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.     

Rapid diagnosis of Candida albicans

The inhibitory action of an antibiotic isolated fromB. subtilis is studied onC. albicans, C. clausenii, C. langeronii, C. stellatoidea, andC. visvanathii, as a possible means forC. albicans rapid identification. Growth inhibition ofC. albicans by the antibiotic was observed when the yeast was preincubated on malt-agar slants, at 30° C, during 24 hours, and tested afterwards for antibiosis on Sabouraud-agar plates. No growth inhibition could be detected withC. clausenii, C. langeronii, C. stellatoidea, andC. viswanathii, under the same experimental conditions.In support of our results are the data reported byPérez-Silva &Gil-Alvarez (1960), where 33 strains ofC. albicans tested against that antibiotic, were shown to be the only sensitive yeast amongst 68 strains of yeast from several species.

---

Zusammenfassung Die Wirkung der Inhibition eines vomB. subtilis isolierten Antibiotiks wurde untersucht anC. albicans, C. clausenii, C. langeronii, C. stellatoides, undC. viswanathii als ein mögliches Mittel für die schnelle Bestimmung vonC. albicans. Wachstumsinhibition vonC. albicans durch das Antibiotik war beobachtet, wenn die Hefe an Maltose-Schrägagar vorher inokuliert, bei 30° C für 24 Std., und nachher für Antibiosis an Sabouraud Agarplatten untersucht worden ist. Keine Inhibitionswirkung konnte mitC. clausenii, C. langeronii, C. stellatoides undC. viswanathii unter den selben experimentellen Bedingungen beobachtet werden. Die Ergebnisse sind durch diejenigen vonPérez-Silva &Gil-Alvarez (1960) unterstützt, in denen 33 Stämme vonC. albicans gegen das Antibiotik getested und gezeigt worden sind, daß nurC. albicans die einzige empfindliche Hefe unter 68 Stämmen von Hefen verschiedener Arten war.

     

Identification of pathogenic fungi in surgical and autopsy specimens by immunofluorescence

Summary A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.     

A comparison of some Ceratocystis species with sporothrix schenckii

Sympodulosporogenous states ofC. minor, C. montia, C. multiannulata, C. narcissi, C. nigrocarpa, C. perparvispora andC. pilifera were compared with typical and atypical strains ofS. schenckii from the aspects of some culture, morphological, serological characteristics, and mouse virulence. With a possible exception, all the former characteristics demonstrated among theCeratocystis species were either the same as, or varied in the same way as those observed amongS. schenckii strains. None of the former hydrolyzed starch, although all of the latter did. All species and strains cross reacted serologically in agglutination and Arthus-rype reactions, and produced similar gross pathological signs when injected with gastric mucin intraperitoneally into mice. Except for some variations in sizes and shapes of sympodulospores and of yeast-like budding forms observed in tissue smears and in cultures incubated at 37°C, theCeratocystis species were not significantly different from typical strains ofS. schenckii.

---

Zusammenfassung Der sympodulosporogene Zustand vonC. minor, C. montia, C. multiannulata, C. narcissi, C. nigrocarpa, C. perparvispora undC. pilifera wurde mit typischen und atypischen Stämmen vonS. schenckii betreffs kultureller, morphologischer, serologischer Charakteristik und auf Virulenz für Mäuse untersucht. Mit einer möglichen Ausnahme waren alle Charakteristiken unter denCeratocystis-Arten dieselben oder sie wechselten wie diejenigen unter den Stämmen vonS. schenckii. Keine Stämme derCeratocystis hydrolysierten Stärke, während die Stämme vonS. schenckii getan haben. Alle die Arten und Stämme zeigten Kreuzreaktionen serologisch und in der Arthus-reaktion und zeigten dieselben groß-pathologischen Veränderungen nach intraperitonealen Injektionen mit Magenmuzin in der Maus. Außer etlicher Abwechslungen in Größe und Gestalt der Sympodulosporen und der hefe-ähnlichen Keimung, die in Gewebeausstrichen und in Kulturen bei 37°C beobachtet worden sind, waren dieCeratocystis-Arten nicht wesentlich unterschiedlich von typischen Stämmen vonS. schenckii.

---

---

The author, Dr.R. W. Davidson (Colorado State University, Ft. Collins, Colorado), and Dr.F. Mariat (Institut Pasteur, Paris) have, in collaboration, identified the ascigerous state ofSporothrix schenckii strain G-118 isolated by the latter to beCeratocystis stenoceras (Robak)C. Moreau.     

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.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.     

Serological cross-reactivity between sporothrix schenckii and various unrelated fungi

The serological cross-reactivity ofSporothrix schenckii with various unrelated fungi was investigated by use of immunodiffusion tests. A rabbit antiS. schenckii serum was obtained, which reacted withCladosporium werneckii, C. carrionii, C. bantianum, Coccidioides immitis, Phialophora jeanselmei, P. gougerotii, P. dermatitidis, Fonsecaea pedrosoi, Aspergillus fumigatus, Histoplasma capsulatum andTrichophyton mentagrophytes, but not withSaccharomyces cerevisiae antigens. The serological determinants responsible for the cross-reactions were suggested to be D-galactosyl residues.     

Effects of surface active agents on the susceptibility of Swiss mice to Candida albicans

Summary The LD50's ofCandida albicans combined with Plurafac B26, Mulsor 224, Pluronic L62, and Polyethylene Glycol 400 Mono Laurate injected intraperitoneally into female white Swiss mice were calculated by the method ofReed &Muench. Each of the previously mentioned surface-active agent —Candida albicans combinations had a lower LD50 than theCandida albicans control.Plurafac B26 was found to intially decrease the number of leukocytes in the peritoneal cavity and thus enhance the invasiveness ofCandida albicans.     

The effect of freezing and the influence of isolation medium on the recovery of pathogenic fungi from sputum

The primary objective of this study was to determine whether freezing sputa in dry ice had any effect on the recovery of pathogenic fungi. Sputa seeded with each of five fungi (Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Coccidioides immitis, and Aspergillus fumigatus) were frozen and stored for 24, 48, and 72 hours on dry ice. H. capsulatum was killed, and only a few colonies of B. dermatitidis and C. neoformans were isolated from these sputa. However, A. fumigatus and C. immitis withstood the effects of freezing. A second objective was to compare the recovery of all five fungi from seeded sputa stored at room temperature for 24, 48, and 72 hours, on yeast extract-phosphate agar with NH₄OH and on Sabhi agar. The yeast extract-phosphate agar with NH₄OH was superior to Sabhi agar, for the isolation of all fungi studied, except A. fumigatus.     

Untersuchungen über Antigengemeinschaften zwischen hefeartigen Pilzen

Zusammenfassung An einer willkürlichen Auswahl von Stämmen aus den GeneraCandida, Torulopsis, Saccharomyces undSchizosaccharomyces konnten mit Hilfe der Immunelektrophorese außer bekannten Beziehungen der Polysaccharidantigene noch einige gemeinsame Eiweißantigene nachgewiesen werden.Übereinstimmungen bestehen mitTsuchiya in Bezug auf die enge Verwandtschaft vonC. albicans, C. tropicalis undC. stellatoidea, sowie zwischenS. cerevisiae undC. robusta.Bei Berücksichtigung aller Antigene fanden wir zusätzlich Antigengemeinschaften zwischenT. glabrata undS. rouxii, sowie weitere antigene Beziehungen beiC. krusei, C. utilis, T. famata undS. cerevisiae, die auf gemeinsamen Eiweißantigenen beruhen.Es wurde der Versuch unternommen, einige Eiweißantigene ausC. albicans zu isolieren. Nach Fällung des größten Teils der Antigene mit Universalpuffer verblieben im Rückstand 2 Eiweißantigene, mit denen Kaninchen immunisiert wurden. Von den beiden induzierten Antikörpern gegen Eiweißantigen vonC. albicans reagierte der eine nur mitC. tropicalis, der andere nur mitC. stellatoidea, jedoch nicht mit anderen Hefen.

---

Summary The serologic relationships of various yeast strains, arbitrarily selected from the generaCandida, Torulopsis, Saccharomyces, andSchizosaccharomyces, were investigated by means of immunoelectrophoretic technique. In addition to serologic crossings ofC. albicans, C. tropicalis, andC. stellatoidea as well as ofS. cerevisiae, andC. robusta, which had previously been reported byTsuchiya and other investigators, we have found, that protein antigens are shared by various strains, i.e.C. krusei, C. utilis, T. famata, andS. cerevisiae. Taking into account the overall picture of their antigenic patterns,T. glabrata andS. rouxii were found to stand particulary close to each other.Attempts were made to isolate some of these proteide antigens fromC. albicans. After partial precipitation of the crude homogenate by the addition ofJohnson &Lindsay's Universal Buffer followed by centrifugation, the supernatant was used to immunize rabbits. Two types of antibodies could be detected; while both reacted in the immunoelectrophoresis withC. albicans antigens, one also precipitatedC. tropicalis and the otherC. stellatoidea. None of the 10 other yeasts which have been tested showed any activity with these rabbit sera.

     

A combination rapid and standard method for identification of Candida albicans

A medium consisting of an aqueous extract of zein, lactose, and Tween 80 is used together with an overlay of 1 % Tween 80 and coverslipping to provide a combined rapid (germ tube) and standard (chlamydospore) method for diagnosis ofCandida albicans. The method is exquisitely sensitive for diagnosis ofC. albicans but lumps chlamydospore-producing strains ofC. tropicalis withC. albicans.     

Vegetation history in the Eastern Romanian Carpathians: pollen analysis of two sequences from the Mohoş crater

Two sequences of about 10.5 m originating from a peat bog in Romania were analysed for pollen (202 and 127 pollen spectra). The vegetation history, supported by 24 ¹⁴C dates is described since the Late Glacial. At the onset of the Holocene Ulmus first appears, together with Betula. Among the main components of the Quercetum mixtum (Quercus, Fraxinus, Tilia, Corylus) that became established almost simultaneously by around 9000 B.P., Quercus frequencies rarely exceed 10%. The local establishment of Carpinus is about 6000 b.p. Its maximum occurred between 4500 and 3000 b.p. Fagus pollen is regularly recorded since 8000 b.p. Its absolute dominance tooks place at about 3000 b.p. Picea pollen is present since the Late Glacial. The first indications of human activities appear at around 6500 b.p.     

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.     

Epidemiological investigations of oral Candida Albicans

Using a gargle-rinse technique, the oral cavities of 103 volunteers were sampled and cultured for the presence ofCandida albicans. Thirty-six (33.95 %) were positive forC. albicans, including 14 females and 22 males. Sixty-four subjects, including negative controls, were placed on treatment regimes of a pre-sleep gargle-rinse with either sterile distilled water (W) or Cepacol® Mouthwash/Gargle (C). The possible effects of ambient temperature, diet, age, sex, and mouthwash use on oralC. albicans levels are illustrated and discussed, including some evidence for familial endemicity. On simulated sporadic or continuous mouthwash use, some individuals showed statistically significant reductions in oralC. albicans flora, whereas others had biologically significant reductions that were not confirmed statistically. A few originally negative individuals developed non-persistent lowC. albicans counts on one or two days. Total bacterial counts were made for 32 subjects, for most of whom biologically significant reductions were obtained, although the counts were highly variable and erratic. The data support the concept that a reduction in oralC. albicans does not lead to an increase in total bacterial flora, and vice versa.with the technical assistance ofAlyce R. Schmitt Paper 741, Department of Botany, The Ohio State University. This investigation was supported by a research grant form the Wm. S. Merrell Co., Cinninnati, O.     

The genus periconia from india

Summary Totally 20 species ofPericonia have been studied, of whichP. ellisii, P. hyderabadensis, P. indica, P. masonii, P. pseudohispidula, P. scrobiculata, P. simplex, P. stilbacearum, P. torulosa, P. warticulata, andP. yadagirensis are new, remaining reported for the first time from these parts. The discussion includes comments on the distribution, nature of colonies, fertility of stipes, their arrangement in colonies and its application in taxonomy. Variation in sporoderm is treated.P. parasitica Tilak,P. ipomoeae &P. xanthicola Vasant Rao are treated conspecific withP. byssoides Pers. exSchweinitz, pending the establishment of their parasitism. An improvised and comprehensive key for 28 species ofPericonia known so far from India is proposed.     

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).     

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     

Inhibition of yeast-mycelium transformation by 2-alkylthio-6-amino-and 2-alkylthio-6-formamidobenzothiazoles and theirin vitro antifungal activity

The antifungal effect of 2-alkylthio-6-amino-and 2-alkylthio-6-formamidobenzothiazoles (22 derivatives in all) onAspergillus niger and variousCandida yeasts was tested. No significant effect was observed withA. niger. With the pathogenic yeast speciesC. albicans andC. guilliermondii, the most efficient derivatives (6-formamido-2-propylthio-, 6-formamido-2-butylthio-, 6-formamido-2-pentylthio-and 6-formamido-2-isopentylthiobenzothiazole) exhibited ED₅₀ values in the range from 2.2 to 21 mg/L and were thus 3–35 times more efficient than 2-mercaptobenzothiazole (Dermacid) that is normally used. 6-Amino-2-pentylthiobenzothiazole was found to be an efficient specific inhibitor of the transformation of the yeast form ofC. albicans to its mycelial one, IC_95(M) being 10 mg/L, which was a concentration 25 times lower than MIC_Y+M.     

Occurrence of atypical lymphocytes following intravenous injection of Candida albicans in mice

The occurrence of atypical lymphocytes has been observed in the course of experimental acute infection withCandida albicans in mice. In animals injected intravenously with 2.5 × 10⁶ ofCandida albicans cells, an increased number of monocytes was seen in 24 hours. Monocytes showed toxic vacuolisation in most instances in protoplasm and sometimes in the nuclei. Only a few atypical lymphocytes could be seen at that time. In the following days the number of monocytes diminished and the number of atypical lymphocytes increased. After four days atypical lymphocytes constituted frequently over 20 % of white cells. The autopsy of sacrificed or dead animals with the presence of such elevated percentages of atypical lymphocytes showed enlargement of cervical lymphnodes in all animals. In mice infected with 1.4 × 10³ ofCandida albicans cells, no level higher than 12% of atypical lymphocytes was seen. Pictures were returning to normal with only a few atypical lymphocytes present among the animals which survived for two months after infection withCandida albicans.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper. Memorial Funds and the Roon Foundation.     

Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.