Adiaspore development and morphological characteristics in a mouse adiaspiromycosis model.

Lesions of adiaspiromycosis, a respiratory disease affecting wild animals, have been found mainly in dead mammals and free-living mammals captured for surveillance. No report has described an investigation of adiaspore formation progress in the lung. After establishing an experimental mouse model of intratracheal adiaspiromycosis infection with the causative agent Emmonsia crescens, we observed adiaspore development. The spores grew and reached a plateau of growth at 70 days post-infection. The median adiaspore diameter showed a plateau of around 40 µm. The characteristic three-layer cell-wall structure of adiaspores was observed in the lung at 70 days post-infection. We examined infection with a few spores, which revealed that adiaspores in the mouse lung progressed from intratracheal infection of at least 400 spores. Moreover, we developed adiaspores in vitro by culture in fetal bovine serum. Although most spores broke, some large spores were intact. They reached about 50 µm diameter. Thick cell walls and dense granules were found as common points between in vitro adiaspores and in vivo adiaspores. These models are expected to be useful for additional investigations of E. crescens adiaspores and adiaspiromycosis.

Disseminated cutaneous chrysosporium infection.

Cutaneous chrysosporium infection is extremely rare and underdiagnosed. We present an immunocompromised patient who presented with recurrent cutaneous abscesses. Histopathology of the abscess showed thick-walled conidia and septate fungal hyphae within the subcutis and fungal culture grew Chrysosporium species.

Biodegradation of feather waste keratin by a keratinolytic soil fungus of the genus Chrysosporium and statistical optimization of feather mass loss.

This paper assesses the ability of strains of Aphanoascus fulvescens and Chrysosporium articulatum isolated from soil (phaesol) to degrade native feather keratin. Strains were identified based on phenotypic traits and nucleotide sequencing. Response Surface Methodology was used to optimize cultivation conditions exhibiting the highest keratinolytic activity. The experiments were based on Box-Behnken designs for the loss of substrate mass (chicken feathers). While substrate mass loss is an "economic coefficient" that reliably indicates feather keratin degradation, it has not been studied before. Stationary liquid cultures of five selected strains were conducted in laboratory conditions at 28 °C using poultry feathers (1 g) as the sole source of carbon, nitrogen and energy. Enzymatic activities, keratin mineralization products and substrate mass loss were determined periodically. The mineralization of keratin proteins by strains yielded a high number of ammonium ions alkalinizing the medium. Increased ammonium ions inhibited the activity of caseinian protease and keratinase. A decrease in the concentration of these ions induced proteolytic enzymes, chiefly the activity of keratinase, at the end of fungal cultivation. Keratinase activity was related to protein- and peptide release and that of caseinian protease to sulfate ions. The highest loss of substrate mass in comparison to the reference strain CBS104.62 (35.4%) was recorded for Aphanoascus fulvescens B21/4-5 (65.9%). Based on a Box-Behnken design, the maximum loss of substrate mass for the Aphanoascus fulvescens strain (71.08%) can be achieved at pH 7.58 and temperature 28.7 °C.

Molecular characterization of reptile pathogens currently known as members of the chrysosporium anamorph of Nannizziopsis vriesii complex and relationship with some human-associated isolates.

In recent years, the Chrysosporium anamorph of Nannizziopsis vriesii (CANV), Chrysosporium guarroi, Chrysosporium ophiodiicola, and Chrysosporium species have been reported as the causes of dermal or deep lesions in reptiles. These infections are contagious and often fatal and affect both captive and wild animals. Forty-nine CANV isolates from reptiles and six isolates from human sources were compared with N. vriesii based on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the family Onygenaceae of the order Onygenales. One lineage represents the genus Nannizziopsis and comprises N. vriesii, N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genus Ophidiomyces, with the species Ophidiomyces ophiodiicola occurring only in snakes, and Paranannizziopsis gen. nov., with three new species infecting squamates and tuataras. The newly described species are Nannizziopsis dermatitidis, Nannizziopsis crocodili, Nannizziopsis barbata, Nannizziopsis infrequens, Nannizziopsis hominis, Nannizziopsis obscura, Paranannizziopsis australasiensis, Paranannizziopsis californiensis, and Paranannizziopsis crustacea. Chrysosporium longisporum has been reclassified as Paranannizziopsis longispora. N. guarroi causes yellow fungus disease, a common infection in bearded dragons and green iguanas, and O. ophiodiicola is an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis.

Keratinophilic fungi recovered from feathers of different species of birds in St Kitts and Nevis / Hongos queratinofílicos obtenidos de las plumas de diferentes especies de aves en St Kitts y Nevis

OBJECTIVE: The aim of the present study was to investigate the occurrence of keratinophilic fungi including dermatophytes on feathers of domestic and wild birds in the islands of St Kitts and Nevis. METHOD: During 2010-2011, samples of feathers from ninety-four birds were examined by hair-baiting technique in Petri-dishes containing sterilized soil. Fungal growths appearing on the feathers and the hair-baits were microscopically examined and the cultures obtained were identified on the basis of their microscopic and colonial morphology. RESULTS: Chrysosporium constituted the majority (86.9%) of the 72 isolates of keratinophilic fungi, represented by mainly C tropicum and C indicum. Sepedonium spp isolates were recovered from nine of the feather samples; two of these were identified as Sepedonium chrysospermum, and the other two as S ampullosporum. CONCLUSION: Recovery of four isolates of the dermatophyte, Microsporum gypseum complex (two each of M gyspeum and M fulvum) from feathers of birds is a finding of public health significance.

OBJETIVO: El objetivo del presente estudio fue investigar la presencia de hongos queratinofílicos, incluyendo dermatofitos, en las plumas de aves domésticas y silvestres en las islas de St Kitts y Nieves. MÉTODOS: Durante 2010-2011, se examinaron muestras de plumas de noventa y cuatro aves, utilizando la técnica de anzuelo queratínico (técnica de Vanbreuseghem) en placas de Petri con tierra esterilizada. Los crecimientos fúngicos que aparecieron sobre las plumas y los anzuelos de queratina de pelos (hair baits) fueron examinados bajo el microscopio, y los cultivos obtenidos fueron identificados sobre la base de su morfología microscópica y colonial. RESULTADOS: Chrysosporium constituyó la mayor parte (86.9%) de los 72 aislados de hongos queratinofílicos, representados principalmente por el C tropicum y el C indicum. Aislados de Sepedonium spp fueron obtenidos de nueve muestras de plumas. Dos de ellos fueron identificados como Sepedonium chrysospermum y los otros dos como S ampullosporum. CONCLUSIÓN: La recuperación de cuatro aislados del complejo M gypseum dermatofito (formado por dos M gyspeum y dos M fulvum respectivamente) de las plumas de aves, es un hallazgo de importancia para la salud pública.

Fungal-mediated nano silver: an effective adulticide against mosquito.

Fungi as such are known to be an effective mosquito control agent. In the present investigation, the effect of silver nanoparticles synthesized with Chrysosporium keratinophilum, Verticillium lecanii, and Fusarium oxysporum f.sp. pisi has been evaluated against the adult mosquito of filariasis vector Culex quinquefasciatus. The silver nanoparticles were characterized by using the UV-Vis spectrophotometer and X-ray diffraction techniques. The micrographs of silver nanoparticles were obtained by transmission electron microscope and scanning electron microscope. Elemental analysis on single particle was carried out by EDX analysis. The characterization study confirmed different shapes and sizes of silver nanoparticles. The efficacy test was performed at five different concentrations for a period of 24 h by the probit analysis. The C. quinquefasciatus has shown higher efficacy against the silver nanoparticles synthesized with C. keratinophilum and V. lecanii (lethal concentration (LC)(50) 0.19 and 0.4 µl/cm(2); LC(90) 2.4 and 3.2 µl/cm(2); and LC(99) 4.0 and 5.6 µl/cm(2)) after 22 h of exposure. While the silver nanoparticles synthesized with F. oxysporum f.sp. pisi were found to be less effective against the C. quinquefasciatus, the silver nanoparticles synthesized by C. keratinophilum and V. lecanii were found to be more effective than those generated with the help of F. oxysporum f.sp. pisi and C. quinquefasciatus. The use of fungus-mediated silver nanoparticles is a rapid, environmentally safer, and greener approach for vector control strategy and is adaptable globally.

Keratinophilic fungi recovered from feathers of different species of birds in St Kitts and Nevis.

OBJECTIVE: The aim of the present study was to investigate the occurrence of keratinophilic fungi including dermatophytes on feathers of domestic and wild birds in the islands of St Kitts and Nevis. METHODS: During 2010-2011, samples of feathers from ninety-four birds were examined by hair-baiting technique in Petri-dishes containing sterilized soil. Fungal growths appearing on the feathers and the hair-baits were microscopically examined and the cultures obtained were identified on the basis of their microscopic and colonial morphology. RESULTS: Chrysosporium constituted the majority (86.9%) of the 72 isolates of keratinophilic fungi, represented by mainly C tropicum and C indicum. Sepedonium spp isolates were recovered from nine of the feather samples; two of these were identified as Sepedonium chrysospermum, and the other two as S ampullosporum. CONCLUSION: Recovery of four isolates of the dermatophyte, Microsporum gypseum complex (two each of M gyspeum and M fulvum) from feathers of birds is a finding of public health significance.

The extreme xerophilic mould Xeromyces bisporus--growth and competition at various water activities.

Little is known about the mould, Xeromyces bisporus, unique in its strong xerophilicity and ability to grow at water activity (a(w)) 0.62, lower than for any other known organism. The linear growth rates of one fast and one slow-growing strain of X. bisporus were assessed at 20, 25, 30 and 37 °C on solid agar media containing a mixture of glucose and fructose to reduce a(w) to 0.94, 0.88, 0.84, 0.80, 0.76 and 0.66. Growth rates of xerophilic species closely related to X. bisporus, viz. Chrysosporium inops, C. xerophilum and Monascus eremophilus, were also assessed. Optimal conditions for growth of both X. bisporus strains were approx. 0.84 a(w) and 30°C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. X. bisporus FRR 2347 even grew well at 0.66 a(w) (0.48 mm/day). C. inops and C. xerophilum were more tolerant of high a(w) than X. bisporus, and could be differentiated from each other based on: the faster growth of C. xerophilum; its preference for temperatures ≥ 30 °C and a(w) ≥ 0.94 (c.f.≤ 25 °C and ~0.88 a(w) for C. inops); and its ability to grow at 0.66 a(w), which is the lowest a(w) reported to date for this species. M. eremophilus grew slowly (max. 0.4mm/day) even in its optimal conditions of ~0.88 a(w) and 25 °C. To investigate the competitive characteristics of X. bisporus at low a(w), both X. bisporus strains were grown in dual-culture with xerotolerant species Aspergillus flavus and Penicillium roqueforti, and xerophilic species A. penicillioides, C. inops, C. xerophilum and Eurotium chevalieri, on glucose-fructose agar plates at 0.94, 0.84, 0.80 and 0.76 a(w) and at 25 °C. Growth rates and types of interactions were assessed. Excretion of inhibitory substances acting over a long-range was not observed by any species; inhibitors acting over a short-range that temporarily slowed competitors' growth or produced a protective zone around the colony were occasionally observed for A. penicillioides, C. inops and C. xerophilum. Instead, rapid growth relative to the competitor was the most common means of dominance. The xerotolerant species, A. flavus and P. roqueforti were dominant over X. bisporus at 0.94 a(w). E. chevalieri was often dominant due to its rapid growth over the entire a(w) range. At a(w)<0.80, X. bisporus was competitive because it grew faster than the other species examined. This supports the concept that its ideal environmental niche is sugary foods with low a(w).

Effect of Chrysosporium keratinophilum metabolites against Culex quinquefasciatus after chromatographic purification.

Chrysosporium keratinophilum is known to be a keratinophilic fungus and an effective mosquito control agent. This fungus was grown on Sabauraud dextrose broth in the laboratory at 25°C, while the relative humidity was maintained at 75 ± 5% for 15 days. Filtration process of metabolites was done using whatman-1 filter paper, column chromatography and flash, chromatography. Larvicidal efficacy was performed against all instars of Culex quinquefasciatus. Larvicidal efficacy was performed at six different concentrations with different effective ratios (ethanol/metabolites: 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9). The mortality values were then subjected by the probit analysis. The larval mortalities were observed for a period of 24, 48, and 72 h, respectively. The first and second instars were highly susceptible to 2:8 ratio. In the first instar after column chromatography, LC(50) =26.66 ppm, LC(90) =121.96 ppm, LC(99) =231.86 ppm were observed after 72 h, while after flash chromatography the LC(50) =20 ppm, LC(90) =123.02 ppm, LC(99) =281.83 ppm were observed after 48 h. In the second instar after column chromatography, LC(50) =18.19 ppm, LC(90) =102.32 ppm, LC(99) =162.18 ppm were observed after 72 h, while doing flash chromatography 100% mortality could be recorded after 24 h. In the third instar after column chromatography, the LC(50) =38.01 ppm, LC(90) =131.82 ppm, LC(99) =245.47 ppm were observed after 72 h, while after flash chromatography the LC(50) =17.78 ppm, LC(90) =100 ppm, LC(99) =151.35 ppm. In the fourth instar, LC(50) =61.65 ppm, LC(90) =181.97 ppm, LC(99) =436.51 ppm, while after flash chromatography LC(50) =40 ppm, LC(90) =120 ppm, and LC(99) =223.87 ppm were observed after 72 h. The extracellular metabolites of C. keratinophilum could be a fungal based larvicides resource for the control of C. quinquefasciatus larvae. This could be another agent for biotechnological exploitation, if found suitable in field trials.

Laboratory and field evaluation of the fungus Chrysosporium lobatum against the larvae of the mosquito Culex quinquefasciatus.

Eleven fungal species in three genera were isolated from the soil at Agra, India by the feather-baiting technique. Out of the 11 species, Chrysosporium lobatum Scharapov, a deuteromycetous (Moniliales: Moniliaceae) fungus, caused high mortality of Culex quinquefasciatus Say (Diptera: Culicidae) larvae in the laboratory. Laboratory and field trials were carried out to study the efficacy of C. lobatum against various instars of C. quinquefasciatus. In the laboratory bioassays, first, second and third instar larvae were assessed separately. Conidia of C. lobatum that were 15 days old were used both in laboratory and field bioassays. Six different concentrations were used in the laboratory bioassays (10, 10(3), 10(4), 5x10(4), 10(5) and 10(6) conidia/mL), and one concentration was tested in the field trials (10(6) conidia/mL). The LC50 values with 95% fiducial limits and probit equations were calculated by the probit analysis. Significant (analysis o.f variance (ANOVA): P<0.0001) mortality difference between the instars of C. quinquefasciatus was observed after 72 h of exposure to conidia of C. lobatum. The third instar C. quinquefasciatus were 319- and 25-fold more susceptible to C. lobatum (0.47x10(3) conidia/mL) than the first and second instars, respectively. The cuticle of the abdominal region and anal papillae were densely covered by the C. lobatum conidia. In the field trials, populations had declined significantly (t test, P=0.003) 15 days after inoculation in the four test pools. Significantly (t test, P=0.03) higher mortality was observed in the pools with water quality that was lower in total dissolved solids, hardness, chemical oxygen demand and conductivity. Based on the performance and survival in the trial, C. lobatum may be considered as a bio-control agent of mosquitoes.

Chrysosporium anamorph of Nannizziopsis vriesii associated with fatal cutaneous mycoses in the salt-water crocodile (Crocodylus porosus).

The Chrysosporium anamorph of Nannizziopsis vriesii, recently identified as the cause of cutaneous infections in chameleons and brown tree snakes, was associated with skin infections and deaths in salt-water crocodile (Crocodylus porosus) hatchlings on two separate occasions 3 years apart. In all, 48 animals died from the infection. All hatchlings came from the same farm in northern Queensland, Australia.

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.

TMC-69, a new antitumor antibiotic with Cdc25A inhibitory activity, produced by Chrysosporium sp. TC1068. Taxonomy, fermentation and biological activities.

A new antibiotic designated TMC-69 has been isolated from the fermentation broth of a fungal strain Chrysosporium sp. TC 1068. TMC-69 exhibited moderate in vitro cytotoxic activity. TMC-69-6H, a derivative of TMC-69 prepared by hydrogenation, possessed more potent in vitro cytotoxicity than TMC-69, and exhibited in vivo antitumor activity against murine P388 leukemia and B16 melanoma. TMC-69-6H was found to specifically inhibit Cdc25A and B phosphatases.

Survey of keratinophilic fungi isolated from city park soils of Pisa, Italy.

A survey of geophilic dermatophytes and related keratinophilic fungi isolated from city park soils of Pisa is reported. Twenty-three (48%) soil samples out of 48 were positive by hair baiting. The following species were isolated: Microsporum gypseum (39%), Trichophyton ajelloi (31%), Chrysosporium keratinophilum (14%), T. terrestre (8%), M. fulvum, Ch. luteum, Ch. indicum (5% each) and M. cookei (2%). The presence of the different species is discussed in relation to the risk of fungal skin infections.

Degradation of chicken feathers by Chrysosporium georgiae.

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin-salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8 pH at 30 degrees C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin-salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracellular keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones.

Czapek casein 50% glucose (CZC50G): a new medium for the identification of foodborne Chrysosporium spp.

A new medium Czapek Casein 50% Glucose agar (CZC50G) has been developed, on which the four foodborne Chrysosporium spp., C. xerophilum, C. inops, C. farinicola and C. fastidium can be distinguished by differences in growth rates and colony morphology. Chrysosporium xerophilum and C. inops both produced dense white colonies, but C. xerophilum grew faster than C. inops, 22 mm in 14 d compared to 9-12 mm in 14 d at 25 degrees C. Some isolates produced a yellow or red reverse due to the reaction of ferric ammonium citrate incorporated in the medium with a fungal metabolite. Chrysosporium farinicola and C. fastidium both grew poorly on this medium and produced sparse colonies: C. farinicola grew faster. Electron micrographs of arthroconidia with a cryo-scanning electron microscope showed thickening of the spore walls in C. inops but not in C. xerophilum. The aleurioconidia of C. farinicola and C. fastidium were different in shape. The differences in colony morphology and growth rate on CZC50G reflected these differences and demonstrated that these four species could be distinguished easily on CZC50G.