Phylogenetic relationships ofCryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences

The genusCryptococcus was found to be heterogeneous on the basis of partial rRNA sequences. The human-pathogenic speciesC. neoformans, comprising 4 serotypes and havingFilobasidiella neoformans andF. bacillispora as teleomorphs, was found at a relatively large distance fromFilobasidium. Serotypes B and C had identical sequences, while in A and D they were different, with D closer to B and C than to A.Filobasidiella depauperata, which lacks a yeast-like anamorph, clustered withF. neoformans.The genusFilobasidium was clearly separated fromFilobasidiella and clustered withC. albidus, C. kuetzingii, C. gastricus, C. lupi, C. vishniaciae, C. bhutanensis, C. aerius, C. terreus andC. ater. The latter may represent the anamorph ofFilobasidium elegans. The organe to red species ofCryptococcus, as well asC. aquaticus andC. yarrowii, were found completely unrelated with these taxa,C. macerans being affiliated toCystofilobasidium capitatum.The genusTrichosporon was found relatively homogeneous; it includesC. humicola, C. curvatus and the filamentous speciesHyalodendron lignicola. Cryptococcus flavus andC. dimennae probably belong to the Tremellales, though distances between these species are large. The positions ofC. laurentii andC. luteolus remains to be determined.     

Un vivo depletion of murine CD8 positive T cells impairs survival during infection with a highly virulent strain ofCryptococcus neoformans

Cell-mediated immunity plays an important but incompletely understood role in host defense againstCryptococcus neoformans. Because of their multiple capacities as cytokine-secreting cells, cytotoxic cells, and antigen-specific suppressor cells, CD8 positive T lymphocytes could potentially either enhance or impair host defense againstC. neoformans. To determine whether CD8 T cells enhance or inhibit host defence during an infection with a highly virulent strain ofC. neoformans, we examined the effect of in vivo CD8 cell depletion on suNival and on the number of organisms in mice infected by either the intratracheal or intravenous routes. Adequacy of depletion was confirmed both phenotypically and functionally. Regardless of the route of infection, we found that survival of mice depleted of CD8 T cells was significantly reduced compared to undepleted mice. Surprisingly, however, CD8 depletion did not alter organism burden measured by quantitative CFU assay in mice infected by either route. These data demonstrate that CD8 positive T cells participate in the immune response to a highly virulent strain ofC. neoformans. By contrast to minimally virulent isolates that do not cause a life threatening infection, the immune response to a highly virulent isolate does not alter the burden of organisms, but does enhance host defense as it is necessary for the optimal survival of infected mice.Abbreviations ³H-TdR ³H-thmidine - CFU colony forming units - FITC Fluorescein isothiocyanate - MLR mixed lymphocyte reaction - PBS phosphate buffered saline     

Gastric colonization withCandida albicans

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

Ultrastructural study ofCryptococcus neoformans by quick-freezing and deep-etching method

The three-dimensional ultrastructure ofCryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method.C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10⁷ cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells ofC. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure ofC. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10–13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions byC. neoformans.     

Sero-systematics ofCytisus sect.Trianthocytisus (Fabaceae)

The serological reaction of seed proteins provides evidence for a partly new systematic arrangement ofCytisus sect.Trianthocytisus and ofCytisus s.l. Proposed modifications agree with recent advances in morphological taxonomy. Sect.Trianthocytisus includes only two species,C. villosus andC. aeolicus. Its position is central within the genus, and this fact agrees with the proposed retypification ofCytisus (type species:C. villosus).C. emeriflorus, formerly included in the same section, constitutes the monospecific sect.Emeroides, which is intermediate towards the genusLembotropis. This is serologically isolated and includes onlyL. nigricans. It is confirmed thatC. sessilifolius should be removed from the genusCytisus as a monospecific genus:Cytisophyllum Lang which is closely allied toHesperolaburnum and toPodocytisus, the most primitive genera ofGenisteae.     

The application of whole-cell protein electrophoresis for the classification and identification of basidiomycetous yeast species

The relationships among 65 basidiomycetous yeast strains were determined by one-dimensional electrophoresis of SDS-solubilized whole-cell proteins. Protein profiles were compared by the Pearson product moment correlation coefficient (r). The strains investigated represented species from the generaCystofilobasidium, Filobasidium, Filobasidiella, Kondoa, Leucosporidium, Mrakia andRhodosporidium. Except for the genusMrakia, all species constituted separate protein electrophoretic clusters. The species of the genusMrakia (M. frigida, M. gelida, M. nivalis andM. stokesii) show highly similar protein patterns, suggesting that these four species may be synonymous. Strains of two varieties ofFilobasidiella neoformans, F. neoformans var.neoformans andF. neoformans var.bacillispora, could not be differentiated by protein electrophoresis.For the delineation of the protein electrophoretic clusters of the yeasts studied, literature data relying on other criteria, such as DNA base composition, carbon source utilization patterns, enzymatic protein electrophoregrams, ubiquinone systems, DNA-DNA homology and rRNA sequence data were used. It was demonstrated that a database of SDS-protein patterns provides a valuable tool for the identification of yeasts.     

The passive cutaneous anaphylaxis (PCA) test in Cryptococcosis

Summary The PCA test appeared to be a useful indicator of past infection due toCryptococcus neoformans when the extracellular starch antigen was used. The whole cell antigen, crude cell wall antigen, ruptured cell antigen, crude carbohydrate antigen and the crude capsular polysaccharide antigen were not found to be effective.Serums from the nine rabbits injected withC. neoformans showed some degree of reactivity in the PCA test with the homologous extracellular starch antigen. Serums from rabbits injected withC. diffluens andC. laurentii gave no response.None of the rabbits exhibited evidence of previous sensitization and none were obviously infected. Six rabbits injected withC. neoformans were skin tested 4 weeks later with all of the antigens. Positive tests were obtained in four rabbits but only with the cell wall antigen or the ruptured cell antigen. PCA activity was not observed in the serums of mice injected with strains ofC. neoformans although some came from mice showing lesions and some came from fatally infected mice.Of the 500 human serums tested for PCA activity using the extracellular starch antigen, only one reacted strongly and with a sufficiently high titer to suggest previous infection. Serums from three cases of cryptococcosis failed to react.This investigation was supported by Communicable Disease Center grant CC 00151-01.The material in this paper is taken in part from a dissertation presented to the Graduate School of The University of Arizona in partial fullfilment of the requirements for the Ph.D. degree.     

Effect of amphotericin B on the lipids of five different strains ofCryptococcus neoformans

Cells of five strains ofCryptococcus neoformans were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin-layer chromatography and by the ultraviolet spectra. Such determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimal inhibitory concentration) levels. Marked alterations of the lipid and sterol contents were observed in the amphotericin B — treated cells. Moreover, ergosterol disappeared in these antibiotic-exposed cells. It is concluded that amphotericin B altered the lipid profiles, especially sterols ofC. neoformans.     

The occurrence of multinucleate giant cells in yeasts

Summary The spontaneous occurrence of giant cells has been observed in young cultures ofLipomyces lipofer and three different genotypes ofSaccharomyces. Stained preparations of all abnormal cultures revealed that the giant cells characteristically contained more than one nucleus, the number ranging from one to six. In both genera the phenomenon was found to be transient, for rapidlygrowing cultures arising from isolated giant cells reverted, at varying rates, to populations of small uninucleate cells which appeared normal in all respects.     

Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

Caprellids (Crustacea: Amphipoda) from Papua New Guinea,with the description of a new species

This is the first study of caprellid amphipods from the coast of Papua New Guinea. Several collections from Madang Lagoon (north) and Bootless Bay (south) have been studied. Seven species in seven genera are recorded, of which Pseudoproto papua sp. nov. is described as new to science. The genus Pseudoproto Mayer, 1903 has consisted, so far, of only one species, Pseudoproto fallax Mayer, 1903. Although only a single male has been found of Pseudoproto papua sp. nov., differences in antennae, mouthparts, gnathopod 2 and pereopods 3 and 4 have revealed that it represents a new species of Pseudoproto. Lateral view figures of all species, together with a key to species level for the Caprellidea from Papua New Guinea are also included. Communicated by H.-D. Franke     

The Growth ofSaccharomycopsis Guttulata

Summary A medium containing an extract of rabbit stomach contents for the growth ofSaccharomycopsis guttulata (Robin) Schi?nning is described. Growth characteristics and tentative fermentation and carbon assimilation patterns are reported. Nitrate assimilation could not be determined with the media available. Sporulation has been observed on artificial media. The growth promoting substance is heat stable and non dialysable, it appears to be destroyed by precipitation of protein from the active extract solution. The possibility of transferringS. guttulata to the genusSaccharomyces and discarding the genusSaccharomycopsis is discussed. A culture ofSaccharomycopsis guttulata has been deposited at the Centraalbureau voor Schimmelcultures, Delft.     

Growth of Cryptococcus neoformans in a thiamine-free medium

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 g/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.     

Four new species of yeast isolated from insect frass in bark ofTsuga heterophylla (Raf.) Sargent

Summary In a study of the yeasts associated with insect frass underneath the bark ofTsuga heterophylla (the Pacific Coast hemlock) four new species of yeast were found. These were described asSporobolomyces singularis, Bullera tsugae, Cryptococcus skinneri andCandida oregonensis. Sporobolomyces singularis is a non-pigmented species, which required an amendment of the genus definition. Ballistospore formation of the new species ofSporobolomyces and ofBullera was absent on malt agar and on potato glucose agar, but positive on corn meal agar. An unusual case of quantitative transgalactosylation by growing cells ofSporobolomyces singularis on lactose has been described. It has been proposed to establish a “Candida parapsilosis Group” of species to whichC. oregonensis was assigned. Supported by a fellowship of the Calouste Gulbenkian Foundation, Lisbon, Portugal.     

Polyamine depletion and growth inhibition ofCryptococcus neoformans by α-difluoromethylornithine and cyclohexylamine

The ability of two known inhibitors of polyamine synthesis,-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and cyclohexylamine, an inhibitor of spermidine synthase, to inhibit thein vitro growth and polyamine synthesis of clinical isolates ofCryptococcus neoformans was examined. Treatment ofC. neoformans with either DFMO or cyclohexylamine resulted in depletion of cellular polyamines and inhibition of growth.Cryptococcus neoformans was shown to lack detectable spermine and to require high concentrations of spermidine, but not putrescine, for growth. The growth inhibition by DFMO and cyclohexylamine was reversed by exogenous polyamines. These findings document the ability of cyclohexylamine and DFMO to inhibit polyamine synthesis and growth in clinically important isolates ofC. neoformans.     

The flagellar root system of zoospores of the green algaChlorosarcinopsis (Chlorosarcinales) as compared withChlamydomonas (Volvocales)

The flagellar root system of zoospores in two species ofChlorosarcinopsis (C. minuta andC. spec.) has been studied in detail. The biflagellate zoospores show a cruciate root system, two of the four microtubular roots containing two microtubules, the other two four microtubules. The flagellar apparatus is otherwise identical with that ofChlamydomonas reinhardi as described byRingo (1967). Evidence is presented that the genusChlamydomonas is characterized by a bilateral symmetric root system (4-2-4-2) rather than a system with four equally numbered roots (i.e. 4-4-4-4). It is suggested that a root system with four identical cruciate roots is not present in any biflagellate algal cell. The taxonomic significance of cruciate root systems in green algae is discussed refering to the identical root systems ofChlorosarcinopsis andChlamydomonas.     

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M _r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations M _r apparent molecular mass - Da dalton - Ig immunoglobulins - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - 2-D gel two-dimensional gel electrophoresis - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.