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.

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.

Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR.

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis. METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results. RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR. CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.

Detection of Snake Fungal Disease Due to Ophidiomyces ophiodiicola in Virginia, USA.

Snake fungal disease (SFD) is an emerging disease of wildlife believed to be caused by Ophidiomyces ophiodiicola. Although geographic and host ranges have yet to be determined, this disease is characterized by crusty scales, superficial pustules, and subcutaneous nodules, with subsequent morbidity and mortality in some snake species. To confirm the presence of SFD and O. ophiodiicola in snakes of eastern Virginia, US, we clinically examined 30 free-ranging snakes on public lands from April to October 2014. Skin biopsy samples were collected from nine snakes that had gross lesions suggestive of SFD; seven of these biopsies were suitable for histologic interpretation, and eight were suitable for culture and PCR detection of O. ophiodiicola. Seven snakes had histologic features consistent with SFD and eight were positive for O. ophiodiicola by PCR or fungal culture.

Rodentborne fungal pathogens in wetland agroecosystem

The past few decades have witnessed an overwhelming increase in the incidence of fungal infections, particularly in immunocompromised individuals. Consequently, zoonotic diseases, especially through rodents constitute a prominent group among the emerging diseases. Rodents are commensal to man and related health risks are common. Water rats (Rattus norvegicus) are typical to Vembanadu-Kol wetland agroecosystems, where they can act as a good carrier nexus for pathogens. The present study evaluates the carrier status of water rats with respect to fungal pathogens. A total of fifty two fungi covering eighteen families were isolated. Among the isolates, eight were dermaptophytes and Chrysosporium sp. (89.18%) was the frequent isolate. The source-wise analyses showed an increased isolation from ventral hair (67 isolates). Water rats of Vembanadu-Kol wetland agroecosystem are potent carrier of dermaptophytes and other opportunistic fungi, and strong carrier paths are existing too.

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).

Fine-needle aspiration may replace skin biopsy for the collection of material for experimental infection of mice with Mycobacterium leprae and Lacazia loboi.

BACKGROUND: Procedures involving the use of Mycobacterium leprae and Lacazia loboi, uncultivated organisms, depend on the collection of material from the lesions of patients or experimental animals. This study compared fine-needle aspiration (FNA) and skin biopsy methods for obtaining bacilli and fungal cells to experimentally infect animals. METHODS: Lepromas from one armadillo and one enlarged footpad of a mouse previously inoculated with L. loboi were submitted to FNA and biopsy. Materials collected were processed for inoculation in mice. RESULTS: Acid-fast bacilli (AFB) collected by two FNA procedures yielded 7.2×10(7) and 5.3×10(6) AFB/ml and biopsies yielded 1.58×10(8) and 3.5×10(8) AFB/ml from each leproma. Yeast-like cells of L. loboi collected by FNA yielded 1.0×10(6) fungal cells/ml and biopsy 1.0×10(7) fungal cells/ml. After 8 months, inoculated animals were sacrificed and the inoculated footpads submitted to histopathological examination and counting of AFB and fungal cells. The results obtained by the two methods were comparable for both microorganisms. CONCLUSIONS: Biopsy may be replaced by FNA during harvesting of material for different purposes, especially for experimental inoculation of mice in leprosy and Jorge Lobo's disease, with the advantage of FNA being a simpler, less invasive, and less costly method.

Immunohistochemical study of Langerhans cells in cutaneous lesions of the Jorge Lobo's disease.

Jorge Lobo's disease is a chronic infection caused by the fungus Lacazia loboi endemic in South America. The infection is characterized by the appearance of parakeloidal, ulcerated or verrucous nodular or plaque-like cutaneous lesions. The histopathological aspect is characterized by poorly organized granulomas with histiocytes and multinucleated giant cells. Little is known about local immune response in lobomycosis skin lesions. Thirty-three skin biopsies from patients with Jorge Lobo's disease were selected from Ambulatory of Dermatology, UFPA. The control group was constituted by ten biopsies from normal skin. Langerhans cells were identified by immunohistochemistry using anti-CD1a antibody (Serotec). The number of positive cells was statistically analyzed. Langerhans cells were visualized along the epidermis in biopsies from Jorge Lobo's disease and the morphology and the number of Langerhans cells did not differ from normal skin (p>0.05). In Jorge Lobo's disease, this cell population probably presents some escape mechanism of the local immune system to evade the antigen presentation by those cells.

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.

Use of sera from humans and dolphins with lacaziosis and sera from experimentally infected mice for Western Blot analyses of Lacazia loboi antigens.

Antibodies in the sera of patients with lacaziosis recognized an approximately 193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.

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.

Immunohistochemical characterization of the cellular infiltrate in Jorge Lobo's disease.

Few studies have been conducted to evaluate the cellular composition of the granulomatous lesions induced by Lacazia loboi. Thus, the objective of the present study was to characterize the mononuclear cell population present in cutaneous lesions obtained from 15 patients with Jorge Lobo's disease. Histological sections were stained with hematoxylin-eosin and methenamine silver and the following mononuclear cells were identified by immunohistochemistry: T lymphocytes (CD3+), helper T lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), B lymphocytes (CD20+), plasma cells (CD79+), natural killer cells (CD57+) and histiocytes (CD68+). This study showed that the inflammatory infiltrate mainly consists of histiocytes and multinucleated giant cells, in addition to the presence of a large number of fungal cells. The identified inflammatory cells showed the following frequency: CD68+ histiocytes > CD3+ T lymphocytes > CD4+ T > CD8+ T lymphocytes > CD57+ natural killer cells > CD79+ plasma cells > CD20+ B lymphocytes. Based on the findings of a large number of fungal cells in the infected tissues and the disorganized cell arrangement in the granuloma, we hypothesize that patients with Jorge Lobo's disease present immunoregulatory disturbances, which are likely to be specific and perhaps responsible for the lack of containment of the pathogen.