticercosis in Mexico.‘ Rev. Inst. salad. y Enferm. Trop., 5, 283-292. Miller, H. M. Jr. (1931a). 'Immunity of the albino rat to superinfes- tation with cysticercus fasciolaris.' J. Prev. Med., 5, 453-464. Miller, H. M. Jr. (1931b). 'The production of artificial immunity in the albino rat to a metazoan parasite.‘ J. Prev. Med., 5, 429- 453. Miller, H. M. Jr. (1932). 'Further studies on immunity to a metazoan parasite, Cysticercus fasciolaris.' J. Prev. Med., 6, 37-46. 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 a metazoan parasite, Cysticercus fasciolaris, 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, cysticercus fasciolaris.' Am. J. Hyg., 20, 424-431. Morse, H. C. III, Bloch, K. J. and Austen, K. F. (1968). 'Biologic properties of rat antibodies. II. Time-course of appearance of antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-Arat); association of this activity with rat IgGa.' J. Immunol., 101, 658-663. Morseth, D. J. (1965). 'Ultrastructure of developing taeniid embryo- phores and associated structures.‘ Exptl. Parasit., 16, 207-216. Morseth, D. J. (1966). 'Chemical composition of embryophoric blocks of Taenia hydatigena, T. ovis and T. pisifbrmis eggs.' Exptl. Parasit., 18, 347-354. 25 Murrell, K. D. (1971). 'The effect of antibody on the permeability control of larval Taenia taeniaefbrmis.‘ J. Parasit.. 57. 875-880. Nash, D. R. and Heremans, J. F. (1972). 'Intestinal mucosa as a source of serum IgA in the rat.‘ Inmunochemistry, 9. 461-464- Nash, D. R., Vaerman, J. P., Bazin, H. and Heremans, J. F. (1969). 'Identification of IgA in rat serum and secretions.‘ J. Immunol., 103, 145-148. Nussenzweig, V. and Binaghi, R. A. (1965). 'Heterogeneity of rat immunoglobulins.’ Int. Arch. Allergy, 27, 355-360. Ogilvie, B. M. (1967). 'Reagin-like antibodies in rats infected with the nematode parasite Nippostrongylus brasiliensis.' Immunology. 12, 113-131. Ohman, J. L. Jr. and Bloch, K. J. (1972). 'Interaction in vivo of homocytotropic antibodies belonging to two different rat immuno- globulin classes: Effect of IgE on the passive cutaneous ana- phylactic reaction mediated by IgG8 antibodies.' J. Immunol., 108, 1637-1646. Orange, R. P., Stechschulte, D. J. and Austen, K. F. (1970). 'Immuno- chemical and biologic properties of IgE. II. Capacity to mediate the immunological release of histamine and slow-reacting substance of anaphylaxis (SRS-A).' J. Immunol., 105, 1087-1095. Orange, R. P., Valentine, M. D. and Austen, K. F. (1968). 'Antigen induced release of slow reacting substance of anaphylaxis (SRS-A rat) in rats prepared with homologous antibody.' J. Exp. Med., 127, 767-782. Oriol, R., Binaghi, R. A. and Coltorti, E. (1971). 'Valence and association constant of rat macroglobulin antibody.' J. Immunol., 104, 932-937. Rickard, M. D. and Bell, K. J. (1971a). 'Successful vaccination of lambs against infection with Taenia ovis using antigens pro- duced 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. taeniaeformis infection in lambs and rats following in vivo growth of their larvae in filtration membrane diffusion chambers.‘ J. Parasit., 57, 571-575. Silverman, P. H. and Griffiths, R. B. (1955). 'A review of methods of sewage disposal in Great Britain, with special reference to epizootiology of Cysticercus bovis.' Ann. Trop. Med. Parasit., 26 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 hexacanth embryo in the penetration of the intestinal mucosa of the intermediate host,and some of its histochemical reactions.‘ Ann. Trop. Med. Parasit., 49, 326-330. Singh, B. B. and Rao, B. V. (1967). 'On the development of Cysticercus fasciolaris in albino rat liver and its reaction on the host tissue.‘ Ceylon Vet. J., 15, 121-129. Smyth, J. D. (1969). The Physiology of Cestoabs, p. 1-279. Oliver and Boyd, London. Smyth, J. D. and Heath, D. D. (1970). 'Pathogenesis of larval cestodes in mammals.‘ Helminthological Abstracts, 39, 1—23. Soulsby, E. J. L. (1963). 'Immunological unresponsiveness to helminth infections in animals.‘ Proceedings 17th International Veteri- nary Congress, (1962), Vol. 1, p. 761-767. Hanover. Soulsby, E. J. L. (1965). Textbook of Veterinary Clinical Pathology, p. 1043-1073. F. A. Davis, Philadelphia. Stanworth, D. R., Humphrey, J. H., Bennich, H. and Johansson, 3. G. O. (1968). 'Inhibition of Prausnitz-KUstner reaction by protec- lytic cleavage fragments of a human myeloma protein of immuno- globulin class E.’ Lancet ii, 17-18. Stechschulte, D. J. and Austen, K. F. (1970). 'Immunoglobulins of rat colostrum.’ J. Immunol., 104, 1052-1062. Stechschulte, D. J., Austen, K. F. and Bloch, K. J. (1967). 'Anti- bodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-A) in the guinea pig and rat.‘ J. Exp. Med., 125, 127-147. Stechsdhulte, D. J., Orange, R. P. and Austen, K. F. (1970). 'Immuno- chemical and biologic properties of rat IgE. I. Immunochemical identification of rat IgE.' J. Immunol., 105, 1082-1086. Sweatman, G. K. (1957). 'Acquired immunity in lambs infected with Taenia hydatigena, Pallas, 1766.' Canadian J. Comp. Med., 21, 65-71. Sweatman, G. K., Williams, R. J., Moriarty, K. M. and Henshall, T. C. (1963). 'On acquired immunity to Echinococcus granulosus in sheep.‘ Res. Vet. Sci., 4, 187-198. Urquhart, G. M. (1961). 'Epizootiological and experimental studies on bovine cysticercosis in East Africa.‘ J. Parasit., 47, 857-869. 27 Vaerman, J. P., Epstein, W., Fudenberg, H. and Ishizaka, K. (1964). 'Direct demonstration of reagin activity in purified YlA globulin.‘ Nature, 203, 1046-1048. Van Breda Vriesman, P. J. C. and Feldman, J. D. (1972). 'Rat YM immunoglobulin: Isolation and some biological characteristics.‘ Immunochemistry, 9, 525-534. THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMIS I. IMMUNOGLOBULIN CLASSES INVOLVED IN PASSIVE TRANSFER OF RESISTANCE R. W. Leid and J. F. Williams Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824, U.S.A. This is journal article No. from the Michigan State University Agricultural Experiment Station. 28 29 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMIS I. IMMUNOGLOBULIN CLASSES INVOLVED IN PASSIVE TRANSFER OF RESISTANCE R. W. Leid and J. F. Williams Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824, U.S.A. Summary. Passive transfer of immunity to Taenia taeniaefbrmis infections in the rat was achieved with serum taken 14, 21 and 28 days after infec- tion, with maximal activity at 28 days. The protective capacity resided in the g1dbulin fraction, which was further fractionated by gel filtra- tion and anion exchange chromatography. The immunoglobulins present in each passively transferred fraction were detected with specific antisera to 7372, 7Sy1, 7M and yA. Protective activity was confined to those fractions containing 78 immunoglobulin. Fractions enriched for yM were unable to confer protection and it was possible to protect recipient rats against challenge with fractions devoid of yA and reaginic antibody activity. 7Syza antibodies were able to confer passive protection when given alone, and probably contributed to the protective capacity of mixtures containing 78y2 and 78y1 immunoglobulins. A mechanism for specific acquired resistance to T. taeniaeformis is proposed based upon the recently established biological properties of 7Syza. Absorption of protective activity from immune rat serum was unsuc- cessful using a variety of techniques, and an explanation is offered for this finding. 30 The results are discussed in relation to the current understanding of acquired resistance in cysticercosis and hydatid disease in domesti- cated food animals. INTRODUCTION Cysticercosis and hydatidosis are cyclozoonotic helminth infections with a widespread distribution and significance in both humans and domesticated food animals. Immunological reactions leading to the occurrence of specific acquired resistance in these diseases remain ill defined, but the laboratory animal model of Taenia taeniaefbrmis infection in the rat has been used by several investigators to approach the phenomenon experimentally. Miller and Gardiner (1932, 1934) and Campbell (1938a,b,c) showed that resistance to challenge infection could be transferred passively with serum of infected rats. Resistance in recipient animals was manifested by a highly significant reduction in the number of cysticerci successfully developing in the tissues. These workers followed contemporary procedures for establishing the role of serum antibody in immunity to infectious disease and their results were the first to demonstrate conclusively the importance of antibody in resistance to helminth infection. Since that time successful passive transfer with serum from infected donors has been achieved in a variety of helminthiases, but only recently has it become possible to identify the immunoglobulins participating in these reactions on the basis of their biological and physico-chemical characteristics (Ogilvie, 1970; Jones, Edwards and Ogilvie, 1970; Wilson, 1966). In the present study we have confirmed the original observations of Miller and Gardiner (1932, 1934) and Campbell (1938a,b) and determined the contribution of identifiable serum immunoglobulins in the passive transfer of resistance to T. taeniaeformis . 31 MATERIAL AND METHODS Maintenance of the parasite T. taeniaeformis occurs naturally as a parasite in the intestine of the domestic cat and is maintained by the predator—prey relationship existing between cats and sylvatic rodents. Eggs released from the terminal segments of the worms appear in the faeces of the cat and are ingested by rats or mice. Embryos hatch from the ingested eggs and migrate to the liver where they establish as a metacestode or "cysti- cercus" stage. This larval form is infective for the cat. ’The strain of T. taeniaeformis used in these experiments was derived from gravid segments obtained from Mr. C. E. Claggett in the Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, Maryland. .Eggs were routinely teased from proglottids into saline containing 50 ug/ml of amphotericin; 2,500 ug/ml streptomycin; 333 U/ml of polymyxin B; and 1,000 U/ml of penicillin G and stored at 4 C. Egg doses were quantitated by a simple dilution method and administered by stomach tube to rats anaesthetized with ether. At least 6 weeks later cysticerci were removed from infected rat livers and 10-15 larvae placed in a gelatin capsule and given orally to each cat. Ten or more weeks thereafter gravid proglottids were obtained either directly from the faeces of the cat or from purged material after dosing with drocarbil (Nemural, Winthrop Labs, New York). Egperimental animals Sprague Dawley rats 28-42 days old were purchased from Spartan Research Animal, Haslett, Michigan. Random source cats were vaccinated against feline enteritis and acclimatised to laboratory facilities and food for 2-3 weeks before experimental infection. Albino guinea pigs were obtained from the Michigan State Health Laboratories 32 (Lansing, Michigan). New Zealand white rabbits were purchased from local suppliers and the sheep were members of a flock maintained by one of the investigators (J.F.W.). A11 laboratory animals were given proprietary brand food and water ad libitum. Passive transfer Recipient animals received intraperitoneal (I.P.) inoculations of serum or immunoglobulin fractions using a tuberculin syringe at the time of oral challenge with 300-600 eggs of T. taeniaefbrmis. Samples of sera, globulin and chromatographic fractions were filtered through a 0.45 u filter (Millipore, Bedford, Massachusetts) prior to inocula- tion. A period of 3 weeks was allowed for migration and establishment of the metacestodes on the surface of the liver. The animals were then sacrificed using carbon dioxide vapor and the total number of cysticerci in each liver determined. The results were statistically analysed using a computerized programme for the student's "t" test on a Monroe 1766 statistical calculator. Preparation of immune sera Rats were infected per as with 300 eggs of T. taeniaefbrmis and sacrificed 28 days later using carbon dioxide vapor. Blood was col- lected from the thoracic cavity after severing the vessels anterior to the heart, allowed to clot for 2-3 hours at 22-23 C and left over- night at 4 C. The serum.was decanted, centrifuged and stored at -20 C without preservatives until used. Immunoelectrophoresis (I.E.P.) and double immunodiffusion (D.I.D.) Immunoelectrophoresis was performed following a slight modifica- tion of the method of Scheidigger (1955) in a Gelman apparatus (Gelman Instrument Co., Ann Arbor, Michigan) with a sodium barbital—HCl buffer, 33 u = 0.038, pH 8.2 (Williams and Chase, 1971). Two percent Noble agar (Difco, Detroit, Michigan) was prepared with barbital buffer diluted 1:2 and contained 1:10,000 merthiolate. Double immunodiffusion was performed according to a micromethod modified from that described by Williams and Chase (1971). Two percent Noble agar was prepared in a 0.1M Tris-H01 buffer, pH 8.1, with a final concentration of merthiolate of 1 in 10,000. Measurement of protein concentration Protein concentrations were generally determined by the method of Lowry, Rosebrough, Farr and Randall (1951). In the case of immuno- globulin solutions, concentrations were calculated from the optical density at 280 nm, multiplied by a factor derived from the extinction coefficient (Oriol, Binaghi and Boussac-Aron, 1968; Binaghi and Oriol, 1968). Rat 7SY2 immunoglobulin levels in passively transferred fractions were quantitated using the radial immunodiffusion technique described by Mancini, Carbonara and Heremans (1965). Chromatography Descending flow gel filtration chromatography was performed on a siliconised 2.5 X 100 cm column of Sephadex G-200 (Pharmacia, Uppsala), equilibrated with 0.1M Tris-HCl, pH 8.0. A modification of the method of Sachs and Painter (1972) was introduced in order to maintain satis- factory flow rates (25-30 ml/hour) through repeated use of the column. Six millimeter glass beads were siliconised and filled the bottom 2 cm of the column and swollen Sephadex G-200 was poured over the bead layer. Samples were dialyzed against the equilibrating buffer before application and eluted fractions collected in 2.8 m1 volumes. Elution profiles were prepared using the optical density of each fraction at 34 280 nm in a Beckman Spectrophotometer (Beckman Instr. Co., Fullerton, California). The procedure for ion-exchange chromatography of rat immunoglObu- line was a modification of that described by Stechschulte, Austen and Block (1967). DEAE Cellulose (Cellex D., BioRad, Richmond, California) was prepared according to the directions of the manufacturer and poured in 1.5 X 30 cm siliconised glass columns. The cellulose was equili- brated against either 0.01M phosphate buffer, pH 7.75, or 0.005M phosphate buffer also at pH 7.75. The 0.005M buffer was used initially where separation of the 7SY2a and 78y2b immunoglobulins was required. Proteins were eluted in a stepwise manner using 0.01M phosphate buffer followed by a 0.05M pH 5.8, 0.1M pH 5.8 and finally 2M NaCl. All phosphate buffers were made 0.015M in NaCl and the samples were dialyzed extensively against the starting buffers before application to the column. Column eluates were collected in 2.8 m1 fractions and the elution pattern monitored by ultraviolet scanning at 280 nm (Gilson, Wisconsin). Protein peaks eluted with each buffer were pooled and concentrated back to the original serum volume. Preparation of antisera Anti-whole rat serum (aWRS) was prepared in rabbits. Whole normal rat serum was diluted 1:5 with phosphate buffered saline (PBS) and emulsified with an equal volume of Complete Freund's Adjuvant (C.F.A., Difco, Detroit). Rabbits received 0.5 ml by I.M. inoculation in each hind leg and 0.1-0.2 m1 portions were injected subcutaneously (S.Q.) at several sites along the back. Booster inoculations of a similar preparation were given 34 and 71 days later and the rabbits were bled out ten days after the last injection. 35 Anti-rat yM (ayM) was prepared in both rabbits and sheep. Normal rat serum immunogldbulins were precipitated 3 times with 50% saturated ammonium sulfate and passed through a Sephadex G-200 column. The ascending portion of the first peak was allowed to react in I.E.P. with aWRS and the precipitin arcs corresponding to IgM were excised from 18 slides. These agar slices were homogenised in a tissue grinder with a minimal volume of PBS and emulsified with an equal volume of C.F.A. Rabbits were inoculated I.M. with 0.5 ml of the emulsion and 0.2 m1 portions injected at several sites over the back. Twenty days later the animals were boosted with a similar preparation and bled out ten days afterward. Sheep anti-rat yM was prepared according to an extensive modifi- cation of the procedure described by Van Breda Vriesman and Feldman (1972). Sheep red cells (2 X 109) were inoculated intravenously (I.V.) or I.P. into rats and the animals bled out 10 days later. The resultant serum was subjected to Sephadex G-200 gel filtration and the ascending side of the first peak heated at 56 C for 1 hour. This protein solution.was mixed with 1 ml of packed sheep red cells in the presence of 10 mg/ml of EDTA. The red cells were washed 3 times and taken up in 1 m1 of PBS. One half of a milliliter of the suspension was mixed with an equal volume of C.F.A. and injected I.M. in the neck. The other 0.5 ml was diluted to a 10 ml volume with PBS and given I.V. to the same sheep. The sheep was bled 20 days later. Both rabbit and sheep anti-1M, prepared as described, reacted in I.E.P. with 7M and several other serum components. After absorption with foetal rat serum polymerized according to the method of Avrameas and Terynck (1969) each antiserum recognized only one arc in I.E.P. with normal rat serum, corresponding to yM (FIG. 1). 36 FIG. 1. ImmunoelectrOphoretic analysis of normal rat serum (NRS) versus Fc specific guinea pig anti-78y (1), guinea pig anti-7571 (2), sheep anti-7M (3) and sheep anti-yA (4). 37 Figure 1 38 Anti-rat secretory VA (aS yA) was also prepared in both rabbits and sheep in a manner similar to that described by Stechschulte and Austen (1970). Stomach contents from freshly suckled l- to 3-day-old rats were homogenised in a tissue grinder, stirred overnight at 4 C and centrifuged at 20,000 X g for 30 minutes. The supernatant was passed firstly through glass wool and then through a 0.45 u filter. The whey was dialysed extensively against 0.1M Tris-H01 and applied, to a Sephadex G-200 gel filtration column. The protein in the first peak was concentrated to the original volume and emulsified with an equal volume of C.F.A. and 1 ml injected I.M. into each hind leg of a sheep. The sheep was bled 30 days later. Rabbit anti-yA was prepared by reacting the first peak of a Sephadex G-200 separation of colostral whey with aWRS in I.E.P. The cathodic precipitin are corresponding to secretory yA was excised from 18 slides. The agar slices were ground in a tissue grinder with a minimal amount of PBS and emulsified with an equal volume of C.F.A. Rabbits were injected I.M. with 0.75 ml in each hind leg and 0.1 ml at several sites along the back. The animals were boosted in a similar manner 20—30 days later and the sera collected 10 days afterward. Both rabbit and sheep anti-secretory yA reacted to I.E.P. against normal rat serum to produce an are recognizable as yA and several other minor arcs. After absorption with the foetal serum immunoadsorbent these anti-sera recognized only yA in normal rat serum (FIG. 1). Anti-y2 was prepared in guinea pigs rather than rabbits or sheep since it has been shown that rats and guinea pigs recognize only the Fc portion of the immunoglobulin molecule (Oriol et al., 1968). The 73 immunoglobulin peak from a G-200 Sephadex gel filtration of normal rat globulin was dialysed against a 0.01M phosphate buffer made 39 0.015M in NaCl and applied to a DEAE cellulose column equilibrated against the same buffer. The initial peak, containing only 7SY2a+b’ was emulsified with an equal volume of C.F.A. Each guinea pig received 30 ug of protein in 1 ml with 0.5 m1 I.M. in each hind leg and the animals were bled out 20-25 days later. The antisera recognised only 78y2 in I.E.P. against normal rat sera (FIG. 1). Anti-7S71 was also prepared in guinea pigs by taking advantage of the method of Henney and Ishizaka (1968) to render the animals tolerant to 7Sy2 immunoglobulins. Guinea pigs were each inoculated with 15 ug of total protein eluted from the DEAE cellulose column with the 0.1M phosphate buffer and emulsified with an equal volume of C.F.A. This preparation contained both 73y2 and 7Sy1 immunoglobulins as revealed by I.E.P. against aWRS. At the same time the guinea pigs were given 5 mg of rat 7Sy2 I.V. to suppress antibody formation to this immuno- globulin class. This antiserum produced only one arc in I.E.P. when tested against normal rat serum (FIG. 1). Anti-rat Fab was prepared in sheep according to a method based upon that described by Oriol et al. (1968). The 78y2 immunoglobulin was purified by DEAE chromatography and 10 mg dialysed overnight against a 0.1M phsophate buffer made 0.003M in EDTA and 0.01M in cysteine. Insoluble papain (20 mg) was added and digestion allowed to proceed for 3 hours while stirring at 37 C. The reaction was stopped by centrifugation and the supernatant dialysed overnight against a 0.005M phosphate buffer pH 6.8. The supernatant was applied to DEAE cellulose equilibrated against the same buffer and the Fab fraction obtained in the first eluted peak while the Fc portion was retained on the column. One milliliter of the eluate containing 0.8 mg Fab was emulsified with an equal volume of C.F.A. and injected 40 I.M. into the hind limbs of a sheep. Thirty days later the sheep was bled. This antiserum reacted with EA, 1M, Y2 and Y1 in I.E.P. with a+b fractions containing these proteins and it therefore served to identify the arcs corresponding to each immunoglobulin class. Metacestode saline extracts Larvae of T. taeniaefbrmis from 2- to 9-month-old infections were washed in distilled water several times and homogenised in a glass tissue grinder with a minimal volume of PBS. The suspension was stirred overnight at 4 C, centrifuged at 17,000 X g and stored at -20 C. In some instances the undissolved residue was taken up a second time in PBS and the above procedure repeated. In preparation for absorption experiments the larvae were homogenised without PBS and the thick suspension added directly to the immunoglobulin solutions. Absorption of immunefglobulins Absorption of the protective capacity of passively transferred globulin fractions of immune serum was attempted using a variety of techniques. Globulins were reacted in one instance with an immuno- adsorbent prepared by crosslinking the larval saline extract with glutaraldehyde (Avrameas and Ternynck, 1969). The mixture was stirred at 22-23 C for 24 hours followed by centrifugation and filtration throughout a 0.45 n filter before passive transfer. In another experi- ment larval extract was coupled to polyacrylamide beads (P-300, -400 mesh, BioRad) using the methods outlined by Ternynck and Avrameas (1972). Sensitised beads were allowed to react with immune globulins for 24 hours at 22-23 C and removed by centrifugation before passive transfer of the filtered supernatant. In the third case the thick larval suspension was added to a solution of immune globulins such that the final concentration was 10% (v/v) and this was stirred for 41 24 hours at 22-23 C. After incubation overnight at 4 C, the suspension was centrifuged and the supernatant filtered. RESULTS Immune serum was obtained 7, 14, 21 and 28 days after infection with 300 eggs of T. taeniaefbrmis. Precipitating antibody activity was detected by day 21 in D.I.D. tests against concentrated larval extract. One milliliter of each sample was used in passive transfer experiments in order to determine at which time the serum contained the greatest protective capacity. Rats receiving 7 day serum harbored a mean of 48.8:15.79 (SD) larvae while those animals given 14 day serum had 12.75111.53. A mean of 4.60:7.02 cysticerci was present in rats which received 21 day serum and no metacestodes were observed in any of the livers of rats receiving 28 day serum. Therefore.serum obtained 28 days after infection with T. taeniaefbrmis eggs was used in all further experiments. These observations on the increased protective capacity of serum taken during the development of the parasite in the liver of infected rats confirm.the observations of Miller and Gardiner (1934) and Campbell (1938a). In the following experiment 0.2, 0.4, and 1 ml quantities of immune serum.were passively transferred to recipient rats in order to determine the quantity of serum required for significant protection. The mean number of cysticerci developing in control animals was 8.62:4.08 (SD) but both the 0.4 and 1 m1 quantities of immune serum completely prevented the establishment of larvae. As little as 0.2 m1 of immune serum resulted in a highly significant reduction in the number of larvae establishing in the recipients (0.60:1.34). In this same experiment the globulin fraction.was removed from.immune serum by 50% (NH4)ZSO4 precipitation (3X) and passively transferred after 42 dialysis against PBS. The mean number of cysticerci in these rats receiving the globulin fraction was 1.86:1.95 (P<0.01). The globulin fraction of immune serum was therefore used for all subsequent chromato- graphic separations. Since the infection levels in this experiment were relatively low, higher levels of challenge were used thereafter as a routine. Globulins were precipitated with 50% SAS 3 times and subjected to gel filtration on Sephadex G-200 to separate 19S from 78 immunoglobu- lins (FIG. 2). Both peaks were concentrated back to the original serum volume. The first peak contained YM detectable by both sheep and rabbit antisera specific for Fc determinants of this immunoglobulin plus a B globulin, detected by aWRS. The second peak from the G-200 column, containing 7S immunoglobulin, was dialysed against a 0.01M phosphate buffer made 0.015M in NaCl and applied to DEAE cellulose equilibrated against the same buffer. Sequential step-wise elution was followed and the results are shown in FIG. 3. The presence of 7872 globulins was monitored in each fraction using guinea pig antiserum specific for the Fc portion of each class. Both 7Syza and 7SY2b were detected in the 0.01, 0.05 and 0.10M phosphate buffer eluates. Rat yA was detected with Fc specific antiserum produced in sheep and rabbits and was present only in the 2M NaCl eluate. Reaginic activity was limited to the 0.05M phosphate buffer eluate and its presence was detected by homologous passive cutaneous anaphylaxis (Leid and Williams, 1973). No monomeric 7M was detected in any of the DEAE cellulose fractions which were passively transferred. An aliquot of the original globulin solution was absorbed with an immunoadsorbent prepared from a saline extract polymerised with glutaraldehyde. 43 FIG. 2. Elution profile at 280 nm of the globulin fraction (50% (NH4)2804) of immune rat serum after gel filtration on Sephadex G-200. Fraction 1 was concentrated and tested for protective activity. Fraction 2 was further fractionated on DEAE-cellulose. 44 N ouswfiw son—:32 on 5... ON« 2: 00 8 3 ON ‘- d- It. vi It. wuoszao 45 FIG. 3. Elution profile at 280 nm of the 78y globulin fraction (50% (NH4)ZSO4) of immune rat serum fractionated on DEAE cellulose with phosphate buffers and 2M NaCl. A11 phosphate buffers were made 0.015M in NaCl. Fractions 1, 2 and 3 were tested for activity in passive transfer experiments. m ouswwm con—5:2 was... om cm. 2.: a.» 8 3 on _ q u - 4 — ll - - ca” _ n _ . u . _ h. _ u h _ _ iv 41 6 -6... +6 a.» r... a.» 1.. n2. re .l 5:5 I .286 J. .23.: I .28.:III1 eoueugwsueu, x 47 Groups of 28-day-old female rats were given 300 eggs per os fol- lowed by an I.P. inoculation of 0.8 m1 quantities of each of the chroma- tographic fractions, PBS or normal 28 day serum. One milliliter portions of the absorbed solution and 0.4 m1 of unfractionated immune serum and globulin were given. All groups were sacrificed 21 days later and the numbers of cysticerci developing in each group compared to that of the controls. The results of the passive transfer of fractions enriched for the various rat immunoglobulin classes and the effects of absorption on protective capacity of globulin solutions are shown in Table 1. The 198 or 1M fraction did not confer protection and the absorption with the polymerised larval extract did not reduce the protective quality of the globulin solutions. All three phosphate buffer eluates (0.01M, 0.05M and 0.10M) produced a highly significant reduction in parasite burdens (P<0.001, 0.001 and 0.01, respectively) when compared to control animals. The levels of 78y2 were determined in each of these phosphate buffer eluates and were as follows: 0.4 mg/ml for 0.01M, 6.6 mg/ml for 0.05M and <0.4 mg/ml for the 0.10M. In view of the fact that 78y23 appeared in all three phosphate buffer eluates a further experiment was performed taking advantage of the technique devised by Stechschulte et al. (1967) for separation of 78yZa from the 7Sy2b by lowering the molarity of the initial buffer in DEAE chromatography. In this case a 0.005M phosphate buffer made 0.015M in NaCl was used as the starting buffer. Both the 198 and 7S peaks were obtained from Sephadex G-200 gel filtration of an immune ' globulin solution and the 19S peak was concentrated and dialysed against PBS extensively before passive transfer. The 75 peak was concentrated, dialysed against the 0.005M phosphate buffer and applied to a DEAE cellulose column. Again step-wise elution was followed 48 Houoofiuwho mo Howwusm vomfiuoahaom 899 a was H :a SE 888%. 538% as, waned 899 a was H a: afienflw ”.3 manage Ho.ov n 00.: H :éa Eamon um.“ museum 8.? a 35 H 2.3 5:33» amass mo 88? mean some 899 a 2.2 H 2.2 ”“33on 8:55 .u5 some? mean 285 80.9 a SS H 3.... ~33on 885 mo SSS name :85 8 a Sr; H 3.3 1533» 835 mo mascara m3 3 a 2.3 H 8.8 St... ”.8 Hgoz we a 3.: M an? madam sandman 3282: and; m 95mm use .n.m H oozed kuuommsmuu mum» mo Honesz mo Hones: coo: hao>fimmmm GOHuomum awououm MHzmskmmHéwqfi qHEMQH mo muum 00m mHHS nmwzmqqmmu mag HZMHMHUmm ZH mZOHHUE ZHADmOAUOZDZ—LH 92¢ SDMMm mg mo MHHUHHUMHomm mammfim H mama 49 (FIG. 4), and the 0.005M and 0.01M eluates were concentrated to the original serum volume and passed through a 0.45 u filter before inocu- lation. In this passive transfer experiment further adsorption pro- cedures were attempted. Preliminary studies using immunoelectrophoresis and radioimmunoelectrophoresis (unpublished observations) had indicated antigen binding activity in the 72 immunoglobulins, and absorption procedures were therefore monitored by D.I.D. tests for removal of precipitins. The globulin fraction.was first absorbed with larval extract coupled to polyacrylamide beads. A total of 1.6 mg of protein was bound to each ml of packed beads. This amount was approximately equivalent to the maximum achieved for the series of protein antigens studied by Ternynck and Avrameas (1972). The absorption was carried out at 22-23 C for 24 hours after which the globulin solution was tested in D.I.D. against concentrated larval extract. All the pre- cipitating antibody activity was not removed by this process and the solution was then absorbed with a freshly homogenised thick larval suSpension as described above. This treatment removed all precipi- tating antibody activity. A second volume of globulin solution was treated only with the thick larval suspension following the procedures outlined previously. Groups of 28-day-old female rats were given 600 eggs of T. taeniaefbrmis orally followed by I.P. injections of the absorbed or unabsorbed immunoglobulin preparations. The animals were sacrificed 21 days later and the results are shown in Table 2 and FIG. 5. Again the IgM fraction conferred no passive protection while absorbed immunoglobulin preparations remained effective in passive transfer. The fraction eluted with the 0.005M phosphate buffer resulted in highly significant protection in recipients (P<0.01) while the 0.01M 50 FIG. 4. Elution profile at 280 nm of the 7SY globulin fraction (50% (NH4)2804) of immune rat serum fractionated on DEAE cellulose with phosphate buffers and 2M NaCl. All phosphate buffers were made 0.015M in NaCl. Fractions 1 and 2 were tested for activity in passive transfer experiments. 51 q ouswwm 50:83,. was... r 2..» emu can 2: 2: on . 4 _ 1 n —# - as J u u cad " u u _ . n u . _ . . _ a. _ c . a. n . . [J u _ .3 ram .. a? van 0 r c an In a.» In. mhfi In an.» In T 53d 1128.6 IT .236 I .236 ltlZMOQO A X aoueugwsueu. .A>\> Noav aowmsoemom Hm>uua no“: ponuomn< Amv .A>\> Noav scamaoemsm Hm>hmH hp uosoaaom .mpmon moaeoahuoozaoe pounce dowfiuam sufia wonuomn< Aav 52 899 a 3.2 H 8.3 g e382... 5153» ”.3 «SSH «.9 s 3.: H $.Nm 3 BESS :23on a... 333 mu m 8.2 w... an? 515on mfiafi mo acetate mama Eod 8.9 a 2.2 H 2.3 5:52» 335 .ua 838E was Hood 8 w 3.: H 8.3 315on 385 mo 838E m3 8.? a 3.8 H :18 afianflm “.3 383 m: o are” H 34: Show um... Hmfioz o=Ho> m asouw Hoe .n.m.H_om>umH wouuommamuu mumu mo monsoz mo gonads sou: %Hm>ammma meowuomum :fiououm mHEEQRMdH2MqH .& mo muwm coo mHHz amvzmaqHHUMBOMm m>Hmm3 on e t. . . .RWJCV 4N6. L. «angaxn asp}, .r. ,. an _. 55 did not. The 0.005M eluate was tested in I.E.P. with anti-whole rat serum and a single arc appeared, corresponding to 75y2a (FIG. 6). It was also tested in D.I.D. against guinea pig anti-y2 and again only a single band formed. DISCUSSION The results of our experiments confirm the role of serum antibody in passively transferred resistance to T. taeniaefbrmis as established by Miller and Gardiner (1932, 1934) and Campbell (1938a,b,c). Pro- tective activity was confined to fractions of immune serum containing 78 immunoglobulins and fractions enriched for 198 antibodies were ineffective. Protection was successfully conferred with fractions devoid of VA and reaginic antibody activity. The results obtained with further fractionation of the 7S component of 28 day immune serum suggest that 78y2a immunoglobulins contain the majority of protective antibodies to infection with T. taeniaefbrmis. We do not exclude the possible participation of 7Sy2b, 7871 or yE in the successful passive transfer of resistance observed with fractions containing these immuno- globulin classes. However, these DEAE eluates were contaminated with 78y2a which was able to confer protection.when given alone. Further- more, the eluate obtained with 0.1M phosphate buffer contained less 7Syza antibodies than the 0.01M and 0.05M fractions and conferred a lesser degree of protection. Enrichment with 78y1 type antibodies was therefore not associated with enhanced protective capacity. A more conclusive demonstration of the quantitative contribution of antibodies of each immunoglobulin class might be achieved by the use of immune serum selectively depleted by absorption with antisera specific for 7SY2a’ 7Sy2b, 7Sy1, yA, YM and possibly yE. This approach has recently been applied successfully by Saif, Bohl and Gupta (1972) in their 56 FIG. 6. Immunoelectrophoretic analysis of normal rat serum (bottom well) and fraction 1, 0.005M eluate (upper wall) from DEAE cellulose fractionation (FIG. 4) versus rabbit anti-whole rat serum (aWRS). 57 Figure 6 58 studies on the immunoglobulin classes containing neutralizing antibody in vitro to transmissible gastroenteritis virus in the pig. we are presently pursuing a similar objective in passive transfer experiments with the T. taeniaefbrmis system. Nevertheless, our present findings on the involvement of 7Sy2 antibodies, especially 7SY2a’ in resistance to infection are of importance both in the context of the biological properties of rat immunoglobulins and their association with protective responses, and also in terms of the relevance of the rat-T. taeniaefbrmis model to immunity to cysticercosis in general. 78y2 antibodies in the rat appear in response to a variety of artificial immunisation procedures (Block, Morse and Austen, 1968; Jones, 1969) and were shown by Jones (1969) to be responsible for short term homologous skin sensitisation. Morse, Block and Austen (1968) demonstrated that 7Sy28 antibodies were capable of preparing rat tissue for antigen induced release of SRS—A, and 7SyZa antibodies inhibited histamine release from mast cells mediated by reaginic antibodies in rats infected with Nippostrongylus brasiliensis (Bach, Block and Austen, 1971). 7872 antibodies were capable of passively transferring resistance to infection.with N. brasiliensis but only after the donor rats had received multiple infections with this parasite (Jones, Edwards and Ogilvie, 1970). Passive protection was conferred pre- dominantly by fractions enriched for 7871 immunoglobulins if serum was obtained from rats after primary infections. Preparations of 7872 which were active in passive transfer did not provoke 5 hour PCA reactions and the authors state that they had never observed anaphy- lactic antibodies of the 73y2a type in rats with N. brasiliensis, although they did not separate this class chromatographically (Jones 59 et al., 1970). We have been unable to demonstrate short-term skin sensitisation at 2, 4 and 5 hours with 78yZa antibodies against T. taeniaefbrmis (Leid and Williams, 1973), but this was readily achieved following artificial immunisation (unpublished observations). Possibly those 7SyZa antibodies which appear in response to helminth infections represent a biologically distinct population within this immunoglobulin class which is not capable of mediating PCA reactions. Alternatively the antigens required for provocation of this short term sensitisation may have been present in challenge solutions at levels insufficient to elicit the PCA reaction. In the latter case a mechanism may be postulated for specific acquired resistance to T. taeniaefbrmis which implicates YZa anti- bodies in the release of vasoactive amines, perhaps at the level of the intestinal mucosa. The destruction of parasites before their establishment in the liver, designated "early immunity" by Campbell (1936), has been suggested to occur within the intestinal mucosa (Leonard and Leonard, 1941). Antigen production by the parasite embryo at or within the intestinal surface might trigger the release of cell bound SRS-A resulting in changes of vascular permeability which permit the increased accumulation of antibody and cells at the site. Complement fixing antibodies are known to occur in the sera of rats infected with T. taeniaefbrmis (Campbell, 1938b; Murrell, 1971) and a complement dependent attack on the embryo could be responsible for immdbilisation or destruction of the parasite. 723 antibodies have also been shown to fix complement (Morse et al., 1968) and chemo- tactic attraction of either specific or non-specific cellular components might also be a factor. 60 The role which secretory yA might play in immunity at the intestinal level is not known, although there is no direct evidence of its involvement in resistance to cysticercosis. However yA secret- ing cells are very prominent in the lamina propria of the small intestine of the rat (Nash, Vaerman, Bazin and Heremans, 1969) and it seems likely that secretory yA antibodies may contribute to specific acquired resistance in this infection. Indirect evidence in favor of this possibility derives from the observations of colostral transfer of protection in rats infected with T. taeniaefbrmis (Miller, 1935) and sheep with T. hydatigena (Gemmell, Blundell-Hasell and Macnamara, 1969) . We were unable to absorb the protective capacity of immune serum using a variety of procedures and this finding is in accord with the results reported by both Miller and Gardiner (1932) and Campbell (1938b). Again the concentration of certain critical antigens in the extracts or preparations used for absorption may have been insufficient to effect complete removal of the protective antibodies. The experi- ments of Rickard and Bell (1971) have some bearing on this suggestion. In their studies the degree of resistance to challenge infection with T. taeniaeformis produced by implanted membrane diffusion chambers containing growing larvae was dependent upon the duration of implan- tation. This might indicate a requirement for the elaboration and release of antigens over an extended period and therefore the concen- tration of such antigens in the developing larvae may not be high at any one time. In vitro maintenance of the cysticerci may offer a means to secure enriched preparations of these important antigens. Little is known of the specific antibodies or antigens involved in immunological events in naturally occurring cysticercosis and 61 hydatidosis in domesticated animals. However, certain features of the biology of these infections support the belief that an acquired resistance analogous to that seen in our experimental model, and possibly mediated by comparable mechanisms, is manifested under field conditions. Resistance to superinfection has been observed with T. saginata in cattle (Urquhart, 1961) and with T. hydatigena and T. ovis in sheep (Gemmell, 1969). Experimentally Sweatman (1957) and Sweatman, Williams, Moriarty and Henshall (1963) have demonstrated acquired resistance in sheep to T. hydatigena and E. granulosus, respectively, and Soulsby (1963) was unable to superinfect calves exposed to T. saginata shortly after birth. Furthermore, resistance to challenge infection with eggs of T. hydatigena in sheep has been shown to be due in part to serum antibodies (Blundell, Gemell and Macnamara, 1968). While direct extrapolation of results obtained with the rat-T. taeniaefbrmis model is not justified, the characterisation of immuno- logical phenomena in this readily manipulated laboratory animal infection may serve to delineate areas for experimental exploration in the more costly domestic animal systems. In this regard we feel that the demonstrated association of protective resistance with an immunoglobulin of rather well defined biological reactivity may be considered an important advance. ACKNOWLEDGEMENTS This work was supported in part by Grant AI-10842-01 and Training Grant GM-01911 from the United States Public Health Service. The senior author was supported by an N.I.H. Pre-doctoral traineeship. The authors are grateful for the excellent technical assistance rendered by Mrs. Anndy Whipple and the expert help of Dr. A. J. Musoke in certain aspects of the work. His thoughtful and constructive criti- cism of the manuscript was very much appreciated. 62 REFERENCES ANRAMEAS, S. and TERNYNCK, T. (1969). 'The cross-linking of proteins with glutaraldehyde and its use for the preparation of immuno- adsorbents.‘ IMmunochemistry,'6, 53. EACH, M. K., BLOCH, K. J. and.AUSTEN, K. F. (1971). 'IgE and IgGa antibody-mediated release of histamine from rat peritoneal cells. II. Interaction of IgGa and IgE at the target cell.‘ J. Exp. Med., .133, 772. BINAGHI, R. and ORIOL, R. (1968). 'Anticorps purifies de type macroglobuline.' Bull. de la Societe de Chimie Biologique, 50, 1035. 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Hyg., 21, 456. MILLER, H. M. JR. and GARDINER, M. L. (1932). 'Passive immunity to infection with a metazoan parasite, Cysticercus fasciolaris 1n the albino rat.’ J. Prev. Med., 6, 479. MILLER, H. M. JR. and GARDINER, M. L. (1934). 'Further studies on passive immunity to a metazoan parasite, cysticercus fasciolaris.' Amer. J. Hyg., 20, 424. MORSE, H. C. III, BLOCH, K. J. and AUSTEN, K. F. (1968). 'Biological properties of rat antibodies. II. Time-course of appearance of antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-Arat); Association of this activity with rat IgGa.' J. Immunol., 101, 658. MURRELL, D. D. (1971). 'The effect of antibody on the permeability control of larval Taenia taeniaeformis.' J. Parasit., 57, 875. OGILVIE, B. M. (1970). 'Immunoglobulin responses in parasitic infec- tions.‘ J. Parasit., 56(Section II. 3). 525. ORIOL, R., BINAGHI, R. and BOUSSAC-ARON, Y. (1968). 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Immunol., 104, 1052. STECHSCHULTE, D. J., AUSTEN, K. F. and BLOCH, K. J. (1967). 'Anti- bodies involved in antigen-induced release of slow reacting sub- stance of anaphylaxis (SRS-A) in the guinea pig and rat.’ J. Exp. Med., 125, 127. SWEATMAN, G. K. (1957). 'Acquired immunity in lambs infected with Taenia hydatigena, Pallas, 1766.‘ Can. J. Comp. Med., 21, 65. SWEATMAN, G. K., WILLIAMS, R. J., MORIARTY, Km M. and HENSHALL, T. C. (1963). 'An acquired immunity to Echinococcus granulosus in sheep.’ Res. Vet. Sci., 4, 187. TERNYNCK, T. and AVRAMEAS, S. (1972). 'Polyacrylamide-protein immuno- adsorbents prepared with g1utara1dehyde.' FEBS Letters. 23, 24- 66 URQUHART, G. M. (1961). 'Epizootiological and experimental studies on bovine cysticercosis in East Africa.‘ J. Parasit. , 47, 857. VAN BREDA VRIESMAN, P. J. C. and FELDMAN, J. P. (1972). 'Rat YM innnunoglobulin: Isolation and some biological characteristics.‘ Immunochemistry, 9, 525. WELLIAMS, C. A. and CHASE, M. W. (1971). Methods in Immunology and Imunochemistry, lst edn., p. 103. Academic Press, New York. WILSON, R. J. M. (1966). ' -antibodies in guinea pigs infected with Y1 the cattle 1ungworm.‘ Immunology, 11, 199. THE IMMUNOLOGICAL RESPONSE OF THE RAT T0 INFECTION WITH TAENIA TAENIAEFORMIS II. CHARACTERISATION OF REAGINIC ANTIBODY AND AN ALLERGEN ASSOCIATED WITH THE LARVAL STAGE R. W. Leid and J. F. Williams Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824, U.S.A. This is journal article No. from the Michigan State University Agricultural Experiment Station. 67 68 THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH.TAEN1A TAENIAEFORMIS II. CHARACTERISATION OF REAGINIC ANTIBODY AND AN ALLERGEN ASSOCIATED WITH THE LARVAL STAGE R. W. Leid and J. F. Williams Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824, U.S.A. Summagy. Skin sensitising antibody or reagin was detected in rats 19 days after infection with Taenia taeniaefbrmis eggs. Peak titres were reached on day 32 and thereafter declined. A second dose of eggs was capable of increasing the levels of circulating reagin even though it was highly unlikely that the embryos had survived more than a transient period of time in the intestinal mucosa. The physico-chemical and biological characteristics of this reagin are consistent with those of the rat immunoglobulin designated yE. In no instance was it possible to demonstrate short-term skin sensitisation by the rat 7SY2a immuno- globulin class even though previous work had shown that antibodies of this type are produced during infection. All short-term reactivity at 2-6 hours appeared to be a result of reaginic antibody fixation. Reagins were not observed to cross the placenta or be transferred by the colostrum from highly immune females to their offspring. The role of reagin in passive and specific acquired resistance to T. taeniae- fbrmis is discussed and the mechanisms whereby it might be contributing to immunological events in this infection are outlined. An allergen was isolated from cysticerci of T. taeniaeformis which was capable of provoking passive cutaneous anaphylaxis (PCA) reactions 69 in sensitised rats in approximately 5 ugm quantities. A single band was obtained in polyacrylamide gel electrophoresis which stained for protein and carbohydrate but not for lipid. Chromatographic and electrophoretic studies indicated that the allergen was very negatively charged at slightly alkaline pH. Activity was completely removed from larval extracts by absorption with a monospecific antiserum prepared against the allergen. The possibility of more than 1 allergen being associated with cysticerci and adult worms of T. taeniaefbrmis is discussed. Cross reactions with other taeniid parasitic extracts were observed but no positive PCA reactions were elicited with extracts prepared from two dissimilar helminths, Sbhistosoma mansoni and Fasciola hepatica although these parasites are known to cause cross reactions in field tests. The potential value of the purification procedure is pointed out in the context of the continuing effort to improve the specificity of clinical diagnostic tests based on intradermal reactions. INTRODUCTION Immediate hypersensitivity reactions are consistently associated with helminthic infections (Sadun, 1972; Ogilvie, 1970) and constitute a particularly prominent and important aspect of the immunological response to cysticercosis in man and domestic animals. Allergic clini- cal signs occur at the time of tissue invasion by Taenia solium in man (Dixon and Hargreaves, 1944) and the pathogenesis of the disease in chronic infections often involves the sudden onset of intense inflamma- tory reactions around degenerating cysticerci (Faust, Russell and Jung, 1970). Humans and pigs infected with T. solium and cattle parasitised with T. saginata develop immediate reactions at the site of intradermal inoculations of crude extracts of these parasites. Attempts to 70 capitalise upon this hypersensitivity in clinical or antemortem diag- nosis have met with very limited success (Froyd, 1963; Machnicka-Roguska and Zweirz, 1970) and the necessity for specific allergen purification has recently been stressed (Euzeby, 1966; Arundel, 1972). In spite of the practical significance of this phenomenon in taeniid infections, there is almost no information on experimental analysis of the reaction in terms of characterisation of the anti- bodies and antigens involved. Our investigations on the rat-T. taeniaeformis model afforded us an opportunity to establish some of the parameters of immediate hypersensitivity in experimental cysticer- cosis and its contribution to protective resistance. Observations on the pattern of appearance of reactivity, the physico-chemical and biological features of reaginic antibodies and the partial purification of one of the allergens implicated are presented in this report. MATERIALS AND METHODS Maintenance of the parasite T. taeniaefbrmis was maintained in rats and cats as described previously (Leid and Williams, 1973). Egperimental animals Sprague Dawley rats were obtained from Spartan Research Animal (Haslett, Michigan). Adult female rats were used as recipients for passive cutaneous anaphylaxis (PCA) tests. New Zealand white rabbits for antiserum production were purchased from local suppliers and the sheep were members of a flock maintained by one of the investigators (J.F.W.). All laboratory animals were given proprietary brand food and water ad libitum. 71 Reagggic serum Whole blood was obtained from the thoracic cavity by severance of the blood vessels anterior to the heart 28-35 days after infection with approximately 300 eggs of T. taeniaefbrmis. The blood was allowed to clot at 22-23 C for 2-3 hours and remained overnight at 4 C before the serum was decanted, centrifuged and stored at -20 C. Immunoelectrophoresis (I.E.P.) and double immunodiffusion (D.I.D.) These procedures were performed as described previously (Leid and Williams, 1973). Preparative electrophoresis Pevikon C-870 (Mercer, New York) block electrophoresis was carried out using a slight modification of the method described by Osterland (1968). Sodium barbital-HCl buffer, u = 0.1, pH 8.4, was diluted 1:2 and used for the inner electrode vessels and a phosphate buffer u = 0.2, pH 7.5 in the outer buffer chambers (Williams and Chase, 1968). The dialysed sample was applied to a slit 8 cm from the cathodic end of the gel. Several drops of a 1% solution of bromophenol blue were added 1-2 cm from the anodic side of the slit to serve as an indicator of the progress of migration. A plexiglass container (32 X 18 cm) was cooled by circulating tap water at approximately 4 C underneath the pevikon layer and electrophoresis performed for 48 hours at 13.75 volts/cm. On completion of the separation, 1 cm segments of the block were removed and the proteins eluted by displacement filtration in phosphate buffered saline (PBS). Each fraction.was concentrated to the original sample volume and the protein concentrations determined. Fractions were dialysed against PBS and stored at -20 C. 72 Measurement of protein concentrations The concentrations of immunoglobulin and antigen solutions were measured as described in a previous paper (Leid and Williams, 1973). Chromatography Gel filtration and ion exchange chromatography of serum samples and parasite extracts were performed as described by Leid and Williams (1973). Anion exchange fractionation of the parasite extract was carried out on DEAE cellulose (Cellex D, BioRad). Step-wiSe elution was achieved with buffers of increasing molarity and decreasing pH, in the following sequence: 0.01M phosphate buffer, pH 7.9; 0.05M phosphate buffer, pH 5.8; 0.10M phosphate buffer, pH 5.8; and finally 2M NaCl. All phosphate buffers were made 0.015M in NaCl and the elution -patterns were monitored by ultraviolet scanning at 280 nm. Fractions were collected in 2.8 ml volumes and the peaks eluted with each buffer were pooled and concentrated to the original sample volume. These pools were dialysed against PBS overnight at 4 C and tested for their ability to provoke PCA reactions in sensitised rats. Cation exchange fractionation of a saline extract of T. taeniae- formis larvae was accomplished using CM-Sephadex A-25 (Pharmacia). The gel was prepared according to directions of the manufacturer, packed in a siliconised 1.5 X 30 cm glass column and equilibrated with 0.0175M phosphate buffer, pH 6.2. Samples were extensively dialysed against this same buffer before application. Step-wise elution was carried out using phosphate buffers of increasing molarity and increasing pH in the following sequence: 0.0175M phosphate buffer, pH 6.2; .03M phosphate buffer, pH 6.6; 0.05M phosphate buffer, pH 6.85; and 0.10M phosphate buffer, pH 7.77. Elution profiles were monitored and the fractions treated as described above. 73 Homologous passive cutaneous anaphylgxis (PCA) The procedure for homologous PCA tests was a slight modification of that described by Ogilvie (1967). Rats were shaved and 0.1 ml quantities of serum or chromatographic fractions were injected intra- dermally (I.D.) over the back. Twenty-four to 72 hours later rats were challenged intravenously (I.V.) with 0.5 m1 of the parasite antigen to be tested together with 0.5 m1 of a 1% solution of brilliant blue R (BioRad). Reactions were read 15-60 minutes after challenge and graded on a scale from 0 to-+++. Positive reactions varied from small areas of intense blueing several millimeters in diameter up to circular zones 15 mm or greater in diameter, which were classified as +++. Positive and negative control sera were included in each recipient and at least 3 rats were used for each sample tested. Whenever doubt- ful responses had occurred the skin was reflected and viewed from the underside. In preliminary experiments brilliant blue R was found to be superior to Evans blue since it was rapidly cleared and background blueing was eliminated. Positive reactions were extremely well delineated by this means. Active cutaneous anaphylaxis (ACA) was used to detect allergenic activity in fractions of parasite extracts. Rats infected for at least 2 months were inoculated I.D. with 0.1 ml of the antigen and I.V. with 0.5 m1 of a 1% solution of brilliant blue R dye 30 minutes later. Reactions were read as described for PCA. In some instances Prausnitz- Kustner (PK) tests were used to monitor the presence of allergens in parasite extracts. The skin site was first sensitised with 0.1 m1 of reaginic serum and then challenged 24 hours later with 0.1 m1 of the antigen I.D. followed by 0.5 m1 of the brilliant blue R dye I.V. 30-60 minutes afterward. Reactions were read as for PCA. 74 Polyacrylamidgyggl electrophoggsis (PAGE) Polyacrylamide gel electrophoresis was performed following the methods of Clarke (1964) for 5% gels and Weber and Osborn (1969) for 10% gels. Separated protein bands were stained with a 1% solution of aniline blue black. Lipid and glycoprotein components of the bands in the gel were detected using the methods of Turner and MacGregor (1969). Parasite extracts Larval extracts of T. taeniaefbrmis were prepared as described by Leid and Williams (1973). Adults of T. taeniaefbrmis were obtained by purging infected cats. These worms were washed extensively in tap water followed by PBS. The washed parasites were homogenised with PBS in a glass tissue grinder immersed in crushed ice. The solution was stirred overnight at 4 C and centrifuged at 17,000 X g. The clarified supernatant was removed and frozen in aliquots at -20 C. Extraction of the insoluble residue was generally repeated to increase the yield of antigen. Antigenic extracts of heterologous parasites were similarly pre- pared from adults of T. pisifbrmis, cysticerci of T. crassiceps and protoscolices of Echinococcus multilocularis. Parasite cYSt f1U1d was obtained from cysticerci of T. hydatigena. These parasites were either maintained in our laboratory according to established procedures or were acquired from natural infections after treatment or at autopsy. In addition extracts were prepared from adults of Sbhistosoma mansoni and Fasciolaghepatica, kindly supplied by Drs. S. W. Berry and Terrence J. Hayes, respectively. 75 In.vitro maintenance of T. taeniaefbrmis Cysticerci were teased from livers of rats infected for 5-6 months and washed in distilled water (3X), sterile distilled water (3X) and finally sterile PBS (4X). Thirty to forty larvae were placed in 200 ml capped culture vials containing Hanks EME (Grand Island Biological Co., Grand Island, New York) with 1,000 U/ml of penicillin, 1,000 ug/ml of streptomycin and 500 U/ml of polymyxin B and incubated at 37 C. The levels of antibiotics were reduced by half at the end of the first week. The pH of the medium was maintained at neutrality by the daily addi- tion of sterile sodium.bicarbonate solution. The medium was replaced at weekly intervals under these circumstances and larvae remained active and grossly normal in appearance for periods up to 6 weeks. The collected culture medium samples were pooled and concentrated 50 fold with Carbowax (Fisher Scientific Co., Pittsburgh, Penn.) before overnight dialysis against PBS (1:10) at 4 C and subsequent lyophilisa— tion. Lyophilised preparations were reconstituted with distilled water before use. ngparation of antisera Anti-T. taeniaefbrmis larval extract (aTLE) was prepared in rabbits. The extract of cysticerci was emulsified with an equal volume of complete Freund's adjuvant (CFA, Difco, Detroit). Each rabbit received intramuscularly (I.M.) and subcutaneously (S.Q.) a total of approximately 2.0 mg protein. Booster inoculations of a similar preparation were administered I.M. 17 and 55 days later. The rabbits were bled 11 days after the final injection and the antisera collected and stored at -20 C. Antisera to adult T. taeniaefbrmis extract (aTAE) and to products excreted or secreted by the cysticerci (anti-in vitro concentrate, 76 aIVC) was prepared in rabbits in a manner similar to the aTLE. Antiserum directed against an allergen prepared by preparative block electrophoresis of T. taeniaefbrmis cysticerci was prepared in a sheep. One hundred micrograms of the allergen contained in 5 m1 of PBS was emulsified with an equal volume of CFA and given by I.M. inocu- lation. The animal was bled 15 days later and this antiserum recognised only one protein when tested in I.E.P. against T. taeniaefbrmis larval extract. RESULTS The reaginic antibody response during infection with T. taeniae- fbrmis was initially detected by testing in PCA pooled serum samples taken on days 7, 14, 21, 28 and 35 post infection. Slight positive reactions at 72 hours post sensitisation were observed at 21 days with strongly positive reactions evident by day 28 and 35. A more detailed account of the pattern of reagin production follow- ing oral doses of eggs of T. taeniaeformis was obtained from PCA tests on serial bleedings taken from the retro-orbital plexus. Rats were bled at frequent intervals particularly during the time period at which reagin was first expected to appear and following the second booster dose of eggs. At least 5 animals were bled for each sample tested and the results are shown in FIG. 1. Reagin first appeared on day 19 post infection, reached a peak titre of 1:8 on day 32 and then declined with activity persisting only with undiluted serum through day 81. A dose of 1,000 eggs was given at this time and within 9 days the titre had reached a peak equivalent to that shown earlier. These high levels were maintained with some fluctuation until a second peak of 1:32 was reached on day 117 post infection. Thereafter a gradual decline occurred. 77 FIG. 1. An analysis of the pattern of appearance of skin sensi- tising antibody (or reagin) to infection with Taenia taeniaefbrmis in the rat. Serial bleedings were obtained at the times indicated and PCA tests done with the sample. A booster dose of 1,000 eggs was given 81 days after primary infection (arrowed). 78 H ouomam ZO_h0mu2_ hmOn— m><0 q FoanGWutamu pawn. 0% pow paw . ON . .1......H..-...fi¥. h