muncmc MECHAMSM 5N RESSTANCE T0 EXPERIMENTAL ENFECTIGN WEIR ‘FAEMA 'FAENEAEOREHS fissertation for the Degree of Ph. D. MiCHIGAN SIME b‘NWERSITY ANTONY j. MUSGKE 1975 — _ — m This is to certify that the A thesis entitled flwVVUWfl/C (0?“ Mfi/Wgam cu AM, 5% 4'0 (AfleW/be/W MA— (ZW) ,./ tum JMLW ‘ M «A.» M ‘ presented by A477 J Mmeam has been accepted towards fulfillment of the requirements for A b degree in A‘fC‘rf b7Q Gj M fax/9C“ dL/Qlll’k_ / 72* W ‘ Major professor Date 1’L/ 213/ 7 5" 0-7639 ‘ imocuorav WAG 8. 80‘78' r *3". \ BINDERY INC. ‘1 ("1 I may BINDERS l v— - urn R‘l’ Ilflllflll - 'I «ill! $f...!‘l. j \ t' ’5‘ ABSTRACT UUK IMMUNOLOGIC MECHANISMS IN RESISTANCE TO EXPERIMENTAL '\ INFECTION WITH TAENIA TAENIAEFORMIS BY Antony J. Musoke Mice were found to be protected against Taenia taeniaeformis infection by passive transfer of serum collected from donors 28 days after infection. The protective activity resided exclusively in the first fraction of 7S immunoglobulins eluting from DEAE cellulose at pH 5.8 with 0.05 M phosphate buffer. This fraction contained 7SY and 7912 immunoglobulins but no detectable yA, 7M or skin 1 sensitizing activity. Fractions containing 78y2 alone were inef- fective in passive transfer. Intraperitoneally implanted metacestodes of either T. taeniae- formis or T. crassiceps in rats provoked a high degree of resistance to oral challenge with eggs of T. taeniaeformis. This resistance was passively transferred to normal recipients with serum. Immuno- globulin fractions of immune serum containing 7SY1 or yM were most effective in passive transfer and little activity was associated with 7872 antibodies. No skin-sensitizing antibodies were detectable in immune sera. These findings are in sharp contrast to previous observations involving protective immunoglobulins and reaginic Antony J. Musoke antibodies in serum from rats with hepatic cysticerci of T. taeniaefbrmis. Cysticerci implanted into normal rats survived for at least 21 days with no sign of host rejection, whereas those implanted into rats with hepatic infections with T. taeniaeformis were killed and encapsulated. Similar results were obtained by implanting cysticerci in normal rats given inoculations of complete Freund's adjuvant. Repeated inoculations of immune serum had no effect on the survival of implanted cysticerci, and it was concluded that exposure to infection by oncospheres provokes cellular defense mechanisms which can be effective against cysticerci in abnormal sites. Why these mechanisms are inoperative against hepatic cysti- cerci remains unclear. Passive transfer of immunity to Taenia taeniaeformis was achieved with serum taken 14, 21, 49 and 63 days after infection. The protective capacity of serum collected at 14 and 21 days resided in the 7872 immunoglobulins and appeared to be exclusively the result of 7SY a antibody activity. However, as the infection 2 progressed the range of chromatographic fractions showing protec- tive capacity was extended to all those containing 78y2 and 78y1 immunoglobulins. Fractions enriched for 7M did not confer protection. Immune serum containing 7SY2a antibodies was able to kill developing parasites after they had left the intestine, and the hepatic postoncospheral forms retained their susceptibility to antibody over the first 5 days of growth. After that time they Antony J. Musoke rapidly became insusceptible to antibody both in vivo and in vitro. Prior to the 5th day their susceptibility to antibody mediated attack was shown to depend on the integrity of the complement system. This appears to be the first time that complement has been demonstrated to play a role in immunity to a helminth infec- tion in vivo. Weanling rats born of mothers infected with Taenia taeniae- formis were found to be passively protected against homologous challenge. Cross fostering of normal suckling rats onto immune mothers established that passive transfer occurred via the colostrum and milk. Immunoglobulin fractions from immune colostrum containing yA were fed to 12- to l4-day-old rats for 4 days via stomach tube. Significant passive protection against challenge with T. taeniaeformis was achieved with 7A from 1 of 3 colostrum pools. The effect of colostral yA preparations on the infectivity of freshly hatched oncospheres of T. taeniaeformis was measured by the intraintestinal inoculation of immunoglobulin solutions into isolated gut loops containing hatched eggs of the parasite. yA from 1 of 3 pools of immune colostrum caused a significant reduc- tion in the number of parasites which reached the liver. This appears to be the first time that protective activity against a helminth infection has been achieved with yA. A fraction of immune colostrum containing both 73y1 and 78y2 immunoglobulins was found to confer passive protection when inoculated parenterally. In view of the prolonged period of absorption (ca. 18 days) of 7S immunoglobulins from the gut by Antony J. Musoke the suckling rat, it seems likely that these antibodies are pri- marily responsible for the natural passive transfer of protection from mother to young. IMMUNOLOGIC MECHANISMS IN RESISTANCE TO EXPERIMENTAL INFECTION WITH TAENIA TAENIAEFORMIS BY “9 Antony J. Musoke 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 1975 Dedicated to my parents ii ACKNOWLEDGEMENTS I wish to express my appreciation for the Assistantship awarded to me by the Department of Microbiology and Public Health, Michigan State University. I wish to express my sincere appreciation to my academic advisor, Dr. Jeffrey F. Williams, for his guidance, encouragement and personal kindness throughout these studies and during the preparation of this dissertation. The advice and encouragement of my guidance committee, Drs. Gordon Carter, Robert Corwin, Harold Miller, Richard Patrick, and Delbert Whitenack, are gratefully acknowledged. The companionship and encouragement and shared self-teaching experiences that I have enjoyed with my fellow students, Drs. Wes Leid, Bruce Hammerberg, Roger w. Cook, Ashraf Aboulatta, Mr. Stephen Hustead, Mr. John Picone, and Miss Martha Calkins, have been enriching, helpful and have led to friendships that will be long remembered. Finally, the excellent technical assistance offered by Mrs. Anndy Whipple and Miss Marla Signs is greatly appreciated. iii TABLE OF CONTENTS I NTRODUCT I ON 0 O I O O O O O O O O O O O O O O O O O L I TE RAN RE REV I Ew O O O O O O O O O O O O O O O O 0 Biology of Taeniid Parasites of Man. . . . . Taenia solium . . . . . . . . . . . . Taenia saginata . . . . . . . . . . . Treatment and Control of T. solium and T. saginata Biology of Taeniid Parasites of Laboratory Animals Taenia taeniaeformis. . . . . . . . . Echinococcus granulosus . . . . . . . Taenia crassiceps . . . . . . . . . . Immunity to Taeniid Parasites. . . . . . . . Antibodies Involved in Immunity to Taeniid Parasites. . . . . . . . . Mechanism of Action of the Antibody . Antigens Involved in Immunity . . . . Immunity and Host-Parasite Relationships. Rat and Mouse Immunoglobulins. . . . . . . . Immunoglobulins of the Rat. . . . . . Immunoglobulins of the Mouse. . . . . Cytophilic Antibodies . . . . . . . . MFEENCES O O O O O O O O O O O O O O O O I O I O 0 ARTICLE 1 - IMMUNOGLOBULINS ASSOCIATED WITH PASSIVE TRANSFER OF RESISTANCE TO TAENIA TAENIAEFORMIS IN THE MOLISE O C O O O O O O O O O O O O O O 0 ARTICLE 2 - IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMIS. III. PROTECTIVE ANTIBODY RESPONSE TO IMPLANTED PARASITES. ARTICLE 3 - IMMUNOLOGICAL RESPONSE TO INFECTION OF THE RAT TO INFECTION WITH TAENIA TABNIAEFORMIS. SEQUENCE OF APPEARANCE OF PROTECTIVE IMMUNO- GLOBULIN AND THE MECHANISM OF ACTION OF 7S'Y2a ANTIBODIES O O O O O O O O O O O O O O 0 iv V. Page 10 11 13 13 14 17 19 21 21 25 26 28 38 %$ 77 Page ARTICLE 4 - THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA THENIAEFORMIS. IV. IMMUNOGLOBULINS INVOLVED IN PASSIVE TRANSFER OF RESISTANCE FROM MOTHER TO OFFSPRING. . . . . . 110 LIST OF TABLES Table Page ARTICLE 1 1 Protective capacity of antiserum and immunoglobulin fractions isolated by column chromatography in recipient mice challenged by mouth with 300 eggs of Taenia taeniaeformis. . . . . . . . . . . . . . . . 48 ARTICLE 2 1 Effects of implanted metacestodes on resistance to T. taeniaeformis . . . . . . . . . . . . . . . . . . . 62 2 Protective capacity of immune implant serum (Pool 1) and immunoglobulin fractions in recipient rats challenged with 200 eggs of Taenia taeniaeformis . . . 68 3 Survival of larvae of T. taeniaeformis implanted in the peritoneal cavities of rats dosed orally with homologous eggs. . . . . . . . . . . . . . . . . . . . 70 ARTICLE 3 1 Passive protective capacity of immune serum in recipient rats inoculated with 700 artificially hatched and activated oncospheres of T. taeniae— fOrmis via a mesenteric vein . . . . . . . . . . . . . 93 2 Effect of depletion of complement (C3) on the establishment of T. taeniaeformis larvae in recipient rats injected with inactivated normal or immune serum. The animals were challenged with 500 eggs and killed 21 days later . . . . . . . . . . . . . . . 100 ARTICLE 4 1 Effect of ingestion of colostrum and milk from immune mothers on resistance to infection with Taenia taeniaeformis in young rats . . . . . . . . . . 120 2 Effect of feeding chromatographic fractions of immune colostrum to normal 14-day—old rats on their resistance to infection with Taenia taeniaeformis. . . 124 vi Table Page Effect of intraintestinal inoculation of chromato- graphic fractions of colostrum enriched for VA on the infectivity of oncospheres of Taenia taeniae— formis in isolated gut segments (sausages) . . . . . . 126 vii LIST OF FIGURES Figure Page ARTICLE 1 l Elution profile at 280 nm of mouse globulins obtained 28 days after infection with T. taeniae- formis and passed through Sephadex G-200 . . . . . . . 41 2 DEAE-cellulose elution profile at 280 nm of 7S immunoglobulins from mice infected with T. taeniae- form-is O O O O O O O O O O O O O O O O O O I O O O 0 O 44 3 Immunoelectrophoretic analysis of fractions of mouse 75 immunoglobulins eluted from DEAE-cellulose with 0.05 M phosphate buffer pH 5.8. . . . . . . . . . 47 4 Representative livers from groups of rats passively immunized with the DEAE fractions indicated. . . . . . 49 ARTICLE 2 1 Anion exchange chromatograph of 3 pools of serum from rats given implanted cysticerci of T. taeniae- forms 0 O O O O O O O O O C O O O O O O O O O O O O O 64 2 Representative livers from groups of rats passively immunized with serum from surgical implants. . . . . . 66 ARTICLE 3 1 Six- (a) and lO-day-old (b) larvae of T. taeniae- formis liberated from liver tissue by a combination of trypsin and collagenase digestion . . . . . . . . . 84 2 Elution profile at 280 nm of cobra venom on DEAE- cellulose with phosphate buffer and 0.05 M NaCl gradient 0 O O O O O O O O O O O O O O O I O O I O O O 87 3 Elution pattern at 280 nm of globulins (50 percent [NH4]2504) of immune rat sera collected 14, 21, 49 and 63 days after infection, on DEAE-cellulose with phosphate buffers and 2 M NaCl . . . . . . . . . . . . 92 viii Figure 4 Percent survival of larvae of Taenia taeniaeformis of different ages after exposure in vivo and in vitro to immune serum containing 78y2a antibodies. 5 Percent inhibition of lytic complement in serum of rats inoculated with 4 units of CoF per rat every 6 hrs for 5 days 0 O O I O O O O O O O O O O O O O 6 Representative livers from four groups of rats, two of which were inoculated with normal or immune inactivated serum. . . . . . . . . . . . . . . . . ARTICLE 4 l Sephadex G-200 gel filtration of globulin fraction of colostrum from the stomachs of 24-hour-old new- mm rats 0 O O O O O O I O O O O I O O O O O O O O 2 Anion exchange chromatography of F1 from gel fil- tration of rat colostrum . . . . . . . . . . . . . 3 Histogram demonstrating the association between susceptibility of suckling rats to infection with Taenia taeniaeformis and age at time of oral dosage with eggs . . . . . . . . . . . . . . . . . ix Page 96 99 102 116 119 123 INTRODUCTION The cysticercoses, affecting domesticated animals and man, result from the tissue migration of larvae of parasites of the family Taeniidae. This group of diseases not only impinges sig- nificantly upon public health and economic production of animal protein in developing countries but, in recent years, has become a cause of concern to the meat producers of industrialized nations. For example, bovine cysticercosis probably reaches its greatest significance in East Africa where Froyd (1960) estimated that up to 35 percent of carcasses were infected with Taenia saginata. However, several diastrous outbreaks of T. saginata infection occurred in feedlots in the Southwest United States in 1968 and the percentage of carcasses showing signs of cysticercosis in federally inspected slaughter houses has risen markedly in the last decade (Schultz, Hatterman, Rich and Martin, 1969). Over the years a variety of methods of control and preven- tion of cysticercosis have been proposed and applied, including programs of health education, improved sanitation and improvement of meat inspection to detect and eliminate infected meat. These control procedures, quite apart from being expensive to operate, particularly in the most severely affected countries, involve complex socio-economic factors and have therefore met with very 2 limited success. In the absence of any chemotherapeutic approaches to treatment of the tissue stages, an immunologic solution to the problem is increasingly called for. Large scale experimentation in domesticated animals is premature at the present stage of knowledge, but fortunately an experimental model for cysticercosis can be found in the natural host-parasite relationship between T. taeniaeformis and laboratory rodents. The work reported herein was designed to attempt to characterize some of the immunological mechanisms operating in resistance to challenge infection with T. taeniaeformis. The literature review has therefore been organized firstly to provide background information on the two important taeniid organisms of man, T. solium and T. saginata. This has been done to point out the analogy between the biology of these important organisms and that of the experimental models which are used. The second section of the literature review deals with protective immune responses to taeniid parasites. Here emphasis is placed on the experimental evidence to date regarding participation of immune mechanisms in resistance to cysticercosis, although evidence from the field is also reviewed. In addition, current theories on the means whereby tissue dwelling metacestodes are able to evade or resist immunological attack have been identified and examined. Lastly a section concerning the current classification and biological functions of rat and mouse immunoglobulins is provided. This information is considered crucial for an understanding of the work reported below much of which deals with exploration of the role of immunoglobulins in immunity. LITERATURE REVIEW Cestodal organisms of the family Taeniidae, order Cyclophyllidea, constitute a group of parasites of both medical and veterinary importance. Parasites of genera Taenia and Echinococcus invade the tissues of man or domesticated animals, which act as intermediate or definitive hosts. In this first section the biology of the important human pathogens is reviewed and an account is given of the principal experimental models used in research. Biology of Taeniid Parasites of Man Taenia solium Taenia solium has a cosmopolitan distribution and is an important parasite wherever man consumes raw or insufficiently cooked pork. It is currently most commonly found in the Slavic people (Czechs, Serbs, etc.) but also occurs frequently in Mexico, many Latin American countries and North China (Soulsby, 1968). Life Cycle. Adults of T. solium live attached to the wall of the small intestine of man. Eggs escape from the branched uterus through a ventral longitudinal slit either before or after the ripe proglottids become free. Like other taeniids, the egg is composed of an outer layer of keratinized blocks with several 3 4 membranes inside of the blocks and finally a 6-hooked or hexacanth embryo within the last membrane. The eggs remain viable on the soil for periods of up to 2 or 3 months. On ingestion by hogs or man, the eggs hatch and activate in the intestine. The initial mechanism consists of the release of the oncosphere which is effected by digestion of the cement substance holding the keratin blocks. The conditions vary with the species of tapeworm: T. taeniaeformis and E. granulosus require pancreatin but not pepsin enzymes; for eggs of T. saginata, pancreatin is ineffective but pepsin is essential (Silverman, 1955; Smyth, 1963). Nothing is known about the hatching and activation conditions for T. solium. With the help of a combination of intestinal juices and other factors yet unknown, the larvae become activated and capable of penetrating the intestinal wall. Penetration into the gut wall by the oncosphere is rapid, occur- ring within 30 minutes. The parasites continue to migrate down the villus until a venule of sufficient size is reached. Penetra- tion and eventual migration through the intestinal wall may be aided by enzymes as several workers (Silverman and Maneely, 1955; Heath, 1971) have noted clear zones around the oncosphere, possibly resulting from lysis of cells around them. The oncospheres of T. solium are then carried to the skeletal musculature where they develop into mature cysticerci, called Cysticercus cellulosae, over a period of 2 months. Muscle infec- tions in man, however, are abortive and represent a dead end. Man is readily infected by ingesting the cysticercus in raw or inadequately cooked pork. The larva is digested free of the 5 host capsule and becomes activated in the small intestine where it attaches to the gut wall. The parasite develops into a mature worm producing large (10—12 mm) proglottids containing up to 40,000 eggs each. Pathology. The adult T. solium in the small intestine may cause considerable irritation at the site of its attachment to the mucosa or may produce intestinal obstruction. Human infection with the cysticerci may occur by the ingestion of eggs in contaminated food or by reverse peristalsis whereby eggs in the bowel are carried forward to the duodenum or stomach and here are stimulated to hatch. Cysticerci may then be found in every organ of the body of man but are most commonly encountered in the muscles, subcutaneous tissue, and the eye. Like other taeniid metacestode infections, the location of the parasite is important. The larvae do not cause a clinical syndrome unless their location impinges on a vital organ. The larvae are occasionally located in the brain. Here the parasite may cause little pathology while alive but upon death a great variety of neurological symptoms may develop, including epilepsy, incoordination, transient paresis, meningoencephalitis and failing vision (Faust, Russell and Jung, 1970). The presence of the growing larvae in the tissue provokes a local cellular reaction which includes infiltration with neutro- ‘phils, eosinophils, lymphocytes, plasma cells, and macrophages. .Fibrosis and necrosis follow this cellular picture with an eventual Caseation and calcification of the cyst. Taenia saginata Taenia saginata has a wide world distribution but occurs most frequently in Africa, Asia, and the USSR. Sporadic outbreaks have been reported in the Southwest United States and cases occur sporadically in most developed countries. Immigration and tourism lead to these occurrences where infection is not endemic in the area. Life Cycle. Taenia saginata resides in the small intestine of man and the life cycle is similar to that of T. solium except that cattle act as the intermediate host. Man is not susceptible to the tissue phase of this parasite. The fully grown "bladder- worm" (Cysticercus bovis) is usually situated in the intermuscular fascial layers surrounded by a connective tissue capsule. In heavy infections, however, organs such as liver, lungs, kidneys and abdominal fat may be infected. After 4-6 months, the cysticerci begin to degenerate and by the ninth month most of them may be dead. This depends on the size of the original infection and also the age of the animal when it was infected. Man becomes infected by eating raw or inade- quatly cooked meat containing the cysticerci of T. saginata and the larvae develop into the adult tapeworm in the small intestine. Gravid segments measuring about 16-20 mm in length and containing about 100,000 eggs each are passed out in approximately 2 months. Pathology. In the intermediate host cysticerci degenerate and eventually die by the ninth month of infection. Because of .its large size, the adult T. saginata is frequently responsible 7 for considerable disturbance in normal functions of the digestive tract. Diarrhea, hunger pains and loss of weight commonly accompany infection with this parasite. Leukocytosis is char- acteristic, with eosinophilia reaching 6-34 percent. Treatment and Control of T. solium and T. saginata Although treatment of infected persons can be carried out with certain taeniicidal agents (e.g., Yomesan), the effect of this effort is not long lasting. Control therefore has to be directed towards infection in domestic animals by way of appro- priate meat inspection procedures. Also corrective hygienic methods have to be encouraged if successful control of the para- sites is to be achieved (Silverman and Griffith, 1955). Until recently chemotherapy of medically important larval tapeworms has been impossible. This may have been a result of the relative inaccessibility of the larvae to the drugs and the diffi- culty and danger of experimentation with some of these infections. Now, however, several chemical compounds have been tested for their activity in laboratory models, and some promising agents have emerged. The cytostatic drug, cyclophosphamide, has been reported to kill the cysticercus of T. taeniaefbrmis in mice provided it is given at a critical time in the early development (Hinz, 1964). Salazar et a1. (1972) showed some evidence that larvae of T. solium are affected by Trichlorfon and may undergo regression. Williams at al. (1973) demonstrated conclusively that eggs of various 8 taeniid parasites become noninfective if treated with Bunamidine hydrochloride in vitro. Benzimidazoles are among the existing anthelmintics which seem to have broad spectrum activity against a variety of para- sites. Their activity against larval cestodes has only recently been investigated. Prophylactic and therapeutic effects of thia- bendazole or cambendazole have been demonstrated by Campbell and Blair (1974). They showed that when mice infected with T. taeniaeformis were fed either of these compounds, there was degeneration or complete destruction of the hepatic cysts. Another related compound, mebendazole, was also successfully used to treat mice infected with T. taeniaeformis (Thienpont, Vanpary and Heremans, 1974). These authors further reported that, apart from regression of the infection, the recovered mice were resistant to challenge infection with eggs of T. taeniaeformis. In separate trials, Heath and Chevis (1974) fed mebendazole to mice and rabbits infected with Echinococcus granulosus and T. pisiformis, respectively. This treatment resulted in complete destruction of all hydatid cysts and cysticerci. In some of these experiments, however, drugs were used at dosages much higher than would normally be tolerated in man or domestic animals. Also the effects of their repeated use needs to be investigated. Nevertheless, the results confirm that meta- cestodes are not necessarily invulnerable to anthelmintic treatment. Trials using cambendazole in cattle infected with T. saginata are already under way (Mann, personal communication) and, if these 9 drugs prove to be as effective as they are in laboratory models, eradication efforts for hydatidosis and cysticercosis may well become oriented toward their use. Biology of Taeniid Parasites of Laboratory Animals Taenia taeniaeformis Taenia taeniaeformis occurs in the small intestine of the cat and other related carnivores and is of cosmopolitan distribution. Life Cycle. The bladderworm stage, Cysticercus fasciolaris, develops in the livers of the intermediate hosts which are chiefly rats and mice and also the rabbit, the squirrel and muskrat. The intermediate host becomes infected by ingesting the egg. The oncosphere of T. taeniaeformis is freed by intestinal enzymes. The predilection site for the parasites is the liver, where the organism continues to migrate to the subcapsular area until encap- sulated with a fibrous tissue layer (Singh and Rao, 1967; Smyth and Heath, 1970). The larvae continue to develop until they reach the infective stage by 60 days (Hutchison, 1958). The cat becomes infected by ingesting livers containing metacestodes and the para- sites then complete their development by maturing in the small intestine. Tapeworm segments containing eggs begin to be passed out approximately 2 months after infection. Pathology. The cysticercus appears to be fairly harmless in the intermediate hosts, even when it occurs in large numbers. A malignant tumor has been associated with this infection in livers 10 of chronically infected rats, but its presence does not seem to cause any clinical syndrome. In the definitive host the head of the adult worm is buried deep in the mucosa causing irritation and, in rare cases, may cause a perforation. Echinococcus granulosus This species is found in the small intestine of dogs and wild carnivores and is widely distributed, reaching its greatest impor- tance in Africa and South America. Life Cycle. Adults of Echinococcus granulosus measuring 3 to 6 mm reside in the small intestine of the dog, jackal (Canis aureus) and wolf (C. lupus). After the eggs have been ingested by the intermediate host, they hatch in the intestine and the embryos migrate in the blood stream to various organs, especially the liver and lungs. Intermediate hosts include man, domestic animals, and numerous wild mammals. The oncosphere continues to grow in these tissues which become infiltrated with giant cells and eosinophils. On the outer layer are fibroblasts with many more eosinophils. By the fifth month the hydatid is approximately 1 cm in diameter. The hydatid consists of an outer friable non-nucleated layer and an inner nucleated germinal layer. From the inner layer scolices develop which may detach, being found free in the cyst fluid. Upon rupture of the mother cyst wall, daughter cysts develOp from these scolices. The larval form of E. granulosus can also be ll propagated in rats or gerbils by intraperitoneal implantation of the cysts or inoculation of the scolices, respectively. This secondary propagation of the parasite in laboratory animals makes it a highly suitable model for laboratory research. The definitive host becomes infected by consuming the viscera of infected larval hosts. Pathology. The damage produced by the hydatid cyst of E. granulosus in humans is both mechanical and allergic. The cysts, which may be lodged in vital centers, may interfere with the func— tion of the organ with damaging, even fatal, results. In some cases the cyst when in relatively unconfined location grows to tremendous size causing a physical burden to the patient and at times may burst precipitating sometimes fatal anaphylactic reactions. Treatment and Control. Surgical intervention when the cyst is located in operable sites is the only available treatment to date as nonsurgical procedures are unsuccessful. Control again involves proper hygienic methods as eggs of E. granulosus are highly resistant to disinfectants (Meymarian and Schwabe, 1962). Dogs should be prevented from eating carcasses of sheep, cattle and hogs in endemic areas. Taenia crassiceps Taenia crassiceps is a common cestode of the red fox (Vulpus vulpes) in Europe and of the Arctic fox (Alopex lagopus invitus). The metacestode, Cysticercus longicollis, has been reported in 12 various small rodents and lemmings (Dicrostomyx groenlandicus) (Freeman, 1962). It has also been found to develop to the adult stage in experimental dogs. The first case of human infection was reported recently in Canada (Shea, Marberley, Walters, Freeman and Fallis, 1973). Life Cycle. The definitive hosts become infected by ingesting cysticerci of T. crassiceps from rodents. The metacestode develops in the small intestine and grows to an egg-producing adult within 5 to 6 weeks. The cycle is then maintained when the eggs are ingested by mice. The oncosphere migrates to the pleural cavity and becomes infective for the definitive host in approximately 2 months. The metacestode can also be maintained by intraperitoneal transfer from mouse to mouse for indefinite periods, but the strain loses its infectivity to dogs (Freeman, 1962). This characteristic does not detract from its value as a laboratory animal mode. Pathology. Nothing is known about the pathology of T. crassiceps in intermediate hosts. Occasional enteritis and digestive disturbances have been reported in the dog and phasic eosinophilia has also been noted (Freeman, 1962). In the one case of human infection so far documented the larvae were located in the eye, causing impaired vision and marked eosinophilia (Shea et a1., 1973). Surgical removal of the cysti- cerci was effective in this case and may be the recommended treatment when possible. l3 Immunity to Taeniid Parasites In this section, the literature review deals with protective immune responses to taeniid parasites. Emphasis is placed on the antibodies involved and their mechanism of action, and the antigens which provoke formation of these antibodies. A section on immunity and host-parasite relationships is also included. Antibodies Involved in Immunity to Taeniid Parasites The early work of Miller and Gardiner (1932) and Campbell (1938) established conclusively the participation of antibody in protection against T. taeniaeformis. Immunity to this larval cestode was first demonstrated by the successful passive immuniza- tion of rats with serum collected 28 days after experimental infection (Miller and Gardiner, 1932; Campbell, 1938a). Similar studies by Kerr (1935) and Hearin (1941) demonstrated passive immunization against T. pisiformis in rabbits and Hymenolepis nana in mice. Blundell-Hassell, Gemmell and Macnamara (1968) were able to transfer immunity to T. hydatigena via serum from artificially immunized lambs. More recently, Leid and Williams (1974a) have demonstrated the ability of antibodies of a single well defined immunoglobulin class, 787 a' to passively transfer resistance to T. taeniaeformis 2 in the rat. A more complete conception of the role of antibody is essential if an understanding of the immune mechanism in this and other species is to be arrived at. The potential importance of locally produced yA antibodies on hatching and penetration of the 14 oncospheres in naturally or passively immunized animals also demands attention. Miller (1931) showed that immunity to T. taeniaeformis could be transferred from immune mothers to the offspring and later Gemmell, Blundell-Hasell and Macnamara (1969) reported some evi- dence that this may occur via colostrum in lambs born of ewes immunized with T. hydatigena. More recently Rickard and Arundel (1974) showed that lambs fed colostrum from ewes immunized with T. ovis were resistant to challenge infection. However, Urquhart (1961) has reported contrary results in identical experiments using animals infected with T. saginata. In rodents the mechanism of passive transfer of immunity to cysticerci has not been studied, although in both rats and mice prenatal and postnatal transfer of some immunoglobulins occurs (Brambell, 1970). It is not known which immunoglobulin types participate in this passive transfer of immunity but, as Gemmell and Macnamara (1972) have pointed out, yA could be implicated in this phenomenon since it predominates in secretory and mucosal surfaces (Heremans and Vaerman, 1971). Almost nothing is known of 7A function in newborn rats, although Hemmings, Jones and Williams (1973) could detect little or no human yA uptake across the gut of lZ-day-old rats. Mechanism of Action of the Antibody Immunity to T. taeniaeformis is believed by some workers to operate during the initial stages of embryo penetration through the lamina propria to the liver (Campbell, 1938a; Miller and Gardiner, 1932). Campbell (1938b) reported that there is an 15 antibody-mediated destruction of parasites before encystment. Leonard and Leonard (1941) extended this observation and showed that, while passively immunized rabbits were resistant to oral infection with T. pisiformis, they were susceptible to the intra- venous administration of hatched oncospheres. Thus they postu- lated that the intestine played a vital role in the mechanism of acquired immunity. Froyd and Round (1960) were able to substan— tiate this proposal by similar experiments in cattle with T. saginata and an "intestinal barrier" has been postulated by several authors in the field. Banerjee and Singh (1969) and Heath (1971) pursued this area of investigation by histopatho- logic studies of oncospheres of T. taeniaeformis in normal rats. They showed that epithelial cells may actually be lysed during penetration of the intestinal wall and that the oncosphere moves into the lamina propria seeking entry into the circulatory system en route to the liver. Heath (1971) further demonstrated that in immune animals oncospheres of T. pisiformis do not attempt to attach to the intestinal wall. This proposal is in agreement with Silverman (1955), who observed an immobilizing effect of immune serum on cestode oncospheres in vitro. There is no experimental evidence to date regarding dependence of this phase of immunity upon such amplification systems as complement activation and vasoactive amine function which could be triggered by antigen-antibody reactions, although Murrell (1971) demonstrated changes in the permeability of larvae of T. taeniae- formis in the presence of heterologous antibody and showed that 16 this effect was complement dependent. The only attempt to impli- cate complement in immunity to a helminth infection was that of Jones and Ogilvie (1971). However, these authors could not involve complement in the sequence of events which cause expulsion of Nippostrongylus brasiliensis worms from the intestine of rats. Smyth and Heath (1970) have reported a rapid and intense inflammatory reaction around degenerating larvae in cysticercosis and hydatidosis infections. It is thought that these reactions may be in part a result of a reaction involving antibody, antigen and mediator cells, particularly mast cells and neutrophils, causing release of substances like histamine, and slow reacting substance of anaphylaxis (SRS-A). The capacity to mediate immunologic release of histamine and SRS-A has been associated with two rat immunoglobulins--yE and 7SY2a--both of which show antibody activity in T. taeniaeformis infection (Leid and Williams, 1974a, 1974b). The release of vaso- active amines mediated by rat yE is dependent upon participation of the mast cell but does not require polymorphonuclear leukocytes or an intact complement system, as is the case in SRS-A release mediated by 7SY2a (Orange, Stechschulte, and Austen, 1972). The presence of accumulations of mast cells around cysticerci of T. taeniaefbrmis has been demonstrated by Varute (1971). It is not known to what extent such reactions may contribute to resistance to T. taeniaeformis, although some work has been done with other helminthic systems. Degranulation of eosinophils and basophils at the site of infection with a nematode, Trichostrongylus l7 colubriformis, has been observed by Rothwell, Dineen and Love (1971), and they have proposed participation of pharmacologically active amines produced by these cells in rejection of the parasite. An experimental approach to this proposal has been made by Keller and Ogilvie (1972) using chemical inhibition of histamine, serotonin and SRS-A and a similar analysis of the situation in T. taeniaeformis should be fruitful. Antigens Involved in Immunity Immunity to T. taeniaeformis can be provoked artificially by the implantation of live parasites or extracts of metacestodes (Miller, 1932; Freeman, 1962). Miller (1932) immunized rats with cysticerci intraperitoneally and produced immunity in recipients and Campbell (1936) was able to provoke a strong resistance to infection by inoculating rats with extracts of T. taeniaeformis. Similarly, Kerr (1934) produced some detectable immunity in rabbits against T. pisiformis by vaccination with extracts. Immunization has generally been much more successful when animals are exposed to live parasite material. Thus rats given intraperitoneal implants of live cysticerci of T. taeniaeformis or T. crassiceps become solidly resistant to challenge (Miller, 1932; Freeman, 1962), although Heath (1973) was unable to do this with cysticerci of T. pisiformis implanted subcutaneously. Campbell (1938a) observed that rats developed some immunity against T. taeniaeformis as early as 7 days after infection. Similar observations were recorded by Hearin (1941) on the early onset of immunity in mice against H. nana. Furthermore, Dow et al. 18 (1962) showed that embryos of T. taeniaeformis attenuated by irra- diation were effective in immunizing rats. Similarly an infection of chemically or physically treated activated embryos of T. hydatigena induced strong resistance in sheep to a challenge infection (Gemmell, 1969). In these two experiments the onco- spheres did not develop into metacestodes. Since killed eggs were ineffective in inducing immunity (Gemmell, 1964, 1969), all these experiments suggest that living organisms, or at least their metabolic products, may be essential for the induction of immunity. It would seem also from these observations that oncosphere survival but not necessarily complete reorganization is important in stimu- lating immunity. Evidence that some of these antigens may be metabolic products is provided by the work of Rickard and Bell (1971a, 1971b). They showed that oncospheres of T. taeniaeformis or T. ovis contained within millipore chambers and implanted intraperitoneally in rats or lambs resulted in a high degree of immunity. Furthermore, antigens collected from in vitro cultures of T. ovis induced a strong resistance to challenge infection in lambs. More recently Rickard and Outteridge (1974) showed that T. pisiformis culture antigen induced a good protective immunity when injected into rabbits, although their results were by no means as clear cut as those of Rickard and Bell (1971a, 1971b). Nothing is known of the chemical and physical characteristics of the protective antigens at this time. 19 Immunity and Host-Parasite Relationships Campbell (1938b) observed that T. taeniaeformis larvae out- grow their susceptibility to antibody by the sixth day of infection and proposed that this evasion of immunological attack by the parasites was derived from the host capsule surrounding the larvae. Recent work, however, indicates that this insusceptibility to antibody seems to result from inherent changes on the part of the parasite (Rickard, 1974). Furthermore, even in the case of the thick-walled metacestode of E. granulosus host proteins including immunoglobulins have been found to have reached the cyst fluid (Hustead and Williams, personal communication; Coltorti and Varela-Diaz, 1972). Since the original proposal of Campbell (1938b) workers in the field of cestode immunology have advanced further theories. These include: (a) coating of the parasite surface with host material (including Specific antibody) so that the cysticercus is not recognized as foreign (Varela-Diaz, Gemmell and Williams, 1972; Rickard, 1974); (b) hiding of the evolutionarily adapted parasite behind a self-made mask of molecular mimicry of host- tissue so that the host recognizes it as self (Sprent, 1959; Dineen, 1963; Damian, 1964); (c) induced synthesis by the parasite of components antigenically identical to host components (Capron et al., 1968). These possible mechanisms of cestode survival in immune hosts are briefly discussed subsequently. (a) Coating of the Parasite. Varela-Diaz et a1. (1972) have proposed a mechanism involving two antibodies and two antigens in juxtaposition. When one of the antibodies having a neutral effect 20 on the parasite combines with the antigenic determinant, the result- ing steric hindrance blocks the action of the lethal antibody. A similar hypothesis, involving one antibody and one antigen, has been advanced by Rickard (1974). He proposed that the parasites become coated with specific antibody which blocks their suscepti- bility to cell mediated immunity. This is analogous to the situa- tion in tumor enhancement whereby cancerous cells are coated with antibody and shielded from cellular attack. For both hypotheses (Varela-Diaz et al., 1972; Rickard, 1974) to be operational one has to assume that metacestodes are capable of inducing formation of a variety of antibodies, some of which have no destructive effects while others have. (b) Molecular Mimicry. This hypothesis suggests that host immune responses exert selective pressure on "fitness" for survival of the parasites. This selection for survival may only favor variants of the parasite which display reduced disparity with the host. A reduction of disparity is only necessary, of course, with those antigenic characteristics of the parasite which stimulate responses adverse to its survival in the host. As Coltorti and Varela-Diaz (1972) pointed out, this hypothesis is inadequate to account for the strict host specificity of the serum components in hydatid cyst fluid, for example. (c) Synthesis of Host Substances by the Parasite. According to this hypothesis, when a parasite enters a given host, the forma- tion of antigens resembling those of the host is induced and rejection of the parasite is avoided (Capron et al., 1968). Chordi and Kagan (1965) and Varela-Diaz and Coltorti (1973) have 21 demonstrated presence of multiple components identical to the host serum in hydatid cyst fluid as well as in membranes in a variety of hosts. If a process of induction were responsible for their synthesis, a considerable portion of the parasite genome would be required in order to code for a variety of molecules specific to the different hosts. In cases, then, where a parasite is capable of becoming established in several hosts, mechanisms of repression and derepression would have to operate to turn on or off wanted or unwanted genes. More recently Capron and his co-workers (Bout, Capron, Dupas and Capron, 1974) have provided some evidence in support of this hypothesis. They showed incorporation of radiolabeled isoleucine and lysine into host-like antigens produced by Schistosoma mansoni parasites in vitro, which suggested that these parasites possessed a system of codes of protein synthesis similar to that of the host. Rat and Mouse Immunoglobulins This portion of the literature review deals with classifica- tion and biological functions of rat and mouse immunoglobulins. Immunoglobulins of the Rat Escribano and Grabar (1962) published the first immunoelectro- phoretic patterns of rat serum and later Arnason et a1. (1963, 1964) distinguished three immunoglobulin classes, 7A, yo and yM. Binaghi and Sarandon de Merlo (1966) further subdivided the yG class into yGa and 7G More recently, Bazin et a1. (1974) have b. recognized 5 major rat immunoglobulin classes, yA, 7Sy1, 7372, yE 22 and 7M, with 7572 having 3 subclasses, a, b and c. Physical and biological properties of each class are discussed subsequently. Z§12_, This 78 immunoglobulin class has been recently divided into 3 subclasses of a, b and c according to their electrophoretic mobility and antigenic differences. The subclasses are separable by DEAE cellulose chromatography, using low ionic strength buffers (Stechschulte, Austen and Bloch, 1967). Like human 7SyG sub- classes (Gergely et al., 1971) they differ in their susceptibility to trypsin digestion, and 7Y2C being susceptible while 7SY2a 7Y2b is not (Nezlin et al., 1973). 7572a antibodies have the capacity to fix for short periods of time in the skin of recipient rats for participation in passive cutaneous anaphylaxis reactions and pre- pare rat peritoneal cells for antigen induced release of slow reacting substance of anaphylaxis (SRS-A) (Stechschulte et al., 1967; Orange, Valentine, and Austen, 1968; Morse et al., 1968). Leid and Williams (1974a) have demonstrated that 78y2 immuno- globulins, especially 7Sy2a, were responsible for passively trans- ferred resistance to T. taeniaeformis. Occasionally 7Sy2 immunoglobulins were found to be protective against Nippostrongylus brasiliensis (Jones, Edwards and Ogilvie, 1970). 2§11_3 The rat immunoglobulin class originally designated yA by Arnason et a1. (1964) and Binaghi and Sarandon de Merlo (1966) has now been shown to be 78y1 (Jones, 1969), although it is not the biological equivalent of 7571 from mice and guinea pigs (Binaghi, 1971). It binds complement and is active in hemolytic 23 assays (Jones, 1969; Morse et al., 1969), although its lytic proper- ties are not as pronounced as those of 7SY2 antibodies. Immuno- globulins of this class have been shown to confer protection against Nippostrongylus brasiliensis (Jones, Edwards and Ogilvie, 1970). 15, An antigenically distinct class of immunoglobulin has now been shown to exist and predominates in all mucosal surfaces and secretions (Nash, Vaerman, Bazin and Heremans, 1969; Stechschulte and Austen, 1970; Bistany and Tomasi, 1970). How- ever, it is present in low concentration in serum (Nash and Heremans, 1972). Electrophoretically yA has a faster mobility than 78y1 or 7572. Other characteristics include a relatively high carbohydrate content, presence in serum as a 78 immunoglobulin, and appearance in secretions as an 113 molecule. However, a poly- peptide chain, termed secretory piece, has not been detected (Stechschulte and Austen, 1970; Bistany and Tomasi, 1970). The absence of the secretory piece is not peculiar to the rat alone,° since it has not been demonstrated in sheep (Sullivan et al., 1969) or horse (Genco et al., 1969). Although yA immunoglobulins predominate on all mucosal surfaces and secretions, their importance in intestinal parasitic infections has not been investigated. IM, This is a slow moving immunoglobulin in an electrophoretic field and has a half life of approximately 3 days (Van Breda Vriesman and Feldman, 1972). Biologically yM has more agglutinating activity and is approximately 300 times more effective in lysis of 24 sensitized red cells than 75 antibodies. These characteristics, together with its valence of 10 and M.W. of 900,000 (198), make it similar to 7M antibodies of other species. Occasionally protective YM antibodies to Nippostrongylus brasiliensis have been found (Jones, Edwards and Ogilvie, 1970). IE, Reagins constitute one of the most striking antibody responses to parasitic infections (Ogilvie, 1964; Leid and Williams, 1974b, 1975). These antibodies were first described for animal parasites by Ogilvie (1964), who used passive cutaneous anaphylaxis tests to demonstrate reagins in rats, monkeys and sheep infected with N. brasiliensis, S. mansoni and T. colubriformis, respectively. Later this antibody was defined by Stechschulte, Orange and Austen (1970) to be rat immunoglobulin yE, similar to the immunoglobulin class designated yE in humans (Ishizaka and Ishizaka, 1967). 7E has the capacity to persist at a skin site in the rat for weeks as assessed by passive cutaneous anaphylaxis (PCA), and the ability to prepare mast cells passively in vitro for antigen- involved release of histamine and serotonin (Becker and Austen, 1966). A possible competition for receptor sites on the peritoneal cells between yE and 7Sy2a antibodies has been demonstrated both in vitro and in vivo (Bach et al., 1971; Ohman and Bloch, 1972). The known physico-chemical properties of this homocytotropic antibody are its fast electrophoretic mobility, molecular size of approximately 88 and susceptibility to inactivation by heat or 2-mercaptoethanol. 25 Although there is no evidence to connect yE antibodies with protective immunity to parasitic infections thus far, it is possible that yE may act in concert with other specific antibodies, for example 7Sy2a (Leid and Williams, 1974a). Immunoglobulins of the Mouse The classification of mouse immunoglobulins owes much to the work of Fahey and co—workers in the early 19605 and also to the availability of myeloma proteins which made further characteriza- tion possible (Askonas and Fahey, 1962; Fahey, Wunderlich and Mischell, 1964; Fahey and Sell, 1965). The nomenclature of these immunoglobulins is similar to that in the rat. As in the rat, five major classes have been recognized, yA, 7Syl, 7Sy2, yE and yM with 7SY2 divided into 3 subclasses, a, b and c (Kalpaktsoglou, Hong and Good, 1973; Prouvost-Danon, Binaghi, Rochas and Boussac- Aron, 1972). 78y2C has sometimes been referred to as IgG (7SY3) by 3 some authors (Grey, Hirst and Cohn, 1971; Bazin, Beckers, Platteau, Mets and Kints, 1973). The major difference in classification seems to be in the short term skin fixing antibody, being 75y2a in the rat and 7571 in the mouse. Physical properties of these mouse immunoglobulins such as molecular weight, electrophoretic mobilities are similar to those of the rat. So far association of purified, well defined classes of immunoglobulins with protective capacity against helminths has not been studied, although effects of immune serum or fractions on parasites have been reported. Haerin (1941) demonstrated passive immunization of mice with immune serum against Hymenolepis nana. 26 Using the same experimental model, DiConza (1969) found that the active serum factors were associated with the 7SyG immunoglobulin fraction of the infected mouse serum. Cytophilic Antibodies The sera of some animals immunized with different antigens, especially sheep red blood cells, contain antibodies with a pro- nounced affinity for macrophages and these antibodies were termed "cytophilic antibodies" by Boyden and Sorkin (1960). They can be demonstrated by autoradiography or by hemoadsorption (Boyden and Sorkin, 1960; Boyden, 1964). Cytophilic antibodies (CA) are known to be produced by rabbits (Boyden and Sorkin, 1960), guinea pigs (Boyden, 1964) and mice (Nelson and Mildenhall, 1967; Lokaj, 1968). However, they have not been described in other species. CA have been shown to belong to 75 globulins (occasionally 198), with electrophoretic mobility of B or fast Y (Brown and Carpenter, 1971). Mercaptoethanol appears to have no effect on the cyto- philic property of these antibodies, although Tizard (1969) had earlier reported contrary results. On the basis of the molecular weight (180,000), mercapto- ethanol resistance and pattern of chromatographic elution, it appears that CA belong to a 7SYG class of immunoglobulin, distinct from 7SyG hemoagglutinin. The significance of CA in vivo is at present unknown, but several suggestions have been advanced by Nelson (1970). These include: (a) CA may function as an Opsonin. In mice the factor was found to be capable of opsonizing erythrocytes for complete 27 phagocytosis. (b) They may serve as a recognition factor in the earliest phase of antibody production. (a) The factor could be involved in the expression of cell mediated immunity, especially delayed type hypersensitivity. CA have not been implicated in any way in parasitic diseases but they represent an important biologic activity of immunoglobulins which should be pursued in infectious diseases, including cysticercosis and hydatidosis. REFERENCES REFERENCES Arnason, B. G., de Vaux St. Cyr, C., and Grabar, P. 1963. Immuno- globulin abnormalities of the thymectomized rat. Nature, 199, 1199-1200. Arnason, B., de 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, 1‘2, 206—224. Askonas, B. A., and Fahey, J. L. 1962. Enzymatically produced subunits of proteins formed by plasma cells in mice. II. 82a myeloma protein and Bence Jones protein. J. Exp. Med., 112, 641-653. Bach, K. M., 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., 111, 771-772. Banerjee, D., and Singh, K. 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Successful vaccination of lambs against infection with Taenia ovis using antigens produced during in vitro cultivation of the larval stages. Res. Vet. Sci., 1_2_, 401-402. Rickard, M. , and Outteridge, P. M. 1974. Antibody and cell- mediated immunity in rabbits infected with the larval stages of Taenia pisiformis. Z. Parasitenk, fl, 187-201. Rothwell, T. L. W., Dineen, J. K., and Love, R. G. 1971. The role of pharmacologically-active amines in resistance to Trichostrongylus colubriformis in the guinea pig. Immunology, 31, 925-938. Salazar, M., Gonzalez, D., and Vega, M. V. 1972. Ensayo de tratamiento de la cisticercosis con metrifonato. Rev. Invest. Solud. Publica, 3_2, 1-7. Schultz, M. G., Hatterman, L. G., Rich, A. B., and Martin, G. A. 1969. An epizootic of bovine cysticercosis. J.A.V.M.A., 155, 1708-1717. Shea, M., Marberley, A. L., Walters, J., Freeman, R. S., and Fallis, A. M. 1973. Intraocular Taenia crassiceps (Cestoda). Amer. Academy of Ophth. and Otol., 33, 0P-778-OP-783. Silverman, P. H. 1955. A technique for studying the in vitro effect of serum on activated taeniid hexacanth embryos. Nature (Lond.), 176, 598-599. Silverman, P. H. , and Griffith, R. B. 1955. A review of methods of sewage disposal in Great Britain with special reference to epizootiology of Cysticercus bovis. Ann. Trop. Med. 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., 4_9_, 326-330. Singh, B. B., and Rao, B. V. 1967. On the development of Cysti- cercus fasciolaris in albino rat liver and its reaction on the host tissue. Ceylon Vet. J., 13, 121-129. 36 Smyth, J. D. 1963. The biology of cestode life cycles. Tech. Comm. No. 34. Commonwealth Bureau of Helminthology. Farnham Royal, England, Commonwealth Agricultural Bureaux. Smyth, J. D., and Heath, D. D. 1970. Pathogenesis of larval cestodes in mammals. Helminthological Abstracts, Series A, 1, 2-23. Soulsby, E. J. L. 1968. Helminths, Arthropods and Protozoa of Domesticated Animals. (Monning.) p. 114-118. The Williams and Wilkins Company, Baltimore. Sprent, J. F. A. 1959. Parasitism, immunity and evolution. The Evolution of Living Organisms. Symposium of the Royal Society of Melbourne, Dec. 1959, G. W. Leeper, ed. Melbourne University Press, p. 149-165. Stechschulte, D. J., and Austen, K. F. 1970. Immunoglobulins of rat colostrum. J. Immunol., 104, 1052-1062. Stechschulte, D. J., Austen, K. F., and Block, 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., 133, 127-147. Sullivan, A. L., Prendergast, R. A., Antunes, L. J., Silverstein, A. M., and Tomasi, T. B. 1969. Characterisation of the serum and secretory immune systems of the cow and sheep. J. Immunol., 133, 334—344. Thienpont, D., Vanpary, 0., and Heremans, L. 1974. Anthelmintic activity of mebendazole against Cysticercus fasciolaris. Tizard, I. R. 1969. Macrophage cytophilic antibody in mice: Differentiation between antigen adherence due to these antibodies and opsonin adherence. Int. Arch. Allergy, 33, 332-346. Urquhart, G. M. 1961. Epizootiological and experimental studies on bovine cysticercosis in East Africa. J. Parasit., 33, 857-869. Van Breda Vriesman, P. J. C., and Feldman, J. D. 1972. Rat IgM immunoglobulin isolation and some biological charac- teristics. Immunochemistry, 3, 525-534. Varela-Diaz, V., and Coltorti, E. A. 1973. The presence of host immunoglobulins in hydatid cyst membranes. J. Parasit., 33, 484-488. 37 Varela-Diaz, V. M., Gemmell, M. A., and Williams, J. F. 1972. Immunological responses of the mammalian host against XII. Observations on antigen tapeworm infections. Exptl. sharing between Taenia hydatigena and T. ovis. Parasit., 33, 96-101. Mast cells in cyst-wall of hydatid cyst of Varute, A. T. 1971. Indian J. Exp. Biol., Taenia taeniaeformis (Batsch). 3, 200-203. Williams, J. F., Colli, C. W., Leid, R. W., and MacArthur, R. 1973. Effects of Bunamidine hydrochloride on infectivity of taeniid ova. J. Parasit., 33, 1141-1144. IMMUNOGLOBULINS ASSOCIATED WITH PASSIVE TRANSFER OF RESISTANCE TO TAENIA TAENIAEFORMIS IN THE MOUSE A. J. MUSOKE AND J. F. WILLIAMS Department of Microbiology and Public Health Michigan State University East Lansing, Michigan 48824 This is journal article No. 6738 from the Michigan Agricultural Experiment Station. SUMMARY. Mice were found to be protected against Taenia taeniae- formis infection by passive transfer of serum collected from donors 28 days after infection. The protective activity resided exclusively in the first fraction of 7S immunoglobulins eluting from DEAE cellulose at pH 5.8 with 0.05 M phosphate buffer. This fraction contained 78y1 and 75y2 immunoglobulins but no detectable 7A, yM or skin sensitizing activity. Fractions containing 7572 alone were ineffective in passive transfer. INTRODUCTION Leid and Williams (1974a) have recently Shown that passive transfer of resistance to Taenia taeniaeformis infection in the rat can be achieved with immunoglobulins of the 78y2a type. The bio- logical characterization of antihapten antibodies of this nature had previously been described by Morse, Bloch and Austen (1968). They demonstrated short-term sensitization of rat skin for passive cutaneous anaphylaxis (PCA) and antigen-induced release of SRS-A from neutrophils and histamine from mast cells. However, 7Sy2a antibodies to T. taeniaeformis were inactive in PCA tests (Leid and Williams, 1974b), suggesting the possibility that subpopulations of this immunoglobulin with distinct biological functions might be stimulated by helminthic infections. We have pursued the peculiar association of protective activity against T. taeniaefOrmis with physico-chemically distinct immuno- globulin types and report here on the localization of the protective capacity of infected mouse serum in an immunoglobulin fraction 38 39 normally associated with Short-term PCA and mast cell sensitization in the mouse (Revoltella and Ovary, 1969; Binaghi, 1971). MATERIALS AND METHODS Parasite 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. The parasite was maintained as described by Leid and Williams (1974a). Experimental Animals Female 28-day-old mice and rats were obtained from Spartan Research Animals, Haslett, Michigan. Antisera Twenty-eight-day-old mice were infected orally with 400 eggs of T. taeniaeformis and 28 days later were exsanguinated by severing the major thoracic vessels under C0 anesthesia. Serum was stored 2 at -20°C. Fractionation of Antiserum Antiserum was centrifuged at 10,000 g for 10 minutes and 4 ml portions were dialyzed overnight against 0.1 M Tris-HCl buffer pH 8.0, and applied to a Sephadex G-200 column (2.5 x 100 cm), equili- brated against the same buffer. The ascending portion of the first peak (Figure l) was taken and this fraction contained yM and one B-globulin arc demonstrable in immunoelectrophoresis. The 40 Figure l. Elution profile at 280 nm of mouse globulins obtained 28 days after infection with T. taeniaeformis and passed through Sephadex G-200. -Fractions were pooled as indicated and F1 was used for passive transfer experiments. F2 was fractionated further on DEAE-cellulose columns. 41 0 I0 o In N "' 1" o “"1083 I. MISNQO ‘IVOIJdO t 100 80 60 b TUBE NUMBER Figure l 42 descending portion of the first peak, together with the 75 peak, were further fractionated on DEAE-cellulose. Stepwise elution was performed using sodium phosphate buffers in the following sequence: 0.005 M, pH 7.8; 0.01 M, pH 7.8; 0.05 M, pH 5.8; 0.1 M, pH 5.8; and finally 2 M NaCl. All buffers were made 0.015 M in NaCl. The pooled fractions under each peak (Figure 2) were concentrated using polyethylene glycol and were tested against rabbit anti-whole mouse serum and anti-IgM and IgA (Meloy Laboratories, Springfield, Virginia) in both immunoelectrophoresis and double diffusion in gel tests, following the methods described by Leid and Williams (1974a). Passive Transfer Fractions 1-5 from DEAE chromatography and the ascending por- tion of the first peak from gel filtration were restored to the original serum volume with phosphate-buffered saline. Each fraction was tested for its capacity to confer protection against a challenge of 300 eggs of T. taeniaeformis in mice. Mice were killed 21 days later and the results were analyzed by a modified Student's E-test. This experimental procedure was repeated using two further batches of serum harvested in a similar manner from other groups of mice. Bassive Cutaneous Anapgylaxis (PCA) All fractions from DEAE-cellulose chromatography were tested for their ability to provoke PCA in sensitized rats and mice following a modification of the procedure described by Revoltella and Ovary (1969). Positive samples were heated to 56°C for 1 hr, 43 Figure 2. DEAE-cellulose elution profile at 280 nm of 7S immunoglobulins from mice infected with T. taeniaeformis. Fractions Fl-FS were tested for protective capacity in passive transfer experiments and activity was localized in F3 (hatched area). PCA activity was restricted to F4. 44 m wuoofim a! a: 92 3. on 3 3 on . . . .nma. Illiill l/Oh. at . “ Qt “WW“ flfi 4 we.“ run... 3... .0 3.... . ........ . .s 8 s 8 on k ON 9n 3%. 31a 3...... as In rENrEud lrlll Small {Iliad L rll Snood IIIL o n:__:no.uo::EE_ 25039... *0 hen-20305020 “(no 0.5.ouoflcouu Seas—.528! coucugwsuu; wowed 45 or reduced and alkylated using the method of Nussenzweig, Merryman and Benacerraf (1964) and retested. RESULTS A typical DEAE-cellulose elution profile for 7S mouse immuno- globulins is shown in Figure 2. Two peaks were consistently eluted with the 0.05 M phosphate buffer, pH 5.8, and these were separated and identified as F3 and F4. These two fractions contained no detectable 7A or 7M, but immunoelectrophoretic analysis using rabbit anti-whole mouse serum showed that they contained distinct populations of 78yl and 78y2 immunoglobulins (Figure 3). The results obtained from a typical passive transfer experi- ment are shown in Table l, where the average numbers of parasites developing in the livers in each group are recorded. Protective capacity was exclusively and consistently associated with F3, and there was no evidence of passive transfer with other fractions from Sephadex G-200 gel filtration (Figure 4). Fractions 1-6 were tested for the ability to produce both homologous and heterologous PCA reactions in mice and rats, respectively. Latent periods of 2 hours and 72 hours were allowed prior to challenge. PCA activity was detected only in F4 (second peak 0.05 M eluate). This serum activity was Shown in homologous and heterologous systems after both 2-hour and 72-hour sensitiza— tion periods, but was destroyed by heating to 56°C for 60 minutes and by reduction and alkylation with 2-mercaptoethanol and iodoacetamide. 46 Figure 3. Immunoelectrophoretic analysis of fractions of mouse 78 immunoglobulins eluted from DEAE-cellulose with 0.05 M phosphate buffer pH 5.8. F3 and F4 were the first and second peaks, respectively, and the troughs were filled with rabbit anti-whole mouse serum. 47 Figure 3 Table 1. Protective capacity of antiserum and immunoglobulin fractions isolated by column chromatography in recipient mice challenged by mouth with 300 eggs of Taenia taeniaeformis Mean no. of + S.E. of P Protein fraction transferred* larvae :_s.d. mean value Normal mouse serum 75.5 i_l9.3 7.9 Immune mouse serum 0.5 :_ 0.83 0.34 <0.001 19S fraction of antiserum 76.0 i_41.6 16.98 n.s. F1-0.005 M DEAE-cellulose 80.3 :_46.78 19.1 n.s. eluate F2-0.01 M DEAE-cellulose 68.8 1 34.86 14.23 n.s. eluate F3-0.05 M (peak 1) DEAE- 3.3 i. 4.5 1.83 <0.001 cellulose eluate F4-0.05 M (peak 2) DEAE- 64.67 i_30.5 12.45 n.s. cellulose eluate F5-0.1 M DEAE-cellulose eluate 74.0 + 21.74 8.88 n.s. n.s. = not significant. * - by intraperitoneal injection. - average number of larvae developing each group. in the livers of 49 Figure 4. Representative livers from groups of rats passively immunized with the DEAE fractions indicated. The animals were challenged per 05 with 300 eggs of T. taeniaeformis and killed 21 days later. 50 w whomem u} I a“. . . ...UI’ . u.. ...H._..._.. 53mmm L<< Panes“. nmsmrnuc: 92 pflle pflla DH53 pflia A 0305" OIHI 005M OJ" 2. yr v w v v a 20- 14DAYS 21DAYS 20' 49mvs 100< 2°‘ sanavs 40 80 ——> TUBE N9 Figure 3 93 Table 1. Passive protective capacity of immune serum in recipient rats inoculated with 700 artificially hatched and acti- vated oncospheres of T. taeniaefbrmis via a mesenteric vein Mean no. of Treatment No. of Route of cysts in SE of (i.v.) rats challenge liver :_SD mean P-value Normal 5 intra- 100.2132.5 14.5 --- serum venous Immune 6 intra- 0.0 0.0 <0.001 serum venous Normal 5 oral 17l.4:17.5 7.8 --- Immune 6 oral 0.0 0.0 <0.001 serum 94 and Froyd and Round (1960) regarding the site of action of pro- tective antibodies, but was consistent with the results of an experiment reported by Campbell in 1938b. He had treated rats with immune serum at daily intervals after oral infection and shown that parasites within the liver remained fully susceptible to antibody for at least 4 days. He had speculated that the development of insusceptibility was due to formation of a fibrous host capsule which isolated the growing parasites from attack. We attempted to confirm his work on the development of insuscepti- bility in growing larvae and to establish if this change was derived from an isolating effect of the host capsule. Ten groups of rats were dosed with 500 eggs of T. taeniaeformis on day 0. Twenty-eight-day immune serum was administered to one group per day from day 0 to 10. All rats were killed 21 days later. The results depicted in Figure 4 showed that the effectiveness of anti- body begins to wane by the sixth day. Postoncospheral stages of T. taeniaeformis were liberated by enzymic digestion from livers of rats dosed orally with 10,000 eggs, at intervals of l, 2, 4, 6, 8 and 10 days and exposed to normal or immune serum in vitro. The parasites were then injected via a mesenteric vein into recipient rats which were sacrificed 21 days later. The results, also depicted in Figure 4, demonstrated that the development of insusceptibility to antibody is derived from inherent changes on the part of the parasites. Attempts were then made to determine the role of complement in the process of immunologic destruction of the parasites prior to 6 95 Figure 4. Percent survival of larvae of Taenia taeniae- formis of different ages after exposure in vivo and in vitro to immune serum containing 7Sy2a antibodies. *M 96 Susceptibility of T.taenioeformis to antibodngvivo 1001 30.4 Petcent. .J smvivel 50-4 ot cysts ~ 9 40* 2‘“) 1 a Age(days) oi cyst at time of exposure to antibody 191M Susceptibility of T.toenioeformis to antibody invitro 100] 80. Pement. . sowint 50.. ot cysts . r 40‘ 20.. A ' I M— ‘.-:-;-;.;.;.;.;.;. 11b 7 8 t Agddays) of cyst at time of exposure to antibody 19ng Figure 4 97 days. In view of the fact that serum complement levels can be effectively depleted using CoF for only 4-5 days (Maillard and Zarco, 1968), it was necessary to establish the optimum time at which to begin the CoF injections. Two groups of rats were inocu- lated with 1 ml of normal or immune serum intravenously and dosed with 2000 eggs of T. taeniaeformis per os. Twenty-four hours later the embryos were liberated from the liver tissue by tryptic diges- tion and injected via a mesenteric vein into normal recipients. The animals were killed 21 days later. The mean number of cysti- cerci in the rats receiving embryos from passively immunized rats was 21 1 3.5 while the control group had 25 1_10.2. These results indicated that there was a lag phase in vivo of at least 24 hrs before a 1 ml dose of antibody resulted in death of the parasites. In the following experiment two groups of rats were dosed orally with 500 eggs of T. taeniaeformis followed by an intravenous inoculation of 1 ml of heat inactivated 28-day immune serum. Two other groups received an equivalent amount of inactivated normal serum. Twenty-four hours later, one of the groups injected with normal or immune inactivated serum began to receive intraperitoneal doses of 4 units per rat of CoF every 6 hours for 5 days. The daily levels of total complement were measured by the method described by Kabat and Mayer (1971) (Figure 5). All rats were sacrificed 21 days later and the mean number of larvae in each group is shown in Table 2. Representative livers from this experi- ment are depicted in Figure 6. These results clearly demonstrate that an intact complement system is required for successful passive transfer of resistance to T. taeniaeformis. 98 Figure 5. Percent inhibition of lytic complement in serum of rats inoculated with 4 units of CoF per rat every 6 hrs for 5 days. ..~___A.— 99 olololloulloullmlluol 1 .962 mar?! LN LN: Ml-i “IO /0 SISA'I :IO NOIJJBIHNI .LNBOHQcH O N 100 Table 2. Effect of depletion of complement (C3) on the establish- ment of T. taeniaeformis larvae in recipient rats injected with inactivated normal or immune serum. The animals were challenged with 500 eggs and killed 21 days later. No. of Avg. no. of cysts SE of P-value IRS Treatment rats in liver :_SD mean x IRS-CoF Inactivated 6 55.3 :_14.2 5.3 --- normal serum + CoF Inactivated 6 2.7 :_ 2.2 0.9 --— immune serum (IRS) Inactivated 6 42.8 :_13.0 5.3 <0.001 immune serum + cobra venom (IRS-CoF) Inactivated 4 91.3 + 13.7 6.9 --- normal serum 101 Figure 6. Representative livers from four groups of rats, two of which were inoculated with normal or immune inactivated serum. The other two groups received inactivated normal or immune serum and CoF. The animals were sacrificed 21 days later. w musmflm mm. + 36.0 bOd imp int ef e: W! in | crc the fom Signl' Surpri ? i 103 DISCUSSION Our results on the sequential appearance of protective anti- bodies in rats infected with T. taeniaeformis have confirmed the importance of 7SY a immunoglobulins during the first 28 days of 2 infection. Not only were those chromatographic fractions from 14- and 21-day serum samples which were enriched for 75y2a the most effective for passive protection, but tryptic digestion of 7572b and 7872c from mixtures containing all three subclasses did not reduce the potency of these preparations. However, a remarkable extension of antibody activity to other chromatographic fractions was apparent with increasing time after infection until all prepara- tions containing 75 immunoglobulins conferred highly significant protection upon recipients. These results indicate either that a diversity of antibody responses develops to the protective antigen(s) produced by the developing parasite or that the antigens produced by older stages of the parasite differ sufficiently to cause the appearance of anti- bodies of distinct immunoglobulin classes. The protective antigens in more mature parasites must nevertheless either be shared or cross react with antigens of the early postoncospheral stages since the resistance mechanism is directed against these antibody labile forms. None of the fractions enriched for yM immunoglobulin showed significant activity in any of our samples. This was particularly surprising since we have found that similar fractions prepared from serum of rats given surgical implants of mature metacestodes of T. 104 taeniaefbrmis are highly effective in protecting recipients (Musoke and Williams, 1975b), whereas fractions enriched for 75y2a were much less effective. It seems likely that these contrasting find- ings derive from the fact that antigens are presented to the immunologic system of the host in a very different manner when live parasites are surgically implanted as opposed to their develop- ing within the liver parenchyma. With regard to the site at which protective antibodies exert their effect on the migrating organisms, our results clearly demon- strate that this effector mechanism can destroy parasites outside of the intestinal environment and that postoncospheral developmental stages in the liver retain a high degree of susceptibility to anti- body for approximately 5 days. We were unable to bypass the effects of circulating protective antibody by administration of challenge doses via the mesenteric vein and a clear pattern of gradually acquired invulnerability of hepatic parasites was demon- strated both in vivo and in vitro. These findings are comparable to the recent observations of Heath (1973) and Rickard (1974) indicating that metacestodes of Taenia pisifbrmis remain susceptible to the effects of immune serum for at least one week, and give little support to the idea of circulating antibody participation in an "intestinal barrier" (Leonard and Leonard, 1941; Froyd and Round, 1960). This is not to say that there is no intestinal component to the resistance mechanism in cysticercosis, however, since we have been able to show that preparations of colostral yA from immune rats can be used to confer protection upon neonatal 105 recipients, and that the action of VA is confined to the intestinal lumen (Musoke, Williams, Leid, and Williams, 1975). It remains to be shown whether or not intestinal secretions containing yA con- tribute to the resistance shown by actively infected animals. The observation that the effectiveness of protective antibody on the postoncospheral stages of T. taeniaeformis wanes as the para- sites develop is in agreement with the results described by Campbell (1938b). However, he postulated that this decrease derived from formation of the fibrous host capsule around the parasite isolating the organism from antibody attack. The results of our experiments do not support this notion since larvae isolated at various stages in vitro, in the absence of the host capsule, also showed this shift toward the antibody-invulnerable phase, especially from day 6 onwards. It appears that the parasites themselves acquire some structural or metabolic characteristics which make them invulnerable to antibody mediated attack. The interesting observation that there is a 24-hour lag phase in vivo before protective antibody exerts its lethal effect is difficult to explain at this time but may indicate a requirement for other elements of the host defense system in destruction of the parasites. The susceptibility of the early postonc03pheral stages of T. taeniaeformis to antibody was shown to be dependent upon the integrity of the complement system in the host. Rats depleted of complement over this critical period were highly significantly deficient in their ability to destroy challenge organisms when given doses of immune serum which resulted in almost total destruc- tion of all parasites in normal challenged animals. We believe 106 that this represents the first instance of the effectiveness of antibody against a helminth being dependent upon complement in vivo, although other workers have used similar experimental approaches in their investigations (Jones and Ogilvie, 1971). The latter authors used CoF to deplete C3 levels in rats but were unable to implicate complement in the sequence of events which results in the expulsion of Nippostrongylus brasiliensis from the intestine. In our system the complement dependent antibody mediated attack on the early stages of T. taeniaefbrmis may be responsible for immobili- zation and destruction of the parasites by lytic effects and could result in the chemotactic attraction of either specific or non- specific cellular components of the defense mechanism. In an attempt to clarify the means whereby older parasites evade antibody attack, we have made repeated efforts to demonstrate the presenceof amounts of hemolytic complement comparable to normal serum levels in the intracapsular fluid bathing the cysticerci of T. taeniaeformis. These have been uniformly unsuccessful (unpub- lished observations). However, a great many other serum proteins were detectable in this fluid in immunodiffusion tests. Although antigenic changes on the surface of the parasite (Varela—Diaz, Gemmell and Williams, 1972) or the masking effect resulting from adhesion of specific antibody (Rickard, 1974) have been postulated for the failure of rejection of cestode parasites in immune animals, it appears to us that anticomplementary factors produced by the metacestodes may be of prime importance in evading immunological damage. We have recently been able to demonstrate the release of 107 anticomplementary substances by cysticerci of T. taeniaeformis both in vitro and in viva (Hammerberg, Musoke, Hustead and Williams, in preparation) and are pursuing the biological significance of this finding in continuing studies in our laboratory. ACKNOWLEDGEMENTS This work was supported by Grant AI-10842-01 from the United States Public Health Service. The authors are grateful for the excellent technical assistance rendered by Mrs. Anndy Whipple and Miss Marla Signs. REFERENCES Ballow, M., and Cochrane, C. G. 1969. 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The migration of oncospheres of Taenia pisi- formis, T. serialis and Echinococcus granulosus within the intermediate host. Int. J. Parasit., l, 45. 108 Heath, D. D. 1973. Resistance to Taenia pisiformis larvae in rabbits. I. Examination of the antigenically protective phase of larval development. II. Temporal relationships and the development phase affected. Int. J. Parasit., 2, 485. Jones, v. F., and Ogilvie, B. M. 1971. Protective immunity to Nippostrongylus brasiliensis: The sequence of events which expels worms from the rat intestine. Immunology, 29, 549. Kabat, E. A., and Mayer, M. M. 1971. Experimental Immunochemistry, 2nd Edn., p. 149. Charles C. Thomas, Springfield, Illinois, U.S.A. Leid, W. R., and Williams, J. F. 1974a. Immunological response of the rat to infection with Taenia taeniaeformis. I. Immuno- globulin classes involved in passive transfer of resistance. Immunology, 21, 195. Leid, W. R., and Williams, J. F. 1974b. Immunological responses of the rat to infection with Taenia taeniaeformis. II. Char— acterisation of reaginic antibody and an allergen associated with the larval stage. Immunology, 21, 209. Leid, W. R., and Williams, J. F. 1975. Reaginic antibody response in rabbits injected with Taenia pisiformis. Int. J. Parasit., (in press). Leonard, A. B., and Leonard, A. E. 1941. The intestinal phase of the resistance of rabbits to the larvae of Taenia pisifbrmis. J. Parasit., 22, 375. Maillard, J. L., and Zarco, R. M. 1968. Decomplementation per un facteur extrait du venin de cobra. Effet sur plusieurs reactions immunes du cobaye et du rat. Annales de L'Institute Pasteur, 114, 756. Morse, H. C., III, Bloch, K. J., and Austen, K. F. 1968. Bio- logic properties of rat antibodies. II. Time-course of appearance of antibodies involved in antigen-induced release of slow reacting substance of anaphylaxis (SRS-Arat); Associa- tion of this activity with rat IgGa. J. Immunol., 191, 658. Musoke, A. J., and Williams, J. F. 1975a. Immunoglobulins associa- ted with passive transfer of resistance against Taenia taeniae- formis in the mouse. Immunology, 28, 97. Musoke, A. J., and Williams, J. F. 1975b. Immunological response of the rat to infection with Taenia taeniaeformis. III. Pro- tective antibody response to implanted parasites. Int. J. Parasit., (submitted for publication). 109 Musoke, A. J., Williams, J. F. Leid, R. W., and Williams, C. S. F. 1975. The immunological response of the rat to infection with Taenia taeniaeformis IV. Immunoglobulins involved in passive transfer of resistance from mother to off-spring. Immunology, (submitted for publication). Miller, H. M., and Gardiner, M. L. 1932. Passive immunity to infection with a metazoan parasite Cysticercus fasciolaris in the albino rat. J. Prev. Med., 6, 479. Nezlin, R. 8., Krilov, M. Yu., and Rokhlin, 0. V. 1973. Different susceptibility of subclasses of rat 1972 to tryptic digestion. Immunochemistry, 19, 651. Rickard, M. D. 1974. Hypothesis for the long term survival of Taenia pisifbrmis cysticerci in rabbits. Z. Parasitenk., 44, 203. Rickard, M. D., and Bell, K. 1971. Immunity produced against Taenia ovis and T. taeniaeformis infection in lambs and rats follow- ing in vivo growth of larvae in filtration membrane diffusion chambers. J. Parasit., 51, 571. Revoltella, R., and Ovary, Z. 1969. Reaginic antibody production in different mouse strains. Immunology, 11, 45. Silverman, P. H. 1954. Studies on the biology of some tapeworms of the genus Taenia. II. Factors affecting hatching and activation of taeniid ova and some criteria for their via- bility. Ann. Trop. Med. Parasit., 48, 207. Varela-Diaz, V. M., Gemmell, M. A., and Williams, J. F. 1972. Immunological responses of the mammalian host against tape- worm infections. XII. Observations on antigen sharing between Taenia hydatigena and T. ovis. Exptl. Parasit., 23, 96. APPENDIX THE IMMUNOLOGICAL RESPONSE OF THE RAT TO INFECTION WITH TAENIA TAENIAEFORMIS. IV. IMMUNOGLOBULINS INVOLVED IN PASSIVE TRANSFER OF RESISTANCE FROM MOTHER TO OFFSPRING A. J. MUSOKE, J. F. WILLIAMS, R. W. LEID AND CHRISTINE S. F. WILLIAMS Department of Microbiology and Public Health and Center for Laboratory Animal Resources Michigan State University East Lansing, Michigan 48824 This is journal article No. from the Michigan Agricultural Experiment Station. SUMMARY Weanling rats born of mothers infected with Taenia taeniae- fbrmis were found to be passively protected against homologous challenge. Cross fostering of normal sucling rats onto immune mothers established that passive transfer occurred via the colostrum and milk. Immunoglobulin fractions from immune colostrum containing yA were fed to 12- to 14-day-old rats for 4 days via stomach tube. Significant passive protection against challenge with T. taeniae- formis was achieved with VA from 1 of 3 colostrum pools. The effect of colostral yA preparations on the infectivity of freshly hatched oncospheres of T. taeniaefbrmis was measured by the intra- intestinal inoculation of immunoglobulin solutions into isolated gut loops containing hatched eggs of the parasite. YA from 1 of 3 pools of immune colostrum caused a significant reduction in the number of parasites which reached the liver. This appears to be the first time that protective activity against a helminth infection has been achieved with 7A. A fraction of immune colostrum containing both 7571 and 7SY2 immunoglobulins was found to confer passive protection when inocu- lated parenterally. In view of the prolonged period of absorption (ca. 18 days) of 7S immunoglobulins from the gut by the sucling rat, it seems likely that these antibodies are primarily responsible for the natural passive transfer of protection from mother to young. INTRODUCTION Natural passive transfer of resistance to cysticercosis from mothers to their offspring has been shown to occur in both 110 lll laboratory and domestic animals. Miller (1935) showed that female rats which were immune to infection with Taenia taeniaefbrmis transferred this resistance to their young. Gemmell, Blundell- Hasell and Macnamara (1969) reported that lambs born of ewes immunized against Taenia hydatigena were passively protected against challenge infection. More recently, Rickard and Arundel (1974) have shown that lambs were protected against Taenia ovis after suckling ewes which were either naturally or artificially immunized against this parasite. In rats the mechanism of natural passive transfer of immunity to T. taeniaeformis has not been studied but serum antibodies in the 7Sy2a immunoglobulin subclass have been implicated in artificial passive protection by Leid and Williams (1974). However, the pos- sible involvement of secretory yA (SyA) antibodies in the natural protection of newborn animals against cysticercosis has been raised by Gemmell and Macnamara (1972), and antibodies of this type have been shown to play an important role in neonatal immunity to a variety of infectious organisms (Porter, Noakes and Allen, 1970; Stone, Stack and Phillips, 1974). Although it has not been shown to be associated with a detec- table secretory fragment, yA is certainly the major immunoglobulin in rat colostrum (Stechschulte and Austen, 1970). We have there- fore investigated the characteristics of passively transferred resistance to T. taeniaeformis in newborn rats and report here on the contributions of colostrally derived antibodies of defined immunoglobulin classes in this process. 112 MATERIAL AND METHODS Parasite The strain of T. taeniaefbrmis used in these experiments was obtained from Mr. C. E. Claggett in the Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, Maryland. The parasite was maintained as described by Leid and Williams (1974). Experimental Animals Sprague-Dawley rats were purchased from Spartan Research Animals, Haslett, Michigan. They were given proprietary brand food and water ad libitum. Immunoelectrophoresis and Double Immunodiffusion Immunoelectrophoresis (I.E.P.) was performed following a slight modification of the method of Scheidegger (1955) in a Gelman apparatus (Gelman Instrument Company, Ann Arbor, Michigan), with a sodium barbital HCl buffer, u = 0.038, pH 8.2 (Williams and Chase, 1971). Two percent Noble agar (Difco, Detroit, Michigan) was pre- pared with barbital buffer diluted 1:2 and contained l:10,000 thiomersolate. Double immunodiffusion (D.I.D.) was performed according to a micromethod modified from that described by Williams and Chase, 1971). Two percent Noble agar (Difco, Detroit, Michigan) was prepared in a 0.1 M TRIS-HCl buffer, pH 8.1 with a final concen- tration of thiomersolate of l:l0,000. Measurement of Protein Concentration Protein concentrations were determined by the method of Lowry et a1. (1951). 113 Harvesting of Rat Colostrum Twenty-eight-day-old female rats were dosed orally with 1000 eggs of T. taeniaeformis, and four weeks later they were mated. Cblostrum was harvested following the method described by Stechschulte and Austen (1970). Three separate pools of colostrum were prepared, each derived from at least 12 litters. The suckling rats were permitted to nurse for 2-3 hours after an overnight star— vation period. They were then killed with carbon dioxide vapor and stomach contents were collected and taken up in phosphate buffered saline pH 7.2 (PBS) before homogenization. The homogenized colostrum was stirred overnight at 4°C and centrifuged at 4°C for 1 hour at 17,000 g. The clear fluid above the pellet and below the floating lipid layer was removed and concentrated with polyethylene glycol and dialyzed extensively against PBS. The globulins were precipi- tated with 50 percent saturated ammonium sulphate and dialyzed free of sulphate ions before chromatography. Chromatography_ Gel filtration chromatography was performed on a siliconized 2.5 x 100 cm column of Sephadex G-200 (Pharmacia, Uppsala), equilibrated with 0.1 M TRIS-HCl pH 8.0. The globulins from colostrum were dialyzed against the equilibrating buffer before application and eluted fractions were collected in 2.8 ml volumes. Elution profiles were prepared using the optical density of each fraction at 280 nm in a Beckman spectrophotometer (Beckman Instru- ment Company, Fullerton, California). 114 The procedure for ion exchange chromatography of rat colostrum was a modification of the method described by Stechschulte and Austen (1970). DEAE cellulose (DE-52, Whatman) was prepared according to the manufacturer's instructions and was poured in 1.5 x 20 cm siliconized glass columns. After initial equilibra- tion against 0.01 M phosphate buffer pH 7.8, proteins were eluted in a stepwise manner using 0.01 M phosphate followed by 0.05 M phosphate pH 5.8 and 0.05 M phosphate + 0.5 M NaCl pH 5.8. The first peak from a Sephadex G-200 fractionation was dialyzed exten- sively against the starting buffer before application to the column. Column eluates were collected in 2.8 ml fractions and the elution pattern followed by ultraviolet monitoring at 280 nm. Protein peaks eluted with each buffer were pooled and concentrated with polyethylene glycol. Preparation of Antisera Antisera to rat immunoglobulins were prepared according to the methods described by Leid and Williams (1974). Analysis of the Chromatographic Fractions Protein peaks obtained by gel filtration on Sephadex G-200 columns were analyzed by IEP and DID, against antisera to rat immuno- globulins. F1 (Figure 1) contained predominantly 7A with minor contamination by 7872 immunoglobulins. F2 contained both 7871 and 7572 immunoglobulins and was not fractionated further. yM immuno- globulins were not detected in colostrum by the techniques employed. F1 was fractionated by anion-exchange chromatography to remove contaminating 7872 immunoglobulins. The fraction eluted with 0.05 115 Figure 1. Sephadex G-200 gel filtration of globulin fraction of colostrum from the stomachs of 24-hour-old newborn rats. Fl contained predominantly 1A with some 7872 immunoglobulins. F2 contained 7Sy1 and 75y; immunoglobulins throughout but no yA. yM was not detectable in any of the fractions tested. --_—_——‘_—-—I~—*-—K_——— 116 wuogz *0 AllSNBO 1V3l1d0<——— —'—'—> TUBE N UMBER Figure l 117 M phosphate + 0.5 M NaCl buffer contained only 7A, with no detec- table 7872 immunoglobulins (Figure 2). RESULTS The first experiment was carried out to confirm Miller's observation (1935) that immunity was passively transferred from mother rats to their offspring, and to determine whether the resistance manifested in young rats at the end of the suckling period was attributable wholly to the ingestion of colostrum and milk or in part to the prenatal transmission of antibodies. Six infected and 6 normal female rats were bred and when close to term 3 from each group were placed in wire floor maternity cages modi- fied from those described by Hollander (1970). The rats littered on one inch (mink cage) mesh and the newborn rats fell immediately through the holes onto the bedding below and were collected. Baby rats from normal mothers were then cross fostered onto immune rats for the entire suckling period. Similarly, baby rats from the immune mothers were cross fostered onto normal rats. The other 3 rats in each group were allowed to suckle their young normally. All the offspring were challenged orally with 200 eggs of T. taeniaefbrmis when weaned at 21 days of age and they were sacri- ficed 21 days later and their livers examined for developing cysts. The results are shown in Table l. The unequal numbers in these groups resulted from cross fostering failures in several litters. However, highly significant resistance was observed in normal rats which had suckled immune mothers (P < .001, Student's 't' test) and in rats which suckled their own immune mothers (P < .001) when 118 Figure 2. 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