(.1 2 ABSTRACT INVESTIGATION OF THE VIRULENCE AND PHYSIOLOGY OF SOME STRAINS AND MUTANTS OF BLASTOMYCES DERMATITIDIS, GILCHRIST AND STOKES 1898 BY Samuel Truman Bass There is very little information on the enzyme activi- ties and virulence of Blastomyces dermatitidus Gilchrist and Stokes 1898. The objectives of this research included the development of less virulent strains of g. dermatitidis, and the study of alkaline and acid phosphatase in relation to virulence. Other factors including lipids in relation to virulence and production of a toxin were investigated. Mutants of g. dermatitidis St. Joseph strain were developed by ultraviolet irradiation from yeast phase cells. Twenty-six of the 45 isolates were selected for study of nutritional requirements, enzyme production, and virulence in mice. The mutants M—25, M—26, M-37 were found to be up to.lOO times less virulent than the parent St. Joseph strain. The basal salts medium of Gilardi and Laffer was used for amino acid and vitamin studies. When asparagine was a Supplement nitrogen source in the medium, the St. Joseph and Samuel Truman Bass Ga-l strains had good growth in the yeast phase only. When proline was added, both the mycelial and yeast phase showed increased amount of growth. In order to maintain viable cul— tures in the yeast phase for more than 5 to 8 days, asparagine was necessary in the medium of Gilardi and Laffer. Glycine, tyrosine, and proline could not be substituted for asparagine. The vitamins, biotin and thramine were not required for the growth of g. dermatitidis. In the study of the alkaline and acid phasphatase activ— ity, an enriched medium, CYPG, consisting of casein, yeast extract, peptone and glucose, was used. The extra cellulose alkaline phosphastase activity was generally higher than the acid phosphatase in the liquid medium at 37 C and at 24 C. The levels of the extracellular alkaline and acid phosphatase activities remained lower at 37 C than at 24 C over a 30 day period. The intracellular alkaline and acid phosphatases were usually much higher than the extracellular enzymes for both phases. Both enzymes were not easily removed from the cell membrane and were found not to be particle bound. There seemed to be no correlation in the level of alkaline or acid phosphatase activity and virulence in mice. The lipid content in yeast cells of strain of g, derma— titidis selected varied with no apparent correlation to viru- lence in mice. When tripsin treated yeast cells at 40 mg/ mouse were injected into mice, the cells were more virulent thanuntreated cells. INVESTIGATION OF THE VIRULENCE AND PHYSIOLOGY OF SOME STRAINS AND MUTANTS OF BLASTOMYCES DERMATITIDIS, GILCHRIST AND STOKES 1898 BY Samuel Truman Bass A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1971 To my wife and children ii ACKNOWLEDGMENTS The writer wishes to express his sincere appreciation to Dr. E. S. Beneke, whose invaluable guidance, interest, patience and encouragement during the course of this in- vestigation has made its completion a possibility. The writer wishes to express his appreciation to Dr. Alvin L. Rogers for his kind criticisms and invaluable assistance in the progress of this problem. The writer also wishes to extend his sincere apprecia- tion to the other members of his guidance committee, Drs. .R. S. Bandurski, J. W. Hooker, H. M. Sell and W. B. Drew, for their time and guidance in this program and in the writing of this thesis. The writer would like to acknowledge the assistance of Dr. Bill Fields for his guidance in selection of mutants. The use of the equipment in the laboratory of Dr. Beneke, Dr. Bandurski and others in the Botany and Plant Pathology Department is greatly appreciated. iii II. III. IV. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . Classification . . . . . . . . . . . . . . Cases of Blastomycosis . . . . . . . . . . Geographic Distribution . . . . . . . . . Natural Habitat . . . . . . . . . . . . . Dimorphorism of Blastomyces dermatitidis . Nutrition of the Organism . . . . . . . . Metabolism . . . . . . . . . . . . . . . . Animal Inoculation . . . . . . . . . . . . MATERIAL AND METHODS . . . . . . . . . . . Blastomyces dermatitidis Cultures . . . . media 0 o o o o O o o o o o o o o u o o 0 Measurement of Changes with the Organism . Mutants of Blastomyces dermatitidis . . . Gel Electrophoresis . . . . . . . . . . Enzyme Studies . . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . Mutants of Blastomyces dermatitidis . . . Media for Growth of Strains of Blastomyces dermatitidis and Mutants . . . . . . . . Virulence of the Mutant Strains . . . . . Alkaline and Acid PhOSphatase Activities . Other Enzyme Activities of Blastomyces derma- titidis, St. Joseph Strain . . . . . . . iv Page 3...; «103w 10 12 13 15 19 26 29 29 31 34 37 39 4O 43 43 48 61 68 87 TABLE OF CONTENTS (Cont.) Page V. DISCUSSION . . . . . . . . . . . . . . . . . 89 Alkaline and Acid Phosphatase Activities . . 95 VI. SUMMARY . . . . . . . . . . . . . . . . . . 100 REFERENCES . . . . . . . . . . . . . . . . . 102 Table II. III. IV. VI. VII. VIII. IX. LIST OF TABLES The mutants of B;_dermatitidis, St. Joseph strain isolated after ultraviolet irradia- tion on CYPG medium at 37 C . . . . . . . Growth of B; dermatitidis St. Joseph strain and mutants M-26 and M-36 on Gilardi and Laffer's medium with asparagine (aBM), cer- tain amino acids, and vitamins for 20 days at 24 C. . . . . . . . . . . . . . . . . Growth of B; dermatitidis St. Joseph strain on several basic media for 15 days at 24 and 37 C . . . . . . . . . . . . . . . . . Growth of B; dermatitidis Ga-l strain on several basic media for 15 days at 24 and 37 C . . . . . . . . . . . . . . . . . . . Growth of St. Joseph strain and Ga—l strain of B;_dermatitidis on modifications of Gilardi and Laffer's medium . . . . . . . Growth rates and viability of B; dermatitidis St. Joseph strain on different media after 15 days incubations at 37 C. . . . . . . . Cell growth and viability of yeast cells of B; dermatitidis St. Joseph strain on the basic salts medium of Gilardi and Laffer with different amino acids. Cultured at 37 C . Virulence of mutants of the St. Joseph strain of B. dermatitidis injected intraperitoneally (IPT—into mice . . . . . . . . . . . . . . The relative virulence of twelve strains of Blastomyces dermatitidis, injected IP in mice The effect of increasing cell concentrations of the mutant M-26 of B; dermatitidis on virulence when injected IP in mice . . . . vi Page 44 49 51 51 52 56 56 62 64 64 LIST OF TABLES (Cont.) Table XI. XII. XIII. XIV. XVI. XVII. XVIII. P Effect of dilutions of cells of different mutants and strains of B. dermatitidis on virulence when injected IP in mice.. . . . A test for toxin production in B. dermati- tidis St. Joseph strain grown at 37 C. Inocu— lation included 2 mg of acetone-dried tubercle bacilli/mouse plus the treatment. Days to 50% kill and lipid concentration of four strains of B. dermatitidis after a culture on BHI plus biotin agar for 10 days at 37 C. The Cells were extracted with chloroform—methanol (2:1) for 48 hr on a soxhlet. . . . . . . . . . . . . . . . . . The lipid concentration of the more virulent St. Joseph strain and the two less virulent strains of B. dermatitidis after culture on CYPG agar for 15 days at 37 C. . . . . . . The lipid content of two strains of B. derma— titidis after culture on CYPG medium for 15 days at 24 C . . . . . . . . . . . . . The extracellular acid and alkaline phos— phatase activities of mutants of B. derma- titidis grown on CYPG medium for 15 days at 24 and 37 C. The activity is expressed in mumolecxfsubstrate changed/hr/plug of agar. Average of two assays. . . . . . . . . . . The extracellular acid and alklaine phos— phatase activities of 8 strains of B. derma— titidis grown on CYPG medium for 15 days at 24 and 37 C. The activity is expressed in mumole13 lalastomyces have been reported in Venezuela (Montemayor, 3‘5’5541) and Arias (1962) in Mexico. Chick g£_al. (1960) reported that 735 cases had <3<=<311rred within the boundaries of the United States, while G'15a‘ndbois in 1963 reported on all published and unpublished cases of blastomycosis in Canada and found 117 such cases 11‘ the country. These cases were primarily in the provinces (bf: Quebec (71 cases) and Ontario (34 cases), while the remaining 12 cases were in four other provinces, Manitoba .-v; y» ..00‘ ab \ ‘v.. v. .. .. ‘5 11 (8 cases), New Brunswick (2 cases) and one case each from Nova Scotia and Saskatchewan. As to the distribution of the disease in the United States, the states with the highest prevalence of cases either border on the Mississippi River or are situated east of that river. The following states had 15 cases or more reported in 1960: Arkansas, 15; Illinois, 74; Indiana, 18. Iowa, 24; Kentucky, 37; Louisiana, 101; Tennessee, 90; mud Wisconsin, 81 (ChiCk, 1960). In another review of V. A. Hospitals in the United 31:5rtes (Bussey, 1964), 198 cases, covering the period of tiJnse from 1946—1957, the disease was reported to be more Prevalent in the Middle Atlantic, South Central, and the (31121<315 «cases in these states may indicate that in the earlier Es‘lit”vey (Chick, 1960) many records of cases of blastomycosis Vvealfe not included, which would in turn indicate that many r“(312‘e cases of blastomycosis may be on hospital records in this country. n‘. 'V 12 Natural Habitat The natural habitat of _B_. dermatitidis remains elu- sive (Ajello, 1967). This is stated deSpite recent re- ports of isolation from soils of B; dermatitidis in Georgia (Denton and DiSalvo, 1964) and Kentucky (Denton et al., 1961). The soil studies of Denton and DiSalvo (1964) found 10"‘of 356 soil samples positive to ]_3_. dermatitidis. The Positive sites were in living quarters of man, mule, cattle, Chickens and rabbits, which certainly would not narrow down the natural habitat to a specific environment. Other prob— lems were encountered with this work, such as repeated Samples for the organism from former positive sites, con— sisstently turned out to be negative. The stored soil samples lost their ability to produce Viable cultures of _B_. dermatitidis, except in one case, aC-‘-<:ording to McDonough et a1. (1961), when the soil was kept at room temperature for 16 months in a screw top bottle. McDonough in other studies (1963, 1965) showed that natural soils would not maintain _B_._ dermatitijés and the yeast phase was lysed. However, if the soil is steri- liZed before inoculation, the fungus has a much better chance of survival. Other workers (Bakerspigel, 1953; Fennell e_t__al_., 1950) in an effort to find a storage media for _B_. dermatitidis used sterilized soil, and re- PQrted similar results. To explain what happens to the organism ...v uu’v -._‘ 11‘ -\ “a. 13 in the soil McDonough suggests that some factor in the soil causes lysis (1970), (Friburg, 1970). Denton and DiSalvo (1968) reported that laboratory animals could become infected with sterilized soils Seeded with the saprophytic form of a highly virulent Strain of B; dermatitidis. The facts reported by the above workers would indicate that somewhere in nature an ecological niche exists that favors B; dermatitidis. Dimophorism of Blastomyces dermatitidis A». Temperature. The interconversion of mycelial (M) and yeast—like (33-) forms in B; dermatitidis and in P; brasiliensis are CiFIEixacterized as examples of thermal dimorphism (Nickerson and Edwards, 1950), (Salvin, 1949). The phenomena are E3IF’Elarently dependent only on the temperature of incubation (>5E3 these species. Salvin (1949) pointed out that tempera- ture is the environmental factor controlling the form of C31:‘<>wth. He also showed that no amino acid, carbohydrate ()3: growth substance was required for the organism in the yeast phase, but noted the presence of some pseudohyphae in ‘tlfiee rich yeast extract and peptone medium. Guidry (1967) reported that the optimum temperature for the yeast-phase growth was 35 C. for 8—12 hours. These results are in agreement with earlier experimenters (Ricketts, 1901; Hamburger, 1907), who reached the same conclusion. 14 B. Other Factors. In 1928, Michelson suggested that unfavorable environ- mental conditions produced by drugs, dyes, bile and incuba— tion temperature caused the organism to revert to yeast stage. He also reported that the yeast—like growth is the resistant form of the organism and the hypha growth is the saprophytic form of the organism. Nickerson (1948a) showed Eunnflavine, cobalt and penicillin promoted Y to M changes, h&Iile cysteine prevented the Y to M change. Weeks (1964) reported the ability for the organism to <311Etnge to the Y form is enhanced in a cottonseed medium VVTIi.le the pH level and the concentration of the glucose liéidi no effect on the conversion process. The 22 isolates C315 B; dermatitidis used in his work were converted at a -nn111:h faster rate than the medium proposed by Kelley (1939). A recent report by Collins and Edwards (1970) is of j-r11:erest since the filamentous form of B; dermatitidis ‘T’Eiss first demonstrated in tissue, while in earlier reports C315 mycelial forms were not well documented. In earlier 1:Eilgborts, the time factor may not have been considered in E31Teaparation of sections from tissues, but in this current report only the tissue of animals that were dead for less tPlan 10 minutes was used. Hence there was no chance for t1leeorganism to revert to the mycelial form after the “‘Ouse had died. 15 Nutrition of the Organism A. Introduction. The nutrition of_B. dermatitidis has been reviewed recently by Gilardi (1965), and earlier byIfickett (1901) and Stober (1914). The areas of nutrition that have been treated with the most effort are: media to be used to isolate the organism (Ricketts, 1901;5flxber, 1914; Kelly, 1939), media with basal requirements for growth, media tr) (Setermine carbon and nitrogen sources and media to determine differences in requirements for the two growth fC>IIns of the organism at 24 and 37 C (Weeks, 1964; Gilardi, 1965). I3 - Vitamin Requirements for Growth. The recent literature is somewhat confusing in that fiiafilnliday and McCoy (1955) have reported biotin is required if<39t‘ growth. They also reported that the only substance that could replace biotin was D-L-desthiobiotin which gave One—half activity of biotin. The investigators who reported no vitamin require- me-I'1ts were needed are Levine and Ordal (1946), Salvin (1949), cs5—1.ardi and Laffer (1962),and Nickerson and Edwards (1950), ‘Ml‘CD may have had biotin in the cotton plugs or in the tubes. fr1Me addition to the medium of vitamins was not stimulatory jer growth of the organism (Salvin, 1949; Gilardi, 1962). Tame answers these latter investigators gave for the results no . .A.~.II ..I a... '.n a V “Av ‘ u a ‘0 A‘ .. I... A - ' 16 of Halliday and McCoy (1955) were that Salvin (1949) used Sealed tubes and extensively washed agar in his experi- ments and Gilardi and Laffer (1962) used liquid culture medium with aluminum caps. CL Other Nutrients. The mycelial form of B; dermatitidis does not require any amino acid for growth (Levine, 1946; Nickerson, 1950). True yeast-phase of the organism grows without the addition 015 organic nitrogen or vitamins (Gilardi and Laffer, 1962). Imexrine and Ordal (1946), and Gilari and Laffer (1962) iiidiicate that amino acids are not required, but are stimu- JLEitzory for growth of both the yeast and mycelial forms of tildes organism. Gilardi and Laffer (1962, 1961) showed the organism ElSBsimilated 67 carbon and 37 nitrogen substrates as the Sole source in liquid cultures. These included hexose, £3th“inc acids and some of the Kreb cycle acids as the sole cinltbon sources. The nitrogen source included ammonium SSEilts; aliphatic amino acids; imino acids, proline and 1“Eiftiroxyproline; acidic amino acids, aspartic and glutamic; IDEissic amino acids, histidine, arginine and lysine, and ()‘lluer amino acid relatives. DzawachiSzwili, Laudau, Newcomer and Plunkett (1964) showed that sea water reduced the growth one-third when Ckbmpared to distilled water or 0.85% NaCl for B: dermati- JEidis. However if 1.7% NaCl is used growth inhibition of 17 50% is observed, at 3.4% NaCl inhibition is about 80-85%, and at 6.8% NaCl inhibition is 100%. However, smith and Furcolow (1969), in studies with NaCl concentrations on the growth of B; dermatitidis, reported an increase of Edmost 2-fold in growth with 1-3% NaCl. The concentration of 5-7% restricted the growth of the organism slightly. In another report, Smith and Furcolow (1964) showed .3: dermatitidis developed the largest number of conidia ‘with a small number of hyphal elements on starling manure irifusion medium. The repeated transfer of B; dermatitidis Cu: starling manure infusion resulted in an increase in the Enannber of total particles, the percentage of microconidia Eirléi the total viability. I) - Basal Media for B. dermatididis. The simplest media used in the literature are the CDIIIes given by Stewart and Meyers (1938) where ammonium Etc3<¥tate or acetamide are the sole source of carbon and 11¢i-tzrogen for the organism in the yeast phase. These E1tl‘l:hors reported that the charps in hydrogen ion concentra- tion (pH) appear to be independent of sugar consumption. “P11ee basal media reported by Levine and Ordal in 1946, Sa.lvin in 1949 and Gilardi and Laffer in 1962, used glucose E155 the sole carbon source and ammonium sulfate as the sole r11Itrogen source. Salvin (1949) suggested that a stimula- tuDry effect can be seen with amino acids. 18 E. Special Media. The media of Salvin (1949) was used to study morph- ology of the dimorphic B; dermatitidis. He concluded that the organism changed from the yeast to mycelial phase solely on the basis of temperature change from 35 to 24 C. Guidry (1967) studied the optimum temperature for the yeast phase and found to be at 35 C, or the lowest tem- .Merature to maintain the yeast phase. Weeks (1946) used a :3pecial type of medium to get B; dermatitidis to change lfIKDm the mycelium to the yeast phase. His cottonseed Huaéiium showed a much more rapid change over three other Ineadiia which had been used to change the mycelium to the §?€3Eist phase of B; dermatitidis. Marwin (1956, 1959) found four non-ionic surface- Ei<21:ive substances that accelerate growth to nine different 1“Human pathogens, and B: dermatitidis was one of these. Another type of nutritional study was reported by ROSenthal (1964) which would allow one to distinguish B_. Eéféggpatitidis from 27 other fungi by the nutrient it was EiIDJJe to hydrolyze. He used the following five substances: t3’1‘osine, casein, urea, gelatin and starch. _B_.. dermatitidis was able to hydrolyze tyrosine, casein and urea and was 1“GTE able to hydrolyze gelatin or starch. ..p"- .-._ n -, ... ,o- 'I‘. ‘ u~~ V . x _n 19 Metabolism A. Lipids Isolated from B; dermatitidis. Peck and Hauser (1938) reported that the lipids of the organism contained two separable types; about two- thirds of the lipid was acetone soluble fat and the other one-third acetone insoluble phosphatide. These workers (demonstrated that the phosphatide contained glycerol pflaosphoric acid, choline and ethanolamine. In the acetone sc>luble fraction they identified glycerol, ergosterol, and palmitic, oleic, and linoleic acids. Two years later the same workers, Peck and Hauser (14940) found that their extraction technique was not re- HKJvdng all the lipid; after exhaustively extracting with zalxzohol, ether, and chloroform the solids contained 5.7% CXE the lipid. Peck (1942), using the lipid from B; derma- jgitidis, made soap and tested it against the inhibition of tide enzyme trypsin. Baker (19429 reported that after repeatedly injecting IQhOSphatide intraperitoneally into mice, cells of the mOnocytic series responded, but this fraction is not re— sPonsible for the polymorphonuclear response and the necrotizing effect related to the living organism. .AleDoory and Larsh (1962) showed that the cell extract Of§;.§l§rmatitidis, as well as other fungi, must be di- gested and reextracted in order to obtain total lipids. The total lipid for _B_. dermatitidis was 16.9% for the ”wcelitun at 15 days and 40.59% for the yeast at 4 days. .0 "‘ .- " t . r~ ‘0‘- r.’ ...,. . ,. -o . - ...a. . 9" ' ... , . . u u‘ I n u IA - --.. ... .- ~v a.» .. A. ‘p.. o I... 0-. no u. .'A| -. -.v, .4 -u II: I” h“ ‘1 20 DiSalvo and Denton (1963) measured the lipid content of four strains of the organism that had differences in virulence, and suggested that the lipid fraction in addi- tion to causing granulomatous reactions might also be related to the virulence of various strains of the organism for mice and, by inference, for man. B. Polysaccharides. Peck and Hauser (1938) isolated polysaccharides from cxell autolyzates of B; dermatitidis. They precipitated tlie polysaccharides with ethanol, removed proteins from tide preparation with chloroform, NH4OH or chloroacetic arzid. The amount of the polysaccharide was about 1% of tine total dried cells. They were able to Show that pa— txients with blastomycosis gave positive allergic reactions tx: the polysaccharides from the organism. Baker (19429, using the polysaccharide of Peck §£_al, (1940) in rabbits, found that a single intraperitoneal injection of the blastomycetic polysaccharide produced, in the first few hours, sterile peritonitis, retrosternal lYmphadenitis and remarkable changes in the blood stream consisting of leukopenia, lymphopenia and increase in the number of immature amphophils. A similar change was ob- Served by Sabin g£_§l, (1938) with polysaccharides from buberc'le bacilli and pneumococci. .l u ‘R o .- .v u w ,- .- ..~ .J n". no”. 1 .'.L- I--.~ is. n. n A. u v. "o‘- '95.. ‘I‘ 21 C. Toxins. A number of saprophytic fungi have been found to pro— duce toxic reactions (Gortner and Blakelee, 1914; Abel and Ford, 1907) when injected into animals. Salvin et al. (1952) was able to demonstrate that Candida albicans has a toxic effect in mice. Baker (1942a)showed that repeated intraperitoneal in- ;jections of heat-killed B; dermatitidis were toxic for mice, arui often lethal. This is thought to explain the final leathal effect in the.experimental disease, in which masses c>fF organisms and intermingled reacting cells become ne- c:r<>tic, and probably permitted the absorption of substances Sticflias those associated with the suspensions of the heat— ](i.11ed organism. He injected 0.1 mg into mice every other day for 8 weeks with no apparent effect. When 10 mg was gyiven every other day, the animals appeared ill after each illjection; most animals died 5 to 11 days, apparently of a tKDXiC effect of the suspension. The body cavity, when ex- Eunined, showed the peritoneum was lined with a friably, iYellow layer, which was composed mainly of killed B: derma- An endotoxin has been demonstrated by Salvin (1952) With acetone dried cells. The addition of 2 mg of tubercle baCilli along with the 1.7 mg of acetone dried cells gave an 11350 in mice for this system in 48 hours for B; derma- titidiES. -He found that the components from 80 mg of acetone ”- pv" v .a“ ~-v‘- on-I It" (1* o n v (1' 22 dried cells, injected intraperitoneally, caused death in about 80% of the mice in 48 hours. Taylor (1964) found a similar toxin released with trypsin treatment of g, germaf titidis cells as well as when preetreated with 1% Hcl solution. D. Enzymes Rosenthal (1964) used a media with a specific sub- 'st;rate to assist in identification of specific pathogenic ftuugi. Today, this technique is used in a routine way to (iiAstinguish between two morphologically similar dermato- phytes, Trichophyton mentagrophytes and E Rubrum. T_._ Inenutagrophytes hydrolyzed urea while T;_rubrum will not hydrolyze this substrate (Philpot, 1967). Urease was shown to be a specific substrate enzyme in E5- dermatitidis by Taylor and Johnson (1962). These workers Sliowed the mycelial and yeast phase exhibited a constitu- txive urease activity, both in growing cultures containing \Lrea as the sole nitrogen source, and in cell extracts. TPhe Optimum production of the enzyme occurred at a pH of '7.0 with urea as the sole nitrogen supply. Taylor (1962) found an enzyme system in one form of the Organism but not in the other phase. .The mycelial phase utilizes uric acid but not the yeast phase. At high PH.S9.0 this activity is inhibited, however the organism grows well and uses uric acid at a pH of 6.5, 7.0, and 8.0 ‘N325‘cz. Taylor could not find evidence of uric acid being 23 used by the yeast-phase of the organism at any hydrogen ion concentration tested. The extracellular enzyme of B; dermatitidis has been examined by Beneke, Wilson and Rogers (1969). These authors found acid and alkaline phosphatases in the culture medium extracts. They noted that with time an increase in activity could be shown. A difference in the amount of these phosphatases in the two dimorphic phases was noted in tflie culture extracts. Other enzymes that showed no activity in the culture Inexiium when tested on substrates by Beneke, Wilson and Iicxgers (1969), were: B-glucosidase, a- and B-galactosidase, hJ-iacetylglucosamidase, and acetate, butyrate, palmitate, stearate and laurylate esterases. Rippon and Varadi (1968) tested three strains of B;_ Charmatitidis and found no activity for the enzyme elastase, Mfldile a number of other fungi did give a positive elastase activity. Rippon and Lorincz (1964) determined the collagenase aCtivity for 37 different pathogenic fungi, with 35 of these negative and the following two positive: 1; schoen- leinii and Streptomyces madurae, Blastomyces dermatitidis Was one of the 35 organisms that gave a negative collagenase aCtiVi ty . .Rcw'et al. (1970) studied the polymorphic enzyme malate CiehYdrOgenase which is found in many fungi. These workers StudieCI the yeast and mycelial phase of B; dermatitidis- I. o.- I'M:— .u .. .Qb . . .no. r ’ ta .-.-;0 .1...» o 4 ct... a .....l- \I . .... a ...... .1. . o--.. v (I) Mp-q. ..¢. h ‘ . .8: F‘ n. I ~‘ i \" t- 3‘ -.,~ .¥‘ ’. \ Y\. V“: ““ I ‘ a c‘ a n . oN . vi’fia ‘5‘Ahi‘ . ‘.-“.~ 5 1": . 0.! v- .‘1. 24 The method used in these studies involved separation of the mitochondria from the cytoplasm by differential centri~ fugation. Gel electrophoresis was used to show the many :finms of the enzyme. Five bands were present in the cyto- Lflasmic fraction, and only two in the mitochondrial frac- tion of the yeast phase of the organism. B. Other Biochemical Properties. Stewart and Meyer (1938) have shown that B: derma- tiqtidis and Coccidioides immitis differ from the metabolism (of? bacteria, in that the production and assimilation of eanunonia is favored in these organisms even in the presence c>fF glucose. This work was supported later by Bernheim (1942) who demonstrated that ammonia was produced from the Inertural and non—natural isomer of the amino acids. These Eunino acids were not deaminated in the process of being metabolized. Bernheim (1942) showed that oxygen uptake increased in tflae presence of amino acids. He also demonstrated that lower fatty acids are oxidized, but higher fatty acids inhibit the control respiration and the oxidation of added sub- Strates. He found that cyanide inhibits the oxidation of all added substrates, but has comparatively little effect on the control of respiration possibly because it fails to Penetrate into the cells. Nickerson and Edwards (1950) Showed that the yeast phase consumes 5 to 6 times more oxy- gen Per unit dry weight than does the mycelial phase. The 25 yeast phase shows exogenous oxidation of acetate and glu— cose while the mycelial phase shows no exogenous oxidation. levine and Novak (1950) studied the respiration of the yeast phase of the organism. They found the carbohydrates, glucose, mannose and xylose, stimulated respiration, while mannitol, glactatol and sorbitol stimulated oxygen uptake. The fatty acids, increasing in size through caprylic acid, stimulated oxygen uptake, larger fatty acids inhibited Inespiration. The respiration was not affected when KI was adhded. Tyrothricin inhibits endogenous respiration and 1/600 M sodium azide increases the endogenous respiration. Taylor (1961) demonstrated that the dimorphic forms of E3. dermatitidis had no difference in total DNA. He found tliat only RNA in the mycelial phase increases at a slightly slower rate, than in the yeast phase. This author found tine trichloroacetic acid (TCA) nitrogen concentration to be HHJCh higher in the yeast phase than in the mycelial phase. Brunelli (1963) tried to associate the amino acids vvith pathogenicity, but no correlation was found. He did, Inowever, show that B; dermatitidis could be characterized 'by the absence of proline and the uniform distribution of the other amino acids. The gas ethylene was shown by Nickerson (1948b) to be released from the cultures of B: dermatitidis. 26 F. Cell Wall and Ultrastructure. The work of Venezuelian workers Carbonell and Kanetsuna (1968), and Kanetsuna and Carbonell (1969) cor- relate structure and cell composition of g; brasiliensis and B; dermatitidis. The isolated or intact cell walls were studied by electron microscopy by use of enzymes and solvents. Both fungi showed fibrils measuring 100 A at tflie external surface and an inner granular layer. Sec— txions showed an external dense layer and an inner stratum (ienIoid of a distinct fibrillar structure. Glucan and <:rxitin were the main components of the cell wall, in addi- 'thDD to small amounts of lipids and proteins. The data Strggest that the external layer is composed mainly of glu— cans. The ultrastructure of the organism has also been described by Brown and Edwards (1968), and Garrison, Lane and' Field (1970) . Animal Inoculation Many of the early workers infected laboratory animals as a confirmatory diagnosis for blastomycosis (Ricketts, 1901: Stober, 1914; Hamburger, 1907). Several different kinds of animals were used, but mainly rats, mice, guinea pigs, and dogs were used for intraperitoneal injection of the organism . lBaker (1938) compared the effect of yeast-phase and "chlizil phase in mice. He was unable to show one form of 27 the organism to be more infective than the other. He noted that regardless of the initial form of the organism, the only form found from the infected animals was the yeast-phase of B; dermatitidis. Baker (1942a) reported that mice were the best laboratory animals for the study of blastomycosis in animals. Smith g£_al., (1966) using viable mycelial particles (144,000) found that hamsters were the most susceptible animals to infections induced by intra- peritoneally injecting the organism. Baker (1939) found that the repeated passage of an organism into a mouse did not increase the virulence nor did it change the cultural characteristic of the organism. Landy et al. (1970) reported that a difference in suscepti— bility exists because of sex in hamsters, and that the male animals are more susceptible than the female animals. Landay et al. (1968) showed that hamsters could be infected by three routes: intramuscular, subcutaneous and intraperitoneally. Heilman (1947), using mice, found that by intravenous injections, the period of time to death could be related to the number of organisms. When 1.84 x 105 cells were used of one strain, death came to the animal in 7-8 days. Conti-Diaz et al. (1970a, 1970b, 1969) found that male hamsters were quite susceptible to Blastomyces dermatitidis if inoculations were intratesticular. A hundred organisms or less for intratesticular inoculation resulted, at the end of a 35-38 day period, in positive cultures of the organism from the animals. 28 Salfelder (1965) was able to infect hamsters with cuta— neous infections. The disease in the hamsters seems to be comparable to the cutaneous disease of man. The organism was not disseminated throughout the body as was true with the intratesticular inoculation reported by Conti-Diaz 33 21- (1970a, 1970b ) . Miner et al. (1969) attempted to lower the error in inoculation of mice. They observed such variables as the size of inoculation needle, site of penetration, individual who inoculates, angle of injection and the speed of injec- tion. None of these factors seemed to improve the system for test of virulence. Denton and DiSalve (1968) treated soils with conidia and allowed the animal only a short time in contact with this soil, then they were put into a new cage. These animals developed a high percentage of blasto- mycosis. This technique may aid in finding additional soils containing the organism. MATERIALS AND METHODS Blastomyces dermatitidis Cultures Eleven isolates of the organism Blastomyces dermatitidis were obtained from three different laboratories. These isolates were from man, dog and sea lion. The organisms were grown on Sabouraud's glucose agar at 24C; and on the medium known as CYPG medium (see Media) at 24 or 37 c. All inoculations were made from the yeast phase of the organism for cultures at 24 and 37 C, as well as for mice inocula- tion. Transfers were made under a transfer hood for cul- tures as a safety precaution. The isolates uSed in these experiments were obtained from the following laboratories: A. The laboratory of Dr. E. S. Beneke, Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan. 1. St. Joseph Strain-—isolated from a patient at St. Joseph Hospital, Ann Arbor, Michigan. 2. Van Camp Strain4-isolated from a dog, in the Veterinary Clinic, Michigan State University, East Lansing, Michigan. 29 30 3. Oklahoma Strain--isolated from a patient in the Oklahoma Medical Center, The University of Oklahoma, Oklahoma City, Oklahoma. 4. Duke Strain--isolated from a patient in Duke Uni- versity, Durham, North Carolina. This isolate had been in culture for more than 20 years. B. CulUnBs obtained from Dr. J. F. Denton, Department of Microbiology, Medical College of Georgia, Augusta, Georgia. 1. Ga-1-—isolated from a human case of chronic cutan— eous blastomycosis at the Talmadge Hospital on December 6, 1958. 2. B-130--isolated from a human patient with a chronic ulcer of the leg at Chester, Pennsylvania. 3. S-182—-isolated from lymph nodes of a seven year old male dog at Cincinnati, Ohio. 4.) SL-1--isolated from a northern sea lion that died in the Chicago Zoological Park. 5. KL-1--isolated in April 1960 from a soil sample collected near Lexington, Kentucky. First soil isolate. C. Cultures received from Dr. E. S. McDonough, Department of Biology, Marquette University, Milwaukee, Wisconsin. 1. ATCC 18187 (A) Dr. McDonough number B-784 (A). 2. ATCC 18188 (a) Dr. McDonough number B-788 (a). .-,.~ .a'ngl . go: do. ‘ H1 1': If) Media A. CYPG Medium. The medium, known as CYPG was used by Beneke, Wilson and Rogers (1969) as a nutrient rich medium. It contains 4 g each, yeast extract, peptone and casino amino acid and 10 g of glucose per liter. Cultures of B; dermatitidis were grown at 24 and 37 C for enzyme studies both on solid and liquid media. B. BHI Medium. Difco brain heart infusion medium, with or without agar was used for growth of yeast phase of strains of B; dermatitidis. C. Basal Media. These media were used to study the nutritional require- ments of the mutant strain. 1. The medium described by Gilardi and Laffer (1962). Basal Medium = BM Glucose 10.0 g Ammonium sulfate 5.0 g KH2P04 1.0 g MgSO4°7H20 0.5 g CaC12:2H20 0.1 9 NaCl 0.1 9 One of the many variations of this medium is the EM \Nith asparagine at 3 g per liter = aBM. Other amino acids were added to this medium at 1 g per liter, also biotin, 3.. 1 _I (I) 32 thiamine and inositol, which had been implicated in PE. dermatitidis nutrition, were added to this medium. 2. Levine and Ordal Medium (1946). Glucose 10.0 g Ammonium Sulfate 5.0 g K2HP04 5.0 g KH2P04 5.0 g MgSO4-7H20 2.0 9 NaCl 0.1 g FeSO4’7H20 0.1 g MgSO4-2H20 0.064 g Water 1,000.0 ml This medium with high buffering capacity was used to see if the pH of medium could be better controlled during the growing period of the organism. 3. The Medium of Salvin (1949). Glucose 10.0 g Ammonium sulfate 5.0 g NazHPO4 5.0 9 NaCl 4.0 g KCl 1.0 g MgSO4-7H20 0.5 g CaClz 0.025 g FeCl3 0.010 9 Water 1,000. ml This medium was also used to see if it would support the growth of B; dermatitidis in both phases. Basic Requirements for Growth .A. Basal Medium. The basal medium reported by Gilardi and Laffer (1962) ‘was used as a basis in a number of nutrient experiments. The medium consisting of inorganic salts plus glucose (EM) and 300 mg/100 ml of asparagine (aBM) was used to determine :1 .a' '.c- - :- .Io“ VP "I! cC .. .n- I u -v .— . I - u- .‘ u - a I ~.' I V‘s . ‘l1.l . \ v~AUI 'VF I u "‘-. ..'. 'v‘. ‘\I-- '1 '- how, a . I. .- 0" H ..,. 33 the requirements for growth of certain mutants. This medium was supplemented with amino acids and with vitamins in various combinations, to check growth requirements of the mutants at 37 and 24 C. B. Basal Media Plus Amino Acids. The basic medium (BM) was supplemented with 100 mg of a single amino acid, or a combination of up to 9 amino acids, at 100 mg each/100 ml. The amino acids, which in preliminary experiments had shown some possibility of sup- porting the growth of the organism, were used in this experiment. The yeast phase of the inoculum was started at 37(30n the BM, and after six days was used to inoculate the flasks for incubation at 24(L The amino acids used in this experiment were creatine, valine, isoleucine, proline, cysteine, hydroxyproline, tyrosine, histidine and serine, which were used individually or in combination. C. Basal Medium Plus Asparagine Plus Supplements. The basal medium plus asparagine had other amino acids and nutrients added in some cases. These were 0.3% glycine and 0.3% tyrosine, 10 ug/liter biotin and 100 ug/liter of thiamine, and inositol (10 mg/liter). These cultures were grown at 24CL At 37<2the cultures appeared to grow no Ibetter than the controls without added amino acid and sup- Plements. we .. :. 1. 5L , -. 0| -. ‘i it . P'v. — -.v... I. ‘ u'En ”‘ ‘ 'A_~ a...“ . A, “N.“ Irv 34 Measurement of Changes with the Organism A. Growth. Three methods were used to determine rates or amount of growth (Oginsky and Umbreit, 1959). 1. Dry Weight. A filter pad was dried for at least 8 hours in an oven at 80—90(L cooled and weighed. The organism was filtered onto the filter pad, washed 3 or 4 times with distilled water, dried at least 12 hours and the difference in weight was the dry weight of the organism. All samples were sterilized before discarding. 2. Wet Weight. In this method the laboratory vacuum line was used as a source of constant vacuum suction, the organism was fil- tered on regular filter paper, washed in water, the water coming through the filter dripped into the suction flask. In order to maintain a constant water content these drips were allowed to continue until the time between drops was greater than 30 seconds. The organism was then removed from the filter pad and weighed immediately. This procedure allowed the cells to be used in other experiments where the active proteins were to be studied. All materials were sterilized before discarding. 35 3. Optical Density Measurements. Several investigators have used optical density (Guidry, 1967), DiSalvo and Denton (1963), measurements as a method to estimate the number of B; dermatitidis cells based on turbidity. The wave length used is quite varied, for instance Holiday and McCoy (1959) used 625 mu, as did Gilardi and Laffer (1962), Guidry (1967) used 500 mu and 550 mu was used by DiSalvo and Denton (1963). In this work the wave length at 550 mu was used to measure turbidity. B. Hemacytometer Bright Line Counting Chamber. A drop of the organism is placed on the hemacytometer, and spread out over the area by the addition of a cover slip. The number of cells in each of the sixteen small squares are counted and totaled in order to calculate the number of organisms in a large square. Calculations are made by multiplying the number of organisms in this large square by 25, then the product by 104 to find the number of organisms per ml. C. Viability of the Organism. 1. The number of viable cells in the inoculum from stock cultures were plated on mycosel agar (Beneke and Rogers, 1970). When the colonies developed, counts were made to determine the number of viable cells that were present in the original inoculum. 1‘. lei . , 36 2. Study of Number of Viable Cells on Varied Media at 37 C. Using the BM and aBM media, the following additions were made: 100 mg/100 ml each of glycine and tyrosine; 100 mg/100 ml each of proline and glycine; 100 mg/100 ml of proline and no additions. The growth of the organism was checked at 5, 8, 12, and 24 days, using the optical density at 550 mu as an index of growth, to establish a growth curve. The viable cell counts were made at 5, 8, 12, and 24 days. In another experiment the number of viable cells in a culture at different times were examined. Four Specific media were used: CYPG, BM, BM + proline, and aBM + pro— line. The optical density of the cell suspension was meas- ured at 550 mu and the viable cells were determined by the Janus Green method (Reca and Campbell, 1967). These two cell functions were measured at 2, 4, 6, 9, 12, and 15 days. On the 15th day other determinations were made, such as pH of supernatant fluid, dry weight, and acid and alkaline phos- phatase. 3. Vital Stain Method. The vital stain was prepared using 20 mg of Janus Green B per 100 ml (0.02%). A 0.1 ml cell suspension or a dilution from the culture was put into a test tube. The 0.1 ml of Janus Green B dye solution was added to the cell suspension and allowed to stand at room temperature for 10 min. A drop of the cell suspension was placed on the hemocytometer for 37 differentiation of viable and non-viable cells under the microscope. The viable cells do not stain while the non- viable cells stain a purple or dark blue. The percent viability can be determined on the basis of the number of stained cells. D. Lipid Determination. -The lipid concentration was determined by the methods of DiSalvo and Denton (1963) and Al—Doory and Larch (1962). The method of DiSalvo and Denton (1963) extracted fats with chloroform—methanol (2:1) as the solvent. The temperature was kept at the boiling point of the solvent in a 250 ml soxhlet apparatus for 48 hours. The Al-Doory and Larch method (1962) gave an additional acid treatment to the cells that were being extracted. These authors called the first extractable lipids the free lipids, and after acid treatment the lipids were called the bound lipids. The method of Al—Doory and Larch (1962) was used to determine the phospholipid. These fats were insoluble in warm acetone. Mutants of Blastomyces dermatitidis A. Technique used to Form Mutants Ultraviolet (UV) light has been used by a number of investigators, Emmons and Hollaender (1939), Fincham and Day (1965), to produce mutation of fungi. The mycelium and 38 the yeast forms are both affected by UV light, Fincham and Day (1965). The UV lamp (2527 A) was set up in the trans— fer hood 12 inches above the table top. The St. Joseph strain, B; dermatitidis, was used in this experiment. The organism was cultured on CYPG agar 10 days at 37 C, harvested and washed with sterilized saline solution. The yeast cells were counted with a hemocytometer and adjusted with saline solution to 1.6 x 108 cells/ml. Thirty ml of cell suspen- sion were placed on the bottom of a flat Petri dish which was placed on the table directly under the UV lamp. A 0.1 ml sample was withdrawn from the dish at 2, 4, 6, 8, 10, 15, 30 and 60 minutes and placed in 10 ml of distilled water (1:100 dilution). This dilution was diluted to 1:10,000 dilutions and 1 and 0.1 ml sample of each dilution (1:100 and 1:10,000) was placed into a Petri dish in which they were mixed into the CYPG and BHI media. The Petri dishes were placed in a 37 C incubation oven for two weeks. After two weeks, 45 cultures were selected for isolation onto CYPG slants from plates of fungi exposed for different times as shown in Table I of the Results. Two weeks later these isolates were examined for variation in cultural char- acteristics as well as for characteristic cell structures under the microscope. The data on the cultures are tabulated in Table I of the Results. Thirteen cultures were unable to grow on the CYPG medium, two other cultures failed to grow in the next transfer, and the remainder were contaminated. The remaining twenty-six cultures were used in the later experiments. 39 B. Morphological Study of Mutants at 24 and 37 C. The cultures of all the UV mutants were placed on BHI agar and incubated at 37C). The colonies were observed for morphological changes. The mycelial phase of these same mutants was studied in culture on Sabouraud's agar and on Gilardi and Lafifer (1962) basal agar medium at 24 C culture temperature for morphological changes. C. Virulence of UV Mutants in Mice. The mutants were tested for virulence in mice. The intraperitoneal injection route was used for inoculation of the mice, with the number of cells injected into the animals ranging from 104 to 108 cells. The number of viable cells were determined by plating techniques, and counting the number of cells with a haemocytometer (Reca and Campbell, 1967). Gel Electrophoresis The preliminary gel electrophoresis of proteins was de- termined using a modification of the method of Davis (1951). In these experiments the 300-400 ug of soluble proteins to be electrophoresed were not put into a sample gel, but were put into 0.2 ml of buffer or into 40% sucrose solution to aid in applying samples onto the gels. The conditions used on the power supply were 5 milliamperes per gel in the 40 electrophoresis. The tubes used to make the gels were 8 mm in diameter and 100 mm in length. The separation gel was 65 mm in length. The gels were stained by either Swartz black (Buffalo Black) or commassie blue (Weber and Osborn, 1969). The commassie blue was the more sensitive of the two stains. Enzyme Studies A. Acid and Alkaline Phosphatase. The assay for phosphatases was made using the method described by Beneke, Wilsonemd Rogers (1969). The samples were taken from near the colony with a 5 mm cork cutter, put into 1 ml of substrate pfnitrophenol phosphate at pH of 5.0 in acetate buffer (0.1 M) for acid phOSphatase and at pH of 8.5 in Tris buffer (0.1 M) for the alkaline phos— phatase. Some of the filtrates of the mutants with the most active phosphatases were assayed after growth in liquid CYPG medium. B. Enzyme Rate Measurements. 1. Acid Phosphatase. The general phosphatase substrate pfnitrophenol phos- phate was used to determine the acid phosphatase activity according to the method of Garen and Levinthal (1960). The method was modified slightly for temperature of incubation to 30C, and a unit of activity is defined as the numberof 41 umoles of pfnitrophenol released per hour at 30 C. The molar absorbancy index for pfnitrophenol in Tris at pH's higher than 8.5 is 1.62 x 104. The acid phosphatase sub- strate was prepared at 1 mg pfnitrophenol phosphate per ml in 0.1 M_acetate buffer pH 5.0. A one ml substrate-enzyme mixture was incubated for a predetermined incubation period. The enzyme activity was stopped by adding to the incubation 2 ml of Tris base at 0.1 M_concentration. The final optical density (OD) was measured at 410 mu. 2. Alkaline Phosphatase. The alkaline phosphatase was determined (Garen and Levinthal, 1960) by use of 1 mg of pfnitrophenol phOSphate as substrate in 1 ml of 0.1 M_Tris pH 8.5. The incubation mixture, made in 1 cm tubes, contained the following sub- stances: 0.8 ml of substrate-buffer mixture, 0.2 ml of enzyme mixture and water. This mixture was incubated for a predetermined period of time. The reaction was stopped by adding 2 ml of Tris base to each tube and the optical density was measured at 410 mu in a spectrophotometer. The molar absorbancy index is the same as for the acid phos- phatase. 3. Other Enzymes Tested. Blastomyces dermatitidis was cultured on CYPG medium at 37C, the cells filtered out of the liquid, and the fil- trate was tested for a number of enzymatic activities. These «I {u .¢v~ [1‘ I) I) a]. 42 were glucose—6—phosphate dehydrogenase, phosphoglucomutase (PGM), and hexokinase. All the methods used the adsorp- tion of NADPH at 340 mu as a measurement of the change of glucose-G—phosphate to 6-phosphogluconic acid as an assay of an enzymatic activity. The molecular change is measured at 340 mu, where NADPH absorbs 6.2 x 103 O.D. units per mole (Worthington, 1966). The enzyme collogenase was assayed by the technique . used by Rippon and Lorincz (1964). The enzyme invertase was determined by the method of Spiro (1966). The reducing sugar was a measure of the amount of activity of the in- vertase. C. Protein Determination The protein content was determined by two methods, 1) the 260-280 adsorption ratios of Warburg and Christian (1942), and 2) the method of Lowry et al. (1951). In many Samples, especially when the CYPG medium or other rich protein media were used, the protein of the solution was of no value to the experiment determined by either method. RESULTS Mutants of Blastomyces dermatitidis The colony of the parent strain, B; dermatitidis St. Joseph strain, was used for comparison with the colonies that developed from the irradiated yeast cells after 10 days on CYPG medium at 37 C. Any morphological changes in the colony or changes in the rate of growth, which may indi- cate a nutritional deficiency, were used as a basis for selection of the isolates. A total of 45 isolates were selected from cells irradiated at 2, 4, 8, 15, 30, and 60 minutes under ultraviolet light were transferred to CYPG or BHI medium for further study. Not all of the 45 isolates grew or continued to grow after transfer. The mutants or variants were selected for isolation on the basis of change in colony morphology. Table I shows the cultures isolated with noted colony variations. Some of the 45 isolates were lost when isolated on BHI or transferred to CYPG medium which indicates unstable characteristics or deficiences in the mutants to maintain colony growth. The remaining 26 isolates were cultured on CYPG medium at both 37 C and room temperature, 24 C. These isolates were able to grow on BHI or CYPG agar media. 43 44 Table I. The mutants of B; dermatitidis, St. Joseph strain isolated after ultraviolet irradiation on CYPG medium at 37 C. Mutant Exposure Colony Appearance min. M-3 60 crenulate edges M-12 2 folded waxy M-13 2 folded waxy M-14 2 folded waxy M-17 2 waxy, folded M-24 4 wrinkled waxy M-25 4 some reddish color M—26 4 slow growing M-27 4 wrinkled, waxy M-28 4 . crenulated edges M-29 4 slow growing M-30 4 small projections M—31 4 normal M-32 4 wrinkled, waxy M-33 4 crenulated M-34 4 crenulated M—35 4 wrinkled, waxy M-36 4 wrinkled, waxy M-37 4 variated yeast M-38 4 wrinkled, waxy M—39 4 wrinkled, waxy M-40 4 whitish-rose, granulated M-41 4 crenulate edge M-42 4 wrinkled edges M-43 4 slow growing M-44 4 small and crenulate edges St. Joseph 0 wrinkled, waxy 45 A. Mutant M-36 Figure 1 will demonstrate the change that has occur- red with the organism isolated from its colony. The di- morphic organism B; dermatitidis has lost its ability to change to mycelium phase. This fact can be confirmed by observing the structure of a room temperature culture (sup- plement aBM) under the microscope (Figure 2). Note the absence of any hypha in M—36; while the parent strain has many true hypha, with dflamydospores and other factors which are typical of B; dermatitidis grown at 24 C. After mutant M-36 was grown on CYPG medium at 24 C and 37C for up to 30 days, the colony appearance at both tem- peratures remained in the yeast-like form with a wrinkled, waxy appearance. The usual downy to granular appearance of the mycelial phase did not appear except for an appear- ance in a sector. Figure 1 illustrates the colony of the parent O B_. dermatitidis St. Joseph strain, and the colony of the mutant, M—36. When the parent strain was compared microscopically with the mutant M-36 (Figure 2), the parent had the usual hyphae, conidia and chlamydospores while the mutant had -typical thick-walled budding cells, 8-15 u in diameter. Some of the cells, not more than 3—4, would remain together. 46 Figure 1. Mycelial phase colony of g; dermatitidis St. Joseph strain (Control) and a yeast-phase colony of mutant M-36 on CYPG medium for 7 days at 24 C. 47 7AIE)_____‘____‘_“_‘_“‘XT320 budding dermatitidis and ; (b) cells of mutant M-36 on aBM medium for 20 days at 24 C. ) Hyphae, conidia and chlamydospores from (a colony of B. Figure 2 48 Media for Growth of Strains of Blastomyces dermatitidis and Mutants A. Amino Acids and Vitamins in Relation to Growth The nutrient requirements of the St. Joseph strain.ofB. dermatitidis and the two morphological mutants from this strain 7 M-26 and M-36, were studied at 24C. When the organisms were grown in Gilardi and Laffer's (1962) basal salts medium containing glucose and asparagine, the rate of growth of M-36 was greater than either of the other strains (see Table II). This was even more marked for M—36 with the addition of biotin and thiamine to the medium. This M-36 strain is in the yeast phase at.24c:which may relate to the more rapid growth rate. When the amino acids, tyrosine and glycine were added at 300 mg/100 ml to the basal salts medium, there was a very marked increase in the growth rate although less for M-26. The addition of biotin and thiamine with the two amino acids to the basal salts medium had no marked effect on an increase in the growth of the strains. Halliday (1955) reported that the vitamin biotin was required for the growth; however biotin may be needed in the case of M-36, especiarly if other nutrients are limiting factors. Mutant strain M—26 showed less growth under these conditions Since the rate of mycelial growth was considerably less for the M-26 strain than either of the two other strains on the basal salts medium plus the two amino acids and the two 49 Table II. Growth of B; dermatitidis St. Joseph strain and mutants M-26 and M-36 on Gilardi and Laffer's medium with asparagine (aBM), certain amino acids, and vitamins for 20 days at 24 c. Strain Medium pH Wet Wt' mg St. Joseph Basal 6.3 104.5 M-36 Basal 7.3 220.0 M-26 Basal 6.7 108.7 St. Joseph Biotin + 6.2 93.0 Thiamine1 M-36 Biotin + 7.4 322.0 Thiamine1 M-26 Biotin + 6.6 114.7 Thiamine1 St. Joseph Glycine + 7.4 1496.0 Tyrosine2 M—36 Glycine + 7.8 1585.3 Tyrosine2 M-26 Glycine + 7.0 1070.0 Tyrosine2 St. Joseph 2 Amino Acids 7.6’ 1596.7 + 2 Vitamins3 M-36 2 Amino Acids 7.8 1426.7 + 2 Vitamins3 M-26 2 Amino Acids 7.0 737.3 + 2 Vitamins3 1One microgram of biotin + 10 ug thiamine/100 ml incubation. 2Three hundred mg of the amino acids tyrosine and glycine per 100 ml. 3Add 300 mg of tyrosine and glycine plus 1 ug of biotin and 10 ug of thiamine per 100 ml. 50 vitamins, it was thought that perhaps one of the other amino acids was required for the mycelial growth. In view of this, a series of 24 amino acids (100 mg) were added to each 100 ml of medium in 24 of these flasks for comparison with controls. The results of these experiments showed that in all cases there was no growth of the organism at 24C. This indicated that strain M-26 requires more than a single amino acid and there are other limiting factors for growth. Additional media were compared with that of Gilardi and Laffer (1962) for rate of growth of the St. Joseph strain and a strain from Georgia, Ga-1. These media in- cluded those of Salvin (1949), Levine and Ordal (1946), and CYPG medium of Beneke, Wilson and Rogers (1969). The re- sults are shown in Table III for St. Joseph strain and Table IV for Ga-1 strain. The-tdata in Tables III and IV indicate that CYPG is the most complete enriched culture medium for the organism at 24 C. The Levin and Ordal (1946) medium is a more nearly complete medium for the growth requirements of the organism at 37C. The BM plus asparagine also shows a good growth at 37 C. However, the CYPG medium will support very good growth of the organisms in stock cultures at both temperatures. B. Proline and Asparagine in Relation to Growth and Viability When proline was added with or without asparagine in the basal salt medium of Gilardi and Laffer, a marked varia— tion occurred for the St. Joseph strain. The cell viability 51 Table III. Growth of E; dermatitidis St. Joseph strain on several basic media for 15 days at 24 and 37 C. Medium Temp. Drngt.1 pH Gilardi and Laffer2 24 13.0 5.8 CYPG 24 815.5 8.1 Salvin 24 113.5 5.6 Salvin 37 126.0 5.3 Levine~and Ordal 37 333.0 5.3 1Usually based on 4 replicates. 2Basal medium. Table IV. Growth of B: dermatitidis Ga-1 strain on several basic media for 15 days at 24 and 37 C. . Dry Wt.1 Medium Temp. mg pH Gilardi and Laffer 24 35.0 5.4 CYPG 24 347.3 8.3 Gilardi and Laffer 24 58.0 6.0 (BM + asparagine) Gilardi and Laffer 37 165.5 7.3 (BM + asparagine) Levine and Ordal 37 270.0 6.0 1Usually based on 4 replicates. 52 was lost rapidly in the basal medium alone at 37C. A great increase in the yeast phase (37cfl growth occurred in the basal medium with asparagine and proline, while without asparagine this did not occur (see Table V). However, there was no marked change ingrowth of the mycelial phase with pro- line and asparagine for the Ga-l strain at.24 pcm . mm0h .um mHUHuHumEHmp .m How UmcHEHmuwp .0 an pmusmmme THTB mmumn SDBOHm one .m musmHm 55 “as .2 ameabau m>ta_m<_>...:. / s I \‘fill VIII! 0” \‘I‘i‘. III] .e/ . .Tww. /4..l-.u\\lou.l \lkh/III/I/I 00—. ial...) : ‘) In N am 09; .LV ALISNJO 1V3|1d0 56 Table VI. Growth rates and viability of B; dermatididis St. Joseph strain on different media after 15 days incu- bations at 37 c. Dry Wt. Medium ‘mg‘ pH CYPG 451 8.7 aBM + Proline 285 5.15 BM + Proline 89 3.6 BM 43 3.5 Table VII. Cell growth and viability of yeast cells of g; dermatitidisffi; Joseph strain on the basic salts medium of Gilardi and Laffer with different amino acids. Cultured at 37 C. Day 5 Day 8 Day 12 Amino Acid on1 %v2 Dw3 OD %v DW OD %v DW None 0.39 71 —— 0.60 11 42 0.60 1 -- Asparagine 0.88 91 —— 1.00 90 179 1.08 77 160 Proline 0.77 95 —- 0.58 49 56 0.70 31 90 ASParagine 0.46 78 -- 0.95 75 166 1.10 82 180 + proline 9‘01199 + 0.23 86 -- 0.50 81 65 0.66 4 -- GlyCIne Asparagine + Proline 0.92 93 -— 0.92 75 298 1.13 97 224 + Glycine GlYCin? + 0.15 94 -— 0.33 78 55 0.36 91 53 TerSIne Asparagine + Glycine 0.28 88 -- 0.47 87 86 0.51 71 73 + Tyrosine 1on 2%V 30w Optical density at 550 mu. Percent viability. Dry weight. 57 .EDHUOE omwo QDHB comHHmmEoo cH Asmmv OCHmmHmmwm mSHm 2m paw AEmv ESHUOE muHmm Hmmmn CH Aommv OCHHOHQ SDHB chuum Lennon .um .mHUHuHumEHOU 4m How UOCHEHODOO mmB mHHmo mo quHHQMH> 0:» pan :8 Own um .D.O we Umusmmma OHTB mmumu LuBOHm one .w musmHm 58 318VIA % 5113:) ON. 0? 00 on - on: .4 325: O or." 5 335:0 923 o.« m... o _ m .55 III in u4Q<_> I I Eunice?! ea>o , ., V 0m>0 ’/\ 1V3lldO' mu 0 99 .LV ALISNJO 59 Figure 5. Growth curves of B. dermatitidis in the mycelium (24 C) and the yeast (37 C) phase on a CYPG medium. 6O GRAIS 5.: u 8 N P N WET WEIGHT 5 IO 15 2O 25 3O FIGURES onvs CULTURED 61 Situation, no cell increase occurred with the asparagine, glycine and tyrosine combination, while good growth occurred when proline was substituted for glycine (see Table VII). This organism was not able to utilize glycine in combina- tion with tyrosine in the medium at 37 C while at 24C excel- lent mycelial growth developed (see Table II) with glycine, tyrosine along with biotin and thiamine. This indicates a marked difference in utilization of the two amino acids at 24 and 37C fer this diphasic organism. Virulence of the Mutant Strains The virulence of the mutant strains of B; dermatitidis St. Joseph was determined by intraperitoneal inoculationcfiffle yeast phase of the organisms. The days to 50% kill were de— termined when possible for each mutant strain, at a higher concentration of cells of about 5 x 106, The usual LD50 for the parent St. Joseph strain would be around 14 to 16 days. At a dilution of 5 x 105 thirty days was necessary for an LD50. Table VIII shows the preliminary test for virulence of 21 of these mutants. These mutant organisms showed a range of virulence; for instance, M-24 and M-42 and M-43 were nearly or slightly more virulent than the parents. A less virulent group of organisms included M-3, M-35, and M-36, with the latter two appearing to be less virulent as higher cell concentrations were used.- The third group of organisms appeared less virulent, as M-26 produced no symptoms in the mice at a cell dilution of 4 x 105. 62 Table VIII. Virulence of mutants of the St. Joseph strain of B. dermatitidis injeCted intraperitoneally (IPT'into mice. Days to No. Mice Mutant Inoculum 50% Kill Killed Observations St. Joseph 5 x 105 30 5/5 granulomas M—3 7.5 x 105 45 4/5 granulomas M-12 6 x 105 36 4/5 granulomas M-13 5.2 x 106 23 5/5 granulomas M-14 9.85 x106 22 5/5 granulomas M-17 2.4 x 106 23 6/6 granulomas M—24 6.5 x 105 24 5/5 granulomas M-26 4 x 105 -- 0/6 no granulomas M-27 1.1 x 107 12 5/5 granulomas M-28 5.2 x 106 16 6/6 granulomas M-29 5.2 x 107 14 4/5 granulomas M-30 1.0 x 106 26 5/5 granulomas M-31 1.04 x107 17 5/5 granulomas M-32 6.4 x 106 14 5/5 granulomas M-35 1.0 x 106 46 3/5 granulomas M-36 4.0 x 106 45 6/8 granulomas M-38 5.65 x106 26 5/5 granulomas M-40 8.0 x 104 —— 1/5 granulomas M-42 1.0 x 105 34 4/5 granulomas M—43 5.0 x 105 39 4/5 granulomas M-25 4.25 x106 -- 2/5 granulomas M-37 5.4 x 106 -- 0/5 no granulomas 63 Two other mutants, M-25 and M-37, were less virulent at a dilution of 4.25 x 106 and 5.4 x 106 respectively. Higher concentrations need to be used to determine the days to 50% kill. Other strains of B; dermatitidis were selected for virulence tests. The 12 strains from patients in different parts of the United States show a variation in virulence when injected into mice (see Table IX). For example, the Ga-1 strain from Georgia had no days to 50% kill at 1.4 x 105 and was selected for further tests at higher cell concentra— tion (see Table XI). The Oklahoma, Kl—l, 130, and S-182 strains are examples of less virulent strains of B. derma- titidis in comparison to St. Joseph strain or several other more virulent strains tested. A. Virulence of the Mutant M-26 A comparison was made on the effect of increasing cell concentration of the mutant M-26 and the parent St. Joseph strain on virulence in mice. Table X shows the difference in virulence of M-26 in comparison to the parent strain of St. Joseph. The parent strain had 29 days to 50% kill with 1.1 x 105 cells of inoculation in the mice, while the M-26 mutant required 30 days with a 1.2 x 107 cells of inocula- tion in the mice. For a 50% kill in 30 days in the mutant strain it is necessary to have over 100 times as much inoculum as is required in the parent strain. This makes the mutant an interesting organism for study of factors related to virulence. Table XI shows a 64 Table IX. The relative virulence of twelve strains of Blastomyces dermatitidis. injected IP in mice. Strain Inoculuation Niilfiége 58%SKIII St. Joseph 9.4 x 105 6/6 22 Wisconsin-788 1.0 x 106 5/6 24 Wisconsin—784 3.2 x 105 4/6 29 Ga—1 1.4 x 105 0/7 -- SL-l 1.2 x 105 4/6 26 KL-l 2.6 x 105 1/6 _- 130 7.5 x 105 1/6 -- S-182 1.5 x 106 0/6 -- Oklahoma 3.75 x107 2/4 89 Van Camp 3.28 x107 4/4 25 Duke 2.1 x 107 7/8 45 Table X. The effect of increasing cell concentrations of the mutant M-26 of B;_dermatitidis on virulence when injected IP in mice. Strain 1:52:13m 3:? Days for No. Mice Observations* mg 50% Kill Killed Parent 1.55 x 107 14.1 14 4/4 Granulomatous Parent 1.1 x 106 1.0 21 4/4 Granulomatous Parent 1.1 x 105 0.1 29 4/4 Granulomatous M-26 1.2 x 107 20.8 30 4/4 Granulomatous M-26 5.8 x 105 1.0 -- 0/4 No granulomas M-26 5.8 x 104 0.1 -— 0/4 No granulomas *- The experiment was terminated after 90 days. NOTE: M-26 at 8.2 x 106 cells per mouse had 6 mice killed out of 16. 65 comparison of serial dilutions of yeast cells of strains Ga-l, M-37, M-25, and St. Joseph. The mutant M-37 was less virulent than the mutant M-25 and the Georgia strain (Ga-1) as indicated by the days for 50% kill at a dilution of 108 cells. Table XI. Effect of dilutions of cells of different mutants and strains of E; dermatitidis on virulence When injected IP in mice. 33375.3: finishes 234555105 237865 Ga-l 6/10* 21 0/10 -- 0/10 __ __ M-37 5/8 78 0/10 —- 0/10 -_ __ M—25 6/7 13 1/10 -- 2/10 —_ __ St. Joseph -— -- 4/4 14 4/4 16 9/9 30 *Number of mice killed/total number of mice. NOTE: M-26 at 8.2 x 106 cells per mouse had 6 mice out of 16lflflled at the end of 90 days. B. Toxin as a Factor A toxin determination was made with the St. Joseph strain by the method similar to that reported by Taylor (1964). He indicated that a toxin may be the cause of death of the mice in his experiments. Table XII shows the results of the toxin experiment indicating that trypsin-treated cells did give a more rapid kill of the mice than did the control. Treatment with 1% HCl sabtion, however, required more time for the kill than did the control. One control for trypsin that was 66 Table XII. A test for toxin production in B; dermatitidis St. Joseph strain grown at 37 C. Inoculation included 2 mg of acetone-dried tubercle bacilli/ mouse plus the treatment. Treatment Inocgéation No. Mice 58%riili 37 C Control 40 7/7 156 37 C Trypsin1 40 7/7 72 37 C 1% HCl2 40 7/7 192 65 C HK3 Trypsin 40 0/7 -- 65 C HK Control 40 0/7 _- Saline 0 0/7 -- 1Two mg of trypsin treatment for 1 hour at 37 C. 2One percent concentrated HCl solution treatment for 1 hour at 37 C. 3HK = Heat kill at 65 C for 1 hour. 67 not run by Taylor was added to these tests to see if the tnfisin-treated heat-killed cells would cause death of mice. No mice were killed under these conditions. The high .number of yeast cells in the trypsin treated mice in com- parison to the control mice does not necessarily indicate toxin later on in the experiment. However, in the time period up to 96 hours after inoculation, a toxin could account for the more rapid death rate in the animals. A second experiment was run in which there was a reduction in the amount of trypsin-treated cells inoculated into the animals. In this case all the animals died at the same rate as the control animals. An experiment similar to Salvin (1952) was tried with 2 mg of heat-killed and 2 mg of acetone-killed cells per mouse. None of these animals died, however, the control animals with 2.8 x 106 cells per ml had an LD50 of 17 days. This evidence suggested that there was no toxin, or not suf- ficient toxin in the strain used to kill the animals as was reported by Salvin (1952). C. Lipids Effect on Virulence A higher lipid Content of strains of B; dermatitidis has been reported by DiSalvo and Denton (1963) to represent an increase in virulence. The organisms were grown in BHI plus biotin agar for 10 days at 37'C. The lipid content of four strains, St. Joseph, Oklahoma, Van Camp, and Duke was extracted with chloroform-methanol (2:1) for 48 hr, for animal inoculation. After intraperitoneal 68 inoculation of cells of the four strains into mice, the Oklahoma strain with the high lipid content had a 50% kill of 89 days (see Table XIII). The strain with the highest lipid content had a 50% kill of 63 days while the other two strains with lower lipid contents had more rapid kill indi- cating greater virulence. Three strains were selected for further comparison of lipid concentration in the cell with virulence in the animal. Two of the strains, M—26 and Ga-l, were low in virulence compared to the St. Joseph strain which higher had higher virulence. The Al-Doory and Larsh (1962) procedure under Methods was used to determine lipids. These organisms were grown on the enriched CYPG medium for 15 days at 37 C. Table XIV shows the lipid content is higher for the two less viru- lent strains, M-26 and Ga-l; lower for the more virulent St. Joseph strain. Table XV shows the lipid content for two of the above strains, St. Joseph and Ga-l strain still has a higher lipid content similar to the St. Joseph strain with a higher lipid content at 24 C than at 37 C. This result differed from that reported by Al-Doory and Larsh (1962‘. Alkaline and Acid Phosphatase Activities A. Cultures at 24 and 37 C Assayed for Enzyme Activity. Variations occurred in the amount’of acid,and alkaline phosphatase activity produced by the various mutants and 69 .mCOHHMCHEHmump OBD mo mmmum>¢ * no «\a toe x oH.m om.HH ao.oa H.Hna o.ooo oxso hm e\e toe x mm.m oe.m mm.n e.oma m.uoo damo cm> om e\m toe x ns.m oe.o SH.NH H.moa m.mom maocmaxo as e\e .oH x so.a em.h em.o h.ome o.oao summon .um. waxmmmm OWWWHMWZ coHumHsoocH *Hmuoe *UHQHA rmum 903 mEchmmHO . mUHmHA R we :H .uz .Dmanom m CO u: me How AHuNV HocmnumEIEHOmouoHno nuHB Umuomuuxm OHOB mHHmO one .0 hMIum mamO;oH How Hmmm cHuOHn msHm Hmm co OHSDHDO m Hmumm mHoHu IHumEHOC .m mo msHmuum Hsom mo coHumHucmosoo UHQHH Dam HHHx Rom on mama .HHHx OHQOB 70 Tablelflflz The lipid concentration of the more virulent St. Joseph strain and the two less virulent strains of E; dermatitidis after culture on CYPG agar for 15 days at 37 C. Wt. in mg % Lipids1H Organism Wet Dry Lipid Free Bound Total %* M-26 2486 461.7 161 14.7 20.1 34.8 Ga-l 2251 331.7 37 9.3 1.9 11.2 St. Joseph 1662 389.9 30 6.7 1.0 7.7 * Based on dry weight. *~)(- Solvent for extraction was chloroform-methanol (2:1). Extraction was for 48 hr. The cells were treated with dilute HCl solution and re-extracted. Table XV. The lipid content<33nm>strains of E3 dermatitidis after culture on CYPG medium for 15 days at 24 C. Wt. in mg % Lipids**7 Organism Wet Dry Lipid Free Bound Total %* Ga—l 3089 340.3 87 22.9 2.6 25.5 St. Joseph 6965 815.5 140 7.7 9.4 17.1 *- Based on dry weight. ** Solvent for extraction was chloroform-methanol (2:1). Extraction was for 48 hr. The cells were treated with dilute HCl solution and re—extracted. 71 strains of B; dermatitidis. In the assay method on solid CYPG medium ultilizing plugs from the medium after growth of the organisms for 15 days, the yeast phase at 37 C, in general, showed a higher acid phosphatase activity than the mycelial phase at 24 C for all the strains checked (see Table XVI). The alkaline phosphatase activity for the organisms in the mycelial and yeast phase were somewhat similar, with some strains higher at 37 C and some higher at 24 C or nearly the same for both temperaturs. The alka- line phosphatase assay was linear for 0-0.5 O.D. at 410 mu. Eight strains of B; dermatitidis were grown on CYPG agar at 24 and 37 C for comparison with the mutant strains, and assayed for alkaline and acid phosphatase. The results are Shown in Table XVII. There was very little difference in most of the eight strains at 24 and 37 C except for Kl-l and 130. These two strains were much higher in alkaline and acid phosphatase activity at 37 C in the yeast phase. Filtrates from eight strains of B: dermatitidis and eight mutants grown on liquid CYPG medium for 15 days at 37 C were used for enzyme assay. The alkaline phosphatase activity was higher than the acid phosphatase in the majority of the strains including all of the mutants. These results are shown in Table XVIII. These groups of organisms were not grown at 24 C for comparison. 72 Table XVI. The extracellular acid and alkaline phosphatase activities of mutants of B; dermatitidis grown on CYPG medium for 15 days at 24 and 37 C. The activity is expressed in mumole of substrate changed/hr/plug of agar. Average of two assays. Mycelialjphase Yeast-phase Mutant Acid Alkaline Acid Alkaline M-3 7.6 7.6 12.6 4.6 M-12 7.2 13.5 3.7 4.1 M—13 2.9 5.0 29.3 3.3 M-14 2.2 5.0 4.6 4.1 M-17 2.9 3.9 6.5 4.6 .M-24 2.6 2.2 17.2 6.7 M-25 4.1 5.2 4.3 3.7 M-26 2.6 3.5 3.9 4.8 M-27 6.7 12.6 10.7 3.7 M-28 6.7 2.5 13.5 14.8 M-29 2.2 5.7 6.3 4.6 M-30 14.6 15.9 15.9 4.6 M-31 8.5 5.7 5.4 4.3 M-32 9.3 4.4 10.0 9.4 M-33 4.1 2.5 10.7 11.4 .M-34 2.5 4.1 4.9 3.9 M-35 3.9 5.0 17.4 13.9 M-36 11.6 2.5 15.0 3.5 M-38 6.5 1.8 20.4 11.4 M-39 2.5 2.2 3.7 3.9 M-43 1.3 1.5 9.3 3.9 M-44 5.7 5.6 13.1 3.9 73 The extracellular acid and alkaline phosphatase activities of 8 strains of B; dermatitidis grown on CYPG medium for 15 days at 24 and 37 C. The activity is expressed in mumole of substrate changes/hr/plug of agar. Average of 2 assays. Table XVII. Mycelium-phase Yeast—phase Strain Acid Alkaline Acid Alkaline St. Joseph 4.1 4.3 1.3 2.4 S-182 2.2 1.3 1.5 4.1 Ga-1 5.2 3.3 3.7 13.1 Sl—l 5.7 15.5 0.7 2.8 Wisconsin 788 3.5 6.3 1.5 2.8 Wisconsin 784 6.5 10.7 1.5 1.8 Kl—1 5.6 3.5 15.7 23.0 130 3.1 3.3 23.0 30.7 Table XVIII. The extracellular acid and alkaline phosphatase activity of 6 strains of E: dermatitidis and 8 mutant strains grown on CYPG medium for 15 days at 37 C. Filtrates were used for assays. The activity is expressed in unitS*/100 ml of filtrate. Average of 2 assays. Strain WetGithIn g. AlkaITizghatasizid Sl-1 1.868 30.0 17.3 Kl-1 2.814 34.7 9.4 Wisconsin 788 2.066 10.6 8.0 Wisconsin 784 1.755 5.9 7.8 130 2.784 13.6 6.1 Ga-l 1.675 13.6 6.1 M-12 2.213 24.1 13.0 M-17 2.073 37.7 15.6 M-25 1.308 30.2 11.8 M-26 1.962 36.4 17.6 M-30 2.386 33.5 14.6 M—35 1.864 42.0 17.5 M-36 1.937 19.2 15.0 M-44 2.122 16.6 8.2 *unit = 1 umole of pfnitrophenol phosphate used per hour. 74 Four additional strains, two mutants and two strains of B; dermatitidis were grown in liquid CYPG medium at 24 and 37C for 20 days. The filtrates were used for alkaline and acid phosphatase activities. The results are illustrated in Table XIX. Under these condition the organisms still produced more alkaline than acid phosphatase at 20 days but at lower levels than at 15 days. However, at.24c:both the alkaline and acid phosphatase activity was higher than at 37C in the 20-day cultures. Four mutants and the parent St. Joseph strain were Checked for alkaline phosphatase activity and virulence in mice after the organisms were grown in CYPG medium for 15 days at 37 C. There was no definite relationship between the amount of alkaline phosphatase activity in these strains and virulence. Two strains, M-26 and M-36, with a some- what higher enzyme activity, were less virulent (see Table xx). B. Growth Curve of B. dermatitidis on CYPG medium at 24 andEW’C in relation to intracellular alkalinegphos- phatase activities. Blastomyces dermatitidis St. Joseph strain was grown on CYPG medium at 24 and 37C for a period of 30 days. The peak of growth for the mycelial phase was between 10 to 12 days, while the alkaline phosphatase activity from cell ex- tracts reached a peak in 20 days (see Figure 6). The peak in growth for the yeast cells was between 9 to 10 days, which was similar time for the highest enzyme activity from cell extracts of yeast cells. Table XIX. 75 The extracellular alkaline and acid phosphatase activity of two strains and two mutants of B. dermatitidis, grown on liquid CYPG medium far 20 days at 24 and 37C. Filtrates were used f assays. The activity is expressed as umoles substrate used/hr/lOO ml. (Average of 2 enzy assays.) or of me Cells, Wet wt. ' Phosphatases . "“in“mg Alkaline (Acid Strains 24 C '37 C 24’C 37 c 24 C 37 g_ Ga-l 2.84 2.5251 98 3 51 3 St. Joseph 3.226 1.742 20 10 15 2 M-26 4.082 2.664 23 14 17 11 M-36 1.4341 2.352 26 17 9 8 1Lost one of 2 replicates by contamination. Table XX. The extracellular alkaline phosphatase in CYPG medium 15 days at 37C and virulence in mice of four mutants and the St. Joseph strain of B; dermatitidis. The activity is expressed as umole of substrate used/hr/lOO ml. . . Wet wt. No. Mice Days to Strain Rep. Alkaline grams Killed Inoculum50% Kill St. Joseph 5 3.6 2.21 5/62 4 x 106 19 M—17 5 11.1 1.94 5/61 2.4x 106 24 M—26 5 13.0 2.84 0/62 4 x 105 -- M-28 5 4.4 2.45 6/6 5.2x 106 16 M-36 2 19.2 1.94 6/82 4 x 106 45 1One animal died of unknown causes. 2Remaining animals were sacrificed after 90 days of treat- ment. Figure 6. 76 Growth curve of B. dermatitidis on CYPG medium at 24 C and 37 C for 30 days. The intracellular phosphatase activity for the yeast phase at 37 and the mycelial phase at 24 C is expressed as umoles of substrate Changed/hr/lOO ml. 77 5 J um< P