Norman F. Conant

Immunologic Studies on the Etiologic Agents of North and South American Blastomycosis. II. Comparison of Serologic Reactions.

This study presents an immunologic comparison of the fungi,B. brasiliensis andB. dermatitidis, by serologic reactions in animals and in humans. The complement fixation test was used to test the sera of rabbits with various homologous and heterologous antigens of the two fungi: yeast-phase, broth filtrates of the mycelial phase, and “purified” protein. A definite cross-reactivity was observed. Sera from humans infected withB. dermatitidis orB. brasiliensis also were tested by complement fixation reaction with homologous and heterologous antigens: yeast-phase, broth filtrates of the mycleial phase, and “purified” protein. Two of nine sera from cases of North American blastomycosis, giving positive reactions with the homologous antigens, cross-reacted at a low titer with the heterologous antigens. Five of eleven sera, positive withB. brasiliensis antigens, reacted with one or more types ofB. dermatitidis antigens. There was no correlation between titer obtained with the homologous antigens and that obtained with the heterologous antigens. Immunization of rabbits was best achieved by use of massive doses of sonic-vibrated yeast-phase cell suspensions. Data were presented indicating the greater fragility ofB. brasiliensis yeast-phase cells as compared withB. dermatitidis cells of the same phase. Evidence was obtained by subjecting cells to sonic vibrations.

The taxonomy of the anascosporous yeast-like fungi.

The species of yeast-like anascosporogenous fungi most frequently isolated from man were found to be the following: albicans, tropicalis, pseudotropicalis, Krusei, parakrusei, stellatoidea and Guilliermondi. A restudy of old cultures maintained in culture bureaus for long periods of time has allowed investigators to add other species to this list. These old cultures, representing single isolations, were thought to be partially dissociated because both rough and smooth colonies developed on blood agar plates at 37°C. when streaked from Sabourauds glucose broth. When the smooth colony was picked for study, the culture could be identified as one of the most frequently isolated species. The rough colonies, however, showed certain differences which allowed them to be classified as distinct species. To avoid characterizing new species on the basis of dissociative changes, freshly isolated cultures should be identified immediately by some rigidly standardized technique giving constant results. A single genus, to be used internationally, has been proposed for these fungi which were obviously too closely related to be separated generically. Since Syringospora Quinquaud 1868 was the first validly published genus, it can be replaced by a more recently published genus only by establishing the latter as a nomen conservandum with appropriate emendation and the selection of the proper type species.

A preliminary note on inter-reaction of skin test sensitivity between histoplasmin and chrysosporin in experimental animals.

Species ofChrysosporium have been isolated from soil in Iran. Guinea pigs inoculated withChrysosporium keratinophilum gave a positive skin test to histoplasmin and vice versa. This would suggest thatChrysosporium species might be reponsible for positive skin tests to histoplasmin in areas known not to be endemic for histoplasmosis.Species ofChrysosporium have been isolated from soil in Iran. Guinea pigs inoculated withChrysosporium keratinophilum gave a positive skin test to histoplasmin and vice versa. This would suggest thatChrysosporium species might be reponsible for positive skin tests to histoplasmin in areas known not to be endemic for histoplasmosis.

Future developments in mycological investigative methods.

Although a fungus was the first recognized infectious agent of man and animal, relatively little is known about fungus diseases in comparison with the accumulated knowledge concerning the epidemiology, immunology, immunochemistry, and specific therapy of bacterial and virus infections. The lack of such information about fungus infections has been the result of methods of study which stressed the clinical aspects of the diseases and the taxonomy of the fungi almost to the exclusion of fundamental studies of the infectious agents and the disease processes which they caused. At the present time, however, there are few disagreements concerning the pathogenicity and the taxonomy of those fungi which produce superficial or systemic mycoses. The dermatophytes are no longer represented by a vast, complicated) and confused group of organisms. Rather, they have been reduced to a small number of easily recognized and identified fungi. The systemic fungi, likewise, have evolved through a period of recognition, classification, and confusion, and now are also known to be represented by a small number of easily recognized and identified fungi. It is hoped, therefore, that investigative methods may now be directed more fully towards gaining a better understanding of the physiological, chemical) immunological, and epidemiological aspects of the human pathogenic fungi and the diseases they produce. A trend toward the use of such mycological investigative methods has already been forecast by recent studies which have made use of these tools. For example, growth requirements of the fungi are no longer studied by changing brands of peptone and types of sugar in the media. Robbins and Ma (1942, 1944, 1945) have investigated the nutritional requirements of Trichophyton discoides, Ahodotorula sp., and Trichophyton mentagrophytes by their behavior on a synthetic basal agar medium or synthetic basal solution to which various vitamins and other growth-promoting substances could be added. Hazen (1 947) has reported preliminary nutritional studies on Mirrosporum audouini and has shown that growth factors present in yeast extract stimulate conidial production and colony formation. A further study of the exact requirements for a better growth of M . audouipai is to be reported later. Since microscopic morphology and gross cultural characteristics are used to identify the pathogenic fungi, the stabilization of such characters by growth on a synthetic medium, to which can be added known substances, would allow duplication and a more stable taxonomy. The value of a method of study that would lead to such a stabilization is, or course, readily apparent. Physiological investigations of the human pathogenic fungi also have made use of methods that provide basic quantitative information concerning the activity of the organisms. Bernheim (1942), Nickerson (1946), and

Immunologic studies on the etiologic agents of North and South American blastomycosis. I. Comparison of hypersensitivity reactions.

This study presents an antigenic comparison of the fungi,B. brasiliensis andB. dermatitidis, by intradermal reactions in animals and humans. Guinea pigs, hypersensitive to the homologous infecting fungus, reacted when skin tested with standardized broth filtrates and yeast-phase vaccines of the heterologous organism. Similar results were obtained in the limited number of human cases of North American blastomycosis when the yeast-phase vaccines were employed. A statistical method, the 50 per cent end-point, was utilized for standardizing broth filtrates of fungi to be employed as skin test antigens.

Coccidioides immitis spherule antigen in a complement fixation test for experimental coccidioidomycosis.

Summary 1. A method is described for the preparation of a serologically active antigen from egg yolk material containing spherules. 2. “Spherule antigen” used in a complement fixation test reacted with sera from animals infected with Coccidioides and animals hyperimmunized with the antigen. 3. The antigen was found to stimulate low titers to coccidioidin in hyperimmunized animals. 4. The “Spherule antigen” completely absorbed anti- bodies to the homologous antigen and coccidioidin from the serum of a hypenmmunized rabbit, but failed to absorb antibodies from the serum of a rabbit infected with Coccidioides immitits.

A serological comparison of the three morphological phases of Coccidioides immitis; complement fixation tests with anti-Histoplasma capsulatum serum

The results of this study indicated that antigens prepared from the three morphological phases ofCoccidioides immitis differed in their complement fixing activity with anti-Histoplasma capsulatum pooled serum. Spherule antigens were serologically less active in tests with the anti-H. capsulatum pooled serum than antigens prepared from arthrospores and from mycelium. Antigenic determinants which are common toC. immitis andH. capsulatum appeared to be located on the intact arthrospore cellular surface but not on the surface of spherule cells.

A serological comparison of the three morphological phases of coccidioides immitis: Complement fixation tests with a Pooled antiserum obtained from rabbits with experimental Coccidioidomycosis

A sonicated spherule preparation was more reactive than a sonicated arthrospore antigen in complement fixation tests with a pooled serum from rabbits with experimental coccidioidomycosis. The reactivity of the sonicated spherule approximated the reactivity of coccidioidin. When the sonicated spherule was separated into its supernatant and sediment fractions, both preparations exhibited serological activity. Ein mit Schallwellen hergestelltes Kügelchen-Präparat war activer in dem Komplement-Tests mit Blutserum von Kaninchen mit einer Coccidioidomycosisinfektion als ein mit Schallwellen hergestelltes Arthrosporantigen. Die Reaktivität der mit Schallwellen hergestellten Kügelchen war der des Coccidioidin ähnlich. Wurden diese Kügelchen in Niederschlag und Lösung getrennt, so hatten beide Präparate serologische Aktivität.

Immunization against experimental lethal simian coccidioidomycosis using whole killed arthrospores and cell fraction.

Immunization against experimental coccidioidomycosis began with the use of a killed, whole-cell antigen. Negroni, Vivoli and Bonfiglioli (8) investigated the immune response in guinea pigs using dead material. Vogel and co-workers (14) at Duke University found that intramuscular immunization with a killed spherule-endospore suspension increased re? sistance of guinea pigs to pulmonary infection. The disease not only occurred less frequently but was less severe in the immunized than in the non-immunized control animals. Later Friedman (1) and Smith (2) reported that killed arthrospore vaccines administered subcutaneously protected mice against lethal disease but not against infection with Coccidioides immitis Rixf. & Gilchr. In the latter experiments the challenge dose was administered intraperitoneally. After Pappagianis and co-workers (10) observed that respiratory exposure appeared to be a more severe test of the resistance of immunized animals than challenge by parenteral inoculation, Levine et al. (5) adopted intranasal instillation as the method of challenge. Killed arthrospores, mycelial and spherule antigens introduced intramuscularly into mice all protected against lethal intranasal challenge. Mice that received the spherule-endospore vaccine appeared to have infections that were milder than mice immunized with other test vaccines. Extending his studies to larger animals, Levine et al (6) tested the efficacy of killed spherule vaccine in cynomolgous monkeys challenged with 200 air-borne organisms. A total dose of 9 mg (dry weight) of vaccine was used for immunizations that were given alternately subcu? taneously and intramuscularly at 1, 11, 28, and 54 days. The animals

Fungus infections of the hair and nails.

Abstract Fungus infections of the hair may be divided clinically into three categories, based on the type of infecting organism: (1) trichomycosis caused by Corynebacterium tenuis, (2) piedra caused by Piedra hortai and Trichosporon beigelii, and (3) ringworm of the scalp (tinea capitis) caused by dermatophytes of the genera Microsporum and Trichophyton. Fungus infections of the nails, tinea unguium, or onychomycosis, appear clinically identical regardless of the fungus etiology, which may be one of several species of Trichophyton, Epidermophyton floccosum, and very rarely, Microsporum canis.