Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Immunological studies of some strains of Trichophyton mentagrophytes Uchida, Midori Jane 1960

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1960_A6_7 U3 I5.pdf [ 6.37MB ]
Metadata
JSON: 831-1.0106507.json
JSON-LD: 831-1.0106507-ld.json
RDF/XML (Pretty): 831-1.0106507-rdf.xml
RDF/JSON: 831-1.0106507-rdf.json
Turtle: 831-1.0106507-turtle.txt
N-Triples: 831-1.0106507-rdf-ntriples.txt
Original Record: 831-1.0106507-source.json
Full Text
831-1.0106507-fulltext.txt
Citation
831-1.0106507.ris

Full Text

IMMUNOLOGICAL STUDIES OF SORE STRAINS OF TRICHOPHYTON MENTAGROPHYTES by MIDORI JANE UCHIDA B.A. University of British Columbia, 1957. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M. Sc. In the Department of Bacteriology and Immunology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH! COLUMBIA October, 1960 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f B a c t e r i o l o g y and Immunology The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 3 , Canada. Date &*d*A,st, .¥-,/?<,()  i ABSTRACT The relatively poor immunogenicity of proteinaceous materials obtained from dermatophytes has been demonstrated. The use of incomplete Freund's adjuvant failed to enhance antibody formation to mold antigens. Many cross-reactions occurred between the antisera of rabbits immunized with various antigenic preparations from dermatophytes, as demonstrated by cross-precipitin tests and skin testing. The occurrence of common antigens among the dermatophytes has been confirmed. Pleomorphic strains did not differ markedly in their immunogenicity as compared to their res-pective non-pieomorphic strains. Skin testing,upon rabbits with dermatophytic antigens elicited responses which were either of the immediate or of the delayed type. Indiea-tions were obtained that toxic principles were present in some of these antigens. Skin tests provided better evi-dence of differentiation between various species and strains than did the precipitin tests. i i TABLE OF CONTENTS Page I. INTRODUCTION, 1 i r . HISTORICAL REVIEW OF THE LITERATURE. 3 A. Classification. 3 B. Immunology of dermatophytes. 8 III. MATERIALS AND METHODS. 16 A. Preparation of antigens: 16 1. Species of dermatophytes. 16 2. Cultivation of organisms: 16 (a) Preliminary tests. 17 (b) Cultivation method. 17 Cc) Method of obtaining pleomorphic growth. 17 3. Method of antigen preparation: 18 Ca) Intracellular antigenic material* 18 (b) Extracellular antigenic material. 20 (c) Alcohol-precipitated antigens. 20 4. Estimation of total, nitrogen. 21 B. Animal experimentation and in vitro antibody tests: 22 1. Rabbit immunization.: 22 Ca) Incomplete Freund's adjuvart and partially purified antigenic material: 22 C.b) Incomplete Freund's adjuvant and mycelial homogenate: 23 Cc) Skin infection. 24 2. Collection and storage of antisera. 24 3. In vitro tests for estimation of antibody: 24 i i i Page (a) Ring precipitin test; 24 Cb) Modification of Hanks' precipitin. method. 25. (a) Hemagglutination technique. 25 (d) Plate diffusion technique. 26 (e) Slide double-diffusion agar precipitin technique. 26 4. Skin tests of rabbits. 26 IV. EXPERIMENTAL RESULTS. 28 A. ' Preliminary observations: 28 1. Cultivation of organisms. 28 2. Method of obtaining pleomorphic growth. 28 3. Method of antigen preparation: 29 (a) Intracellular antigenic material. 29 Cb) Extracellular antigenic material. 29 Cc) Alcohol-precipitated antigens.. 30 4. Estimation of total nitrogen. 30 5. In vitro tests for estimation of antibody: 31 (a) Ring precipitin test., 31 (b) Modification of Hanks' precipitin method. 31 (c) Hemagglutination technique. 31 (d) Plate diffusion technique; 32 (e) Slide double-diffusion agar precipitin technique. 32 B. Final experimentation: 33 1. Rabbit immunization: 33 Ca) Series 1: 33 iv Page (b) Series 2: 35 Cc:) Series 3: 36 (d) Series 4: 39 2. Cross-precipitin. tests. 40 3. First skin tests. 41 4. Second skin tests. 59 V. GENERAL DISCUSSION.. 63 VI. SUMMARY. 71 VII. BIBLIOGRAPHY. 74 VIII. APPENDICES . 77 DIAGRAMS Page I. 27 PMTES i . i i . i n . n r . 55 56 57 58 ACKNOWLEDGEMENTS The author wishes to express her appreciation to Dr. J . J . Stock for his guidance and supervision during the course of this project. The kind advice of Dr. D.C.B. Duff, Department of Bacteriology and Immunology, in the preparation of this thesis, the photography of Mr. G.E. Rasmussen, and the financial assistance of the Medical Research Council, are gratefully acknowledged. I. INTRODUCTION. Compared to the volume of information about the Immunology of bacteria, there is very l i t t l e known about the immunology of the group of pathogenic fungi called the dermatophytes. The purpose of this study was to obtain more information about the antigenic structure and immuno-specificity, i f any, of various strains of fungi in the species Trichophyton mentagrophytes. Various protein-free liquid media were investigated to determine the.one providing the most rapid as well as the heaviest growth. Partially purified proteinaceous materials, of both intracellular and extracellular origin, were examined as possible strain-specific sources of anti-genic material, which could lead to the production of strain-specific antibodies in rabbits. With the use of incomplete Freund's adjuvant, i t was hoped to develop more satisfactory antibody responses of the test animals to the antigenic material. The antisera obtained were examined in various ways, which included tests for the presence of cross-reactions. The antisera of rabbits, immunized with certain strains of this species which had undergone pleomorphism, were obtained for the purpose of testing differences in their reactivity, as compared to homologous and hetero-logous non-pleomiorphic growth. In this project i t was hoped that more information 2 concerning antibody production to various antigenic ex-tracts of the dermatophytes would be obtained, as well as some clarification of .the classification of these fungi. 3 II. HISTORICAL REVIEW OF THE LITERATURE. A. Classification. Dermatophytes are a group of fungi which parasitize the superficial keratinized areas of the mammalian body e.g. skin, nails, hair, but rarely attack the subcutaneous tissues or cause systemic diseases. They appear as myce-l i a l fragments in skin and nails, or as spores and mycelial fragments inside or outside the hair shaft. On suitable solid media in vitro, dermatophytes develop filamentous colonies which may produce various types of spores. On the basis of spore forms, the ringworm fungi are now divi -ded into 3 genera: Microsporum, Trichophyton, and Eplder-mophyton. Before this classification was generally accepted, many workers had attempted by other approaches to classify the dermatophytes. Sabouraud (45) conducted investigations of clinical cases of ringworm and published, on the basis of these, his monumental work "Les Teignes". His classification, inf lu-enced by clinical considerations, placed the dermatophytes into 4 genera: Epidermophyton, Achorion, Microsporum, and Trichophyton. However, when the botanical characters of the fungi were considered, i t was found that species morphologically resembling each other could be placed in different groups in Sabouraud's classification. The characteristics on 4 which the scheme was based, such as ectothrix or endothrix behaviour on or in the hair, were inconstant. The develop-ment of the fungus upon the hair depended upon the structure of the hair and the nature of the fungus species. In some cases, a species behaved differently in different animal species. For example, Kato (29) isolated a dermatophyte which was endothrix in man, but was ectothrix when inocula-ted into guinea pigs, dogs, and rabbits. Langeron (36) raised other objections. He stated that advances in the knowledge of structure and relationship of certain dermatophytes made Sabouraud's scheme obsolete, and that the new species described resulted In the genera being too large in number to be practical. Matruchot and Dassonville (39), (40) concluded that the dermatophytes were Gymnoascaceae which had lost the ability to produce asci. The evidence that they presented to support this view was as follows: 1. Both dermatophytes and Gymnoascaceae produced a similar type of external spore or aleuriospore. . 2. Spindles were found in the Ctenomyces, a genus of of Gymnoascaceae, as well as in the dermatophytes. 3. All. Gymnoascaceae had spiral hyphae analagous to spirals of some Trichophyton species. 4. Ctenomyces serratus produced lesions of a tricho-phytic nature in a dog. Ota and Langeron (42) f i rs t attempted a truly botanical 5 classification based upon mycological characteristics. They placed the dermatophytes in the family Conidiosporaceae under a new sub-family, Closterosporeae. This sub-family was divided into 6 genera: Trichophyton, Sabouraudites, Epidermophyton, Grubyella, Bodinia,. and Endodermophyton. They placed a l l Microsporum and Trichophyton species pro-ducing macroconidia in the genus Sabouraudites. This did not result in a natural division because differences in types of macroconidia were ignored. Grigorakl (23) made 3 major groups of dermatophytes: the f i rs t 2 conformed with those of Matruchot and Dassonville and were classed as Gymnoascaceae. The third group inclu-ded a l l the species in. which only arthrospores were formed, and was called Arthrosporaceae. Rivalier ('43) classified the dermatophytes in 5 genera: Sabouraudites. Ctenomyces, Megalosporon, Trichophyton and Epidermophyton. The ideal classification for dermatophytes was thought to be a mycologic one i . e . , one based on the mor-phology of these fungi in culture. However, i t was stated by Emmons (13) that these fungi did not lend themselves to such a classification. He also thought that the der-matophytes were as amenable to a mycologic classification as other groups of the Fungi Imperfecti, and such a scheme ran counter to the clinical groupings of Sabouraud in minor points only. 6 According to Emmons (13), there were 3 groups of dermatophytes following natural lines of relationship. These were Trichophyton, which reproduced i n culture prin-cipally by conidia; Epidermophyton, which in clinical i n -fections could not be distinguished from Trichophyton jj but which did not, however, invade the hair; and Microsporum, which produced spindle-shaped macroconidia and clavate conidia in culture. In hairs infected by Microsporum, there was a mosaic sheath of arthrospores around each hair. The classification at present accepted by most my-cologists is similar to the one proposed by Emmons (13;). This, in turn, is similar to the one proposed by Sabouraud(45). Genus Microsporum The characteristic spore form of this genus is the large (8-15 >c wide by 40-150 long), spindle-shaped, pointed, multicellular, rough, thick-walled macroconidium with a broad base. These spores are ' numerous, whereas the microconidia are scarce in primary cultures., Conant( 10) recognizes .3 species - M. audouini Gruby, 1843, M. canis Bodin, 1902, and M. gypseum (Bodin) Guiart and Grigoraki, 1928./ Genus Trichophyton Unlike the genus Microsporum. the predominant spore form in. this genus is the microconidium. These microconidia are subspherical, pyriform or 7 clavate (1.5-2A by 2-5 / \ ) and occur on the sides of hyphae (en thyrse) or on conidiophores In grape-like clusters. The macroconidia are more rarely seen, but are characteristic, being long, thin-walled, multi-septate, clavate spores with blunt ends (4.-6 A wide by 10-50.A, long). Racquet mycelia, nodular bodies, spiral hyphae, favic chandeliers, and chlamydospores are produced by some species. Conant(lO) follows Malmsten*s classification for the Trichophyton. There are 5 groups with typical species representing them., A. Gypseum group 1. T. mentagrophytes - synonyms Achorion  quinckeanum, T.. felineum, T. gypseum, T. equinum, T. granulosum, T. lacticolor, T. radians, T> persicolor, T. farinulentum, T. asteroides, T. interdigitale. B. Rubrum group 2. T. rubrum - synonym T. purpureum. C. Crateriform group 3. T. tonsurans - synonyms T. epilans, T. Sabouraudi,. T . sulfureum.. D. Faviform group 4., T. Schoenleini. 5. T. coneentricum 6. T. ferrugineum 7. T. violaceum 8. T. verrucosa - synonym T. discoides E. Rosaceum group 9. T. gallinae - synonym M. gallinae 10. T.. megnini - synonym T. rosaceum  Genus Epidermophyton This genus has one species,. E. floccosum. The only form of spore seen is the macroconidium. These spores are oval to egg-shaped, 2- to 6-celled,. smooth, and have slightly thickened walls* They are occasion-ally grouped in dense clusters, but in most strains are scattered. Old cultures contain many chlamydo-spores and racquet mycelia. B. Immunology of dermatophytes. According to Rivalier (44), an animal parasitized by a certain dermatophyte became refractory to a second infec-tion by the same species, and even, to one or several other species. The dermatophytes did not seem to produce such a refractory state in humans, although he reported that a ringworm infection causing intense inflammation protected against subsequent attack with the same parasite. There were cases, however, where suppurative Trichophyton infec-tions were not followed by a state of immunity. Rivalier stated that Bloch and Massini (5) had found that a dermatophyte causing strong inflammation protected 9 guinea pigs against the same ringworm or against a l l other species of ringworm. This was not the case with non-inflammatory dermatophytic fungi. Therefore, the refractory state in guinea pigs was multivalent, and was conditioned by the degree of response to the primary inoculation. This refractory state coincided with a particular con-dition of acquired hypersensitivity or allergy which the animal showed by a sudden inflammatory reaction upon contact with the pathogenic parasite. Bruno Bloch (4) showed this experimentally by infecting guinea pigs with T. mentagro-phytes spores and reinfecting them 20 days after healing with the same organism. Upon primary infection, the organ-Ism" was visible in the scales and hairs on the seventh day, but mycelia could not be detected in the scales or hairs during the second infectious process. This is called the accelerated trichopnytic reaction or Bruno Bloch phenomenon, and Is comparable to Koch's phenomenon of tuberculous rein-fection. De Lamater and Benham (1.2) confirmed the results of Bloch. Using T. gypseum strain 1 from a squirrel, they reinfected guinea pigs which had recovered from their f irst infection. No fungi were found in the lesions produced upon reinfection. Upon third and fourth inoculations, the inflammatory reactions became more severe, indicating a sensitization. Similar diseases to the one produced by strain 1 were caused by reinoculation of 2 other T. gypseum 10 strains, one from a monkey, the other from a human. Bloch (4) could not obtain the allergic state in gui-nea pigs brought about by cutaneous inoculation, of a dermato-phyte by any other method, but De Lamater and Benham (.12) infected guinea pigs by cardiac puncture and intravenous injection. Many in vitro, methods have been used to determine titres of antisera obtained during a course of immunization, or during an infection by these dermatophytes. They Include precipitin, collodion particle agglutination, complement fixation, and hemagglutination techniques. Citron (9) reported that agglutination and precipitin reactions took place between fungus extracts and the sera of rabbits injected with living fungus spores. Wharton,. Reiss, and Wharton (55) demonstrated antibodies by preci-pitin tests, as did Ito and KIrita (26)„ Miller, Stewart and Kimura (41) demonstrated cross-reactions between T. interdigitale and T. rosaceum by the precipitin method. Sharp (48), (49), using the precipitin technique, reported titres of 1/1,000 to 1/8,000 between antisera and their homologous antigens. Wharton et a l . (55) found that pre-cipitating antibodies in immunized animals persisted for periods up to 18 months. Keeney and Eriksen (30) injected rabbits with live fungal spores, and by the use of" the collodion, agglutina-tion technique found an absence of cross-reactions between 11 2 strains of T. mentagrophytes., Huppert (25) using "single cel l " suspensions for immunization, and the collodion agglutination technique, demonstrated cross-reactions between a l l members of the Gypseum group which were tested. The complement fixation test was used by Miller et al . (41) without success. On the contrary, Gotz (22) reported that Kishi- and Seeliger obtained results with this test. The latter worker found that even with a highly diluted T> rubrum antiserum,; a positive complement fixation test was obtained with polysaccharide components of various species of Micro sporum,, Trichophyton:, and Epidermophyton as antigens. The successful use of the hemagglutination method with the polysaccharide fraction of Trichophytons has been reported by Kishi (33). According to this worker, the agglutination test was a more sensitive one than that of complement fixation. It was believed by many workers that common antigens or antigenic structures occurred in a l l hyphomycetes., Using the Schultz-Dale reaction,, Jadassohn, Schaaf, and Wohler (27) found that one or more common antigens were present in 4 different pathogenic hyphomycetes. Sharp (49) also discovered by cross-reaction experiments in vitro that common antigens occurred in the Microsporum, Trichophy-ton, and Epidermophyton genera. Similar antigens were found in different Trichophyton species by Miller et al* (41) and 12 also by Huppert (.23).* Seeliger (47) discovered that T. mentagrophytes spores were agglutinated by T* rubrum antiserum, and that this antiserum produced positive preci-pitin tests with polysaccharides of various species of Microsporum, Trichophyton, and Epidermophyton. However, as stated before, Keeney and Eriksen (30) found no reactions between 2 strains of T. mentagrophytes. Antigens which were characteristic of the individual fungus, as well as antigens common to certain groups of hy-phomyeetes, have been reported by Jadassohn et a l . (27). An indication of species-specificity between members of the Microsporum group was found by Sharp (49). Fischer (14), using the same method of Jadassohn and his co-workers, found that the Epidermophyton species possessed a different antigenic formula than that of the Trichophyton species. Keeney and Eriksen (30) showed species-specific components in T. mentagrophytes and T. rubrum. Citron's (9) early experiments indicated that an antiserum would precipitate better with the homologous than with a heterologous Tricho-phyton antigen* According to Sharp (49) the tendency to precipitate with another fungal species approached closer to the homologous when the immunization schedule was inten-sive or prolonged. Up to the present, Sharp (48) seems to have been the only worker to determine the effect of pleomorphism upon the yield of fungal antigenic protein. He stated that equal 13 amounts of protein were obtained from pleomorphic and non-pleomorphie strains of M. felineum (M. l a n o s u m ) T h e spores contributed the same amount of protein as the hyphae. The poor immunogenic a c t i v i t y of the dermatophytes i s well known. An antiserum t i t r e of 1/200 i s generally con-sidered high f o r an antigen-antibody reaction i n which the an-ti g e n i c agent i s a dermatophyte.. M i l l e r et a l . (.41.). Huppert (25), and Keeney and Eriksen (30) were unable to demonstrate higher t i t r e s consistently, and usually not at a l l , but Sharp (48), (49) obtained t i t r e s of up to 1/8,000 using the p r e c i p i t i n method. I t seems that the age of the culture has an effect upon the immunogenicity of fungal, antigens.. Link and Wilcox C37) found that immunogenic a c t i v i t y was strongest with very young cultures, while Sharp (49) showed that the older the culture, the less the amount of antigenic material obtained. Foster (15) postulated that there could be l o s s of antigenic material through autolysis. In an acid medium, there was l i t t l e or no l o s s of weight, indicating s l i g h t , i f any, auto-l y s i s ; but the amount of c h i t i n i n the mycelium increased with time, and t h i s may have masked any autolytic a c t i v i t y , especially of the non-chitinous material. In an alkaline medium, the c h i t i n content decreased with age, and In a neutral pH medium, autolysis was great. Various antigenic materials from dermatophytes have been used by d i f f e r e n t workers.in the immunization of 14 animals. Keeney and Eriksen (30) injected l i v e fungal spores into rabbits while Sharp C48), (49) injected par-t i a l l y p u r i f i e d proteinaceous material. Adjuvants have been used by 2 workers. Huppert (25), using one-celled suspensions of the fungus, and an incomplete Freund's adju-vant, was unable to obtain h i g h - t i t r e d antisera. Wharton et al.(55) employed the k i l l e d mycelial mat of T. purpureum incorporated i n a mixture of k i l l e d tubercle b a c i l l i , peanut o i l , and "Falba" (20) and found that rabbits developed com-( plete immunity to i n f e c t i o n with the l i v i n g fungal material. P a r t i a l success was obtained by substituting mineral o i l f o r peanut o i l and jojoba o i l f o r "Falba". Actual i n f e c t i o n of rabbits was experimentally carried out by Wharton et a l . (55). They found that 11 out of 16 rabbits developed p r e c i p i t a t i n g antibodies as a result of the i n f e c t i o n . The animals, immunized according to the method mentioned i n the previous paragraph, also demonstrated p r e c i p i t a t i n g antibodies. In both the case of the immunized animals and the infected animals, when the antibody t i t r e s became low or were l o s t , the rabbits became susceptible to i n f e c t i o n or r e i n f e c t i o n as the case might be. Keeney and Eriksen (.30) carried out skin test reactions on animals, using ammonium sulphate-precipitated fractions and the alcohol precipitate of the l i q u i d medium i n which T. mentagrophytes #599 had been grown. The test animals had been immunized previously with T. mentagrophytes #599 15 and #640, and T. rubrum antigens. Cross-reactions occurred in a l l cases and were recorded as a measurement of response in diameters. The 1 control animal seemed to react as much as immunized animals. De Lamater and Benham (12) perform-ed skin tests on guinea pigs at intervals varying from 2 days to 2 months after they had recovered from primary i n -fections with T. gypseum strain 1* Two-tenths of 1 ml. of a 2% extract of the dried fungus powder was used as the skin test antigen. Reactions to the homologous and heterologous extracts were practically identical, and a l l were of the delayed type. Tests after recovery from second and third infections showed definite increases in skin reactivity, but with T. gypseum strains 2 and 3 there were no such increases. 16 III.. MATERIALS AND METHODS.. A. Preparation of antigens: 1. Species of dermatophytes. The following dermatophytes were obtained through the courtesy of Dr. P . Blank, Department of Bacteriology and Immunology, MeGill University, Montreal, Quebec, and of Dr. C.W. Emmons, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. M. canis-felineum M. quinckeanum #7 - source - mouse M. quinckeanum #8 - source - mouse M.. quinckeanum #13 - source - mouse T. asteroides T. farinulentum #2 T. granulosum T. interdigital.e T. mentagrophytes #666 - source - dog #675 - source - chinchilla rabbit #687 - source - monkey T. persicolor T. radians Stock cultures of these dermatophytes were maintain-ed on cerelose agar slopes (34). Subcultures were made to fresh slopes and a check for characteristic morphology, both microscopic and macroscopic, was carried out every 6 weeks. Cultures in which growth had been obtained were refrigerated between subculturings. 2. Cultivation of organisms: 17 Ca) Preliminary tests. Three liquid culture media were tested to deter-mine the one best suited for quick typical growth of mycelia. Four species of ringworm dermatophytes, M.. canis-felineum, T. asteroides, T. interdigitale, and T. farinulentum were inoculated separately into flasks containing modified Lednian's medium C41), Sabouraud's cerelose broth, and a partially defined medium ("see Appendix A) described by Keeney and Eriksen (30). It was found that the latter medium, referred to henceforth as Keeney*s medium, provided the largest amount of surface growth in the shortest time. As this medium contains no proteins, any pro-tein found i n the culture f luid would have been syn-thesized and liberated by the organism. Cb) Cultivation method. Each organism was inoculated separately into 20 one-litre flasks which contained 250 ml. of sterile liquid Keeney's medium. These were incu-bated at 25°C until the mycelial mat in each flask covered the surface of the medium, the incubation periods varying from 4 to 6 weeks for the 7 organisms used. CcO Method of obtaining pleomorphic growth. Pleomorphism in T. persicolor and in T. menta-grophytes #675 was obtained by incubation at 37°C. 18 These species were judged pleomorphic by a l t e r a -t i o n i n macroscopic morphology and by the lack: of macroconldial formation. The i n t r a c e l l u l a r and i e x t r a c e l l u l a r antigenic materials from these c u l -tures were prepared as follows. Method of antigen preparation: Ca) I n t r a c e l l u l a r antigenic material. (1) Concentration and p a r t i a l p u r i f i c a t i o n . The contents of the f l a s k s were poured into cheesecloth supported on a glass funnel. The f i l t r a t e was collected and treated accor-ding to procedure (b) f o r recovery of extracel-l u l a r antigenic material.. The mycelial, mat was autoclaved f o r 10 minutes at 121°C and immediately refrigerated. When c h i l l e d , the mat received three 500 ml. s t e r i l e water wash-ings and three 500 ml. washings with s t e r i l e Locke's solution (see Appendix B), and then was macerated i n a Waring biendor equipped with special cutting blades and with a Cenco surface cooler. The macerated mycelia were then ground In an Abbe b a l l m i l l for 24 hours at 6°C. Upon microscopic observation at t h i s time, i t was seen that the mycelia had been re-duced to 1- or 2-celled fragments. The slurry from the m i l l was centrifuged at 2,000 r.p.m. 19 at 6°C. The supernatant f luid was passed through a pre-treated (.8), (54), (see Appendix C.) Seitz sterilizing pad to remove any mycelial fragments. The filtrate was saturated with ammonium sulphate at 6°C and allowed to stand overnight. The resulting pre-cipitate was- then dissolved in a minimal amount of physiological, saline, re-precipitated with ammonium sulphate, re-dissolved In saline and dialyzed under toluene through cellophane against tap water until free of the sulphate ion (7)., The material was then dialyzed overnight against 0.425% saline at 6°C. The dialysate was passed through a pre-treated Seitz clarifying pad to remove any material not in solution. This filtrate was lyophiiized to dryness and the material stored under partial vacuum over calcium chloride at 6 ° C (ii) Modification of procedure. The mycelial mat was collected and immed-iately washed with sterile water and sterile Locke's solution to which had been added merthio-late in ratio of 1:50,000. It was then macera-ted for 15 minutes in a VirTis "23" homogenizer at 23,000 r»p>m., with the aid of glass beads, according to a technique reported useful for the breaking of bacterial and yeast cells (35). The container was immersed in a brine and ice bath to retard rise of temperature during the 20 homogenizing process. This rapid treatment produced a slurry consisting essentially of 1- ot 2-celled frag-ments. The material was centrifuged and the remainder of the procedure was the same as given, in procedure (i) (see Concentration and partial purification,, page 18). (b) Extracellular antigenic material.. Concentration and partial purification: After collection of the mycelial mat, the cul-ture medium was sterilized by fil tration through pre-treated Seitz clarifying and sterilizing pads. The filtrate was then concentrated by flash-evaporation until a 25- to 50-fold concentration was achieved. This concentrate was filtered through a pre-treated clarify-ing pad, twice-precipitated with ammonium sulphate, dialyzed, and freeze-dried in the manner previously described for the intracellular antigenic material.* (e) Alcohol-precipitated antigens. The lyophilized material, obtained after ammonium sulphate precipitation, was dissolved in a minimal amount of distilled water and centrifuged to remove any un-dissolved particles. The supernatant was cooled to -25°C and an equal volume of chilled 95% alcohol was added. If precipitation did not occur, more alcohol was added until definite turbidity appeared. Twenty-four hours * Because of the relatively crude nature of the protein-containing precipitates employed as antigens, and because of the strong probability that they contain carbohydrates and non-protein nitrogen, these prepara-tions shall be referred to as "proteinaceous material". 21 later,, the precipitate was recovered by centri-fugation. The alcohol was removed by decantation, with the residual alcohol being drained off as completely as possible without actual drying of the precipitate. The material remaining was dissolved in a minimal amount of 0.85% saline. Total nitrogen determinations were carried out, and the solutions standardized to contain 0.25 mg. of nitrogen per ml., the material then being stored in the frozen state. Estimation, of total nitrogen. Determinations of the total nitrogen content of the freeze-dried materials were carried out by the semi-micro-Kjeldahl method (.28)., Fifty mg. amounts of the specimen were dissolved in 2.5 ml. of distilled water. One ml. of the solution was placed into each of two 100 ml. Kjeldahl flasks and treated according to the procedure of Kabat and Meyer (28). The dis-t i l l i n g apparatus used was the Kirk-type. The calcu-lation of the amount of nitrogen was modified slightly to include a correction factor for instrument error: (a) Ml. N/70 HG1 x 0.2 = mg. nitrogen (b) Actual amount = correction factor x mg. nitrogen of nitrogen (c) Correction factor .= 1  amount of N/70HCKin ml.) to titrater standard x 0. ammonium sulphate solution 22 Animal experimentation and in vitro antibody tests: 1. Rabbit immunization: Injections of antigens into rabbits were made sub-cutaneously in the scapular region. The immunization schedules were carried out as follows: (a) Incomplete Freund's adjuvant and partially  purified antigenic material: (i) The antigen was emulsified into incomplete Freund's base (20) as described below: One part "Falba" Two parts o i l CMarcol GX) These ingredients were placed in serum vials, stoppered with non-absorbent cotton, and s ter i l -ized in the autoclave at 121°C for 30 minutes. These materials were held at 37°C until the re-maining steps were completed. Two parts of pro-teinaceous material dissolved in 0.85% saline were added to the Freund's base and the cotton plug replaced by a rubber stopper. The contents of the vials were then emulsified, being shaken for 5 minutes by a mechanical agitator. The an-tigenic materials prepared in this way were stored in a freezer until used, and half an hour before being injected into rabbits were placed in a 37°C water bath in order to reduce viscosity. The ma-terials prepared in this manner contained 0.8 mg. 23 nitrogen per ml. (ii) Immunization schedule: Week Volume injected Nitrogen injected (in ml.) ~~ (in mg.) 1 0.5 0.4 2 1.0 0.8 3 1.0 0.8 5 2.0 0.8 7 2.0 0.8 8 Test bleed. 9 No injections given. 10 No injections given. 11 Test bleed. 14 1.5 1.2 15 1.5 1.2 16 1.5 1.2 17 1.5 1.2 18 ,1.5 1.2 19 1.0 0.8 20 Two 50.0 ml. bleeds on intermittent days. Approximately 12.0 mg. of nitrogen were injected into each rabbit during a period of 5 months, (b) Incomplete FreuhdVs adjuvant and mycelial homogenate: (i) The antigenic materials were prepared as before. (ii) Immunization schedule. Week Volume injected Nitrogen injected (in ml.) (in mg.) 1 .0.5, 1.0 0.4, 0.8 2 1.5, 2.0 1.2, 1.6 3 2.0, 2.0 1.6, 1.6 4 2.0, 2.0 1.6, 1.6 5 No injections given. 24 Week 6 7 9 8 Volume injected. Nitrogen injected Cin ml.) (In mg.) 2.0, 2.0 1.6, 1.6 Trial bleed. 2.0, 2.0 1.6, 1.6 Trial bleed. 11 Eleven days after the 1.6 bleed,; a 2.0 ml. i n -jection was made. 12 Two final bleeds on intermittent days. A total of 18.4 mg. of nitrogen was injected into each rabbit over a 3 months period. Cc) Skin infection. Four rabbits were infected with T* granulosum according to the method of Bruno Bloch, as reported by Rivalier (.44). 2. Collection and storage of antisera. Bleedings were made from the anterior marginal ear vein in as aseptic a manner as possible, and the sera were stored separately In the frozen state with no addition of preservative. 3. In vitro tests for estimation ofantibody; (a) Ring precipitin test. Varying dilutions of antiserum in saline, e.g. 1/10, 1/20, 1/50, etc., were placed separately in precipitin tubes in 0.5 ml. quantities. Then 0.5 ml. of antigen, containing 0.5 mg. proteinaceous material per ml. or 1.0 mg. material per ml. , was carefully overlaid onto the antiserum. The tubes were read , 25 at 30 minutes and 1 hour at room temperature. They were then refrigerated overnight and re-read the following day. Control tubes consisted of antigen and saline, and antibody and saline, at the respective least dilutions of each material, (fe) Modification of Hanks' precipitin method. It was thought that incorporation of the antigen into a gel would facilitate the observation of reac-tivity of precipitin test materials. Accordingly, Miller 's modification (,41) of Hanks' precipitin method was carried out. Antigenic material was incorporated into the gelatin base in a ratio; of 1:1 and then put into precipitin tubes ;in 0.2 ml. amounts, which were placed overnight in the refrigerator. Then 0.3 ml. of an antiserum dilution was overlaid onto this anti-gen. The tubes were incubated at 30°C for 3 hours when results were recorded. They were then placed in the refrigerator and re-read at 24 and 48 hours, • after allowing them to stand at room temperature for 15 minutes. Control tubes were prepared in the usual manner. (c) Hemagglutination technique. Since the hemagglutination technique was believed to be a more sensitive test to determine the presence of antibody, this method was assessed for its value in an estimation of rabb.it antibodies formed to fungal 26 antigenic materials. The method used was that of Hoyden, as modi-fied by Stavitsky (50). The concentration of tannic acid used was 1/30,000 and that of the antigen was 3.0 mg. proteinaceous material per ml., (d) Plate diffusion technique. The plate diffusion method of Bjorklund (1) was attempted, using various dilutions of antisera e.g. 1/10 and 1/20. Ce) Slide double-diffusion agar precipitin technique. The slide precipitin technique of Crowle (11) was used with varying dilutions of antisera e.g., 1/2, 1/10, 1/20, 1/50. 4. Skin tests of rabbits. Since the observations concerning precipitins lack-ed significance, a skin test technique was used to deter-mine whether or not more findings could be obtained. Rabbits injected with the mycelial homogenates were tested after the immunization schedule was completed and the final bleedings had been made. A booster injection was given 1 week prior to the skin tests. Results were compared with those obtained in non-Immunized rabbits. After most of the hair on the rabbit's back had been removed by electric clippers, a barium sulphide de-pilating agent (38) was applied for 2 minutes, and was then removed by thorough washings with soap and water. 27 After 24 hours, the depilated area was subdivided Into 18 or 22 sections. Partially purified proteinaceous antigenic material (0.025 mg. nitrogen in 0.1 ml*) in sterile saline, to which had been added merthiolate in ratio of 1:50,000, was Injected separately in 0.1ml.. amounts into each of the sections as shown below. The control was sterile saline containing merthiolate. The "staggered" method of injection was found adequate to prevent reactions from becoming too conflu-ent. Where a spreading type of reaction occurred, the area was so great as to be indistinguishable from neigh-bouring reactions, unless the injections were placed from 4 to 6 inches apart. Observations were made" at 10 to 15 minutes, 1 hour, 18 to 24 hours, 48 hours, and 6 days. Diagram I. control injection 28 IV. EXPERIMENTAL RESULTS. A. Preliminary observations: 1. Cultivation of organisms. Of the cultures used, T. persicolor required the longest growth period on Keeney's medium, and this took as long as 6 weeks in duration. A l l other strains and species showed maximum growth within 4 weeks, this being mostly surface growth. TABLE I CULTURAL CHARACTERISTICS OF ORGANISMS Species and strains used Color of Color and Color of liquor af- texture underside ter growth of organ-ism. of too of surface growth. of sur-face growth. T. mentagroDhytes #666 yellow yellow, granular yellow £. mentagroDhytes #675 dark brown buff, granular dark.'.: .. • brown T. mentagroDhytes #687 yellow buff, granular reddish-brown T. persicolor yellow pinkish, fluffy white M. auinckeanum (#7.#8, & #13) yellow white, fluffy yellow white 2. Method of obtaining pleomorphic growth. Pleomorphic growth was obtained from T. mentagro-phytes #675 and T. persicolor by incubation at 37°G. The growth of the pleomorphic strains differed from the non-pleomorphic state by the absence of macrocon-29 idia and the appearance of white, fluffy, surface growth. 3. Method of antigen preparation: (a) Intracellular antigenic material. Animal experiments showed that the precipitin levels of these antisera were comparatively low after immunization with the intracellular material incorpor-ated into incomplete Freund's adjuvant. After an immu-nization period of 5 months, during which each rabbit received approximately 12 mg. nitrogen, the highest titre was 1/50. Therefore a modification of the method for obtaining intracellular antigenic material was made (see Modification of procedure, page 19) in the hopes that a more immunogenic product could be obtained. Upon dialyzing against tap water, some of the saline-soluble ammonium sulphate-precipitated material, from most of the organisms used, re-precipitated. Dialysis against 0.425% saline usually re-dissolved most of this precipitate. To ensure the absence of any undissolved particles in the dialyzed material, f i l tration through pre-treated Seitz clarifying pads was carried out. (b) Extracellular antigenic material. The method of obtaining extracellular antigenic material was the same throughout the course of experi-ments. The extracellular, material of T. persicolor had 30 a tendency to foam when being flash-evaporated.. A l l other extracellular concentrates did not foam. •(e) Alcohol-precipitated antigens. Alcohol precipitation did not seem to produce as much material as did treatment with ammonium sulphate. It would appear, therefore, that this extra step pro-duced additional purification. 4» Estimation, of total nitrogen. In nearly a l l cases, the extracellular antigenic material showed more total nitrogen on a dry weight basis than did the Intracellular. Since the medium contained no protein,, the ammonium sulphate-precipitable material in the culture medium must have been synthesized, and resulted from the growth of the organism in this fluid medium. There are 2 possible explanations for this pheno-menon.. Proteins may have escaped from the mycelia during growth, or the mycelia may have undergone autolysis, thus liberating the internal contents of the cells . According to Foster (15),, in an acid medium,, there was no loss of mycelial weight,, indicating no autolysis. However,, he also stated that i t was likely that autolysis would occur in cultures 36 to 170 days old. Since most lots prepared for obtaining antigenic material were 28 days old,, perhaps this phenomenon does not explain the higher nitrogen values of the extracellular material. 31 5. In vitro tests for estimation of antibody: (a) Ring precipitin test. Except for the disadvantage mentioned below, this test proved satisfactory for antibody estimation* If the reactions were observed over a period of days, eventual mixing of the antigen and the antiserum was unavoidable, since both components were in the liquid state. (b) Modification of Hanks' precipitin method. This method was adopted finally as the technique of choice. The incorporation of the antigen in a gel aided in preventing mixing of the antigen and anti-serum in most cases until the last reading was taken after 48 hours. A positive reaction was readily ob-served as a demarcated line or area between the 2 layers; occasionally i t was seen as a cloudiness throughout the tube. The results of these tests were reported on. a combination of observations based upon the thickness of the band of precipitate, the number of bands, and the density of the precipitate. Cc) Hemagglutination technique. The hemagglutination technique was unsatisfac-tory. No positive results could be obtained, even with sera showing a high titre with the precipitin test. According to Gotz (22), qualitatively different antibodies might be present in the serum of an.Immun-32 ized animal, such that one antibody could be de-monstrable only by the precipitin test, one only by the hemagglutination, etc. KIshi (33) showed the presence of hemagglutinating antibodies using the polysaccharide fraction of Trichophytons with human red blood cells as the receptor. It may be that the "tanned" red cells did not possess the receptor sites necessary for the ad-sorption of the fungus antigens, or a much higher concentration of antigen may have been needed for complete coverage of the red cells* surfaces. (d) Plate diffusion technique. This method also was found unsatisfactory.. No bands of precipitate appeared at any time during the 7 day period of observation. (e) Slide double-diffusion agar precipitin technique. This technique was satisfactory with some anti-sera but not with others. Its main advantage con-sists in the small amounts of materials needed. There are a few disadvantages which negated its use in the case of my experiments. Where more than 1 band of precipitate was obtained, there was no clear delin-eation between bands. Also, some of the antigens which showed a positive tube precipitin test produced no results with this technique. 33 . B, Final experimentation: 1. Rabbit immunization: (a) Series I: TABLE II. RESULTS OF RING PRECIPITIN TESTS USING ANTISERA  OF RABBITS IMMUNIZED WITH INCOMPLETE FREUND1S ADJUVANT  AND PARTIALLY PURIFIED ANTIGENIC MATERIAL ' .(AMMONIUM SULPHATE-FRACTIONATED). Number of rabbits Immunogenic material Titre 1 T» persicolor intracellular -2 Tfc persicolor extracellular 1/50 1/50 2 T» mentagrophytes #666 intracellular 1/10 1/20 1 T. mentagrophytes #666 extracellular 1/10 1 T.. mentagrophytes #675 intracellular 1/10 1 T. mentagrophytes #675 extracellular 1/50 2 T. mentagrophytes #687 extracellular 1/50 1/100 Readings were taken after 24 hours. According to the results obtained in this series, i t seemed that the extracellular antigenic substance was more immunogenic than the intracellular in a l l cases, except T., mentagrophytes #666 extracellular. It cannot be stated, however, that T. mentagrophytes #687 intracellular antigen was less immunogenic than its corresponding extracellular material. Single rab-34 bits were used only when there was insufficient material for the immunization of 2 animals. Since the immunizing material was standardized to contain 0.8 mg. of nitrogen per ml.,, the amount of nitrogen inoculated into each rabbit could not have influenced the results, except perhaps in the case of the rabbit receiving T. mentagrophytes #675 intracellular. This animal was not injected after the fifteenth week because of the lack of immunizing material. As too few animals were used in this series, no specific correlation of observations could be made from these results,, other than the fact that partially purified mold proteins are comparatively poor immunogenic materials when used in rabbits. Before the initiation of immunization, sera from a l l rabbits in this and subsequent series dem-onstrated negative precipitin reactions with the respective antigenic materials used for injection. Series 2: 35 TABLE III RESULTS OF RING PRECIPITIN* TESTS WITH ANTISERA  OF RABBITS BMUNIZED WITH INCOMPLETE FREUND'S ADJUVANT  AND MYCELIAL HOMOGENATES? AND TESTED WITH HOMOLOGOUS INTRACELLULAR AND EXTRACELLULAR ANTIGENS. Number of Rabbits Immunogenic material Titre Intracellular Extrac ellular antigenic substance antigenic substance 2. T.. mentagrophytes #666 l/IOO 1/1.00 -2. T. mentagrophytes #675 1/200 1/200 — 2. T. mentagrophytes #687 — 1/50 1/100 2. M. qulnckeanum #7 1/20 1/20 1/50 1/50 2. T. persicolor 1/100 1/10 — 2. T. mentagrophytes #675 pleomorphic 1/20 1/20 1/200 1/200 2. T. persicolor pleomorphic 1/50 1/20 1/50 1/10 Readings were taken after 24 hours. Titres of antisera tested with the intracellular antigens were expected to be higher than or comparable with those of the extracellular antigens, as the latter material was not injected into the rabbits. This does not appear to be the case with certain strains of these dermatophytes. Perhaps the extra-36, cellular antigen is not truly extracellular, but most-ly intracellular because of liberation of cellular con-tents into the medium during growth. If. such is the case,, i t seems peculiar that the other organisms which do not show high extracellular titres do not behave in a comparable manner. Since pleomorphism was initiated by growing the cultures at 37°C rather than at room temperature, the higher incubation temperature might have promoted more autolysis with the 1 strain, T.. mentagrophytes #675. This incubation temperature, however, did not affect, the other organism. T. persicolor. (c;) Series 3: TABLE IV; RESULTS OF RING PRECIPITIN TESTS WITH ANTISERA OF RABBITS IMMUNIZED WITH INCOMPLETE FREUND'S  ADJUVANT AND MYCELIAL HOMQflENATSS, AND TCTEITWITH  HOMOLOGOUS INTRACELLULAR AND EXTRACELLULAR ANTIGENS. No. of rabbits Immunogenic material I n t r -acellular antigenic substance Extra-cellular antigenic 1 substance 2 T. mentagrophytes #666 1/200 1/50 1/100 1/100 2 T. mentagrophytes #675 1/100 1/20 1/20 1/20 2 T. mentagrophytes #687 1/20 1/20 1/20 2 M* quinckeanum #7 1/400 1/400 1/20 1/50 1* M.. quinckeanum #8 1/50 -2 T. persicolor 1/100 1/200 1/20 Readings were taken after 24 hours at 6 C. * Another rabbit receiving M.. quinckeanum #8 died after the fourth week of injections.. 37 For the most part, t i t r e s obtained through the use of i n t r a c e l l u l a r materials were higher than those found with e x t r a c e l l u l a r materials. Upon comparison with Table I I I , i t may be seen that the same immunogenic agent produced di f f e r e n t r e s u l t s i n di f f e r e n t series of rabbits. Only with mycelial homogenates of T. persicolor and T. mentagro-phytes #675 were the results similar, though the de-grees of r e a c t i v i t y d i f f e r e d . In a l l other cases, the r e s u l t s appeared to be reversed, i . e . antisera which gave good t i t r e s with the e x t r a c e l l u l a r antigens i n one series showed poor t i t r e s with the same antigens i n the other series and vice versa. This may be ex-plained only on the basis that d i f f e r e n t l o t s of organisms were used i n the 2 series. Although the incubation time and the method of preparation of the antigenic materials were the same, s u f f i c i e n t d i f f e r -ences occurred to produce diverse r e s u l t s . Foster (18) stated that protein synthesis and content of molds were not constant, but even single strains fluctuated within wide l i m i t s , according to the c u l t u r a l conditions during growth. The re s u l t s given i n Table V seem to indicate that with c u l t u r a l conditions controlled as much as possible, great v a r i a -b i l i t y i n nitrogen content occurred. The t o t a l n i t r o -gen values might not, however, be indicative of 38 protein alone since the fungal cell contains many 'nitrogenous polysaccharides, nitrogen-containing lipids, coenzymes, vitamins, amino acids, purine and pyrimidine bases, amino sugars, (16), (17). TABLE V. TOTAL NITROGEN VALUES OF DIFFERENT LOTS OF  ANTIGENS OBTAINED DURING THE PERIOD BETWEEN SEPTEMBER 1959 AND MAY I960, Antigen Total nitrogen/20 mg. proteinaceous material (In mg.). T. mentagrophytes #675 intracellular 0.031 0.069 T. mentagrophytes #675 extracellular 0.074 0.102 T. mentagrophytes #675 pleomorphic intracellular 0.040 0.035 T. mentagrophytes #675 pleomorphic extracellular 0.014 0.516 0.093 M. quinckeanum #7 intracellular 0.268 0.419 M. quinckeanum #7 extrac ellular 0.117 0.013 T. persicolor pleomorphic intracellular 0.041 0.033 0.053 T. persicolor pleomorphic extracellular 0.065 The use of mycelial homogenates incorporated into modified Freund's adjuvant as antigens, in series 2 and 3, seemed to provide higher precipitin 3 9 t i t r e s with most antisera than did the use of the p a r t i a l l y p u r i f i e d i n t r a c e l l u l a r material i n s e r i e s l l . (a) Series 4; TABLE VI ANTISERA OF RABBITS IMMUNIZED WITH. PLEOMORPHIC  MYCELIAL HOMOGENATE AND TESTED WITH CQRRSS-PONDING NON-PLEOMORPHIC AND PLEOMORPHIC ANTIGENS. No. of rabbits Immunogenic material . (mycelial homogenate) In v i t r o antigen* Intra- Extra c e l l u - c e l l u -l a r a n t i -genic l a r a n t i -genic subs-stance subs-stance 2 T. mentagrophytes #675 pleomorphic T. mentagrophytes #675 non-pleomor-phic 1/20 1/20 -T. mentagrophytes #675 pleomorphic 1/20 1/20 1/200 1/200 2 T. persicolor pleomorphic T., persicolor non-pleomorphic 1/50 1/20 1/50 T. persicolor pleomorphic 1/50 1/20 1/50 1/10 Readings were taken after 24 hours at 6 C * Twice-precipitated with ammonium sulphate. Antisera of rabbits receiving injections of T. mentagrophytes #675 pleomorphic and T» persicolor pleomorphic Immunogenic material did not d i f f e r i n t i t r e s when tested with t h e i r corresponding pleomor-phic and non-pleomorphic i n t r a c e l l u l a r i n v i t r o a n t i -gens. However, there was a difference between the t i t r e s of antisera of rabbits immunized with T. men-tagrophytes #675 pleomorph'ico mycelial homogenate when 40 T.. mentagrophytes non-pleomorphic, and #675 pleomor-p h i c e x t r a c e l l u l a r test antigens were used. In a l l cases, the t i t r e s of the antisera cannot 'be considered high i n the l i g h t of Sharp's work (48), (49). But the re s u l t s as described here are analagous to those of most other workers (25), (30), (41).. The comparatively poor immunogenicity of mold proteins re-sulted i n low c i r c u l a t i n g antibody formation, which probably accounted f o r the low t i t r e s . 2. Cross-precipitin t e s t s . The results of the cr o s s - p r e c i p i t i n reactions (see Tables VII, VIII, IX, and X) indicated that the immuni-zing antigen did not necessarily provide the strongest reaction, and that a great deal of crossing occurred. This seems to support the view that there are common antig among the dermatophytes. I t i s in t e r e s t i n g to note that the M. quinckeanum i n t r a c e l l u l a r and ex t r a c e l l u l a r antigens cross-reacted with many of the T., mentagrophytes antisera, thus indi c a -t i n g common antigens between the Microsporum and Tricho-phyton genera. Antisera from i n d i v i d u a l rabbits d i f f e r e d i n the degree of cross - r e a c t i v i t y with the antigens. Some rabbits produced antisera which cross-reacted to a t i t r e of 1/100 or 1/200 with nearly a l l antigens tested (e.g. one of the rabbits immunized with M. quinckeanum #7, 41 one immunized with I", persicolor) • Other animals show-ed poor antibody titres with nearly a l l antigens (e.g. the rabbit immunized with M. quinckeanum #8, one immun-" ized with T. mentagrophytesv#687, and one with T. persi-color) . 3. First skin tests. Skin tests upon rabbits were carried out to deter-mine relationships between circulating and skin-reactive antibodies. The results are given in Tables XII, and XIV. Measurements of reactive sites were taken, but were not considered relevant. In some cases a 1+ red-dening reaction measured 24mm. while a 4+ necrotic reac-tion measured only 8 mm. The degrees of severity of the positive reactions differed (compare Plate II, Figure i and Plate II, Figure i i ) . Table XII shows that testing with the 5 materials, ammonium sulphate-precipitated T. mentagrophytes #675 pleomorphic intracellular, #675 intracellular, #675 extracellular, #687 intracellular, and T. persicolor pleomorphic intracellular antigens (see Plate I, Figure i ) , produced maximum positive reactions in al l rabbits tested. Thes-e positive results appeared as early as 10 minutes after injections were given (see Plate I, Figure i i ) . It was necessary to determine whether the reac-tions were due to the presence of strong antigen-anti-TABLE VII CROSS-PRECIPITIN REACTIONS OF ANTISERA OF RABBITS  IMMUNIZEITWITH VARIOUS DERMATOPHYTE ANTIGENS AND TESTED WITH PARTIALLY PURIFIED MATERIALS.I Test Tube Antigen Immunogenic Material (mycelial homogenate) T. mentagrophytes #666 T. mentagrophytes #687 Rabbit 1 Rabbit 2 Rabbit 3 , Rabbit h Rabbit 1 Rabbit i 2 Rabbit 3 T. mentagrophytes #666 intracellular 1/200 1/50 . 1/100 l/IOO 1/20 1/20 1/50 T. mentagrophytes #666 extracellular l A o o 1/200 1/50 1/50 1/50 1/50 1/100 ft mentagrophytes #675 intracellular - NT NT NT - NT NT T. mentagrophytes #'675 extracellular - 1/10 - 1/10 1/10 -T. mentagrophytes #675 pleomorphic intracellular 1/10 NT NT 1/50 1/20 - 1/50 T. mentagrophytes #687 intracellular NT NT- NT NT 1/20 T. mentagrophytes #687 extracellular 1/20 NT NT 1/10 1/20 i/?o 1/100 1/50 TY"persicolor 1/50 . 1/20 _1Z5D.__ _1/2Q _. _ mm mm _ . intracellular T. persicolor extracellular 1/10 - mm - - - -T. persicolor pleomorphic intracellular 1/100 NT NT 1/100 1/10 1/50 -T. persicolor pleomorphic extracellular 1/200 1/20 1/20 1/50 1/50 1/200 1/100 T. asteroides extracellular 1/20 1/20 MB 1/20 1/10 1/20 -T. radians extracellular 1/20 1/10 - 1/20 1/10 1/20 -M. quinckeanum #7 intracellular 1/100 NT NT i/5o 1/50 1/100 1/100 M. quinckeanum #7 extracellular 1/20 - - - 1/10 ' M. quinckeanum #8 intracellular 1/20 NT NT NT - - -M. quinckeanum #8 extracellular 1/20 1/20 - 1/10 - - -M. quinckeanum #1^ extracellular 1/20 - NT 1/10 1/20 - -NT - not tested because of lack of antigen. Readings taken between 3 hours-and ^8 hours. Concentration of antigen per tube - 0.025 mg. total.nitrogen. & Antigens twice-precipitated with ammonium sulphate. TABLE V I I I CROSS-PRECIPITIN REACTIONS OF ANTISERA OF RABBITS  IMMTOIZED"WITH VARIOUS DERMAT0PHYTIC ANTIGENS ANTT"fISTED * WITH PARTIALLY PURIFIED MATERIALS.A Te s t Tube A n t i g e n Immunogenic M a t e r i a l ( M y c e l i a l homogenate) T. mentagrophytes #675 T. mentagrophytes # 6 7 5 pleomorphic Rabbit 1 Rabbit 2 Rabbit 3 Rabbit 1 Rabbit 2 T. mentagrophytes # 666 i n t r a c e l l u l a r 1/100 1/20 1/50 1/100 1/10 T. mentagrophytes #666 e x t r a c e l l u l a r 1/100 1/100 1/20 1/20 1/10 T. mentagrophytes #675 i n t r a c e l l u l a r 1/100 1/20 1/200 M l 1/10 T. mentagrophytes #675 e x t r a c e l l u l a r 1/50 1/20 1/20 -T. mentagrophytes #675 pleomorphic i n t r a c e l l u l a r 1/10 1/20 1/10 1/100 1/20 T. mentagrophytes #687 i n t r a c e l l u l a r NT NT NT 1/20 -T. mentagrophytes #687 e x t r a c e l l u l a r 1/20 1/10 - 1/10 tma T. p e r s i c o l o r i n t r a c e l l u l a r V 5 0 1/100 1/20 1/50 1/200 T. p e r s i c o l o r e x t r a c e l l u l a r 1/10 - 1/20 1/50 . T. p e r s i c o l o r pleomorphic i n t r a c e l l u l a r 1/100 1/100 1/50 1/100 1/20 T. p e r s i c o l o r pleomorphic e x t r a c e l l u l a r 1/200 1/50 1/20 1/100 1/50 T. a s t e r o i d e s e x t r a c e l l u l a r 1/20 1/20 mm) 1/50 1/20 T. r a d i a n s e x t r a c e l l u l a r 1/20 1/20 - 1/20 1/20 M. quinckeanum #7 i n t r a c e l l u l a r 1/100 1/50 1/50 1/200 1/100 M. quinckeanum #7 • ' e x t r a c e l l u l a r 1/20 - 1/10 1/20 -M„ quinckeanum #8 i n t r a c e l l u l a r . 1/20 ** - 1/20 -M„ quinckeanum #8 e x t r a c e l l u l a r . 1/20 1/10 - 1/20 1/10 M. quinckeanum #1"} e x t r a c e l l u l a r 1/20 1/10 - 1/20 1/20 NT - not t e s t e d because o f l a c k of a n t i g e n . Readings taken between 3 hours and *f8 hours. C o n c e n t r a t i o n of a n t i g e n per tube - 0.025 mg. t o t a l n i t r o g e n . & Antigens t w i c e - p r e c i p i t a t e d w i t h ammonium sulp h a t e . TABLE I X CROSS-PRECIPITIN REACTIONS OF ANTISERA OF RABBITS  IMMUNIZED WITH VARIOUS DERMATOPHYTE ANTIGENS AND TESTED ; ' ' . ' WITH PARTIALLY PURIFIED MATERIALS.A Test Tube A n t i g e n Immunogenic M a t e r i a l ( M y c e l i a l homogenate) M. quinckeanum #7 •M. quinckea-num #8j my-c e l i a l foomo-genate ' Rabbit 1 Rabbit 2 Rabbit 3 Rabbit h Rabbit Ltft T. mentagrophytes #666 i n t r a c e l l u l a r 1/20 1/10 1/100 1/200 1/20 T. mentagrophytes #666 e x t r a c e l l u l a r 1/100 1/50 1/100 1/100 1/50 T. mentagrophytes #675 i n t r a c e l l u l a r 1/10 NT NT 1/100 -T. mentagrophytes #675 e x t r a c e l l u l a r 1/10 - 1/20 1/200 -T. mentagrophytes #675 pleomorphic i n t r a c e l l u l a r 1/50 1/50 1/50 1/100 -T. mentagrophytes #687 i n t r a c e l l u l a r NT NT NT 1/100 NT T. mentagrophytes #087 e x t r a c e l l u l a r 1/20 1/10 1/50 1/100 1/10 „ -i' u J • - -. , . -T. p e r s i c o l o r i n t r a c e l l u l a r 1/50 - 1/50 1/200 1/20 T. p e r s i c o l o r e x t r a c e l l u l a r 1/200 1/10 1/20 1/100 1/20 T. p e r s i c o l o r pleomorphic i n t r a c e l l u l a r 1/50 1/100 1/20 1/100 T. p e r s i c o l o r pleomorphic e x t r a c e l l u l a r 1/50 1/50 1/100 1/100 1/20 T. a s t e r o i d e s e x t r a c e l l u l a r 1/20 , 1/20 1/100 1/200 -T. r a d i a n s e x t r a c e l l u l a r 1/20 - 1/20 1/100 -M. quinckeanum #7 i n t r a c e l l u l a r 1/50 1/20 1/100 1/100 1/20 M. quinckeanum #7 e x t r a c e l l u l a r 1/50 1/10 1/50 ., 1/100 -M. quinckeanum #8 i n t r a c e l l u l a r 1/20 NT 1/100 1/50 M. quinckeanum #8 e x t r a c e l l u l a r 1 A 0 0 1 A 0 0 1/50 1/100 -M. quinckeanum #1"} e x t r a c e l l u l a r 1/20 1/10 1/20 1/100 -NT - not t e s t e d because of l a c k o f a n t i g e n . Readings taken between 3 hours and hB hours. C o n c e n t r a t i o n of a n t i g e n per tube - 0.025 mg. t o t a l n i t r o g e n . k A n t i g e n s t w i c e - p r e c i p i t a t e d w i t h ammonium s u l p h a t e . kk Rabbit 2 d i e d before f i n a l b l e e d i n g . TABLE X CROSS-PRECIPITIN REACTIONS OF ANTISERA OF RABBITS  IMMUNIZED WITH VARIOUS DERMATOPHYTE ANTIGENS AND TESTED  WITH PARTIALLY PURIFIED MATERIALS".A Test Tube Antigen Immunogenic Material (mycelial homogenate) T. persicolor T. persicolor pleomorphic Rabbit 1 Rabbit 2 1 Rabbit 3 1 Rabbit 1 Rabbit 2 T. mentagrophytes #666 intracellular - 1/20 1/10 1/10 1/100 T. mentagrophytes #666 extracellular 1/20 1/100 1/100 1/50 1/100 T. mentagrophytes #675 intracellular - NT NT mm 1/100 T. mentagrophytes #675 extracellular - - - 1/10 1/200 T. mentagrophytes #675 pleomorphic intracellular 1/10 mm 1/20 1/10 1/100 T, mentagrophytes #687 intracellular - NT NT 1/20 1/100 T, mentagrophytes #687 extracellular mm 1/10 - 1/10 1/200 T. persicolor intracellular 1/200 1/10 1/100 1/10 1/200 T, persicolor extracellular 1/20 - - 1/10 1/200 NT 1/50 T. versicolor _ j y i o o 1/200 1/100 intracellular T. persicolor pleomorphic extracellular 1/50 NT 1/100 1/20 1/200 T. asteroides extracellular 1/10 mm - - 1/200 T. radians extracellular - 1/10 - - 1/200 M0 quinckeanum #7 intracellular 1/100 1/100 1/100 1/20 1/100 M. quinckeanum #7 extracellular - - 1/20 - 1/200 M. quinckeanum #8 intracellular mm - - 1/10 1/100 M. quinckeanum #8 extracellular - - - 1/10 1/100 M, quinckeanum #13 CJ extracellular — - - 1/20 1/100 NT - not tested because of lack of antigen. Readings taken between 3 hours and k-8 hours. Concentration of antigen per tube - 0.025 mg. total nitrogen. k Antigens twice-precipitated with ammonium sulphate. 46 body r e a c t i v i t y in. the tissues,, to toxic substances, or to high sodium chloride concentrations i n the antigenic material.. In order to maintain equivalent t o t a l nitrogen i n a l l skin tests, i t was found necessary to use larger amounts of the l y o p h i l i z e d preparation i n cases of low nitrogenncontent. This resulted i n a r e l a t i v e l y high Sodium chloride concentration.,, due to the conditions of d i a l y s i s during preparation, especially i n the cases of pleomorphic antigenic material,, T. mentagrophytes #675 i n t r a c e l l u l a r and ex t r a c e l l u l a r and #687 i n t r a c e l l u l a r (see Table XI). Preliminary experiments with alcohol-precipitated antigenic material showed that the removal of sodium chloride eliminated the reaction i n the cases of T. men- tagrophytes #675 i n t r a c e l l u l a r and #687 intracellular,,. and T.. persicolor pleomorphic i n t r a c e l l u l a r . With T. men- tagrophytes #675 ext r a c e l l u l a r , a s l i g h t reaction occurred with the undiluted material. These r e s u l t s pose a question. Did the alcohol f a i l to prec i p i t a t e a toxin or did the a l -cohol modify certain antigenic groupings which occurred i n the ammonium sulphate-precipitated material? Of the 5 antigens which produced maximum reactions t as shown i n Table XII, only one showed a positive reaction after alcohol p r e c i p i t a t i o n (see Table XIV). The sodium chloride concentration of the other four may have produced a 47 TABLE XI REPRESENTATIVE VALUES FOR TOTAL NITROGEN OF TWICE-PRECIPITATED AMMONIUM. SULPHATE ANTIGENIC MATERIAL FROM DIFFERENT STRAINS, Antigenic Material Mg. nitrogen /20 mg. of proteinaceous.material T. mentagrophytes #666 intracellular 0.^43 T. mentagrophytes #666 extracellular 0.234 T. mentagrophytes #675 pleomorphic intracellular 0.035 T. mentagrophytes' #675 intracellular 0.031 T. mentagrophytes #675 extracellular 0.074 T. mentagrophytes #687 intracellular 0.060 T. mentagrophytes #687 extracellular 0.316 T. persicolor intracellular 0.369 T. persicolor extracellular 0.226 T. persicolor pleomorphic intracellular 0.033 T. persicolor pleomorphic extracellular 0.065 M. quinckeanum #7 intracellular 0.117 M. quinckeanum #7 extracellular 0.268 M. quinckeanum #8 intracellular 0.117 M. quinckeanunn #8 extracellular 0.193 M. jquinckeanum #13 extracellular 0.716 T. asteroides extracellular 0.147 T. radians extracellular 0.437 48 hemorrhagic cutaneous effect. . In the second series, this reaction did not occur, probably due to the absence of the salt. The reaction did occur with T. mentagro-phytes #687 intracellular, thus indicating that a toxic effect was produced by this antigen, and that this material may have been a toxin which was precipitable by alcohol. i Observation of results obtained from skin testings of immunized rabbits with alcohol-precipitated and ammon-ium sulphate-precipitated antigens, and non-immunized controls, revealed the following: :-.)(a) M. quinckeanum #13 extracellular produced posi-tive reactions in nearly a l l immunized rabbits (see Tables XII and XIV), but few or none at a l l in non-immunized controls (see Table XIII).. (b) Polysaccharides of Nbcardia asteroides, T. gran-ulosum, and Candida albicans produced no skin reactions in both the immunized (see Table XIV) and non-immunized animals (see Table XIII). Cc) The toxic reactions of alcohol-precipitated T.mentagrophytes #687 intracellular and extracellular were negligible in non-immunized rabbits (see Table XIII), but positive in nearly a l l Immunized rabbits (see Table XIV). The latter reactions were observed as early as 10 minutes after injection of the skin test antigen. (d) The alcohol-precipitated antigens of M. quinckeanum TABLE XII SKIN REACTIONS OF RABBITS IMMUNIZED WITH VARIOUS DERMAT0PHYTIC ANTIGENS AND TESTED WITH PARTIALLY PURIFIED MATERIALS £ - 2M- AND M-8 HOUR READINGS COMBINED. Skin Test Antigen Immunogenic Material (mycelial homogenate) T. mentagrophytes r r. persicolor - M.quin-cknea-num #666 #675 #675 pleo-morphic #687 pleo-morphic #7 Rab 1 bits 2 Rabl 1 oits 2 Rabl 1 bits 2 Rab 1 bits 2 Rab 1 bits 2 Rab 1 bits , 2 Rabl 1 J i t s 2 T. mentagrophytes #666 intracellular 2 + R 2 + R T. mentagrophytes #666 extracellular if + NH 4-4 N - - - - 3 + H 3 + H 1 + R 3 + H - - - 34-H T. mentagrophytes #675 intracellular >f + N 4- + N 4-4 N if 4-N 3 + N 4-4-N h+ N 4-4-N if 4-N if + N 3 + N 3 + N 3 + N 4-4-NR T. mentagrophytes #6!75' extracellular h¥ N 4-4 N 4- 4 N 1+ 4 N 3 + NR 3+-N 4-4 N 4-4-N 4-4 N 4-4-N - 3 + N 3+ N 3+ N T. mentagrophytes #675 pleomorphic intracellular if 4 N 4-4 N If + N h 4 N 4- +• N 4- + N if 4 N if +-N 1+ + N if 4-N 3+-N 3 + N 3 + N 3 + N T. mentagrophytes #687 intracellular h 4 N 4- + N 1+ 4-N If 4 N 4- + N 4- + N 4- + N 4-4 N 4-4-N if + N 2 4 N 4-4 N if + NR if + NR T. mentagrophytes #687 extracellular 2+-H 3 + H 2 + H k + H - - 3 + H 4-4-N 4-4-. N 4-4-N - mm - 3t H T. persicolor intracellular - - - - - - - 1 + R - - - - -T. persicolor extracellular if + N if 4-N 4-4 NH If 4-N - 24-R 4-f N N 4-4-N if+• N -N - --T. persicolor pleomorphic - -intracellular 4-4 1 4--+-N 4-4 N k + N 4-4-N 3 + N 4- + N 2 4-H 14-R 2 + H 3 + N 3 + N J 4 _ N _3_4^ N T. persicolor pleomorphic extracellular 3 + H 3 + N 2-f H 3 + N 3 + NR 2 + R mm 1 + R if + N 2 + N 34-N 3+ N 2 + N T. asteroides extracellular - • 2 + H 24 H - - 3 + H mm 1+ R 23-H 24 H 14 R 34 NR T. radians extracellular 3 + H h + H 3 + H 3 + H 2 + H - 4-4-H 3 + NR 2+ R 2 4-H - - - 24 H M. quinckeanum #7 intracellular 4- + N 2 4-H ^ + NH 4- + NH 24-R - 4- + N 4-4-:N - - - - 24-N M. quinckeanum #7 extracellular 4-+-N 4- 4 N >+ + NH 4- + NH - - 4-4-N 4-4 N if 4 N 2 4-R - 14-R - 4-4-H M. quinckeanum #8 intracellular 2 + H 2 + H 2 + R 4- + H 3 + NR 3 + NR 3 + NH 3 + H - 2+ N - 24-N 3 + N 2+-NH M. quinckeanum #8 extracellular 3 + N 4- + N - 3 + NH 3+ ::R 24-NR 2+ NR 3 + N 1+ R 2 + H - 24-R - 24-H M. quinckeanum #13 extracellular 3 + H 4--+-H 3 + H 3 + H 3 + H 2 + R 4-4 H -3 + H 1 + R 3 4 H - 14-R 24-R 3 + H - 83 no reaction, N = necrosis, R » redness, H = hemorrhage. 4-+- • I t - a severity of reaction, (see Plate II , Figure i , for various ranges of reactions. For close-ups, see Plate III, Figure i ) . k Antigens twice-precipitated with ammonium sulphate. TABLE XIII SKIN REACTIONS OF CONTROL (NON-IMMUNIZED) RABBITS TESTED WITH PARTIALLY PURIFIED MATERIALS-24 AND M-8 HOUR READINGS. COMBINED. Skin Test Antigen GROUP A GROUP B Tested with' ammonium sulphate-precipi- Tested with alcohol-tated antigens precipitated antigens 1 2 3 labbit Numbe] ,5 6 7 1 Rabc 2 )it Num 3 * ber . T. mentagrophytes #666 intracellular - - mm - - - - - - - -T. mentagrophytes #666 extracellular - - - - - 1 + R 1 + R M i 1 + R -T. mentagrophytes #675 intracellular N 3 + N 3 + NR - 3 + N 3 + NR h + NR 1 + H - - -T. mentagrophytes #675 extracellular h + N >* + N -' 3 + N 3 + NR »* + NR - - - -T. mentagrophytes #675 pleomorphic intracellular h + N h + N 3 + NR 3 + NR 3 + NR 3 + NR 3 + NR 1+-R 1 + R T. mentagrophytes #687 intracellular h + N h + N h + NR 4 + NR 3 + H k + NR - 1 + H - 1 + R T. mentagrophytes #687 extracellular - - - - - 1 + R - - - -T. persicolor intracellular - - - - - - . - - - -T. persicolor extracellular. h + . N « a - • mm - - - - - -N N 3 + NR 3 t NR 2 + R 2 + R T-. persicolor pleomorphic intracellular 3 + NR " 3 + NR 3 + NR T. persicolo  pleomorphic extracellular k + N 2 + R 3 + NR 3 + NR ** 2 t R 2 + R 1 f 1 R M i T. asteroides extracellular - - - - - - - - - - -T. radians extracellular 1 + R - M l - - - - -M. quinckeanum #7 intracellular r mm} r - - -M. quinckeanum #7 extracellular h + N 1 + R - - - - M T - - -M. -quinckeanum #8 intracellular - 2 f N 2 + R 3 + N - 3 + NR 3 + NR M, quinckeanum #8 extracellular 3 + H - 1 + R M l - mm • - - - -M. quinckeanum #13 extracellular 1+ R 2 4 R mm - - - 1 + R - - M l T, mentagrophytes* #675 intracellular - - - -T. mentagrophytes* #675 extracellular M l 1 + R 1 + R Polysaccharides A Nocardia asteroides - mm. -T. granulosum - - -Candida albicans - - . - -- = no reaction, N = necrosis, R » redness,. H = hemorrhage, *f+ —>1+ = severity of reaction, k Dialyzed against water (contain no sodium chloride and are not alcohol-precipit. A Supplied by Dr. Blank. TABLE XIV SKIN REACTIONS OF RABBITS IMMUNIZED WITH VARIOUS  DERMATOPHYTE ANTIGENS AND TESTED WITH ALCOHOL PRECIPITATED ANTIGENS - 24 AND 4bHiOUR READINGS COMBINED. Skin Test Antigen Immunogenic Material (mycelial homogenate) T. mentagrophytes M. quinckeanum T. persicol-#666 #7 or Rabbit 1 Rabbit 2 Rabbit 1 Rabbit 2 Rabbit 1 k T. mentagrophytes #666 intracellular 2 4 R 1 + H -H -T. mentagrophytes #666 extracellular k 4 H k + NH 2 4 -H 3+-H -T. mentagrophytes #675 intracellular — - - - — T. mentagrophytes #675 extracellular - - - M i T. mentagrophytes #675 pleomorphic intracellular T. mentagrophytes #687 intracellular h + NR h 4 -N h + N k +-N N T. mentagrophytes #687 extracellular h + H h 4 NH k + H h + H -T. persicolor intracellular 1 4 R — - — — T. persicolor extracellular 1 + R - - 1 + H -"1. persicolor " ' ' ' -Pleomorphic •"" - _ Intracellular 1 4 " R - 2 4 : -H T. p e r s i c o l o r pleomorphic extracellular 1 + R CM mm T. asteroides extracellular 1 4 H 1 + H 3 + H -T. radians extracellular 1 4 R •a* - 2 + H -Mi. quinckeanum #7 intracellular 1 4 R - 1 + H 2 + H mm M. quinckeanum #7 extracellular 2 4 R — 2 4 H 2 4 -H -M. quinckeanum #8 extracellular - -• > + 4 -H 3 + H 1 + R M. quinckeanum #13 extracellular 3 4 -H 1 4 R k 4 H k + H 1 4 R T. mentagrophytes #675 intracellular 2 4 H mm 1 + R - -T. mentagrophytes Ai #675 extracellular 1 _ 3 + N - 3 + H 1 4 R Polysaccharides Nocardia asteroides - - - M l mm T. granulosum - - - - -Candida albicans - - nm - mm - 3 no reaction:,: N » necrosis, R = redness, H * hemorrhage, 4+ —>1+ = severity of reaction. k Rabbit 2 died before final bleeding. kk Dialyzed against water (contain no sodium chloride and are not alcohol-precipitated). 52 #7 from intracellular and extracellular sources, reacted more intensely with the sera of rabbits immunized with #7 mycelial homogenate than with the sera of rabbits immunized with T. mentagrophytes #666 and T. persicolor Csee Table XIV). ; (e) In the case of T. mentagrophytes #675 extra-cellular antigen, i t seemed that alcohol-precipitation resulted in the loss of a toxic factor. Injections of material dialyzed against water gave a positive reac-tion in 3 out of 5 rabbits (see Table XIV), whereas the alcohol-precipitated material produced negative skin reactions in a l l 5 animals. (f) In general, the rabbits tested with the alcohol precipitated antigens reacted to a lesser degree (see Table XIV) than did animals tested with ammonium sul-phate-precipitated antigens (see Table XII). Results of skin tests on rabbits infected with T. granulosum are given in Table XV. Individual rabbit gave varying degrees of reactivity to the skin test materials. The 3 M.. quinckeanum species produced pos-itive reactions upon skin testing. Polysaccharides elicited no responses, even the homologous one. Rabbit infected with one species of dermatophyte exhibited cross-reactivity with many different .:species as demon-strated by skin tests. 53 T. mentagrophytes #675 extracellular antigen, which was dialyzed against water, contained no sodium chloride, and was not alcohol-precipitated, produced positive results in 2 out of 4 rabbits. The corres-ponding alcohol-precipitated antigen was negative in a l l 4 rabbits. TABLE XV SKIN REACTIONS OF RABBITS INFECTED KTH T. GRANULOSUM AND TESTED WITH ALCOHOL-PRECIPITATED ANTIGENS - 24 AND 48 HOUR READINGS COMBINED. Skin Test Antigen Rabbit Number 1 2 3 h T. mentagrophytes ^666 intracellular - - - 1 + R T. mentagrophytes #666 extracellular - 1 4 R - -T. mentagrophytes #675 intracellular — - 1 + R T. mentagrophytes #675 extracellular - - an -T. mentagrophytes #675 pleomorphic intracellular 1 4 R mm 1 + R T. mentagrophytes #687 intracellular - - - 1 + R T. mentagrophytes #687 extracellular 1 4 R -T. persicolor intracellular ' — — - 1 + R T. persicolor extracellular - - 1 4 R -T. persicolor pleomorphic intracellular - 1 +-R - 1 + R ' T . persicolor pleomorphic extracellular 1 4 R I f R T. asteroides extracellular 1 4 R - mm -T. radians extracellular - - - -M. quinckeanum #7 intracellular 1 4 -R 1 4 R - 1 -f R M. quinckeanum #7 extracellular - 1 4 R - 1 + R M. quinckeanum #8 extracellular 1 4-R 1 4 R - 1 + R M. quinckeanum #13 extracellular 1 + R 1 4 R - 1 + R T. mentagrophytes #675 intracellular k — - -• -T. mentagrophytes #675 extracellular k - - 2 + R 1 4 R Polysaccharides Nocardia asteroides -- - -T. granulosum - - - -Candida albicans - - - -Skin tests were carried out 36 days after the animals were experimentally i n -fected, during the convalescent stage. - = no reaction, R = redness, 4 4 — K L 4 = severity of reaction. k Dialyzed against water (contain no sodium chloride, and are not alcohol-precipitated) • 55 PLATE I F i g u r e i i : Showing n e c r o t i c areas which sometimes appeared as e a r l y as 10 minutes a f t e r the i n j e c t i o n s were given. F i g u r e i i : L ess severe degrees o f r e a c t i o n s . (Compare wit h F i g u r e i ) . 57 58 PLATE IV Fi g u r e i : S k i n r e a c t i o n s o f a r a b b i t immunized w i t h T. mentagrophytes #666 m y c e l i a l homogenate to 2 d i f f e r e n t antigens. 1-M. quinckeanum #7 i n t r a c e l l u l a r , 2-T. mentagrophytes #687 e x t r a c e l l u l a r . (Compare with F i g u r e i i ) . F i g u r e i i : S k i n r e a c t i o n s o f a r a b b i t immunized with T. p e r s i c o l o r m y c e l i a l homogenate to 2 d i f -f e r e n t antigens. 1-M. quinckeanum #7 i n t r a -c e l l u l a r , 2-T. mentagrophytes #687 e x t r a c e l l u l a r . 59 4. Second skin tests* At different time intervals, a second injection was given near the site of the f irs t in order to observe whether (I) the f irs t injection showed any change under this condition, and (2) whether the results of the second injection were lesser, equal to, or more severe than- that of the f i rs t . The results of second skin tests on con-trols are given in Table XVI. There was no "flare-up" or increase in degree of reactivity of the f irs t injection sites after second in-jections. As may be seen from Table XVI, the second injection;: did not necessarily produce a greater response than the f i rs t ; In fact, there were some cases where the second injection was negative i . e . , a possible demon-stration of desensitization. In some cases there appear-ed to be a sensitization i . e . , the f irs t injections gave no response but the second injections were positive. In other instances, neither sensitization nor desensitiza-tion occurred e.g., in rabbits tested with alcohol-precipitated T. mentagrophytes #675 extracellular an-tigen and with 4 out of 5 rabbits tested with ammonium sulphate-precipitated T. mentagrophytes #666 Intracellular antigen. The amount of time elapsing between f irs t and second injections did not seem to influence the intensity or the presence or absence of the second reaction. TABLE XVI RESULTS OBTAINED IN WON-IMMUNIZED CONTROL RABBITS ON FIRST  AND SECOND INJECTIONS OF SOME OF THE SKIN TEST ANTIGENS. GROUP A GROUP B Skin Test Antigen Tested .with ammonium sulphate- Tested with alcohol-precipitated antigens. precipitated anti-gens. Rabbit 3 Rabbit Rabbit 5 Rabbit 6 Rabbit 7 Rabbit 1 1-Rabbit 2 Rabbit 3 1° 2° 1° 2° 1° 2° 1° 2° 1° 2° 1° 2° 1 1° 2° 1° 2° T. mentagrophytes #666 intracellular - - 2+ N - - mm - . - - -T. mentagrophytes extracellular 1+ R 3 + H - 3 + H 1+ 1:R 1 + R T. mentagrophytes #675 extracellular T. mentagrophytes #675 pleomorphic intracellular 3+ NR 3 + NR-3+ .NR 3 + NR 3 + NR 3 + N 3 + NR 2 + R 3 + NR 3 + NR T. mentagrophytes #687 extracellular - It R - 2 + R - 1 + R T. persicolor intracellular 1+ R T. persicolor extracellular - 1+ R - - - 2 + R T. persicolor pleomorphic intracellular 3 + NR 3 + NR 3 + NR 3 + NR 3+ NR 3 + NR 3 + NR 3 + NR 3 + NR 2 + R T. persicolor pleomorphic extracellular 2+ R 3 + NR 3 + NR mm 3 + NR 1 + R 3 + NR 2 + R 1 M. quinckeanum #8 intracellular 2+ R - 3 + NR 2 + NR 3 + N 3 + NR - 4 + NR 3 + NR 3 * NR Mo quinckeanum #8 extracellular mm 2 + NR - - 1+ R - - 2 + R - 1 + R M. quinckeanum #13 extracellular 1+ R 2 + R - 2 + R mm 3 * R T. mentagrophytes k #675 extracellular - 2i R 1+ tR 1 + R 14 :-R 1+-R k Dialyzed against water (contains no sodium chloride, and is not alcohol-precipitated). 1° - f i rs t skin test. 2° - second, skin test. Group A, rabbits 3 and *f - second injections administered k-8 hours after f i r s t . Group A, rabbits 5? 6 and 7, and Group B - second injections administered 1 week after f i r s t . 6 1 Results of second injections of certain antigens on immunized rabbits are given in. Table XVII. TABLE XVII, RESULTS OBTAINED IN. IMMUNIZED RABBITS ON FIRST AND SECOND INJECTIONS OF SOME OF THE SKIN. TEST ANTIGENS. Skin Test Antigen, Immunogenic Material (mycelial homogenate) T. mentagro- M. ouinekea-Dhvtes #666 num #7 T. ner-sicolor Rabbit 1 Rabbit 2 Rabbit. 1 Rabbit 2 Rabbit 1 1 ° 2 ° 1 ° 1 ° 2 ° jo 2 ° 1 ° 2 ° T. mentagrophytes #666 extracellular 44 H 44 H 44-NH: - 2+ H. 34-H 34 H - •• - 44 H T. mentagrophytes #675 extracellular — — — — — — T. mentagrophytes #687 extracellular 4+ H 2+ H: 44 N H — 4+ H 4 + 44 K 44 H — 2 4 R T. persicolor intrac ellular 1+ R-24-H: -- 3 + H — — — 2 4 H. - 2 + R T„ persicolor extracellular, 1+ R 1 4 R ... — — — — 14 H: 2 4 H — 3 4 R M. quinckeanum #13 extracellular 3 4 R 44 H 14 R 24-H 4+ H 44-E 4+ H 44-H 14 R 3 + R T.: mentagrophytes* #675 extracellular — 3 4 H 3 4 N - - 2+ NR 3 4 H: 44 H 1 4 R 3 4 R * Dialyzed against water (contains no sodium chloride and is not alcohol-precipitated). 1 ° - f i rst skin test. 2 ° - second skin test. Second injections administered 1 week after f i rs t . Results were again obtained which may be likened to desensitization. and sensitization. It is interesting to note that the rabbit immunized with T. persicolor gave consis-tently higher readings upon second injections, except in the case of T. mentagrophytes #675 extracellular antigen, than did the other rabbits. 62 M. quinckeanum #13 extracellular material seemed to sensitize the skin so that a second injection of the same antigen, caused a greater reaction than the f i r s t . 6 3 V. GENERAL DISCUSSION. C e r t a i n o b s e r v a t i o n s obtained d u r i n g the course of t h i s p r o j e c t have been d i s c u s s e d i n the Experimental R e s u l t s . I n t h i s i n v e s t i g a t i o n of the immunology of v a r i o u s dermatophytes, immunogenic m a t e r i a l s of a proteinaceous na-t u r e were used as a n t i g e n s . These m a t e r i a l s were s e p a r a t e l y obtained from an i n t r a c e l l u l a r source i . e . , the m y c e l i a l mat, and from an e x t r a c e l l u l a r source i . e . , the l i q u i d c u l -t u r e medium i n which the organism had grown. P r e l i m i n a r y experimentation demonstrated t h a t d i f f e r -ent c u l t u r e media produced marked d i f f e r e n c e s i n the r a t e and the amount of growth of dermatophytes. Keeney"s medium was chosen as i t p r o v i d e d the most s u r f a c e growth i n the s h o r t e s t time. S i n c e t h i s medium co n t a i n e d no p r o t e i n , any proteinaceous m a t e r i a l found i n the l i q u i d must have been s y n t h e s i z e d by the mold growing i n pure c u l t u r e . A method f o r the p a r t i a l p u r i f i c a t i o n of proteinaceous m a t e r i a l from an i n t r a c e l l u l a r m y c e l i a l source was developed through a m o d i f i c a t i o n of methods used by other i n v e s t i g a -t o r s (25), (^ 1)> C+8). A m o d i f i c a t i o n of Keeney and E r i k s e n 1 s (30)method f o r the p a r t i a l p u r i f i c a t i o n of e x t r a c e l l u l a r p roteinaceous m a t e r i a l was used t o o b t a i n t h i s a n t i g e n i c substance. S i n c e the nature of the n i t r o g e n - c o n t a i n i n g ma-t e r i a l present i n the ammonium s u l p h a t e - p r e c i p i t a t e d a n t i -gens was unknown, p r e v i o u s l y washed f i l t e r i n g s u r f a c e s were 64 used d u r i n g the i n v e s t i g a t i o n , because u n t r e a t e d S e i t z pads have been shown t o produce a l t e r a t i o n and l o s s of c e r t a i n p r o t e i n s (46). The d i f f e r e n c e s between the v a r i o u s s p e c i e s and s t r a i n s of dermatophytes employed i n t h i s p r o j e c t were not pronoun-ced except t h a t they d i f f e r e d i n c o l o r and i n type of growth i . e . , f l u f f y or g r a n u l a r . The growth p e r i o d s were s i m i l a r , except f o r one s p e c i e s , T. p e r s i c o l o r . which r e q u i r e d about 2 weeks longer, than the o t h e r s . T o t a l n i t r o g e n determinations of the i n t r a c e l l u l a r and e x t r a c e l l u l a r a n t i g e n i c m a t e r i a l of a l l s p e c i e s and s t r a i n s v a r i e d i n value from one l o t to another (see T a b l e V), although c u l t u r a l c o n d i t i o n s were c o n t r o l l e d as much as p o s s i b l e . I n n e a r l y a l l cases, however, the e x t r a c e l l u l a r p a r t i a l l y p u r i f i e d substance had a h i g h e r t o t a l n i t r o g e n content than the i n t r a c e l l u l a r product. Some of t h i s n i t r o -gen may have come from the m y c e l i a l mat f o l l o w i n g a u t o l y s i s , but i t i s improbable t h a t a u t o l y s i s was the s o l e cause. As r e p o r t e d i n the Exp e r i m e n t a l R e s u l t s (see page 3 9 ) , t h e r e was a d i f f e r e n c e between t i t r e s of a n t i s e r a of r a b b i t s immunized w i t h T. mentagrophytes #675 pleomorphic m y c e l i a l homogenate when t e s t e d w i t h T. mentagrophytes #675 non-pleomorphic and w i t h #675 pleomorphic e x t r a c e l l u l a r i n v i t r o a n t i g e n s . Perhaps t h i s may be e x p l a i n e d on the b a s i s of pleomorphism. F o s t e r (19) s t a t e d t h a t about 1/2 of the amino a c i d content of the my c e l i a was t r a n s f e r r e d t o spores upon 65 their formation. Assuming that the amino acids referred to were present as protein, then i t might be envisaged that the intracellular protein of the non-spore-forming pleomorphic strain not required for spore-formation,, might be passed through the mycelial wall. If this is the case, then the increased antigenicity of the extracellular preparation from the pleomorphic organism could be explaimed simply on the basis of a greater content of originally intracellular immu-nogenic: material. Tests on antisera of rabbits immunized with T. persi-color pleomorphic mycelial homogenate demonstrated l i t t l e difference in titres when both T. persicolor non-pleomorphic and pleomorphic extracellular antigens were used. In a non-pleomorphic culture of this species, macroconidial formation is sparse, and therefore a significant amount of protein would not be diverted for spore-production. It may be expected in this example, that the non-pleomorphic culture might contri-bute an amount of extracellular protein approaching that formed with the pleomorphic variety. The partially purified ammonium sulphate-precipitated immunogenic material, in combination with incomplete Freund's adjuvant, produced rabbit antisera titres ranging from 1/10 to 1/100. The extracellular substance appeared to possess more immunogenicity in most cases, except perhaps for the T. mentagrophytes #666 extracellular material. Subsequent investigations showed that mycelial homogenates, with the 66 use of incomplete Freund's adjuvant, on the average, pro-duced slightly higher antisera titres than did the partially purified antigens. These results may, however, be due to the larger amount of nitrogen injected into the rabbits, along with the use of a shorter immunization period* As these antibody concentrations are approximately the same as those reported by other workers (30), (41) who did not employ Freund's adjuvant,, the use of this material does not seem to enhance antibody formation to mold antigens. In this project, Hanks' precipitin technique appeared to be the best, as compared with 3 other methods', for the in vitro determination of circulating antibodies. The com-plement fixation reaction was not used as other investigators have reported conflicting observations (33), (41), (47). Results reported in this thesis confirm the view that proteinaceous substances from molds are poor antigens in stimulating the formation of circulating antibodies. This may be due to an inherent characteristic of mold,antigens as yet unknown, or to the low total nitrogen content of fungal proteinaceous materials (see Table V), or to the possibility that some of the nitrogen may not be in the form of protein (16), (17). The results of cross-reactions using the precipitin test confirm prior reports that the dermatophytes possess many antigens in common. The intensity of some of these cross-reactions may, in part, have been due to the use of a com-67 paratively long course of immunization. This may have pro-duced a broadening of antibody-specificity as has been demon-strated with molds (.49) as well as with other antigens (24), (51), (53). As the results of the precipitin, tests were inconclusive, intracutaneous skin tests were performed on immunized and non-immunized rabbits. These tests with partially purified pro-teinaceous materials revealed that normal rabbits differed markedly in their degree of reactivity to such antigens. The animals may have become sensitized to certain species of dermatophytes during their l i fe or they may have reacted to non-dialyzable protein-precipitable toxic principles from these fungi. Immunized rabbits differed in the nature of their res-ponse to partially purified proteinaceous materials acting as skin test antigens. Some materials produced a reaction which could be observed within 10 minutes to 1 hour, and could be referred to as an immediate or anaphylactic type of response. Others produced results which were only obser-ved 24, 48, or 96 hours after administration. This delayed response may be likened to a tuberculin reaction. Cross-reactivity to many antigens were observed in nearly a l l Immunized rabbits. The results of the precipitin tests further demonstra-ted the differences in individual rabbits. Animals which re-ceived the same immunizing material differed .in the amount of 68 c i r c u l a t i n g antibodies produced, and als~b i n the degree of cro s s - r e a c t i v i t y of t h e i r antisera. Boyd (6) has stated that the role of animal i n d i v i d u a l i t y was important i n deter-mining the heterogeneity of antibodies which could be produced. Re-precipitation of the p a r t i a l l y p u r i f i e d proteinaceous material of some molds with equal amounts of 95% alcohol, altered the nature of the skin r e a c t i v i t y of these substances when used as antigens. With some stra i n s and species, toxic p r i n c i p l e s and/or antigenic factors were l o s t ; i n others, there wasvno apparent l o s s . For example, alcohol-precipita-t i o n seemed to r e s u l t i n the loss of a toxic factor i n the case of T. mentagrophytes #675 extracellular. This antigen produced no skin reactions i n rabbits (see Tables XIV and XV). Of nine animals used, 5 gave positive r e s u l t s when tested with the ammonium sulphate-precipitated material dialyzed against water and containing no sodium chloride. .Partially p u r i f i e d polysaccharides from Nocardia aster-oides, Candida albicans, and T. granulosum were employed as skin test antigens oil 13 rabbits. There were no positive reactions. T. granulosum polysaccharide did not produce a reaction on rabbits infected with T. granulosum.. Three species- of M. quinckeanum demonstrated strong i n v i t r o and irt vivo cross-reactions with various strains and species of the genus Trichophyton. These l a t t e r r e s u l t s support the view (3) that the c l a s s i f i c a t i o n of the various genera of dermatophytes should be based primarily on morphological c h a r a c t e r i s t i c s 69 and on c l i n i c a l f i n d i n g s . The s k i n r e a c t i o n s o f r a b b i t s which had been, i n f e c t e d e x p e r i m e n t a l l y w i t h T. granulosum were not as severe as those o f r a b b i t s immunized w i t h m y c e l i a l homogenates. S ince p o s i t i v e r e a c t i o n s d i d o c c u r , t i s s u e - b o u n d a n t i b o d i e s were presumably p r e s e n t , o r an a l t e r e d c e l l u l a r r e a c t i o n had taken p l a c e as a r e s u l t o f the i n f e c t i o u s p r o c e s s . Other workers have demonstrated t h a t immunity or at l e a s t p a r t i a l immunity to the homologous organism was observed i n humans and guinea p i g s i n f e c t e d or t r e a t e d w i t h dermatophytes. Keeney and Huppert (31) a p p l i e d T. mentagrophytes t o p i c a l l y to guinea p i g s and found t h a t an i n c r e a s e d r e s i s t a n c e to I n f e c t i o n w i t h the homologous fungus o c c u r r e d . The same workers (32) t r e a t -ed 1 human v o l u n t e e r s w i t h an' ointment c o n t a i n i n g T.. mentagro-p h y t e s . A f t e r 4 weeks, the v o l u n t e e r s were cha l lenged w i t h t h i s organism and were found to be p a r t i a l l y r e s i s t a n t . Friedman and Derbes (.21) observed humans who had become n a t -o i r a l l y i n f e c t e d w i t h M. a u d o u i n i , and came to the c o n c l u s i o n t h a t the ones who escaped subsequent i n f e c t i o n were r e s i s t a n t as a r e s u l t o f a p r e v i o u s i n f e c t i o n . They d i d not know whether the immunity observed was due to an undetected a n t i -body or to a s p e c i f i c and p r o t e c t i v e c e l l u l a r a l t e r a t i o n i n -duced by an I n f e c t i o u s agent. Second i n j e c t i o n s of s k i n t e s t a n t i g e n i c m a t e r i a l made at d i f f e r e n t i n t e r v a l s a f t e r the f i r s t i n j e c t i o n s , a l s o p r o -duced d i v e r s e r e s u l t s . These r e a c t i o n s appeared to depend upon 70 the nature of the antigens, and the strain or species with which the rabbit had been immunized. In some cases, reac-tions similar to desensitizations occurred; in other'Cases, an increased ^hypersensitivity was observed. No correlation could be made between the results of precipitin tests and those demonstrated by skin test reac-tions, with materials derived from any given strain or species. In many instances, contradictions were observed between invvitro and in vivo results. If the majority of the reactions produced by skin tests are due to tissue-fixed antibodies, then, i t appears that these antibodies are of a different nature or concentration than are the circu-lating antibodies. Skin tests appear to present evidence of immunospecific reactions, and give a better indication of specific reactivities between closely related species and strains of molds than do the precipitin tests. 71 V I . SUMMARY. As a result of the experiments conducted in this i n -vestigation,, the following observations may be presented: 1. Partially purified proteinaceous materials from dermatophytes appear to be poor antigen's for the stimulation of circulating antibodies in the rabbit. 2. Total nitrogen values per mg. of partially purified intracelllular and extracellular proteinaceous material from one organism vary from one lot to another. 3. Extracellular proteinaceous material usually poss-esses a higher total nitrogen content per mg. than that obtained from an intracellular source. 4. Mycelial homogenates produce higher rabbit antisera titres than do partially purified antigens. 5. The use of incomplete Freund's adjuvant with mold antigens does not seem to enhance antibody forma-tion to any marked extent. 6. Cross-precipitin reactions confirm prior reports of common antigens among the dermatophytes* 7. Skin tests upon rabbits immunized with various mold antigens demonstrate cross-reactions with many heterologous strains and species of dermato-phytes.. 8. Reactions obtained on skin testing with partially purified fungal antigens were either of the immed-72 late or the delayed type of response. 9!. Rabbits differ greatly in their ability to produce circulating antibodies to dermatophytic antigens,, and in the nature of their localized cellular responses to skin testing with these materials. 10., Partially purified proteinaceous materials of molds may lose certain antigens when precipitated with alcohol., II., Partially purified polysaccharides of Nocardia aster-oides,, T. granulosum, and Candida albicans do not cause skin reactions in rabbits Immunized with an-tigenic materials obtained from various dermato-phytes or in rabbits infected with a species.of dermatophyte. 12. Second injections of skin test antigenic materials adjacent to the f i rs t sites,, produce results which may be likened to a desensitization or to an In-creased hypersensitivity., 13. Skin, tests appear to provide better evidence of differentiation between closely related species and strains of molds than do precipitin tests. 14. Intracellular partially purified antigenic material appears to be a better source of strain—specific antigens than does the antigenic substance from, an extracellular source. 15. Rabbits experimentally Infected with a mildly 73 pyogenic strain of dermatophyte do not react as markedly to skin tests as do animals Immunized with mycelial homogenates* 16. Serological tests, using partially purified fungal proteinaceous materials as antigens, do not appear to aid in the classification of various genera of dermatophytes.. 74 VII;. BIBLIOGRAPHY. 1., Bjorklund, B, , Proc. Soc. Exp.. Biol . Med., 1952, 79, 319. 2. Blank,, F . , Biochim., Biophys. Acta, 1953, 10, 110. 3. Blank, F.,, "Current Problems in Dermatology", Karger Basel,, Hew York, 1959,. 1, 349. 4. Bioch,, B., from Rivalier, R., Path.-Biol. , 1960, 8, 307. 5. . Blojch,. B ;., and Massini, R., Z. Hyg. Infekt. Kr.,, 1909, 63, 68. 6* Boyd, W.C., "Mechanisms of Hypersensitivity", Lit t le .Brom&Co*, 1.959, p. 47. 7. British Pharmacopoeia, The Pharmaceutical Press, London, 1958, pp. 740 and 849. 8. Browne, H.H., J . Bact., 19.42, 43, 315. 9. Citron, J.,, Z.Hyg. Infekt Kr . , 1905, 49, 120. 10. Conant, H.F. , Smith, D.T., Baker, R.D., Callaway, J*L. , and Martin, D.S.., "Manual of Clinical Mycology", W.B. Saunders Co.,.Philadelphia, 1958, pp.330-351. 11. Crowle, A.J .„ J . Lab. Clin. Med., 1958, 52, 784. 12. De Lamater, E.D., and Benham, R.W.,, J . Inves. Derm*, 1938, 1, 469. 13., Emmons, C.W., Arch. Derm. Syph., 1.934, 30, 337.. 14. Fischer, E . , Arch. Klin. Exp. Derm.,, 1956, 203, 270. 15. Foster, J.W., "Chemical Activities of Fungi", Academic Press Inc.,. Hew York, 1949, p. 93. 16. Foster J.W., i b i d . , p.1.01. 17. Foster J.W., i b i d . , p.102. 1.8. Foster J.W., ibid, p.1.03. 19.. Foster, J.W.., i b i d . , p . l l l . 20. Freund, J . , ; Ann. Rev., Microbiol.., 1947, 1, 291. 21. Friedman, L . , and Derbes, V..J., Ann.. H..Y. Acad. S c i . , / I960,. 89, 1.78. 75 22. Gotz, H.» "Current Problems in Dermatology", Karger Basel, New York, 1959, 1, 234. 23. Grigoraki, L,.., Ann.. Sci. Nat. Bot. Ser., 1925, 7, 165. 24. Heidelberger, M.., and Kendall, F .E . , J., Exp. Med., 1935, 62, 697. 25. Huppert, M., Ph.D. thesis, Columbia University, Doctoral Dissertation Series, 1955, Publication no.12441. 26. Ito, K , and Kirita, K., Bul l . Pharm. Res. Inst.. (Osaka), 1956,, 11, 10. 27. Jadassohn., W., Schaaf, F . , and Wohler, G. .J.Immunol., 1937, 32, 203. 28., Kabat, E .A. , and Mayer, M.,M., "Experimental Immuno-chemistry", Charles C. Thomas, I l l inois , 1948, pp.282-287. 29. Kato, Jap. J . Derm. Urol..,, 1926,, 27, 4. 30. Keeney, E .L . , and Eriksen, N.., J . Allergy, 1949, 20, 172. 31. Keeney, E .L . , and Huppert, M.., J . Inves. Derm., 1959, 32, 7. 32. Keeney, E .L . , and Huppert, M., i b i d . , p).15. 33. Kl'shi, S., Jap. J . Derm. Urol..,, 1957, 67, 197. 34. Kolmer, J .A . , Spaulding, E .H. , and Robinson, H..W., "Approved Laboratory Technic", Appleton-Century-Crofts, Inc., New York, 1945,, p.403. 35. Lamanna, C. , and Mallette, M.F., J . Bact.., 1954, 67, 503. 36. Langeron, M.,, Ann. Parasit.,, 1926, 4, 193. 37. Link, G.K. and Wilcox, H.W.,'Bot. Gaz., 1933, 95, 1. 38. Mackie, T..J., and McCartney, J . E . , "Handbook of Practical Bacteriology", E. & S. Livingstone Ltd. , Edinburgh, 1950, p.222. 39. Matruchot, L„, and Dassonville, Ch., Bull . Soc. My col . France, 1899, 15, 240. 40. Matruchot, L . , and Dassonville, Ch., Bul l . Soc. Mycol.. France, 1900, 17, 123. 41. Miller, H.E. , Stewart, R.A., and Kimura, F . , Arch. Derm. Syph., 1941, 44, 804. 76 42:* Ota,, KL,. and Langeron, M.» Ann. Parasit., 1923, I* 305. 43. Rivalier, E.,, from Blank,, F.,, "Current Problems in Derma-tology",,, Karger Basel, New York. 1959,, 1,, 349. 44. Rivalier, E.,, Path.-Biol., 19.60, 8 „ 30.7, 45. Sabouraud, R . " L e s Teignes", Masson et Cie, 1910, 46. Sanders, H.D.,, and Stock* J . J . , Canad. Pharm. J . ,,• I960,, 93* 67. 47* Seeliger, H.P.R., "Mykologische Serodiagnostik", S.118, Joh. Ambrosia Barth-Verlag, Leipzig,, 1958. 48* Sharp, W.B., J . Inves. Derm., 1941,, 4„ 205. 49* Sharp, W.B*, Texas Rep. Biol* Med.,, 1945,, 3, 159. 50. Stavitsky,, A.B.,, J . Immunol.,, 1954,,. 72, 360. 51. Tiselius, A.,, and Kabat, E.A.., J . Exp. Med. „ 1939, 69, 119* 52* Todd,, J .C*, Sanford, A.,H.,„ and Wells, B.B., "Clinical Diagnosis by Laboratory Methods", W.B., Saunders Co., Philadelphia,. 1953,,, p.931. 53* Treffers,, H*P.,. Heidelberger,, M*, and Freund. J . , J . Exp* Med., 1947,, 86,, 83* 54. Webb,, H*B*,, Irish, O.J.,, and Lyday,, V.,1., J . Bact. 1944, 48, 429. 55. Whartom, M..L. * Reiss, F . , and Wharton,, D.R.A., J . Inves. Derm*, 1950,, 14„ 291. 77 VIII. APPENDICES. Appendix A. Keeney's medium (30) Casamino acids (Difco) 5.0 gm. Dextrose 10.0 gm. K2HP04 0.75 gm. NaCl 1.0 gm. MgS04«7H20 1.5 gm. Ferric citrate 0.02 gm. Distilled water to make 1,000.0 ml. The pH of the medium was adjusted to 6.0. Appendix B. Locke's solution (52) NaCl 0.9 gm. CaCl 2 0.024 gm. KC1 0.042 gm.. NaEOg 0.02 gm. Dextrose 0.25 gm. Distilled water 100.0 ml. Appendix C«, Washing of Seitz f i l t e r pads (46) The Seitz f i l te r pads were washed with appropriate amounts of a 3.0% W/V citric acid solution to rid them of alkaline impurities. The washing with acid was followed by several rinses of distilled water until the pH of the filtrate was equal to the pH of the water used. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0106507/manifest

Comment

Related Items