Molecular Characterization of Reptile Pathogens Currently Known as Members of the Chrysosporium Anamorph of Nannizziopsis vriesii Complex and Relationship with Some Human-Associated Isolates

Table 1.

Isolates examined in this study

Nucleotide sequence accession numbers.

The GenBank accession numbers for the newly generated ITS and SSU sequences are listed in Table 1. Additional ITS sequences were deposited in GenBank under accession no. KF477239 for Amauroascus albicans (strain UAMH 3102), KF477241 for A. glomerata (strain UAMH 3551), KF477243 for N. mirabilis (strain UAMH 7712), and KF477240 and KF477242 for the Nannizziopsis-like isolates UAMH 3124 and UAMH 4036.

Phylogenetic analyses of the SSU rRNA gene and ITS rRNA gene partitions.

The SSU data matrix comprised 60 taxa and 1,702 characters, of which 208 were parsimony informative. The ITS data matrix comprised 96 taxa and 857 characters, of which 416 were parsimony informative. Maximum parsimony analyses resulted in 3,641 most parsimonious trees (MPT) of score 692 for the SSU and 6,543 MPT of score 3,161 for the ITS (Fig. 1 and 2). The large number of trees found was due to the uncertainty of relationships within the Onygenaceae (SSU) and the treatment of the multiple identical sequences for the target groups by the branch-swapping algorithm (ITS). The topology for the MPT shown in Fig. 1 was retained in the corresponding strict consensus of all trees, except for the arrangements of some taxa within the uppermost clade of the Onygenaceae. Although the genetic diversity sampled for the ITS analyses was broad, the resulting tree structure was congruent with that derived from the SSU data for the target taxa. Bayesian analyses resulted in a single consensus tree for each gene region. In both the SSU and ITS analyses, the reptile isolates grouped into three individually strongly supported clades (BS of 98 to 100 and PP of 100 for each clade) designated A, B, and C that represent the genus Nannizziopsis and two new lineages. There was strong support for the three lineages within the family Onygenaceae of the Onygenales (Fig. 1) (BS, 93; PP, 100), and the lineages were distinct from all Chrysosporium species and other taxa (Fig. 1 and 2). Clade A was strongly supported as a sister to clade B (BS, 100; PP, 100) by the SSU analyses but not by ITS analysis. Clades A and B were additionally supported, individually and as sister clades, by long branches in the SSU tree, indicating substantial genetic differentiation. Both A and B clades comprised several subclades. Clade A included eight subclades encompassing the ex-type culture of N. vriesii (I), chameleon and gecko isolates (II), crocodile isolates (IIIa), isolates from agamid and iguanid lizards (IIIb and IV), and humans (V, VI, and VII) (Fig. 2). The ITS sequence for strain UAMH 5875 (subclade VII), confirmed using DNA extracted from two independently grown stock cultures, was unusually long, with a large insertion of approximately 150 nucleotides in the ITS1 spacer region. No strictly anamorphic reptile isolate grouped with the teleomorphic N. vriesii. An isolate from the leopard gecko (UAMH 11231) was an intermediate between subclades I and II. Subclade IV included the ex-type culture of N. guarroi and all unclassified isolates from inland bearded dragons and iguanas. An isolate from a coastal bearded dragon (UAMH 11185) was excluded and grouped as a sister to subclade IIIa. Clade B comprised three subclades. Subclade VIIIa included isolates from acrochordid snakes, tuataras, and a coastal bearded dragon. Tentacled snake isolates from a Canadian zoo and those from an American zoo grouped in separate subclades (VIIIb and IX). A sequence from the ex-type culture of C. ophiodiicola and all unclassified isolates from semiaquatic and terrestrial snakes were placed in clade C. A relationship of clade C with clades A and B was unresolved. Excluded from Nannizziopsis clade A were all species currently or formerly classified in Arachnotheca or Nannizziopsis, including A. glomerata, A. albicans (formerly N. albicans), and N. mirabilis. Moreover, there was no close relationship between these species (Fig. 2). Similarly, the two unclassified Nannizziopsis-like isolates did not group with any sampled species, so their sequences were deposited as Onygenaceae species.

Based on the new international code of nomenclature for algae, fungi, and plants, which ends the separate naming of different states of fungi (Article 59) (33), we assigned members of clade A to Nannizziopsis and described six new species for the lizard and human subclades. We propose the new genus Paranannizziopsis for members of clade B, with three new species encompassing isolates from highly aquatic squamates, tuataras, and a coastal bearded dragon. Clade C included C. ophiodiicola and all unclassified isolates from aquatic, semiaquatic, and terrestrial snakes that are described here as Ophidiomyces ophiodiicola.

Morphology.

Although isolates from reptiles may be suspected as belonging to one of the genera described here based on the macro- and micromorphological characteristics described below, the morphological distinctions within and between clades A, B, and C were generally insufficient for a reliable identification of isolates to the species level without the use of DNA sequencing. Table 2 provides a summary of the morphological and physiological characteristics for these species. All isolates were moderately fast growing on PDA at 30°C and had yellowish-white, velvety to powdery, dense, and sometimes zonate, colonies, with uncolored to yellowish reverse (Fig. 3 and Table 2). Isolates streaked on PDA sometimes demonstrated a mixture of mycelial colonies and moist-to-glabrous transitory yeast-like colonies (Fig. 3E and K). Exudate droplets occasionally formed on the colony surfaces, but diffusible colored pigments were not produced on PDA. Thermotolerance distinguished a few species that demonstrated good growth at 35°C (e.g. Fig. 3B, J, M, P, and S). All isolates were tolerant of cycloheximide and perforated hairs. All isolates produced aleurioconidia, which are solitary conidia released by lytic dehiscence (Fig. 4 to 9). The aleurioconidia were commonly sessile, sometimes subtended by slightly swollen cells, or formed at the ends of short stalks. They were clavate or pyriform with truncate bases, occasionally subglobose or obovate, mostly single celled, and occasionally two celled. The aleurioconidia resembled those of some Chrysosporium and Trichophyton species, and this similarity has led to the misidentification of isolates in the past; however, two morphological characteristics distinguished the fungi described here. Nannizziopsis and Ophidiomyces species commonly have chains of adjacent cylindrical arthroconidia that are produced by schizolytic fragmentation of the hyphae (Fig. 4 to 7 and 9C and D). This type of arthroconidial development occurred also in Paranannizziopsis crustacea (Fig. 9B) but was lacking in the two other Paranannizziopsis species. These and other species occasionally produced rhexolytically dehiscing intercalary arthroconidia, a type of arthroconidium produced by many onygenalean fungi (Fig. 8B). The second notable characteristic, occurring in members of all three genera but not in Chrysosporium species or dermatophytes, was the formation of short, solitary, undulate, lateral branches that were occasionally sparsely septate (Fig. 4 to 9). Arthroconidia sometimes demonstrated budding and were found especially in the moist yeast-like colonies (Fig. 3E, 4D, and 5D). Arthroconidia were also produced in vivo in the epidermises of infected animals (Fig. 10A and Table 1). Some isolates produced ascomatal initials (Fig. 6J, 7D, and 7F), typically within cottony sectors, or developed infertile ascomata (Fig. 5E and 6D). With the exception of N. vriesii, isolates failed to produce ascospores in cultures that were grown on OAT or other media and incubated for several months. Isolates demonstrated varied responses on BCP-MS-G and in urea broth (Table 2). Variation sometimes occurred among isolates of a species or between subcultures from the powdery and cottony sectors of an individual isolate. Most O. ophiodiicola isolates demonstrated alkalinization, as well as strong clearing of the medium. Isolates were weakly to strongly urease positive except for a single isolate from a human source. O. ophiodiicola isolates often produced a mercaptan (skunk-like) odor.

Nannizziopsis Currah.

Mycotaxon 24:160, 1985. MycoBank accession no. MB 25725). Type species: Nannizziopsis vriesii (Apinis) Currah, Mycotaxon 24:164, 1985 (subclade I) (MycoBank accession no. MB 104542). N. vriesii was described and illustrated by Currah (42) and Apinis (13) and is distinguished from all the other Nannizziopsis species described below by the production of ascomata. Ascomata (gymnothecia) produced on OAT at 30°C were composed of hyaline anastomosing asperulate hyphae containing small globose punctate-reticulate ascospores measuring 2.2 to 3 μm in diameter (Fig. 4A). Colonies on PDA were 4.5 to 5.5 cm in diameter after 21 days, velvety to slightly cottony, and furrowed. Growth was inhibited at 35°C, with colonies attaining 2.5 cm in diameter (Fig. 3A and B). Aleurioconidia were 2.5 to 6 μm long (in rare cases up to 8 μm long) and 1.5 to 2.7 μm wide (Fig. 4B). Isolates produced undulate hyphae and cylindrical fission arthroconidia measuring 2.7 to 7.3 μm long and 1.7 to 2.7 μm wide and sometimes showing yeast-like budding (Fig. 4C and D). Notes: The ex-type strain was isolated from the skin and lungs of an Ameiva lizard and was the only isolate in this study from a teiid lizard. No details are available on the course of infection. The isolate from soil (UAMH 3526) was the only one obtained from an inanimate substrate. Although Stchigel et al. (30) identified an isolate from human infection (RKI 04-0104) as N. vriesii, we suggest that it is closer to Nannizziopsis obscura, a human-pathogenic species (see Notes for N. obscura).

Nannizziopsis dermatitidis Sigler, Hambleton, and Paré sp. nov. (subclade II).

MycoBank accession no. MB 804604. Colonies on PDA attained 3.8 to 4.7 cm diameter after 21 days and were strongly zonate and powdery with a thin margin (Fig. 3C). Most isolates failed to grow at 35°C. Aleurioconidia were clavate to pyriform and measured 2.8 to 7.5 μm long if single-celled, up to 9 μm long if 2-celled, and 1.2 to 3 μm wide (Fig. 5A). Undulate branches and cylindrical- to slightly barrel-shaped fission arthroconidia were formed with arthroconidia measuring 2.8 to 9 μm long and 1.5 to 3 μm wide (Fig. 5B and C). Transitory yeast-like colonies grown on PDA at 30°C were composed of ovoid-to-cylindrical yeast-like cells and arthroconidia (Fig. 5D). Similarly, arthroconidia and yeast-like cells predominated in cultures of an isolate from day gecko (UAMH 6610) that were glabrous and cerebriform centrally but cottony at the periphery. Colonies of this isolate grew slowly at both 30°C (2.5 cm in diameter) and 35°C (0.8 cm in diameter). Holotype: Canada, Ontario, ex-liver and kidney of male F. lateralis, 24 November 1993, UAMH 7583, dried specimen and living culture. Etymology: of skin lesions. Notes: Infections in chameleons and geckos are recorded (Table 1) (3, 15, 18). In an experimental challenge of veiled chameleons, the ex-type culture of N. dermatitidis induced lesions confirmed by histopathology and culture (4). A day gecko isolate confirmed here as N. dermatitidis was reported originally as a Trichophyton species that caused deep dermatitis in geckos imported to Germany (15). An isolate from leopard gecko (UAMH 11231) (18) appeared to be similar to N. dermatitidis, but its intermediate placement between N. vriesii and N. dermatitidis in the ITS tree (Fig. 2) suggests that an analysis of additional isolates is required to resolve its taxonomic position.

Nannizziopsis crocodili Sigler, Hambleton, and Paré sp. nov. (subclade IIIa).

MycoBank accession no. MB 804605. Colonies on PDA were 4 to 5.5 cm in diameter, velvety to powdery, slightly to strongly zonate, and sometimes with exudate droplets after 21 days (Fig. 3F). Growth was slow at 35°C (1 to 2.7 cm diameter after 21 days). Aleurioconidia were subglobose, measuring 1.5 to 2.5 μm long and 1.3 to 2.4 μm wide, and sessile or borne on swollen cells either on the vegetative mycelium or within ascomata-like structures (pseudogymnothecia) (Fig. 5E, F, and G). The pseudogymnothecia developed within 2 to 3 weeks on both PDA and OAT and were composed of branched septate, anastomosing, and asperulate hyphae surrounding masses of conidia. No ascospores were produced in cultures incubated for several months or when available isolates were mated. Arthroconidia measuring 3.7 to 7.5 μm long and 2 to 3 μm wide were produced at low frequency and often showed germination (Fig. 5H and I). Undulate hyphae were formed (Fig. 5H, inset). Holotype: Australia, Gulf of Carpentaria, skin lesion on C. porosus, 1999, A. Thomas, UAMH 9666, dried specimen and living culture. Etymology: of the crocodile. Notes: This species is known only as the cause of fatal dermatitis in farmed Australian saltwater crocodiles, with hatchlings developing plaque-like lesions (25). It is distinguished from all other Nannizziopsis species by the small subglobose conidia often subtended by swollen cells, and by the formation of pseudogymnothecia.

Nannizziopsis barbata Sigler, Hambleton, and Paré sp. nov. (subclade IIIb).

MycoBank accession no. MB 804606. Colonies on PDA were 5.5 to 6 cm in diameter, powdery, flat to slightly raised and cottony at the center, but otherwise zonate after 21 days (Fig. 3H). There was no growth at 35°C. Aleurioconidia were pyriform to clavate, measured 3 to 6.5 μm long and 1.8 to 2.5 μm wide, and were sessile or borne on slightly swollen cells (Fig. 6A). Fission arthroconidia measuring 4.4 to 8.5 μm long and 1.7 to 3.5 μm wide, as well as undulate hyphae, were commonly produced (Fig. 6B). Moist colonies on PDA demonstrated budding (Fig. 6C). Infertile pseudogymnothecia composed of hyaline, anastomosing, asperulate hyphae were produced on OAT (Fig. 6D). Holotype: Australia, New South Wales, Penrith, skin lesion on P. barbata, 10 June 2009, UAMH 11185, dried specimen and living culture. Etymology: of the species P. barbata. Notes: N. barbata is thought to be the cause of an outbreak of infection in four P. barbata housed together in an outdoor enclosure at the Taronga Zoo (10). Histopathology confirmed hyphae in the skin of all four animals, the liver in two cases, and the heart of one animal. Cultures of a Chrysosporium-like fungus obtained from two animals were not held for follow-up investigation. However, a fifth wild-caught captive animal presented at the same clinic with ulcerated skin lesions in which histopathology sections revealed hyphae. The ex-type isolate obtained from a skin specimen at necropsy demonstrated the highest ITS similarity (92% similarity) with N. crocodili isolates from Australia (Fig. 2).

Nannizziopsis guarroi (J. Cabañes and Abarca) J. Cabañes, Abarca, Stchigel, and Cano (30) (subclade IV).

Holotype CBS 124553. Colonies on PDA were 2.7 to 4.7 cm in diameter, powdery, sometimes sectoring to cottony, often strongly zonate, sometimes with exudate droplets (Fig. 3I). Growth at 35°C was similar, with colonies attaining 2.3 to 4 cm diameter (Fig. 3J). Aleurioconidia were clavate to pyriform and measured 3.2 to 6.5 μm long and 1.5 to 2.5 μm wide (Fig. 6E). Undulate hyphae were common (Fig. 6F). Arthroconidia in chains measured 2.8 to 7 μm long and 2 to 3.7 μm wide and sometimes showed budding in young cultures (Fig. 6G). Notes: N. guarroi is distinguished from other reptile-associated Nannizziopsis species by its slightly lower growth rate at 30°C and good growth at 35°C. Our results differ from those of Stchigel et al. (30) with respect to BCP-MS-G responses, in that repeated testing of isolates showed no pH change or slight acidification (trace yellow) rather than alkalinization, as was reported in that study. In the ITS tree (Fig. 2), N. guarroi groups with Nannizziopsis hominis and other species from human sources, all of which grow well at 35°C. N. guarroi has been recorded frequently from inland bearded dragons and green iguanas that are commonly kept as pets, as well as from sungazers (C. giganteus) and common agama (Agama agama) (7–9, 11, 12, 16, 30, 34). This species is considered the etiologic agent of yellow fungus disease in inland bearded dragons, a contagious and progressive necrogranulomatous dermatomycosis first observed about 15 years ago. Two isolates from coastal bearded dragons (P. barbata) were excluded from N. guarroi and grouped in N. barbata (subclade IIIb) and Paranannizziopsis australasiensis (subclade VIIIa), respectively.

Nannizziopsis infrequens Sigler, Hambleton, and Paré sp. nov. (subclade V).

MycoBank accession no. MB 804608. Colonies attained 6 cm in diameter and were flat, velvety to powdery (Fig. 3L). At 35°C, colonies were 5 cm in diameter and more radially furrowed (Fig. 3M). Clavate to pyriform aleurioconidia were 2.2 to 5.5 μm long and 1.5 to 2.7 μm wide (Fig. 6H). Occasional intercalary arthroconidia, undulate hyphae, and ascomatal initials were produced but fission arthroconidia were not observed (Fig. 6I and J). Holotype: United States, Iowa, human bronchial wash specimen, 3 November 2003, UAMH 10417, dried specimen and living culture. Etymology: infrequent (in occurrence). Notes: The ex-type strain was isolated from a bronchial washing obtained from an HIV⁺ male with pneumonia; however, the isolate was not considered to be contributing to the lung infection, and therefore no antifungal therapy was given (27). Because the patient resided in an area that is endemic for histoplasmosis, an AccuProbe test (Gen-Probe, San Diego, CA) was performed and tested positive in two different tests, albeit at lower relative light unit (RLU) values than are typically obtained with Histoplasma capsulatum controls (27) (Table 2). Tests performed at the submitting laboratory indicated that the isolate grew at 40°C. The human-associated N. infrequens differs from the recently described reptile-associated species N. chlamydospora at 12 positions in the ITS region and by the absence of chlamydospores; it differs from N. draconii at 18 positions in the ITS region (Fig. 2), by its ability to grow at 40°C, and by the absence of alkalinity on BCP-MS-G (30).

Nannizziopsis hominis Sigler, Hambleton, and Paré sp. nov. (subclade VI).

MycoBank accession no. MB 804609. Colonies on PDA were 4.2 to 4.8 cm in diameter and were velvety to powdery, flat, radially furrowed or slightly zonate, and sometimes sectoring (Fig. 3O). At 35°C, colonies were 3 to 3.5 cm in diameter, velvety, and flat to strongly furrowed (Fig. 3P). Growth was similar on peptone yeast extract (PYE) after 21 days at 30°C, but a slight yellow diffusible pigment was produced (Fig. 3Q). Aleurioconidia were single-celled, rarely 2-celled, and measured 2.3 to 7 μm long and 1.5 to 3.2 μm wide (Fig. 7A). Undulate hyphae and fission arthroconidia were produced (Fig. 7B and C). Arthroconidia were cylindrical- to barrel-shaped, with rounded ends, and measured 2.4 to 9 μm long by 1.4 to 3.1 μm wide. Some ascomatal initials were present in cottony sectors (Fig. 7D). Holotype: United States, California, human thigh mass, 1994, UAMH 7859, dried specimen and living culture. Etymology: of man. Notes: N. hominis was isolated in 1994 from a deep muscle mass on the right thigh, right groin, buttock, and lung of an HIV⁺ male who died 8 months after the initial isolation, presumably of complications from AIDS. The patient received itraconazole for the fungal infection. He resided in the Sacramento, CA area, worked in business, had no history of drug use, and had no pets. The isolate from the lungs was tentatively identified initially as a Trichophyton species. In 2000, a white mold was isolated three times from the swollen lymph nodes of an immunocompetent Nigerian man from the Boston, MA area who presented with disseminated adenopathy following a trip to Nigeria. One of the isolates from this patient tested positive in the AccuProbe Blastomyces culture identification test (Table 2) and was sent to the Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio, TX, for evaluation. Because of its Chrysosporium-like morphology, the isolate was then forwarded to the UAMH for further review. The patient was readmitted to the hospital 6 months later and found to have a disseminated fungal infection involving the heart (endocarditis), lungs, spleen, and kidneys. The fungus was not regrown, but the patient had been on itraconazole since the initial diagnosis and remained on the drug for 2 years. An isolate from disseminated disease in a Nigerian-American (GenBank accession no. HF547876, isolate UTHSC R-4317) that was identified as N. guarroi by Stchigel et al. (30) appears to represent another N. hominis isolate. The ITS sequence differs in only 3 positions, and the isolate grew at 40°C, whereas N. guarroi isolates did not grow at this temperature.

Nannizziopsis obscura Sigler, Hambleton, and Paré sp. nov. (subclade VII).

MycoBank accession no. MB 804610. Colonies growing at 30°C and 35°C were similar, attaining 5 cm in diameter, thin, often sectoring, with sectors glabrous to felty or thinly cottony, flat, or furrowed (Fig. 3R and S). Conidia were sessile or on short stalks, clavate, or pyriform, occasionally 2-celled, and measured 2.5 to 7 μm long and 1.6 to 2.7 μm wide (Fig. 7E). Ascomatal initials and undulate hyphae were produced (Fig. 7F and H). Arthroconidia measuring 3.1 to 7.7 μm long and 1.6 to 3.2 μm wide were produced in glabrous sectors and sometimes showed budding (Fig. 7G). Holotype: United States, New York, human leg abscess, 1984, UAMH 5875, dried specimen and living culture. Etymology: hidden (occurrence). Notes: N. obscura was isolated from two successive biopsy samples of a large tibial abscess in the right leg of a 24-year-old African-American male with a 2-month history of osteomyelitis (28). The patient's history included a trip to Africa and exposure to dust during home renovations. Histologic sections of the specimen revealed septate hyphae and budding yeasts within and around giant cells (28). The fungus was reported to grow at 37°C and on medium with cycloheximide and to produce numerous arthroconidia initially thought to resemble Geotrichum. It was identified as a Chrysosporium sp. when it was observed to also produce typical aleurioconidia (28). The patient was treated with amphotericin B over a 4-month period. An isolate causing a brain infection in an HIV⁺ Nigerian male in Germany was identified as the CANV, but no details on the methods used to identify the fungus were provided (35). Stchigel et al. (30) identified the isolate (RKI 04-0104; GenBank accession no. HF547869) as N. vriesii, but its ability to grow at 40°C, low ITS similarity with other N. vriesii isolates (93%), and the low support for the grouping with N. vriesii in both phylogenetic analyses make this identification questionable. Their sequence groups with N. obscura, also a human pathogen, but differs at 14 positions along the ITS region.

Paranannizziopsis Sigler, Hambleton, and Paré gen. nov.

MycoBank accession no. MB 804611. Colonies were pale and moderately fast growing. Vegetative hyphae were narrow, branched, and septate, sometimes with racquet mycelia. Conidia (aleurioconidia) were sessile or produced on slightly swollen cells or on short stalks and were released by rhexolytic dehiscence. They were hyaline, smooth, pyriform, and clavate to obovate. Arthroconidia were absent, intercalary, or produced in adjacent chains. Undulate lateral branches were produced. No teleomorph was produced. Paranannizziopsis species are distinguished from Nannizziopsis and Ophidiomyces species by the uncommon occurrence or absence of fission arthroconidia (Table 2). Type species: P. australasiensis Sigler, Hambleton, and Paré.

Paranannizziopsis australasiensis Sigler, Hambleton, and Paré sp. nov. (subclade VIIIa).

MycoBank accession no. MB 804612. Colonies on PDA attained 4.5- to 5-cm diameter and were powdery or sometimes cottony, flat, or faintly zonate (Fig. 3U). There was no growth at 35°C. Aleurioconidia were sessile or were subtended by slightly swollen cells from which one or two conidia were produced (Fig. 8A). The conidia were pyriform to clavate and measured 3.5 to 8 μm long and 1.5 to 2.7 μm wide. Occasional intercalary arthroconidia and undulate hyphae were produced (Fig. 8B and C). Ascomatal initials occurred in cottony sectors and appeared as inflated cells with secondary proliferations (Fig. 8D and E). Some mycelia surrounding the ascomatal initials demonstrated swollen intercalary cells (Fig. 8F). Holotype: New Zealand, Auckland, skin lesion of S. punctatus punctatus, C. Harvey, 2011, UAMH 11645, dried specimen and living culture. Etymology: from Australasia. Notes: All isolates have been obtained from animals housed in zoos in Australia or New Zealand. Two aquatic file snakes (Acrochordus species) from the Melbourne Zoo presented with rapidly developing multifocal necrotizing skin lesions. Three isolates were associated with skin lesions in northern tuataras held in the Auckland Zoological Park, Auckland, New Zealand (36). A coastal bearded dragon (P. barbata) from the same zoo was diagnosed postmortem when the fungus was grown from a stored frozen biopsy specimen.

Paranannizziopsis californiensis Sigler, Hambleton, and Paré sp. nov. (subclade VIIIb).

MycoBank accession no. MB 804614. Colonies on PDA attained 4.5- to 5.2-cm diameter and were powdery and flat to slightly zonate (Fig. 3V). Growth at 35°C was strongly inhibited. Aleurioconidia were clavate to pyriform or obovate, measured 4 to 8.5 μm long and 1.8 to 2.6 μm wide, and were sessile or borne on a slightly swollen cell (Fig. 8G, arrow). Undulate hyphae were uncommon, and arthroconidia were not observed. Ascomatal initials occurred in cottony sectors and were associated with large irregularly shaped cells (Fig. 8H and I). The latter measured 10 to 36 μm long and 3.5 to 9.5 μm wide. Holotype: United States, California, San Diego, skin lesion of Erpeton tentaculatum, L. Sigler, 8 May 2006, UAMH 10693, dried specimen and living culture. Etymology: from California. Notes: P. californiensis was isolated from the skin or scales of three of five aquatic captive snakes (E. tentaculatum) housed at the San Diego Zoo. Several snakes died in the outbreak. Although both P. californiensis and P. crustacea were isolated from E. tentaculatum, they differ genetically (Fig. 2), morphologically (Table 2), and potentially, histopathologically. While typical cylindrical arthroconidia were observed in the tissues of lesions caused by P. crustacea (Fig. 10A), aleurioconidia were observed at the surface of a lesion caused by P. californiensis (Fig. 10B) A review of additional cases will be required to determine if the formation of aleurioconidia in tissue is a consistent finding in P. crustacea infections.

Paranannizziopsis crustacea Sigler, Hambleton, and Paré sp. nov. (subclade IX).

MycoBank accession no. MB 804615. Colonies on PDA attained 5.8 to 6.5 cm diameter and were powdery, flat, occasionally with dense downy overgrowth (Fig. 3W). There was no growth at 35°C. Aleurioconidia were clavate to pyriform or obovate, sessile or formed on short stalks, and measured 4 to 7.5 μm long and 2 to 3.5 μm wide (Fig. 9A). Undulate hyphae, fission arthroconidia, and occasional intercalary arthroconidia were produced (Fig. 9B). Arthroconidia measured 3.8 to 9.2 μm long and 1.9 to 2.7 μm wide. Holotype: Canada, Ontario, skin lesion of E. tentaculatum, L. Sigler, 11 June 2002, UAMH 10199, dried specimen and living culture. Etymology: crusty (lesion). Notes: P. crustacea caused fatal dermatitis in four captive tentacled snakes (24). On the surface of the ulcers was a crust of cellular debris containing clusters of arthroconidia. In culture, the isolates produced powdery and cottony sectors that when subcultured, differed in their preponderance of aleurioconidia and arthroconidia, respectively, and in the physiological reactions they elicited. The powdery aleurioconidial colony type was strongly urease positive and showed strong clearing of milk solids on BCP-MS-G medium, in contrast to the cottony arthroconidial type that was weakly urease positive and showed less clearing on BCP-MS-G (Table 2).

Paranannizziopsis longispora (Stchigel, Deanna A. Sutton, Cano, and Guarro) Sigler, Hambleton, and Paré comb. nov.

MycoBank accession no. MB 805156. Basionym: Chrysosporium longisporum Stchigel, Deanna A. Sutton, Cano, and Guarro, Persoonia 31:93, 2013. MycoBank accession no. MB 801990. The sequence of C. longisporum (GenBank accession no. HF547873, isolate UTHSC R-4380) groups closest to P. crustacea but differs at 9 positions in the ITS region. This level of sequence difference, combined with morphological differences, including the absence of growth at 30°C, absence of fission arthroconidia in chains, and longer conidia (3 to 13 μm long), provides support for the retention of both species (30).

Ophidiomyces Sigler, Hambleton, and Paré (see reference 29) (IF550166).

MycoBank accession no. MB 550166. Colonies were yellowish-white and moderately fast growing. Vegetative hyphae were narrow, branched, and septate, occasionally with racquet mycelia. Conidia were sessile or borne on short stalks and released by rhexolytic dehiscence (aleurioconidia). Aleurioconidia were hyaline, smooth, and cylindrical to clavate. Arthroconidia were formed in chains by schizolytic fragmentation of hyphae or were sometimes intercalary. Short, undulate, and sparsely septate lateral branches were common. No teleomorph is known. Type species: O. ophiodiicola (Guarro, Deanna A. Sutton, Wickes, and Rajeev) Sigler, Hambleton, and Paré (see reference 29) (IF550167). MycoBank accession no. MB 550167. Basionym: C. ophiodiicola Guarro, Deanna A. Sutton, Wickes, and Rajeev, J. Clin. Microbiol. 47:1208. MycoBank accession no. MB 506606. Holotype: United States, Georgia, Sparta, black rat snake with mycotic granuloma, 2009, CBS 122913 (isotypes FMR 9510, UTHSC 07-604 [R-3923]). Colonies on PDA were 4 to 6 cm in diameter and were velvety to powdery, dense, flat, frequently zonate, and sometimes with cottony sectors (Fig. 3X). Clear exudate droplets were often present. Most isolates failed to grow at 35°C, but two isolates (UAMH 10949 and UAMH 11295) attained a diameter of 2 to 3.5 cm after 21 days. Aleurioconidia were sessile or borne at the ends of short stalks, cylindrical to clavate, and 2.5 to 7.5 μm long and 1.5 to 2.5 μm wide (Fig. 9C). Arthroconidia were 3 to 12.5 μm long (in rare cases up to 15 μm long) by 1.5 to 3.5 μm wide (Fig. 9D). In young cultures on PDA, arthroconidia sometimes showed budding or germination. Undulate hyphae were commonly produced (Fig. 9D). Rarely, these fragmented to form chains of arthroconidia. Some isolates produced ascomatal initials in cottony sectors. Most isolates produced a strong to weak mercaptan-like odor. Notes: In addition to the mycotic granuloma in a black rat snake (26), published reports concerning O. ophiodiicola, and confirmed here by the sequencing of isolates, include fatal dermatitis in brown tree snakes attributed to the CANV (isolate UAMH 6642) (19), disseminated infection in a garter snake attributed to Chrysosporium queenslandicum (isolate UAMH 9832) (20), disseminated dermatitis in green anacondas (isolate UAMH 10949) (21), and moist dermatitis in an Australian broad-headed snake (isolate UAMH 11295) (23). Fungal dermatitis in wild-caught captive carpet snakes (Morelia spilotes variegata) was attributed to Geotrichum (37), but the development of arthroconidia at the surface of the lesions is highly characteristic of O. ophiodiicola infection (19). A diagnosis of CANV in a boa constrictor was based on the appearance of the skin lesions and of the hyphae in tissue (22). Most cases involve captive animals, but O. ophiodiicola is also associated with infections in wild snakes. The isolates from Nerodia species in the present study were from wild-caught snakes that were part of a zoology research study in Florida (Table 1). Six of 30 snakes became infected. A sequence (GenBank accession no. JX878608) from an isolate from one of 11 wild timber rattlesnakes (Crotalus horridus) from Massachusetts showed high homology with the O. ophiodiicola sequences described here (38) (Fig. 2). Similarly, direct PCR of five skin biopsy samples from eastern massasauga rattlesnakes yielded amplicons that showed >99% homology with O. ophiodiicola (GenBank accession no. EU715819) (5). Most isolates that we studied came from the United States, with others from Australia, Germany, or the United Kingdom (Table 1). Only one isolate (UAMH 9985) was not associated with infection and came from skin scales of a captive African rock python (P. sebae) in a southwestern zoo during a survey of shed reptile exuviae (6).

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