Ophidiomycosis, an emerging fungal disease of snakes: Targeted surveillance on military lands and detection in the western US and Puerto Rico.

Wildlife disease surveillance and pathogen detection are fundamental for conservation, population sustainability, and public health. Detection of pathogens in snakes is often overlooked despite their essential roles as both predators and prey within their communities. Ophidiomycosis (formerly referred to as Snake Fungal Disease, SFD), an emergent disease on the North American landscape caused by the fungus Ophidiomyces ophiodiicola, poses a threat to snake population health and stability. We tested 657 individual snakes representing 58 species in 31 states from 56 military bases in the continental US and Puerto Rico for O. ophiodiicola. Ophidiomyces ophiodiicola DNA was detected in samples from 113 snakes for a prevalence of 17.2% (95% CI: 14.4-20.3%), representing 25 species from 19 states/territories, including the first reports of the pathogen in snakes in Idaho, Oklahoma, and Puerto Rico. Most animals were ophidiomycosis negative (n = 462), with Ophidiomyces detected by qPCR (n = 64), possible ophidiomycosis (n = 82), and apparent ophidiomycosis (n = 49) occurring less frequently. Adults had 2.38 times greater odds than juveniles of being diagnosed with ophidiomycosis. Snakes from Georgia, Massachusetts, Pennsylvania, and Virginia all had greater odds of ophidiomycosis diagnosis, while snakes from Idaho were less likely to be diagnosed with ophidiomycosis. The results of this survey indicate that this pathogen is endemic in the eastern US and identified new sites that could represent emergence or improved detection of endemic sites. The direct mortality of snakes with ophidiomycosis is unknown from this study, but the presence of numerous individuals with clinical disease warrants further investigation and possible conservation action.

Diversity of Onygenalean Fungi in Keratin-Rich Habitats of Maharashtra (India) and Description of Three Novel Taxa.

Extensive survey was carried out in the state of Maharashtra, India, as part of a 3-year project to explore keratinophilic fungal diversity for conservation and biotechnological potential. A total of 578 soil samples were collected from keratin-rich habitats across 24 districts of Maharashtra State. Hair-baiting technique and micro-dilution drop-trail method were employed for isolation and purification of keratinophilic fungi from soil. A total of 66 species belonging to 17 genera of order Onygenales were recorded in hair baits. Eleven taxa were found to be new to science, most of which were rare as they were recorded in only one sample out of the > 500 samples analyzed. Three novel taxa have been characterized at morphological and molecular level and described here as new to science. These taxa include Currahmyces indicus gen. et sp. nov., Canomyces reticulatus gen. et sp. nov., Ctenomyces indicus sp. nov. All these novel taxa are morphologically and phylogenetically distinct from known taxa of order Onygenales. The study indicates that systematic sampling of a larger area is needed to uncover the hidden (unknown) diversity of keratinophilic fungi which is overlooked in sporadic samplings as evident from previous studies.

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.

Emydomyces testavorans, a New Genus and Species of Onygenalean Fungus Isolated from Shell Lesions of Freshwater Aquatic Turtles.

The fungal order Onygenales includes many pathogens of humans and animals, and recent studies have shown some onygenalean fungi to be significant emerging pathogens of reptiles. Although many of these fungi have similar morphological features in histologic tissue sections, recent molecular analyses have revealed a genetically complex and diverse group of reptile pathogens comprising several genera, most notably Nannizziopsis, Ophidiomyces, and Paranannizziopsis Infections by members of these genera have been previously reported in a variety of reptile species, including crocodilians, lizards, snakes, and tuataras, with negative impacts on conservation efforts for some reptiles. Despite the well-documented pathogenicity of these fungi in all other extant reptile lineages, infection has not yet been reported in aquatic turtles. In this study, we report the isolation of an onygenalean fungus associated with shell lesions in freshwater aquatic turtles. The morphologic and genetic characteristics of multiple isolates (n = 21) are described and illustrated. Based on these features and results of a multigene phylogenetic analysis, a new genus and species, Emydomyces testavorans, are proposed for these fungi isolated from turtle shell lesions.

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.

Xanthothecium peruvianum isolated from human stratum corneum: A case report, characterisation and short review that suggest emendation of Arachnomyces peruvianus.

From stratum corneum samples of a palmar eczema, a fungus was isolated that developed white colonies with a yellowish dark reverse, suggestive of dermatophytes. The isolate produced numerous chlamydospores and sparse aleuroconidia, was resistant to cycloheximide, grew well on human stratum corneum samples and was positive in tests for urease production and hair perforation, but no dermatophyte could be identified. After several weeks, cleistothecia with delicate asci and disc-shaped ascospores were formed, suggesting Arachnomyces spp. The analyses of the ribosomal ITS and LSU (D1/D2 domains) nucleotide sequences proved a good match with the ex-type strain of Xanthothecium peruvianum (family Onygenaceae, order Onygenales), and LSU sequence showed 99% similarity with Arachnomyces glareosus. This is the first report of X. peruvianum isolated from human skin. The description of our isolate provides new information about this species and proposes its transfer to the genus Arachnomyces with the subsequent emendation of the description of Arachnomyces peruvianus. Morphologically and physiologically it mimics dermatophytes and other species of the genus Arachnomyces. Although the clinical situation did not suggest any relevance for A. peruvianus as a primary pathogen, this fungus may act as a secondary pathogen under suitable conditions due to its keratinolytic capacity.

Novel taxa of thermally dimorphic systemic pathogens in the Ajellomycetaceae (Onygenales).

Recent discoveries of novel systemic fungal pathogens with thermally dimorphic yeast-like phases have challenged the current taxonomy of the Ajellomycetaceae, a family currently comprising the genera Blastomyces, Emmonsia, Emmonsiellopsis, Helicocarpus, Histoplasma, Lacazia and Paracoccidioides. Our morphological, phylogenetic and phylogenomic analyses demonstrated species relationships and their specific phenotypes, clarified generic boundaries and provided the first annotated genome assemblies to support the description of two new species. A new genus, Emergomyces, accommodates Emmonsia pasteuriana as type species, and the new species Emergomyces africanus, the aetiological agent of case series of disseminated infections in South Africa. Both species produce small yeast cells that bud at a narrow base at 37°C and lack adiaspores, classically associated with the genus Emmonsia. Another novel dimorphic pathogen, producing broad-based budding cells at 37°C and occurring outside North America, proved to belong to the genus Blastomyces, and is described as Blastomyces percursus.

A novel dimorphic pathogen, Emergomyces orientalis (Onygenales), agent of disseminated infection.

A novel dimorphic fungus, Emergomyces orientalis sp. nov. a close relative of systemic pathogens in the family Ajellomycetaceae (Blastomyces, Histoplasma). The fungus is reported in a 64-year-old male from Shanxi, China. The patient developed disseminated skin lesions, productive cough with fever and showed nodular opacities in his left lung on chest radiography. The patient had no identified cause of immunodeficiency apart from type-2 diabetes mellitus. Clinical, histopathological and mycological characteristics of the agent are given, and its phylogenetic position is determined with multilocus sequence data.

Xerotolerant fungi in house dust: taxonomy of Spiromastix, Pseudospiromastix and Sigleria gen. nov. in Spiromastigaceae (Onygenales, Eurotiomycetes).

During a global investigation of fungi in house dust, we isolated six novel arthroconidial fungi. Phylogenies from combined analysis of nuc rDNA 18S, 28S and internal transcribed spacers sequences demonstrated that these fungi and two species preserved in culture collections represent undescribed species of Spiromastigaceae, Onygenales. Seven of the eight species lacked sexual states and only characters of asexual states and growth rates on different media could be used to characterize them. The eighth species produced ascomata only on water agar. We introduce six new species and one new combination in Spiromastix and validate the recently proposed family Spiromastigaceae, genus Pseudospiromastix and combination Ps. tentaculata. The new genus Sigleria is proposed for two new species that differ from Spiromastix by conidiophore branching patterns, slower growth and a limited ability to utilize nitrate as a sole N source. A key to the three genera of Spiromastigaceae, Spiromastix, Pseudospiromastix and Sigleria, is provided. Phylogenetic analyses support the placement of Spiromastigaceae within Onygenales.

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.

Introduction of Non-Native Pollinators Can Lead to Trans-Continental Movement of Bee-Associated Fungi.

Bees are essential pollinators for many flowering plants, including agriculturally important crops such as apple. As geographic ranges of bees or their host plants change as a result of human activities, we need to identify pathogens that could be transmitted among newly sympatric species to evaluate and anticipate their effects on bee communities. We used PCR screening and DNA sequencing to evaluate exposure to potentially disease-causing microorganisms in a pollinator of apple, the horned mason bee (Osmia cornifrons). We did not detect microsporidia, Wolbachia, or trypanosomes, which are common pathogens of bees, in any of the hundreds of mason bees screened. We did detect both pathogenic and apathogenic (saprophytic) fungal species in the genus Ascosphaera (chalkbrood), an unidentified species of Aspergillus fungus, and a strain of bacteria in the genus Paenibacillus that is probably apathogenic. We detected pathogenic fungal strains in asymptomatic adult bees that therefore may be carriers of disease. We demonstrate that fungi from the genus Ascosphaera have been transported to North America along with the bee from its native range in Japan, and that O. cornifrons is exposed to fungi previously only identified from nests of other related bee species. Further study will be required to quantify pathogenicity and health effects of these different microbial species on O. cornifrons and on closely-related native North American mason bees that may now be exposed to novel pathogens. A global perspective is required for pathogen research as geographic ranges of insects and microorganisms shift due to intentional or accidental introductions.

Emmonsiellopsis, a new genus related to the thermally dimorphic fungi of the family Ajellomycetaceae.

Two interesting fungi were isolated from fluvial sediments collected in the North of Spain. They were morphologically related to the thermally dimorphic fungi of the family Ajellomycetaceae, but the analysis of the internal transcribed spacer region of the rDNA, and the domains D1 and D2 of the 28S rRNA gene sequences confirmed that they were different from all the species described in that family. They were accommodated in the new genus Emmonsiellopsis as E. coralliformis sp. nov. and E. terrestris sp. nov. The two species are distinguished mainly by the maximum temper-ature of growth (up to 33 °C for E. coralliformis and to 42 °C for E. terrestris), the dendritic mycelium of E. coralliformis and the conidial ornamentation (verrucose in E. coralliformis and spinulose in E. terrestris). In addition, the phylogenetic data demonstrated that Ajellomyces griseus also represents a new genus within the Ajellomycetaceae, namely Helicocarpus. This new genus is easily distinguished by the lack of asexual morph, the production of brownish gymnothecial ascomata and oblate to lenticular, sparingly pitted ascospores. The proposal of both new genera was confirmed by the analysis of actin gene sequences.

Auxarthron ostraviense sp. nov., and A. umbrinum associated with non-dermatophytic onychomycosis.

Auxarthron is a genus within the Onygenales encompassing keratinophilic species with typical ascomata (gymnothecia) consisting of anastomosing network of thick-walled hyphae and small globose or oblate ascospores. No association of this genus with clinically relevant cases of human or animal infection has been reported. This paper describes the isolation of an undescribed Auxarthron species as an agent of proven onychomycosis affecting almost all fingernails in a man with psoriasis. The causality of the isolated fungus was verified by repeated sampling and direct microscopy revealing irregular septate hyphae. Based on micro- and macromorphological features and unique sequence data (ITS region, benA and RPB2 gene), the isolated fungus is proposed as the new species A. ostraviense. The sibling species of A. ostraviense, A. umbrinum, was isolated from three patients with suspected onychomycosis and a detailed clinical history is provided for one of these patients. All four isolates were tested for susceptibility to selected antifungal agents. Terbinafine and clotrimazole appear to be effective in vitro. The morphological identification of Auxarthron spp. is non-trivial, time-consuming and requires cultivation media other than Sabouraud glucose agar which is routinely used in dermatomycology.

Isolation and characterization of a new fungal genus and species, Aphanoascella galapagosensis, from carapace keratitis of a Galapagos tortoise (Chelonoidis nigra microphyes).

A new fungal genus and species, Aphanoascella galapagosensis, recovered from carapace keratitis in a Galapagos tortoise residing in a south Texas zoological collection, is characterized and described. The presence of a pale peridium composed of textura epidermoidea surrounded by scarce Hülle cell-like chlamydospores, and the characteristic reticulate ascospores with an equatorial rim separates it from other genera within the Onygenales. The phylogenetic tree inferred from the analysis of D1/D2 sequences demonstrates that this fungus represents a new lineage within that order. As D1/D2 and ITS sequence data also shows a further separation of Aphanoascus spp. into two monophyletic groups, we propose to retain the generic name Keratinophyton for species whose ascospores are pitted and display a conspicuous equatorial rim, and thereby propose new combinations in this genus for four Aphanoascus species.

A multi-gene phylogeny provides additional insight into the relationships between several Ascosphaera species.

Ascosphaera fungi are highly associated with social and solitary bees, with some species being pathogenic to bees (causing chalkbrood) while others are not, and proper identification within this genus is important. Unfortunately, morphological characterizations can be difficult, and molecular characterizations have only used one genetic region. We evaluated multiple phylogenies of the Ascosphaera using up to six loci: the Internal Transcribed Spacer (ITS) region, 18S rRNA, 28S rRNA, Elongation Factor-1α (EF-1α) the RNA polymerase II largest subunit (RPB1), and the second largest subunit (RPB2). The ITS sequence alone produced an inadequate phylogeny, and the addition of both the 18S and 28S rRNA loci to the ITS sequence produced a phylogeny similar to that based on all six genetic regions. For all phylogenies, Ascosphaera torchioi was in a separate clade that was the most basal, with a strong genetic similarity to Eremascus albus, introducing the possibility of paraphyly within Ascosphaera. Also, based on this new phylogeny, we now suggest that the Apis mellifera (honey bee) pathogens arose within a group of saprophytes, and the Megachile (leafcutting bees) pathogens arose separately.

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis.

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A. apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A. apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.

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).

Molecular phylogeny of animal pathogen Lacazia loboi inferred from rDNA and DNA coding sequences.

Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.