First Report of Ophidiomycosis in a Free-Ranging California Kingsnake (Lampropeltis californiae) in California, USA.

Ophidiomycosis (snake fungal disease) is an emerging threat to snake health worldwide. We report a case of disseminated ophidiomycosis in a California kingsnake (Lampropeltis californiae) from Plymouth, Amador County, California, US, which is the first report of the disease in this species and in a free-ranging snake in California.

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.

Histopathology, Molecular Identification and Antifungal Susceptibility Testing of Nannizziopsis arthrosporioides from a Captive Cuban Rock Iguana (Cyclura nubila).

Chrysosporium-related fungi, the cause of superficial and deep mycoses, are an emerging infectious disease affecting not only reptiles but also immunocompromized humans. However, the information on Nannizziopsis arthrosporioides is extremely scarce. We herein characterized N. arthrosporioides isolated from a Cuban rock iguana (Cyclura nubila). Three skin ulcers were found in a Cuban rock iguana after captivity for 8 years. Microscopic examination revealed hyperplastic, hyperkeratotic, and ulcerative dermatitis coupled with numerous branched, septate fungal hyphae. The fungal culture yielded growth of zonate, felted cottony-powdery colonies with lobate margins on medium. Maximum-likelihood phylogeny tree based on the combined partial actin and partial ß-tubulin genes demonstrated that current isolates were mostly close to N. arthrosporioides. Furthermore, antifungal susceptibility test demonstrated that N. arthrosporioides had lowest minimal inhibitory concentration (MIC) values to isavuconazole, efinaconazole, and luliconazole, which may be the potential treatment of choice for N. arthrosporioides infection. The current study describes the first confirmed case of dermatomycosis caused by N. arthrosporioides of a captive reptile in Asia with detailed descriptions of the clinical, histopathological, and mycological features. The current findings provide new information on global distribution and host range of N. arthrosporioides and can raise the concern on the transboundary or emerging disease of N. arthrosporioides in Asian region.

Prevalence of Ophidiomyces ophiodiicola, the Causative Agent of Snake Fungal Disease, in the Interior Plateau Ecoregion of Tennessee, USA.

The fungal pathogen Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, has been implicated in declines of North American snake populations since 2006 and the geographic range of this pathogen is still not fully known. In Tennessee, US, O. ophiodiicola has been detected since 2012, but large portions of the state have not been surveyed for this pathogen. Our primary objective was to monitor the prevalence of O. ophiodiicola in the Interior Plateau ecoregion of Tennessee by swabbing all snakes that were encountered during road cruising survey efforts in 2017 and 2018. Eleven snakes of four species, copperhead (Agkistrodon contortrix), common water snake (Nerodia sipedon), black kingsnake (Lampropeltis nigra), and smooth earthsnake (Virginia valeriae), tested positive for the presence of O. ophiodiicola. Overall, 9.2% (11/120) of snakes sampled tested positive for the presence of O. ophiodiiola, and we further observed a seasonal trend in detections with summer months having the greatest frequency of detections. Our results extend the known geographic range of O. ophiodiicola in Tennessee by adding four previously unconfirmed O. ophiodiicola-positive counties. Further sampling will need to be conducted across west Tennessee because this is the most data-deficient region of the state. Our results offer additional evidence of the presence of this pathogen in Tennessee and will help researchers further understand the geographic distribution and host range.

HOST FACTORS AND TESTING MODALITY AGREEMENT ASSOCIATED WITH OPHIDIOMYCES INFECTION IN A FREE-RANGING SNAKE POPULATION IN SOUTHEAST OHIO, USA.

Snake fungal disease (SFD) is an emerging mycotic disease caused by Ophidiomyces ophiodiicola, and has been demonstrated to impact snake populations of conservation concern in the United States negatively. Although Ophidiomyces has been shown to affect diverse taxa and to have a broad distribution, host factors associated with infected individuals and optimal testing protocols are not yet well characterized. The purpose of this study was to evaluate host factors and agreement across testing modalities associated with Ophidiomyces infection in a free-ranging snake population in southeast Ohio. Wild-caught snakes were swabbed and biopsied to test for Ophidiomyces via quantitative polymerase chain reaction (qPCR), culture, and histopathology. The host parameters assessed were species, sex, snout-vent length, body weight, month captured, and presence of gross skin lesions. A total of 8/30 individuals across three species-Black Racers (Coluber constrictor), Grey Ratsnakes (Pantherophis spiloides), and Eastern Gartersnakes (Thamnophis sirtalis sirtalis)-tested positive via at least one testing modality for Ophidiomyces infection. There were no associations between sex, snout-vent length, or weight and Ophidiomyces infection status. A higher proportion of individuals with gross lesions tested positive for Ophidiomyces than those without gross lesions, and most individuals that tested positive were caught in April or May. A low level of agreement was observed across testing modalities. Swab qPCR identified the most Ophidiomyces-positive individuals, and fungal culture identified the fewest at 0 individuals. Although there are limitations associated with a sample size of 30, these findings support the potential of this pathogen to infect individuals broadly across species and size, highlighting the relevance of this disease for snake conservation efforts. They also suggest that although little agreement was observed across test modalities, the concurrent use of multiple modalities is a more sensitive method for characterizing prevalence and distribution of Ophidiomyces.

MYCOTIC DERMATITIS IN JUVENILE FRESHWATER CROCODILES (CROCODYLUS JOHNSTONI) CAUSED BY NANNIZZIOPSIS CROCODILI.

Nannizziopsis crocodili, a contagious, keratinophilic fungus, was identified from biopsied tissue in a captive juvenile freshwater crocodile during an outbreak of severe multifocal dermatitis affecting four of five crocodiles. Lesions progressed from superficial, well-demarcated ulceration of scales, to black pigmentation, localized edema, erythema, and flattening of the scales. Treatment with topical enilconazole provided clinical improvement in three of four crocodiles but all developed terminal gout. One crocodile did not develop clinical disease despite long-term exposure. This is the first report of N. crocodili in freshwater crocodiles and in a location remote to the index Australian case.

Evidence of Vertical Transmission of the Snake Fungal Pathogen Ophidiomyces ophiodiicola.

Snake fungal disease (ophidiomycosis) is an emerging infection of snakes caused by Ophidiomyces ophiodiicola. Little is known about mechanisms of this pathogen's transmission and its implications for conservation of wild snake populations. We report four cases with evidence of vertical transmission of O. ophiodiicola from dam to offspring.

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.

Field Diagnostics and Seasonality of Ophidiomyces ophiodiicola in Wild Snake Populations.

Snake fungal disease (SFD) is an emerging disease caused by the fungal pathogen, Ophidiomyces ophiodiicola. Clinical signs of SFD include dermal lesions, including regional and local edema, crusts, and ulcers. Snake fungal disease is widespread in the Eastern United States, yet there are limited data on how clinical signs of SFD compare with laboratory diagnostics. We compared two sampling methods for O. ophiodiicola, scale clip collection and swabbing, to evaluate whether collection method impacted the results of polymerase chain reaction (PCR). In addition, we evaluated the use of clinical signs to predict the presence of O. ophiodiicola across seasons, snake habitat affiliation (aquatic or terrestrial) and study sites. We found no significant difference in PCR results between sampling methods. Clinical signs were a strong predictor of O. ophiodiicola presence in spring and summer seasons. Snakes occupying terrestrial environments had a lower overall probability of testing positive for O. ophiodiicola compared to snakes occupying aquatic environments. Although our study indicates that both clinical signs of SFD and prevalence of O. ophiodiicola vary seasonally and based on habitat preferences of the host, our analysis suggests that clinical signs can serve as a reliable indicator of O. ophiodiicola presence, especially during spring and summer.

POSTNATAL MORTALITY IN NEONATE RATTLESNAKES ASSOCIATED WITH OPHIDIOMYCES OPHIODIICOLA.

Ophidiomycosis, historically referred to as snake fungal disease (SFD), caused by Ophidiomyces ophiodiicola, is a significant disease of snakes characterized by crusty scales, pustules, subcutaneous nodules, and death. Ophidiomycosis is a proposed threat to sustainability of free-ranging snake populations throughout the United States and Europe, but the clinical progression during periods of reproductive activity (gravid females, neonates) is unknown. In spring 2012, five apparently healthy gravid eastern massasauga (Sistrurus catenatus) rattlesnakes from Clinton County, Illinois, were brought into captivity to give birth and be returned into the population. While in captivity, one adult female and 21 neonates died. Five individuals were subsequently confirmed positive for O. ophiodiicola by using quantitative polymerase chain reaction (qPCR). In 2016, a gravid timber rattlesnake (Crotalus horridus) with ophidiomycosis from Jackson County, Illinois, gave birth in captivity to 13 neonates. Skin swabs were taken from all neonates immediately after birth and confirmed negative for O. ophiodiicola by using qPCR. The neonates remained housed with the positive female for 10 days before all animals were reswabbed and released back into the wild. One neonate was O. ophiodiicola positive at time of release. The initial negative result followed by a positive result several days postpartum suggests that the neonate was infected by the female after direct contact. Both case series represent natural infection of neonates after parturition and highlight the importance of this disease in a demographically important age class.

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.

Molecular detection of airborne Emergomyces africanus, a thermally dimorphic fungal pathogen, in Cape Town, South Africa.

Emergomyces africanus is a thermally dimorphic fungus that causes a systemic mycosis in immunocompromised persons in South Africa. Infection is presumed to follow inhalation of airborne propagules. We developed a quantitative PCR protocol able to detect as few as 5 Es. africanus propagules per day. Samples were collected in Cape Town, South Africa over 50 weeks by a Burkard spore trap with an alternate orifice. We detected Es. africanus in air samples from 34 days (10%) distributed over 11 weeks. These results suggest environmental exposure to airborne Es. africanus propagules occurs more commonly in endemic areas than previously appreciated.

Attempted molecular detection of the thermally dimorphic human fungal pathogen Emergomyces africanus in terrestrial small mammals in South Africa.

The ecological niche of Emergomyces africanus (formerly Emmonsia species), a dimorphic fungus that causes an AIDS-related mycosis in South Africa, is unknown. We hypothesized that natural infection with E. africanus occurs in wild small mammals. Using molecular detection with primers specific for E. africanus, we examined 1402 DNA samples from 26 species of mole-rats, rodents, and insectivores trapped in South Africa that included 1324 lung, 37 kidney, and 41 liver specimens. DNA of E. africanus was not detected in any animals. We conclude that natural infection of wild small mammals in South Africa with E. africanus has not been proven.

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.

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.

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.

Cutaneous blastomycosis. An imported case with good response to itraconazole / Blastomicosis cutánea. Caso clínico importado con buena respuesta al itraconazol

Background. Blastomycosis is a subacute or chronic deep mycosis caused by a dimorphic fungus called Blastomyces dermatitidis, which generally produces a pulmonary form of the disease and, to a lesser extent, extra-pulmonary forms such as cutaneous, osteoarticular and genitourinary, among others. Cutaneous blastomycosis is the second clinical presentation in frequency. It is considered as primary when it begins by inoculation of the fungus due to traumas, and secondary when the lung fails to contain the infection. Case-report. We present the case of a 57 year-old male who had a 5 year-history of an irregularly shaped verrucous infiltrative plaque related to and insect bite and posterior trauma due to the manipulation of the lesion. B. dermatitidis was identified using direct examination, stains, isolation in culture media, histopathology, and molecular studies. An antifungal susceptibility test was performed using method M38-A2 (CLSI). Clinical and mycological cure was achieved with itraconazole. Conclusions. This cutaneous blastomycosis case acquired in the United States (Indianapolis) is rather interesting and looks quite similar to other mycoses such as coccidioidomycosis or sporotrichosis. The presented case shows one of the multiple issues concerning migration between neighboring countries (AU)

Antecedentes. La blastomicosis es una micosis profunda, subaguda o crónica, causada por el hongo dimorfo Blastomyces dermatitidis, que generalmente produce una enfermedad pulmonar y, en menor proporción, formas extrapulmonares (cutánea, osteoarticular y genitourinaria). La blastomicosis cutánea es la segunda forma clínica más observada; es de tipo primario cuando se presenta por la inoculación del hongo a través de traumatismos, y de tipo secundario cuando se disemina a partir de un foco pulmonar. Caso clínico. Se presenta el caso de un varón de 57 años con blastomicosis cutánea verrugosa de 5 años de evolución, relacionada con la picadura de un insecto, que empeora con la manipulación de la lesión. El caso fue confirmado por la identificación de B. dermatitidis en exámenes directos, tinciones, aislamiento en medios de cultivo habituales, análisis histopatológico y estudios moleculares. Se realizó una prueba de sensibilidad antifúngica por el método M38-A2 (CLSI). La curación clínica y micológica se logró con itraconazol oral. Conclusiones. Este caso de blastomicosis cutánea adquirida en Estados Unidos (Indianápolis) es de gran interés y resulta similar a los de otras micosis, como la coccidioidomicosis y la esporotricosis. Este caso refleja uno de los múltiples factores relacionados con la migración entre países cercanos (AU)