FLUORESCENT-ANTIBODY STUDIES OF THE MYCELIAL FORM

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1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 1, pp January, 1964 Copyright X 1964 by the American Society for Microbiology Printed in U.S.A. FLUORESCENT-ANTIBODY STUDIES OF THE MYCELIAL FORM OF HISTOPLASMA CAPSULATUM AND MORPHOLOGICALLY SIMILAR FUNGI LEO KAUFMAN AND BRENDA BRANDT Communicable Disease Center, U.S. Public Health Service, Department of Health, Education, and Welfare, Atlanta, Georgia Received for publication 29 August 1963 ABSTRACT KAUFMAN, LEO (Communicable Disease Center, Atlanta, Ga.), AND BRENDA BRANDT. Fluorescentantibody studies of the mycelial form of Histoplasma capsulatum and morphologically similar fungi. J. Bacteriol. 87: Fluorescentantibody reagents were developed to differentiate the mycelial form of the pathogen, Histoplasnma capsulatum, from several morphologically similar but saprophytic species of the genera Sepedonium and Chrysosporiurn. Cross-staining and adsorption procedures revealed antigenic relationships among certain species of these three genera and other fungi. The significance of these observations to the diagnostician and taxonomist is discussed. The gross and microscopic similarities of the mycelial form of Histoplasma capsulatum and some species of Sepedonium and Chrysosporium (Howell, 1939; Carmichael, 1962) pose problems in the identification of these fungi. The fact that H. capsulatum, unlike members of the other genera, has a yeastlike form is not always of diagnostic value. Some strains of H. capsulatum require repeated time-consuming attempts by a multiplicity of methods to convert them to the yeast form. The possibility also exists that some strains of atypical and typical H. capsulatum that cannot be converted are, in reality, H. capsulatum but cannot be conclusively identified as such. For these reasons, it was considered worthwhile to attempt to employ the fluorescent-antibody (FA) procedure for the rapid and specific differentiation of the tuberculate sporebearing species of Chrysosporium and Sepedonium from H. capsulatum. It was also hoped that such a study would reveal any antigenic relationships that might exist among these morphologically similar fungi. Similarly, because of the dubious relationships of H. duboisii and H. farciminosum to H. capsulatum, FA staining of these species was also undertaken. An additional objective was the development of a conjugate that could be used to rapidly detect H. capsulatum in soils. MATERIALS AND METHODS Antigens used for the production of antisera were prepared from mycelial elemeilts of H. capsulatum strain 28, Chrysosporium sp. strain R-53 [the Chrysosporium state of Arthroderma tuberculatum (Carmichael, 1962)], and S. chrysospermum strain 130 that had been grown in Sabouraud dextrose broth. The Sepedonium and Chrysosporium cultures were shaken on a reciprocal shaker for 1 week and the H. capsulatum cultures for 3 weeks, while incubating at 25 C. All cultures were treated with 0.5% formalin and, after appropriate sterility tests, were washed three times in 0.85% sodium chloride treated with 0.5% formalin. The washed mycelial elements were broken by sonic vibration (Sonifier, Bronson Instruments, Inc., Stamford, Conn.) for 30 min, and standardized so that the resulting suspension yielded 42% light transmission at 550 m,u in a Coleman spectrophotometer (model 6A). Antisera were prepared by injecting rabbits intravenously on 3 consecutive days for 3 consecutive weeks with the following volumes of antigen: 1.0, 2.0, and 2.0 ml. Each rabbit was bled a week after the final injection. The anti globulin fractions were obtained from each antiserum by three precipitations with halfsaturated ammonium sulfate. In each instance, conjugation was with fluorescein isothiocyanate. The conjugation, adsorption, and staining methods used were described in an earlier paper (Kaufman and Kaplan, 1963). A total of 19 isolates were studied: ten mycelialform cultures of H. capsulatum (strains 28, 105, 582, 681, 748, 1497, A811, A222, A827, and L261); five cultures of Sepedonium (S. ampul- 120

2 VOL. 87, 1964 FLUORESCENT-ANTIBODY STUDIES OF FUNGI 121 losporium, S. chlorium, S. chrysospermum, Sepedonium sp. strain UAMH-847, and S. xylogenium); four of Chrysosporium [C. asperatum, Chrysosporium sp. strain , the Chrysosporium state of A. tuberculatum and the Chrysosporium state of Ctenomyces serratus (Carmichael, 1962)]. Smears of soil for examination by the FA procedure were prepared by mixing 1 teaspoon of soil in 30 ml of physiological saline. After thorough stirring, the soil was allowed to settle for approximately 1 hr, after which smears were made from each supernatant fluid (Ajello et al., 1960). RESULTS Staining properties of the H. capsulatum conjugate. The staining characteristics of this unadsorbed reagent and that of the adsorbed conjugates are listed in Table 1. That H. capsulatum shares antigens with the Chrysosporium and Sepedonium species is evident (Table 1). The H. capsulatum conjugate gave staining titers of 1:32 with the homologous strains and Sepedonium species and a 1:4 titer with the Chrysosporium species. Adsorption of the H. capsulatum conjugate five consecutive times with elements of Chrysosporium sp. strain R-53 produced a reagent (H-S conjugate) that brightly stained the ten H. capsulatum isolates, the five Sepedonium species, and none of the four Chrysosporium species. These results indicated that the H. capsulatum strains and the Sepedonium species shared an antigen(s) not possessed by the Chrysosporium species. The intensity of staining exhibited by the H. capsulatum and the Sepedonium species indicated that the shared antigens were present in good quantity. Adsorption of the H. capsulatum conjugate four consecutive times. twice with elements of S. ampullosporium and twice with elements of S. chrysospermum, produced a reagent that stained only H. capsulatum. Its staining activity, however, was weak, giving only a i to a + intensity. The reactivity of the specific conjugate was so low that it would appear to be of little or no practical value. Staining properties of the Chrysosporium sp. conjugate. The FA staining reactions of the labeled anti-chrysosporium sp. strain R-53 globulins are shown in Table 2. In addition to TABLE 1. Staining reactions of unadsorbed and adsorbed conjugated antimycelial-form Histoplasma capsulatum globulins with homologous and heterologous antigens* H. capsula- Sepedon- Chrysospo- Adsorbing antigen H.m stainsu.a seium rieum tunsris species species None.. 3 to 4+ 3 to 4+ 3 to 4+ Chrysosporium sp. strain R to 3+ 2 to 3+ - S. ampullosporium and S. chrysospermum _ to 1 + * Staining reactions are expressed as follows: reasonable staining = 2 to 4+; poor staining = i to 1+; negative staining = -. TABLE 2. Staining reactions of unadsorbed and adsorbed conjugated anti-chrysosporium sp. strain R-58 globulins with homologous and heterologous antigens* Histo-Chyo Adsorbing antigen capsul plasmna alum Sepedonium spce Chryso- specium strains None... 3 to 4+ 3 to 4+ 3 to 4+ H. capsulatum... 4 to 1+1 to 2+ S. ampullosporium... _ - 2 to 3+ * Staining reactions are expressed as follows: reasonable staining = 2 to 4+; poor staining = i to 1+; negative staining = -. staining the species of Chrysosporium, the unadsorbed reagent cross-stained all the H. capsulatum and Sepedonium cultures. Staining titers of 1:8 to 1:16 were obtained with both the homologous and heterologous fungi. Adsorption of the labeled antiglobulin solution four times with the mycelial elements of H. capsulatum produced a reagent that stained both the Sepedonium and Chrysosporium species. The Sepedonium elements, however, stained very faintly: most filaments showed a i or a rare 1 + staining. Thus, it appeared that the Chrysosporium species shared a factor with the Sepedonium species, which was absent in the mycelial form of H. capsulatum. Adsorption of the labeled Chrysosporium sp. antiglobulin four times with S. ampullosporium removed all the cross-staining reactivity and apparently rendered the conjugate specific for

3 122 TABLE 3. Staining reactions of unadsorbed and adsorbed conjugated anti-sepedonium chrysopermum globulins with homologous and heterologous antigens* None... 3 to 4+ 3 to 4+ 3 to 4+ H. capsulatumn. 1 to 2+ - to 4 Chrysosporiumn sp. strain R to 2+ * Staining reactions are expressed as follows: reasonable staining = 2 to 4+; poor staining = + to 1+; negative staining = -. TABLE 4. Differential staining patterns shown by Histoplasma capsulatunt, Chrysosporium species, and Sepedonium species with adsorbed conjugatesa Histo- Sepedo- Ckryso- Adsorbing antigens plasma nium sporium capsulatumspce strains spce seis Histoplasma- Sepedo- Chryso- Fungi stained Sepedo- nium sporium nium conjugate" conjugated conjugateb H. capsulatum strains (10) _ - Chrysosporiumn species (5)... + Sepedonium species (4) Symbols: + = 1 to 4+ staining;- = 0 to + staining. I H. capsulatum conjugate adsorbed with Chrysosporium sp. strain R-53. c S. chrysospermum conjugate adsorbed with Chrysosporium sp. strain R-53. d Chrysosporium sp. strain R-53 conjugate adsorbed with S. ampullosporiurn. KAUFMAN AND BRANDT the genus Chrysosporium. Specific staining intensities ranging from 2 to 3 + were consistently obtained with this reagent. This reagent will henceforth be referred to as the C conjugate. Staining properties of the S. chrysospermum conjugate. The labeled S. chrysospermum antiglobulin stained the H. capsulatum strains and the various Sepedonium species with titers of 1:4 and 1:8, respectively. A lower staining titer of 1: 2 was obtained with the Chrysosporium species. The data in Table 3 indicate again the possession of common factors by the three genera. Adsorption of the labeled antiglobulin with elements of H. capsulatum produced a reagent reactive only J. BACTERIOL. with the Sepedonium and Chrysosporium species. The Chrysosporium species, however, stained poorly ( + intensity). Adsorption of the conjugate with elements of Chrysosporium sp. strain R-53 eliminated the staining of H. capsulatum and the various species of Chrysosporium without significantly impairing the conjugate's capacity to stain the Sepedonium species. This reagent consistently stained the Sepedonium species and not the members of the other two genera, and will be referred to as the S conjugate. The staining characteristics of the adsorbed conjugates indicated that the H. capsulatum isolates and the Chrysosporium and Sepedonium species could be differentiated and identified through the appropriate use of a combination of FA reagents. The adsorbed conjugates and their differential staining patterns are presented in Table 4. With these conjugates, H. capsulatum will stain only with the Histoplasma-Sepedonium (H-S) conjugate. If a culture reacts only with the Chrysosporium (C) conjugate, it would be considered to be a species of Chrysosporium. The staining of an isolate by both the H-S and Sepedonium (S) reagents would identify the fungus as a Sepedonium. Staining properties of the three reagents with additional homologous and heterologous mycelial antigens. Our three adsorbed conjugates permitted the recognition and separation of H. capsulatum, the Sepedonium, and the Chrysosporium species. However, we realized that the effectiveness of these "specific" fluorescent antibodies could not be fully evaluated until we had studied and evaluated their reactivity with a larger number of fungi with tuberculate and nontuberculate spores (Table 5). The data shown in Table 5 indicate that the H-S conjugate, in addition to staining H. capsulatum, cross-stained both intra- and extrageneric species of fungi. None of these species, however, produced tuberculate spores and, consequently, would not pose a problem in the identification of H. capsulatum. All of the 15 other strains of H. capsulatum reacted with the H-S reagent. It is noteworthy that the two additional Sepedonium species did not stain with the H-S reagent, a reaction contrary to that observed with the species of Sepedonium studied earlier (Table 4). It is apparent from the data that the S conjugate also stained fungi other than the species

4 VOL. 87, 1964 FLUORESCENT-ANTIBODY STUDIES OF FUNGI 123 of Sepedonium. However, aside from the two strains of the Chrysosporium state of Thielavia sepedonium, none of the reacting fungi bore tuberculate spores. The C conjugate showed the least cross-staining activity of the three conjugates listed in Table 5. Except for Alternaria strain R-57, only tuberculate spore-bearing species of Chrysosporium reacted with the C reagent. The C reagent, however, did not stain three tuberculate spore-producing chrysosporiums: two strains of the Chrysosporium state of T. sepedonium and one strain of Chrysosporium strain B-352. None of the strains of C. keratinophilum and C. pannorum, which produce smooth aleuriospores, reacted with this reagent. Diagnostic application. To determine the efficacy of the FA reagents as diagnostic tools, we requested the Diagnostic Laboratory of the Mycology Unit to give us all the tuberculate spore-bearing cultures received for identification. With our three reagents, we independently identified the seven cultures marked with an asterisk in Table 5. Five of these cultures were identified as H. capsulatum, one as a species of Sepedonium, and one as a Chrysosporium. In every instance, the identities determined by the FA procedure were verified by conventional diagnostic procedures. Soil examinations. Although the data presented in Table 5 indicated that the H-S reagent cross-stained various nontuberculate sporebearing fungi commonly found in soil, we attempted to use this conjugate to reveal elements of H. capsulatum in various soil samples shown by conventional procedures to contain this pathogen. Examination of ten soil specimens with the H-S conjugate failed to reveal tuberculate spores. It did however, brilliantly reveal numerous mycelial filaments, microconidia, and smooth-walled macroconidia. These structures were morphologically similar to elements produced by H. capsulatum, but, regrettably, they, of course, are not of definitive diagnostic value. DISCUSSION Our attempts to produce a useful specific FA reagent for H. capsulatum were unsuccessful. The data shown in Table 1 indicate that, although an antibody specific to the mycelial form of the pathogen is present in the H. capsulatum antisera, it occurs at a level too low for practical use in the diagnostic laboratory. Attempts to boost the staining capacity of this antigen by concentration failed. The identification of H. capsulatum, however, was achieved by use of the H-S and S conjugates; 25 strains of H. capsulatum tested with the H-S and S conjugates (Tables 4 and 5) reacted with the H-S reagent but not with the S conjugate. The S conjugate reacted with all seven species of Sepedonium and with two strains of the Chrysosporium state of T. sepedonium. That the latter strains reacted with the S reagent does not indicate that the reagent is nonspecific. The taxonomic position of the Chrysosporium state of T. sepedonium is questionable. Carmichael (1962) classified the imperfect form of T. sepedonium in the genus Chrysosporium. Since our serological studies revealed that the C reagent did not react with the two strains of the Chrysosporium state of T. sepedonium, Carmichael's classification becomes questionable in our opinion. Emmons (1932), in his original description of T. sepedonium, stated that the asexual spores produced by T. sepedonium were typical of the genus Sepedonium. The two imperfect forms we studied are antigenically closely related to the sepedoniums and, for practical diagnostic purposes, may be considered as such. On the basis of our findings, it is suggested that tuberculate spore-bearing fungi that react either with both the H-S and S reagents or with the S reagent alone (Tables 4 and 5) be classified in the genus Sepedonium. The C conjugate appeared to be an effective agent for identifying Chrysosporium cultures. This reagent reacted with 10 of 11 tuberculate spore-bearing cultures identified by conventional procedures as being members of the genus Chrysosporium. On the basis of the data presented (Table 5), it is apparent that the identification of H. capsulatum may be greatly facilitated by the use of two FA reagents as follows. Staining with the H-S conjugate would indicate that an isolate that produced tuberculate spores was either H. capsulatum or a species of Sepedonium. If this isolate then stained with the S reagent, it was a Sepedoniumn; if not, the isolate was probably H. capsulatum. With appropriate FA reagents in stock, these tests could be performed within 1 hr. With conventional procedures, which involve

5 124 KAUFMAN AND BRANDT J. BACTERIOL. TABLE 5. Reactions of pathogenic and saprophytic fungi to staining with the Histoplasma-Sepedonium, Sepedonium, and Chrysosporium conjugates ~~~Histoplasma- Sepedoisium Chrysosporium Fungus stained Sepeaonium conjugate conjugate conjugate Aspergillus fumigatus A A. niger Alternaria sp. R Blastomyces dermatitidis B B. dermatitidis B B. dermatitidis B B. dermatitidis B B. dermatitidis B Botryotrichum state of Chaetomium piluliferum A Chrysosporium keratinophilum I C. keratinophilum I C. kerotinophilum I-ll C. pannorum B C. pannorum B C. pannorum B C. pannorum B Chrysosporium state Of Arthroderma tuberculatum UG-25-A Of A. tuberculatum Ct-50* Of A. tuberculatum A Of Ctenomyces serratus Of Thielavia sepedonium A Of T. sepedonium B Chrysosporium sp. UG-25-B Chrysosporium sp. UG Chrysosporium sp. UG Chrysosporium sp. B Curvularia sp. B Fusarium sp. A Gliocladium roseum Emmonsia parva E. parva E. parva A E. parva A Helminthosporium sp. A Histoplasma capsulatum 157* H. capsulatum 159* H. capsulatum H. capsulatum H. capsulatum 324* H. capsulatum T-1-3d* H. capsulatum T-2-2d* H. capsulatum A H. capsulatum A H. capsulatum A H. capsulatum A H. capsulatum A H. capsulatum A H. capsulatum A H. capsulatum A H. duboisii B H. duboisii B

6 VOL. 87, 1964 FLUORESCENT-ANTIBODY STUDIES OF FUNGI 125 TABLE 5. Continued Hseopedonima Sepedonium Chrysosporiumn Fungus stained Sepedoniug conjugate conjugate H. duboisii B H. farciminosum A H. farciminosum A H. farciminosum A Masoniella grisea Penicillium caseocolum A Penicillium sp Paecilomyces sp. A Rhizopus nigricans A Scopulariopsis sp Sepedonium sp. UAMH Sepedonium sp * Streptomyces sp. R Trichoderma sp. R * Cultures from the diagnostic laboratory. attempts at conversion of an isolate from mycelial to yeast form, identification would require 1 week or more. The results of this and other studies (Kaufman and Kaplan, 1963) have shown that H. capsulatum shares antigens with numerous nontuberculate spore-bearing fungi, a fact that limits the use of fluorescein-labeled anti-h. capsulatum globulins for detecting H. capsulatum in soil. Unless one uses the H-S and S reagents to stain filaments with attached tuberculate spores, an unequivocal identification of H. capsulatum cannot be achieved. To date, we have not encountered any tuberculate spores in numerous soil specimens examined. Thus, the inference of Kaplan et al. (1961) that H. capsulatum was stained in soil specimens is definitely equivocal. Our cross-staining and adsorption data reveal some interesting antigenic relationships among the fungi studied. All members of the three genera have at least one antigen in common. In addition, H. capsulatum and the Sepedonium species share another antigen (Table 1), while the Chrysosporium species share an additional antigen with the Sepedonium species (Table 2). The FA data also suggest that H. capsulatum possesses a minute quantity of a specific antigen (Table 1), whereas Chrysosporium and Sepedonium species have high levels of their respective specific antigens (Tables 2 and 3). These studies have given an insight to the antigenic relationships of the genera Chrysosporium, Histoplasma, and Sepedonium which, in conjunction with other data, supports the taxonomic concept that these genera are separate and distinct. In studies on the morphological relationship between H. capsulatum and similar forms, Howell (1939) observed that the tuberculate macroconidia of H. capsulatum were similar in appearance to those of S. chrysospermum. However, he did not consider that this morphological similarity merited placement of H. capsulatum in the genus Sepedonium. Because of the association of H. capsulatum with dung-enriched soil and its production of hyaline spores, Carmichael (1962) suggested that H. capsulatum is more closely related to Chrysosporium than to Sepedonium. Our serological findings suggest that H. capsulatum is allied more closely to Sepedonium than to Chrysosporium. H. capsulatum not only possesses a weak specific antigen but also shares at least two other antigens with Sepedonium. The proposal of Vanbreuseghem (1953) that differences between H. duboisii and H. capsulatum, especially the tissue forms, justify the recognition of H. duboisii as a distinct species has not been accepted by several investigators (Drouhet and Schwarz, 1956; Taschdjian, 1959; Carmichael, 1962). They consider H. duboisii to be no more than a variety of H. capsulatum. Our investigations revealed that H. duboisii is antigenically different from H. capsulatum. The mycelial form of H. duboisii does not share with H. capsulatum the antigens necessary to react with the H-S conjugate. Furthermore, the

7 126 KAUFMAN AND BRANDT J. BACTERIOL. organisms differ in that the yeast form of H. duboisii lacks the antigen(s) specific for the yeast form of H. capsulatum (Kaufman and Kaplan, 1961). Therefore, on the basis of antigenic components, H. duboisii appears to be quite distinct from H. capsulatum and merits species rank. Of further interest is the antigenic relationship of the mycelial form of H. farciminosum to the mycelial form of H. capsulatum. The mycelial form of H. farciminosum does not produce spores of any kind and macroscopically does not resemble H. capsulatum. Antigenically, however, it appears to be more closely related to H. capsulatum than to H. duboisii. Of additional taxonomic interest is the antigenic relationship of Blastomyces dermatitidis and Emmonsia parva to Chrysosporium. Carmichael (1962) considers these fungi to be classified better in the genus Chrysosporium. This radical concept is not supported by our serological data (Table 5). None of the strains of B. dermatitidis or E. parva examined by us reacted with the C or S conjugates; all, however, reacted with the H-S reagents. It is not our intention at this time to revise the classification of any of the fungi studied in this investigation. However, we do feel that taxonomists should consider antigenic characteristics, as well as morphological and biochemical properties, in determining the taxonomic position of a fungus. ACKNOWLEDGMENTS The authors are grateful to C. T. Rogerson, Curator, Cryptogamic Botany, the New York Botanical Garden, for supplying several of the Sepedonium cultures, and to J. W. Carmichael, Provincial Laboratory of Public Health, University of Alberta, for supplying and identifying many of the cultures used in this study. LITERATURE CITED AJELLO, L., T. BRICENO-MAAZ, H. CAMPINS, AND J. C. MOORE Isolation of Histoplasma capsulatum from an oil bird (Steatornis Caripensis) cave in Venezuela. Mycopathol. Mycol. Appl. 12: CARMICHAEL, J. W Chrysosporium and some other aleuriosporic hyphomycetes. Can. J. Botany 40: DROUHET, E., AND J. SCHWARZ Comparative studies with 18 strains of Histoplasma. J. Lab. Clin. Med. 47: EMMONS, C. W The development of the ascocarp in two species of Thielavia. Bull. Torrey Botan. Club 59: HOWELL, A Studies on Histoplasma capsulatum and similar form species. I. Morphology and development. Mycologia 31: KAPLAN, W., L. AJELLO, D. B. DI BITETTO, AND E. S. McDONOUGH The discovery of Histoplasma capsulatum in Connecticut soil incidental to the investigation of a case of feline cryptococcosis. Mycopathol. Mycol. Appl. 14:1-8. KAUFMAN, L., AND W. KAPLAN Preparation of a fluorescent antibody specific for the yeast phase of Histoplasma capsulatum. J. Bacteriol. 82: KAUFMAN, L., AND W. KAPLAN Serological characterization of pathogenic fungi by means of fluorescent antibodies. I. Antigenic relationships between yeast and mycelial forms of Histoplasma capsulatum and Blastomyces dermatitidis. J. Bacteriol. 85: TASCHDJIAN, C. L Hyphal fusion studies on Histoplasma capsulatum and Histoplasma duboisii Vanbreuseghem Mykosen 2:1-6. VANBREUSEGHEM, R Histoplasma duboisii and African histoplasmosis. Mycologia 65: