Immunity to histoplasmosis induced in mice by components of Histoplasma capsulatum

Twenty-three strains of Histoplasma capsulatum were studied. The data indicated that each strain had individual characteristics with regard to: macroscopic appearance of colonies, measurement of spore sizes, relative number of spores, virulence as determined by intravenous LD50 in mice, immunization capacity of vaccines, and chemical composition of the vaccines prepared from four strains. Strains G10, G17M, 2645, and 3330 were used to study the immunogenicity of the fungus, A cell free extract prepared from the mycelial phase of strain G10 and a cell free extract prepared from the yeast phase of strain G17M produced similar protection in immunized mice, G10 and G17M vaccines also had similar chemical composition with high carbohydrate content and low protein content. Strains 3154 and G17M were found to be the most virulent by the method employed. The sporulation number, as determined by a rank rating system, showed that each strain produced various numbers of both types of spores, The results indicated that Sabouraud's medium with phosphate ion produced abundant and numerous spores. The macroscopic description of each strain on various media indicated that each strain varies in morphological characteristics depending on the nutrition available.

IMMUNITY TO HISTOPLASMOSIS INDUCED IN MICE BY COMPONENTS OF HISTOPLASMA CAPSULATUM by Kenneth Leroy Anderson A thesis submitted to the faculty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology University of Utah August, 1968 Ijl'!!VERSIW Of U1 AH lIBMR~ This ThesIs for the Doctor of Philosophy Degree by Kenneth Leroy Anderson has been approved chairman, Supervisory Committee Reader, Supervisory Committee Reader, Supervisory Committee Reader, Supervisory Committee Reader, Supervisory Committee Reader Reader Head, Major Department Dean, Graduate School I am deeply indebted to Dr. Stanley Marcus who has critically reviewed this manuscript and for his assistance? guidance~ and encouragement during these studies. Grateful acknowledgement is made to Dr Gebhardt~ Dr. Paul S. Nicholes~ Louis P, Dr. Bill B. Wiley, Dr. Douglas Wo Hill, Oro Gilbert A. Hill, and Dr. Charles Co Nabors who have made extensive and important suggestions during my association at the University of Utah. Special thanks is extended to Dr. Thomas F. Dougherty and Dr. Marvin Rogolsky for their help and interest. Particular recognition is extended to numerous colleagues for their encouraging words during these studies and to my wife, Jeanne 9 without whom this would have been impossible, The author wishes to thank Mrs. Diane Ward 9 Mrs. Helen Wyatt, and Miss Arlene Imada for help in typing and proofreading of this thesis. Special appreciation goes to Mrs. Priscilla Maydens, Medical Librarian, who aided the author in the format and in checking the bibl iographical material. The research reported herein has been supported by Publ ic Health Service Grant (AI-K6-l4,924) from the National Institutes of Health. iii TABLE OF CONTENTS TABLES.............................................. FIGURES ..........................• ABSTRACT .•••• 0 . . . . . . . . . . . . . . . . . . . . . I NTRoDUCT ION •..•..•. ? ••••••••••••• 0 ? 0 0 • •• • 0 • 0 ........ • I I. I I I. IV. V. VI. I I. I I I. I V• V. 0 ••••••• 0 ..... History of Histoplasmosis .......... ••••• 0 ••• 0 vii ix 0 " • ••• • • • •• •••• 0 3 3 Morphological and Physiological characteristics of Histoplasma capsulatum ..••...•... 7 A. Mycelial Phase •..••••••..••••.....••... 7 B. Sporulation .............................. 11 C. Yeast Phase .•..••••••.•••..•••••••••.• 13 Demonstration of Immunity to Histoplasmosis .............................. 15 Manifestation of the Immune Response in Histoplasmosis .•••.••••..••.. " ........... 28 Immunogens of Histoplasma capsulatum .••••. o 30 Mechanisms of the Immune Response to Histoplasma capsulatum •••.•.••••.•••••..••• 32 MATER I ALS AND METHODS ................... I. • ................. REV I EW OF LITERATURE ..................... I. 0 v ar g ani sm s ., Mice. 0 •..•.• " • " .• " ..••• ••••••••••••••••••• Med i a. " •.•.•.• 0 0 0 • ••• ••••••••••••• " L D5 0 de term ina t i on s •....••.• " 0 ••••••• " •••• • • • • • 0 ••••• .. • • • • " • • •• •••• 0 •• ••••• ., • • • • • • •• 0 • • • • • • • •• • • • • 34 34 36 36 39 Strain description ••••.•••••••••••••••••••. 39 iv VI. Spore measurements ..... VI I. VI I n a. DO •••• Vaccine preparation .. ........ 0 00 D •••••••••• •••••• •••• Tests used to characterize vaccine ..... 10 I X. I mmu n i z at i on .. 0 EXPERIMENTAL RESULTS .... •• o o. • 00.0 ••• , •• a 0 •• 0 0 0 0 ••• 0 o •• 0 0 •• ,> Spore size measurements. I I. I I I. 0.0 0 ••• ••••••• 0 V. 46 ••• 47 57 60 Virulence studies ...... ~ .............. 67 I mm u n i z a t ion . . . . 0 44 Relative numbers of spores . . . . . . . ..... .0. 0 • 0 • 0 • • • • 0 • • • " • • a ". o.o •• 0 • BIB L I OG RAP HY 0 0 0 • 0 0 • • • 0 0 0 0 •• " •••••• 0 • • • • • • • • • • • • • •• ••••••• DISCUSSION ...................................... SUMM AR Y • D. 0 IV. 42 47 00 Macroscopic appearance of colonieso ...... I. 40 • •• • 0 •• 0 o. 70 88 107 1 10 TABLES 1. Induced immunity to Histoplasma capsulatum: Summary of findings on vaccination and challenge routes and morphological phase of 1 ive and ki lled preparations ... " 21 Origin of stock cultures of Histoplasma cap s u 1at um. 35 00.0 2. a 3. • 0 • 0 0 • 0 • 'j • 0 •• 0 •• •• 0 •• o. 0 • 0 " ••••••••••••• •••• 0 • • • • • • • a •• Strains of Histoplasma capsulatum grown on Sabouraudis dextrose medium with phosphate ion for ten weeks....... . .... 48 Strains of Histoplasma capsulatum grown on modified Sauton1s medium for ten weeks ..... 50 00 4. <> ' v ••••• 0 ••••••• a •• o ••• •••• 5. Strains of Histoplasma capsu1atum grown on modified Sautonis medium without dextrose for ten weekso .. oo. 6. 000000000000000.000"0""00 Strains of Histoplasma capsu1atum grown on synthet i c med i um 1 for ten weeks 0 7. a a 000. 0 9. 10. 13. 14. 000 •• J • 00. 17. 55 59 Media supporting the best sporulation of Hist lasma capsu1atum . . . 68 •••• 0 ••• 0 •••••••••••• Mouse LD50 determinations of yeast phase Histoplasma capsu1atum strain G17M .. ••••••••• Mouse LD50 determinations of yeast phase Histoplasma capsulatum strain 3154 ....... 0 Mouse LD50 determinations of yeast phase Histoplasma capsu1atum of various strains ..... o 0 ••••••••••••••• a Strains of Histoplasma capsulatum util ized i n i mmu nit y stu die s .. ., 0 0 • 0 0 ••• , 0. •••••• 69 71 72 73 Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-killed cell free extract of the mycel ium and macroconidia of G10 and challenged with 3154 ............. 75 Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-kil led cell free extract of the myce1 ium and macroconidia of G10 and challenged with 3154.......... ...... 77 Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-kil led cell free extract of the mycel ium of G75 and challenged with 3154 . . . o. 78 ao 16. '0 53 64 0 15. 0 Histoplasma capsu1atum relative numbers of macroconidia and microconidia on five media.... 0 12. " Histoplasma capsu1atum macroconidia and microconidia spore sizes on five media . . . . . . . 0 11. 0 Strains of Histoplasma capsu1atum grown on synthet i c med i um 2 for ten weeks ...... 00' 8. • 52 000 vi •• o •••• 0000............. 18. 190 Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-killed cell free extract of the mycel ium of G75 and challenged with 3154.000000000.0 .. 00000.0 ..... 0 79 Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-killed cell free extract of the myce1 ium and microconidia of 3330 and challenged with 3154 .. 81 o. 20. 0 •••• 0 " " • a • 0 • 0 •••••• 0 • 0. •• 82 83 84 Comparison and summary of potency ratios of vaccines from various strains of Histoplasma capsulatum.ooo .... 86 Chemical composition of five strains of Histoplasma capsu1atum.a ...... 87 • 0 " 0 0 0 0 0". oo 24. " o. Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-killed whole spores or yeast phase cells and challenged wit h 3 154" 0 0 ....... o. 0 23. •• Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-killed cell free extract of yeast phase G17M and cha 1 1enged wit h 3 1540 . 0.... 0 22. O. Vaccination of mice against histoplasmosis. Mice vaccinated with formal in-kil led cell free extract of the myce1 ium and spores of 2645 and challenged with 3154.0" ...... 0 .. 00 21. Q. 0 •• coo • " •••••••••• 0 0 0 ••• 0 6 •••••••••••••••• 0.0 ••• 0 ••• 0.0.0 FIGURES 1. Diagrammatic representation of the 1 ife cycle of Histoplasma capsulatum and Gymnascus demon b r e un n i i ......... 0 2. • 0 • Q •• " 0 ••••• 0 • " " • 0 •••• Four strains of Histoplasma capsulatum grown on Sabouraud's dextrose medium with phosphate ion at room temperature for ten wee k s . . . . . .. "0."'. 0 • " • 0 vii •• 0 •• 0 ••• " 0 no •••• 0 0 0 0 ••• • 0 5 58 3. Histoplasma capsulatum macroconidial and microconidial spore size on synthetic medium 1 ... 61 ~. Histoplasma capsulatum macroconidial and microconidial spore size on synthetic medium 2 ... 62 5. Histoplasma capsulatum macroconidial and microconidial spore size on synthetic medium 30.0 63 vi i i ABSTRACT Twenty-three strains of Histoplasma capsulatum were studied. The data indicated that each strain had individual characteristics with regard to; macroscopic appearance of colonies, measurement of spore sizes 9 relative number of spores j virulence as determined by intravenous LD50 in mice, immunization capacity of vaccines, and chemical composition of the vaccines prepared from four strains. Strains G10} G17M9 2645, and 3330 were used to study the immunogenicity of the fungus. A cell free extract prepared from the mycel ial phase of strain G10 and a cell free extract prepared from the yeast phase of strain G17M produced similar protection in immunized mice. G10 and G17M vaccines also had similar chemical composition with high carbohydrate content and low protein content. Strains 3154 and G17M were found to be the most virulent by the method employed. number~ The sporulation as determined by a rank rating system, showed that each strain produced various numbers of both types of spores. The results indicated that Sabouraudis medium with phosphate ion produced abundant and numerous spores. The macroscopic description of each strain on various media indicated that each strain varies in morphological characteristics depending on the nutrition available. ix IMMUNITY TO HISTOPLASMOSIS INDUCED IN MICE BY COMPONENTS OF HISTOPLASMA CAPSULATUM INTRODUCTION The extent and mechanisms of resistance to histoplasmosis represent an area of significant basic and appl jed effort in mycology. The problems associated with the antigenic analysis of Histoplasma capsulatum are as complex as the fungi themselves. In its natural habitat of soils or on agar incubated at room temperature intricate network of interlaced 1 the fungus forms an hyphae~ from which a special ized complex of spores are produced. The large, globose macroconidium, characteristic of H. capsulatum, has a thick wall, internal structure~ and many short finger-l ike appendages emanating from all sides. j In addition there exists a second type of spore called the microconidium. The smooth-wal led microconidium is spherical or pyriform in shape. It is possible that the constitution of the macro- conidium is not identical with that of the microconidium. Two other elements exist which may also be different in composition: the yeast phase of the organism and the hyphal elements. The objectives of the work presented in this thesis have been: a) preparation of vaccines from various elements 2 of ~o capsulatum in order todetermine immunogenic capacity; b) characterization of the virulence of selected yeast phase strains of the organism by intravenous LDSO; c) differential spore production on various media; and d) description of the macroscopic appearance of strains of the fungus on various media. 3 REVIEW OF LITERATURE Increased awareness in recent decades of the prevalence of airborne fungi and the high frequency of these organisms as etiological agents of pulmonary disease has stimulated wide-spread interest in their pathogenicity and immunogenicity. The defense mechanisms are 1 ittle understood in the mycoses. Antibodies arising during the course of disease have high diagnostic and prognostic values but the protective effect of such antibodies has not been demonstrated Cellular responses to infection with these organisms produce granulomas and delayed hypersensitivity. sal ient features 9 Such reactions are the former is associated with heightened immunity but the role of the latter is undetermined, I. HISTORY OF HISTOPLASMOSIS The causal agent of histoplasmosis was first observed by Darl ing (1906, 1908~ 1909). He bel ieved the organism to be a protozoan and gave it the generic name, Histoplasma. The species name 9 capsu1atum, was used to describe a capsular-appearing halo surrounding the organism in stained tissue sections. Da Rocha-Lima (1912) was the first to suspect that the organism was actually a fungus and not a protozoan. ~. capsulatum was obtained in culture by Hansmann and Schenken in 1934 and a complete description of the 4 organism was publ ished by De Monbreun in 1934. De Monbreun clearly demonstrated the diphasic nature of the fungus and accurately described the cultural characteristics, morphology Ciferri and Radael 1 i (1934) and 1 ife cycle of the organism. stated the systematic position of genus Histoplasma 9 ~. capsulatum as fol lows: order Blastosporales, superfamily Atelosaccharomycetaceae, and family Histoplasmaceae no sexual stage could be demonstrated for ~Q capsulatum Conant (1941) placed the organism in the Fungi as a member of the form-family Monil iaceae. Since 9 Imperfecti, However sexual reproduction has been demonstrated by Ajello and Cheng (1967), The development of an ascigenous state was stimulated on soil overlaid with feathers. The organism is homothall IC and is given the perfect state name of Gymnoascus demonbreunni i. A diagrammatic representation of the 1 i cycle of capsulatum and Go demonbreunni i is shown in Figure 10 ~. The three cycles represent the mycel ial and yeast phase of the asexual ~. capsulatum and the sexual stage of the fungus G. demonbreunni i. are depicted. The various structures, forms, and spores Recently the val idity of the species G. demonbreunni i as the perfect state of H. capsulatum has been questioned (Kwon-Chung, 1968). Knowledge of the growth of ~. capsulatum in nature dates from the first isolation by Emmons, Morlan and Hill 5 Various morphological forms of Mycelial phase phase Conversion Germination of macroconidium and 0 microcon id i um 0 0 \ . ~ ~~ o;;? . . Histoplasma ca~sulatum --~- __ ----I~as~~~~51 ____ ~~---j llymnascys emon reynn I I~. (sexual) A -, AV-/f ':~;""" ascospores ~ cii ';:;." II) 'f,: '1": ir' ~ ;' ... (f!1 ~<> .. • '.. ':,,_ ~""" --__ . . . . . ___ • 1i.1!t.~%~\~1&~:'~' Cross section of ~~~~w~~ cleistothecia ~~~~~~~ and asci Figure 1. yeast phase Convers i on ' t o the mycel ial phase .... '-'It_ .·~i\~~~: @ J~ring \ ~ .l~," Somat i c hyphae " .... Cleisto~heCium __ A diagrammatic representation of the 1 ife cycle of Histoplasma capsulatum and Gymnascus demonbreunni i 6 (1949). They isolated the organism from the soil about a rodent burrow under a chicken coop. Grayston, Loosl i, and Alexander (1951) reported the first isolation of the organism in connection with an epidemic of histoplasmosis which occurred in a silo in Northern Indiana. This fungus grows in the soil and is inhaled after the soil some activity which creates an aerosol, is stirred by In general, these organisms are smal 1 ~ 5 u or less in size (Cozad and Furcolow, 1953). Thus j in an aerosol these organisms can readily penetrate to the alveolar bed and be retained there (Brown, Cook, Ney, and Hatch, 1950), Dodd and Tompkins in 1934 described the first antemortem description of histoplasmosis. Following the publ ication of this work, fatal cases were recognized with increasing frequency, However? it was still regarded as a rare and fatal human disease. In the 1940ls a series of events conspired to change this situation. Christie and Peterson (1945) reported on pulmonary calcification with negative tubercul in tests in the Mississippi Valley area. Palmer (1945) incorporating histoplasmin skin testing in a broad survey on tubercul in sensitivity demonstrated a strong epidemiologic correlation between the incidence of pulmonary calcifications and positive histoplasmin skin test in individuals with negative tubercul in skin tests. These discoveries permitted a new 7 evaluation and understanding of the epidemiology of the disease produced by ~. capsulatum. Histoplasmosis has been found to be an intracellular mycosis of the reticuloendothel ial system. Usually respira- tory in origin, histoplasmosis may be asymptomatic or benign, acute ~. or chronic, and widely disseminated or fatal. capsulatum parasitism of reticuloendothel ial cells probably results in early and wide dissemination even in mild or inapparent disease (Emmons~ Binford and Utz~ 1963), Histoplasmosis is a disease of worldwide distribution. The hypothesis has been suggested that all of the river valleys in the temperate and tropical zones of the world, between 45 0 north and 45 0 south latitude are endemic areas (Furcolow, 1960). It has been estimated that in the United States alone 30 mill ion people have been infected with this fungus? and half a million a year acquire the infection (Furcolow I I. A. j 1965). MORPHOLOGICAL AND Mycel ial Phase When mycel ial fragments are streaked over Sabouraud's dextrose agar, small white, cottony colonies develop which increase in size. At the time maximum growth is reached~ the central portion and upper drier parts of the culture assume a yellow to buff color which slowly turns to tan or 8 dark brown. Gradually the entire culture will become brown in color and will take on a drier more fragile or powdery appearance. Usually sporulation is associated with the tan coloration and aging of the culture (Pine, 1960)0 In general, the mycel ium varies from 1 - 5 u in diameter. The hyphae are usually refractile, branched, and multicellular (septate). Each mycel ial cell component has one or more nuclei (Negroni j 1940). When the mycel ium becomes older, its protoplasm is displaced toward the walls and vacuoles and oil globules are seen in the central portions of the cells. At this time 9 the walls thicken and a few cells may develop into pecul iar appearing swell ings of assorted shapes and sizes which in some cases reach a diameter as great as 15 u. Racquet hyphae and occasionally coiled hyphae and nodular bodies may be formed while intercalary chlamydospores, single and in chains, and ranging from 5 - 8 may be found in most cultures. ~ in diameter As the hyphae spread in radiating fashion from the center of the colony, parallel hyphae lying at the periphery may be observed to undergo anastomoses. These hyphal fusions do not result in the apparent formation of any special ized structures, ti. capsulatum produces two types of ale uri os pores, macroconidi p and microconidia (Howell ~ 1939). The macroconidia are typically large tuberculate 1 spherical to 9 pyriform spores described as ranging from 10 - 25 u (Pine, 1960), or 8 - 14 u (Emmons~ Binford j Utz, 1963). These macroconidia are formed in the aerial portions of the mycel ium generally at the end of short pedicels but may be sessile or formed at the end of long hyphae (Pine? 1960)0 As described by Howell (1939) the large aerial spores begin their development as bulbous enlargements on the ends of lateral branches and are called spore initials o These branches may be simple with a single spore on the end of each branch~ or one to several spores may be produced acro- peta1ous1y and directly on a short branch. As these spore initials or prespores increase in size, the spores become spherical to pyriform in shape and their walls gradually increase in thickness. The character of the spore wall varies greatly with the strain examined, the medium used, or the part of the mycel ium in which it is produced (Howell, 1939). In addition to tuberculate and smooth macroconidia, there are forms having only several large bulbous or convoluted swell ings or numerous spindly or warty projections (Negroni, 1940). Preponder- antly smooth-wal led macroconidia have been noted deep in the mycel ial mat next to or embedded in the agar (Howell, 1939). Certain spores formed in these regions exhibit a halo of substance surrounding the body of the macroconidium and have 10 Evans~ been cal led nymbospores (Nielsen and 1954)0 The spines or finger-1 ike projections may be 1 - 8 u in length (Conant, 1941) but average about 5 u It is the tuberculate o spore which serves to identify the fungus. The optimum pH for growth of the myce1 ia1 phase on a synthetic medium is approximately pH 6 5 (Howell, 1941). 0 At pH 505 to 6.5 considerable aerial myce1 ium and macroconidia were formed on this medium whereas at pH 7.7 - 8.6 there was 1 ittle or no aerial mycel ium and sporulation was negl igiblea The optimum temperature for growth ranges from 25 0 to 30 0 C9 and temperatures greater than 320 C are usually inhibitory (Howell, 1940). Based on optical density measurements~ a generation time of approximately 12 hours has been obtained at 25 0 C in shake culture (Pine, 1954)0 A high humidity is recognized as usually being beneficial on agar media (Menges, Furcolow~ Larsh and Hinton y 1952). The organism is a strict aerobe and growth will be greatly inhibited by merely closing off the culture tube with a rubber stopper or by attempting to grow the organism beneath the agar surface (De Monbreun 1934)0 Vitamin requirement studies of results o Scheff (1945) has reported biotin and niacin. li. capsu1atum show variable li. capsulatum required However, Salvin (1949) studied different strains of the fungus and found that no vitamin supplement 11 was necessary. The amino acid requirement of the mycel ial phase has been reported to be complex and variable for different strains (Rowley and Pine, 1955)0 Scheff (1945) obtained dry weights of mycel ial mass with various carbohydrates; glucose, maltose and sucrose yielded the greatest weight increases. Although the consensus of opinion is that the mycel ial phase is readily grown at 25 0 C on a variety of media with relatively few nutritional requirements, the need for additional growth factors at 37 0 C is indicated (Howell, 1948; Scherr, 1956; 1957). The simplest basal medium for the growth of the mycel ial phase is the glucose-asparagine synthetic medium used by Salvin (1949). However, general- izations should be guarded because each strain has its own pecul iar characteristics regarding nutritional requirements, responses to temperature, and other environmental conditions. 8. Sporulation Sporulation is dependent upon the strain of the organism and upon the environmental conditions of the culture. The addition of whole blood and temperatures greater than 320 C inhibit sporulation (Howell, 1940)Q Some strains sporulate better at pH values ranging from 6.5 to 7.5. Such increased sporulation may be correlated with greater growth. Negroni (1940) has shown that 12 sporulation may be greatly affected by the carbohydrate or nitrogen source in the medium. For example~ in a glu- cose containing medium, asparagine supported best growth at room temperature, but only macroconidia were formed. When KN03 was substituted for asparagine as the nitrogen source less growth occurred and many smooth and rough macroconidia were formed. Util izing (NH4)2S02 as the nitrogen source, this inorganic chemical resulted in 1 ittle growth with only smooth macroconidia and ~icroconidia developed, and when no nitrogen source was added~ being Negron! found abundant microconidia and a few smooth macroconidia with scant mycel ial growth. Artis and Baum (1963) studied 32 strains of H capsulatum focusing on the abil ity of these strains to form tuberculate spores. Nine strains did not produce tuberculate spores on Sabouraud's agar 9 on corn meal agar 9 on "spent" or media with pH values ranging from 405 to 7.0 medium~ Tuberculate spore production did occur in these nine strains when Sabouraud's medium was enriched with phosphate especially KH2P04° Smith and Furcolow (1964) suggested that the addition of infusions of starl ing manure to soil had the effect not only of producing more total particles but of producing a large number of microconidia and of increasing the viabil ity 13 of these spores to at least twice that observed in other media Q Of particular interest was the observation that although large numbers of particles are produced on Sabouraud~s medium, 84% of these particles were hypha1 elements and not spores 9 and the overall viabi1 ity was only 3%. Smith (1964) has reported that 0.6% yeast extract and 2% agar in distilled water stimulated rapid growth and sporulation of H. capsu1atum similar to sporulation in star1 ing manure extract medium. This rapidly grown fungus developed many viable microconidia despite a scant growth of vegatative myce1 ium. Ho capsulatum spores are resistant to drying and will remain viable in dry soil during an observation period of 4 years (Pine and Peacock, 1958). They are resistant to a temperature of 45 0 C for 30 minutes (Negron!, 1940). All spores of these strains were killed at a temperature of 50 0 C for 1 hour or at a temperature of 60 0 C for 5 minutes. The spores will survive over an observation period of 600 days in water at 4 0 C but rapidly decrease in viabil ity as the temperature is increased to 37 0 C (Cooke and Kobler, Ritter~ C. 1953~ 1954). The Yeast Phase The yeast phase cells of young cultures are oval bodies which are approximately 1.5 to 2.0u by 3.0 to 3.5u 14 (Negroni, 1965) with as many as three buds formed on a single mother apolar~ cell. Budding occurs at either pole or may be The cells usually appear round or oval, but elongated, swol len, or dumbbell shapes are also observed (Pine, 1960) The cells have a thin cell wall) the cytoplasm contains oil droplets and the nuclei of yeast cells appear peripherally as crescent shaped masses (De Monbreun, 1934). A halo about the cells appears to be a capsule, however India ink preparations do not reveal a true capsule (Conant, 1941), Ribi and Salvin (1956) were unable to find any electronmicroscopic evidence for the presence of a capsule. The optimum pH for yeast phase growth is considered to be between 6.5 and 7.5. Cross (1948) tested the effect of pH on the yeast-phase growth in three different media. Best growth was supported by brain-heart infusion broth at a pH range of 7.2 to 7.6. Salvin (1947) found that the maximum growth of the yeast-phase occurred between pH 603 and 8.1. blood 1 On basal agar medium supplemented with whole the rate of growth was similar at pH 5.5, 6.5, and 7.5 (Pine, 1954). In general, the growth of the yeast phase on blood agar or serum media occurs at temperatures between 340 and 37 0 C. At lower temperatures conversion of the yeast phase to the mycel ial phase occurs. However} growth of the yeast 15 phase of H. capsulatum of strains studied may be maintained at temperatures of 25 0 C (Pine, 1957; Scherr 1 1956). Growth oft hey e a s t p has e iss t ric t 1y a e rob i c (P i ne, 1954) Din 1 iquid shake tubes the generation time ranges from 9 to 11 hours under optimal conditions (Pine and Peacock, 1958) while on blood agar media the generation time varies from 6 to 8 hou r s (P i ne, 1955 ) . Row 1e y and Hub e r (1 956 ) inoculated mice with non-lethal doses of the yeast phase and estimated the generation time to be 15 to 19 hours. Howard (1964) determined the intracellular generation time in mouse histocytes to be 10.3 ~ 1.5 hours. As a carbon source, Negroni (1940) found glucose mannose, or mannitol supported best growth. j Salvin (1949) in a survey on the abil ity of the yeast phase to assimilate various nitrogen compounds found that only cysteine, cystine and glutathione were assimilated. The vitamins necessary for growth were reported to be biotin (Salvin, 1949), thioctic acid (Pine, 1957) thiamine, inositol and niacin (McVeigh and Morton, 1965). I I I. DEMONSTRATION OF IMMUNITY TO HISTOPLASMOSIS A prerequisite for the demonstration of enhanced resistance is the use of a susceptible host. The subject of host range of H. capsulatum has been reviewed recently 16 (Salvin, 1963). The most widely used laboratory animal has been the mouse. The extent of immunity as measured by survival after challenge has been frequently employed, especially when high challenge doses are used. The mortal ity endpoint generally requires observation periods of several weeks to months. Marcus and Rambo (1952) reported on the determination of LD50 values for yeast phase cells injected intravenously and noted differences in virulence of two strains of H. capsulatum. In growing bacteria and viruses, the organisms obtained are generally quite uniform in size and morphology. contrast, cultures of Coccidioides immitis and ~. in capsulatum vary not only in size and shape but are often multiform in a given medium. Wit~ C. immitis for example 7 four distinct morphological forms may be found: 1) spherules, 2) endospores, 3) arthrospores, and 4) myce1 ia1elements. Likewise H. capsulatum exists in four forms: 1) yeast phase cells, 2) mycel ial elements, 3) macroconidia, and 4) microconidia. It is now known that antigenicity varies with the morphological phase of different fungi. Kaufman and Blumer (1966) in studies with a fluorescent antibody for the yeast phase of H. capsu1atum determined the occurrence of yeast phase serotypes. The yeast phase cells of H. capsulatum are highly immunogenic (Hill and Marcus, 1959). Mycel ial culture filtrates of this fungus also induce a low order 17 of resistance (Salvin, 1953). It is apparent, then, that there are resistance-inducing immunogens common to both phases. Marked difference in protective immunogenicity was observed between the saprophytic and parasitic phases of C. immitis. Mycel ial elements and arthrospores killed with formal in protected mice against low challenge doses by the intraperitoneal route (Converse, Castleberry, Besemer and Snyder, 1962; Friedman and Smith, 1956; Pappagianis, Miller, Smith, Berman, Kobayashi, 1961). Levine 9 Cobb, and Smith (1960; 1961) observed that spherules and endospores afforded stronger protection to mice against intranasal challenge doses than did either mycel ial elements or arthrospores, Using mycel ial elements and spherules grown simultaneously in the same flasks of medium, Kong and Levine (1967) observed that mice vaccinated with spherules were protected against a challenge of approximately 200 LD50 compared with approximately 20 LD50 in mice vaccinated with mycel ial elements. Employing C. immitis arthrospores, Friedman, Smith and Gordon (1955) studied three strains for comparative virulence in mice. The animals were inoculated intraperitonea11y; using three different parameters, the strains showed differing virulence with respect to each other. The authors concluded that virulence is relative and the ideal comparison is made 18 if the same number of viable particles of each strain has been inoculated into the animals. Inocula containing 100 viable arthrospores per mill 11 iter were used The order of virulence from the greatest to the least was Silveria Perry? and Mauser strains" Using spherules harvested from mice infected with arthrospores Pappagianis, Smith? and Kobayashi (1956) determined the virulence of the in vivo form of Co immitis, The spherules were ruptured releasing their endospores, The endospores of both strains studied did not show a significant difference in the virulence of endospores and arthrospores. Levine, Cobb 1 and Smith (1960) described aspects of protective immunity induced in mice by formal in-ki1 led and purified, mycel ial, arthrospore or spherule-endospore vaccines. Intramuscular immunization with 1.6 mg of each preparation resulted in the spherule-endospore vaccine conferring superior protection to that of the mycel ial or of the arthrospore preparations? to intranasal challenge doses of 79 to 318 arthrospores. The spherule-endospore preparation was administered in several doses. Better protection was obtained than the same amount given in a single dose. Papp ian i set a 1 ., (1961) i mmun i zed mice wit h vi ab 1e 19 C. immitis. Mice vaccinated with dead arthrospores survived intraperitoneal challenge doses but not the intranasal challenge, Mice immunized with viable arthrospores developed a high level of protection surviving both intraperitoneal and intranasal challenge doses. The c u 1min a t ion 0 f the stu d yon i mm un i t y 0 f C. i mm i tis has been the study of the immunogenic properties of nondisrupted and disrupted spherules by Kong, (1963). Levine~ and Smith Studies on the cellular fractions demonstrated that the soluble fraction did not afford protection against a challenge dose of 44 arthrospores. The particulate fraction which contained spherules cell walls afforded protection against a challenging dose of 10 2 arthrosporeso The recombined fraction showed that the primary immunogenic substance of the spherule was in the cell wall. The maturation cycle of the tissue phase, pecul iar to C. immitis illustrates an additional relationship between morphology and immunogenicity. Levine, Kong, and Smith (1965) showed that immunogenicity increased as the spherule matured and endosporulated. The increase in immunogenicity apparently reflected concomitant biosynthetic developments in the wall, which was shown to contain virtually all the immunogen (Kong, Levine and Smith, 1963). It seems apparent that induced immunity to fungal 20 diseases is strongly influenced by the morphological attributes of the vaccine preparation, as well as the routes of vaccination and of challenge. As is evident in Table 1 there has been general agreement that 1 ive H. capsulatum is effective in immunizing animals to subsequent challenge. Agreement concerning the efficacy of 1 ive vaccines by different investigators does not extend to killed vaccines. Reasons for discrepancies observed with killed vaccines or immunogenic extracts may include variables which become highly critical with killed vaccine or extracts. Resistance to infection with H. capsulatum can be induced in experimental animals by sublethal infection or by immunization with killed yeast phase cells or polysaccharides derived from these cells. Marcus and Rambo (1952) reported on the determination of LD50 values for yeast phase cells injected intravenously and noted differences in virulence of two strains of ~. capsulatum by this technique. They also reported that animals infected with a sublethal number of yeast phase cells became resistant to subsequent challenge with lethal numbers of organisms. Salvin (1953) demonstrated a method for obtaining constant death rates in white Swiss mice. Various routes of inoculation were examined by infecting mice with live cells, Intracerebral inoculation seemed most successful in producing Table 1 Induced immunity to Histoplasma ca~sulatum: Summary of findings on vaccination and cha lenge routes and morphological phase of 1 ive and kil led preparations Organism Live or Killed Grow Phase Host nation Route Live Yeast Mice i cer"i'\" i p"i'{' icer icer ip iv Strong Strong Increas Increased i V"i~ icer iv Strong Increased sc"{' it r"i'{' icer ip icer itr icer icer ip iv Histo~lasma capsulatum Live Kil led Mycel i al Mice Mice Yeast lenge Route Resistance Strong Increased increased Strong Increased Increased inc r. iv Increased Strong iv icer increased iv No inc r. sc icer Strong iv Increased fp,,;l{' icer Strong oral icer Increased iv No inc I sc subcutaneous im itr :::: intratracheal fp 0 ';t icer :::: intracerebral ip ritoneal intr :::: intravenou:; v Reference Salvin 1955 Salvin 1955 Schaefer et al. 1954 Rambo & Marcus 1955 Salfelder 1964 Salvin 1955 Hi 11 & Marcus 1959 Rambo & Marcus 1955 Row 1ey et a 1. 1956 Salvin 1955 Farrell et a 1~ 1953 Salvin 1953 Salvin 1953 Schaefer et al. 1954 Hi 11 & Marcus 1959 Rowley & Huber 1956 Hi 11 & Marcus 1959 Salvin 1953 Hi 11 & Marcus 1959 Rowley & Huber 1956 Salvin 1953 Hill & Marcus 1959 Salvin 1953 Salvin 1953 Hi 11 & Marcus 1959 = intramuscular = foot pad N 22 constant death rates. A total of 220 mice were inoculated intracerebrally with yeast phase cells in physiologic sal ine~ The average LD50 at 21 days was determined in three separate experiments to be 3.0 x 10 4 organisms for H. 1atum strain 6515. Immunization of mice for short periods of time was accomp1 ished by intraperitoneal inoculation with 5 mg of acetone-dried yeast phase cells, the broth filtrate from the mycel ial growth, or the broth filtrate from the growth of the yeast phase cells. Subsequent intracerebral challenge revealed protection against 300-400 LD50 of the fungus. Seve r ali mm un i z at i on r ou t e s ~ i n t rap e r ito n e a 1, i n t r a ve no us, and subcutaneous, were effective. Salvin (1955a) demonstrated that mice immunized with acetone-dried yeast phase cells had less infected tissues (spleen, 1 iver and kidney) than non-immunized mice. The presence of the fungus in tissues was determined by tissue section. The difference between the two groups was most noticeable during the first three weeks after intracerebral challenge with a lethal dose of yeast phase cells. In non- immunized mice very rapid growth of the fungus occurred during the first few hours after chal1enge 9 and then a slow and gradual decrease in numbers of yeast phase cells occurred in the tissue cells during the next few months. Immunization 23 was relative in that it tended merely to lower the number of cells in various tissues. Resistance to reinfection in experimental histoplasmosis was shown by Salvin (1955b)o Mice previously infected with H. capsulatum were found to be more resistant to subsequent intracerebral challenge than normal mice. This resistance was manifested by a decrease in death rate, mice that developed resistance after a sublethal peritoneal In intra- inoculation, subsequent intraperitoneal challenge resulted in the greatest number of fungus cells developing in the spleen, 1 iver, and at the site of injection. In intracerebral1y challenged mice which were immunized by a sublethal intracerebral injection, the fungus cells occurred in the 1 iver~ spleen, and brain. In control animals after intracerebral chal1enge 9 the organisms were found in lungs and kidney, In general, previous in tion tended to inhibit the growth of the fungus in the sp1een g 1 iver, and at the site of inoculation. Rowley and Huber (1956) studied the growth of 1a um in norma 1, II H. capsu- supe r i n fec ted ' ! (recha 1 1enge of in fec ted animals), and immunized mice. Chronic infections were produced in mice by injections of small numbers of viable yeast phase cells, approximately 70 aggregates. were resistant to the lethal ef These mice ts of 10 6 aggregates 24 injected intravenously. Growth of the fungus was inhibited in superinfected mice whereas in controls 9 after initial rapid multipl ication, there was a progressive decl ine in the number of viable organisms in the tissues. Mice immunized with a killed yeast phase cel 1 vaccine~ 10 9 cel Is given intraperitoneal ly or 10 8 cells given intravenously} failed to show any effect on the growth of tissues. Ho capsulatum in It was apparent that immunization with kil led yeast phase cells did not inhibit growth of the fungus in the 1 iver and spleen after intravenous challenge of 80 cells, Survival of Ho capsulatum in experimental histoplasmosis was demonstrated by Saslaw and Schaefer (1956). Mice were immunized and challenged by the intraperitoneal route of injection. The fungus was recovered frequently from the reticuloendothel ial tissues as late as 45 weeks after infection. Mice surviving previous sublethal challenge or those previously given one dose of heat-killed organisms showed 1 ittle variation. The only difference observed was a greater number of positive organ cultures in the sublethal group between 16 and 30 weeks. Hill and Marcus (1959) studied the quantitative aspects of resistance induced against H. capsulatumo Using the intravenous route, LD50 determinations were studied using the yeast phase of the fungus. Strains exhibiting low and 25 high virulence were found by intraperitoneal of each strain. Groups of mice were immunized injection of formal in-kil led yeast cells The animals were challenged intravenously with varying numbers of yeast phase cells of the high lence strain. viru~ Either strain of the fungus was as effective as the other in immunization when compared to non-immunized control animals. The effect of route of immunization using either subcutaneous, intramuscular~ intravenous routes was studied. intraperitoneal or It was found that similar resistance levels were achieved by each route. Infection- immunity was studied for its effect upon subsequent challenge. The group that was sublethally infected showed the same type of dose-dependent mortal ity relationship that was observed in the other groups of immunized animals. An important consideration was the dose of vaccine. The extent of immunity induced by kil led yeast phase Ho capsulatum was found to be dose-dependent (Schaefer and Saslaw, 1954; Salvin~ 1955) and at high doses the extent of resistance was comparable to that fol lowing sublethal infection (Saslaw and Schaefer, 1954; Salvin and Marcus, 1959). to~. t 1955b; Hill Using 1 ive organisms the immune response capsulatum was found to be dose-dependent (Salvin 9 1955a). Although the dependency was less pronounced than with killed organisms, the presence of the immune response 26 demonstrated that strong stimulation occurred only after a threshold of immunogens was attained. Speculations concerning the high immunogenicity of 1 ive organisms have been discussed by Salvin (1960). One speculation is that the 1 ive organisms multiply and thereby increase their immunogen content in the host. Such mu1tip1 ication was evident after sublethal infection with Histoplasma (Rambo and Marcus~ 1954; Hill and Marcus, 1959). Resistance evoked by 1 ive organisms was 1 ittle influenced by the parenteral immunization route used (Table 1). The vaccination route appeared to be of major importance in fungal immunity induced by non1 iving vaccines. Aside from varia- tions in the virulence of fungus strains (Drouhet and Schwarz j 1956; Howell and Kipkie, 1950) it is well documented that the capacity of a given strain to kill animals varies with the route of in ~. ction and with the host. The LD50 of capsulatum for mice increases, in terms of numbers of or g ani s ms, when a dm i n i s t ere din the f 0 1 1ow i n g 0 r de r : i n t r a - cerebrally, intravenously, intraperitoneally (Salvin, 1955a), However, using the intramuscular, intraperitoneal, subcutaneous, and intravenous routes for immunization~ Marcus and Hill (1959) found similar resistance achieved by each immunization route to subsequent intravenous challenge. In monkeys (Macaca mu1atta) intratrachael and intravenous injections of 27 ~. capsulatum yeast phase appeared to produce a more severe dis e a set han t hat f 0 1 1ow j n gin t ran a sal Carl isle and Sparks, 1960). i n oc u 1a t I on (S a slaw In mice the intranasal route generally caused more severe histoplasmosis (Salvin} 1955a) than the intraperitoneal route Immunized mice showed a lowered resistance to respiratory challenge than to challenges by other routes (Grayston and Sal vin J 1956) 0 These workers found that mice given killed yeast cells of H. capsulatum showed less severe pathological changes after intracerebral challenge than after intranasal challenge, The foregoing reports indicate the importance of the challenge route in vaccine studies The intracerebral intra- venous? and intraperitoneal routes generally al low the organisms to disseminate rapidly to various organs~ respiratory route? in appropriate doses, whereas the initiates primarily a pulmonary disease (Grayston and Altman 9 1954; Procknow~ Page? and Loosl i? 1960) with extra-pulmonary dissemination as a later occurrence. It seems apparent from the reports concerning ~. capsulatum that immunity induced by 1 ive organisms persists longer than that induced by killed organisms. The duration of immunity evoked by 1 ive organisms may be attributed to prolonged stimulation provided by the persistence of the fungus in the host tissue (Salvin~ 1955a). Salvin (1955b) found that 28 resistance to histoplasmosis induced by alive vaccine was when the mice were challenged three days first detect after immunization and that it remained strong for at least 14 days, Immu nit y f 0 1 1ow i n g sub 1e t hal also effective against ~. in t ion \tJas capsu1atum (Schaefer and Sas1aw) 1954) at 40 days and at 90 days (H ill and Marcus ~ 1959) 0 However, with kil led vaccines 9 immunity to histoplasmosis decl ines over a relatively short period. Using a vaccine from yeast phase of Ho capsu1atum, Salvin (1955b) reported a decl ine with time, in resistance to intracerebral challenge. Mice were protected against a chal len of 320 LD50 adminis- trated six to f6urteen days postvaccination but at 24 days they were protected only against 10 LD500 IV. MANIFESTATION OF THE IMMUNE RESPONSE IN HISTOPLASMOSIS The most stringent tests of efficacy in experimental immunization are the prevention of overt in ctlon after challenge or the induction in the host of resistance sufficient to clear infection organisms without the development of serious illness. Live yeast phase (Salvin~ 1955b) preparations have enabled animals to withstand severe challenge and to survive for extended periods. Suppressed growth of ~o capsulatum has also been reported in mice immunized with 1 ive or killed organisms and cha1len d intranasallY9 intraperitoneallY1 intravenouslY9 29 or intracerebrally, and in guinea pigs challenged intraperitoneally (Salvin, 1955a; 1955b)o Initially after challenge, mUltipl ication was most pronounced at the site of infection in both control and immunized mice j and the extent of brain and kidney involvement varied with the route of infection ( Sal v i n 9 195 5 b ) 0 Grow tho f !:!. 0 cap s u 1a tum 0 c cur s 1a r gel y within cells of the reticuloendothel ial system and the organisms or lesions containing the organisms have been found invariably in the 1 iver and spleen regardless of the challenge route (Grayston and Altman, 1954; Grayston and Salvin, 1956; Larsh and Cozad, 1965; Procknow, Page? and L00 s 1 i, 1960; Sal v i n ~ 1965 ) 0 Row 1e y and Hub e r (1 956 ) indicated that growth of the organisms in tissues was inhibited in intravenously challenged mice only by the use of 1 ive cell vaccines. The six-day post challenge interval employed by Rowley might have been too short to detect low levels of inhibition. In the study by Grayston and Salvin (1956) brain involvement in control mice was extensive after intracerebral challenge, but there were only minimal inflammatory infiltrates in mice vaccinated with 1 ive or kil led organisms. Oif rences in the 1 iver were less marked but granulomas were few and well organized in immunized mice. The spleens in both groups contained many organisms but few lesions. 30 The killed cell vaccine appeared to be less effective against intranasal challenge than against intracerebral challenge. Pathological involvement after int anasal challenge was 1 imited and lung and 1 iver lesions in the control group were resolved spontaneously. Delayed dermal hypersensitivity reaction to H? capsulatum has been el icited in infected guinea pigs (Johnson and Scherago 9 1960; Knight and Marcus Hill ~ 1958; Marcus, Aoki 1965 ), r a b bit s (M ark ow i t z ~ 1964) j ~ mo n key s (S a slaw and J Carl isle? and Sparks, 1960) and rats (Okudaira and Schwartz} 1962). Killed organisms or their fractions also were effective in sensitizing guinea pigs (Larsh 9 1960; Salvin, 1955a)n Because of the relative difficulty in skin testing in the mouse~ delayed hypersensitivity in this animal was tested by injecting histoplasmin either intravenously or into the foot pad. Early deaths in mice previously vaccinated with 1 ive or killed ~o capsulatum were produced either by intra- venous injection of organism (Box and Briggs~ 1961) or by intraperitoneal injection of organisms in mucin (SalvinJ 1958)0 The state of hypersensitivity persisted for 14 months (Box and Briggs, 1961). v. I MMUNOGENS OF HIS OPLASMA CAPSULATUM The antigens which are protective in been found in the cell wall. ~o capsulatum have The yeast phase walls were as 31 immunogenic as killed whole cells and the protoplasmic fraction contained virtually no activity (Salvin and Ribi 1955). The cell wall of the dimorphic fungus contains 1 ipids (Blank~ polysaccharides, proteins, and chitin-l ike substances 1954; Pine, Boone, and McLaughl in!t 1966)0 It is presumed to be the locus of protective and serologically active antigens in the yeast phase of the organism. Salvin and Smith (1959) isolated a protein-carbohydrate complex from culture filtrates of autolyzed yeast phase Histoplasma cells and it was as immunogenic as kil led cells, In contrast, the protective capacity of a predominantly polysaccharide-containing preparation used by Knight, Hill and Marcus (1959) was inferior to that of kil led cells. The two findings were not necessarily contradictory; neither the isolation and purification procedures were the same nor were the immunization and challenge regimens similaro The specificity of immunity induced by ~o capsulatum has been examined by cross challenge both with heterologous organisms having antigens in common and with those not so constituted. Live yeast phase H. capsulatum increased resistance against challenge with Blastomyces dermatitidis (Salfelder and Schwartz, 1964) in miceo Kil led yeast cells increased resistance to Candida albicans challenge (Hasenclever and Mitchell 1963)0 Hedgecock (1961) found that j 32 immunization with a vaccine prepared from the mycel ial phase of H. capsulatum increased resistance to challenge by Mycobacterium tuberculosis. Induced immunity is not strain- specific in experimental histoplasmosis (Hill and Marcus 1959; Ramb 0 VI 51 Mar c us, and Gun n, 1955 ) 0 MECHANISMS OF THE IMMUNE RESPONSE TO HISTOPLASMA CAPSULATUM Mechanisms and development of immunity to the mycoses have received 1 itt1e study. The significance of antihisto- plasma antibodies in resistance is still obscureo Such antibodies conferred no passive protection on recipient mice (Rowley and Huber, 1956; Salvin, 1960). The importance of a cellular role in immunity to H. capsu1atum was demonstrated by accelerated infiltration of inflammatory cells and associated development of granulomatous lesions (Grayston and Salvin, 1956). Concerning the role of phagocytosis in immunity with H. capsulatum, an intracellular results have been reported. organism~ In one study~ contradictory the extent of fungal growth in macrophages from vaccinated mice was the same as that in macrophages from control mice (Howard, 1965). I not her stu die s 1 ( Hill and Mar c u s 1 1960) p rio r i mm un i z a t j on increased the digestive capacity of macrophages (Miya and Marcus, 1961; Wu and Marcus, 1963). Studies in animals designed to elucidate the patho- 33 genesis of Ho capsulatum infection and to develop potent vaccines have been hampered by intrinsic and extrinsic factors. The hazards involved in handl ing this pathogenic spore-forming fungus, and its morphological variabil ity present difficulties in experimentationo inroads into the understanding of fungal Nevertheless, immunity have been made; enhanced resistance to the fungus has been achieved by immunization. Also the site of the immunogenic substance(s) appears to have been local ized in the yeast phase cell wall? Immunomycology has received less attention than viral and bacterial immunology. ecological knowledge However expands~ j as epidemiological and publ ic health statistics point increasingly to the desirabil ity of fungal vaccines, especially in regions of endemic systemic mycotic disease. MATERIALS AND METHODS I ORGAN ISMS 0 Cultures of Ho capsulatum in the yeast phase were maintained at 37 0 C on antibiotic human blood agar, These were transferred at weekly intervals. Mycel ial cultures of ~o capsulatum were maintained at room temperature on SabouraudBs dextrose agar and were transferred every three to six months. Strains were also maintained under sterile mineral oilo Table 2 1 ists the twenty-three strains of H. capsulatum used in these studies and indicates the origin of the culture" These strains were originally isolated from human and animal sources j and from soil samples The yeast phase of the strains of the fungus were obtained by J...!l vitro or i.!l vivo conversion of mycel ial elements, In vitro conversions were obtained by transferring mycel ial elements onto blood agar slants containing antibiotics and after incubation at 37 0 C. Close-growing moist mycel ial colonies developed which were transferred until the yeast phase appeared. Mice were injected either intraperitoneally or intravenously with a sal ine suspension of the mycel ial elements to obtain ~ vivo conversion. The mice were autopsied at the end of two and four weeks and specimens 35 Table 2 Origin of stock cultures of Histoplasma capsulatum Strain number 21 71 a 2247 2584 2585 2586 2645 2779 2813 2870 2888 3014 3021 a b c d Strain number Or i gin human, undated human, undated human, 1957 so i 1 , 1957 human, 1954 human, 1957 from Japan, 1957 human, undated mouse, undated human, undated so i 1 , 1960 opossum, 1960 obtained obtained obtained obtained from from from from 3072 a 3154 3289 3321 3330 G10 b G56 G72 G75 6651 .~ G17M Origin undated from India, 1965 human? 1965 human, 1965 human 1965 human, 1949 var duboisi, undated human, 1959 human, 1960 human, undated human 1955 human~ j a j Dr. N. F. Conant, Duke University Dr. C. Campbe1 1, Harvard University Dr. C. Emmons, NIH University of Utah stock culture collection 36 from 1 iver 1 lung, and spleen inoculated onto antibiotic blood agar plates. The resulting yeast phase organisms were then transferred onto blood agar slants. I I. MICE Adult albino mice (Mus musculus) of mixed sexes, obtained from local sources were used in all experiments. Mice were maintained on Purina Laboratory Chow or Rockland mouse dieto I I I. MEDIA All cultures were grown and maintained on one of the f 0 1 1ow i n g me d i a: Liquid. Yeast phase organisms for vaccine production were grown in the fo 11 ow i ng 1 iquid culture medium ( Hil1 Tryptose phosphate broth (Difco) Yeast extract Maltose Cystine Distilled water Sol id~ 9 1958) . 29.4 g 4 0 g 0 10 0 g 0 005 mg 1000 ml The medium used for in vitro conversion 1 maintenance and plate counts was antibiotic blood agar (Hill and Marcus, 1959). Tryptose phosphate broth (Difco) 29.5 g Agar (Difco) 20.0 g Human blood 15-20% Distil led water 800 m1 37 A final concentration of 25-50 units of penicill in and 25-50 ~g of streptomycin per ml of medium were added. The medium used for maintenance of the stock culture was Sabouraud's dextrose agar (Ajell0, Georg, Kaplan, and Kaufman 1 1963). Dextrose 40 9 Peptone 10 9 Agar 20 9 Distilled water 1000 ml Final pH of 5.6. Five media were employed in studying the sporulation characteristics and colony appearance. Modified Sauton1s medium (Will is and Cummings~ 50.0 9 Dextrose Asparagine 4.0 9 Citric acid 2.0 9 50.0 9 G1yce r i n KH2P04 MgS04 3 H20 7H20 NH4 ferric citrate 0.5 9 0.5 9 0005g Agar 20 Dis tilled water 1000 ml F i na 1 pH of 704. 9 1952). 38 Sabouraud's medium with phosphate (McVeigh and Morton, 1965) 0 Dextrose 40,0 9 Peptone 10.0 9 KH2P04 1.5 9 Agar 20 Distilled water 1000 ml 9 Final pH of 5.6. The fol lowing three synthetic media were modified after Artis and Baum (1963). Synthetic Medium One Dextrose 10.0 9 KH2P04 1,5 9 MgS04 · 7H20 0,5 9 CaC12 . 2H20 ( NH 4)2 S0 4 0.15g 2,0 9 Agar 20.0 9 Di st ill ed water 1000 ml Synthetic Medium Two Medium two was identical to one except 2 9 of asparagine was substituted for the (NH4)2S04. Synthetic Medium Three Medium three was identical to medium one except for the (NH4)2S04 which was excluded. 39 A final pH after steril ization of 7 2 was obtained in these synthetic media. IV. LDSO DETERMINATIONS Animals were injected intravenously with 0.5 ml of the yeast cell suspensions. The organisms used were grown at 37 0 C upon antibiotic blood agar The inoculum was prepared by suspending the organisms in 1% tryptose phosphate broth (Difco) in OQ9% NaCl (PSS). After aseptically filtering through glass wool, total numbers of cells were estimated from counts made in a hemocytometerQ phase cells were counted as one. Aggregates of yeast The concentration of the yeast phase organisms was adjusted with 1% tryptose phosphate broth so that each dose of organism was injected intravenously in 0.5 ml volume. The mortal ity ratio results after 30 days were analyzed by the use of either the method of Miller and Tainter (1944) or the method of Litchfield and Wilcoxon (1949). V. STRAIN DESCRIPTION Twenty-three strains of.!::!.o capsulatum maintained on Sabouraud's dextrose agar in the mycel ial phase were described as to their macroscopic appearanceo The media employed were: modified Sauton's medium, Sabouraud's medium containing phosphate ion, and synthetic media l? 29 and 3, The nitrogen source was asparagine in Sauton's medium, peptone in 40 Sabouraudls medium, and (NH4)2S04 in synthetic medium 1 ~ asparagine in synthetic 2, and no added nitrogen in synthetic medium 3. Plates were poured from each agar medium and inoculated with mycel ial elementso The plates were sealed with parafilm and incubated at room temperature. The plates were observed frequently for contamination as well as type of growth? Af te r 10 weeks of growth the cu 1 tu res we re k ill ed by exposu re to formaldehyde fumes for 2-7 days at room temperature, This procedure killed all cells as demonstrated by failure to multiply on Sabouraud's agar. The macroscopic appearance of the colonies was described in mycological nomenclature and documented with photographso VI. SPORE MEASUREMENTS Microscope sl ides were made of each strain grown on each medium The preparations were observed for the pro- duct ion of microconidial and macroconidial spore types? The method of preparation of the microscope sl ides was based on a procedure described by Oro N. F. Conant (personal comm un i cat i on) . Api e ce 0 f t ran spa r en t cut to fit under a coversl ip. II S tic k y tap e II was The sticky tape was then pressed against the formal in-killed growth of the funguso The tape was removed with the spores and hyphal elements 41 adhering and placed sticky side up onto a microscope sl ideo Lacto-phenol cotton blue was dropped onto the tape and the coversl ip appl ied. The preparation was pressed with the eraser end of a pencil to remove any trapped air bubbles from beneath the coversl ip. Lacto-phenol cotton blue mounting fluid was prepared according to the following formulation: (Ajell0 9 Georg, Kaplan, and Kaufman 9 1963). Phenol crystals 20.0 g Lactic acid 20.0 g G1yce r i n 40.0 g Cotton blue (Poirrier's blue) Distilled water 0005g 2000 ml The diameters of the microconidia and of the macroconidia were measured using an ocular micrometer. The diameter was measured from cell wall surface to surface not including the tuberculations of either spore type. The ocular micrometer was standardized for the optics of the microscope in use in this laboratory using a stage micrometer. Spore sizes are averages of six to ten measurements. The relative numbers of the spores were determined o A numerical rating was employed as follows: none observed (0), rare (1), few (2), and numerous (3)0 is self explanatory. "None observed" The sl ides were observed for two to 42 five minutes using the low power objective (total magnification, 60X) for macroconidia, and then the high dry objective (total magnification, 264X) for macroconidia o IrRare" means the spores were difficult to find; only by moving to various oil immersion fields (total magnification, 600X) were any observed. II Few" is defined as observing one field in which one or two spores could be found. "Numerous" is defined as more than two spores per observed oil immersion field. Using this rating system, a number was calculated to represent macroconidial and microconidial spore production in the various media. A ratio of macroconidia to microconidia was determined for each strain on the five media. VI I. VACCINE PREPARATION Strains of H. capsu1atum were grown in the mycel ial phase of Sabouraud's dextrose agar containing phsophate ion? After 10 weeks of growth the cultures were killed by exposure to formaldehyde fumes a~ described previously. The mycel ial grow t h was r emov e d as e p t i ca 1 1 y by s c rap i n g t he colon y sur -, face with a scalpel after covering the surface of the large p 1a s tic pet rid ish e s (1 5 0 x 15 mm) wit h sal i n e con t a i n i n g 0.1% Tween 80. and mycel ial The resulting suspension, composed of spores elements~ was concentrated by centrifugation 43 (800 x g) and then washed in sal ine solution. After the final wash, the spores and mycel ial elements were suspended in sal ine. Yeast phase suspensions of the organisms were grown in 1 iquid culture medium. One hundred ml of med um was placed in 500 ml Erlenmeyer flasks. Suspensions of yeast phase organisms grown on antibiotic blood agar were used as inocula o The cultures were incubated at 37 0 C until maximum growth was obtained (3-7 days) The flasks were agitated by a mechanical platform shaker (16 rpm) during the incubation period. Formal in was added to a final concentration of 0.5% and the flasks were kept at 37 0 C overnight. This procedure killed all cells as demonstrated by culture on antibiotic blood agar. The cells were concen- trated by centrifugation (800 X g) and then washed in sal ine, After the final wash the cells were suspended in sal ine, After the final wash, the suspension of yeast phase or mycel ia1 phase cells in sal ine was transferred to a 450 ml solution bottle (3" x 7" diameter, height) containing numerous glass marbles (16 mm diameter) and stainless steel balls (10 mm and 6 mm diameters). placed upon revolving rollers. The bottle was then The ball mill was al lowed to run continuously for 48 hours (myce1 ia1 phase) or 168 hours (yeast phase) in the refrigerator (ca. 50 C) 0 After 44 grinding the cellular debris was removed by centrifugation at low speed (400-500 X g) and the supernate fluid employed as vaccine (designated a cell free extract). Macroconidia were separated from the mycel ium and m.icroconidia using a floation method (Stewart and Meyer, 1932)0 The spores and hyphal elements were removed after covering the surface of the petri dish with sterile distil led water. The surface was scraped 1 ightly with a scalpel. The suspension was removed and pipetted into a large glass tube (7 3/4" x 1 1/4"). The upper layer was removed after a 20 minute settl ing period, The procedure was repeated twice to further remove the mycel ium and microconidia from macroconidia. The vaccine preparations were weighed by pipetting 1,0 ml of each preparation onto tared steel planchetso Three al iquots of each vaccine preparation were dried in an oven at 87 0 C until constant weight was reached. weights were obtaine~ After the dry appropriate dilutions were made for immunization procedures. VI I I. TESTS USED TO CHARACTERIZE VACCINES The vaccine preparations were tested for nitrogen total hexose concentration. and The methods were found in Kabat and Mayer (1962) and will only be summarized. 45 Estimation of protein with Fol in-Ciocalteu phenol reagent The method used is a modification for the estimation of 10-100 ug of nitrogen. determinations is about! 1 The error reported for repeated ~g nitrogen. Reagents: 1. Folin reagent (Fisher Scientific Coo). 2. 12.5% solution of Na2C03 (anhydrous). 3. 0.1% solution of CuS04 . 5H20. Procedure: Two ml of solut ion were measured into a tube. Six ml of the Na2C03 solution and 100 ml of the CUS04 solution were added, mixed and allowed to stand for one hour at room temperature. One ml of 1:3 freshly diluted Fol in reagent was slowly added with constant mixing. After 20 to 30 minutes, the determinations (standard solution and unknown solutions) were read at 750 mu against a blank of 2 m1 of distilled water to which all the reagents had been added. The Coleman Model 30 spectrophotometer was used. Anthrone reaction for total hexoses The reaction depended upon the formation of furfural derivatives and was best used in the range of carbohydrate from 50-250 ug. Reagent: 1 c Two grams anthrone in one 1 iter of concentrated H2S04. 46 Procedure: Ten ml of anthrone reagent were pipetted into test tubes in a water bath at 10-15 0 C. carefully layered above the reagent. of glucose were included. mixed. The sample (5 ml) was Blanks and standards Each tube was agitated until The tubes were warmed to room temperature and heated at 90 0 C for 16 minutes, cooled and read in the Coleman Model 30 spectrophotometer at 625 mUg I X. I MMUN I ZAT I ON Groups of mice were immunized by using the prepared formal in-kil led vaccines. Each animal received an intra- peritoneal injection of 0.25 ml using various schedules as noted in the experiment. 47 EXPERIMENTAL RESULTS I. MACROSCOPIC APPEARANCE OF COLONIES Description of each strain of H. capsulatum on five different media grown at room temperature for 10 weeks is presented in detail. Each strain was described according to the following: 1) Sabouraud's dextrose medium with phosphate ion, (Table 3); 2) modified Sauton's medium, (Table 4); 3) modified Sauton1s medium without dextrose, (Table 5); 4) synthetic medium 1, (Table 6); and 5) synthetic medium 2, (Table 7). The macroscopic colonial characteristics were not described for synthetic media 3. Growth was scant on this medium which contained no added nitrogen source. Growth on synthetic medium 1 was similar to that obtained in synthetic medium 2. Sabouraud's and Sauton1s media both grew colonies differing in appearance. The nitrogen source in each medium was: 1) Sabouraud's, peptone; 2) Sauton's, asparagine; 3) synthetic 1, (NH4)2S04; and 4) synthetic 2j asparagine. The carbohydrate source in all of the media was dextrose. Sabouraud's and Sauton1s media supported best growth in these experiments. growth. Synthetic 2 also supported good On synthetic 1, synthetic 3 and Sauton1s without 48 Table 3 Strains of Histoplasma capsulatum grown on Sabouraud's dextrose medium with phosphate ion for ten weeks Strain G-1O: colony flat, granular, with tan and white concentric rings. Strain G17M: colony uniformly tan with concentric rings and lobate edges. Strain G-56: colony cottony white and tan concentric rings. Strain G-72: colony cottony, flat, tan with white variation Strain G-75: colony white and cottony, flat, Strain 2171 : co 1on y va rie gat B d whit e and tan cottony. Strain 2247: entire colony tan, wooly, with concentric rings. Strain 2584: colony center raised and folded, surface wooly, central color 1 ight tan, concentric ring of darker brown. Strain 2585: colony tan with powdery surface raised from agar surface and folded with numerous radial grooves J f 1a tan d o Strain 2586: entire colony heaped, glabrous ridges and powdery valleys. Strain 2645: colony flat, powdery, with cottony white peripheral growth and tan to brown center. Strain 2779: colony surface smooth and wooly with few folds, gray to tan in color. 49 Table 3 (continued) Strain 2813: colony center heaped, central area gray, edges tan to brown Q Strain 2870: colony center heaped with irregular folds, entire colony powdery and tan. Strain 2888: colony cottony with heaped center, center gray, peripheral growth 1 ight tan. Strain 3014: colony heaped and irregular folds, surface powdery, peripheral growth tan. Strain 3021 : colony dark tan, center raised and cottony, outside margin powdery. Strain 3072: colony center heaped, periphery with irregular folds, color of center white to tan with edges tan and powdery. Strain 3154: colony gray with brown concentric rings, center heaped with many cracking ridges. Strain 3289: colony divided, one gray and cottony with ridges, the other tan and powdery with heaped and ridged areas. Strain 3321: entire colony heaped with irregular folds, colony uniformly gray and powdery. Strain 3330: colony center crateriform, edges with irregular folds, folds powdery white, colony center glabrous. Strain 6651: colony with white cottony center, peripheral growth white to tan, entire colony flat. 50 Table 4 Strains of Histoplasma capsulatum grown on modified Sauton1s medium for ten weeks Strain G-10: colony with white powdery peripheral growth, surface at the center is glabrous, waxy with radial folds and steel-gray in coloro Strain G17M: colony crateriform with steel-gray center which is waxy and glabrous, peripheral growth granular to cottony. Strain G-56: colony heaped with a white fluffy surface, Strain G-72: colony white and powdery, center heaped and crateriform. Strain G-75: no growth. Strain 2171 : colony with raised center with radial folds, color even throughout, surface tan and granular. Strain 2247: colony heaped and irregularly folded, surface powdery to downy, center glabrous and steelgray with color following some of the radial grooves. Strain 2584: colony center cottony, concentric rings of g ray, ou t sid e r i n g whit e, wit h mar gin p owd e r y and gray. Strain 2585: colony center raised and purple with peripheral growth gray with powdery appearance. Strain 2586: colony glabrous, center raised, color 1 ight brown. Strain 2645: colony powdery and granular with steel-gray glabrous center. 51 Table 4 (continued) Strain 2779: colony uniformly white, center raised with radiating ridges. Strain 2813: colony variegated brown and white, center raised, peripheral growth with dark brown concentric ring. Strain 2870: colony center grayish black with ridges, peripheral growth with brown concentric ring, Strain 2888: colony center heaped with ridges, dark gray in color, peripheral growth 1 ight gray, colony uniformly wooly. Strain 3014: colony center raised with numerous ridges, peripheral growth powdery and brown. Strain 3021: colony center raised and dark brown, outside margin gray and powdery. Strain 3154: colony center heaped and glabrous, dark brown in color, peripherally colony white radially furrowed and wooly. Strain 3321: colony center raised with many radial ridges, colony white to 1 ight gray except for concentric ring which is dark gray. Strain 3330: colony center brown and raised, edges powdery and white. Strain 6651: colony flat with folds, peripheral growth powdery to cottony, center raised, steel-gray and waxy_ 52 Table 5 Strains of Histoplasma capsulatum grown on modified Sauton1s medium without dextrose for ten weeks Strain G17M: colony uniformly gray and wooly_ Strain G-75: colony uniformly dark tan and powdery. Strain 21 71 : colony white with raised center. Strain 2584: colony uniformly tan and cottony. Strain 2645: colony center raised and 1 i ght tan, peripheral growth brown. Strain 2870: colony grayish white and woo 1 y. Strain 2888: colony dark brown with 1 i ght tan concentric ring. Strain 3321: colony center raised and dark gray, peripheral growth grayish white. Strain 3289: colony center raised with ridges, color grayish white. 53 Table 6 Strains of Histoplasma capsulatum grown on Synthetic medium 1 for ten weeks Strain G-1O: colony center sl ightly raised, entire colony glabrous and smooth, colony cream colored. Strain G17M: colony center raised, surface cottony, color creamy. Strain G-56: colony center elevated, surface cottony and whi teo Strain G-72 : colony center heaped and cracked, surface wooly and cream colored. Strain G-75: colony white, raised center. Strain 21 71 : colony gray, entire colony shallowly wrinkled. Strain 2247: colony tan, entire colony deeply wrinkled. Strain 2584: colony center raised, entire colony glabrous and dark tan. Strain 2585: growth scant, tan and crusty. Strain 2586: colony white with tan raised central area, crescent shaped tan surface area peripheral, remainder of colony wooly. Strain 2645: colony glabrous and smooth, tan in color, center raised. Strain 2779: colony 1 ight tan, center raised, uniformly powdery. 54 Table 6 (continued) Strain 2813: colony center raised and 1 ight tan, colony cottony to powdery and 1 ight tan, Strain 2870: colony white with raised center, cottony, Strain 2888: colony center raised, glabrous, and gray, peripheral growth wooly and white. Strain 3014: colony center crateriform, edges lobed} entire colony powdery and tan. Strain 3021 : colony center heaped, periphery wooly and 1 i ght tan. Strain 3072: colony center sl ight1y raised, entire colony wooly and 1 ight tan. Strain 3154: colony center heaped, entire surface powdery and dark tan. Strain 3289: colony glabrous and crusty, entire surface tana Strain 3321 : colony center heaped, uniformly gray and powdery. Strain 3330: colony center raised 1 entirely white and powdery. Strain 6651 : colony whitish, raised center, glabrous margin. 55 Table 7 Strains of Histoplasma capsulatum grown on Synthetic medium 2 for ten weeks Strain G-10: colony center white raised with furrows) peripheral growth scant, tan; and adherent. Strain G17M: colony center crateriform with radial furrows, growth white, peripheral growth tan and adherent. Strain G-56: colony white with patches of tan growth scant~ cottony. Strain G-72: colony whitish with diffuse tan throughout1 raised center 1 radial grooves near periphery, growth generally granular. Strain G-75: colony uniformly white, with raised center} growth wooly. Strain 2171: colony variegated white and and furrowed. Strain 2247: colony center gray-white, raised and furrowed} peripheral growth white and cottony. Strain 2584: colony uniformly grayish white, smooth and powdery. Strain 2585: colony center heaped~ and folds few and irregular, entire colony white and cottony. Strain 2586: colony center crateriform, edges glabrous, center powdery white edges smooth and tan. tan~ peripheral center raised 9 Strain 2645: colony center crateriform, edges smooth and powdery, central colony dark tan, periphery 1 ighter. 56 Table 7 (continued) Strain 2779: colony tan with white concentric ring, uniformly powdery. Strain 2813: colony center heaped, entire colony powdery and 1 ight tan. Strain 2870: colony cottony, smooth and white. Strain 2888: colony with white central region with wide peripheral, band of center powdery, ba~ glabrous. Strain 3014: colony gray with 1 ight tan center, cottony and raised from agar surface Strain 3021: colony center heaped, and gl istening irregular folds to edges, entire colony white. Strain 3072: colony center heaped, powdery and white, peripheral growth tan and wooly. Strain 3154: colony center raised sl ightly, entire colony smooth and cottony, colony center 1 ight tan, edges dark tan. Strain 3289: entire colony dark tan, smooth and powdery. Strain 3321: colony center sl ightly heaped, central colony 1 ight tan, edges tan and wooly. Strain 3330: colony center raised with few folds, center tan and powdery, edges white and cottony. Strain 6651: colony flat entirely white, with scant growth in peripheral areas. smooth~ 1 57 dextrose the strains of Ho capsulatum grew poorly and after 10 weeks, only covered the central area of the petri dishes. Figure 2 shows color photographs of macroscopic . colonies of four strains of -H. capsu1atum grown at room temperature for 10 weeks on Sabouraud's medium with phosphate ion. The strains shown are: I I. G10~ G17M? G56, and 2779. SPORE SIZE MEASUREMENTS Spore size measurements were made from observations on 23 strains of ~. capsu1atum grown on Sabouraud's dextrose media with phosphate ion, modified Sautonis medium, and synthetic media 1, 2, and 3 (Table 8). and microconidia were measured. Both macroconidia The values were reported as averages for both of the spore types. The blanks in the table represent cultures which failed to grow or were contaminated. The zeros indicate that no spores were found of that type. The macroconidia1 and microconidial spores ranged in size from 6.5 - 14.3 u and 2.3 - 5.8 ~o The averages of the measured spores were: 1) Sabouraud's macroconidia, 12.2 ~ and microconidia, 3.8 u; 2) Sauton's, 10.5 u and 4.0 u; 3) synthetic 1, 9.4 u and 3.8 U; 4) synthetic 2? 9.7 u and 3.2 ,u; and finally 5) synthetic 3? 10.9 The largest macroconidia (12.2~) ~ and 3.5 u. were formed on Sabouraud's Figure 2. Strain G10 Strain G17M Strain GS6 Strain 2779 Four strains of Histoplasma capsulatum grown on Sabouraudls dextrose medium with phosphate ion at room temperature for ten weeks" V1 00 59 Table 8 Histoplasma capsulatum macroconidia and microconidia spore sizes on five media Strain No. SAB C P04 G10 G17M G56 G72 G75 2171 2247 2586 2585 2585 2645 2779 2813 2870 2888 3014 3021 3072 3154 3289 3321 3330 6651 - 'k 'I','i~ Sauton1s _...)( 13.1/5. ]"i'o'\ 0/2.7 10.1/3.7 0/0 0/0 0/3.0 11.8/0 0/0 14.3/4.5 10.7/3.1 12.1/4.0 11.0/0 0/0 0/3.5 12.0/3.0 13.5/5.7 10.2/0 0/2.5 13.7/3" 1 0/3.8 0/0 10.0/4.2 0/0 12.8/0 10.5/5.8 0/0 0/0 0/0 10.3/4.5 9.5/4.0 12.5/3.8 10.0/3.3 8.5/0 11 • 1/0 #1 #2 Synthetic #3 9.8/0 7.3/3.0 11.6/5.2 9.7/4.0 0/3.5 0/0 9.3/2.3 0/4.7 9.1/4.3 0/3.7 8.9/3.8 12.0/4.2 9.8/2.7 0/0 9.1/3.8 10.4/5.0 9.3/4.3 10.5/4.9 10.5/3.7 9.0/2.7 6.5/2.9 0/3.7 7.0/4.5 10.9/0 9.0/3.0 10.4/306 0/3.2 9.5/0 0/2.8 9.9/3.5 0/3.0 11 .6/3 0/2.3 0/3.5 0/0 8.5/4.9 9.3/3.0 9.1/2.9 0/0 0/0 0/0 0/0 0/3.5 11.0/3.1 9.5/3.3 9.3/2.7 0/3.0 8.5/2.8 (data not obtained) macroconidia/microconidia, diameter i n microns 0/2.7 10.6/3.2 0/0 8.3/3,3 0/0 12.6/5.7 9.0/4.0 11.7/4.5 11.0/4.3 0/0 12.0/0 14.2/3.8 9.7/2.9 10.0/.3.3 13.6/3.8 0/3.3 0/2.7 0/3.2 60 with phosphate ion. Sautonis (4.0 ~). The largest microconidia were formed on Mean sizes for microconidia on Sabouraud1s with phosphate and synthetic 1 were both 3.8 ~. The results shown in Figures 3, 4, and 5 were the average and the range of each spore size for each strain represented. Each point represents the macroconidial and microconidial spore size for each strain with the macroconidial size increasing along the abscissa. The results of spore size measurements of synthetic media l? 2~ and 3 showed that no correlation existed between the size of the macroconidia and microconidia on synthetic media. I I I. RELATIVE NUMBERS OF SPORES Table 9 shows the relative numbers of macroconidia based on the rank system previously described. For each strain grown on each of the five media, the rank ratios given were for macroconidia over microconidia. Discarded cultures were indicated by blanks. The last column in the table shows a number which represented the sporulation character of the strains of H. capsulatum studied. A number above 1.00 indicated that the strain had produced large numbers of macroconidia, a number below 1.00 indicated that the microconidia predominated. number of 1.00 indicated that the strain produced an equal A 17.0 16.0 .,., • -0- macroconidia microconidia - LO C """I (D 15.0 . w 14.0 3::J: 13.0 () 12.0 T (/') 11.0 ""0 0 """I (D (/') (1) ....1:: -.- 8.0 7.0 6.0 ! 5.0 2.0 J 61 4.0 3.0 t I 0 1 T 1 1 1 •I I 1 I 1 I !1 j I1 1• 10.0 -. 9.0 N I I -- • rt 0 0 0 ---' -0 (/') () ::J ill 3 -. ill ill 0... ---' ill ""0 (/') 0 C l (D T -. c3 I1 1 J T T I : ---' ill rt (/') (D 1r 1 1 I () (/') N r (/') """I I 1 I ,.. 1 :3 ill o () ::J l 0 (/') () -<0 ::J ::J rt -< ::::r 0... (D -. rtOJ -.- () 3 ill ::J (Do... 0... 1 .0 3321 6651 G17M 2645 3289 2585 2888 3021 G72 G10 2813 3014 3072 3154 G56 c :3 Histoplasma c3psu1atum strain number (j'\ 15.0 macroconidia microconidia 14.0 --- ." -0- , ro 13.0 12.0 I 11.0 I (j) 10.0 ,o ro 9. 0 ""0 I I (j) N ro -2 -..- 8.0 7.0 6.0 5.0 4.0 3.0 2.0 ~ o I1 I1 1 T r 1 +::3 I :::c O(j} ,M o o o 0 :J OJ -.(j) 0..3 --OJ OJ --0 OJ U'l ""0 ,o (fl C roOJ !.fl rt C --:3 I r N T 1r CD :3 o UJ 0 :::J I o o (flO :::J M -. ::::r-Q. ro -. MOJ 1 .0 o 6651 G17M 2813 2779 Histoplasma 3321 3289 2645 G56 strain n Gl0 er 3154 2247 UJ :3 :::J roo... 0... c: :3 N ()'\ N 15.0 macroconidia microconidia 14.0 ~ ~ " I 13.0 12.0 I 1 1 .0 lJ'l -0 o 'I ro 10.0 N ro '2 -- I 9.0 III 8.0 7.0 I 1 1O C , ro \n 3 1:::C 1 lJ . . :2 6.0 5.0 4.0 3.0 2.0 - I1 N I 1 ro :3 :3 w o () :::J "'\ o () o ::J It -. :::r Cl. (1) - . rtOJ 1.0 () 2585 2779 Gl0 3072 G56 3154 2247 t r 3 2645 3014 i 11 nLI(llbe r 3289 3 OJ :J (1)CL a.. c 3 w 0'\ w 64 Table 9 Histoplasma capsu1atum relative numbers of macroconidia and microconidia on five media Strain No. G10 G17M G56 G72 G75 2171 2247 2584 2585 2586 2645 2779 2813 2870 2888 3014 3021 3072 3154 3289 3321 3330 6651 a -;'(' SAB c P04 Sauton1s 3/3 a 2/2 3/3 3/3 0/3 3/3 0/0 0/0 2/0 0/3 3/0 0/0 3/2 2/2 3/2 1/0 0/0 0/1 2/2 3/1 1/0 0/3 3/2 0/1 2/3 2/3 0/2 0/0 2/2 0/0 3/2 2/0 3/2 0/0 0/0 0/0 3/2 2/1 3/1 3/1 2/0 1/0 2/3 #1 #2 Synt het i c media 2/0 3/2 3/2 2/2 0/ 1 0/0 2/2 0/1 1/2 0/1 2/2 1/2 3/3 0/0 3/3 1/2 1/2 2/3 3/3 2/3 2/3 0/2 1/2 3/0 2/1 3/2 0/3 0/0 0/0 3/3 0/2 0/3 0/0 3/3 3/2 2/3 0/0 0/0 0/0 0/0 0/1 3/3 3/2 1/3 0/1 3/2 1/0 2/ 1 3/2 0/1 0/0 0/2 3/3 0/0 1/2 0/0 3/2 2/2 3/2 1/1 0/0 1/0 3/2 3/3 1/ 1 3/1 0/2 0/1 0/2 Sporulation numbe r"" 2.00 1 00 1 . 25 0.33 0.00 0.80 0.62 o 00 0.78 0.00 1.27 1 . 25 1 . 17 2.00 1.00 0.67 1 . 13 1 . 11 1 .38 1 . 10 0.80 0.20 0.67 macroconidia rank number/microconidia rank number relative numbers of macroconidia to microconidia 65 number of macroconidia and microconidia. The figures for strain G10 served as an example for the calculations of the spore number: SAB C P04 Sauton 3/3 3/3 G10 Syn. 2/0 Syn, 2 3/0 Syn. 3 1/0 For the macroconidial number each numerator was added: 3 + 3 + 2 + 3 + 1 = 12. Similar calculations determined the number for the microconidia, i.e. a denominator of six. The ratio, sum of the numerators over sum of the denominators, i.e., 12/6 or 2.00 was the sporulation number and was a relative measure of the type of spores produced by the straino The value did not give any indication of the abundance of spores produced. The data presented suggested that a correlation existed between nutritional properties of the media employed and sporulation abil ity. This is based on the following obser- vations: two of 23 strains (G10 and 2870) produced a predominance of macroconidia as indicated by the sporulation number. However, the number of spores produced by G10 was abundant on four of the five media, whereas strain 2870 produced only scant numbers of spores on two of the five media. Two of 23 strains (G75 and 2586) produced only microconidia. Synthetic medium 1 yielded low numbers of microconidia from strains G75 and 2586, but no sporulation 66 occurred on the remainder of the media examined. Two of 23 strains (G17M and 2888) produced an equal number of both spore types as indicated by the sporulation number. On Sautorls medium G17M did not produce macroconidia and on the remaining media the microconidia and macroconidia were produced in varying numberso Both (G17M and 2888) of these strains produced varying numbers or no spores on the media on which they were grown. Two strains (G72 and 3330) produced a greater number of microconidia than macroconidia, as indicated by the sporulation number. The remaining strains yielded sporulation numbers that varied between the two extremes (0.20-2.00). Although the sporulation number characterized the relation between total macroconidia and microconidia formed, it gave no estimate of the abundance of spores formed. For example, strain G10 formed only macroconidia on the synthetic media but the number of macroconidia formed falls into the classification "rare." On the other hand this strain forms equal and large numbers of both spore types on Sabouraud's medium with phosphateo Calculations were made similar to those described above but designed in this case to describe sporulation for each medium. The rank numbers of each strain on each medium were added, i.e. adding vertically in Table 9. Media which 67 supported best macroconidia1 or microconidial production could be determined using this method. Results are recorded in Table 10. The results suggested that the medium which supported the best macroconidial sporulation was Sabouraud's with phosphate. Synthetic media 1 and 2, and Sauton's medium supported better microconidial production, Synthetic medium 3 supported formation of equal numbers of the two spore types. IV. VIRULENCE STUDIES In order to study the resistance of mice to infection with yeast phase~. capsu1atum~ it was desirable to know the virulence of selected strains and the effect that prolonged maintenance on artificial culture media had upon this virulence. Therefore, LD50 determinations were performed at various times throughout the course of this study. Data are presented in Table 11 which were obtained from LD50 determinations of H. capsu1atum strain G17M, at different periods. Data in the table prior to 1956 were taken from Hill (1958). The virulence of this strain of H. capsulatum did not change significantly over a period of 15 years. Furthermore, other factors such as mouse passage, human passage, and conversion did not change the virulence of this strain. 68 Table 10 Media supporting the best sporulation of Histoplasma capsulatum Medium Sporulation number Sabouraud's wi th phosphate ion 1 .30 Modified Sauton1s 0.83 Synthetic medium 0.79 Synthetic medium 2 0.88 Synthetic medium 3 1.00 69 Tab 1e 11 Mouse LD50 determinations of yeast phase Histoplasma capsu1atum strain G17M 1 LD50 ' Strain Da G17 a) 12/ 17/53 1 • 1 x 10 6 (0.76 - 1.6 G17 a) 5/11/54 2.7 x 10 6 G17-5 b) 10/18/54 9.0 x 10 5 ( 1 . 0 - 7. 0 x 10 6 ) (0.35 - 2.3 x 10 6 ) G17-10 b) 11/04/54 7.5 x 10 5 (0.28 - 2.0 x 10 6 ) G17-15 b) 1/31/55 1 .2 x 10 6 (0.66 - 2.2 x 10 6 ) G17 M c) 4/21/55 6.5 x 10 5 (0.33 - 1.3 x 10 6 ) G17 M c) 8/19/56 1.2 x 10 6 (0.80 - 1.8 x 10 6 ) G17 M a) 6/20/66 5.0 x 10 5 (0.02 - 9.8 x 10 5 ) G17 M a) 7/08/66 1 • 1 x 10 6 G17 M a) 7/10/66 6.4 x 10 5 (0.92 - 1.3 x 10 6 ) (3.4 - 6.4 x 10 6 ) G17 M a) 5/19/68 7.0 x 10 5 ( O. 18 - 1. 2 x 1 0 6 ) X 10 6 )+ LD50 determined by method of Miller and Tainter (1944) + 95% confidence 1 imits a) conversion from mycel ial phase b) mouse passage, one/week for 15 weeks; determination at 5, 10, and 15 weeks c) after human passage 70 Strain 3154 of H. capsulatum was found to be of the same virulence for mice over an 18 month period. Table 12 presents data obtained from LD50 determinations with this strain. Table 13 presents data obtained from virulence determinations on various strains of H. capsu1atum at different times throughout this study. The virulence of three of these strains (2645, 3330, and 6651) appeared to be similar. Strain 2813 was the least virulent of the six strains that were studied. V. IMMUNIZATION Using the sporulation number as a basis for determining the morphological characteristics of a strain, four strains were chosen to represent the mycel ial phase of tl. capsulatum, (Table 14). Strain G17M was used as the source of yeast phase cells. Mycel ial phase cultures were grown on Sabouraud1s dextrose agar for 10 weeks at room temperature and harvested. The harvest from the cultures was placed in a ball mill and a cell free extract was prepared. The cell free extract was preserved with formal in and used as a vaccine. A whole macroconidial vaccine prepared with spores from strain G10 was also used to immunize mice. 71 Table 12 Mouse LDSO determinations of yeast phase Histoplasma capsulatum strain 31S4 Strain Date 31S4 March, 1967 1 .4 x 10 4 t(88, 900)+ 31S4 Octobe r, 1967 9.6 x lOS :!"(60, 000) 31S4 January, 1968 6.0 x 10 4 ~(82, 31S4 Ap r i 1 , 1968 3.4 x lOS t( 160, 000) 31S4 June, 1968 6.2 x lOS ~( 1 70, 000) 31S4 May, 1968 3.0 x lOS :!"(200, 000) .,', 000) LDSO and standard deviation determined by method of Miller and Tainter (1944) + LDSO"k one standard deviation 72 Table 13 Mouse LD50 determinations of yeast phase Histoplasma capsu1atum of various strains Strain Date 2645 June, 1966 2.4 x 105 ::(2.0 x 105) 2645 December, 1967 :t(2,9 x 105) 2813 March, 1967 7.6 x 105 3.0 x 10 8 3330 Ap r i 1 , 1968 7.0 x 105 :±-(4.5 x 105) 6651 October, 1965 5.0 x 105 ~(9.7 ,', ~(4.3 x 10 8 ) x 105) lethal dose 50 :!" standard deviation after Miller and Tainter ( 1944) 73 Table 14 Strains of Histoplasma capsul atum util ized in immunity studies Strain Element produced SporulatIon number G10 Macroconidia 2 OO G75 Mycel ium 0.00 3330 Mi'croconidia OD20 2645 Mixture of both spores 1.27 D 74 Yeast phase cultures were grown on 1 iquid medium and harvested after five days at 37 0 Co The cells were either pulverized in a ball mill or were left intact and util ized as a vaccine. All groups of mice in these reported experiments were immunized by intraperitoneal injection of either formal in- killed cell free extracts or formal in-kil led whole cells. The doses of the challenging strain were also similar in each experiment, i.e., 1 x 105, 1 x 10 6 , and 1 x 10 7 population units injected intravenously. After termination of each experiment the remaining mice were autopsied and the lungs were cultured on antibiotic blood agar. The data were analysed by the method of Litchfield and Wilcoxon (1949) and the LD50 and potency ratios are given in each experiment. In the first experiment (Table 15) the cell free extract prepared from strain G10 was used. The sporulation number for this strain of H. capsulatum indicated that the strain produced mainly macroconidia. Mice in one group received a 3 mg (dry weight) injection of the vaccine. in the second group were injected with 2 mg. of mice were injected with 1 mg. injected with sal ine (PSS). Mice The third group The control group was Ten days after the initial Table IS Vaccination of mice against histoplasmosis Mice vaccinated with formal in-kil led cell free extract of the mycel ium and macroconidia of GIO and challenged with 31S4 30 day mortal ity ratios, per cent deaths, and culture results challenge dose 1 x lOS PSS control 3 mg 2 mg 1/9 (6/8 11% a 7S%)b 0/10 (1/10 0 10%) 0/10 (1/10 8/10 (2/2 80% 100%) 0/10 (1/10 0 10%) 1 x 10 7 10/10 (0/0 100% 0 3/10 (4/7 30% S7%) Totals 66% 80%) 4 . 2 x lOS :±"(2.S x lOS) 1 x 10 6 LOSO 19/29 (8/10 c P.R. d 10% 22%) 1.3 x 10 7 7 :±"(1.2 x 10 ) 31 (10-9S) 3/30 (6/27 1 mq 0/10 (2/10 0/10 (6/10 0 10%) 0 20%) 0 60%) 1/10 (S/9 0/30 (9/30 0 30%) 2/31 (10/29 0/11 (2/11 1/10 (3/9 a 18%) 10% 33%) 10% SS%) 7% 3S%) 6 1.0 x 10 6 :(9.4 x 10 ) 24 (7-83) a mortality ratio and per cent deaths b culture positive lungs/total survivors c LOSO = lethal dose SO and standard deviation d potency ratio and 9S per cent confidence 1 imits -........J \Ji 76 injections all mice were challenged intravenously with strain 3154. The results of immunization with G10, macroconidia1 vaccine are shown in Table 15 and suggest that there was a difference between 3 mg and 1 mg in one injectiono The per cent deaths and culture results indicated no difference between the two groups immunized with either 2 mg or 1 mg. A lower frequency of culture positive lungs occurred in the group immunized with 3 mg. The potency ratio comparison between 3 mg and 1 mg was 31 and 24 respectively. The results in a second experiment involving immunization with G10 and challenge after 10 days with 3154 are shown in Table 16. The culture results indicated that as the dose of the challenge was increased the number of culture positive lungs increased. Although the total culture positive per- centage was similar in the 0.8 mg and 0.4 mg groups, the potency ratio for 0.4 mg was 18.5 and the ratio for 0.1 mg was 5.0. Results in two experiments involving mice immunized with a cell free extract of the myce1 ium of G75 (a strain producing no spores) are recorded in Tables 17 and 18. The sal ine controls for both experiments have equal LD50 values of 1.3 x 10 6 . The number of culture positive lungs for yeast phase growth, varied from 12% in the 0.8 mg group to Table 16 Vaccination of mice against histoplasmosis Mice vaccinated with formal in-kil led cell free extract of the mycel ium and macroconidia of G10 and challenged with 3145 30 day mortal ity ratios, per cent deaths, and culture results challenge dose 1 x 10 5 1 x 10 6 - 1 x 10' PSS control 1/8 13% a 43%)b {3/7 3 mg not done 3/8 (4/5 8/8 not done COlO Totals LD50c 38% 90% ) 100% 0) 12/24 50% (7/12 58%) 7.0 x 105 ::(4.0 x 10 5 ) 1/5 (3/5 20% 60%) {1/5 20% 60%) (3/5 P.R. d 2 mg 1 mq 11% 67%) 0/8 (2/8 0% 25%) 1/9 (6/8 1/9 (6/8 4/9 ! (4/5 11 % 67%) 45% 90%) 3/8 38% (4/5 90%) 6/10 60% (4/4 100%) 5/26 38% (12/21 57%) 1.3 x 10 7 2-(1.6 x 10 7 ) 18.5 (6-57) .- 10/27 37% (14/17 38%) 3.5 x 10° ±(7.2 x 10 6 ) 5.0 (1.6-15) a mortal ity ratio and per cent deaths b culture positive lungs/total survivors c LD50 = lethal dose 50 and standard deviation d potency ratio and 95 per cent confidence 1 imits -.......J -.......J Table 17 Vaccination of mice against histoplasmosis Mice vaccinated with formal in-kil led eel 1 free extract of the mycel ium of G75 and challenged with 3154 30 day mortal ity ratios~ per cent deaths9 and culture results cha 11 enge dose 1 x 105 1 x 10 6 1 x 10 7 Totals LD50 c PSS control O%a 0/10 70%)b (7/10 7/10 70% 100%) (3/3 8/10 80% (2/2 100%) 15/30 50% (12/15 80%) 1. 3 x 10 6 :: ( 1 . 1 x 10 6 ) 1/9 (0/8 1/9 (1/8 1/9 (5/8 3/27 (6/24 3 mg 11 % O~) 11 % 13%) 11% 63%) 11 % 25%) P.R. d 2 mg 0°1/f) 0/8 (1/8 13%) ODIo 0/8 0) (0/8 1/7 14% (4/6 67%) 1/23 4% (5/22 23%) 2.6 x 10 7 ~(1.6 x 10 7 ) 1 mg 1/7 14% (2/6 ~~%) 0/7 0% (3/7 43%) 0% 0/7 (6/7 86%) 1/21 5% (11/20 55%) 20 (7.6-53) a mortal ity ratio and per cent deaths b culture positive lungs/total survivors c LD50 ~ lethal dose 50 and standard deviation d potency ratio and 95 per cent confidence limits ........, (X) Table 18 Vaccination of mice against histoplasmosis Mice vaccinated with formal in-killed cell free extract of the myce1 ium of G75 and challenged wIth 3154 30 day mortal ity ratios, per cent challenge dose 1 x 10 5 1 x 10 6 PSS control 10% a 1/10 89%)b (8/9 7/10 70% (2/3 67%) 1 x 10 7 7/10 (3/3 Totals 15/30 (13/15 LD50 P~R. 70% 100%) 50% 87%) 1 . 3 x 10 6 -r(1.8 x 10 6 ) c d 0.8 mg 2/10 20% 07'0' (0/8 0/11 0'-0 (0 111 9%) 0/7 0'0 (2/7 29%) 2/28 (3/26 7% 12%) -- -- deaths~ and culture results 0.4 mg 0/7 (2/7 1/7 (2/6 O~o 29%) 14% 33%) 6/10 60% (4/4 100%) 7/24 (8/17 29% 47%) 6.1 x 10 6 6 ::(6.5 x 10 ) 4.7 (1,.3 18) 0.1 mg 0/8 (1/8 1/8 (4/7 4/10 (3/6 0'0 13%-) 13% 57%) 40% 50%) 5/26 (8/21 19% 38%) 1.3 x 10 7 ::(1.7 x 10 7 ) 10 _(1. 3= 43 )_ a mortal ity ratio and per cent deaths b culture positive lungsltotal survivors c LD50 = lethal dose 50 and standard deviation d potency ratio and 95 per cent confidence 1 imits '-l \...0 80 80% in the sal ine control group. The potency ratios are as follows: 2 mg, 20; 0.4 mg, 4.7; and 0.1 mg , 10. The injection of 2 mg of vaccine gave the highest protection based on the potency ratio. The cell free extract vaccine prepared from strain 3330, was used to immunize mice and yielded the results shown in Table 19. The potency ratios were: 1 mg, 11.8; 0.4 mg9 7.6 and 0.1 mg, 1.5. The culture results indicated that as the dose of the vaccine was increased the number of culture positive lungs decreased. Using the cell free extract of strain 2645, which yielded a mixture of both types of spores, as a vaccine the potency ratios were: 15.4 for 3 mg and 9.3 for 2 mg and 1 mg. The results are shown in Table 20. The total per cent culture positive lungs for each group were: controls, 42%; 3 mg, 23%; 2 mg, 14%; and 1 mg, 31%. Vaccination of mice with the cell free extract prepared from the yeast phase of strain G17M gave results shown in Table 21. The potency ratios are: for 0.4 mg a value of 23 and for 0.1 mg a value of 11.5. The culture result totals ranged from 11-92%. Mice immunized with whole cells of either macroconidia of strain G10 or yeast cells of G17M gave the results shown in Table 22. The potency ratio calculated for each group of Table 19 V3ccination of mice against histoplasmosis Mice vaccinated with formal in~killed cell free extract of the mycel ium and microconidia of 3330 and chal1eng with 3 30 day mortal ity ratios, per cent deaths, and culture results cna' 1enge dose I PSS controll 1 x 105 not done 1 x 10 6 1 x 10 7 ta1s LO 50 c I 5/7 (2/2 7/7 (0/0 72% 100%) 100% 0~6) 1 mg not done I 12/14 86% (2/2 100%J 4.6 x 105 5 x 10 ) :±" 2 mg 0/5 (2/5 0/5 (1/5 0/10 (3/10 0% 40%) OPa 20%) O~a 30%) 004 mg 0/7 I (4/7 2/9 14% o I 22% 4/8 57%) (3/4 I 0.1 mg 2/7 50% J 7/8 75%) (1/1 29% 40o/n) 38% 60%) 88% 100%) 12/23 52% 3/23 12% 7/23 30% (3/20 40%) (6/16 37% (6/11 55%) 5.4 x 10 6 , 305 x 10 6 700 x 105 ±(5.5 x 106~ ±(l Q3 xl0 6 ) ±(1~2 x 10 6 ) PoRod 11.8 (4.2-31) I 7.6 (NC) 1.5 (0.43=5a2) a mortal ity ratio and per cent deaths b culture positive lungs/total survivors c L0 50 = lethal dose 50 and standard deviation d potency ratio and 95 per cent confidence 1 imits (X) T 1e 20 Vaccination of mice against histoplasmosis Mice v3ccinated with formal in-kil led cell free extract of mycel ium and spores of 2645 and challenged with 3154 30 day mortal Ity ratio5~ per cent deaths, and culture results cha 1 1enge dose 1 x 105 1 x 10 6 1 x 10 7 Totals LD50c P.R. d I PSS control 2/10 20% a (2/8 25%)b 7/10 70% (2/3 67%) 9/10 90% ( 1/1 100%) 18/30 60% L51J1_ 1.t2%) 5.6 x 10 5 ±(5.9 x 10 5J 3 mg 0/8 (0/8 0~;1 O~) 0/9 (3/9 0 0'10 33%) 2 mg 0/10 0% (1/10 10%) 0/9 o (0/9 o 5/9 (2/5 55% 40%) 7/10 (2/3 5/26 21% (5/22 23%) 7/29 (3/22 8.6 x 10 6 :!"16.2 x 10 6 ) '70% 67%1 24% 14%) 5.2 x 10 6 !(3.2 x 10 6 ) 15.4 (4.7-50) 9.3 (2.9-29) 1 mg 0/9 (0/9 I (3/9 0/9 O~i.,,' O~l 0';0 33%) I (3/3 7/1 0 70% 100%) 7/28 (6/21 25% 31%) 5,,2 x 10 6 ±(3.2 x 10 6 ) 9.3 (2a9-29) a mortal ity ratio and per cent deaths b culture positive lungs/total survivors c LD50= lethal dose 50 and standard deviation d potency ratio and 95 per cent confidence 1 imits co N Table 21 Vaccination of mice against histoplasmosis Mice vaccinated with formal in-ki1 led cell free extract of yeast phase G17M and challenged with 31S4 30 day mortal ity ratios, per cent deaths, and culture results cha 11 enge dose 1 x lOS 1 x 10 6 1 x 10 7 Totals LDSO c P.R. d PSS control 1/8 13% a (7/7 100%)b 3/8 38% (4/S 90%) 8/8 100% oj:, ) (0/0 12/24 SO% (11/12 92%) 7 x lOS i"(4.4 x lOS) 1 mg 004 mg 0/8 0 (0/8 o ) O~ 0/9 (0/9 0/8 (1/8 0/8 (2/8 0%) 0'"'13%) 0 2S%) 0/27 (3/27 11 %) "":';" -- --- o ,", 0/8 (2/8 1/8 (6/8 2S%) 13% 67%) 1/24 (3/23 106 x 1. 4 47% 34%) 10 7 x 10 7 ) ::J 0.1 0/9 (1/9 1/8 (2/7 3/8 (3/S 23 (7.8-69) mg 0 11%) 13% 28%) 38% 60%) 4/2S (6/21 16% 28%) 8.0 x 10 6 ~(9.4 x 10 6 ) 11 S (3.6-3 8 1 Q -, a mortality ratio and per cent deaths b culture positive lungs/total survivors c LOSO = lethal dose SO and standard deviation d potency r3tio and 9S per cent confidence 1 imits 00 w 1e 22 Vaccination of mice against histoplasmosis Mice vaccin3ted with formal in-killed whole spores or yeast phase cells and challenged with 3154 30 day mortal tty ratios, per cent deaths, and culture results challenge dose 1 x 105 1 x 10 6 1 x 10 7 Totals L050 P.R" c PSS control 2/10 (6/8 5/10 (5/5 9/10 ( 1/1 20% a 67%)b 50% 100%) 90% 100%) 16/30 (12/14 53% 86%) 7.0 x 105 6 x 10 ) !(1~7 d -- GlO MacroconiSia 7 0/7 (5/7 3/8 (3/5 6/8 (2/2 ~ x 10 0 71%) 38% 60%) 75% 100%) 9/23 39% (10/14 72%) 204 x 10 6 :!"(2.1 x 10 6 ) 3~4 (0.9-12.5) a mortal ity ratio and per cent deaths Gl7M whole cells 10 6 cells 5 x 10 7 cells 0/8 0 0 0/9 (4/8 (4/9 45%) 50%) 4/9 1/7 14% 45% (2/6 60%) (3/5 33%) 2/10 20% 3/9 33% (6/8 (5/6 84%) 75%) 7/27 25% (12/20 60%) 6 6 5~0 x 10 ±(4.9 x 10 ) 7.2 (2- 28) 3/25 12% (12/22 55%) 8.0 x 10 6 6 ±-(809 x 10 ) 1 1" 4 (206~51 ) b culture positive lungs/total survivors c L050 = lethal dose 50 and standa d deviation d potency ratio and 95 per cent confidence 1 imit Q) ..j::'- 85 immunized mice were: G10 (7.3 x 10 4 cells), 3,4; G17M (1 x 10 6 ce 11 s), 7 2; and G17M (5 x 10 7 ce 1 1s) ~ 11 04) g The culture results were similar: G10 macroconidia, 72%; G17M (1 x 10 6 ), 60%; and G17M (5 x 10 7 ), 55%, Eighty-six per cent of the sal ine controls were culture positiveo Results recorded in Table 23 show a summary of the potency ratios for the vaccines prepared from the various strains of Ho capsulatum o The results indicated that cell free extracts either of strain G10 (mycel ial phase) or of strain G17M (yeast phase) had similar potency ratios. The ratios are: G10 - 0.4 mg, 18 5; O. 1 mg, 5.0 and G17M - 0 4 mg, 23; O. 1 mg, 1 1 05, 0 for identical vaccine doses. Strains G75, 2645 1 and 3330 cell free extract vaccines had potency ratios which varied from 1.5 to 20.0. The whole cell vaccines prepared from strains G17M and G10 had ratios between 3.4 and 11.4. Determinations of the chemical composition of the vaccines prepared from certain shown in Table 24 ~o capsulatum strains are Strains G10 and G17M have similar carbo- hydrate-protein ratios of 57 and 56 respectively. The remaining vaccines had ratios that varied between 6 7 and 0 86 Table 23 Comparison and summary of potency ratios of vaccines from various strains of Histoplasma capsulatum Amount a Injected Potencyb Ratio Gl0 3.0 1 0 0.4 0.1 31.0 24.0 18? 5 5.0 G75 Strain (10-95) (7-83) (6-57) (1.6-15) myce 1 i a 1 phase CFE c 2.0 mg 0.4 mg 0.1 mg 20.0 (7.6-53) 4.7 (1.3-18) 10.0 (2.3-43) myce 1 i a 1 phase CFE 2645 3.0 mg 2.0 mg 1 .0 mg 15.4 (4.7-50) 9.3 (2.9-29) 9.3 (2.9-29) myce 1 i a 1 phase CFE 3330 1 . 0 mg 0.4 mg 0.1 mg 1 1 .8 (4.2-31) myce 1 i a 1 phase CFE G17M 0.4 O. 1 10 6 5 x mg mg 23.0 (7.8-69) 11.5 (3.6-38) yeast phase CFE (0.53 mg) 107 (2.7 mg) 7.2 (2-28) 1 1 .4 (2.6-51 ) formal inized yeast cells G17M Gl0 mg mg mg mg Description 7.3 x 10 4 7.6 (NC) a 1 . 5 (0.43-5.2) 3.4 (0.9-12.5) formal inized macroconidia a dry weight of suspension injected b potency ratio and 95% confidence 1 imits determined by Litchfield and Wilcoxon (1949) c CFE (ce 11 free ext ract) d not calculated 87 Table 24 Chemical composition of five strains of Histoplasma capsulatum Dry %CHO a ) %Protein b ) Ratio c ) Weight Strain Vaccine Description G10 myce 1 i a 1 phase, CFE"'- 34% 0.6% 57.0 2308 mg G75 myce 1 i a 1 phase, CFE 47% 7.1% 6.7 6.3 mg G17M yeast phase, CFE 39% 0.7% 56.0 9.4 mg 2645 myce1 ia1 phase, CFE 35% 2.9% 12.0 10.9 mg 3330 myce1 ia1 phase, CFE 33% 4.4% 7 5 1701 mg G17M yeast phase, whole cells 39% 3.2% 9. 1 10 5 mg * CFE (ce 11 free extract) a) ~arbohydrate (CHO) determined by anthrone method b) protein determined by Fo1 in and Cioca1teu method c) ratio % CHO/% protein . / 0 88 DISCUSSION H capsulatum is composed of a thallus which may be considered as both vegetative and reproductiveo The vegetative thallus is composed entirely of mycel ial filaments of hypha, which are generally branched and spread in all directions from which the spores arise. The reproductive part includes all structures which lead to the formation and dispersal of the various spore types. The extreme polymorphism of fungi to students of mycology. is a major challenge Doctors, veterinary surgeons, and cl inical laboratory and publ ic health workers famil iar with the 1 ife cycles of protozoa and helminths which are parasitic on man and animals will appreciate the significance of the range of fungal formso Morphological reduction in size associated with parasitism may be regarded as a particular case of fungal polymorphism (Lan ron and Vanbrueseghen, 1965). parasite, ~. Thus, the fungal capsu1atum, has a saprophytic stage composed of varied forms and a parasitic state which is yeast-1 ike, The occurrence of histoplasmosis in man and animals is related to the colonization of suitable natural substrates in the environment by histoplasma. Apparently, histoplas- mosis is contracted by inhalation of spores after exposure 89 to the fungus growing in the environment. H. capsu1atum has been isolated from this environment which includes soil and debris from: chicken houses 9 hollow trees, caves~ barnyards 1 river bottoms including water tissues. ~. Available laboratory specimens human and animal 9 identified as capsu1atum by observing typical macroconidia, have n given various strain designations by individuals who isolated the fungus. The designations range from letters and numbers to the name of the patient or locale of the isolateo Identification is currently based solely on morphology. This situation leads to some strange results. First, available isolates must always be identified as H. capsulatum after transfer to a new laboratory. This can be a tedious procedure when a relatively asporogenous strain is being investigated (ego~ G75). The current arbitrary designation of strains may be based on locale of isolation? in which case it is not clear whether the organism was isolated from soil ~ a lesion (human or animal) or fomites. Again9 strain designation may be based on whether the organism was isolated from a particular area, For example, one strain used in this study, 3154, is labeled "India." The origin of the strain is again unclear. The large number of strains each 1aboaratory maintains results in much cumbersome dup1 ication of effort. It must 90 be granted that 1 ittle is known out~. capsulatum~ and that some means for identifying strains of the fungus in terms of origin as well as species j must exist. The procedures used in the identification of pathogenic bacteria might well be fol lowed. The classification of pathogenic b act e ria fa 1 1sin the f 0 1 1ow i n g g e n era 1 g r 0 up i n g s: 1) morphology and staining, 2) cultural characteristics such as fermentation and pigment production~ 3) serology including antigenic structure, and 4) production of a disease process. A keystone in the classification of higher plants and animals is the definition of a species as a group of individuals capable of continued fertile interbreeding (Davis et alo, 1968). However, this keystone does not apply to bacteria and fungi for which the situation is quite different. The number of characteristics available which enable species identification for bacteria and fungi is no known example 'of ontogeny to rec there is no fossil ordinarily asexual is 1 imited. There itulate phylogeny; record; and finally, reproduction is a The definition of Davis, et al.? 1968, for species is therefore not usually fulfil led o The grouping of individual strains into the same kind or species is usually settled without benefit of the interfertility test. Moreover the mutabil ity and the rapid growth of these organisms? combined with strong selection pressures from changes in the 92 peptone, 2) modified Sauton1s contained asparagine, 3) synthetic 1 contained (NH4)2S04, 4) synthetic 2 contained asparagine and 5) synthetic 3 contained no nitrogen source, Sautonis and synthetic 2 contained the same nitrogen source, but macroscopically the colonies appeared dif rento Factors other than carbohydrate and nitrogen sources must playa role in the color and texture of the colonyo The mycel ial phase of ~o capsulatum grows well at 25 0 C on synthetic media having a single nitrogen source~ although some strains demonstrate individual amino acid requirements (Rowley and Pine, 1955). Of the carbohydrate sources dextrose gives optimal growth (Scheff~ 1945). The growth on synthetic 1 was less in comparison to growth on Sabouraud's and Sauton1s. Synthetic 3 supported growth which was scant, del icate and about the size of a dime, This latter medium contained no added nitrogen. The endo- genous nitrogen present in the medium and the nitrogen present in the inoculum was apparently a 1 imiting growth factor o Growth on synthetic medium 2 was similar in size to growth on Sabouraud's with phsophate; however~ growth on synthetic 2 medium differed in color and textureo In addition to the differences noted above~ there are differences in the sporulation characteristics of each strain of Ho c sulatum and growth on the various media. 93 Apparently there are certain 1 imitations of interpretation with regard to the sporulation data due to the methods of study. It is probable that measurements made from colonies grown on media other than those studied might have given different results. It might also have been better to have emulsified the whole culture and then measured the numbers and sizes of the spores rather than selecting an arbitrary area as was done. It does not appear~ however, that these criticisms would 1 imit comparison between strains where all were treated in 1 ike manner. The measurements of the spore sizes suggest that there are differences among media which induce differences in spore sizes. For example, macroconidia grown on Sabouraud's with phosphate were larger than the spores observed in the remaining media employed in the study. Mycel ial phase cultures grown on Sauton1s medium produced microconidia which were larger than those formed on the other media. The difference in size of the macroconidia is more striking than is the difference in microconidial sizes on the six media util ized in this study. The figures showing the sizes of microconidia and macroconidia of different strains of ~. capsulatum yielded no overlapping between the two spore types. However, these figures represent only three of the six media studied, i.e., synthetic media 1, 2, and 30 These 94 media contained similar constituents except for the additional nitrogen source or lack of nitrogen. Apparently there was a size difference in macroconidia formed on synthetic 1 as compared to synthetic 3 media. of the data yielded no additonal Although sporulation of ~. Analysis of the remainder information. capsulatum is primarily a strain dependent phenomenon (Howel l, 1939), some investigators have noted variation in degree of sporulation in supplemented media o For example, addition of phsophate ion to Sabouraud's medium (Artis and Baum, 1963) and variations of media containing different nitrogen or carbohydrate sources (Negroni, 1940) have been noted to enhance sporulationo The data that have been presented in this thesis suggest that there is a correlation between nutritional properties and sporulation abil ity. The factor(s) or characteristic(s) of the culture media tested which contributed to the production of either macroconidia or microconidia were not examined. The enrichment of Sabouraud's medium with a source of phosphate such as KH ZP0 4 was reported by Artis and Baum (1963) as being useful in stimulating tuberculate spore production (macroconidia production). The results of their study demonstrated that tuberculate macroconidia are not in a resting or survival stage since depleted media and nutritionally poor media did 95 not stimulate ore production. Using calculations from the sporulation numbers, the Sabouraud's medium with additional phosphate ion supported relatively more macroconidia. Similar calculations suggested that the synthetic media 1 and 2, and modified Sauton1s medium produced relatively more microconidia. Synthetic medium 3 supported formation of approximately equal numbers of both spore typeso Smith and Furcolow (1964) suggested that the addition of an infusion of starl ing manure to soil has the effect of producing more total particles and of producing a large number of microconidia as well as increasing the viabi1 ity at least twice that observed in other media. Of particular interest was the finding that although large numbers of particles are produced on Sabouraud's medium, 84% of these particles are hyphal elements and not spores, but the overall viabil ity was only 3%. It is clear that some essen- tial element(s) in the extract of bird manure augments the orulation potential of the organism in soil. However 1 the presence of large amounts of extraneous materials makes the use of this medium undesirable for immunological work with the derived myce1 ial phase. Smith (1964) has reported that a medium composed of 0.6% yeast extract and 2% agar in distilled water stimulated rapid growth and sporulation of H. capsulatum similar to 96 that of star1 ing manure extract medium. The rapid growth consisted of many viable microconidia and a low ratio of vegetative myce1 ium. These findings suggest that the spore- stimulating substances are nutritional in nature. The results of sporulation experiments reported in this thesis support the premise that sporu]·ation is related to the nutritional environment on which the Ho capsu1atum is grown. The measurement of the abi1 ity of each strain to produce spores was based on microscopic appearance of the myce1 ium and spores. The measurement was relative and rank statistics were used in assigning a number value to a set of defined terms. Using this method the sporulation number of each strain on each medium was obtained. This sporulation number gives no indication of the abundance of the spores but only the abi1 ity of each strain to produce spores. The sporulation number was used to indicate the abi1 ity of a strain to produce spores and the type of spore for preparation of vaccines. Sabouraud's medium with phosphate ion was chosen as the medium upon which the mycel ia1 phase organisms were to be grown. This medium was chosen from among the media studied for the following reasons: 1) ease of preparation, 2) abundance of growth, and 3) abi1 ity to support spore production in abundant numbers. Harvest of the myce1 ia1 phase of H. capsu1atum occurred after 10 weeks. 97 This period was chosen because maximum growth and numerous spores were obtained at this time. Data from the experiments described in this thesis suggest that sporulation by H. capsulatum is dependent upon the strain of the organism and the medium on which it grows. Some strains will sporulate in such a manner that only well defined large macroconidia are produced (strain G10). Other strains, for example 3330 and 6651, formed large numbers of microconidia and few macroconidia. In many strains there is a marked size and morphological differentiation between microconidia and macroconidia. In a few strains the gradation of size of the spores progresses so smoothly from microconidia to macroconidia that no real distinction can be made between the two forms. A fundamental problem in medical mycology is the question of variation in virulence between strains of H. capsulatum. Drouhet and Schwarz (1956) and Howell and Kipkie (1950) have reporte of H. capsulatum. these differences in strains In only one available report (Rowley and Huber, 1955) has it been observed that different strains of this organism were not significantly different with regard to mouse virulence. Using methods described in this thesis, significant differences in virulence were noted among the tested strains of ~. capsulatum, i.e. an LD50 for 98 strain 31S4 of 3.0 x lOS, an LDSO for strain G17M of 1.1 x 10 6 , and for strain 2813 of 3.0 x 10 8 (Table 13). It can be recalled that yeast phase organisms of each strain were injected intravenously. These differences in LDSO could be due to differences in the mouse virulence of the strains of organisms used but this difference might be unrelated to human disease. There was no morphological or physiological explanation apparent for the wide variance in mouse virulence observed with the different strains. The 23 strains of H. capsu1atum studied were isolated from human and animal sources, and from soils. The strains differed in myce1 ial phase morphology both in macroscopic colony appearance and in microscopic appearance. The yeast phase cells of the organisms observed were morphologically similar. Results showed that the virulence of strain G17M did not change to any significant degree when maintained in the yeast phase on antibiotic containing blood agar for long periods of time at a temperature of 37 0 C. Similar results were demonstrated with strain 31S4 but over a 1.S year time period as opposed to lS year period for G17M. Furthermore, other factors such as rapid passage on cultural medium, mouse passage, human passage, and conversion did not change the virulence of strain G17M. 99 The susceptibil ity of the mouse to infection by small doses of H. capsulatum, either spores or yeast phase? makes this host a significant one in which to study problems in ! mm un i t Y~ Let hal i n f e c t i on s wit h I n r e 1at i vel y s h0 r t per I od s are produced only by parenteral inoculation of 10 5 or more yeast phase organisms depending on the virulence of the s t r a i nus e d ~ Sin c e a m0 r tal i t yen d po nth a s c e r' t a i n advantages, challenge doses of strain 3154 were employed with doses as great in magnitude as 10 7 organisms administered by the intravenous route, that is, about 100 LD50 doses. The strains of H. capsulatum ut,l ized in immunity studies were chosen because of the fol lowing characteristics: strain G10 produced abundant macroconidia on Sabouraud's medium with phosphate. Strain G75 became pleomorphic after culti- vation in the stock culture collection. Pleomorphism is defined in mycology as a degenerative change in a fungus that converts the colony into one that is completely void of characteristic spores required for identification (Ajello) et al.) 1963). Pleomorphism is apparently irreversible. Strain G75 produced no spores~ only mycel ium. Strain 3330 exhibited good growth on Sabouraudus with phosphate ion, many macroconidia and few microconidia were formed. This same strain on synthetic medium 1 showed less growth than on Sabouraudis, but the number of microconidia formed was 100 higher, Because of the greater amount of growth on Sabouraudis with phosphate~ grow this strain of ~. this medium was selected to capsulatume Strain 2645 of He capsulatum was chosen because it produced an abundance of both types of spores. Strain G17M was chosen to represent the species in the yeast phase because of previous characterization of the organism at the University of Utah o The intravenous route of challenge for infection of mice was chosen because of its superiority to other routes of infection for producing deaths in micee For our studies intracerebral injections have not been used because of the fol lowing problems: 1) a meningitis is produced which is rarely found in hosts naturally infected or in animals experimentally infected via other routes; 2) the volume of inoculum must necessarily be small; 3) the location of injections is not exact and deaths may be produced by focal brain lesions; 4) intracerebral injections are not made easily in most other species of laboratory animals e The intraperitoneal route of immunization was used. Previous workers (Hill and Marcus, 1959) had found the intraperitoneal route as effective as intramuscular, subcutaneous or intravenous inoculations. The potency ratio for the comparison of normal groups to the other four groups 101 was found to be 2.4 with 95% confidence I imits of 1.25 to 45. In this thesis data were presented which permitted calculation of an estimate of the extent of resistance induced by immunization. This estimate is defined as the potency ratio, i.e., the ratio of the LD50 estimates for the control and to that of the immunized groups. The graphic method employed was modified by Litchfield and Wilcoxon (1949) from a method previously described (Miller and Tainter, 1944) for estimating lethal dose 50 and standard deviation. The procedure involved fitting dose-response I ines by eye and calculation of LD50 and its error. By means of graphs and nomograms potency ratios and their errors were calculated. Challenge of the accuracy of the method by Finney (1952) was effectively answered by Litchfield and Wilcoxon (1953). Since the method of analysis was based on the assumption of a dose-response relationship, the adaptation to data presented here was val id only if there existed a direct relationship between challenge dose of organisms and mortal ity. Although the dose-response characteristic following organism challenge was clearly evident for normal mice, this parameter was less apparent for the immunized animals. The data have indicated that a challenge dose-mortal ity response relationship did exist with immunized as well as normal mice: therefore the appl ication of the method of analysis was 102 justified. However, the shal low slopes of dose-response 1 ines lead to a wide range in the confidence 1 imits. Intraperitoneal vaccination of the mice with cell free extract of strain GIO, a strain whose sporulation number indicated macroconidia production, and intravenous challenge (administered ten days postvaccination) gave the following potency ratios with the vaccination amounts in mil 1 i gram s 0 f dry wei g h t: 3 mg, 3 1; 1 mg, 24 ; and O. 1 mg9 5.0. o. 4 mg ~ 18 . 5 ; Analysis of the potency ratios for this strain depended on the finding that the LD50 values of the sal ine controls were 4.2 x 105 and 7.0 x 105. There was a definite dose-response relationship between the potency ratios. Careful observation of the mortal ity ratios com- paring sal ine controls and various immunized groups yielded other dose-response relationships. Finally, analysis of results of culture of organs from survivors showed a doseresponse dependence. From this information one could deduce that as the dose of the vaccine is increased the mortal ity ratio~ per cent deaths, culture results decrease and the LD50 values increase, i.e., it required greater numbers of organisms as a challenge dose to achieve the same effect. For example at a challenge dose of 1 x 10 6 the per cent deaths in the sal ine control group was 38% and it was also 38% for the group immunized with 0.1 mg dry weight cell 103 free extract. For the group immunized with 0.4 mg of the cell free extract, the deaths reached 11%. Looking at a challenge dose of 1 x 10 7 , the per cent deaths in the sal ine control group was 100%, 20% for 0.8 mg, 45% for 0.4 mg and 60% for 0.1 mg. The per cent culture positive data yielded similar results as follows: 60% for 0.8 mg, 90% for 0.4 mg and 100% for 0.1 mg. In animals vaccinated with 7.3 x 10 4 whole macroconidia of strain GIO the potency ratio was 3.4. This ratio was sma1 ler than any of the values obtained with the cell free extract vaccine. The comparison can be made only with the understanding that the dry weight of the whole macroconidia was unknown. Similar dose-response relationships were observed in the vaccination with strain G75. The potency ratios that could be calculated were for 2 mg a value of 20; for 0.4 mg, 4.7 and for 0.1 mg a value of 10. The dose-response relationship was unapparent in this case since the 0.1 mg ratio was higher than the 0.4 mg. Strain 3330 and strain 2645 of H. capsulatum yielded similar dose-response relationships with the potency ratios increasing as the dry weight of the vaccine increased. The cell free extract of yeast phase cells of strain G17M yielded potency ratios of 23 for 0.4 mg and 11.5 for 104 0 1 mgo Whole formal in-killed yeast cells used as a vaccine gave potency ratios of 7.2 for 10 6 cells and 11.4 for 0 5 x 10 7 cells. Hi 11 and Marcus (1959) using a vaccine prepared from 4 x 10 7 cells prepared from strain G17M or 6651, obtained a potency ratio of 5.3 (1.21-23) when the mice were challenged with G17M. These two results do not necessarily conf1 ict since the immunization procedures used differ and also the strain of H. capsu1atum used to challenge the immunized mice. Knight and Marcus (1958), in animals vaccinated with approximately 10 7 whole killed organisms, found a potency ratio of 7.6. The difference in the data reported in this thesis, between 1 x 10 6 cells and 5 x 10 7 cells, i .e o approximately a 50 fold increase in the number of cells used to immunize, deserves further study. Two of the strains of ~o capsulatum studied, G10 and G17M, had similar potency ratios with regard to vaccine prepared from cell free extract. Strain G10 had a potency value of 18.5 (6-57) for 0.4 mg, whereas strain GJ7M had a potency ratio of 23 (7.8-69) for 004 mg. Values were similar for 0.1 mg dry weight of vaccine: strain G10, 5.0 (1.6-15) and strain G17M, 11.5 (3.6-38). The vaccine prepared from strain G10 was from the mycel ial phase and according to the sporulation number this strain produced mainly macroconidia. The vaccine from strain G17M was 105 prepared from the yeast phase of the organism. Using the carbohydrate to protein ratio, strain GIO and strain G17M had similar chemical composition. of 57 and strain G17M a ratio of Strain GIO had a ratio 56~ There was a suggestion of a relationship between chemical composition and immunizing capacity. Knight, Hill and Marcus (1959) studied the immunogenicity of a vaccine prepared from a polysaccharide moiety of the yeast phase growth of H. capsulatum. The polysaccharide was capable of stimulating active resistance against intra~nously induced histoplasmosis in mice. Fol lowing intra- peritoneal vaccination of mice with graded doses or a single dose, both of which totaled 1 mg of polysaccharide, the potency ratio for both groups was approximately 4.3. The remaining mycel ial phase vaccines prepared from strains G75, 2645, and 3330 had low carbohydrate to protein ratio, i.e., the percentage of protein found in these vaccines was higher. Strain G75 had both the highest carbohydrate and protein percentage. The relationship between dry weight, protein, and polysaccharide content of the mycel ial phase cell walls of H. capsulatum has been previously reported by McNall (1962). The protein content was 11.2% of the dry weight of isolated cell walls. The polysaccharide was reported as 30.0%. 106 Studies on six strains of yeast phase~. capsu1atum by Pine, Boone and McLaugh1 in (1966) showed protein content ranging from 7-38% and carbohydrate content ranging from 7-31%. The values for cell free extracts of myce1 ial phase of the fungus reported in this thesis ranged from 33-47% carbohydrate for four strains. The protein content ranged from 0.6% to 7.1% based on dry weight of the mycel ial phase. The yeast phase cell free extract of G17M contained 39% carbohydrate and 0.7% protein whereas the whole cells from the same strain were composed of 39% carbohydrate and 3.2% protein. 107 SUMMARY Twenty-three strains of H. capsulatum were characterized as to: 1) macroscopic appearance of the fungus on various media; 2) differential spore production on various media; 3) intravenous LDSO of the yeast phase of certain strains of the organism; and 4) immunogenic capacity of vaccines prepared from various elements of the selected strains of the fungus. Results of experiments reflected apparent strain differences which exist among the species H. capsulatum. The first difference was morphological and might be related to the nitrogen source contained in the media. medium with phosphate supported the best growth. Sabouraud's The remaJnder of the media supported mycel ial growth in the following comparative order from good to scant: modified Sautonls, synthetic 2, synthetic 1, and synthetic 3. Synthetic 3 supported growth which was scant and del icate. Variations were also noted in the sporulation characteristics of each strain grown on the various media. The measurements of spore sizes were consistant with values reported by other workers. Sabouraud's medium with phosphate produced larger macroconidia than the other media studied. Larger microconidia were produced on Sauton's medium than spores formed on the remaining media. The macroconidial 108 and microconidial spores ranged in size from 6.5 to 14.3 u and 2Q3 to 5.8 u respectively. The relative measurement of numbers of spores produced by each strain were based on a rank system. Strain G10 and 2870 produced a predominance of macroconidia as indicated by the sporulation number. Two of the 23 strains (G75 and 2586) produced only microconidia. Two strains, G17M and 2888, produced equal numbers of both spore types. The remaining strains yielded sporulation numbers that varied about those of the above described strains. The medium which produced the macroconidia best was Sabouraud's with phosphate. Synthetic 1 and 2, and modified Sauton's produced mainly microconidia. The remain- ing medium, synthetic medium 3, produced sparse but equal numbers of both spore types. The intravenous L050 of selected yeast phase strains of H. capsulatum were studied. Significant differences in viru- lence were noted. Strain 3154 had an L050 value of 3.0 x 105. At the other extreme, the L0 5 0 for strain 2813 was 3.0 x 10 8 , The values of the remaining strains fell between these two L050 values. The yeast phase of strain 3154 was used as the challenge organism in the immunization experiments. Vaccines prepared from G10, G17M, 2645, and 3330 were used to imunize mice. After challenge with the yeast phase of 3154, the potency ratios were determined. The vaccine 109 prepared from the mycel ial phase of GIG cell free extract and the cell free extract prepared from the yeast phase of G17M had similar potency ratios. These two vaccines, GIG and Gl7M had similar chemical composition also, with high carbohydrate and low protein content. The remaining vaccines, prepared either from cell free extracts or whole cells, yielded potency ratios that varied from 1.5 to 20.0, with no correlation between chemical composition and potency ratio. The 23 strains of H. capsulatum studied were isolated from human and animal sources, and from soils. The strains differed in mycel ial phase morphology, mouse virulence, and immunogenic capacity as determined by the results of these experiments. 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Proceedings of the Society for Experimental Biology and Medicine 29:937-938 Will is, H. S., and M. M. Cumm i n g s . 195 2 . 0 jag nos tic and Experimental Methods in Tuberculosis. C. C, Thomas, Springfield, 111. Wu, W. G., and S. Marcus. 1963. Humoral factors in cellular resistance, I. The effects of heated and unheated homologous and heterologous sera on phagocytosis and cytopepsis by normal and "immune ll macrophages Journal of Immunology 91 :313-322. RESEARCH PROPOSALS submitted by Kenneth Leroy Anderson in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology University of Utah August, 1968 RESEARCH PROPOSALS 10 Investigation in depth should be undertaken of the chemical composition and morphologic and antigenic structure of the soluble, extracellular growth products~ cell walls, membrane and cytoplasmic fractions of Histoplasma capsulatum. 2. The relationship of diagnostic and protective antigens with cell composition and structure of H. capsulatum remains unknown and requires studYa 3. Further studies on the critical separation of the spores and mycel ium of H. capsulatum would yield specific information with regard to the chemical composition of these elements of H. capsulatum. 4. The use of aerosol challenge, using purified spore and mycel ial elements, might yield more specific information concerning the mechanism of infection in experimental pulmonary histoplasmosis. 5. Immunity studies util izing vaccines produced from various elements of He capsulatum and subsequent challenge of immunized animals with either purified viable microconidia or macroconidia would yield significant basic immunomycologic information. 2 6. Epidemiological studies on a significant sample of Utah residents to determine the incidence of skin test sensitivity to~. capsulatum and Blastomyces dermatitidis should be accompl ished correlating skin test data with attempts to isolate these fungi. 7. Relatively few pertinent data are available to indicate the potential cl inical usefulness of antifungal vaccination. Possibly such information could be obtained by immunity trials in a variety of hosts challenged under conditions that simulate or approximate natural exposure. 8. The significance of delayed hypersensitivity in the chronic fungal diseases in unknown and merits further study. 9. Studies must be pursued involving the relationship of the use of immunosuppressive drugs and opportunistic mycotic pathogens in patients that have received or are about to receive transplanted organs. 10. Further studies are indicated of the relationship of the various strains of vl,ulence j ~. capsulatum and the morphology, and spore production of these strains.