A survey of zoo aviaries for the presence of Histoplasma capsulatum and Cryptococcus neoformans

A zoo's aviaries were surveyed for the presence ofH. capsulatum andC. neoformans. Dried feces samples were collected in the 14 aviaries and tested forC. neoformans by using direct Cultural methods. This pathogen was isolated from an indoor aviary housing one White-breasted Toucan, and from another indoor aviary housing two Concave-casqued Hornbills. Soil samples were collected in 12 outdoor aviaries and two other areas of the zoo premises and tested forH. capsulatum andC. neoformans by direct and indirect cultural methods. Neither fungal pathogen was isolated from the soil samples. Fresh feces samples were collected from the two aviaries whereC. neoformans had been isolated in dried feces. Tests for the presence ofC. neoformans were made by direct culture methods, however the organism was not isolated.Contribution No. 154, Department of Infectious Diseases, College of Veterinary Medicine, Agricultural Experiment Station, Manhattan, 66502.     

The possible role of uric acid in the ecology of Histoplasma capsulatum

Summary The results of this study indicate thatHistoplasma capsulatum in its saprophytic form is able to utilize the major nitrogenous constituent of avian manure as a nitrogen source. In addition, the enzymes responsible for the pathway of uric acid degradation to inorganic nitrogen have been demonstrated in cell-free systems. These enzymes include uricase, allantoinase, allantoicase, and urease. The uricase ofHistoplasma appears to be a cell wall or cell membrane-associated enzyme, while the other enzymes were located in the soluble portion of cell-free extracts. Cell-free extracts ofCryptococcus neoformans are actively uricolytic.It is suggested that this ability ofH. capsulatum hyphae to utilize uric acid and related compounds as growth substrates may in part explain the indisputable ecologic association of this pathogenic fungus with avian and possibly chiropteran-associated soils and habitats in those areas endemic for histoplasmosis.From the Research Laboratories, Veterans Administration Hospital, Kansas City, Missouri, the Department of Biology, University of Missouri at Kansas City and the Department of Microbiology, University of Kansas School of Medicine, Kansas City, Kansas. Supported by VA-8200 funds.Portion of a Thesis presented by the senior author to the Graduate Faculty of the University of Missouri at Kansas City as partial fulfillment of the requirements for the Degree of Master of Arts.     

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.     

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.     

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.     

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.     

Isolation of fungi from judean desert soil

Summary 1. Results of culture of thirty soil samples obtained from the Judean Desert on the western side of the Dead Sea are reported. These soil samples were obtained from caves found in the walls of the cliff leading to the plateau Massada, the level of the caves varying from sea level to 300 feet below.2. Two strains ofCryptococcus neoformans were recovered from soil obtained from a bat cave 300 feet below the top of the plateau.3. No evidence ofHistoplasma capsulatum orCoccidioides immitis was found.4. This evidence, coupled with negative skin test data reported previously, implies thatHistoplasma capsulatum andCoccidioides immitis are probably not inhabitants of soil in this part of Israel.     

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.     

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.     

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.     

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.     

Additional studies of histoplasmin formation

Summary Culture filtrates of 20 strains ofHistoplasma capsulatum were studied to determine the effect of certain growth conditions on histoplasmin formation. The presence of histoplasmin was denoted by an antigenic titer of 1:4 or higher with the complement fixation test.The data indicated that, in addition to verifying that the strain used affected histoplasmin formation, the morphological condition of the inoculum was extremely important. It was found that most strains which converted readily to the yeast phase at 37° C produced histoplasmin poorly. Tests with different volumes of media also showed that 500 ml volumes of culture media produced histoplasmin with higher titers than 3 liter volumes when cultured at 25° C for six months.Some additional histoplasmin could be liberated by sonification of the mycelial pad from culture filtrates which contained histoplasmin. A few strains produced high titer histoplasmin by the shake method if incubated for three months, but they had low titers after only six weeks.Complement fixation tests with sera from proven cases of histoplasmosis indicated that histoplasmin from a single strain ofH. capsulatum can give identical results with those obtained with histoplasmin from a pool ofH. capsulatum strains if H and M antigen components are present.     

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.     

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.     

Preparation and standardization of antigens of Histoplasma capsulatum and Coccidioides immitis

Methods have been developed for the separation and purification of various antigenically active cellular components ofH. capsulatum (34) andC. immitis (35), and chemical procedures have been employed in the characterization of these antigens. The concluding remarks illustrate the current trends in immunological studies ofH. capsulatum andC. immitis. Hopefully, these approaches will provide knowledge and insight into the basic biological properties ofH. capsulatum andC. immitis and should, in the future, culminate in the physicochemical characterization of their important antigens.     

Relation of the pigeon to cryptococcosis: Natural carrier state,heat resistance and survival of Cryptococcus neoformans

Summary The feral pigeon in New York City was found to serve as a mechanical carrier of pathogenic strains ofCryptococcus neoformans. Of 94 feral pigeons freshly trapped in the city, 7 were found to carryC. neoformans on their beaks and feet, while their rectal swabs were negative. Following crop instillation ofC. neoformans in 3 feral pigeons, the fungus survived passage through the gastrointestinal tract and appeared in the fresh feces within one hour after inoculation and was still present 24 hours later. The internal body (rectal) temperatures of 57 feral pigeons recorded soon after capture in two seasons of the year, ranged from 41.5° C to 43.3° C and averaged 42.5° ± 0.39° C. The birds were able to maintain their high temperatures in the face of sustained cold, indicating the presence of a strong thermoregulating mechanism. A study of the growth and survival of 60 human strains ofC. neoformans on Sabouraud dextrose yeast extract agar slants revealed that 100 % of the cultures were able to grow at 39° C, 92 % at 40° C, 30 % at 41° C, 17 % at 42° C and 8 % at 43° C. Despite exposure to these temperatures for 7 days, 100 % of the human strains survived at 40° C, 97 % at 41° C, 95 % at 42° C, 91 % at 43° C, 88 % at 44° C, 47 % at 45° C and none at 47° C. Seventy strains ofC. neoformans of pigeon excreta origin showed a similar pattern of heat resistance, except that 11 % of these strains survived 47° C. The inability ofC. neoformans to grow at 44° C, a property shared by all 130 strains, provides a new species characteristic.C. neoformans was found to multiply rapidly in moist pigeon excreta extract, reaching counts up to 60 million per ml within 3 weeks, and to be still viable in moist as well as dessicated pigeon excreta extract after more than two years of storage at room temperature.Presented in part at the 26th VA-Armed Forces Pulmonary Disease Research Conference, Cleveland, Ohio, Jan. 23–26, 1967, and the 67th Annual Meeting American Society for Microbiology, New York, April 30–May 4, 1967. Supported by Grants GB-4722 from the National Science Foundation and U-1367 from The Health Research Council of the City of New York.Acknowledgment is gratefully given toThomas J. Dalton, Division of Environmental Sanitation, Department of Health, New York City andMabel Halloran for their valuable assistance and to the ASPCA of Manhattan and Brooklyn for their cooperation in this study.     

Ultrastructure of the motile cells of the prostrate filamentous green algaeProtoderma sarcinoidea andChamaetrichon capsulatum

The biflagellate zoospores ofProtoderma sarcinoidea and the quadriflagellate zoospores ofChamaetrichon capsulatum are each covered by an amorphous, mucous material and a single layer of square scales, and the pyrenoid matrix is traversed by one or more thylakoid membranes. In the flagellar apparatus the basal bodies ofP. sarcinoidea and the upper basal bodies ofC. capsulatum are displaced in the counterclockwise absolute orientation, while the lower basal bodies ofC. capsulatum are directly opposed. Other components of the flagellar apparatus observed in each alga include: cruciately arranged d and s rootlets, each associated with an electron-dense component; simple terminal caps comprised of large and small subunits; a terminal electron-dense mass located near the proximal end of each basal body inP. sarcinoidea and near the upper basal bodies inC. capsulatum; and two rhizoplasts. Components specific to one or the other species include a single accessory basal body inP. sarcinoidea and a fibrous, electron-opaque band that links the upper and the lower basal bodies inC. capsulatum. The flagellar apparatus architecture ofP. sarcinoidea resemblesGayralia oxysperma, while that ofC. capsulatum is similar toTrichosarcina polymorphum andUlothrix species, all of which are included in theUlothrix-group,Ulotrichales, Ulvophyceae.     

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.     

Inhibition of Histoplasma capsulatum by Garlic

The mycelial phase ofHistoplasma capsulatum was inhibited by both the volatile and water soluble components of garlic,Allium sativum L. Garlic extract at a concentration of 254 parts per billion (ppb) was inhibitory, while 8.1 parts per million (ppm) were lethal to pure cultures ofH. capsulatum. The role of garlic as an eradicent is discussed.The work was conducted while the author was a graduate student at the University of Kentucky, Lexington, Kentucky.     

Ultrastructural changes produced by ketoconazole in the yeast-like phase of Paracoccidioides brasiliensis and Histoplasma capsulatum

The ultrastructural changes produced by ketoconazole on the yeast-phase ofH. capsulatum andP. brasiliensis were studied by means of scanning and transmission electron microscopy.The observed alterations on both fungi were very similar to those induced by the same drug on the ultrastructure ofC. albicans. These alterations include surface changes, abnormal membrane proliferation, fatty degeneration of the cytoplasm and lysis of subcellular organelles. P. brasiliensis seems to be more sensitive to ketoconazole thanH. capsulatum, since the necrosis of most of the cells was obtained in the former at a concentration of 0.1 gmg/ml and in the latter at 1 g/ml.