Novel Paranannizziopsis species in a Wagler's viper (Tropidolaemus wagleri), tentacled snakes (Erpeton tentaculatum), and a rhinoceros snake (Rhynchophis boulengeri) in a zoological collection.

We report several cases of fungal infections in snakes associated with a new species within the genus Paranannizziopsis. Three juvenile Wagler's vipers (Tropidolaemus wagleri) presented with skin abnormalities or ulcerative dermatitis, and two snakes died. Histologic examination of skin from the living viper revealed hyperplastic, hyperkeratotic, and crusting epidermitis with intralesional fungal elements. The terrestrial Wagler's vipers were housed in a room with fully aquatic tentacled snakes (Erpeton tentaculatum), among which there had been a history of intermittent skin lesions. Approximately 2 months after the biopsy of the viper, a skin sample was collected from one tentacled snake (TS1) with skin abnormalities and revealed a fungal infection with a similar histologic appearance. Fungal isolates were obtained via culture from the Wagler's viper and TS1 and revealed a novel species, Paranannizziopsis tardicrescens, based on phenotypic characterization and molecular analysis. P. tardicrescens was cultured and identified by DNA sequence analysis 8 months later from a dead tentacled snake in an exhibit in an adjacent hallway and 13 months later from a living rhinoceros snake (Rhynchophis boulengeri) with two focal skin lesions. Antifungal susceptibility testing on three of four cultured isolates demonstrated potent in vitro activity for terbinafine and voriconazole.

Spore morphology and ultrastructure of an Ascosphaera apis strain from the honeybees (Apis mellifera) in southwest China.

Ascosphaera apis is an intestinally infective, spore-forming, filamentous fungus that infects honeybees and causes deadly chalkbrood disease. Although A. apis has been known for 60 y, little is known about the ultrastructure of the spores. In this study, the fine morphology and ultrastructure of an isolate, A. apis CQ1 from southwest China, was comprehensively identified by transmission electron microscopy, confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy. The high sequence similarity and phylogenetic data based on nuc rDNA ITS1-5.8S-ITS2 (ITS) supported the hypothesis that the CQ1 strain is a new member of the A. apis species. Morphological observation indicated that the mature spores are long ovals with an average size of 2 × 1.2 µm and are tightly packed inside spherical spore balls. More than 10 spore balls that were 8-16 µm in diameter were wrapped and formed a spherical, nearly hyaline spore cyst of 50-60 µm in diameter. Ultrastructural analysis showed that mature spores have two nuclei with distinctly different sizes. A large nucleus with double nuclear membranes was found in the center of the spore, whereas the small nucleus was only one-fifth of the large nucleus volume and was located near the end of the spore. Numerous ribosomes filled the cytoplasm, and many mitochondria with well-defined structures were arranged along the inner spore wall. The spore wall consists of an electron-dense outer surface layer, an electron-lucent layer, and an inner plasma membrane. Chitin is the major component of the spore wall. The germinated spore was observed as an empty spore coat, whereas the protoplasts, including the nuclei, mitochondria, and ribosomes, had been discharged. In addition to these typical fungal spore organelles, an unknown electron-dense regular structure might be the growing mycelium, which was arranged close to the inner spore wall and almost covered the entire wall area.

Characterization of Nannizziopsis guarroi with genomic and proteomic analysis in three lizard species.

Fungal infections in captive as well as in free-living reptiles caused by emerging obligate pathogenic fungi appear with increasing frequency and give occasion to establish new and fast methods for routine diagnostics. The so-called yellow fungus disease is one of the most important and common fungal dermatomycoses in central bearded dragons (Pogona vitticeps) and green iguanas (Iguana iguana) and is caused by Nannizziopsis guarroi. The aim of this study was to prove reliability in identification of N. guarroi with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in comparison to molecular biological analysis of ribosomal DNA genes. In seven lizards from three different species, including central bearded dragons, green iguanas, and a European green lizard (Lacerta viridis), dermatomycoses caused by N. guarroi were diagnosed by isolation of the fungal pathogen as well as histopathological confirmation of the granulomatous inflammatory reaction in deep skin biopsies. With this survey, we proved that MALDI-TOF MS is a diagnostic tool for accurate identification of N. guarroi. Besides small subunit 18S rDNA (SSU) and internal transcribed spacer (ITS)1-5.8S rDNA, a large fragment of the large subunit of the 28S rDNA (LSU), including the domain (D)1 and D2 have been sequenced, for phylogenetical analysis. Large fragment of the LSU from N. guarroi has been sequenced for the first time. Yellow fungus disease in a European lizard species is described for the first time to our knowledge as well, which could be of importance for free-ranging populations of European lizards.

New Species Spiromastigoides albida from a Lung Biopsy.

The new species Spiromastigoides albida (Onygenales, Eurotiomycetes, Ascomycota), from a lung biopsy in USA, is proposed and described based on morphological data and the analysis of rRNA, and fragments of actin and ß-tubulin gene sequences. This species is characterized by white colonies and a malbranchea-like asexual morph with profusely branching curved conidiophores forming sporodochia-like structures. Moreover, new combinations for Gymnoascus alatosporus, and for some new species recently described under the generic name Spiromastix, are provided.

Halophilic and thermotolerant Gymnoascus species from several special environments, China.

This study introduces three new Gymnoascus species (Gymnoascaceae, Onygenales), G. halophilus, G. stercorarius and G. thermotolerans, isolated from sediments in Chaka Salt Lake, compost and cornfield soil, respectively, in China, based on a polyphasic characterization including morphology, physiology and molecular phylogeny. Phylogenetic relationships were assessed based on the nuclear internal transcribed spacer (ITS = ITS1 + 5.8S + ITS2) region and a combined multilocus alignment of the ITS, 18S subunit rRNA gene and 28S subunit rRNA genes. Our study identified phylogenetic and phenotypic characters that differentiated the three new species from known species in the genus. Salinity and temperature tolerance tests revealed that G. halophilus was an obligate halophile while G. stercorarius and G. thermotolerans were halotolerant and thermotolerant. A key to accepted species of Gymnoascus is provided.

Ascosphaera subglobosa, a new spore cyst fungus from North America associated with the solitary bee Megachile rotundata.

Ascosphaera subglobosa (Eurotiomycetes: Onygenales) is newly described from the pollen provisions and nesting material of the solitary leaf-cutting bee Megachile rotundata in Canada and the western United States. This new species, related to A. atra and A. duoformis, is distinguished from other Ascosphaera species by its globose to subglobose ascospores, evanescent spore balls and unique nuclear ribosomal DNA sequences (ITS and LSU).

Enzymatic isolation of Lacazia loboi cells from skin lesions of lobomycosis.

Lacazia loboi is the etiologic agent of Jorge Lobo's disease, a cutaneous and subcutaneous mycosis endemic to Latin America tropical regions and characterized by chronic nodular or keloidal lesions which develop after traumatic events. A new method for the extraction of L. loboi yeast cells from biopsies of lobomycosis skin lesions is presented. The method is based on the proteolytic action of the enzyme dispase which is known for its action against fibronectin and collagen type IV. Fungal identification was based on histological examination of the biological material and molecular analysis based on 18S ribosomal sequences. Observations under optic and fluorescence microscopy proved the efficacy of enzymatic isolation of the lobomycosis etiologic agent, as well as identifying the organism's main parasitic characteristics. Molecular phylogenetic analysis corroborated the histological examination and indicated L. loboi relationship with other members of the Onygenales. Use of dispase proved to be ideal for the isolation of L. loboi from human biopsies, shows promise as an important tool for improving biological studies of this peculiar fungus.

Onygenales (Eurotiomycetes, Ascomycota) queratinofílicos en suelos de establecimientos educacionales urbanos y rurales de la V Región, Chile / Queratinophilic onygenales (Eurotiomycetes, Ascomycota) in urban and rural soils of eduacational center of V Region, Chile

Con la finalidad de estudiar la presencia de Onygenales queratinofílicas potencialmente patógenas para el hombre y los animales, se analizaron mediante la técnica del anzuelo queratínico (Marzo – Diciembre, 2006), suelos de establecimientos educacionales urbanos y rurales de la V Región, Chile. Se colectaron un total de 64 muestras, de las cuales, la mitad se obtuvieron en Valparaíso-Viña del Mar (urbano) y la otra en Olmué-Limache (en zonas rurales). En zona urbana se aislaron 112 cepas (7 géneros y 12 especies) y en la rural 147 (11 géneros y 18 especies). Los géneros de mayor prevalencia en la zona urbana y rural en orden decreciente fueron: Chrysosporium y su teleomorfo 32 por ciento versus 45 por ciento; Keratinomyces y su teleomorfo, 26 por ciento y 16 por ciento; Microsporum y su teleomorfo 23 por ciento y 16 por ciento y Myceliophthora y su teleomorfo con un 13 y 10 por ciento. Amauroascus mutatus, Auxarthron umbrinum, Gymnoascus reessii, Chrysosporium charmichaelii, Ch. merdarium, Ch. tropicum, Geomyces pannorum var. pannorum fueron detectados sólo en la zona rural; mientras que Malbranchea flava sólo en la zona urbana. Mediante la técnica de Takashio, se pudo identificar separadamente las especies del complex Microsporum gypseum, determinándose que M. gypseum (Arthrodema gypseum) y M.fulvum (A. fulvum) obtuvieron una frecuencia de aislamiento similar en ambas zonas, siendo el primero un oportunista potencialmente patógeno para el hombre y los animales.

With the purpose of studying the presence of keratinophilic Onygenales that are potentially pathogenous for man and animal, urban and rural soils from educational centers in the V Region were examined with the keratinic bait technique (march-december 2006). A total of 64 samples were collected, 32 of them being from Valparaíso-Viña del Mar (urban) while the rest in Olmué-Limache (in rural zones). One hundred and twelve strains (7 genera and 12 species) were collected in the urban zone and 147 strains in the rural zone (11genera and 18 species). Genera with the highest prevalence both in the urban and the rural zones were, in decreasing order: Chrysosporium and its teleomorph 32 per cent vs. 45 per cent; Keratinomyces and its teleomorph 26 per cent and 16 per cent; Microsporum and its teleomorph 23 per cent and 16 per cent and Myceliophthora and its. teleomorph with 13 per cent and 10 per cent. Amauroascus mutatus, Auxarthron umbrinum, Gymnoascus reessii, Chrysosporium charmichaelii, Ch. merdarium, Ch.tropicum, Geomyces pannorum var. pannorum were detected only in the rural zone, whereas Malbranchea flava only in the urban zone. Species of the complex Microsporum gypseum, could be identified separately by means of the Takashio technique, coming to the conclusion that M. gypseum (Arthrodema gypseum) and M.fulvum (A. fulvum) achieved a similar frequency of isolation in both zones and that the former is a potentially opportunistic pathogen for man and animal.

Hongos geofílicos en la zona occidental de la región chaqueña (Argentina) / Geophilic fungi in western zone of Chaco Region, Argentina

Mediante las técnicas de Orr y de dilución, se estudiaron muestras de tierra de la zona occidental de la región chaqueña recogidas durante las estaciones de invierno y verano del año 2006. Mediante el anzuelo queratínico se aislaron 22 géneros y 25 especies fúngicas. Los Onygenales estuvieron representados con sólo 8 géneros, principalmente Chrysosporium indicum, Aphanoascus fulvescens y Myceliophthora vellerea, mientras en los no Onigenales, destacan: Paecilomyces lilacinus y Aspergillus fumigatus. En las muestras procesadas por dilución en PDA, se obtuvieron 24 géneros y 43 especies. Entre éstas, el género más frecuente y con mayor diversidad fue Aspergillus, siendo A. fumigatus la especie más representada, siguiendo en importancia los micelios hialinos y dematiáceos sin fructificar, los Penicillium del sub género Biverticillium y los Trichoderma de la sección Trichoderma. Se destaca la presencia de Corynascus verrucosus y C. setosus. Con ambas técnicas no se obtuvieron diferencias significativas respecto del número total de aislamientos entre invierno y verano.

Soil samples from the western zone of Chaco Region and collected during winter and summer of 2006 were studied by means of Orr and dilution techniques. Twenty two genera and 25 fungal species resulted with the use of the keratinic bait. Onygenales were represented by only 8 genera, mainly Chrysosporium indicum, Aphanoascus fulvescens and Myceliophtora vellerea, while Paecilomyces lilacinus and Aspergillus fumigates were present in the not Onygenales group. In samples processed by PDA dilution, 23 genera and 43 species were obtained. Among these, Aspergillus was the most frequent genus and which showed the highest diversity, being A. fumigatus the highest represented species, followed in importance by fructification-free hyaline and dematiaceous mycelia, Penicillium of the sub genus Biverticillium and Trichoderma from the Trichoderma section. The presence of Corynascus verrucosus and C. setosus is also pointed out. There were no significant differences detected under both techniques as regards the overall number of isolations within winter and summer.

Molecular study of archival fungal strains isolated from cases of lacaziosis (Jorge Lobo's disease).

Lacazia loboi, the aetiological agent of lacaziosis (Jorge Lobo's disease), is an uncultivated anomalous fungal microbe closely related to Paracoccidioides brasiliensis, both restricted Latin American Pathogens. Early reports suggesting that L. loboi had been isolated in pure culture from cases of lacaziosis, only added more confusion to the already confusing aetiology of this disease. These strains were later identified as unusual contaminants and some of them as P. brasiliensis. Recent phylogenetic analysis grouped L. loboi as the sister taxon to P. brasiliensis, thus it was postulated that the original P. brasiliensis strains recovered from cases of lacaziosis, could be the aetiological agent of the disease. Using molecular methodologies, we investigated the archival P. brasiliensis isolate No. 525 from a case of lacaziosis, as well as other archival isolates, identified earlier as common contaminants, all recovered from similar cases of the disease. Phylogenetic analysis, using the 18S small subunit rDNA sequences of these isolates showed that strain No. 525 was a typical P. brasiliensis isolate and the other studied strains were indeed contaminants. This study unequivocally indicates that the aetiological agent of lacaziosis is yet to be cultured.

Algunos microhongos geofílicos de las planicies semiáridas del noroeste de la provincia de San Luis (Argentina) / Some geophilic microfungi from Norwest semiarid plains in the province of San Luis, Argentine

Por las técnicas del anzuelo queratínico y de dilución, se estudiaron tierras de las planicies semiáridas del noroeste de la provincia de San Luis (Argentina), en invierno y verano. Con la técnica del anzuelo queratínico se aislaron 12 géneros y 15 especies fúngicas, mientras en las procesadas por dilución en PDA, 15 géneros y 29 especies. La mayoría de las detectadas con la primera técnica pertenecieron a los Onygenales (65,4 por ciento), siendo Chrysosporium indicum el de más alta frecuencia, mientras que en las diluciones, la mayoría correspondió a hongos mitospóricos (>75 por ciento), dominando los integrantes del género Aspergillus (35,8 por ciento). Con ambas técnicas la riqueza de especies fue levemente superior en verano y aún cuando la diversidad general fue baja, se aprecia la capacidad de adaptación y sobrevivencia de ciertos géneros y especies en ambientes edáficos adversos.

Soils from northwestern semiarid plains in the province of San Luis (Argentina) were examined by means of keratinic bait and dilution techniques both in winter and summer. The first technique yielded 12 genera and 15 fungal species whereas those processed in PDA dilution resulted in 15 genera and 29 species. Most fungi detected with the former technique belonged to Onygenales(65.4 percent), being Chrysosporium indicum the most frequently occurring, while in dilutions mitosporic fungi(<75 percent) showed the highest occurrence, those of the genus Aspergillus having dominance (35.8 percent). The richness of species was fairly higher with both techniquesand even though general diversity was low, the ability of certain genera and species to adapt and survive underadverse edaphic environments could be clearly seen.

Molecular phylogeny and taxonomy of medically important fungi.

Some recent advances in study of molecular evolution and taxonomy of human pathogens are discussed. In systemic Onygenales as well as in Chaetothyriales, pathogenic species are phylogenetically intermingled with non-pathogenic taxa. When a teleomorph of Coccidioides immitis is eventually found, it is predicted to resemble Uncinocarpus, a genus otherwise comprising environmental species. In the dermatophytes, Trichophyton and Microsporum are paraphyletic, whereas Epidermophyton is polyphyletic. On the basis of 18S and ITS rDNA sequencing data, Exophiala anamorphs (black yeasts) are confirmed to be closely related to the ascomycete genus Capronia. The related neurotropic species Cladophialophora bantiana is remarkable in consistently having introns in its 18S rDNA gene.