Specific fluorescent antiglobulins for the detection and identification of blastomyces dermatitidis yeast-phase cells

Summary Two lots of rabbit anti-Blastomyces dermatitidis globulins were conjugated with fluorescein isothiocyanate. These reagents stained brightly elements of the yeast and mycelial phases of 10 strains ofB. dermatitidis. In addition, the labeled antibodies cross-reacted with elements of the yeast and mycelial phases of 7 strains ofHistoplasma capsulatum and cells of numerous other heterologous fungi. Adsorption of one lot of labeled antibodies twice with yeast cells ofH. capsulatum and once with elements ofGeotrichum candidum rendered the conjugate specific for the yeast phase ofB. dermatitidis. Three adsorptions with yeast cells ofH. capsulatum followed by a single adsorption with elements ofG. candidum rendered the second conjugate specific for yeast-phase cells ofB. dermatitidis. The specific reagents did not react with the mycelial phase of this fungus.     

Protoplasts of systemic dimorphic fungal pathogens: Histoplasma capsulatum and blastomyces dermatitidis

Summary Protoplasts have been released fromH. capsulatum in the mycelial and yeast phases and from the mycelial and incompletly converted yeast phase ofB. dermatitidis by the enzymatic action of snail digestive juice. There is great variation in the mode of protoplast formation not only between species but between the two morphological forms, particularly inH. capsulatum These studies were supported in part by grants from the American Thoracic Socicty and the United States Public Health Service National Institute of Allergy and Infectious Diseases (1-R1-AI-7520-01).     

Isoelectric focusing and ELISA evaluation of a <Emphasis Type="Italic">Blastomyces dermatitidis</Emphasis> human isolate

Blastomyces dermatitidis is a dimorphic fungal organism and the causative agent of blastomycosis. This organism is endemic east of the Mississippi river as is the fungal organism Histoplasma capsulatum. This study was performed to determine if sensitive and specific antigens from the B. dermatitidis yeast phase lysate (human isolate 592) could be separated using isoelectric focusing (IEF) to eliminate antigens that are cross-reactive with H. capsulatum. Indirect enzyme linked immunosorbent assays were performed to test for reactivity and cross-reactivity and indicate that certain fractions (4–6) were highly reactive. Fraction 16 exhibited a high degree of cross-reactivity with H. capsulatum. This study indicates that IEF may be a useful method for the separation of B. dermatitidis proteins.     

Studies in histoplasmosis III. Cross reactions and characterization of histoplasma and blastomyces inhibitory factors

Summary The Histoplasma tissue inhibitory factor HIF has been demonstrated in tissues of hamsters and mice infected with four strains ofHistoplasma capsulatum. A similar HIF has been demonstrated in liver homogenates from hamsters infected withBlastomyces dermatitidis.High titer HIF completely inhibits growth of yeast cells. Lower relative proportions of HIF: yeast cells results in diminution in number of colonies and delay in initiation of growth.The Histoplasma and Blastomyces HIF show cross-inhibition with the heterologous yeast cells, but do not inhibit the respective mycelia from these organisms although HIF is adsorbed from the supernatant by both yeasts and mycelium.Histoplasma HIF adsorbs, without inhibiting growth, toCandida albicans, Cryptococcus neoformans, Bacillus subtilis andEscherichia coli.Because of the non-specific adsorption of HIF, it is probable that it may often be present in tissue but not demonstrable because bound and probable that production of an HIF in infectious diseases may be a general phenomenon.Supported by Public Health Service Grant No. 1-SO-IFR-05359-01.     

Experimental canine histoplasmosis and blastomycosis

Normal adult beagle dogs were experimentally infected withHistoplasma capsulatum orBlastomyces dermatitidis. Clinical signs of histoplasmosis and blastomycosis were similar to those seen in natural infections in dogs, although diarrhea was not seen in dogs with experimental histoplasmosis. Significant radiographic changes were seen in the lungs of all dogs inoculated with one of the organisms but not in the control dogs.Amphotericin B treatment of the dogs infected with mycelia ofBlastomyces dermatitidis resulted in clinical improvement and prevented death, but did not cure all of the dogs. Four of five dogs randomly selected for placebo treatment died within 34 days of inoculation, whereas all five dogs in the amphotericin B treated group were alive 14 weeks after inoculation.Since no deaths occurred in dogs inoculated with the mycelia ofHistoplasma capsulatum, weight loss was used as a measure of the degree of illness. No difference could be demonstrated between weight losses of three dogs treated with amphotericin B and of three dogs treated with a placebo. Four other dogs inoculated withH. capsulatum did not have a 20% weight loss, a criterion for treatment. The 10 control dogs maintained their preinoculation weight.From the Ecological Investigations Program, Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, United States Department of Health, Education, and Welfare, Kansas City, Kansas.     

Effect of yeast extract concentration on growth ofSchizoccharomyces pombe

Summary The effect of yeast extract on the growth ofSchizosaccharomyces pombe was investigated using a complex-synthetic medium. Batch cultures at low-glucose concentration show that a too low concentration of yeast extract may limit the biomass formation. On this medium kinetics and yields were found to be similar to those obtained on a synthetic-defined medium under both aerobic and anaerobic conditions.     

Effect of prior vaccination on experimental Blastomycosis

Immunogens were found associated with particular fractions prepared from spherules ofCoccidioides immitis (Kong, Levine &Smith, 1963) and from yeast cells ofHistoplasma capsulatum (Salvin &Ribi, 1955). However,Blastomyces dermatitidis, another dimorphic systemic fungal pathogen was shown to elicit a minimal immunogenic response in experimental animals (Kong &Levine, 1967). It was therefore deemed pertinent to study factors which might enhance the resistance of mice to infection withB. dermatitidis.     

Efficacy of brain heart infusion-egg albumen agar,yeast extract phosphate agar and peptone glucose agar media for isolation of Blastomyces dermatitidis from sputum

The efficacy of brain heart infusion (BHI)-egg albumen agar, yeast extract phosphate agar and several modified peptone glucose agar media was evaluated for isolation of Blastomyces dermatitidis from sputum concomitantly seeded with the yeast form of the pathogen and Candida albicans. Based upon high per cent culture positivity of sputum, improved recovery (CFU/ml) of the seeded inoculum, faster growth rate of B. dermatitidis and low level of contamination, BHI-egg albumen agar, followed by yeast extract phosphate agar are recommended as the media of choice for the isolation of B. dermatitidis from contaminated clinical specimens.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Cross-protection between Histoplasma capsulatum and Oidiodendron kalrai in mice

Summary Cross-protection studies were carried out by immunizing mice intraperitoneally with live and formalin killed yeast cells ofHistoplasma capsulatum andOidiodendron kalrai. Immunized and non-immunized mice were challenged intravenously 21 days later with the yeast cells ofHistoplasma capsulatum. The greatest protection was observed in mice immunized with live cells ofH. capsulatum and was definitely superior to that obtained with killed cells ofH. capsulatum. Significant protection against challenge byH. capsulatum was observed in mice immunized with killed but not with live cells ofO. kalrai.This work was supported from a research grant from the Bremer Foundation.The authors wish to thank Professor CharlotteC. Campbell for the supply ofHistoplasma capsulatum culture.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Environmental factors and growth of Histoplasma capsulatum in soil

Summary Environmental factors influencing the growth ofHistoplasma capsulatum in soil have been studied. The role of temperature and moisture in the growth of the fungus was found to be critical. The fungus can tolerate very low temperatures, if the soil moisture content is high, but cannot withstand temperatures of 40° C or above for an extended period.Dry, sterile chicken manure and an extract of unsterile chicken manure showed an inhibitory effect on the growth of the fungus. However, the relationship between bird manure andH. capsulatum has not been satisfactorily clarified.Morphological studies ofH. capsulatum in soil showed that the fungus grows within the upper two inches of the soil and a majority of sporulation occurs within the upper one-half inch of soil. Morphological studies of the fungus, in the presence of chicken manure and chicken manure extract, showed an increased number of macroconidia and microconidia and decreased mycelial production.The growth ofH. capsulatum in soil is markedly affected by soil pH above 10 and below 5. No growth was observed on soil cultures outside these values. There was no definite pH range within these values in which the fungus grew more abundantly.     

Fluorescent antibody techniques for identification of Coccidioides immitis and Histoplasma capsulatum

This is a review of practical uses of immunofluorescence in detection of the two fungi in host and environment and in identification of their cultures, as well as in serologic case finding. Reagents directed at the yeast phase ofHistoplasma capsulatum have been fairly successful in differentiating this species from others, the main difficulty being the tendency to cross-react withBlastomyces dermatitidis andH. duboisii. Conjugates for the mycelial phase ofH. capsulatum tend to cross-react withSepedonium andChrysosporium, but careful absorption may yield specific reagents. Anti-yeast-phase conjugates are a valuable adjunct to cultural and clinical methods when used to detect and identifyH. capsulatum in sputum and other clinical specimens. Conjugates specific for the spherules or tissue phase ofCoccidioides immitis have yielded false negative results when applied to clinical specimens. The fluorescent-inhibition procedure is useful for serologic case finding in histoplasmosis and the same technique has shown fairly good agreement in coccidioidomycosis with complement-fixation and tube-precipitin methods. Immunofluorescence reagents for the two species have been useful in screening surgical and autopsy specimens, animal tissues, and soils.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Solid-state cultivation ofPenicillium capsulatum on beet pulp

Summary Extracellular enzyme production byP. capsulatum during solid-state fermentation on beet pulp is maximal at 30°C with initial moisture contents of 60–75% and when growth medium is supplemented with corn steep liquor, ammonium sulphate and yeast extract. On balance, solid-state co-cultures ofP. capsulatum withT. reesei orT. emersonii do not yield greater overall enzyme activity thanP. capsulatum alone.     

Inhibition of pathogenic fungi in vitro by p-hydroxy methyl benzoate

Summary p-hydroxy methyl benzoate is fungistatic, in rather low concentrations, to pathogenic fungi. 0.1 %p-hydroxy methyl benzoate was required to inhibit growth ofCandida albicans andMonosporium apiospermum on a Sabouraud's agar medium.Trichophyton mentagrophytes, T. tonsurans, Geotrichum sp.,Sporotrichum schenckii, Blastomyces dermatitidis andCryptococcus neoformans failed to grow in the presence of 0.05 %p-hydroxy methyl benzoate. Growth ofEpidermophyton floccosum, Microsporum audouini, M. canis, M. gypseum, Trichophyton ferrugineum, T. rubrum, Hormodendrum compactum, H. Pedrosoi, Phialophora verrucosa, Nocardia asteroides, Coccidioides immitis, Haplosporangium parvum andHistoplasma capsulatum was suppressed by 0.025 % but not by 0.0125 % of this compound.     

Comparative fluorescent antibody staining of histoplasma capsulatum and histoplasma duboisii with a specific anti-yeast phase H. Capsulatum conjugate

Summary The specific anti-yeast phaseHistoplasma capsulatum conjugate has been tested against 13 yeast phase strains ofH. capsulatum and 9 ofH. duboisii. The conjugate was specific forH. capsulatum, no yeast phase form ofH. duboisii obtained in vitro or in vivo reacted with it. The taxonomic implications of these results are discussed.     

The uptake of low molecular weight sulfur-containing compounds by Histoplasma capsulatum and related dimorphic fungi

A number of low molecular weight organic sulfur-containing compounds were tested for their effect on the respiratory activity of yeastlike and mycelialH. capsulatum. Of the compounds tested, L-cyst(e)ine was found to give maximum stimulatory effect on yeastlike phase respiration. The D- and meso isomers of cyst(e)ine as well as substituted derivatives were much less effective in the stimulation of respiratory activity of yeastlikeH. capsulatum. Respiration of homologous mycelial phase cell suspensions was depressed in the presence of L-cystine as substrate, while respiratory activity of yeastlikeB. dermatitidis andS. schenckii was unaffected.Whole cell suspensions of yeastlikeH. capsulatum actively transported S³⁵-labeled L-cystine and methionine but apparently not -mercaptoacetate-S³⁵. Mycelial phaseH. capsulatum and the yeastlike and mycelial phases ofB. dermatitidis andS. schenckii were observed to take up S³⁵-labeled L-cystine to a much lesser degree than yeastlikeH. capsulatum as determined on a dry weight basis. These results suggest significant differences in the transport and subsequent intracellular mechanisms of metabolism of low molecular weight sulfur-containing -amino acids and related compounds by yeastlikeH. capsulatum and its corresponding mycelial phase as well as the dimorphic fungiB. dermatitidis andS. schenckii.

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Zusammenfassung Eine Anzahl organischer, Sulfur-enthaltender Verbindungen mit niedrigem Molekulargewicht sind betreffs ihrer Wirkung an der Atmungsaktivität von hefeähnlichem und myzelialemH. capsulatum untersucht worden. Von den untersuchten Verbindungen gab L-cyst(e)ine die größte Reizwirkung an der Atmung der Hefephase. Die D- und Meso-Isomers von Cyst(e)ine so wie auch die substituierten Derivatives waren in der Reizung der Atmungsaktivität der Hefephase vonH. capsulatum weniger wirksam. Die Atmung der Suspension von Zellen der homologen Myzelphase war in der Gegenwart von L-cystine als Substrat unterdrückt, während die Atmungsaktivität der Hefephase desB. dermatitidis und die desS. schenckii unbeeinflußt blieb. Suspensionen von ganzen Zellen der Hefephase desH. capsulatum transportierten wirksam S³⁵ L-cystine und Methionine, aber anscheinend nicht beta-mercaptoacetate-S³⁵. Myzelphase-H. capsulatum und Hefeund Myzelphasen desB. dermatitidis undS. schenckii nehmen S³⁵-L-Cystine zu einem geringeren Grade auf denn Hefephase-H. capsulatum wie es am Trockengewicht festgestellt worden ist. Diese Ergebnisse legen es nahe, daß wesentliche Unterschiede im Transport und in dem nachfolgenden Intracellularmechanismus des Stoffwechsels von den Sulfurenthaltenden alfa-Aminosäuren mit niedrigem Molekulargewicht und verwandten Verbindingen durch die Hefephase desH. capsulatum und der bezüglichen Myzelphase, so wie auch durch die Doppelphasenpilze:B. dermatitidis undS. schenckii bestehen.

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This study has been supported by Part I VA-8200 Funds.     

Behavior of certain fungi injected in a mixed suspension into mice

Summary A total of 40 mice was injected with mixed suspensions ofC. albicans, C. neoformans, B. dermatitidis andH. capsulatum intraperitoneally, subcutaneously, intravenously and intracerebrally. Cultures from spleen, liver, kidney, lung and brain were made at intervals of 1, 4, 8, 15 and 30 days after inoculation. Results were recorded and compared with those obtained from a similar experiment where the organisms were injected separately.This project was aided in part by grant E-986 of the N.I.H.     

Agrobacterium tumefaciens integrates transfer DNA into single chromosomal sites of dimorphic fungi and yields homokaryotic progeny from multinucleate yeast

The dimorphic fungi Blastomyces dermatitidis and Histoplasma capsulatum cause systemic mycoses in humans and other animals. Forward genetic approaches to generating and screening mutants for biologically important phenotypes have been underutilized for these pathogens. The plant-transforming bacterium Agrobacterium tumefaciens was tested to determine whether it could transform these fungi and if the fate of transforming DNA was suited for use as an insertional mutagen. Yeast cells from both fungi and germinating conidia from B. dermatitidis were transformed via A. tumefaciens by using hygromycin resistance for selection. Transformation frequencies up to 1 per 100 yeast cells were obtained at high effector-to-target ratios of 3,000:1. B. dermatitidis and H. capsulatum ura5 lines were complemented with transfer DNA vectors expressing URA5 at efficiencies 5 to 10 times greater than those obtained using hygromycin selection. Southern blot analyses indicated that in 80% of transformants the transferred DNA was integrated into chromosomal DNA at single, unique sites in the genome. Progeny of B. dermatitidis transformants unexpectedly showed that a single round of colony growth under hygromycin selection or visible selection of transformants by lacZ expression generated homokaryotic progeny from multinucleate yeast. Theoretical analysis of random organelle sorting suggests that the majority of B. dermatitidis cells would be homokaryons after the ca. 20 generations necessary for colony formation. Taken together, the results demonstrate that A. tumefaciens efficiently transfers DNA into B. dermatitidis and H. capsulatum and has the properties necessary for use as an insertional mutagen in these fungi.     

Estimation of the number of viable particles of Histoplasma capsulatum in soil

Summary Serial dilutions of suspensions of soil samples positive forH. capsulatum were made and injected intravenously into mice. The dilution producing infection in 50 % of the mice injected (ID₅₀) was determined for each sample and provided a measure for quantitative comparisons. A known number of viable particles ofH. capsulatum was added to soil, and serial dilutions were made of the suspension and injected into mice to determine that dilution containing an ID₅₀. One ID₅₀ was calculated to contain 1.6 viable particles ofH. capsulatum per ml of inoculum. With the assumption that one ID₅₀ of unknown samples contained 1.6 viable particles per ml inoculum, the total number of viable particles per gram of soil in several sites was calculated. The total number of viable particles ofH. capsulatum per gram of soil in different sites ranged from 101 to 201,900, almost a two thousandfold difference. Now that the number of viable particles ofH. capsulatum in positive sites can be determined, it may be possible to determine the concentration of particles necessary to make sites significant sources of infection.From the Ecological Investigations Program, National Communicable Disease Center, Bureau of Disease Prevention and Environmental Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Kansas City, Kansas.Presented in part at the annual meeting of the American Society for Microbiology, New York, N.Y., April 30-May 4, 1967.