AN ELECTROPHORETIC STUDY OF SERA FROM RATS ARTIFICIALLY INFECTED WITH AND IMMUNIZED AGAINST THE LARVAL CESTODE, CYSTICKRCUS FASCIQLArtlS By Nathan Kraut A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1955 ProQuest Number: 10008667 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008667 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 ACKNOWLEDGEMENTS To: Dr, William D. Lindquist, for the guidance aria advice that made this work possible; Dr, Charles H , Cunningham, who so kindly co-operated in making available the space and facilities for con­ ducting the electrophoretic analyses; Dr, Jack J. Stockton, for making available and in­ structing in the use of the lyophilizing apparatus; Dr, Evelyn R. Sanders, whose valuable criticisms of the electrophoretic data and aid in calibrating the electrophoresis cells were ino3t helpful; Dr, George H, Burch, Pitman-boore Company, who so generously supplied the feline distemper vaccine; and Dr, William D. Baten, for his invaluable advice and assistance with the statistical methods used,,. The author wishes to express his sincere appreciation. TABLE OF CONTENTS Page I. INTRODUCTION 1 II . LITERATURE III. MATERIALS AND METHODS A* B. C. D* E. V. 33 Artificially Infected R a t s ..................... 33 Artificially Immunized Rats* • • • • • • • • 53 DISCUSSION A* B. VI. Artificially immunized rats * * . * * * * 3 1 Experiment IV ................... 31 ........................... 31 Experiment V RESULTS A. B. 19 Animal Care and M a n a g e m e n t ..................... 19 Bleeding, Infection and Immunization Procedures • • ......................... . . . 20 Total Protein Determinations • ................. 2l| Electrophoresis ..............................25 Experimental P r o t o c o l s ............... .. • • • 28 1, Artificially infected rats . . . . * • • 2 8 Experiment I . . ..................... 28 Experiment II • ......................... 29 Experiment III • • ....................... 29 2* IV. 1*. 7k Artificially Infected R a t s ..................... 7k Artificially Immunized Rats * ..................79 SUMMARI AND CONCLUSIONS BIBLIOG-RAPHI ............................ 82 ...................................... 86 LIST OF TABLES TABLE I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. Electrophoretic analysis Page of experiment I • • • • • • 35 Larvae in livers of rats bled for serum analysis of experiment I ..................................... 36 Larvae in livers of rats reinfected to determine presence of immunity Induced from initial infection .......... of experiment I . . . . . . • • • • • • • • Electrophoretic analysis 37 of experiment II . . . . . . ij.0 Larvae in livers of rats bled for serum analysis of experiment II . • • • • • • • • • • • . . • • • • lj.1 Larvae In livers of rats passively immunized with serum from rats bled on thirty-fifth day of experiment II ....................... Electrophoretic analysis of experiment III • • • • • 5^4- Larvae in livers of rats bled for serum analysis of experiment III ............................. 55 Larvae In livers of rats passively Immunized with serum from rats bled on twelfth and thirty-fifth days of experiment III • • . • • • * . . • • • • . • 60 Electrophoretic analysis of experiment IV . . . . . . 65 Larvae In livers of artificially immunized rats to test for presence of immunity in experiment IV ... 66 Electrophoretic analysis of experiment V 68 . . . . . . Larvae in livers of rats passively immunized with serum from rats bled on twenty-eighth day of experiment V •• 72 INTRODUCTION Following the improvements In the moving boundary electrophoresis apparatus introduced by TIselius (1937), there ensued a rapid and large number of applications of electrophoresis to biological, chemical, and physical prob­ lems. The extensive Electrophoresis Bibliography of Henley and Schuettler (1953) attests to the magnitude and diversity of electrophoretic studies. Reviews by Stern and Reiner (191+6), Luetscher (19I|.7), Gutman (19^8), Lewis (1950), Antweiler (1952), and Fisher (1953) deal with the significant contributions to medical and biological problems. The theory and methods have been well summarized by Longsworth and Maclnnes (1939), Longsworth (19i|-2, 1952), Abramson et al. (19li2), Alberty (l9lj-8) and Moore and Abramson (1950). In immunological studies, if the assumption is correct that all antibodies are proteins, then antibody production ultimately resolves itself Into a study of protein metabolism (Cannon, 1945)* Electrophoresis affords an excellent tool for the study of the protein content of body fluids, which, in turn, reflects the physiological state of the animal (Reiner, 1952). It is the consensus of opinion among para­ sitologists that the basic mechanisms operative in the immune response against animal parasites are essentially the same as those functioning against other infectious agents 2 (Taliaferro* 19l4»Oa; Culbertson, 1951) • Hence, electrophoresis should prove a valuable method in elucidating the many prob­ lems in parasitic immunology, as it has in other fields of immunological study. Luetscher (loc. cit•), for example, points to the significant contributions made by employing this method in establishing the relationship of antibody to the plasma proteins. In spite of its great potential for the clarification of many questions in parasitic immunology (e.g., hostparasite relationships, host response to parasitic organisms, parasite physiology and biochemistry) the field of parasitology has sorely lagged in taking advantage of this technique. planations for this, noted by Stauber (195^-) Ex ­ in his review of the applications of electrophoresis in parasitology, are the high cost of the apparatus and its inaccessibility in areas rich in clinical material in medical parasitology. The development of zone electrophoresis should stimulate efforts in this direction by virtue of its economy, simplicity and requirements for smaller amounts of sample (Tiselius and Plodin, 1953). Prom Stauber*s (loc. cit*) review it is readily evident that the majority of electrophoretic studies in parasitology have been concerned with protozoan infections. Furthermore, these studies were primarily directed towards clinical consider­ ations. The paucity of electrophoresis applications to the helminths is particularly striking and no work is known to 3 have been reported on host serum protein changes induced by a larval cestode infestation. In the above review, aside from the quantitative and qualitative changes noted in the serum proteins of infected hosts, the specific antibody has not yet been properly demonstrated and isolated in the Increased gamma-globulin fraction for any animal parasitic infection... .f/ The closest approach to the latter has been made by Wright and Oliver-G-onzalez (191-1-3 ^ • They demonstrated the presence of antibodies against Trichine11a spiralis adults and larvae in the Increased and electrophoretically isolated gamma-globulin of immune rabbit serum by the In vitro pre­ cipitin test. However, following in vitro absorption of the immune serum, no significant change occurred in any of the electrophoretic components* By an increased application of the electrophoretic method in the field of parasitology the resolution of such situations as encountered by Wright and Oliver-Gonzalez (loc. cit.) and other questions may be markedly hastened. k LITERATURE Immunological studies in parasitology during the first thirty years of the present century were dominated by a search for efficacious clinical diagnostic tests# Taliaferro*s (1929) important monograph collated the early significant literature In the field of parasitic immunology up through this period# In the subsequent period, an increased and vigorous attack has been made on the mechanisms and processes of animal parasite immunity. The resultant of these efforts has been the accrual of a body of evidence establishing the humoral and cellular aspects of animal parasite immunity# Through the study of cestodes, "the larval infections with the taenoid tapeworms have furnished the clearest-cut evidence of antibody action of any of the metazoan parasites" (Taliaferro, 19i^0b)• Dealing in general with the immunologi­ cal results of this latter period are the resumes of Talia­ ferro (I93lf, 19i*0b, 1914-8 ), Culbertson (1938, 1914-1, 1951) and Chandler (1953)• Summaries dealing with more specific seg­ ments of parasite immunity are those of Taliaferro (1940a) for helminth acquired immunity and Ackert (191+2^ for natural resistance In helminths. Peters (1938) and Larsh (1914-5* 1951) have reviewed the important phases of cestode immunity. An Immune response requires stimulation of the host tissues by foreign antigens# Parasites possessing a parenteral 5 stag© meet this criterion. Intestinal lumen-dwelling forms ©licit little, If any, immunological response by virtue of the failure of their antigens to reach the host tissues or to do so In an effective concentration. Most of the evidence demonstrating that lumen-dwelling forms initiate a negligible, or no, immune response comes from studies on adult tapeworms. Living adult Hymenolep3is nana introduced directly into the mouse Intestine failed to protect against a superimposed in­ fection with ova (Hearin, 19lj-l) • When the worms of an initial infection with H. dlminuta were eliminated from the rat intestine by chemotherapeutic or direct surgical means, the results suggested that the resistance of young rats to a second infection was a manifestation of the "crowding” effect (Chandler, 1939)• Oral and parenteral attempts to immunize other rats were negative (Chandler, 19i^0). Cats and kittens were not protected against a superinfection with cysts of Taenia taeniaeformis (Miller, 1931©* 1932d). The presence of the adult Multlceps glomeratus conferred no immunity against a second feeding of scolices to dogs (Clapham, I9I4.O)* Young chickens reinfected with Raillietina cesticillus had apparently little or no acquired immunity against the super­ imposed Infection (Luttermoser, 1938). Chandler (19)4-8) feels that the bulk of the evidence supports the contention that lumen-dwelling tapeworms induce little, If any, Immunity. In a discussion of Chandlerfs view, Stoll (19)i8) takes issue with the negative reports on 6 immunity In lumen-dwelling cestodes* The basis of his argument is the failure of workers to take cognizance of the fact that, In nature, there Is a constant reinfection of hosts with in­ fective forms. The eventual result of this repeated reinfec­ tion is a gradual quantitative build up of antigenic materials from the parasite. By being absorbed into the host system, the antigens ultimately Induce an immune response. It is his opinion that more attention must be given to the adequate reinfection of hosts In testing for the presence of immunity. There have been successful attempts to effect an immune response against adult cestodes residing In the gut lumen. Dogs that were heavily Infected with Taenia pisiformis re­ ceived intravenous injections of worm extracts. They appeared to have developed an Immunity to the toxic reaction that follows such an Injection in non-Infected dogs (Essex 1930). e_t a l . There is evidence that, in lambs under one year of age, Immunity develops against Moniezia exp ansa reinfection in cases where the lamb has recovered from an infection and continues to graze in infected regions. A local immunity involving the intestinal mucosa is its suggested basis (Seddon, 1931). A report of the resistance of rats to superin­ fection with Hymenolepis diminuta has been made, though only three rats were used (Palais, 19314.)* Kittens were partially immunized against T. taeniaeformis and dogs against Dlbothri ocephalus latus by subcutaneous Injections, and the feeding of homologous larval emulsions (Ohira, 1935). Attempts to 7 immunize adult and young dogs against the adult Kchlnococcus granulosus resulted in partial protection following immuniza­ tion with hydatid cyst material (Turner e_t al*. 1936) * It is apparent that the question of immunity against intestinal-dwelling cestodes is far from conclusively clari­ fied* This may be extended to lumen-dwelling helminths in general, for Burlingame and Chandler (19i|-1) present similar negative conclusions for an acanthocephalid intestinal worm, Moniliformis dublus* in rats* However, for the present, we may accept with reservations the view that worms living in the intestinal lumen elicit little, or no, immune response by the host. Larval cestodes present another situation. Since they undergo a parenteral phase or reside in the host tissue, there has been conclusive evidence of a humoral response against them. Rabbits infected with Coenurus glomeratus exhibited positive intradermal tests against the cyst fluid antigen (Clapham, 19^0). In four cases of human Cysticercus cellulosae, and in C. cellulosae infected pigs, precipitins were demonstrated in the serum of each (Trawinski and Rothfeld, 1935; Trawinski, 1936). Monkeys infected with Sparganum mansonoides had their sera tested against the specific anti­ gen by the complement fixation test with positive results* Sera from normal humans and monkeys gave negative tests (Mueller and Chapman, 1937). When monkeys were Immunized with adult S* mansonoides injections, evidence of protection against infection with the larvae was manifested by a failure 8 to develop elephantiasis and the prompt encapsulation of the spargana (Mueller, 1938). Hymenolepls nana is unique among cestodes in that it can undergo its adult intestinal lumen phase and parenteral larval, cysticercoid, stage in the same host. It passes its larval cycle within the villi of the small intestine. Resistance to superinfection has been re­ ported in mice and rats (Shorb, 1933; Hunninen, 1935)* How­ ever, it has been reported possible to superimpose an infec­ tion in both mice and rats, (Shorb* loc. cit.). A 75 percent reduction in the number of cysticercoids resulted following repeated injections of fresh adult worm material into mice (Larsh, 19Api+) • The humoral basis of immunity against H. nana is evident from the positive results obtained by the passive transfer of immunity to mice with sera from Infected animals and by in vitro serological tests with infected mouse serum (Hearin, 19lll; Larsh, 191+3) • Oxen previously infected with ova of Taenia saginata exhibited a resistance to a second infection when com­ pared to controls. A group of oxen allowed to graze on a pas­ ture known to be infested with T. saginata eggs showed no evidence of a secondary infection applied by drenching though control animals showed normal infections. The proposition is made, on the basis of these results, that cattle be naturally Immunized by allowing them to graze on known infested pastures (Penfold and Penfold, 1937)# Similar demonstrations of larval cestode immunity have been made In sheep artificially immunized against hydatid cysts (Turner e_t al.* 1937); ana for Cysticercus pisiformls in artificially and passively protected rabbits (Miller and Kerr, 1932; Kerr, 1935). 9 £* fasciolarls, which has been subjected to intensive immunological studies in rats, is the larval stage of the cyclophyllidean cestode, JT* taeniaeformis* The adult is the common cestode in the small Intestine of cats, and also occurs in dogs, foxes and other closely related carnivores* Rats, mice, other rodents and also rabbits harbor the larval stage in their livers, though extra-hepatic sites have been recorded in rats (Bullock and Curtis, 1925) and rabbits (Mahon, 19514-)# Wepfer in 1688 and Hartmann in 1695 were the first to report the finding of fasciolaris* The life cycle was experimentally established by Kuchenmeister in l85l by feeding cysts to cats* This work was confirmed and extended by Leuckart in 18514- by feeding eggs to white mice and the recovered larvae to cats (Sambon, 192l|.) • fasciolaris is morphologically characterized by the presence of several immature segments between the scolex and terminal bladder* The anatomy of the fully developed larvae has been studied by Rees (1951)# Sambon (loc* cit.) suggested the term “strobilocercus" which is commonly used for this larval cestode. The early development of .C* fasciolaris in experimentally infected rats has been studied by Crusz (19l|-8). Ten days after infection, the larva is an elongate bladder and no cells appear specifically differentiated* On the twenty-fifth day the scolex-anlage appears in most larvae, and by the thirty-sixth day the final phase of the bladder and invaginated scolex differentiation occurs. 10 Bullock and Curtis (192lf, 1926) have described the host tissue reaction to the larva. Upon the ingestion of the eggs by the rat, the onchospheres are liberated in the small in­ testine (Bullock et a_l.j 193U) • They proceed to penetrate the intestinal mucosa, enter into the blood vessels of the gut wall, and become lodged in the capillaries of the liver via the portal circulation. The localization of the larvae in the liver is not necessitated by any particular requirements found therein by the parasite. It has been adequately shown to be the result of mechanical filtration by the liver (Bul­ lock and Curtis, 1925)• Within twenty-one hours following infection, a larva has been observed in the liver capillary. By the sixth day t h e .larva are grossly visible on the liver surface• Liver reaction to the parasite can be conveniently summarized into two main stages: first, the exudative inflam­ mation and necrosis of liver tissue bordering the larvae that lasts about eight to ten days following infection with varying degrees of cirrhosis and fatty infiltration occurring in heavy infections; and secondly, the active proliferative stage that begins about the time the first stage ends, reaches its maxi­ mum phase between the fifteenth to twentieth day and then gradually subsides with the resultant formation of fibrouswalled cysts. The cells involved In the proliferative reac­ tion appear to arise from liver vascular endothelium and connective tissue. 11 Leonard (19I4.O) has studied the liver tissue response of rabbits non-immunized and passively immunized against £• pisiformls. An extension of the above work on C^* fasciolaris to immunized rats would be extremely interesting* fasciolaris is considered to be a benign parasite in rats (Miller and Dawley, 1928)* The larvae do cause sarcomas arising from their cyst walls (Bullock and Curtis, 1926), with evidence of the causative agent being associated with the calcium carbonate corpuscles of the parasite (Dunning and Curtis, 1914-6). Adenomatous lesions of the rat stomach have been attributed to the larval infection (Blumberg and Gardner, 1914.0 ), and myeloid changes in the spleens of rats, mice and hamsters appear 1933)* related to this organism (Hoeppli and Feng, Evidence presented in this thesis suggests liver dysfunction in heavily infected rats. However, the infection does not appear to be particularly detrimental to the host*s existence under conditions in nature. Significant contributions to parasite immunology have been rendered from studies utilizing fasciolarls» Co n ­ clusive demonstrations of acquired (Miller, 19318), artificial (Miller, 1931c) and passive (Miller and Gardiner, 1932c) immunity against a metazoan parasite have emanated from these efforts. Campbell (1938a) pointed to the suitability of C. crasslcollls (= fasciolarls) to immunological studies on the basis of its life cycle. He stated, (a) the degree of infection 12 can be easily regulated, (b) an accurate measure of the in­ tensity of infection can be obtained, due to the localization and size of the parasite, (c) any infections of _C* crassiccllis other than the experimental one, can be detected by variation in the size of cysts, and (d) during the early stage of its migration the parasite is in direct contact with the host's blood and therefore with any humoral antibodies that might be present#11 Miller (1931&, 3) showed that rats infected with G_* fasciolarls of from two to six months duration exhibited pro­ tection against a superinfection# Even a single large cyst in the liver of rats is protective against a second invasion. When the larvae were surgically removed from the liver cysts, the acquired immunity was still virtually complete sixty days following their removal (Miller and Massie, 1932). Artificial immunization is also effective in rats, though less so than in acquired immunity (Miller, 19313)• Using fresh larval and powdered adult worm suspensions, Miller (1930* 1931c) showed that either, injected usually intraperitoneally, was equally effective in producing immunity. Infections in control rats develop normally, whereas complete or nearly complete inhibition was effected in the injected animals# Miller (1931b, 1932e) immunized his animals with a series of six intraperitone&l injections given on alternate days with a rest period between the third and fourth injection. Immunity was found to be complete after the third injection 13 and still present 167 days after the last immunizing dose. However, initiating the injections following infection of the rats with eggs resulted in no protection. Living larvae and fragments thereof were protective when placed in the peritoneal cavity. Lipid extracted antigen was successfully employed, but negative results were given by powdered Taenia pisiformis, the common dog tapeworm* Further evaluation of non-specific worm materials was made by Hiller (1932b, 1935>&)* He found that though, as already noted, powdered T. pisiformis was ineffective, the introduction of living whole worms or portions thereof into the peritoneal cavity resulted in a high degree of immunity* The results on this and other related species of tapeworms suggests some degree of common antigenicity as a significant immune response was initiated by other related species* There is also conclusive evidence of the humoral nature of the immune response encompassed in the passive immunity studies. Sera collected from infected and artificially im­ munized rats were shown to protect rats when injected intraperitoneally (Hiller and Gardiner, 1932a, b, c). Some evi­ dence is also presented of the slight protective property of normal serum (Miller and Gardiner, 1932a, c)* Miller (1932c, 1934) has shown that the effectiveness of immune serum has decreased when injected nine days after infection and was no longer protective when given on the tenth day. This is attributed to the effective walling off Ik of the larvae by the host at this time, making them inacces­ sible to the immune serum* It also corresponds to the early proliferative stage described by Bullock and Curtis (192k)* Complete immunity persisted up to 36 days after injection of anti-serum In some rats (Miller and Gardiner, 1932c)* Immune serum in the amount of 1 ml* per 525 grams body weight afforded complete protection in rats. A direct quan­ titative relationship exists between the size of the infection and protectiveness of the serum. Serum from rats with eighteen or more cysts was most effective, and those with only dead or less than eighteen living cysts had a lesser value. Within four days following Infection, serum from the donor rats exhibited some immune capacity, and serum from a rat containing 123 living cysts of ten days duration prevented the develop­ ment of living cysts in the recipient of the anti-sera (Miller and Gardiner, 193^)* Passive transfer of immunity to the young of infected and artificially immune mother rats has been noted. in the young for about six weeks. It lasts Whether it Is passed on to the young in the milk and/or transplacentally is not known (Miller 1932a, 1935b). Important points brought out In the extensive work of Miller and his group briefly review above are as follovs: 1) rats already infected are immune to a superimposed infec­ tion* The degree of immunity is proportional to the size of the infection and is detectable within four days after 15 infection. 2) Active immunization can be induced with intra- peritoneal injections of adult and larval worm material. Immunity is evident after the third injection of an immunizing schedule of six injections given on alternate days and skipping one between the third and fourth. 3) Protection can be passively transferred if the anti-serum is given to the recipient animal within nine days after infection. Campbell (I93^a ) has extended the work on £. fasciolaris. demonstrating an "early” and "late” immunity involving two antibodies. He confirmed the quantitative relationship be­ tween the size of infection and degree of serum protective­ ness. In rats averaging 38 cysts, the serum showed some pro­ tective ability by the fifteenth day and was maximally effective by the twenty-eighth day after Infection. Serum from very heavily infected rats collected on the seventh day was about 75 percent protective, and maximally protective by the four­ teenth day. Two antibody mechanisms were evident. One destroys the larvae prior to encystment ("early immunity11) and is present within serum collected on the eleventh day after infection. The other destroys larvae after they have encysted ("late immunity11) and Is found in serum collected from animals on the twenty-eighth day. Campbell (1938b) was able to absorb out the "early1* immune factor from serum collected from rats eleven days after Infection, and from artificially immunized rat and 16 rabbit serum* He obtained negative results with serum of rats obtained on the twenty-eighth day following infection and containing the “late immunity" antibody. The latter probably explains Miller and Gardiner's (1932c) negative absorption experiment. The non-absorbable antibodies that are responsible for the "late immunity11 in a natural infection are probably a response to metabolic products or some other antigen elaborated by and necessary to the growth of the larvae (Campbell 1933b, c)* A cysticercocidal factor has been re­ ported from infected rat serum, though a similar property is exerted by physiological saline, and normal rat and guinea pig serum* It is suggested that it may be the “late immunity" factor (Chen, 1950)• Campbell (1936, 1937, 1939a) has shown that different chemical fractions of the parasite are variable In their ability to produce artificial immunization. Polysaccharide fractions from the larvae produced non-protective antibodies in injected rabbits. Intraperitoneal injections in rats of whole worm material and chemical fractions such as “globulin", nucleoprotein and “albumins" from fresh worm material pro­ duced a strong resistance, whereas albumins from dried worm material were of little value. Different fractions also pro­ duced different degrees of “early" and "late" immunity. However, no definite conclusions relative to the latter are drawn• 17 Dual antibody production is far from exclusive to this parasite. Similar results have been reported for Cysticercus pisiformis in rabbits (Leonard and Leonard, 19l|.l) • Among other helminths, convincing evidence has been obtained that the protective antibodies against .Nippostrongylus murls are those produced against its excretions and secretions, and not those against the somatic antigens (Thorson, 1951+)• Rabbits infected with Trichinella spiral!s show evidence of producing antilarval and antiadult humoral factors (OliverGonzalez, 19lpl) * These demonstrations leave little doubt that the host produces multiple humoral factors against the complex of antigens present in animal parasites. Not only are somatic antigens available, but also parasite metabolic products to the host antibody synthesizing mechanism. The relative im­ portance of these antigens to the resistance manifestations in animal parasite immunity is still an inadequately explored region (Chandler, 1953)* The purpose of the study herein reported was twofold. The first was to ascertain the effects of a fasciolaris infection in rats as reflected in the protein metabolism. Liver tissue involvement in this infection would be ex­ pected to influence the h o s t ’s tissue metabolism. Secondly, an attempt was made to relate the r a t ’s humoral response against the parasite to the serum proteins. That a dual antibody production against this infection occurs in rats 13 has been established (Campbell, 1933a). By analyses of sera from infected and artificially immunized rats, it was hoped to determine in which protein fraction(s) the antibodies are produced. Such information would enlighten our knowledge of the immune mechanisms against metazoan parasites. It was felt that electrophoresis afforded an excellent tool for these purposes. 19 MATERIALS AND METHODS A* Animal Car© and Management Animals utilized in this study were cats and albino rats. Cats of unknown age were acquired from private sources in and around East Lansing, Michigan. Upon arrival in the labora­ tory, the cats were placed in wire-bottomed cages and immunized against distemper with feline distemper vaccine.* Immunization was accomplished by giving two 2 ml. injections of vaccine intraperitoneally about 7-10 days apart. The cats were fed ground meal** mixed with about an equal amount of warm water, canned cat food***, and pas­ teurized, homogenized milk. Rats used were of the Wistar or Sprague-Dawley strain and were purchased from commercial sources. Since females have been shown to be more resistant than males to an infection with Cysticercus fasciolaris (Campbell, 1939b; Campbell and Melcher, 19l|.0), only males were utilized. Five to six weeks old rats were shipped with litter mates kept separate. Upon receipt, they were placed in wire-bottomed cages and kept in a room isolated from all other laboratory animals. This isolation was particularly Important insofar as it reduced ^ i t m a n - M o o r e Company, Indianapolis, Indiana. ^ K e n -L-Meal, Quaker Oats Company, Chicago, Illinois *** "Three Little Kittens" 20 the danger of accidental infection with eggs from the adult worm in the feces of infected cats. Food and water were available ad libitum. All rats were fed a pellet ration*. B. Bleeding, Infection and Immunization Procedures For each species of animal, the serum protein electro­ phoretic pattern is relatively constant with respect to the quantity and number of components present (Deutsch and G-oodloe, 191+5; Moore, 1914-5). However, a number of factors such as age (Heim and Schechtman, 19514-) > sex (Moore, 191+8), strain (Thompson e_t al.., 1951+)# season of the year (Hill and Trevorrow, 191+2), severe Injury (Gjessing et al v X9l|7) # and protein depletion induced by plasmapheresis and protein deficient diets (Chow et al., 1914-8 } cause significant vari­ ations in the serum pattern of an animal. In order to obviate such factors and to take into consideration the relatively small total blood volume of rats (Burke, 19514-)* the following procedure was used to obtain an adequate amount of serum. All litter mates were segregated equally, when possible, into control and experimental groups. The evening prior to the day of bleeding, food was withdrawn from the cages of the animals to be bled to reduce serum lipid turbidity. Th© experimental animals and the corresponding litter mate controls ’^Miller Bs Eaties, Battle Creek Dog Food Company, Battle Creek, Michigan. 21 were lightly anaesthetized by a 0 *05-0.10 ml# intraperitoneal injection of Halatal.-Jfr This was supplemented with ether to accomplish complete anesthesia* The rats were immobilized in a supine position on a board by means of rubber bands attached to the board. Under aseptic conditions, each rat was maximally bled by intracardiac puncture and then dis­ carded# The blood was placed in sterile test tubes, allowed to clot overnight at room temperature in a slanted position and the serum then collected# To insure an adequate amount of serum for testing, generally 2-3 rats were bled per ex­ perimental and control group# The serum collected from each rat was pooled, centrifuged for a minimum of 30 minutes at about 2,000 r.p.m* and the clear serum drawn off. Analysis of the serum was made immediately or it was stored in a l±° C* cold room until it could be analysed* In order to have a source of Taenia taeniaeformis eggs, cats were infected with encysted cysticerci maintained in stock rats# The cyst was introduced on to the back of the tongue of the cat who was forced to swallow it whole by gently restraining the cat from chewing# 3-1| cysts. Each cat received Adults with gravid segments developed in about six weeks. On the day rats were to be infected with eggs, an infected cat was sacrificed using ether or chloroform* The adult worms were recovered from the small intestine and the terminal ^Halatal, City, Mo. -Jensen-Salsbery Laboratories, Inc#, Kansas 22 gravid proglottids cut off. Eggs were recovered from the gravid proglottids by teasing them apart with dissecting needles in 0.85 percent NaCl. Quantification of the eggs was accomplished by using a hemacytometer. After adequate mixing of the eggs to insure a uniform suspension a sample was drawn off with a pipette drawn out to a fine point. same pipette was used for all determinations. The The chamber was charged and the number of eggs in the entire ruled area was counted. The average of ten such counts was taken and converted Into number of eggs per ml. of egg suspension. Rats were Infected intragastrically by injecting 1 ml. of the evenly dispersed egg suspension through a No. 8 French catheter that was passed orally into the stomach. Cysticerci, whole and lyophilized, were used as antigen for artificial immunization. Whole cysticerci were obtained from infected stock rats and washed 3-^i- times with sterile 0.85 percent N&C1 . The excess saline was blotted off with filter paper and the larvae then stored at minus 30° C. until used. When needed for immunization, the cysticerci were ground into a paste with a mortar and pestle and made up into a 10 or 20 percent suspension in 0.85 pereent NaCl. Cysticerci were lyophilized by placing them In 2 ml. ampoules. They were then quickly frozen at minus 70-75° C. in a 95 percent alcohol- dry ice mixture and then placed under vacuum (50-100 microns Hg.) for fy.8 hours at room temperature. At the end of this time, the necks of the ampoules were sealed with an oxygen 23 torch* Until used, the lyophilized material was stored at minus 3 0 ° C* For immunizing injections, the lyophilized larvae were ground into a fine powder and made up into 2*5 or 5*0 percent suspensions in 0*85 percent saline* Rats were immunized according to the schedule given by Miller (1931c)* Intraperitoneal injections of 1 ml* of worm suspension were given on alternate days for a total of six injections* A rest period was interposed between the third and fourth injection. Hence, if the first injection was made on the first day of a month, the schedule for the six injections would be as follows: 1-3-5-9-11-13* Injec­ tions given subsequent to the above standard series of injections will be noted under the experimental protocols of this section* Immune (from infected or artificially immunized rats) and control sera for passive immunization were obtained by pooling the sera from 8-10 rats for each group. Since the sera were used no later than 3 days after they were collected, they were stored at !+° C* with no preservative added. Each recipient of Immune or normal serum was injected with 1 ml* of serum intraperitoneally within 21+ hours before or after being Infected with Taenia taeniaeformis eggs. Since a limited amount of serum was available for passive transfer of immunity, the amount of serum injected was not based on body weight. Miller and Gardiner®s (19314-) and C a m p b e l l ^ (1938a, b) studies suggested that injections of serum not 24 based on body weight would serve the purpose of this in­ vestigation* The number of larvae present in the livers of infected rats was determined by counting the total number of cysts visible on the surface of the entire liver* All animals were routinely autopsied and the livers examined for the presence of cysts from an accidental infection* Any animal found to be accidentally infected was discarded* C* Total Protein Determinations Serum total protein was determined by the biuret reaction* Weichselbaum1s (l9lj-6 ) biuret reagent was used* A standard curve was constructed on semi-log graph paper by using a standard Bovine Albumin solution* whose nitrogen content was specified (about 10 mg* of protein nitrogen per ml*)* Con­ version into grams percent of protein was effected by multi­ plying the nitrogen content by the factor 6.25* The use and advantages of this standard solution were presented by Bernhard and Scher (1951)* Sera and standard solutions to be analysed for total pro­ tein were diluted 1:20 by adding 0*5 ml* of serum to 9*5 ml* of distilled water and mixing by gently inverting several times. To l+.O ml* of diluted serum were added i^.O ml. of biuret reagent and then well mixed by inversion. A blank ^Protein Standard Solution (Crystalline Bovine Albumin), Armour and Company, Chicago, Illinois. 25 was prepared by substituting ip.O ml. of distilled water for the diluted samples. As a check on the standard curve, the known albumin solution was frequently run with the sera being determined. The solutions were allowed to stand one hour at room temperature for maximal color development and then read on a photeloraeter*- at a wave length of 525 millimicrons “JHf * -n From the percent transmission of each sample, the total serum protein could be obtained directly from the standard calibrated curve. D* Electrophoresis All sera were analysed by the moving boundary electro­ phoresis technique, using the Perkin-Elmer Model 3$ Tiselius Electrophoresis Apparatus. Moore and White (19^8) have described this instrument and further descriptive and pro­ cedural particulars are available in the Perkin-Elmer Instruc­ tion Manual (1951)* The barbiturate (veronal and sodium veronal) buffer of pH 8 .6 , 0.1 ionic strength described by Longsworth (19^2) was used throughout this study. The particular electrophoresis procedure employed in this study was as follows: Sera to be analysed were diluted with buffer 1:2, generally 2 ml. serum and 1+ ml. buffer. The diluted serum *Cenco-Sheard-Sanford Photelometer, Cat. No. 1^1000, Central Scientific Company, Chicago. ^ G r e e n Filter, Cat. No. 87309B. 26 was placed into a dialyzing membrane of seamless regenerated viscose process cellulose-** and dialyzed by the mechanical stirring method of Reiner and Fenichel (191+6) at room temper­ ature. Dialysis was carried out for one hour against 1+00 ml. of buffer; and, was followed by further dialysis for four hours against 600 ml. of fresh buffer. The equilibrated system was then left overnight in a [|.° C. cold room (15-lS hour s ) • The 2 ml. capacity cell was greased, assembled and also allowed to stand overnight at 1+° C. Pilling and complete ^ assembling of the cell and buffer bottles were done in the i+° C. cold room. The complete assembly was then ready to be placed in the water-bath of the electrophoresis apparatus. All analyses were made at 7*5 milliamperes with an average voltage of 112 volts for 7600 seconds. The potential gradient or electric field strength, in volts/cm., averaged 8.18 v/cm. and the bath temperature averaged 0 .5 ° C. Electrophoretic patterns were obtained by photographing the ascending and descending limbs of the cell by the modified scanning method of Longsworth (1939* 191+6). To aid in es­ tablishing the base line for area measurements, a scanning photograph of each cell limb was made prior to shifting the initial boundary into view. By superimposing this photograph on the base of the pattern photographed after the boundaries have migrated, the base line of the latter photograph could *Visking Corporation, Chicago, Illinois. 27 be more accurately established (Longsworth and Maclnnes, 19li-0) • The electrophoretic patterns were photographed with Kodak M Plates and developed by standard photographic methods* Component areas and mobilities were calculated from the descending limb patterns (Longsworth and Maclnnes, 1940)• A two-fold enlargement of the pattern was projected from a photographic enlarger and the outline traced on paper* Com­ ponent areas were delineated by dropping an ordinate between the minimal point of adjacent areas to the base line (Tiselius and Kabat, 1939)* Each area was measured with a planimeter# in arbitrary units* The relative percentage of each component was obtained by dividing the area of each component by the total area, exclusive of the epsilon-boundary anomaly. Conversion into grams percent protein for each component was readily accom­ plished by relating the relative percentage of each component to the total protein of the serum sample. Mobilities of each component were ascertained by measuring, in centimeters, the distance of an ordinate dividing the com­ ponent area in half from the midpoint of the initial boundary (Longsworth and Maclnnes, loc* cit.; Moore and Abramson, 1950). The latter boundary is photographed just prior to turning on the current. By fitting this distance into the equation given K and E No. k-2^6 Compensating polar planimeter, Keuffel and Esser Company, New York* 28 by Longsworth and Maclnnes (loc. cit.) the mobility (Jl) was 2 -1 obtained in x 10 cm. volt sec* * • The average component mobilities determined for the sera analysed in this study were as follows: albumin •........ 5*7 alpha 1 -globulin ... 1|_•8 alpha 2 -globulin .. 3»9 beta-globulin ....... 2.6 g a m m a - g l o b u l i n ... 1*6 For conductivity measurements, the conductance of the equilibrated buffer was determined in lieu of the dialyzed sample as suggested by Moore, et al. (191+9) • E. !• Experimental Protocols Artificially Infected Rats Experiment I * Wistar strain rats, all of the same age, were grouped into experimental and control groups by segre­ gating litter mates. Rats from the experimental reservoir were initially Infected with about 1,200 Taenia taeniaeformis eggs at 8 weeks of age. Infected rats and their litter mate controls were bled at 3 -day intervals following infection up to 15 days. Final bleedings were made 21, 28 and 35 days after initial Infection. To relate the protective capacity of the rats to the results obtained from the electrophoretic analysis of serum, animals initially infected when 8 weeks of age were reinfected with about 1,800 eggs on the day of the last bleeding. Their 29 litter mates were also infected to serve as a control on the viability of the eggs used in the second infection* All were autcpsied Ij. weeks later. Experiment I I . The results of experiment I suggested that a repeat be made in order to obtain a higher level of infection in the rats. Wistar strain rats were again separ­ ated, by litter mates, into control and experimental reser­ voirs. When 8 weeks old, the experimental rats were infected with about 7,000 eggs. Bleedings were made, of experimental rats and corresponding litter mate controls, at ip-day inter­ vals up to 28 days* A final bleeding was done on the thirty-fifth day after infection. To test again the protective capacity of the sera col­ lected on the thirty-fifth day by the in. vivo method, sera collected from infected and control rats were used for passive immunization of other rats. Rats, 13 weeks old, were separated into three groups so that litter mates were distributed as evenly as possible. One group received normal rat serum, the second infected rat serum and the third no serum. Each rat was injected intraperitoneally with 2 ml. of the respective sera within 2L\. hours after being infected with about 1,200 eggs. They were autopsied 9 weeks after infection with the eggs. Experiment I I I . In order to determine in which fraction(s) of the seruin proteins the Hearlyl! and "late" immune factors 30 are found, this experiment was undertaken. About 3*000 eggs were given to 8 week old Sprague-Dawley rats. Infected and control litter mates were bled at ip day intervals up to the twelfth day. The sera collected from normal and infected rats on the twelfth day were absorbed, according to Campbellfs (1938b) method, by adding enough fresh larval paste to make a final 2 percent suspension. Both pre- and post-absorbed sera were analysed electrophoretically. To correlate any changes in the absorbed sera electro­ phoretic patterns with the protective capacity of the sera, the sera were tested in vivo by passive immunization. For this purpose, 5> groups of 10 weeks old rats were set up by distributing litter mates as equally as possible. They were grouped as follows: 1 . received 2 . received 3 . received Ip. received 5 * received Each rat received no serum normal rat serum normal rat serum that was absorbed infected rat serum infected rat serum that was absorbed 1 ml. of serum intr&peritoneally and within 2lp hours after receiving the serum they were all infected with about 3,000 eggs. All rats were necropsied 4 weeks after Infection. The procedure outlined for the 12 day bleeding was re­ peated on the thirty-fifth day following the initial infection. It was hoped to discern the "late” immune factor from the "early" immune factor by relating any changes obtained here to the results on the sera from the 12 day bleeding. The 31 only details that differed were in the use of lyophilized cysts for sera absorption and a new lot of eggs (dosage of 3#500)♦ Also, the rats were 13 weeks and days old when infected. 2. Artificially Immunized Rats Experiment I V , Since ’’early immunity’1 is apparently a response against larval somatic antigens (Campbell, 1938b, c) it seemed reasonable to establish the protein fraction in which these antibodies are present by immunizing rats with worm material. Hence, Wistar rats received a standard series of 6 intraperitoneal injections of 1 ml. of a 2.5 percent saline (0.85 percent sodium chloride) suspension of lyophilized cysticerci. weeks did. Injections were started when the rats were 7 Bleedings of immunized and control litter mates were made at weekly intervals after the first Injection for a total of 6 weeks. Two days before the third and fourth weekly bleedings, each rat received an extra injection of 1 ml. of a 5 percent suspension of lyophilized cysticerci. To test the effectiveness of the immunization, on the day of the last bleeding injected and litter mate controls were each Infected with about 2,000 eggs. They were necropsied 5 weeks later. Experiment V . From the results of experiment IV, it was decided to use whole larvae, stored at minus 30° C«, as 32 the antigen* A 10 percent saline (0.85 percent sodium chloride) suspension of ground larval paste was injected intraperitoneally (1 ml./injection) for the standard series of 6 injections* Injections were initiated when the Sprague- Dawley rats were 8 weeks old. A final injection of a 20 percent suspension was given L|. days prior to the last bleeding. Immunized and control litter mates were bled at 1+ day intervals following the first injection for a total of 28 days* Sera collected on the twenty-eighth day were utilized for absorption studies* III was followed* The procedure outlined in experiment Lyophilized adults were used for absorbing the sera. In the passive Immunization study, rats 12 weeks and Ij. days old were infected with about 1,500 eggs within 214. hours after receiving the serum (1 ml. intraperitoneally). They were autopsied I4. weeks after infection. 33 RESULTS A. Artificially Infected Rats Electrophoretic analysis of the sera from infected rats of experiment I revealed no significant changes in the total protein, A/G ratio (albumin/total globulin) or serum protein components. All of the electrophoretic data, in terms of both grams percent protein and relative percent composition, as well as the total proteins and A/G ratios presented in this thesis were analyzed statistically. The "t" value was determined for the mean of the differences between the experi­ mental and control groups. Any Mt ’* value occurring at or beyond the 5 percent level of probability was considered significant. A value at or exceeding the 1 percent level of probability was considered highly significant. The re­ sults of the electrophoretic analyses are presented in Table I. A look at the average values shows minor differences between the control and Infected groups. No consistent trend was obtained for any of the serum protein components. Eggs used to infect the animals had apparently a low percentage of viable onchospheres. A total average of 23-5 living and 3.9 dead cysts were found in the livers of the animals bled for serum analysis. The largest average number of larvae were found in the rats bled on the fifteenth day. Table II shows the number of cysts found in the rats bled for serum analysis. Though no changes were discernible in the serum, rats reinfected on the day of the last bleeding were almost com­ pletely immune to the second infection. It is of further Interest that the average number of cysts from the initial Infection was nearly a third less than that of the rats bled for serum analysis (Table III, Figures 13 and lijJ ♦ Hence, it is evident that the initial infection had rendered the animals resistant to a superimposed infection in spite of the failure of any changes to appear In the serum proteins. The results of experiment I led to the natural desire to determine the results of a heavier Infection in the rats. From the changes in the serum proteins of experiment XI, it seems apparent that the results of experiment I are ex­ plainable on the basis of the small number of cysts resulting from the initial infection. Comparison of Tables II and V shows that the rats bled for serum analysis in experiment II had a total average number of cysts nearly seven times that of the animals of experiment I. Results of the. sera analyses of experiment II are pre­ sented in Table IV. No significant differences were obtained between the total protein and alpha 1 -globulin. The gamma­ globulin of the infected group was significantly increased. 35 0 «s! i Et(>nC\l<^l^COsD HOfMlA^OJOH • •••••»• r*-\ f\j » r l H H H r l r l r l H H rH ro O H D CA rH l f \ CM CM rH CM <0 CM rH CA O » • • • • • • • rH rH rH r H rH rH O r H © g o £5 © ^ (NJvO C M A O C N I N oOlACM C MroCM sO co CO o'** O CMGO O O rOsD (A ^J-cO cOvO (A rH U \ cO C 'C O o c o O v Os CO Ch C M O O Er* r-» C5 at o Eh *• • • • • • « O^sOCO t^.CM-J-CMUN #•• •• • •• a-co co a- cacm co 00 l f \ N <><0 OO M3 r-t CM crOM) (Ofs-fs. OvO ■ • • • • ■ • • • • - _d~ _d-prH_d-o~d‘ CMr^ oO-d*-d-VAM5 VA co _df• •• • • oOsO ••• t © G O £5 O • O POVA0OCA rOt"^ rH CA CO CAcO A-v£> O O O OO © G O £3 O • co vO sO » • • • • • ■ • * • • oorOvO CU N O N H 0O \A - d " - d < O OO .d " O -d" © • o £5 >• * • • • « • * A- CM CM CM CM CM CM r o r o CM CO CAoOvO A- _drH O O H H C M O \D U \n VA CM rH A -d C O O CACQ CQ COCO (ACM A - O CM O rO x> CM CM rO CO CO cO co co oo CM CM CM r o r o oo r o oO oo g— ■H’fe*. \T\CO O O C O (\JOlA rH CA • VA 4 0 0 O A- CAVO(A ArH _d'_d'A-’U> _d CM a ■H s H sO rH CM vO CM CM CM A - CM CM CM CM CM CVJ CM CM ANALYSIS OP EXPERIMENT at d a. rH & Q, O G +3 o M rH rH 0 © £ o £5 1 CM rH C5 •H 03 a> u ELECTROPHORETIC at -P © PQ * ’P £ o o U '- ' CM € O Tl © +5 OQ O © CMV A O O -rf-C M O O • * ■• • • • • « UNIAvO vO vO v0 nO VA I I • I I • I I 03 c p • • # • » « • • rH• • • ■• ■ • • • • • U W A lA V A vO lA vO HD VA rOvO CA CMVArHOO VA rH «H CM CM 00 *» G a Um Q TOTt © © hQ OS rH < Q fO O £3 •P at 03 O 03 O {=> (X, CA CM1ACO rH A ~ _ d -H 'LTsnO © sO N ("-CO O' A- o rH rH CM rH rH rH CMVACO rH •* » rH rH H c^vO 0 s CM •* •*. O rH rH rH CM rH i—! i—IrH rH rH rH rH H H •»*> * •* •* «* %CM U>,CO rH O <"OvO O ' rH rH rH CM H H H H H H H H loa^uoo • CAC M1ACO rH A -^4 rH XAM3 vO\ON A-OO -< G © G O £5 © G o Id © o c © o *H •H G Wi •H OQ © td 36 TABLE I I LARVAE IN LIVERS OP RATS BLED POR SERUM ANALYSIS OP EXPERIMENT I Days Since Infected Rat No. Total Living Dead 3 12k 125 126 6 127 128 129 9 12 15 21 28 35 Cysts Average Living Dead ■M■£Hr -Jtt* ■{HHJ 130 131 132 20 23 36 133 131* 135 10 7 9 136 137 138 50 26 22 8 Ij5 32.7 5.7 139 llj.0 141 36 3k 15 k 5 0 28.3 3.0 llj.2 11*3 11^ 16 19 * 1 0 17.5 0.5 11*5 llj.6 1U7 12 9 3 26.5 6.0 23*5 3*9 Average Discarded - accidental infection ■SHJ- Not yet grossly visible into- Not distinguishable from living 26 •3 iHBc 8.7 **hh* 37 TABLE I I I LARVAE IN LIVERS OP RATS REINFECTED TO DETERMINE PRESENCE OF IMMUNITY INDUCED FROM INITIAL INFECTION OF EXPERIMENT I Cysts Reinfected Group Rat No* From initial infection Living Dead 1 ■M- 2 20 3 From second Infection Dead Living Control Group For second infection Living Dead k 7 12k 13 0 2 1 120 5 16 0 0 7 177 3 k 12 0 1 0 72 10 5 & 2 k 62 li 6 ■K-iS* 1U3 15 7 9 0 0 i 173 21 8 k 0 1 2 92 3 9 11 0 1 0 119 10 10 k 0 0 1 78 2k 8.6 0 1,2 2.6 Average 116.0 No large cyst typical of initial infection seen* ■SHi- Discarded - accidental infection. 11.5 38 Total globulin, alpha 2 -globulin and beta-globulin showed highly significant increases in the infected animals* Highly significant decreases were obtained In the albumin and A/G ratio of the Infected rats. Figures 1 to 10 are the serum electrophoretic patterns showing the changes occurring at various days following in­ fection* Of particular interest are the marked changes occurring on the eighth, twelfth and sixteenth days after infection. On the fourth day, the only noticeable differ­ ence between the infected and control groups was the in­ creased alpha 2 -globulin (Table IV, Figures 1 and 2)• The alpha 2 -globulin of the infected rats persisted at a fairly constant level above the control animals throughout the entire experiment (Table IV)* Sera on the eighth day showed a sharp increase in the beta-globulin and total globulin, with a marked decrease in the albumin and A/G ratio (Figures 3> U-j 11 and 12). By the twelfth day the beta-globulin and total globulin dropped sharply to a lower level while the albumin and A/G ratio rose markedly. The albumin was at about a con­ trol level on the twelfth day, had essentially the same value £s the controls on the sixteenth day and maintained Itself below control values throughout the remainder of the experi­ ment (Figures 5 to 8 , 11 and 12). Referring again to Figures 3 to 8 and 12, there was a sharp rise in gamma-globulin that occurred on the twelfth day with a marked drop to a lower lev^l by the sixteenth day. 39 From Figures 7 to 12 and Table IV it can be seen that the differences between the serum proteins of the infected and control groups from the sixteenth day on are fairly constant and maintained themselves in the sera obtained on the thirty-fifth day following infection* Also, it appeared that protein metabolism was most drastically affected for ap­ proximately two weeks; between the fourth and sixteenth days following infection* Table V shows that there were marked quantitative differences in the average number of larvae in the rats bled for serum analysis on the various days* but these differences per se appeared to have no effect be­ yond the sixteenth day* However, before definite conclusions can be made on this point further studies are indicated. That a protective humoral factor was present in the serum of the infected rats obtained on the thirty-fifth day was clearly evident from the complete protection afforded rats passively immunized with this serum. Though rats re­ ceiving serum from non-infected rats had fewer living cysts than the rats receiving no serum, the total number (living plus dead) of cysts in both groups were about the same (Table VI, Figures 15 to 17)# The slight protective capacity of normal rat serum was observed in the other passive immuni­ zation studies conducted herein and Miller and Gardiner (1932a, c) have also reported this* Also, it should be noted that the rats utilized throughout this study appeared to show an age resistance to infection with the tapeworm eggs* In 91 Xt to o GO G O lA 'O CM H c O £) H O C \|C A j^ _ tH H • H H H H H H r H r l > vO ^ f ^ s o J O O H U\_^_H‘(\J C\J OJ • rH _d -co rH -o o CO CM t" - rH _ r K c CO rH CO COCO o CO HD CM O H C O CACO CO O rH CA • • • X* to • * co c^CM CM CM CM r o c o c o CM * M CO ao I A CA cO V Y L O rH CA CA CM O A - O d - A CM rH A - to O rH rH -H ^ ^ P-. © O xt C © •H CO o © 03* £ «JM « W Tt to >t»to 60 <0 rH Hj Q CQ • O 525 -P at oc; OKODP^ • ••••••• • IfMAlTsUMAvO HDHD "LA © u at I l l l l I l I H to > < O vD 1 A M D X A I A H D MD O vO to! w aj X. _y±GO CM HD 0 _ H < 0 -LA to rH rH CM CM CM CO > < o IS © o C at o •H © 1*0 TABLE V LARVAE IN LIVERS OP RATS BLED FOR SERUM ANALYSIS OF EXPERIMENT II sfn °e Infected Rat No. __________________ Cyste_____________________ m , Total Average Living Dead Living Dead k 22k 225 226 8 227 22 8 160 12 229 230 219 212 11 9 16 231 232 98 110 7 5 20 233 23k 235 207 2k 235 236 28 35 ** 160 215.5 10 IOI4. 6 lk 3 221 8.5 312 110 33 kk 211 38.5 237 238 132 1*4-6 2 8 139 239 240 2kl 2k2 2k3 2kk 2*4-5 2*4-6 211-7 2I4.8 56 19 60 128 127 117 209 182 275 12 2 10 18 7 1 20 16 10 Average l55*7 12*2 ■a Not yet grossly visible Discarded - accidental Infection Not distinguishable from living 130.3 5 10.7 PLATE X Serum electrophoretic patterns of rats bled on fourth day of experiment II. Fig. 1 . Control rats Fig. 2. Infected rats PLATE I Figure 1 Figure 2 P LA T E I I Serum electrophoretic patterns of rats bled on eighth day of experiment II* Fig. 3* Control rats Fig. Infected rats k3 PLATE II Figure 3 Figure 4 P LA TE I I I Seruxn electrophoretic patterns of rats bled on twelfth day of experiment II* F ig * 5* C o n tro l ra ts F ig . 6. In fe c te d ra ts PLATE III Figure 5 Figure 6 P LA TE I V Serum electrophoretic patterns of rats bled on sixteenth day of experiment II# F ig . 7* C o n tro l r a ts F ig # 8# In fe c te d ra ts Figure 7 Figure 8 PLATE V Serum electrophoretic patterns of rats bled on thirty-fifth day of experiment II. F ig . 9. F ig . 10# C o n tro l r a ts In fe c te d ra ts Figure 10 1*7 F ig . 11 Relation of Albumin, Total Globulin and A/G Ratio to Infection of Experiment II 3.00 9 2.00 — o 1.00 Ratio 2.00 k /C r Gm. Percent Protein I4.OO Control 4 8 12 O Total Globulin — — — Albumin — ■■1 1 1 — L 1 16 20 2i+ 28 32 36 Days After Infection kQ P ig . 12 Relation of Beta- and Gaircma-Globu lints to Infection of Experiment II Infected 1.6 Control o BetaGamma- Gm*. Percent Protein 1.2 1.0 * 0.8 0*6 0.2 I______I k 8 12 J 1------ 1______ I______ I 16 20 2k 28 32 Days After Infection I 36 k9 TABLE V I LARVAE IN LIVERS OP RATS PASSIVELY IMMUNIZED WITH SERUM FROM RATS BLED ON THIRTY-FIFTH DAY OF EXPERIMENT II Injected with Serum from rats: Infected Non-infected Cysts Cysts Living Dead Living Dead No Serum Cysts Living Dead 1 0 0 8 7k 23 23 2 0 0 15 35 2 16 3 0 0 7 50 « k 0 0 1 29 58 13 5 0 0 6 k 22 31 6 0 0 1 20 18 28 7 0 0 ■ * 1 30 0 0 6.3 Average # Discarded 55 accidental infection 35.3 20.7 23 *5 P LA TE V I Livers from rats used in reinfection study of experiment I. Fig# 13. Liver of rat (No# 1) that received eggs of initial and second infection# Fig. lip* Liver of rat (No. 1) that received eggs only of second infection* Livers of rats used in passive immunization study of experiment II. Fig. 15. Liver of rat (No. 1) that received serum from Infected rats. Fig# 16. Liver of rat (No# 7) that received serum from non-infected rats. Fig. 17. Liver of rat (No. 5) that received no serum. PLATE VI 50 51 the passive immunizatlon study just noted, the rats receiving normal or no serum had cysts that were smaller than those ob­ tained in rats infected at an earlier age. Older rats used for infection showed smaller numbers of cysts than young rats. A decrease in susceptibility to this infection with an Increase in age of rats has been reported by Curtis (1933) and Greenfield (l9i|2). e_t al* However, Miller and Massie (1932) questioned the existence of age immunity in rats against this parasite. In an effort to demonstrate in which protein fraction(s) the antibody against the larvae are produced, experiment III was undertaken. Though comparison of experiments II and III was limited by the fewer analyses made In the latter experi­ ment, the same general results were evident in experiment III as were obtained In experiment II. The results of experiment III were not as significant as those of experiment II. Total protein and alpha 1 -globulin were not significantly changed. The total globulin and beta-globulin were significantly in­ creased in the infected animals and the A/G ratio was reduced significantly. However, the albumin, alpha 2- and gamma­ globulins showed no significant di fferences whereas in experi­ ment II they did. The electrophoretic results are given in Table VII and Figures 18 to 25* An explanation would appear to lie in the smaller number of cysts present In rats bled on the eighth and twelfth days following infection (Table VIII). 52 The average number of cysts present were about half that In the rats bled on the corresponding days of experiment II (Tables V and VIII). Campbell (1938b) demonstrated that the "earlyM Immune factor is absorbable from the serum collected on the eleventh day following infection. Sera collected from infected and non-Infected rats on the twelfth day after infection were analyzed electrophoretically before and after absorption with fresh larval paste. No significant alterations between the pre- and post-absorbed patterns were obtained. In Table VII, only the A/G ratios are given for the post-absorbed sera and serve to show the lack of any significant change between the patterns. That the sera collected on the twelfth day contained protective humoral bodies was evident from the results ob­ tained In the passive immunization study. Table IX and Figures 26 to 30 show that the serum from the infected rats was almost completely protective. of the antibodies was accomplished. Furthermore, some absorption However, it would appear that the absorption was inadequate and possibly accounts for the failure to induce any change in the electrophoretic pat­ tern. Sera collected on the thirty-fifth day also failed to show any difference between the pre- and post-absorbed serum patterns (Table VII). The pre-absorbed serum from the infected rats, when used for passive immunization, was also 53 found to contain antibodies as It afforded complete protec­ tion to the recipient rats* There was no evidence of any absorption of antibody in rats that received the absorbedimmune serum. They were also completely protected (Table IX, Figures 31 to 35)* The non-absorbability of the "late” immune factor that has been shown to be present in 35-day serum would agree with Campbell1s (1938b, c) results. On the basis of these absorption experiments, no con­ jecture is possible with respect to the protein component(s) in which the ’•early” and ’’late” Immune factors are produced. Though the "late” immune antibody is not absorbable by the method employed, it was felt that it might be possible to relate the changes that were hoped for in the absorbed 12and 35-day sera. Thereby, definite Information might have been procured on the protein-component site of the "early” immune antibody with some evidence presenting itself for the localization of the "late” immune factor. B. Artificially Immunized Rats Using larval worm material, it has been shown that rats could be artificially immunized (Miller, 1930, 1931c)• immunity thus induced was absorbable Also, (Campbell, 1938b). Therefore, the next logical approach to the problem of ascer­ taining in which serum protein fraction the "early" immune factor is produced would be to follow the serum changes in such immunized rats. A £ * O HIS' 4 •P CD CQ -—«, co 0 • rH rH X vO CO ^ 0 o n on o n • « -• GO vO vOOO ca CO • « ••- Aon • Aon • -C±CM H r*H CO C— ArH CM C O • • 0 CO • 0 CO OO • • 1 •9 CM G •«H cd rH .G G a P i—1 o I G •H CO < E-i rH i n -G A - O • 0 • CM CM CM G " -* •rH ^ . © • 43 E O o G s—' Oh © O 73 G © •H ■P CO O CD 03 V h A• CM A—-=t in rH sO O • • • cm on on 0 ® # on 0 Ar— • CM rH CA onon -=J- on vO © 0 on r— in vO G -• ♦ • •O oncm CM CM CM P C'-r- c a CM ons0 CA in • • •- ♦ u\u> in in ~=± nO A~ CA r— M3 © vO • • 0 ■ 0- sO• G O in in in in inS3 • CM • in in-on QO ©I bil f i d I I 4) C M -=tCO © > £» G ctf M Q bfl 1A on O -it co vO nO vO CA cd U G *< 03 T j © >-» © bO Cd rH O Tl • 4) on © X w sO CM O O CA O •■• ~• CM CM on is i—1 *< rH at -P O Eh in tH rH p p o rH C5 5U oa at 1 O t o nvO 0 cm rH HI CM 00 O O O ' • • • • • • rH rH rH rH U\ 1 « : S * i } s'O » } t$ 3 } e£ ) o n r— n ~ sO r H in O 0 > rH A- co cocor— ca a • • • •• • • 0 O _d“ CO vD vO vO at O *r4 'in ca •H g *H CO • rH ("A ■P ai Ph o p C M on on I • I on o n J- rH rn o n on on O cij cc; i-4 H ph Xoaq.uoQ Ch onin _rj- CM CM on nn on d * i CM^t vO CM CM CM on on on m i In on on paq.oejux O i—I © > OJ CO CO OJ U \ CO CO • • • • OJ O ' d c o • • O' CO « <3 • £ at P ■M PQ | O- c o d O O CO • • • rH © © 03 •H © © d O a o d o -d o A -C O • • t" CO * O O C O [m IA O -IA • • • vO IA • rH O ' lA d • • CO IA * rH © rH A as M 1 © d O' o * d ca . 1 I 1 -p sO o O 'C O • • © oj d d o ♦ 35 1 •H OJ rH d as xi a o Or rH iH c5 < A CO CO • 1 d 1 O co c o oo • • • ia h v Or rH d 00 • rH d GO O'' • • IA oo • I < 1 a •h rH oo d o vQ rH vO • • * OJ c o OJ i— ! oJ +3 X3> O o E-< rH o d _C±rH O rO O J c o • • • co co co X> r—t < Ert CO O l d A • • • OJ o j O 1 o « C\J I 1 •H x i—i sO • OJ rH t—1 •CO -d c o OJ o • ♦ COCO iH OJ • CO I 1 1 d rH *H ^ aj A O O £ © O 3 **”9<0 tX d K H d H © +* *H I I II I bO >> © O CQ ©1 u aS d © © > O «< S o *H •H I I I I I rH OJ GO\A OJ OJ c o d d fd PnxO f P O p— P> U A v O t"- t'- O (X c! j p O co^O O' * rH OJ •» rH rH p a oj ij^oo d%- d ^ j - j“ •P cd © bO «tf d © > 3O as d P *h co 1 p o O rH _H“F— rH Od d d d _zj~rH GO O' H d> O vO co O cr: ia v o r - c5 ptj N 0s d fesd d ft -dA rH rH d d M rH C"-dfH f-GQ o *IA nO iH d b£ •H cO _zJ"oj f'- O' rH O J d Ol OJ CO ‘ ‘ ' NJL>CO O OJ OJ OJ OJ CO O rH © > © 66 TABLE X I LARVAE IN LIVERS OP ARTIFICIALLY IMMUNIZED RATS TO TEST FOR PRESENCE OF IMMUNITY IN EXPERIMENT IV R A T Immunized Cysts Living Dead N 0. Control Cysts Living Dead 0 0 1 322 127 68 77 2 21*6 72 1 68 3 202 76 k 180 60 # 8 1*6 5 111* 39 1 17 6 22 21+ 25 90 7 35 20 92 82 8 Average 27.9 5L*3 ^Discarded - accidental infection •i* 160.1 59.7 P LA TE X I I I Livers from rats infected after artificial immunization of experiment IV. Pig. 36* Liver of immunized rat (No. 7) Fig. 37. Liver of immunized rat Fig. 38* Liver of control rat Fig. 39. Liver of control rat (No. 3)« (No. 1) (No. 5)* 67 PLATE XIII Figure 36 F ig u r e 38 Figure 37 Figure 39 68 M r - l rH rH rH 1 H1 —i S OP EXPERIMENT ANALYSIS 03 C M 03 £ •rH as c a rH XI © £ cu £ A rH o o < XJ rH a CJ 1 rH o £ 4 > as O X © CL, rH rH << £ H rH i—1 O S£ -P O o EH rH © cA OOO CO CA-ztO rH CAcA_d"-d" CAIA vO ca a ~d O •i3 I f \ CA_dCA O f — rH A H O AGO CA CA • • • • • • * rH rH CA O rH OO_d*c—c a ia r— CA O CACO CA CA CA x0 CA vO cAC'—'AsQ sO• IA A -d-^rlAlA • • • • • • IA IA CM_d-_d^o ca o _d" 'O'DvO tA -d lA IA D—CO CA O vO ^H-QO CACO O H O n O CA O O CMrH OJ CM_zt CO O O H O r l H O • • » • • ca A- » • ca • •«••• ■ • • • IA rH rAO) A- CACA NOO'£)NHOn C i > £ tH • • • • • • • * • • • •Hr-' © ©^ ■H -P 6 o oo Eh 0) A- £ o o •53 cA © <"A53 • * « CM CMcA CACMCM ca CA lA lA '-O h± 0 IA-d" rH O CMCO O CA H rH rH GO-d'CM A-xO -LA CO CACA O A~ rH C\J fv_ cA CACAC\J CA ca ca IN- © IA £ • o 53 CAOJ A-CO _d'0' O CO rH H CO CA O CM ■• o c • o CA ca C Mca CM cAcA CM _d © o £ •o CA 53 CM rHsO C A lA O -^t CO OJ o H O H A CA OO © O £ • 1 <11 • rH C\J cA CACMCMCA C\J r~ © £ o 53 H H H H H H • • « • • ■• ■• • • n ELECTROPHORETIC XII V C3 CA ca vO CA t^CO CA O CMlAN--drH• •• • • • • • I A v O 'vO I A I A v O P H vO © -P -H A O © S © o 3 'Hj © O• V0 • • • • • • • O O O IAHD xO IA 0) fcsd at £ © CMCAlAvO O O N > £ o • o H-> CCS 03 © w © aS £ © > o £ CO H © ra »o to l>» © H! (tfrH p ca •O vo 53 •rH CO I I I I • I I _d<0 CMvO O _d*£> nQ vO C - A -C O CO O 03 O rH rH OJ U\CO IA •'C A lA -d O O rH i— I OJ 03 |H _d-p- rH IA IA IA h^IAIAIA EHIAIAIAU\ •» •* I £5 •> •» %CM _HnO O O CAvO 0s rH rH rH O lA lA IA lA lA lA lA f£ {Z2 o o-=tGO o j o o d tUvDOO t^-P—COCO P P A- O CAvO 0 CMCACA CA CMIA IA IA IACO O O * * ** ^CA-d'lA O OCM IA IA IA IJ\ CMOJ CACA * * I lA lA lA lA A- CA rH «kCAcA-rt H IACO rH _ d lA lA lA CMCMCACA lA lA lA lA © > © p average O at -P © PQ 1 • TABLE Nrl to calculate © * % t <>d-iAi)c-ir\0 -o o eont., 619-625. Antweiler, H. J. (Edit.). 1952. Die Quantitative Elektrophorese in der Medizin. Springer, Berlin, 212 pp. Bernhard, A*, and Scher, Y. 1951* Bovine albumin standard for serum protein determinations. Science 114: 674* Bersohn, I., and Lurie, H. I* 1953* Experimental bilharziasis in animals. II. Correlation of biochemistry (,fliver function tests1') and histopathological changes in the liver in early bilharziasis. South African Med. J. 27: 950-954* Blumberg, H . , and Gardner, R. E. 1940* Adenomatous stomach lesion of the rat associated with heavy Cystlcercus faseiolaris infestation. Proc. Soc. Exper. Biol, and Med. 45: “673-677. Bullock, F. D., and Curtis, M. R. 1924* A study of the reactions of the tissues of the ratfs liver to the larvae of Tenia crasslcollis and the histogenesis of cystlcercus sarcoma. J. Cancer Res. 8: 44^'"46l* Ibid. 1925. On the transplantability of the larva of Tenia crasslcollis and the probable role of the liver In cystlcercus disease of rats. J. Cancer Res. 9: 444-452. Ibid. 1926. Further studies on the transplantation of the larvae of Taenia crasslcollis and the experimental pro­ duction of subcutaneous cystlcercus sarcomata. J. Cancer Res. 10: 393-421. Bullock, F. D., Dunning, W. F., and Curtis, M. R. 1934. Observations on the digestion of the shells of the eggs of Taenia taeniaeformis. Am. J. Cancer ?r>: n. 397. Burke, J. D. 1954* Blood volume In mammals. Zool. 27: 1-21. Physiol. Burlingame, P. L . , and Chandler, A. C. 1941. Host-parasite relations of Moniliformis dubius (Acanthocephala) in albino rats, and the environmental nature of resistance to single and superimposed infections with this parasite. Am. J, Hyg. 33: 1-21 (Section D) Campbell, D. H. 1936. Active immunization of albino rats with protein fractions from Taenia taeniaeformis ana Its larval form Cystlcercus fasciolaris. Am. J. H v e . 23: 104-113. Ibid. 1937. Active Immunization of albino rats with chemical fractions from the cat tapeworm (Taenia taeniaeformis) and Its larval form (Cystlcercus fasciolaris ^crasslcollisj ). Arch. Path. 2 3 : 751. Ibid. 1936a* The specific protective property of serum from rate infected with Cystlcercus crasslcollis. J. Immunol. 35: 195-204* Ibid. 1936b. The specific absorbability of protective antibodies against Cystlcercus crasslcollis in rats and £* pisiformis in rabbits from Infected and arti­ ficially immunized animals. J. Immunol. 35: 205-216. Ibid. 1938c. Further studies on the "nonabsorbable” pro­ tective property in serum from rats infected with Cystlcercus crassicolli5 . J. Immunol. 3 5 : 465-476. Ibid. 1939a. A polysaccharide fraction from Cystlcercus crasslcollis and its role in immunity. J. Infect. Dis. 65: 12-15* Ibid. 1939b. The effect of sex hormones on the normal resistance of rats to Cystlcercus crasslcollis. Science 8 9 : 415-416* Campbell, D. H . , and Melcher, L. R. 1940* Relationship of sex factors to resistance against Cysticercus crasslcollis In rats. J. Infect. D i s * 66:184-188• Cann, J. R., and Kirkwood, J. G. 1949* The fractionation of proteins by electrophoresis-convection. Cold Spring Harbor Symp. 1 4 1 9-23* Cannon, P. R. 1945* The relationship of protein metabolism to antibody production and resistance to infection* Adv. Prot. Chem. 2: 135-154. Chandler, A. C* 1939. The effects of number and age of worms on development of primary and secondary infections with Hymenolepls diminuta in rats, and an Investigation Into the true nature of "Preraunition" in tapeworm infections. Am. J. Hyg. 29: 105-114 (Section D). Ibid. 1940* Failure of artificial immunization to influence Hymenolepls diminuta Infections in rats. Am. J. Hyg. 31: 17-22 (Section D). Ibid. 1946* Factors modifying host resistance to helminthic infections. Proc. Fourth Internat. Cong, on Trop. Med. and Malaria 2: 975-981. Ibid. 1953* Immunity in parasitic diseases. Assn. 36: 811-834* J. Egypt. Med. Chen, H. T. 1950. The In vitro action of rat immune serum on the larvae of Taenia taeniaeformis. J. Infect. Dis. 86: 205-213. Chow, B. F., Seeley, R. D., Allison, J. B., and Cole, W. H. 1946. The effect of repletion on plasma proteins in the dog measured by electrophoretic analysis. Arch. Biochem. 1 6 : 69-78 . Clapham, P. A. 1940. Studies on Coenurus glomeratus. Helminth. 18: 45-52. J. Crusz, H. 1948. Further studies on the development of Cystlcercus fasclolaris and Cysticercus plsiformis, with special reference to the growth and sclerotization of the rostellar hooks. J. Helminth. 22: 179-198. Culbertson, J. T. 1938. Recent contributions to the immuno­ logy of helminthic infections. Arch. Path. 25: 85-117, cont.*256-280. Culbertson, J. T. 1941* Immunity Against Animal Parasites. Columbia University Press, New York, 274 PP* Culbertson, J. T. 1951* Chapter 3* Immunological Mechanisms in Parasitic Infections. Most, H., Editor. Parasitic Infections in Man. Columbia University Press, New fork, 229 pp. 89 Curtis, M. R., Dunning, W. P., and Bullock, F. D . 1933. Genetic factors in relation to the etiology of malig­ nant tumors. Am. J. Cancer 17: 89lj-923. Deutsch, H# F#, and. Goodloe, M. B, 19l(-5* An electro­ phoretic survey of various animal plasmas. J. Biol. Chem. 161: 1-20. Dole, V. P., Emerson, Jr., K, and Braun, E. 19l|5. Electro­ phoretic changes in the plasma protein patterns of patients with relapsing malaria* J. Clin. Invest. 2 l\.i 6Ljlj-6l|7* Dunning, W. P., and Curtis, M. R. I9I4.6 . Multiple peritoneal sarcoma In rats from intraperitoneal injection of washed, ground Taenia larvae. Cancer Research 6 : 668-670. Enders, J. F* 19i4-iLp• Chemical, clinical, and immunological studies on the products of human plasma fractionation. X. The concentrations of certain antibodies in globulin fractions derived from human blood plasma. J. Clin. Invest. 23 : 510-530. Essex, H. E., Markowitz, J., and Mann, P. C. 1930. Immunity in dogs to aqueous extracts of Taenia pisiformis. J. Parasitol. 17: 107. Fisher, B. 1953* Recent contributions of electrophoresis to clinical pathology. Am. J. Clin. Path. 23: 214.6-262. Franklin, M . , Bean, W. B., Paul, W. D., Routh, J. I., Huerga, J. d© la, and Popper, H. 1951* Electrophoretic studies in liver disease. II. Gamma globulin in chronic liver disease. J. Clin. Invest. 30:729-737. Gjessing, E. C. Ludewig, S., and Chanutin, A. 19lp7• Fractionation, electrophoresis, and chemical studies of proteins in sera of control and injured dogs. J. Biol. Chem. 170: 551-5&9* Gray, S* J . , and Barron, E. S. G. 19^3* The electrophoretic analyses of the serum proteins in diseases of the liver. J. Clin. Invest. 2.2: 191-200. Greenfield, S. H. 19^2. Age resistance of the albino rat to Cystlcercus fasclolaris. J. Parasitol. 2 8 : 207-211* Gutman, A. B. 19i|.8. The plasma proteins in disease. Prot. Chem. I4.: 155-250* Adv. 90 Hearin, J* T. I9I4.I. Studies on the acquired immunity to the dwarf tapeworm, Hymenolepls nana var. fraterna. in the mouse host. Am. J. Hyg. 33*'"71^87 (Section D). Heim, W. G*, and Schechtman, A. M. 19SH-* Electrophoretic analysis of the serum of the chicken during development. J. Biol. Chem. 209: 214.1-21^7. Henley, ^., and Schuettler, C« I». 1953* Electrophoresis Bibliography. American Instrument Comp., Inc., Silver Spring, Md., 228 pp. Hill, R. M * , and Trevorrow, V # 1914-2 . Normal variation in the concentration of fibrinogen, albumin, and globulin in blood plasma. J. Phys. Chem. I46: 1117-1129. Hoeppli, R., and Feng, L. C. 1933* Myeloid changes in the spleen of experimental animals due to Infection with Cystlcercus fasciolaris and to emulsions prepared from tapeworms. Chinese Med. J. I4 . 7 : 1114-6-1153_[Seen in abstract only, Biol. Abst. 10: I2I4.O (1938)], . Hunninen, A. V. 1935* Studies on the life history and host parasite relations of Hymenolepls fraterna (H. nana, var. fraterna. Stiles) In white mice. Am. J. Hyg. 22: Jplij-I^jJ. Kabat, E. A. 1914-3* Immunochemistry of the proteins. Immunol. 14.7 : 513-587. J. Kabat, E. A., and Mayer, M. M. 1914-8 . Experimental Iramunochemistry. Charles C. Thomas, Pub., Springfield, 111. 567 pp. Kass, E. H. 1914.5 * The occurrence of normal serum gamma­ globulin in human lymphocytes. Science 101: 337-338. Kerr, K. B. 1935. Immunity against a cestode parasite Cystlcercus pisiformis. Am* J. Hyg. 22: 169-182. Lamirande, G. de, and Cantero, A. 1952. Electrophoretic analysis of plasma protein constituents In rats bearing regenerating and pre-neoplastic livers. Cancer Re­ search 12: 330-33 3 * Larsh, Jr., J. E. 1914-3* Serological studies on the mouse strain of the dwarf tapeworm, Hymenolepls nana var. fraterna. Am. J. Hyg. 37: 289-293* 91 Larsh, Jr., J.E. 1944* Studies on the artificial immunization of mice against Infection with the dwarf tapeworm, Hymenolepi 3 nana var. fraterna. Am. J. Hyg. 3 9 : 129Ibid. 1945* Im m u n ity r e la t io n s in human cestode in f e c t io n s . J. Elisha Mitchell Scient. Soc. 61: 201-210. Ibid. 1951* Host-parasite relationships In cestode infec­ tions, with emphasis on host resistance. J. Parasitol. 3 7 : 3 4 3 -3 5 2 . Leland, Jr., S. E. 1953* An electrophoretic and chemical fractionation study of sera from rats immunized against the nematode. Njppostrongylus muris. Unpublished Ph. D. Thesis, Michigan State College, 93 numb, leaves. Leonard, A. B. 1940• The accelerated tissue response to Cystlcercus pisiformis in passively immunized rabbits. Am. J. Hyg. 3£: 117-124 (Section T>) • Leonard, A. B., and Leonard, A. E. 1941* The intestinal phase of the resistance of rabbits to the larvae of Taenia pisiformis. J. Parasitol. 27: 375-378. Lewis, L. A. 1950. Electrophoresis in Physiology. Charles C. Thomas, Publisher, Springfield, 111., 66 pp. Longeworth, L. G. 1939. A modification of the schlieren method for use In electrophoretic analysis. J. Am. Chem* Soc. 61: 529-530. Ibid. 1 9 4 2 . Recent advances In the study of proteins by electrophoresis. Chem. Rev. 30: 323-340* Ibid. 1946. Optical methods in electrophoresis. Chem., Analyt. Ed., l8: 219-229* Ind. Eng. Ibid. 1952. Chapter on Electrophoresis. Methods in Medical R e s e a r c h (Corcoran, A. C., Editor), Vol. 5# PP* 63-106. Year Book Pub., Inc., Chicago. Longsworth, L. G., and Maclnnes, D. A. 1939. Electrophoresis of proteins by the Tiselius method. Chem. Rev. 24* 271-287. Ibid. 1940. The Interpretation of simple electrophoretic patterns. J. Am. Chem. Soc. 62* 705-711. 92 Luetscher, Jr., J. A. 1914-7 .' Biological and medical appli­ cations of electrophoresis. Physiol.Rev. 27: 621-61^2 . Luttermoser, G. W. 1938. Susceptibility of chickens to reinfection with R a i H i e t i n a cesticillus as determined by the presence of the original terminal segment. J. Parasitol. 2k (Suppl.): U 4-1 5 * Hadden, S. C., and Whipple, G. H. 1914-0 . Plasma proteins: their source, production and utilization. Physiol. Rev. 20: 1914--2 1 7 . Mahon, J. 19514-# Occurrence of larvae of Taenia taeniaef ormis (Batsch, 1786) in the American rabbit, LeTpus americanus. J. Parasitol. I4.O: 6 9 8 . Martin, N. H. 1914-6 . The components of the serum proteins in infective hepatitis and in homologous serum jaundice Can electrophoretic study). Brit. J. Exper. Path. 27: 363-368. Ibid. 1914-9# An electrophoretic study of the components of the serum proteins in cirrhosis of the liver. Brit. J. Exper* Path. 30: 231-236. Miller,. Jr., H. M. 1930. Experiments on Immunity of the white rat to Cysticercus fasciolaris. Proc. Soc. Exper. Biol, and Med. 2*/: 926. Ibid. 1931a. Immunity of the white rat to super Infestation with Cysticercus fasciolaris. Proc. Soc. Exper. Biol, and Med. 2 8 : I4.67 -I4.6 8 . Ibid. 1931b. Further experiments on artificial Immunity to 1 a. larval cestode. Proc. Soc. Exper. Biol, and Med. 2 8 : 8814.-885# Ibid. 1931c. The production of artificial immunity in the albino rat to a metazoan parasite. J# Prevent. Med. 5 : I4.29-I4. 52. Ibid. 1931d. Immunity of the albino rat to superinfestation with Cysticercus fasciolaris. J# Prevent. Med. 5 s 1453-14.^5^ ~ Ibid. 1931e. S u p e r infection of cats J. Parasitol. 1 8 : 1 2 6 . with Taenia taeniaef ormis. Ibid. 1932a. Transmission to offspring of immunity against infection with a metazoan (cestode) parasite. Proc. Soc. Exper. Biol, and Med. 29: 112l|.# 93 Milier, Jr., H .M. 1932b* Acquired immunity against a metazoan parasite by use of non-specific worm materials* Proc. Soc* Exper* Biol* and Med. 29: 1125-1126. Ibid. 1932c. Therapeutic effect of specific immune serums against a metazoan parasite (Cysticercus fasciolaris). Proc. Soc. Exper. Biol, and Med. 30: 52-831 Ibid. 1932d. Superinfection of cats with Taenia taeniaeformis. J. Prevent. Med. 6 : 17-29. Ibid. 1932e. Further studies on immunity to a metazoan parasite, Cysticercus fasciolaris* J. Prevent. Med. 6* 37-1+6. “ -------Ibid* 1931+• Specific immune serums as inhibitors of In­ fections of a metazoan parasite (Cysticercus fasciolaris)* Am. J. Hyg. 19: 270-277. Ibid. 1935a*. Experiments on acquired immunity to a metazoan parasite by use of non-specific worm materials. Am. J. Hyg. 21: 27-31+. Ibid* 1935b. Transmission to offspring of immunity against infection with a metazoan (cestode) parasite. Am. J* Hyg. 21: 1^56-lj.6l. Miller, Jr., H. M., and Dawley, C. W. 1928. An experimental study of some effects of Cysticercus fasciolaris Rud. on the white rat. J. Parasitol. lf>: 87-103. Miller, Jr., H. M., and Gardiner, M. L. 1932a. Passive immunity to infection with a larval tapeworm of the albino rat. Science 7 5 : 270* I bi d . 1932b. Protection of the rat against infection with a larval tapeworm by serum from immune rats. Proc. Soc. Exper. Biol, and Med. 29: 779-780. Ibid* 1932c. Passive immunity to Infection with a metazoan parasite, Cysticercus fasciolaris. in the albinp rat. J* Prevent ♦ M e d . 6 : 1+79-1+96• Ibid. 1931+. Further studies on passive immunity to a metazoan parasite, Cysticercus fasciolaris. Am. J. Hyg. 20: 1+21+-1+31. Miller, Jr., H. M., and Kerr, K. B. 1932. Attempts to immunize rabbits against a larval cestode, Cystioercus pisiformis. Proc. Soc. Exper. Biol, and Med. 29* o*0—o/l. 9k Miller, Jr., H. M . , and M*ssie, E. 1932. Persistence of acquired immunity to Cysticercus fasciolaris after removal of the worms. J. Prevent. Med. 6 : 31-3 6 . Moore, D. H. 19l|5* Species differences in serum protein patterns. J. Biol. Chem. 161: 21-32. Ibid. 19^.8• Effect of reciprocal steroid treatment on the electrophoretic patterns of fowl sera. Endocrinology ij.2: 38-1+5. Moore, D. H., and Abramson, H. A. 1950. Chapter on Electro­ phoresis. Medical Physics (Glasser, 0 ., Editor), Vol. II, pp. 327-335* Year Book Pub., Inc., Chicago. Moore, D. H., Roberts, J. B., Costello, M . , and Schonberger, T.hi. 19^9* Factors influencing the electrophoretic analysis of human serum. J. Biol. Chem. 180: lli|.7-ll58* Moore, D. H., and White, J. U. I9I4.8 . A new compact Tisellus electrophoresis apparatus. Rev. Sci. Instruments 19: 700-706. Mueller, J. F. 1938. Studies on Sparganum mansonoides and Sparganum proliferum. Am. J. Trop. Med. lb: 303-328. Mueller, J. F., and Chapman, 0 . D. 1937* Resistance and immunity reactions in infections with Sparganum mansonoides. J# Parasitol. 23* 581-582# Ohira, T. 1935** On the active immunization of animals against tapeworms. Far Eastr Ass. Trop. Med. Tr. 9 Cong. Is 601-6014.. Olberg, H. 1955. Dber die bluteiweissveranderungen bei experimenteller infektion von mausen mit Trypanosoma brucei. Zentralbl. Bakteriol., I. Abt., O r i g . 162: 120-135* Oliver-Gonzalez, J. 19i|.l* The dual antibody basis of acquired Immunity in trichinosis. J. Infect. Dis. 69^ 25^-*270. Palais, M. 193k♦ Resistance des rats a 1 *infestation d* Hymenolepis diminuta (Rud.). Compt. Rend. Soc. Biol. 117: 1016-1017. Penfold, W. J., and Penfold, H. B. 1937. Cysticercus bovis and its prevention. J. Helminth. 15* 37-^0. Perkin-Elmer Corporation. 1951* Instruction Manual, Portable -Tisellus Electrophoresis Apparatus, Model 3 8 . Norwalk, Conn., 37 pp* ** Original article not seen. 95 Peters, B. G. 1936. Some recent developments in helminthology. Proc. Roy. Soc. Med. 29: 1071^-IO8I4.. Peters, Jr., T*, and Anf insen, C* B. 1950. Production of radioactive serum albumin by liver slices. J. Biol. Chem.182: 171-179. Popper, H., Bean, W. B . , Huerga, J. de la, Franklin, M., Tsumagari, Y., Routh, J. I., and Steigmann, F. 1951. Electrophoretic serum protein fractions in hepatobiliary disease* Gastroenterology 17: 138-1 6 2 . Rees, G. 1951The anatomy of Cysticercus taeniae-taeniaeformis (Batsch 1786) (Cysticercus fasciolaris Rud. l8o8 ), from the liver of Rattus norveglcus (Erx.). including an account of spiral torsion in the species and some minor abnormalities in structure. Parasitology I4.I: lj.6-59. Reiner, M. 1952. Application of electrophoresis to medical problems. Med. Ann. Dist. Columbia 21: 11-16. Reiner, M., and Fenichel, R* L. 1914-8 . Dialysis of protein solutions for electrophoresis. Science 108: I6I4.-I6 6 . Roberts, S., and White, A. 191+9. Studies on the origin of the serum proteins. J. Biol. Chem. l8 0 s 505-51&. Sambon, L. W. 19214.. The elucidation of cancer. Med. and Hyg. 27: 1214-—17i+* J. Trop. Seddon, H. R. 1931. The development in sheep of immunity to Moftiezia expanse. Ann. Trop* Med. and Parasitol. 25:' lt.31-l4.35. Seibert, F. B., Seibert, M. V., Atno, A. J., and Campbell, H. W. 1914.7 * Variation in protein and polysaccharide content of sera in the chronic diseases, tuberculosis, sarcoidosis, and carcinoma. J. Clin. Invest. 26: 90-102. Shedlovsky, T., and Scudder, J. 1914-2. A comparison of erythrocyte sedimentation rates and electrophoretic patterns of normal and pathological human blood. J. Exper. Med. 7 5 s 119-1 2 6 . Shorb, D. A. 1933. Host-parasite relations of Hymenolepls fraterna in the rat and the mouse. Am. J. Hyg. lb: 7I4-— 113* Stauber, L. A. 19514-. Application of electrophoretic techniques in the field of parasitic diseases* Exptl. Parasitol. 3: 5MJ.-568. 96 Stern, K. G., and Keiner, M. 1946. Electrophoresis in medicine. Yale J. Biol. Med. 19: 67-99. Stoll, N. R. 191^8. Abstract of discussion. Proc. Fourth Internat. Gong, on Trop. Med. and Malaria 2: 98I-983. Taliaferro, W. H # 1929. The Immunology of Parasitic Infec­ tions. The Century Co., New York. 4 14 pp. Ibid. 1934. Some cellular bases for immune reactions in parasitic infections. J. Parasitol. 20 : 149- 161. Ibid. 1940a. The mechanism of acquired Immunity in infec­ tions with parasitic worms. Physiol. Rev. 20: 469-492. Ibid. 1940b. The mechanism of immunity to metazoan para­ sites. Am. J. Trop. Med. 20: 169-132. Ibid. 194^. The inhibition of reproduction of parasites by immune factors. Bacteriol. Rev. 12: 1-17. Thompson, S., Foster, J. F., Gowen, J. W., and Tauber, 0. E. 1954. Hereditary differences in serum proteins of normal mice. Proc. Soc. Exper. Biol, and Med. 8 7 : 315-317. Thorson, R. E. 1954* Absorption of protective antibodies from serum of rats immune to the nematode, Nippostrongylus m uris. J. Parasitol. I4.O: 300-303* Tisellus, A. 1937. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc. 33: 524-531. Tisellus, A., and Flodin, P. 1953Adv. Prot. Chem. 8: 431-4^6. Zone electrophoresis. Tisellus, A., and Kabat, E. A. 1939. An electrophoretic study of immune sera and purified antibody preparations. J. Exper. Med. 69: 119-131* Trawirfskl, A. 1936. Ueber anwendung der prazipitationsreaktion zum nachweis der schweinezystizerkose. Zentralbl. Bakteriol., I. Abt., Orig. 136: 116-120. Trawinski, A., and Rothfeld, J. 1935. Ueber anwendung der prazipitationsreaktion zum nachweis der gehirnzystizerkose beim menschen. Zentralbl. Bakteriol., I. Abt., Orig. 134- 472-474* 97 Tumen, H . , and Bockus, H. L. 1937. The clinical significance of serum proteins in hepatic diseases. Compared with other liver function tests. Am. J. Med. Sc. 193: 788-800. Turner, E* L., Berberian, D. A., and Dennis, E. W. 1936. The production of artificial immunity in dogs against Bchinococcus granulosus. J. Parasitol. 22* 11^-28. Turner, E. L., Dennis, E. W . , and Berberian, D. A. 1937. The production of artificial immunity against hydatid disease in sheep. J. Parasitol. 232 ij.3-6l. Weichselbaum, T. E. 1914-6 . An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Am. J. Clin. Path., Technical Section, 10: i4.0-l|.9. Wright, G. G., and Oliver-Gonzalez, J. 1914-3. Electrophoretic studies on antibodies to Trichine11a spiralis in the rabbit. J* Infect. Dis. 72: 2I4.2-2lj.J• AN ELECTROPHORETIC STUDY OF SERA FROM RATS ARTIFICIALLY INFECTED WITH AND IMMUNIZED AGAINST THE LARVAL CESTODE, CYSTICERCUS FASCIOLARIS By Nathan Kraut AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health Year 1955 N a t h a n hrant 1 This study was undertaken to: (1) ascertain the effects of a larval cestode, Cystlcercus fasciolaris. infection on rats as manifested by serum protein metabolism, and (2) to attempt to relate the humoral factors produced against this parasite to the serum proteins. Serum protein analyses were made by the moving boundary electrophoresis method. Rats were experimentally infected with the larval cestode and their sera analyzed electrophoretically on various days following infection. A similar procedure was followed for rats artificially immunized against the parasite by a series of intraperitoneal Injections of larval worm material. The following results were obtained from the studies on the experimentally infected rats: 1. In lightly infected rats having about 38 cysts as their highest average total number (living plus dead), no significant quantitative differences were obtained be­ tween infected and non-infected rats in any of the serum protein components. A qualitative change was evidenced by the ability of the lightly Infected rats to resist a second infection. 2. Heavily infected rats having a total number of cysts averaging 66 or more showed significant quantitative changes among the serum protein components. Qualitative changes in the serum proteins were also evident among this Nathan Kraut 2 group as use of their sera for passive immunization rendered the recipient rats resistant to an infection. These general results obtained In the lightly and heavily infected animals leads to the expected and obvious conclusion that quantitative changes in serum protein metabolism are related to the degree of infection. 3# Specific changes In serum protein components obtained in the heavily infected rats were significant in­ creases in the total, alpha 2-, beta-, and gamma-globulins. Significant decreases for the albumin and A/G ratio were ob­ tained. The total protein and alpha 1 -globulin showed no significant differences. I4.. As related to time following infection, the following were noted In the heavily infected rats: a) Four days after Infection, the only apparent difference In serum protein components between Infected and control rats was an increased alpha 2 -globulin In the former. b) On the eighth day, the albumin showed a sharp decrease and beta-globulin a marked increase. cj Twelve days after Infection, the albumin had risen, beta-globulin dropped sharply, gamma-globulin showed a marked increase and dropped to a lower level on the six­ teenth day* d) From the sixteenth day after infection to the last day of the experiment (thirty-fifth), the quantitative Nathan Kraut 3 differences between the control and Infected rats maintained themselves at a fairly constant level. 5* In an effort to determine in which serum pro­ tein fraction(s) the antibodies against the parasite were produced, sera obtained 12- and 35“ days after infection were absorbed with larval worm material. Electrophoretic patterns of the sera made before and after absorption revealed no sig­ nificant differences. Evidence of some absorption of anti­ bodies from the 12-day sera was obtained in passive immuni­ zation studies. The following conclusions were drawn from the results secured on the Infected rats: 1. None of the serum protein changes can be specifically attributed to the parasite. 2. The serum protein changes are typical of that resulting from liver tissue Involvement, induced in this case by the parasite infection. 3. It appears that the protein changes were corre­ lated with the liver tissue response to the infection. If. For a period of about two weeks, between the foufth and sixteenth days after infection, serum protein metabolism appeared to be most drastically altered due to the liver involvement* 5. From the sixteenth day following infection throughout the remainder of the experimental period (to Nathan Kraut it thirty-fifth, day after infection) the protein metaoolism seemed to have adjusted to a stable level of activity. From the studies on the animals artificially immunized with larval worm material, 1* the following resulted: Significant changes in the serum proteins were not obtained. 2. Antibody production was effected as evidenced by the in vivo studies. Hence, though quantitative changes were not noted, qualitative changes had occurred in the serum proteins• 3. Antisera absorbed with homologous antigen re­ vealed no reduction in any serum protein component when preand post-absorbe d sera electrophoretic patterns were analyzed. If. Some absorption was effected as the in vivo studies showed. It was concluded on the basis of the results obtained with the artificially immunized rats that: 1. The antibody response produced may have been quantitatively Insufficient to be detected by the electro­ phoresis apparatus. 2. Similarly, absorption was In an amount too small to be detected by the method employed.