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.     

The discovery ofHistoplasma capsulatum in Connecticut soil incidental to the investigation of a case of feline cryptococcosis

Summary The seventh case of cryptococcosis in a cat is described. The animal involved was a five-year-old Maltese short hair that had lived in Connecticut all of its life. An investigation was carried out to discover the point source of this feline infection. This search was unsuccessful but it did result in the isolation ofH. capsulatum for the first time from Connecticut soil.In addition, the preliminary findings of a study to evaluate the use of the fluorescent antibody technique for the rapid detection ofH. capsulatum in soil are reported. Round to oval forms measuring 1.5 to 3.5 microns in diameter were demonstrated in smears of the positive Connecticut soil that had been stained with fluorescein labeled anti-H. capsulatum globulins. The morphology of these elements was similar to that of the microconidia ofH. capsulatum. Through the use of this immune conjugate five additional positive soil samples from other areas were also shown to contain these forms. None of these six soils showed stained elements when treated with normal conjugates. The implications of these findings are discussed.     

II. Evidence of the presence in yeast extract of substances which stimulate the growth of Histoplasma capsulatum and blastomyces dermatitidis similarly to that found in starling manure extract

Summary A medium consisting of agar plus yeast extract contained the necessary metabolites for rapid growth and sporulation ofHistoplasma capsulatum andBlastomyces dermatitidis. H. capsulatum when harvested after 10 or 30 days incubation period from this medium was shown to have a similar number of spores as well as total particle viability for each period of growth.The growth characteristics ofH. capsulatum and four different isolates ofB. dermatitidis on yeast extract medium were similar to that obtained previously using starling (Sturnis vulgaris) manure extract medium. These characteristics are rapid growth consisting of many viable spores and a low ratio of vegetative mycelium.Several isolations ofH. capsulatum from naturally contaminated soil specimens were made using yeast extract medium.From the Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare.     

Induction of novel protein synthesis by opsonizedHistoplasma capsulatum ingested by murine peritoneal macrophages

It is known thatHistoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages. This resistance can be closly related to its pathogenicity. The mechanism of this resistance has been investigated, but it has not been clarified as yet. To learn about the metabolic condition of the yeast-form ofH. capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingestedH. capsulatum with [³⁵S]-methionine labeling. Cycloheximide at 5 to 10 µg/ml was used to preferentially inhibit macrophage uptake of [³⁵S]-methionine without affectingH. capsulatum uptake. Protein synthesis byH. capsulatum in medium alone served as a positive control. The negative control consisted of macrophages with ingested heat-killedH. capsulatum. Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingestedH. capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins. Ten of these novel or increased proteins were apparently common to both strains. These metabolic changes in ingestedH. capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there.     

Second Case of Histoplasmosis in a Captive Mara (<Emphasis Type="Italic">Dolichotis patagonum</Emphasis>): Pathological Findings

A second case of histoplasmosis in a captive mara (Dolichotis patagonum) from a colony at the wildlife park Africam Safari, Puebla, Mexico, is described, and the mara died with disseminated clinical form of the disease, affecting mostly the large intestine and adrenal. The pathological findings of this case 2 revealed severe granulomatous typhlocolitis and moderate granulomatous gastrohepatic lymphadenitis with numerous yeast-like cells, 2–4 μm in diameter, with a clear halo surrounding them inside the cytoplasm of macrophages, suggesting the parasitic form of Histoplasma capsulatum. Adrenocortical cells had abundant similar microorganisms in their cytoplasm without any associated lesion. Gomori’s methenamine silver and periodic acid Schiff stained positively these microorganisms. Immunohistochemistry, using a rabbit anti-H. capsulatum serum, and transmission electron microscopy supported the diagnosis of H. capsulatum infection.     

Insertional mutagenesis enables cleistothecial formation in a non-mating strain of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Background  

Histoplasma capsulatum is a pathogenic ascomycete fungus that rapidly loses mating ability in culture. Loss of mating ability, as well as the organism's low rate of targeted gene replacement, limits techniques available for genetic studies in H. capsulatum. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in H. capsulatum strains, introducing a variety of classical genetics techniques to the field. We generated a strain, UC1, by insertional mutagenesis of the laboratory strain G217B, and found that UC1 acquired the ability to form mating structures called cleistothecia. The aim of this study was to determine the mechanism by which UC1 gained the ability to form cleistothecia. We also present initial studies demonstrating that UC1 can be used as a tool to determine molecular correlates of mating in 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.     

Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires,Argentina

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

     

Suppressive effect of streptomycin on the phagocytic activity of mouse peritoneal macrophages for Histoplasma capsulatum

Streptomycin inhibited the phagocytic activity of mouse peritoneal macrophages forHistoplasma capsulatum. The inhibitory effect was demonstrable following both in vitro and in vivo administration of drug. The observations from examination by direct smear were confirmed by culturing for viable phagocytized organisms. A simple and reproducible technique for the counting of viable phagocytized organisms was developed. Forty-eight hours in vitro treatment of macrophage cultures with 10 to 200 µg/ml of streptomycin produced a graded inhibition of phagocytic activity, minimal at 10 µg/ml and maximal at 200 µg/ml of streptomycin. The parenteral administration of streptomycin significantly reduced phagocytic activity of mouse peritoneal macrophages forH. capsulatum. Mice were treated daily with the subcutaneous injections of 5, 2.5 or 1 mg streptomycin or saline. At 7, 14, 21 and 28 days post-treatment phagocytic activity of macrophages obtained from these mice was tested. There was a progressive, dose-dependent decrease in the phagocytic activity of macrophages derived from streptomycin-treated mice.     

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.     

A survey to determine the occurrence of Histoplasma capsulatum and Cryptococcus neoformans in air-conditioners

Summary A survey of 84 dust samples from 42 air-conditioners and controls was conducted at Kansas State University, Manhattan. Isolations ofC. neoformans andH. capsulatum were attemped using various mycological procedures. H. capsulatum was not recovered in this survey. One isolate ofC. neoformans was obtained. The contaminated air-conditioner yielding the organism contained considerable bird feces, feathers, and dust.Contribution No. 102, Department of Infectious Diseases, Kansas Agricultural Experiment Station, Manhattan, Kansas.     

Resistance of Histoplasma capsulatum to killing by human neutrophils

The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.Abbreviations CFU colony forming units - PMN polymorphonuclear neutrophil - CTCM complete tissue culture medium - CL chemiluminescence - HPO horseradish peroxidase - P-L lysosomal peroxidase positive material     

A natural focus ofHistoplasma capsulatum var.duboisii is a bat cave

The natural reservoir ofHistoplasma capsulatum var.duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known. We report the isolation ofH. capsulatum var.duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria. Eight of 45 samples of soil admixed with bat guano yieldedH. capsulatum var.duboisii. Of the 35 bats belonging to the speciesNycteris hispida andTadirida pumila examined, only one (N. hispida) yielded this fungus from its intestinal contents. Identification of the isolates asHistoplasma was confirmed by exoantigen tests and by mating with tester strains ofH. capsulatum. In vitro conversion to large yeast from suggestive ofH. capsulatum var.duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine. Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8–15 µm in diameter) within giant cells in the infected tissues. Investigations on the possible occurrence of human infections in the area are in progress.A poster based on this work was presented at the 11th ISHAM Congress in Montreal, Canada (22–28 June 1991), La-Hoffman Roche, Basel, Switzerland kindly financed the trip of one of us (H.C.G) for the Congress.     

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.     

Histoplasmosis in India (a case report)

Summary A case of generalised infection withH. capsulatum is described in which the main stress was on the lungs and in the latter stage an ulcer in the oral cavity developed. The fungus was recovered from the sputum as well as the ulcer.     

Identification of the infectious source of an unusual outbreak of histoplasmosis,in a hotel in Acapulco,state of Guerrero,Mexico

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel’s ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92–99%) between them.     

Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule‐degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non‐conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.     

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.     

Detection of Environmental Sources of Histoplasma capsulatum in Chiang Mai, Thailand, by Nested PCR

Histoplasmosis is a systemic mycosis caused by inhaling spores of Histoplasma capsulatum, a dimorphic fungus. This fungus grows in soil contaminated with bat and avian excreta. Each year, patients with disseminated histoplasmosis have been diagnosed in Chiang Mai, northern Thailand. No published information is currently available on the environmental sources of this fungus in Chiang Mai or anywhere else in Thailand. The aim of this study was to detect H. capsulatum in soil samples contaminated with bat guano and avian droppings by nested PCR. Two hundred and sixty-five samples were collected from the following three sources: soil contaminated with bat guano, 88 samples; soil contaminated with bird droppings, 86 samples; and soil contaminated with chicken droppings, 91 samples. Genomic DNA was directly extracted from each sample, and H. capsulatum was detected by nested PCR using a primer set specific to a gene encoding 100-kDa-like protein (HcI, HcII and HcIII, HcIV). Histoplasma capsulatum was detected in seven of 88 soil samples contaminated with bat guano, one of 21 soil samples contaminated with pigeon droppings and 10 of 91 soil samples contaminated with chicken droppings. The results indicate the possibility of the association of bat guano and chicken droppings with H. capsulatum in this area of Thailand.