Application of flow cytometry to differentiating Exophiala dermatitidis E. moniliae and E. jeanselmei from each other

We applied a flow cytometry apparatus (FCM) to differenciating Exophiala dermatitidis, E. moniliae and E. jeanselmei from each other. The wavelength of the argon laser emitted from the FCM was 488 nm and the aperture of nozzle from which the stream of fluid containing single cells was blown out was 100 m. By irradiating the stream with laser by either the forward light scatter (FLS) or by the perpendicular light scattr (PLS), we were able to get two pieces of informations. Histograms displayed by the FLS indicate the cell size, while dot displays by the PLS reflect the cell structure. As a result, E. dermatitidis was clearly differenciated from either E. moniliae or E. jeanselmei by their histograms by FLS. In addition, dot displays by the PLS differenciated E. moniliae from E. jeanselmei.In conclusion, flow cytometry is available for differenciating E. dermatitidis, E. moniliae and E. jeanselmei from each other.     

Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind. III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 15 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.     

Mitochondrial DNA analysis ofExophiala moniliae

Mitochondrial DNA(mtDNA) analysis with restriction enzymes, Hae III, Hind III and Msp I was performed in 17Exophiala moniliae strains. The results were as follows: (1)E. moniliae could be classified into 10 types based on restriction patterns, (2)E. moniliae is suggested to be a complex organism because of extensive mtDNA polymorphism among strains likeE. jeanselmei and (3) two types ofE. moniliae are identical with two types ofE. jeanselmei. These results suggest thatE. moniliae is not genetically defined fromE. jeanselmei and the taxonomical status ofE. moniliae requires reevaluation     

Degradation of monohydroxylated benzoates by strain KUFI-6N of the yeast-like fungus Exophiala jeanselmei

Strain KUFI-6N of Exophiala jeanselmei, a cyclohexanol-utilizing yeast-like fungus, was found to grow on 3 isomers of hydroxybenzoate that functioned as the sole carbon sources. Distinct and highly specific hydroxylases converted p- and m-hydroxybenzoate to protocatechuate and o-hydroxybenzoate to catechol.     

Invasion of a soft contact lens by Exophiala jeanselmei

The invasion of a soft contact lens by Exophiala jeanselmei is documented. All species in this genus are pathogenic. In humans E. jeanselmei is a recognized cause of mycetoma, phaeohyphomycosis and keratomycosis. This fungus has not been previously listed among lens invaders.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

Pleoanamorphic life cycle ofExophiala (Wangiella) dermatitidis

The anamorph life cycle of the black yeastExophiala (Wangiella) dermatitidis is described. The fungus is dimorphic, yeast cells being the prevalent form of propagation. The fungus is strongly hydrophilic, probably completing its anamorph life cycle in submersion. Adaptation to dry conditions is slow. Types of conidiogenesis comprise annellidic, phialidic and sympodial reproduction, in addition to isotropic development. Phialoconidia fail to germinate under the conditions tested, and thus may have a function other than dispersal. Sterile, multicellular bodies resembling aCapronia teleomorph are described.     

Mitochondrial DNA analysis of Exophiala jeanselmei var. lecanii-corni and Exophiala castellanii

A Japanese clinical isolate (KU-A-0094) which was identified by de Hoog et al. as Exophiala jeanselmei var. lecanii-corni with difficulty, was compared with 5 strains including the type cultures of E. jeanselmei var. lecanii-corni, var. jeanselmei and E. castellanii using RFLP (restriction fragment length polymorphism) patterns of mtDNA (mitochondrial DNA). RFLP patterns of KUA-0094 were identical with those of E. jeanselmei var. lecanii-corni and different from those of E. castellanii with restriction enzymes of HaeIII, MspI and hindIII. Therefore, de Hoog et al.'s identification of KU-A-0094 was confirmed. Additionally, mtDNA-RFLP patterns of E. jeanselmei var. lecanii-corni and E. jeanselmei var. jeanselmei were also different from each other. Consequently E. jeanselmei var. lecanii-corni seem to be a species in its own right rather than a variant of E. jeanselmei. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fungi isolated from skins and pens of healthy animals in Nigeria

The mycoflora of 220 skin scrapings, hair, nail samples and pens’ materials of apparently healthy animals including cows, sheep, goats, rabbits, pigs and dogs were determined. Twenty eight species of fungi belonging to ten genera were recovered. Chrysosporium spp. were the most common and C. keratinophilum was recovered from all animals. Dermatophytes which are known causal agents of dermatophytosis were also isolated indifferent frequencies (Microsporum gypseum, M. canis, Trichophytonmentagrophytes, T. rubrum). This revised version was published online in June 2006 with corrections to the Cover Date.     

An outbreak of blastomycosis in Eastern Tennessee

Most cases of blastomycosis are sporadic and only nine outbreaks representing a total of 112 cases have previously been reported. Less than half of these have been culture proven cases. Outbreaks have previously occurred in North Carolina, Minnesota, Illinois, Wisconsin and Virginia. We report three culturally confirmed cases of blastomycosis from Elizabethton, Tennessee, who had onset of illness within a one-week span of time. The patients presented with fever, chest pain, weight loss, poor appetite and myalgia. Each initially had a dry cough which became productive of purulent sputum as the illness progressed. Mild hemoptysis occurred during each patient's course. Serologic testing by immunodiffusion and enzyme immunoassay were positive and testing by complement fixation was negative in each case. The diagnosis was made by histopathology on transbronchial biopsy or transthoracic needle aspiration material. Each patient improved on ketoconazole therapy.     

Cytologic findings in 43 cases of blastomycosis diagnosed ante-mortem in naturally-infected dogs

A retrospective study of the cytology of blastomycosis was undertaken. Sixty-one samples from 43 naturally infected dogs diagnosedante-mortem by means of cytology were reviewed. Skin and lymph nodes rendered the highest number of positive samples with 17 out of 18 and 14 out of 17 respectively. Transtracheal washes contained the tissue forms ofBlastomyces dermatitidis in 3 out of 5 samples. Pyogranulomatous inflammation was diagnosed in 38 of the 61 samples, and of these, 25 cases contained multinucleated giant cells. Epithelioid cells were found in all 38 cases. Purulent inflammation was seen in 15 out of 61 samples. Three cases had a minimal or virtual lack of inflammation. The cytologic findings are described and photographically illustrated.     

Frequency and Diversity of Fungi Colonizing Tissues of Upland Cotton

A study of the mycoflora of upland cotton in Alabama was conducted throughout the 2000 and 2001 growing seasons. Plants were sampled at seedling, first bloom, full bloom, and maturity stages of development. Thirty-seven genera representing 58 species of fungi were isolated, including 9 species of Fusarium. Fusarium oxysporum, F. solani, and F. equiseti were the most common members of this genus occurring at all four sampling stages in both years. Eight species accounted for 67% of the total fungi isolated during the two-year study. Alternaria alternata was the most common fungus encountered, accounting for 19 and 10% of the total fungi isolated in 2000 and 2001, respectively. Twenty species of fungi are reported for the first time colonizing upland cotton tissues.     

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

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

Studies on the molecular ecology of Blastomyces dermatitidis

The microecology of Blastomyces dermatitidis, the dimorphic etiologic agentof the potentially fatal systemic fungal infection, blastomycosis, is not well defined.Blastomyces dermatitidis may occur periodically at natural sites, perhaps aided by rotting organic material, animal droppings and physical changes. Semi-quantitative growth studies of B. dermatitidis on 2% agar plates determined the ability toutilize or tolerate a variety of substrates including simple and complex molecules as carbon source, and organic and inorganic nitrogen sources. Allantoin, creatinine, quanidoacetic acid, guanidine and cysteine may be used as sole nitrogen source. Allantoin in combination with dextrose, glycerol, lichenen, celloboise and other wood by-products support growth of B. dermatitidis at room temperature. The nutritional conversion of the fungus to the yeast form at room temperature, well demonstrated on allantoin/glycerol/yeast extract media, appears to be affected by certain inorganic compounds. The organism tolerates low to moderate levels of alpha-pinene, tannic acid, shikimic acid, veratryl alcohol, vanillic acid, and polyethyleneglycol-200. There are significant differences among isolates regarding growth on various substances at 20° and 37° centigrade. It appears that a variety of wood by-products and animal waste substrates, in combination, support the growth of B. dermatitidis. Their role in the ecological niche of B. dermatitidis, and the importance of nutritional dimorphism in the natural environment warrants further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Clinical Characteristics and Outcomes in Patients with Pulmonary Blastomycosis

Background  Blastomycosis is an uncommon granulomatous infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. The most frequent clinical infections involve the lung, skin, and bone. Pulmonary manifestations range from asymptomatic self-limited infection to severe diffuse pneumonia causing respiratory failure. Objectives  To establish the clinical characteristics and outcomes of patients with pulmonary blastomycosis diagnosed at hospitals in Manitoba and northwestern Ontario, Canada. Methods  A retrospective review of medical records was done for 318 patients with blastomycosis in these regions. Results  The majority of patients were Caucasian (198 (62.5%) patients), male (193 (61%) patients), and residents of Ontario (209 (65.7%) patients). Most patients were treated in an inpatient hospital ward (266 (84%) patients) and survived (294 (92%) patients). Pulmonary involvement, either alone or associated with other sites, was present in 296 (93%) of the 318 patients; 22 (7%) patients had no evidence of pulmonary blastomycosis. The majority of patients had localized lung disease (1–3 quadrants on chest radiograph involved; 225 (82%) patients). Of 294 (92%) patients requiring hospitalization, 266 (90%) patients received all inpatient care on a general medical ward; 28 (10%) patients received some care in the intensive care unit (ICU). Factors associated with ICU admission included diffuse pulmonary disease (four quadrants involved on chest radiograph), diabetes, and prior use of antimicrobial therapy. Twenty-four (8%) patients died, and multivariate analysis showed that older age and Aboriginal ethnicity were the significant risk factors for death from blastomycosis. Conclusion  Blastomycosis is a cause of serious, potentially life-threatening pulmonary infection in this geographic region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fungi associated with terrestrial orchid mycorrhizas,seeds and protocorms

The identity and ecological role of fungi in the mycorrhizal roots of 25 species of mature terrestrial orchids and in 17 species of field incubated orchid seedlings were examined. Isolates of symbiotic fungi from mature orchid mycorrhizas were basidiomycetes primarily in the generaCeratorhiza, Epulorhiza andMoniliopsis; a few unidentified taxa with clamped hyphae were also recovered. More than one taxon of peloton-forming fungus was often observed in the cleared and stained mycorrhizas. AlthoughCeratorhiza andEpulorhiza strains were isolated from the developing protocorms, pelotons of clamped hyphae were often presents in the cleared protocorms of several orchid species. These basidiomycetes are difficult to isolate and may be symbionts of ectotrophic plants. The higher proportion of endophytes bearing clamp connections in developing seeds than in the mycorrhizas is attributed to differences in the nutritional requirements of the fully mycotrophic protocorms and partially autotrophic plants. Most isolates ofCeratorhiza differed enzymatically fromEpulorhiza in producing polyphenol oxidases. Dual cultures with thirteen orchid isolates and five non-orchid hosts showed that some taxa can form harmless associations with non-orchid hosts. It is suggested that most terrestrial orchid mycorrhizas are relatively non-specific and that the mycobionts can be saprophytes, parasites or mycorrhizal associates of other plants.     

Composition and Nutritional Characteristics of Fungi Consumed by Callimico goeldii in Pando, Bolivia

Though ≥22 species of Primates consume fungi, most do so at low rates, comprising <5% of their feeding time. Goeldi's monkeys (Callimico goeldii), spend up to 29% of their feeding time year-round consuming fungal sporocarps, the fruiting bodies of fungi. We provide comprehensive data on the nutritional characteristics of 4 species of fungi consumed by Callimico goeldii (Ascopolyporus polyporoides, Ascopolyporus polychrous, Auricularia auricula, and Auricularia delicata). The composition of the fungi is similar to that of other fungi: predominantly fiber (66.2–83.0% dry matter) with small amounts of sugar (2.0–5.6% dry matter) and crude fat (0.9–1.6% dry matter). Though the crude protein content is substantial (5.5–13.4% dry matter), much of the nitrogen in the fungi is not likely to be available to Callimico goeldii because it is associated with indigestible food components or is in nonprotein form. The mineral content of the fungi are within the normal range for fungi generally and the calcium-to-phosphorus ratio is low (0.07–0.25). Fungi appear to be a low-quality food resource for Callimico goeldii and may contribute to their relatively large home ranges and low population density compared to other Callitrichinae. Research on the ability of Callimico goeldii to digest fungi is needed to understand fully the nutritional value of fungi to them. We discuss adaptations Callimico goeldii may have for improving their ability to obtain nutrients from fungi and potential ecological correlates of mycophagy.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.     

An Imported Case of Blastomyces Dermatitidis Infection in Mexico

Blastomycosis is an acute or chronic primary infection of the respiratory system, endemic in North America (United States of America and Canada), Africa and Asia. We report a case in Mexico, in a three years old child who had been born in California and lived in Chicago, U.S.A. The patient presented pulmonary symptoms prior to development of a skin ulcer. Blastomyces dermatitidis was identified by mycological and molecular procedures. The patient was successfully treated with amphotericin B, oral ketoconazole and itraconazole.