av‘vb'v'f'l”.¥V v unyvuuy-rv'uwvv pAmoemcrrY AND nu games; or mama-11.1111 '_scH£Nc1<11 TRITIONAL AND 111.121me 1995) air. (MATRUCfl 1 OBTAINED av ULTRAVIOLET LIGHT mmmmmmnwtgmwzfifiurmmnheywmp. , . 33... . a 1.31.9. ...fisuflfinfiung: .2 finflfinhiwwmgmwil a. .-.1 . .un. . $191.. 121......“21Lwfluwfi 59A 2a..-. AT... .. h. .12 K » v D’I‘” I!!! s n .. .. . . . .. . ...! J.- vélvv 0.. I. _ . . 5.. , 2.. ... . . .. ...... . an... .... an. Gazing . . 1 . . . . . kg!) . .W .efla... I..." . . v u; I 1 A . .. . . . u. . ., U . .. . . 1 . . . . . H... 3. . . 1 .... 0.4.. . ...fluxl. . .h 1 . hum-l ...... u: . Q .. . a. . .e'x*-*:‘~' r . .,-. . ‘. .fi ‘. 1. ~. r-‘ . v '1 .. ... .~' ... . , ..;_ 1 . 1:511:11nvmsiifiu Jukfi..§!.fifiurmmh. ...Hfiniflxhrz; RmYmYWiflfiur. .511! , “*1“de ..... ...... ..1 J. .1. Ir... . . . . .. .. .. .. .....1. 3. 11.1.... ”11.1. i. tux. 1 . . I. 1 x 1 ...... . .1 1 111......11...r..v11.1|x.1x 1.5.1.11... .11 16.111311 1 .. . ...1... 1 . -... .. ..-. 121.. 11 11:11} ..1: 1.- .21 .11.. 51...”... -... 1 . ...1 . .... ......n....fl.l.1...aa1.! .1.§1§Jflu§. . . 1 1 11 1 11.11.|U .u1.11\or..n.ll.|1. 1 1|- 1::- . 1 1. J «14:31:: This is to certify that the thesis entitled pathogenicity and Nutritional studies of soorotrichum schenckii (Matruchot 1905) and—{Variants Obtafned by Ultraviolet Light Irradiation. presented by Se th S. 2.21 zuba has been accepted towards fulfillment of the requirements for __ P__h.D_ _ degree in__BOtan._y__ _(L:y0010gy) .2 £24,112 14, “K Major professor [hm,January 20, 1956 0-169 -“Y ’5’ LIBRA 1.: ‘1' k“ 1. M ich 1:33 11 S ta 11: University ,j 1 -“’/' PATHOGENICITY AND NUTRITIONAL STUDIES OF SPOROTRICHUM SCHENCKII (MATRUCHOT 1905) AND VARIANTS OBTAIN- ED BY ULTRAVIOLET LIGHT IRRADIATION By Seth S. Mizuba AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology / Approved €:LML£249> gifiglpuazifilp- F1. t!’ 1 rr ,. 1 sh.) 11‘ ABSTRACT Morphological, pathogenetic, and nutritional studies of three strains of pathogenic Sporotrichum schenckii, their mutants and variants and of Sporotrichum epigeum, a non-pathogenic species, were conducted. The variant and mu— tant strains were obtained by irradiation of the fungous strains under ultraviolet light. Several yeast and pleomor— phic forms of the parent strains were obtained which ordi- narily exist only in the living animal or human host or on special enriched media at 37°C. Those strains that sur- vived continual transfer without change were used in the morphological, pathogenetic, and nutritional studies along with several mycelial variants. On Sabouraud dextrose agar plus animal and human bloods at 37°C. variations in morphological types from mycelium to mycelial-yeast or yeast forms and from pleomorphic to yeast forms were obtained. Anti-fungal factors in several animal bloods for specific strains were noted. In the pathogenetic tests, the mycelial strains were found to infect mice more readily than the yeast or pleo- morphic strains. Carbon source studies showed that no one carbohydrate source was able to promote excellent growth of all strains of fungi and in most of the cases, the mutant strains showed inability to grow as vigorously as the parent strain. The nitrogen source studies indicated that potassium nitrate, ammonium nitrate and dl-isoleucine were utilized poorly by the various strains. No one nitrogen source was able to support excellent growth of all strains of these fungi and as in the carbon source study, the mutant strains were unable to grow as vigorously as the parent strains. MicroKJeldahl tests did not show that strains used in the nitrogen studies were able to fix nitrogen. PATHOGENICITY AND NUTRITIONAL STUDIES OF SPOROTRICHUM SCHENCKII (MATRUCHOT 1905) AND VARIANTS OBTAIN— ED BY ULTRAVIOLET LIGHT IRRADIATION By ( , '~~.‘~ ' 01K; Seth S: Mizuba A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1956 ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. Everett S. Beneke under whose inspiration, supervision, and unfailing interest this investigation was undertaken and to whom the results are herewith dedicated. He is also greatly indebted to Dr. Lloyd Wilson and Mr. Samuel Ringel for their valuable suggestions on the nu- tritional studies, and to Dr. A. R. Danes and others for making available the different animal bloods for the blood agar studies. Grateful acknowledgment is also due to Dr. Constantine J. Alexopoulos for his helpful criticisms in writing the thesis, and to Drs. William Drew and Henrik S. Stafseth who served as advisors on my committee. Special acknowledgment is given to Miss Ruth Knight- linger of Bauer and Black (Research Division) of Chicago, who very graciously gave so much of her time in typing the manuscript. Finally, my thanks go to fellow graduate students who offered valuable suggestions and criticisms throughout this investigation. \N TABLE OF CONTENTS Introle-Ction.........OOOOOOOOOOO Ultraviolet Light Irradiation Introduction................ Method and Materials..................... Results and Discussion................... ConClUSionOOOOOOOI......OOOOOOOOOOOOOO... Study of Morphology Introduction.. ...... ..................... Method and Materials..................... Results and Discussion................... Conclusion............................... Pathogenicity IntlflOductiOHOOOOOOOOC0.0.000...00.0.00... [VI-ethOd and l\‘1aterialSooooooooo000000000000 ( Results and Discussion...... ..... ........ conCluSionOOOOOOOOOOOOOOOOOOOOOOOOOO..... Carbohydrate Metabolism IntrOdUCtionooooooooooooooooooooooooooooo MEthOd and Illaterialsooooooeoo0.0000000coo - Results and DiscuSSiOnOOOO......OOOOCOOOO COnCluSiOnOOOOOOOOOO00...... Nitrogen Metabolism Introduction................ I’IethOd and P'ZaterialSOOOOOOOOO0.0.0.000... Results and Discussion................... COHClUSj-OHOOOO.....OOOOOOOOO Bibliography..................... 52 55 57 72 TABLE ...—...... |< Growth Studies On 5 Per Cent Sabouraud Dextrose Blood Agars Of Sporotrichum Schenckii Strains And Sporotrichur Hv3 g.I Human, Horse, Cow, and Chicken Blood Agars...... Pig, Goat, Mink, Dog, and Sheep Blood A 3ars..... Study Of Pathogenicity Of See rotrichum Schenckii Strains And Soorotrichum Epil eUI1 In Michigan Department Oi Health Mouse Strain 26 Days After Inoculation....................... In Michigan State University Zoology Department Strain 2o Days After Inoculation.............. Growth Study Of Sporotrichum Schenckii Strain 1% In L-Asparagine Medium dith Variations In Amount OI InOCUluni-OOOOOOOOO.....OOOOOOOOOOO......OOOOOOOO Co parison Of Dry Myc celial weights Of Soorotrichum Schenckii Strain l and SnorotIIchuI LII eum From L—Asparagine and DL-AspaIa gine NIOOIa Incubate d At 2A°C For 25 Days nei hts Represent Averages of GPO“ rths In Tripliéat87000000000......OOCCOOOOOOOOC Carbohydrate Utilization And Change of PH BY Sporotrichum Schenckii Strains and Snorotrichum ”OIUBUI After 23 Days Incubation At AETC IData Represent Avera ges From Cultures In Triplicate) Basal Medium and L— Arabinose.................. D—Kylose and D- Fructose....................... L-Glucose..................................... L-Galactose and L-Sorbose..................... D-Mannose and L-Rhamnose...................... Glycerol and Sucrose.......................... Lactose and Maltese........................... Starch and Raffinose........ Dulcitol and Inulin...... Degree Of Sporulation In Carbohydrate Media For Sporotrichum Schenckii Strains and Sporotrichum E Ieeum After 2“ Da"s Incubation at ZZVC.......... 2 .>’ Nitrogen Utilization And Change Of PH By Soorotri— chum Schenckii Strains and Spo oretrichum LOL euu Aiter 25 Days Incubation at 23TC IData Represent Averages of Cultures in Triplicate) Basal Medium and Potassium Nitrate............ Ammonium Nitrate And Ammonium Sulfate......... Ammonium Tartrate And Ammonium Sulfate........ L-Asparagine And DL-Ornithine................. DL-Proline and DL-Alanine..................... Arginine And Methionine....................... Continued on next page a. PA GE 21 22 [U CD [\3 KC (Ax)? U: m 1—4 QKO (9 0“ 0.! ('1‘ ~. _ \sé LIST OF TABI3S(continued) TABLE PAGE VII L(+) Glutamic Acid And DL—Leucine.............. O "—' L-Tryptophane And Threonine.................... 6b L-Histidine And Casamino Acid.................. O DL—Isoleucine.................................. 67 VIII Degree of Sporulation in Nitrogen Media For Sporotrichum Schenckii Strains and Snorotrichum Epigeum After 23 Days Incubation at AAIC.......... 71 PLATE I II III LIST OF PLATES - Growth On Sabouraud Dextrose Agar Of Different Strains Of Sporotrichum Schenekii and Sporotrichum Epigeum Strains 2 and l2............- Strains l} and 14............ Strains 15 and 24............ Strains 34 and Al............ Strains 56 and 60............ Strains O1 and OA............ Strains SP............. Growth Of Sporotrichum Schenckii Strain 6+ On Sabouraud DeAtrose Blood Agar Goat and Human Blood Agars... Horse and Cow Blood Agars.... Cigar—Shaped Bodies (Sporotrichum Sehenckii Strain 15) As Found In Mice And zone of Henolysis On Sa- bouraud Dextrose Blood Cigar Caused By Sporotrichum Epigeum............. PAGE [\D U 1 FIGURE LIST OF FIGURES PAGE Growth Curves of Sporotrichum Schenckii Strain 14 Anngporotrichum epigeum in L-Asparagine Medium Uhen Incubated at 24°C; Change of PH By Sporotrichum Schenckii Strain 14 and Sporotrichum Spggeum In L- Asparagine Medium When Incubated at 24°C.......37 INTRODUCTION Eleven species of Sporotrichum, most of which were de- rived from decaying wood, were described by Link (1809). An organism resembling one described by Link causing a human disease was reported by Schenck (1899) in 1899. This organism was named Sporotrichum schenckii in 1900 by Helston and Perkins (1900) who isolated a fungus from a patient and found it to be identical with that of Schenck's. Since 1900, this organism has been reported as the cause of sporotrichosis in man and animals, such as the rat, mouse, rabbit, guinea pig, pig, horse, cow, sheep, and goat (Saunders 1948). De Beurmann and Ramond (1903) in France described the disease in 1903 and the fungus was named Sporotrichum beurmanni by Natruchot and Ramond (1905) in 1905. 'S; beurmanni and other pathogenic sporotricha de- scribed since are now thought to be variants of Schenck's fungus and are considered to be synonyms of S; schenckii. (Dodge.l936). The disease was brought into world-wide prominence in 1947 when it reached epidemic proportions among workers handling infected timber in South African gold mines. (Brown SE a; 1947). Sporo- trichosis has been reported from all continents and seems to be most prevalent in the North Central portion of the United States and in France among the European countries (Conant g3 a; 1944). Lurie (1950, 1951) investigated the carbohydrate and nitrogen metabolism of the pathogenic sporotricha. She found that the differences in utilization of carbohydrate and nitrogen were useless as a means of identifying and classifying pathogenic sporotricha. The phenomenon of filamentous growth in nature or in laboratory media and growth of the yeast form in living ani- mals or human beings or on special enriched media at 37°C has intrigued many investigators. Kurung and Yejian (1954) reported growing Sporotrichum schenckii, Blastomyces derma- titides and brasiliensis, and Histoplasma capsulatum in the yeast phase at room temperature and their maintenance for lengthy periods using a potato—flour—egg mixture medium. The purpose of this paper is to present the results of studies on Sporotrichum schenckii strains and their mutants and variants obtained by ultraviolet light irradiation. Em- phasis is placed on the pleomorphic (mixture of yeast and mycelium) and yeast phases obtained through ultraviolet light irradiation. Changes in morphology, pathogenicity, and in the ability to utilize different nitrogen and carbohydrate sources are investigated. OJ ULTRAVIOLET LIGHT IRRADIATION INTRODUCTION ultraviolet light has been known to have destructive effects on microorganisms in lethal doses and variational and mutational effects in sublethal doses. Lacasgne (1930) found that Saccharomyces ellipsoides eXposed to radiations of wave-lengths between 2800 and 3800 Angstroms units fell into three classes according to their susceptibility to the rays. Those in the first class died instantly. Those in the second class died upon continued periods of irradiation, while those in the third class un- derwent a temporary.stationary period of no cell division but ultimately recovered their power of reproduction. A- mong those that survived the exposure to the rays, Oster (1934- 1955) in his work with saccharOmyoes cerevisae observed cer- tain abnormalities in cell growth and reproduction. He ob- served cells of giant size, cells more spherical in shape, cells showing a long filament-like process constricted at intervals along the length, and colonies which had their sizes increased at a rate roughly prOportional to the increase in incident energy. Emmons and Hollaender (1939) studies the production of a wide variety of mutants in fungi by ultraviolet irradia- tion. Horowitz g£_g£ (1946) caused mutations in fungi by the use of mustard gas, Layers and Hanson (1945) by the use of neutrons and Hollaender and Zimmer (1945) by the use of X-rays. The wide variety of mutants and variants in fungi caused by the above mentioned methods has brought into fo- cus the question of a fungus being a new species or merely a variant. Hollaender and Emmons (1946) found some of the Tricho- phyton mentagrophytes mutants closely resembling naturally occurring Species. They found other mutants to be of types which could be classified as new "species" if one did not know that they had originated from specific fungous cultures. A mutant of Trichgphyton interdigitale and T;_violaceum. Another question discussed by Hollaender and Emmons (1946) is the possibility of producing mutants by irradia- tion with sufficiently intense wave lengths, such as are pre- sent in the ultraviolet spectrum of sunlight. Emmons (1932), in a report of spontaneously occurring mutations of Microsporum gypseum, hypothesized that many of the dermatophytes now known as species are only varieties of a single unstable species. This is probably the situation in the numerous so-called species of pathogenic sporotricha. METHOD AND MATERIALS The following is a history of cultures used in irra- diation of spores: (l) Sporotrichum schenckii. Duke University. Isolated February 1934. (2) Sporotrichum schenckii. Kalamazoo, Michigan. Dr. H. R. Prentice. Isolated August 1953. (3) Sporotrichum schenckii. Michigan Department of Health. Lansing, Michigan. Dr. J. Roberts. Isolated 1953. Spores of a standard Sporotrichum schenckii culture grown for 14 days on Sabouraud agar plates were washed off with a physiological saline solution containing 1 to 100,000 parts of aerosol (diamyl sodium sulfo succinate), and filter- ed through non—absorbent cotton. Spore counts were made with a haemacytometer and the sport number was adjusted to 50,000, 000 spores per cc. Twenty cubic centimeters of spore suspen- sion were placed in a flat-bottom Petri dish which was mounted 6 inches from a General Electric Lamp emitting 80 to 95 per cent ultraviolet light of 2527 A°. The suspension was stirred with a glass rod and 1 cc. of spore suspension was withdrawn from the dish at 2, 4, 6, 8, 10, 12, 16, and 20 minutes af- ter initial exposure. A non-irradiated 1 cc. control sample was taken previous to irradiation to check the possibility of spontaneous variants and mutants occurring among the spores. These 1 cc. spore suspensions were diluted serially from 1-10 to l-l0,000,000 at the shorter exposures and 1-10 to l-lO0,000 at the longer exposures. The diluted spore suspensions were plated into Sabouraud dextrose agar plates and incubated at room temperatures. Morphologically different colonies were chosen and cultured for observation for stability during the year. RESULTS AND DISCUSSION Irradiation caused the following variations and muta- tions to occur. From the Duke strain 13 morphologically different strains deveIOped. When the Kalamazoo strain was exposed to ultraviolet light irradiation, 43 morphologically different forms resulted while from the Michigan Department of Health strain, 12 variants and mutants resulted. From the Duke strain, 6 yeast forms were obtained at room temperature on Sabouraud agar. However, in the course of continued semi-weekly transfers, they lost their reproductive powers and only the mycelial forms survived. From the Kalamazoo strain, two yeast forms were obtained. These survived but later developed pleomorphic*. These pleomorphic forms were used in later nutritional and pathogenetic tests. From the Michigan Department of Health strain, one yeast form was obtained as a result of irradiation. This, too, was used in later nutritional and pathogenicity tests. Initially, it was observed that all irradiated spores required a longer period before germination and growth into a colony. The variants and mutants were observed to take even longer. However, after six months of continued trans- fer, the variants and mutants were observed to have regained most of their ability to reproduce at the more normal rate. The following is a list of organisms and code numbers which were assigned and which were used in all later patho- *Pleomorphic denotes a mixture of yeast and filamentous forms genetic and nutritional studies. IRRADIATION CODE NUMBERS STRAINS TIME 2 Duke University. Mycelial form. 2 minutes 12 Duke University. Mycelial form. 4 minutes 15 Duke University. Mycelial form. 4 minutes 14 Duke University. (Original) 0 Minutes Mycelial form. 15 Kalamazoo. (Original) Mycelial O minutes form 24 Kalamazoo. Pleomorphic form. 6 minutes 54 Kalamazoo. Mycelial form. 12 minutes 41 Kalamazoo. Pleomorphic form. 6 minutes 58 Kalamazoo. Mycelial form. 4 minutes 60 Michigan Department of Health. 4 minutes Yeast form. 61 Michigan Department of Health 4 minutes Mycelial form. 64 Michigan Department of Health O minutes (Original) Mycelial form. SP Sporotrichum epigeum. American 0 minutes Type Culture Kssociation Photographs of the colonies (after 20 days) and mycelia (after seven days) of the above strains, grown on Sabouraud dex- trose agar, appear in Plate I. Strain 41 shows the yeast phase. Both strains 24 and 41 shortly after transfer grew as yeast phases with the mycelial phase gradually developing with the advancing age of the colony. The yeast-like appear— ance of the somatic structure may readily be seen in strain 60. Strains l2 and 15 had a clustering of hypae as in a PLATE I GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS 0F SPOROTRICHUM SCIENCKII AND SPOROTRICHUM EPIGEUM DOLafiffig: 50 DAYS OLD MYCELIA: 7 DAYS OLD 20x azuox STRAIN 2 2240K STRAIN 12 PLATE I (continued) GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS OF “ SPOROTRICHUM SCHENCKII AND SPOROTRICHUM EPIGEUM 20X 22hOX 10 PLATE I (continued) GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS OF SPOROTRICHUM SCHENCKII AND SPOROTRICHUM EPIGEUM ‘3' . 20x . 221w): STRAIN 15 11 PLATE I (continued) GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS OF SPOROTRICHUM SCEENGKII AND SPOROTRICHUM EPIGEUM 20X STRAIN 14,1 12 PLATE:I (continued) GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS OF SPOROTRICHUM SCEENCKII AND SPOROTRICHUM EPIGEUM mx 221(0): STRAIN 58 STRAIN 6O 1 PLATE I (continued) 5 GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS OF SPOROTRICHUM SCHENCKII AND SPOROTRICH'UM EPIGEUM 20x zauox l4 PLATE I (continued) GROWTH ON SABOURAUD DEXTROSE AGAR OF DIFFERENT STRAINS OF SPOROTRICHUM SCHENCKII AND SPOROTRICHUM EPIGEUM 20X ZZHOX STRAIN SP coremia with relatively few spores which were not limit- ed to the top of the structure. Also, strains 61 and 64 (original culture) produced mostly sessile conidia along the sides of the hyphae. CONCLUSION Strains morphologically different from the hyphal form of parent isolates ranging from true mycelial forms, pleo- morphic forms, to true yeast forms of Sporotrichum schenckii and ordinarily present in the yeast form only in infected hosts or on specialized media at 57°C. were obtained by ultraviolet light irradiation. STUDY OF MORPHOLOGY INTRODUCTION Sporotrichum schenckii when grown on Sabouraud dex- trose agar at room temperature appears as a small white colony lacking aerial mycelium. With increase in growth, the surface of the colony becomes folded and leathery with the color varying from white to tan, or brown to black, de— pending on the strain and upon the media. When examined microscopically, delicate, branching, septate hyphae 1.5 to 2 microns in diameter bearing a cluster of pyriform conidia, 2 to 4 by 2 to 6 microns in size at the tips of the conidiophores, can be observed. In some strains, the conidia are sessile, being borne directly on the hyphae. When cultured on cystine blood agar at 57°C., the growth remains soft and yeast-like, and is composed of fusi- form to oval or oblong bodies and short mycelial fragments. In the human or animal bodies, this organism exists as a fusiform single-cell in giant cells or polymorphonu- clear cells in lesions or exudates. In this phase of the investigation, a study was made of the colonies and the microscopic characteristics of the strains obtained by irradiation of spores when grown under the following conditions: (1) on Sabouraud dextrose agar at 25°C., and (2) on Sabouraud dextrose agar containing either 5 per cent animal or human bloods plus 20 units per cc. of penicillin and 40 units per cc. of streptomycin and l7 incubated at 57°C. METHOD AND MATERIALS Colonial morphology was studied by inoculation of Sa— bouraud glucose agar plates and Sabouraud glucose agar plates containing 5 per cent human or animal bloods. One set of the plain Sabouraud agar plates was incubated at 24°C. and the other set at 57°C. The blood agar plates were incubated at 57°C. and the presence or absence of hemolysis was noted. Mycelial characteristics were studied by allowing the organisms to grow on blocks of Sabouraud glucose agar on sterile glass slides with a cover-slip on the agar block in a moist chamber. The mycelia were allowed to grow out on the slides and on the cover—slips. The mycelia on the cover-slips were mounted by removing the cover-slip, plac- ing a drOp of lactophenol-cotton blue on a clean slide, placing the cover-slip with mycelial growth on the drop, and sealing with fingernail polish. RESULTS AND DISCUSSION The §;_schenckii strains along with S; epigeum are pic— tured in Plate 1. Strains 24 and 41 were yeast forms upon initial transfer to Sabouraud dextrose agar, but later de- veloped into pleomorphic forms while strain 60 has remain- ed a yeast form. The photographs show strains 41 and 60 as yeast forms, while strain 24 is shown as a pleomorphic form. The irradiated forms from strains 14, strains 2, 12, and 15 were observed to be typical mycelial forms with pyri- form conidial clusters. The same is true for strain 15 and its irradiated form, strain 54. Strain 64 is an atypical form Of.§; schenckii along with its irradiated form, strain 61 in which the conidia are sessile on the hyphae (Table I). None of the strains grown on Sabouraud agar at 57°C. showed any change in microscopic characteristics from my- celial to yeast or yeast to mycelial growths, although the production of conidia was impaired and some of the mycelial colonies assumed a moist yeast-like growth. On horse blood, strains 2, l2, l5, 14, 24, 54, and 61 were mycelial and budding forms, whereas, strains 15, 24, 60, and 64 (Plate II) were pure yeast forms. Only strain 58 was a strict mycelial form. On cow blood agar, both strains 15 and 15 were unable to reproduce. It is thought that an inhibitory substance, or substances were present in this particular blood since repeated inoculation upon blood agar containing blood from the same cow gave similar results. Strains 24, 41, and 60 were strict yeast types, whereas, strains 54, 58, 61, and 64 (Plate II) were pleomorphic types. Strain 2, 12, and 14 were mycelial types. On chicken blood agar, strains 15, 24, and 60 were 19 PLATE II GROWTH OF SPOROTRICHUM SCHENCKII STRAIN 6h ON SABOURAUD AGAR COLONIES: 1h DAYS OLD SOMATIC STRUCTURES: 14 DAYS OLD 20X 22hOX GOAT BLOOD AGAR ,- ". ,u L": ‘ 20X _ 22uox HUMAN BLOOD AGAR PLATE II (continued) GROWTH OF SPOROTRICHUM SCHENCKII STRAIN 61). ON SABOURAUD “'”'DEXTRDEETBEDUD AGAR COLONIES: 1’4 DAYS OLD SOMATIC STRUCTURES: 111 DAYS OLD 20x ° azuox 20x ' zzuox cow BLOOD AGAR TA BLE I — (continued) GROWTH STUDIES ON 5% SABOURAUD DEXTROSE BLOOD AGARS OF SPOROTRICHUM 21 Schenckii Strains and Sporotrichum Epigeum (Y)=Yeast-like Colony PIG GOAT TYPE OF STRAIN TYPE OF GROWTH HEMOLYSIS (10 Days Old) GROWTH HEMOLYSIS Yeast - 2 Mycelium + Yeast . + 12 Pleomorphic + Mycelium + 15 Mycelium (chlamy- . Spore - Pleomorphic + 14 Pleomorphic + Pleomorphic - 15 Pleomorphic + Yeast - 24 Yeast + Pleomorphic - 54 Pleomorphic + Yeast — 41 Pleomorphic + Pleomorphic - 58 Pleomorphic + Yeast + 60 Yeast + Pleomorphic - 61 Pleomorphic + Pleomorphic + 64 Pleomorphic + No growth - Sp No growth - MINK DOG TYPE OF STRAIN TYPE OF GROWTH HEMOLYSIS (10 Days Old) GROWTH HEMOLYSIS No grthh - 2 No growth - H II _ 12 II II _ II II _ 1:)? H H _ " " - 14 Pleomorphic - Mycelium (poor growth) - 15 Yeast - No growth - 24 No growth __ " " - 54 Pleomorphic - Pleomorphic - 41 Yeast Mycelium - 5d Pleomorphic - Yeast - 6O Yeast - Mycellium ,— 61 Pleomorphic - Mycelium - 64 Yeast — No growth - Sp No growth + Sheep = No growth or hemolysis Control = Cultures on Sabouraud blood agar at 57°C, with no change in original growth types. , -..-‘, -“aa ‘- - - --.-r— 0-. .p‘- — GROWTH STUDIES ON 5? Q.‘EOURAUD DEXTROSE l’lS , JU’X Schenckii Strai and Sporotrichum Epigeum T ‘LE I :1) [‘1 _/ BLOOD AGARS (Y)=Yeast-like Colony OF 22 SPOROTRICHUM (10 Days old) HUMAN STRAIN TYPE OF GROWTH HEMOLYSIS _ Mycelium + 2 Mycelium + 12 Mycelium i 15 Mycelium (Y; + 14 Mycelium Y + 15 Yeast — 24 Pleomorphic — 54 Yeast + 41 Mycelium (Y) + 55 Yeast - 60 Mycelium i 61 Yeast f 64 No growth f SP COW TYPE OF GROWTH HEMOLYSIS Mycelium — 2 Mycelium + 126 No growth - l5 Mycelium - 4 No growth - l5 Yeast - 24 Pleomorphic - 54 Yeast - 41 Pleomorphic - 5O Yeast (Growth Microscopic) — 6O Mycelium EY — 61 Mycelium Y - 64 No growth - Sp HORSE TYPE OF GROWTH HEMOLYSIS Mycelium Pleomorphic Mycelium (Y) Mycelium Y Yeast Yeast Mycelium (Y; Mycelium Y Mycelium Yeast Pleomorphic Yeast No growth CHICKEN TYPE OF GROWTH Mycelium Pleomorphic Mycelium Mycelium Yeast chelium Pleomorphic Mycelium Yeast Myfielium No growth HEMOLYSIS of the yeast type, whereas, strains 14, 54, 58, 61, and 64 were pleomorphic types. Strain 15 was the only mycelial type. On goat blood agar, strains 24 and 60 were yeast types, whereas, strains 12, 14, 15, 34, 41, 53, 61, and 64 (Plate II) were pleomorphic types. Of the two mycelial forms, strain 15 was observed to have more chlamydospore-like growth, while strain 2 had none. On mink blood agar, strains 2, 15, l4, 14, 24, and 54 were unable to grow. Only strain 60 was a yeast type, where- as, strain 41 was a pleomorphic type. In the mycelial types, strains 15 and 58 were able to grow only very poorly while strains 61 and 64 showed relatively good growths. On human blood agar, strains 24, 41, 60, and 64 (Plate II) grew as yeast forms while the others grew as mycelial forms. On dog blood agar, strains 2, 12, 15, and 24 were unable to grow. Strains 41 and 60 were yeast types, while strains 14, 54, 58, and 61 were pleomorphic types. There was no strict mycelial form. Usually sheep blood agar supported growth of the organ- ism, but this particular sheep blood agar did not support the growth of any of the strains. There was no correlation be- tween the ability of the strains to hemolyze the different types of bloods and the types of growth. Human and goat bloods were hemolyzed by most of the strains. Pig blood was hemolyzed by a few strains, notably by the Duke University strains and the Michigan Department of Health strains. Cow and dog bloods were hemolyzed only by a single strain. It was interesting to note that human and dog bloods were hemolyzed by strain SP (Plate 5) in spite of the fact that no mycelial growth could be observed. CONCLUSION Dog, mink, and cow bloods were found to contain a certain substance or substances which inhibited the growth of certain strains of S; schenckii. On the different blood agars where growth did occur, the strains varied from mycelial to pleomorphic to yeast forms with only strain 60 remaining consistently, a yeast form. From this brief study, it can be seen that animal and human bloods contain certain factors which enable strains of S; schenckii to grow as yeast or pleomorphic forms at the appropriate temperature, Just as in blood-cysteine or brain-heart infusion agars which are extremely rich in cer- tain growth factors. Although sporotrichosis has been reported in animals and humans and the ability of an organism to hemolyze blood is regarded as one of the factors in pathogenicity, only in the human, goat, and pig blood agars was hemolysis found to occur quite consistently. 25 PLATE III CIGAR—SHAPED BODIES (SPOROTRICHUM SCHENCKII STRAIN 13) AS FOUND IN MICE AND ZONE OF HEMOLYSIS ON SABOUHAUD DEXTROSE BLOOD AGAR CAUSED BY SPOROTRICHUM EPIGEUM Cigar-shaped bodies 2240): Zone of hemolysis 20x 26 PATHOGENICITY INTRODUCTION Sporotrichosis causes a subacute or chronic infec- tion which is usually confined to the skin and subcutan- eous tissues but may occur in internal organs. The organ- ism is introduced by trauma and develops by causing a pus- tule, ulcer, or abscess, which falls to heal under ordinary treatment. Often, the regional lymphatics are invaded and cord-like thickening of the lymph vessels and subcutan- eous abcesses result. In the mouse or rat, intraperitoneal or intratesticu- 1ar inoculation of spores results in abcesses in the pel- vic region and mesenteric lymph nodes, and in the spleen and liver. The legs may swell, and ulceration may develop along the tail (Baker, 1947). The purpose of this study was to determine the effects of Sporotrichum schenckii strains and Sporotrichum epigeum upon mice when introduced intraperitoneally. METHOD AND MATERIALS One-half milliliter of spore or yeast suspension (10 million cells per m1.) of 14-day old virulent cultures of Sporotricha in saline solution was injected intraperi- toneally into mice six to 12 months old. The avirulent strain SP was made into a suspension by macerating the washed mycelium from a 14-day old Petri dish culture. One-half milli- liter of the dense milky-colored suspension was injected intraperitoneally. The mice were observed daily for deaths until the 23th day, at which time they were all sacrificed. At that time, the general external as well as internal conditions of the mice were recorded. The presence or absence of ci— gar-shaped bodies was noted by making Gram stains of smears from pus and nodules, and examining the stained smears mi- croscopically. assume AND DISCUSSION V The results of mouse inoculation have been tabulated in Table II. Mice from the Michigan Department of Health when inoculated with the yeast or pleomorphic strains, 24, 41, and 60 remained non-infected. However, the same cul- tures infected one out of three inoculated mice which were obtained from the Zoology Department. Except for strain SP which was definitely known as a non-pathogen, the other strains caused infection in mice upon inoculation of the spores. A difference in the morphological appearance of the organisms in the infected mice was noted in several of the strains. Normally, cigar-shaped bodies were observed upon Gram staining of the pus or crushed nodules, while a ten- dency toward formation of oval to round cells was observed in strain 2. TAB E II (continued) 29 STUDY OF PATHOGENICITY OF SPOROTRICHUE SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM IN MICHIGAN DEPARTMENT OF HEALTH MOUSE STRAIN 28 DAYS AFTER INOCULATION Culture Sex of External Infectivity Micro.Exam. Inocu- Mouse Condition Lesions Nodules (Cigar lated Bodies) 2 Female Normal No pus Numerous on mesent. + Female Normal No pus Numerous on mesent. + Female Normal No pus Num.on mesent.,liver + 12 Female Normal No pus Numerous on mesent. + Female Normal No pus Numerous on mesent. + Female Normal No pus None - 15 Female Emaciated Pus in PC Few on liver + Female Emaciated Pus in PC Few on liver,spleen + . Female Normal No pus Few on mesentery + 14 Female Normal No pus Numerous on mesent. + Female Normal No pus Numerous on mesent. + Female Normal No pus Few on mesent.,liver + 15 Female Emaciated Pus in PC Num.on mesent.liver + Female Normal No pus Few on liver + Female Normal No pus Few on mesentery + 24 Female Normal No pus None - Female Normal No pus None - Female Dead; 7 days No pus None - 54 Female Normal No pus Few on mesent.spleen + Female Normal No pus Few on mesentery + Female Normal No pus Few on mesent.liver + 41 Female Normal No pus None - Female Normal No pus None - Female Normal No pus None - 58 Female Normal No pus Few on mesentery + Female Normal No pus Few on mesentery + Female Normal No pus Few on mesentery + 60 Female Normal No pus Few on mesent.liver + Female Normal No pus Few on mesent.liver + Female Normal No pus Few on mesent.liver + 61 Female Normal No pus Numerous on mesent. + Female Normal No pus Few on mesentery + 64 Female Normal No pus Num.on spleen,liver + Female Fur Rough Pus in PC Num.on mesent.liver + Female Fur Rough Pus in PC Num.on mesent.liver + Sp Female Normal No pus None - Female Normal No pus None - Female Normal No pus None - CN Female Normal No pus None - Female Normal No pus None - Female Normal No pus None - If the external appearance of the mice and the pre- sence of cigar-shaped bodies were taken as indication of high virulence, strain 15 could be regarded as highly viru— lent since all the mice that were inoculated with it were emaciated with roughened fur after 28 days. Strain 64 al- so seemed quite virulent. However, differences in biolo- gical resistance to this strain were noted. It was observed in making Gram stains of smears that crushed nodules invariably gave positive results as indi- cated by cigar—shaped bodies, but that pus smears more of- ten gave negative than positive results. CONCLUSION Strains 24, 41, and 60 (pleomorphic and yeast forms) could infect none of the mice from the Michigan Department of Health, and only one out of three of the mice from the Michigan State University Zoology Department strains was infected. Strains l5 and 64 were found to infect the greatest percentage of mice and to cause the appearance of the sever- est symptoms of sporotrichosis. The tendency toward formation of oval to round cells instead of the regular cigar—shaped bodies in the host was noted for strain 2. CARBOHYDRATE METABOLISM INTRODUCTION The carbohydrate fermentations of several pathogenic species of Sporotrichum have been reported. Schenck (1898) and Hektoen and Perkins (1900) reported on_§; schenckii. Gougerot and Blanchetiere (1909) reported on the fermentation reaction differences of S; beurmanni and S; schenckii. Su- crose was fermented but not lactose by S. beurmanii while .S; schenckii was found to hydrolyze and ferment lactose but not sucrose. Meyer and Aird (1915) reported on 15 Sporotrichum species. They found that acid but not gas was produced by all strains in glucose media; lactose and mannose were not fermented and other sugars gave variable results. They were not able to differentiate species by carbohydrate fermen- tation. Lurie (1950) studied nutritional requirements of seven pathogenic sporotricha and found that the organisms ferment glycerol, glucose, maltose, and fructose but not mannose or lactose to any appreciable extent. She confirmed Mayer and Aird's (1915) conclusion that the differentiation of species by carbohydrate fermentations is not possible. This section on carbohydrate utilization was conducted to determine the change in pH, amount of growth with utili- zation of various carbon sources, and the possible correla- 52 tion to pathogenicity. Sporulation was also to be ob- served. METHOD AND MATERIALS Strains 2, 12, 15, 14, 15, 24, 41, 58, 60, 61, 64, and SP were used in this study. These strains were main- tained on Sabouraud glucose agar slants and transferred bi-weekly. Five—day old cultures were used for the fol- lowing inoculation of media. The basal medium was of the following compositions: BASAL MEDIUM KHgPO4 1.0 gm. MgSO4.7H20 0.5 gm. KCl 0.5 gm. Trace elements 10 ml. stock solution Vitamin mixture 10 ml. stock solution Asparagine 2.0 gm. Glass distilled H20 1000 ml. .Trace Elements (stock solution) CuSO4.5H20 0.059 gm. FeSO4. 7H20 0.010 gm. MCCngiHeO 0.007 gm. ZHC12.7H20 0.8530 gm. NagB407.5H20 0.026 gm. (NH4)6M0--Cg4.4H20 0.004 gm. Glass distilled H20 1000 ml. Vitamin Mixture (stock solution) Thiamine hydrochloride 0.10 gm. Riboflavin 0.05 gm. Pydridexine 0.05 gm. Calcium penthothenate 0.02 gm. Paraamino benzoic acid 0.05 gm. Nicotinamide 0.02 gm. Choline chloride 0.02 gm. Inosite 0.04 gm. Folic acid 4 micro gm. Glass distilled H20 1000 ml. To this basal medium was added a different carbohydrate source equivalent in carbon weight to 10 gm. of d-glucose as prescribed by Lilly and Barnett (1951). Carbohydrate sources used were d-ribose, d-xylose, d-glucose, d-fructose, l-sor- bose, l-arabinose, d—mannose, l-galactose, maltose, sucrose, lactose, raffinose, rhamnose, dulcitol, glycerol, strach, and inulin. The pH was adjusted to 6.4 by the use of 1 normal sodium hydroxide solution. This pH was chosen to avoid loss of nitrogen from the media in later nitrogen studies. The media were dispensed in 25 ml. amounts into acid-washed tubes (200 mm. x 25 mm.) which were twice rinsed in distilled water and once in glass distilled water. The media were heated for an hour on three successive days in flowing steam. The method of inoculation of media in triplicate was as follows: The fungus growing as a mycelial mat was asep- tically removed and placed in a Petri dish. Agar was scraped off the mycelial mat and the mat was chopped into approxi- mately 1 mm. squares. These mycelial squares were then rinsed twice in sterile distilled water and a tube of liquid medium was inoculated with a single square. In preliminary tests conducted on the size of mycelial squares treated in the a- bove manner and used as inoculum, if the squares were varied from 1 to 4 square millimeters, growth of the different squares was very nearly of the same weights (Table III). since both l-asparagine and dl-asparagine were utilized equally well, l-asparagine was chosen as the nitrogen source. (Table IV). In the case of yeast cultures, a mass of organ- isms approximately equal to 1 cubic mm. was used as the inoculum. The inoculated tubes, containing 25 ml. of broth medium, were placed into wire baskets, slanted at a 15° angle and incubated at 24°C. for 25 days. (Figure 1). Number 1 Whatman filter paper, used in the harvest of mycelium, was prepared by drying in a 60°C. drying oven for 2 days, plac- ing the paper in a dessicator containing calcium sulfate for 24 hours, and then weighed. The mycelial mat was harvested using a Buchner funnel containing the filter paper and apply- ing slight suction. The yeast cells were filtered using double layers of filter paper in the Buchner funnel and without applying suction. Each filter paper was rinsed twice with distilled water, dried for two days in a 60°C. drying oven and then placed in a dessicator containing calcium sulfate for 24 hours. The weight of the mycelial growth and yeast cells was taken by subtracting from the total weight, the weight of the growths in the basal medium for each strain. During the filtration process, the pH of the filtrate of each culture was taken with a Beckman pH meter. The filtrate from each culture was saved for the carbohydrate analysis. The amount of carbohydrate left in the medium after 25 55 TABLE III GROWTH STUDY OF SPOROTRICHUM SCHENCKII STRAIN 14 IN L-ASPARAGINE MEDIUM WITH VARIATIONS IN AMOUNT OF INOCULUM AFTER 25 DAYS AT 24°C. Amount of Original Final Dry Wgt.of Mycelium Inoculum PH PH in Mg.after 25 days 1x1 mm. 6.40 7.1 84.0 1x2 mm. 6.40 7.3 67.0 1x5 mm. 6.40 7.5 87.4 1x4 mm. 6.40 7.1 89.4 56 TABLE IV COMPARISON OF DRY MYCELIAL GROWTHS IN L-ASPARAGINE AND DL-ASPARAGINE MEDIA OF SPOROTRICHUM SCHENCKII STRAIN 14 AND SPOROTRICHUM EPIGEUM INCUBATED AT 24°C FOR 25 DAYS (Weights Represent Averages 0f Growtns in Triplicate) ‘1 'L-Asparagine ‘DL-Asparagine Days Strain I4 Strain Sp Strain l4 Strain SP 0 0 Mg. 0 Mg. 0 Mg. 0 Mg. 5 1.8 " 2.1 " 0.5 " 0.9 " 10 5.5 " 35.1 " 1.70 " 5.5 " 15 26.9 " 45.0 " 24.0 " 20.9 " 20 48-9 " 49.0 " 55.9 " 45-3 " 15 84.5 " 54.3 " 79.0 " 46.6 " 50 85.0 " 52.0 " 60.1 " 49.9 " 100 57 FIGURE 1 GROWTH CURVES 0F SEOROTRICHUM SCHENCKII STRAIN IN AND SPOROTRICHUM EPIGEUM IN L-ASPARAGINE MEDIUM WHEN IN- CUBATED'AT 2b” 5. L ———-§porotrichum schenckii Strain 14 —-- _porotrichum cpigeum 15 2‘0 55 50 Days CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAIN IN AND SPOROTRICHUM EPIGEUM IN L-ASPARAGINE MEDIUM WHEN IN- CUBATED Af‘2h° C. 7.00 ) ‘\\ F 6000 i \\ PH -\ \ s \ \ 00 h \\ 5' s , , ‘\ , ‘-- Opprotrichum schenckii \‘ ,’ Strain 14 \\ ,1 --- _porotrichum cpigcum 4000 I \ I’ \ I \ I \ I ’ \ \\\/’,’ $000 1 J l + J 0 5 10 15 90 25 50 days was determined in the manner prescribed by Lurie (1950) which is a modification of Benedict's and of the A. 0. A. C. (1946) methods for monosaccharides, polysaccharides, and polyhydric alcohols. Monosaccharide, polysaccharide, or polyhydric alcohol in the medium was calculated and the results appear in Table V. It must be pointed out that the carbohydrate titrated and present in the filtrate includes that actually left in the media of the original and whatever compounds that may have been produced by the fungi which are able to reduce alkaline copper reagents or are capable of being oxidized by potassium dichromate. Therefore, an unavoidable source of error exists and a lower utilization of carbohydrate would be indicated than actually occurred. However, this method of direct determination of carbohydrates before and after growth of the fungus should provide useful informa- tion as to their utilization. RESULTS AND DISCUSSION The results as they appear in Table V are the composite averages of each strain tested in triplicate. Carbohydrate sources found to favor the growth of equal or greater weight of mycelium than a medium with glucose as the carbohydrate source, and with l-asparagine as the nitrogen source, were dl-xylose, dl-fructose, l-galactose, l-sorbose, l-rhamnose, maltose and inulin. Strains 24, 54, 41, and 60 gave poor to fair growths with most carbohydrates employed which were utilized readily by other strains. Strain 41 grew well in 59 TABLE V CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data Represents Averages of Triplicate Tubes) Basal Medium STRAIN PH After Dry Ngt.of Carbohydrate Carbohydrate 25 Days Growth in Solution in Utilized in Mg. Mg. Mg. 2 6.40 l 7 0 0 12 6.40 2.0 0 0 15 6.45 0.5 0 0 14 6.50 1.2 0 0 15 6.50 0.8 0 0 24 6.50 1.5 0 0 54 6.42 1.2 0 0 41 6.52 2.0 0 0 58 6.40 1.8 O 0 60 6.45 0.0 0 0 61 6.42 1.2 0 0 64 6.40 1.7 0 O Sp 6.40 0.9 0 0 Control* 6.40 0.0 0 0 LeArabinose 2 “6.90 56.8 210.7 '39.6 12 6.90 46.85 216. 5 -55.8 15 6.89 45.0 221.1 —29.2 14 6.01 65.8 199.0 —51.5 15 6.80 24.5 5.8 —244.5 2+ 5.97 6.8 242.1 -8.2 54 6.11 7.5 250.5 -0.0 41 6.70 18.8 252.8 -l7.5 58 6.91 26.8 224.6 ~25.7 60 6.39 15.0 247.9 - 2.4 61 6.40 29.5 222.5 -20.0 64 6.78 27.9 256.5 -l4.0 Sp 6.91 50.5 228.1 -22.2 Control 6.4 0 250.5 0 *Control for all sources of nitrogen indicates uninoculated tubes. TABLE v (continued) CARBOHYDRATE UTILIzATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data Represents Averages of Triplicate Tubes) D-Xylose Strain PH After ‘Dry Ngt.of Carbohydrate Carbohydrate 25 Days Growth in Solution in Utilized in Mg. Mg. Mg. 2 6.90 ‘66.2 2.9 —247.4 12 6.79 75.2 21.5 -229.0 15 6.90 84.1 2.5 -248.0 14 6.70 69.7 28.5 -222.0 15 6.80 79.6 28.5 -222.0 24 6.59 1.5 220.7 - 25.6 54 6.40 10.5 725.6 - 25.6 41 6.40 25.0 196.2 — 24.7 58 6.22 69.1 112.8 — 54.1 60 6.40 17.0 225.6 —157.5 61 6.65 72.2 56.4 - 24.7 64 7.10 79.2 2.9 —195.9 Sp 5.80 54.1 225.6 -247.4 Control 6.40 0 25 .5 0 D-Fructose 2 5.67 105.7 69.5 ~180Q8’ 12 5.72 102.4 85.5 —164.8 15 6.80 105.7 12.1 -258.2 4 5.60 88.4 60.6 —ici.7 15 5.90 100.8 45 4 -206.9 24 6.00 2.9 224.5 — 25.0 54 5.60 7.5 224.5 — 25.8 41 5.90 25.5 225.7 — 26.6 58 5.50 67.3 184.5 - 66.0 60 6.20 14.6 255.5 - 15.0 61 6.00 56.4 96.4 -155.9 64 5.80 66.5 160.1 - 90.2 Sp 7.20 2.4 10.9 —259.4 Control 6.40 0 25.05 0 41 TABLE v (continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATIoN AT 25°C (Data Represents Averages of Triplicate Tubes Glucose Strain PH After Dry Wgt.of Carbohydrate Carbohydrate 25 Days Growth in Solution in Utilized in Ag. 15g. Mg, 2 7:62 59.0 201.0 - 46.5 12 5.50 50.1 227.0 - 25.5 15 7.60 87.0 105.5 - 45.0 14 6.05 84.5 122.1 -126.2 15 6.20 65.8 198.0 - 52.5 24 6.55 4.6 247.5 - 5.0 54 6.01 18.1 250.0 - 20.5 41 6.40 1.4 248.0 - 12.5 58 5.80 15 0 245.5 - 5.5 60 6.80 7.5 222.1 - 7.0 61 5.10 26 4 211.1 - 28.2 64 6.00 45.6 200.8 - 59.2 Sp 5.81 56.4 250.5 - 49.5 Control 6.45 0 0 0 42 TABLE V (Continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data §epresents Averages of Triplicate Tubes L-Galactose Strain PH After Dry wgt.of Carbohydrate Carbohydrate 25 Days Growth in Solution in Utilized in M3. Mg. N3. 2 '6.93 59.5 201.1 — 49.2 12 7.10 65.5 250.1 - 20.2 15 6.20 47.6 85.8 -166.5 14 5.77 92.9 2.7 -247.6 15 7.10 51.5 222.5 - 20.0 24 6.50 5.2 248.0 - 2.5 54 6.52 9.1 242.4 - 7.9 41 6.60 14.6 224.5 - 25.8 58 6.82 21.7 252.4 - 17.9 60 6.60 4.2 229.0 — 21.5 61 5.95 29.5 251.1 — 19.0 64 6.90 55.0 226.8 - 25.5 Sp 6.80 77.9 2.7 —247.6 Control 6.40 0 250.5 0 L-Sorbose 2 7.50 104.7 206.1 - 42.2 12 6.59 105.1 148.5 -102.0 15 7.50 115.9 250.0 - 0.5 14 6.55 74.6 159.5 -101.0 15 6.80 84.9 217.1 - 55.2 24 6.4 4.5 209.5 - 4o 8 54 6.50 16.8 212.5 — 58.0 41 6.61 79.6 151.7 — 90.6 58 6.72 60.5 182.5 - 68.0 60 6.50 16.0 210.6 - 59.7 61 6.52 55.9 199.5 — 51.0 64 6.65 79.5 176.6 - 75.7 Sp 7.10 8.5 214 0 - 56.5 Control 6.40 0 250 5 0 45 TABLE V (continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data iiepresents Averages of Triplicate Tubes) D-Mannose STRAIN PH After Dry Wgt.of Carbohydrate Carbohydrate 25 Days Growth in Solution in Utilized in M0. M3. M3. 2 7.20 47io 22. 0 - 29. 9 12 5.70 59.9 149. 5 —101. 0 15 6.96 59.5 190.7 - 59.6 14 7.20 59.6 200.6 - 49.7 15 5.80 55.2 228.7 - 21.6 24 6.40 5.4 250.5 0 34 6.05 5.0 246.9 — 5.4 41 5.10 56.9 162.5 - 87.8 58 4.68 66.4 7.5 242.8 60 6.16 9.5 165.0 - 87.5 61 4.90 64.1 149.5 —101.0 64 6.55 70.8 249.0 - 1.5 Sp 5.20 120.6 1 5 -248.8 Control 6.40 0250.5 0 L-Rhamnose 2 6:57 129.67 250. 4 — 22.7 12 5.92 51.0 97 5 -155.5 15 6.50 49.5 5. 0 -250.0 14 5.08 46.2 75.1 179.9 15 6.12 57.0 166. 9 - 641 24 5.90 5.7 240.8 - 12. 2 54 5.42 24.9 207. 2 - 45.8 41 6.60 9.2 79.2 -175.o 58 6.18 55.9 207.2 - 45.8 60 6.55 25.8 207.2 - 45.8 61 5.60 54.5 201.1 - 51.9 64 5.65 40.6 176.7 - 76.5 Sp 7.12 5.9 219.4 - 55.6 Control 6.42 0 255.0 0 TABLE v (continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data Represents Averages of Triplicate Tubes) Glycerol Strain PH After Dry Wgt.of Carb6hydrate Carbohydrate Growth in Solution in Utilized in M3. M3. M3. 2 5.70 29.2 52.1 -205.4 12 6.70 18.2 9.8 -247.7 15 5.85 24.2 40.5 -217.2 14 6.0 24.2 128.1 -129.4 15 6.2 15.5 177.3 —160.2 24 6.4 5.0 172.9 - 85.6 34 6.4 1.2 173.1 - 84.4 41 5.90 50.1 189.2 - 68.5 58 5.50 50.7 90.7 -186.8 60 6.5 6.1 95.5 -164.0 61 6.52 21.0 74.7 -182.8 64 6.50 8.2 68.1 -189.4 Sp 6.42 27.8 000 -257.5 Control 6.45 0 257.5 0 Sucrose 2 5.9 46.5 208.2 - 5133 12 7.1 39.3 227.6 - 12.2 15 6.2 46.6 214.1 - 25.9 14 6.8 45.5 222.0 - 18.0 15 6.8 20.6 227.5 - 12.7 24 6.5 5.7 234.1 - 5.9 54 6.6 12.4 229.9 - 10.1 41 6.7 18.6 254.1 - 5.9 58 6.7 18.6 234.6 - 5.4 60 6.8 6.1 256.8 - 5.2 61 6.8 20.1 252.0 - 8.0 64 5.87 40.2 228.8 - 11.2 Sp 4.1 79.1 1.1 —258.9 Control 6.41 0 240.0 0 45 TABLE V (continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data Represents Averages of Triplicate Tubes) Lactose Strain PH After Dry Wgt.0f CaFBohydrate Carbohydrate 25 Days Growth in Solution in Utilized in M3. M3. M3. 2 7.05 15.5 244.5 + ‘4.5 12 7.37 14.2 193.1 -239.6 15 7.55 15.2 15.7 -226.5 14 7.15 17.5 255.7 + 15.7 15 7.20 14.8 243.4 + 3.4 24 6.42 5.7 258.2 - 1.8 54 7.00 12.7 178.2 - 61.8 41 7.25 15.6 192.5 - 47.5 58 7.25 25.8 246.7 - 5.5 60 7.17 14.4 239.4 - .6 61 7.10 20.1 185.7 — 54.5 64 7.10 18.7 255.4 - 4.6 Sp 7.55 19.0 191.4 - 48.6 Control 6.45 0 240.0 0 Maltose 2 '6.5 100.5 trace 0 12 5.5 95.6 trace 0 15 6.72 101.1 trace 0 14 5.2 99.6 trace 0 15 7.0 5 .8 159.4 - 80.6 24 4.84 5.7 519.8 + 79.8 34 4.92 14.9 346.0 +106.0 41 6.8 24.4 321.3 + 81.5 58 7.1 42.2 299.1 + 59.1 60 7.1 24.7 518.2 + 78.2 61 4.5 50.7 507.8 + 67.8 64 7.0 31.2 241.8 + 1.8 Sp 7.1 59.6 trace 0 Control 6.4 0 240.0 0 46 TABLE v (continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data Represents Averages of Triplicate Tubes) Starch Strain PH After Dry Wgt.of CarbOhydrate Carbohydrate 25 Days Growth in Solution in Utilized in M3. M3. M3. 2 6:787 25.0 21619 - 10.1 12 5.99 67.7 144.2 - 82.8 13 6.9 17.9 194.9 - 32.1 14 5.79 52.0 225.5 - 5.7 15 6.70 29.5 216.1 - 10.9 24 6.18 11.7 256.7 + 9.5 34 5 95 19.2 222.8 - 4.2 41 6 65 32.2 220.8 - 6.2 58 6.52 25.5 221.4 - 4.6 60 6.59 16.6 222.2 - 4.8 51 5.85 25.5 199.1 - 27.9 64 5.70 42.2 222.8 - 4.2 Sp 6.70 27.1 45.8 -118.1 Control 6.40 0 227.0 0 Raffinose 2 6170 9.7 268.0 — 7.2 12 7.10 12.6 229.5 - 45.9 15 7.20 4.5 257.9 - 57.5 14 6.90 15.6 225 0 - 50.2 15 6.90 11.4 295.8 + 17.6 24 6.42 5.5 2’8.0 + 12.8 54 6.90 8.5 285.2 + 10.0 41 7.10 5.9 295.8 + 81J+ 58 7.00 9.7 275.2 0.0 60 6.70 5.2 222.1 - 55.1 61 6.80 12.6 296.6 + 21.4 64 7.10 10.2 286.6 + 11.4 Sp 6 50 21.8 239.3 + 35.9 Control 6.40 0 275.2 0 4‘7: TABLE V (continued) CARBOHYDRATE UTILIZATION AND CHANGE OF PH BY SPOROTRICHUM SCHENCKII STRAINS AND SPOROTRICHUM EPIGEUM AFTER 25 DAYS INCUBATION AT 25°C (Data Represents Averages of Triplicate Tubes) Dulcitol Strain PH After Dry Wgt.of Carbohydrate Carbohydrate 25 Days Growth in Solution in Utilized in M3. Mg. Mg. 2 6.65 12.6* Could Could 12 7.20 25.6 15 7.25 10.7 not not 14 6.95 21.9 15 6.90 14.2 be be 24 6.50 5.1 54 6.90 8.6 determined determined 41 6.85 5.5 58 6.60 5.5 by by 60 6.92 7.5 61 6.80 11.5 64 6.90 9.4 titration titration Sp 6.92 46.7 2.9 -250.1 Control 6.40 0 255.0 0 Inulin 2 6100 65.6 122.2 -107.1 12 6.50 67.2 2.0 —227.5 15 6.00 82.9 1.0 -228.5 14 6.70 69.1 5.0 -226.5 15 6.02 84.0 1.0 -226.5 24 6.20 6.90 27.5 —201.6 54 6.50 27.1 59.7 -189.6 41 6.41 55.4 5.0 -226.5 58 6.75 27.0 122.2 —107.1 60 6.40 26.1 91.7 —l57.6 61 6.72 119.7 106.9 -122.4 64 6.6 59.9 1.0 ~228.5 Sp 7.2 45.9 91.7 -157.6 Control 6.4 0 229.5 0 sorbose, glycerol and starch. In basal medium, which was lacking in any carbohydrate, there was very little growth (maximum yield of 2.10 mg.) with very little change in pH by the strains. The pathogenic sporotricha strains in media containing d-xylose, 1-sorbose, lactose (except strain 24), raffinose, and dulcitol caused a shift toward alka- linity, whereas, media containing d-fructose, d-mannose (except strains 2, 15, and 14), glycerol (except strains 12 and 61), and l—rhamnose (except strains 2, l5, and 14) became more acid. With other carbohydrate sources, there was no consistency in a shift of pH either toward alkalinity or acidity. This is in agreement with the studies of other investigators on glycerol, d-glucose, and d—fructose media for the non-irradiated strain 14 (Duke University), but not for several of the other non—irra— diated and irradiated strains. The results with maltose remain erratic. Among the media showing consistent lowering of the carbohy- drate, but with no consistency in the lowering of pH after the growth of the organism were sucrose, starch (except strain 24), inulin, l-arabinose, d-xylose, l-galactose, and 1-sorbose. All except two investigators have reported mannose not to be fermented by pathogenic sporotricha. However, the irradiated strains and also two non—irradiated strains, 15 and 64, fermented small amounts of mannose. It was of interest to notethat while there was reduction of 119 the carbohydrate in most media, in media containing maltose, or lactose, there was an actual increase in the amount of reducing substance, probably representative metabolic by-products of the fungus. The ability of the organisms to grow and reproduce, seems to be the most important factor in relation to pathogenicity. These strains did not grow well in the majority of the carbohydrate media. Another one which was weakly pathogenic in mice was strain 2 which grew, however, as well as other more strongly pathogenic strains. Later nitrogen studies reveal that this strain was un— able to grow well in some of the media containing nitrogen. Of the carbohydrates tested, maltose, and sucrose came closest to being excellent promoters of Sporulation of S; schenckii strains (Table VI). No carbohydrate allowed excellent Sporulation in all strains tested, just as no carbohydrate showed luxuriant growth in all of the strains tested. The non-pathogenic strain, §L_ epigeum,which was non-sporulating on Sabouraud dextrose agar, showed no Sporulation in any of the media containing carbohydrates. The abundance of mycelium was not necessarily correlated with Sporulation since growth in inulin was poor, while Sporulation was good. CONCLUSION D-fructose and maltose were found to produce the most luxuri- ant growths of most of the strains tested. oomMSSm co mde whomm omcoo u +++ mmpoam spas pmpm>oo momMLSm mo m\a u - moLOQm spa; omuo>oo oomwnzm go m\H u ++ SHHwOAQoomopoae oHQHmH> mopoam o: n O I I I I I I I I I I I I I I I I I am +++ +++ +++ ++ ++ +++ +++ +++ ++ ++ + ++ ++ ++ ++ ++ I 4m + +++ + + ++ + + + + + + ++ + + + + I am ++ +++ ++ + +++ ++ ++ + I + + I ++ + + + I am I + ++ + + + ++ I ++ I I + I + + + I He ++ ++ I + +++ ++ I I ++ + + + + ++ + + I Tn + + + + + I ++ I + I + + + + + + I :4 ++ ++ ++ ++ +++ + I +++ ++ +++ + +++ ++ ++ ++ ++ I ma ++ ++ ++ ++ +++ ++ +++ +++ ++ ++ + +++ ++ ++ ++ ++ I :H +++ ++ ++ ++ +++ ++ ++ + +++ +++ +++ +++ ++ +++ ++ ++ I ma +++ ++ ++ ++ +++ ++ +++ +++ ++ ++ I +++ ++ ++ ++ ++ I NH + +++ ++ ++ +++ ++ +++ + ++ +++ +++ +++ ++ ++ ++ ++ I m I G H S N n1 S O G u n e 4 e e n T. 41 _ J. J. 4. fl fl fl m. m n T. J e T. o o .A H w 9v no no #1 V X s J I o I. J a. 1. J o u. e o e T. 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000o00 cpaz 0000>oo 0000030 mo m\H u++ 0H0H00> 000o00 09 "I I I I I I I I I I I I I I I I I I I 00 + +++ + ++ +++ +++ + + + + + + ++ + + + + I 00 ++ + + ++ + ++ + + + I + + I + ++ + + I H0 I I I I I I I I I I I I I I I I I I 00 ++ I + ++ + ++ + + + + ++ + + ++ + + + I am I I I + + + I I ++ + I I + + I I + I H0 + I + ++ + + I I + + I + I + + + I I m I I I + I I I I I + I I + I I I + I 00 +++ ++ ++ +++ +++ +++ + + +++ ++ ++ +++ ++ ++ + ++ + I 0H +++ + ++ ++ + +++ + + + ++ ++ + + + + ++ + I :H +++ ++ +++ +++ + +++ ++ +++ +++ +++ +++ +++ ++ +++ ++ ++ ++ I mH +++ +++ + +++ + +++ + + + + +++ + + + + + ++ I 0H +++ +++ ++ ++ + ++ ++ I +++ + +++ +++ ++ ++ ++ + + I 0 L T M V. G G ”U mi 00 V uv W :0 H 25 m fl "M 4. fl \1 a J W. wi I . ,L w w mm o e 1 bus H“ J mm H 9.? n4 on _ _ _ H. R“ 0+U QUU U U U24 8 J five I. e .a He Au/\ "q _I V. a O. s o ego nuo 7.0 1.9 e e tum S 0 fl I. 1.9 I. u .L J J d _L. Ju .Lu 1U 18 ...I _L. Um» «w U ad u D;L no Tr E O u E u 14» Tat. JVI 708 D u u _k H; 47 a n u U u T. I J a Jmm can now B_L a o p. u 0 4+ .I e T. .I a. e ecu «em n+ qwm p. T H0 d B u u u U 00 4+ 8 8 H01 I u u. w e a e I I e m e e I u u I w 0 8 8 0900 00 00H0003000 0000 00 00000 2000000 200000000000 020 0200000 000020000 000000000000 000 00002 20000002 20 20000000000 00 000000 HHH> Mdm<9 72 CONCLUSION L(+) glutamic acid and l—asparagine were assimilated most easily by the fungous strains. Proline, found to be a "primary" amino acid for A; 22322 studies conducted by Steinberg (1942) gave relatively good growth but did not promote the most abundant growth. The amino acid sources together with ammonium sulfate and ammonium tartrate were good sources of nitrogen but am- monium nitrate and potassium nitrate were found to be poor sources of nitrogen. Ammonium compounds have generally been regarded as being utilized before other nitrogen sources. In the case of ammonium nitrate, the possibility of antago- nistic substances being present in the medium is very likely. The percentage of nitrogen in the mycelium ranged from 1.0 to 3.8 depending upon the strain and nitrogen source. Under these experimental conditions, §L_schenckii strains and §; epigeum do not appear capable of fixing atmospheric nitrogens. No single amino acid is indispensable when the organisms are provided with essential elements and vitamins. No Specific strain always produced mycelium abundantly in the nitrogen media used. However, strains 24, 41, and 60 were observed to produce sparse growth in many of the media containing different types of nitrogen. 75 Sporulation was observed to be dependent upon strain variation as well as on the type of nitrogen in the medium. 74 BIBLIOGRAPHY Baker, R.D. 1947. Experimental sporotrichosis in mice. Amer. Jour. Trop. Med. 27; 749 Beneke, E.S. l9 5. Detection of mycotic infection in animals. MSC Veternarian..1:; 219—251. Brown, R.D., Weintraub, M.W. Simpson, F.w. Simson, M.A.F. Helm, J.N. Bowen, F.A.Brandt and C. Berman. 1997. Sporotrichosis infection in mine of the Witwaters- rand, Johannesburg. Published by Transvaal Chamber of Mines. Burris, R.H., and P.W. Wilson. 1947. Comparison of the metabolism of ammonia and molecular nitrogen in Azotobacter. Ann. Rev. Biochem. 1“; 005-700. Conant, N.F., D.S.Martin, D. T. Smith, R. D. Baker, and J.L. Callaway. 1954. Manual of Clinical MycolOg y. Philadelphia. W. B. Saunders Co. De Beurmann and Ramond. 1905. 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