Medical Mycology, 2018, 56, 610–620 doi: 10.1093/mmy/myx083 Advance Access Publication Date: 9 October 2017 Original Article Original Article Characterization of Nannizziopsis guarroi with genomic and proteomic analysis in three lizard species Juliane Schneider1, Tilo Heydel2, Linus Klasen1, Michael Pees1, Wieland Schrodl¨ 2 and Volker Schmidt1,∗ 1Clinic for Birds and Reptiles, University of Leipzig, Leipzig, Germany and 2Institute of Bacteriology and Mycology, University of Leipzig, Leipzig, Germany ∗To whom correspondence should be addressed. Volker Schmidt, Dr. med. vet., Dip ECZM (avian & herp), Clinic for Birds and Reptiles, University of Leipzig, An den Tierkliniken 17, D-04103 Leipzig, Germany. Tel: 0049 341 97 38 421; Fax: 0049 341 97 38 409; E-mail: [email protected] Received 12 February 2017; Revised 11 April 2017; Accepted 24 August 2017; Editorial Decision 10 May 2017 Abstract 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. Key words: CANV, Lacerta viridis, Pogona vitticeps, Iguana iguana, yellow fungus disease. Introduction Nannizziopsis guarroi is an ascomycetous, filamentous fun- gus of the family Nannizziopsiaceae (Eurotiomycetes: Ony- Fungal infections caused by different emerging obligate genales) identified as a member of the Chrysosporium and facultative pathogenic fungi in captive as well as in anamorph of Nannizziopsis vriesii complex (CANV).3–5 free-living reptiles are increasing the last two decades.1,2 610 C The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: [email protected] Downloaded from https://academic.oup.com/mmy/article-abstract/56/5/610/4372450 by Georgetown University Library user on 04 June 2018 Schneider et al. 611 Different reports of recent years have indicated several ker- nary practice, it is important to generate new and supple- atinophilic CANV as a reason for often fatal superficial or ment existing databases.23 deep dermatomycosis in a number of reptile species whereas The aim of this study was to prove accuracy in identifi- human-associated species exist as well.3,4,6,7 Nannizziopsis cation of N. guarroi with MALDI-TOF MS in comparison guarroi appeared mainly in central bearded dragons (Pog- to nucleotide sequence analysis. ona vitticeps) and green iguanas (Iguana iguana) world- 3,4,6,8,9 wide causing the so called ‘Yellow fungus disease’. Methods Besides skin lesions, rare cases of systemic infection ex- ist.10 Nannizziopsis chlamydospora and N. draconii have Animals and sampling procedure recently been described in central bearded dragons with Nannizziopsis guarroi isolates were obtained from a to- 8,11 dermatomycosis. Another primary pathogenic species of tal of seven lizards out of three collections; four central the Onygenales order is Ophidiomyces ophiodiicola, caus- bearded dragons out of two collections, two green iguanas, 12,13 ing snake fungal disease. While snake fungal disease and one European green lizard (Lacerta viridis) from a third is well known in captive snakes all over the world, it is collection. All lizards were adult. Central bearded dragons also emerging in free-ranging snake populations across the were bred in Germany. The other lizards came from an 1,14 United States. animal shelter; their origin is unknown. Contact between Cytological and histopathological examination of the the collections was not reported. Lizards displayed a severe skin and underlying tissue biopsy samples is advisable in dermatitis, which was also seen in other members of their re- 2 order to diagnose deep fungal dermatitis. Voriconazole spective collection. Swabs from the oral cavity, tongue, tra- applied orally at 10 mg/kg bodyweight once every 24 h chea, skin, and cloaca were sampled sterile with individually up to 70 days is recommended as an efficient and safe packed sterile microbiological swabs (Applimed, Chatel-St-ˆ treatment option of CANV-complex-induced dermatitis in Denis, Freiburg, Switzerland) during the thorough clinical 15 bearded dragons. The fungus may be difficult to identify examination. Four of the seven lizards were euthanized due without isolation and differentiation, because the aleurio- to the severity and expansion of dermatitis, and swabs were conidia are similar to those of other Chrysosporium or Tri- taken from the oral cavity, tongue, lungs, intestines, and chophyton species, and the arthroconidia resemble those of liver with individually packed sterile microbiological swabs 4 Geotrichum or Trichosporon species. (Applimed) during post mortem examination. Skin sam- Identification of these fungal species capable of induc- ples as well as visceral organs from the euthanized lizards ing fatal disease is of great importance to initiate targeted were assessed by cytological and histopathological exam- antifungal therapy and reduce mortality in reptiles. Next ination followed by mycological examination of the re- generation sequencing is considered the gold standard for spective swabs. The remaining three central bearded drag- accurate identification of human pathogenic yeasts and fila- ons, all from one collection, were treated successfully with 16 mentous fungi. Phylogenetic studies on fungal pathogens voriconazole as recommended.17 of the CANV-complex focus on fragments of the small sub- unit ribosomal DNA (SSU), DNA sequencing of the nu- Isolation, cultivation and antifungal susceptibility clear ribosome regions ITS1-5.8S-ITS2, and domains 1 and 2 (D1/D2) of the large subunit ribosomal DNA (LSU), as The collected swabs were plated onto Sabouraud- well as actin and β-tubulin genes.4,5,11 Chloramphenicol-Gentamycin-Agar (SAB-CHL/GEN) Matrix-assisted laser desorption/ionisation time-of- (Oxoid, Wesel, Germany) as well as on Potato-Dextrose- ◦ flight mass spectrometry (MALDI-TOF MS) is established Agar (PDA) (Oxoid) and incubated at 30 C. All sample ◦ as a time-saving, accurate, affordable, and feasible diag- cultures were stored at −70 C in cryotubes (Roti-Store nostic tool for identification of fungi and bacteria in clin- cryotubes, Carl Roth, Karlsruhe, Germany) in order ical microbiology.17–20 Species differentiation of dermato- to preserve the isolates for further investigation. After phytes grown in culture by MALDI-TOF-MS, especially re-cultivation of the isolates, their colonial features and Trichophyton species, has recently been reported as fast growth rates were documented on SAB-CHL/GEN and ◦ ◦ and very specific method.21 Currently, identification of op- PDA and incubated at 30 C and 35 C for 10 days. Growth portunistic pathogenic fungi can be unsatisfactory due to of the cultures was observed and recorded after 5 and restrictions of commercially obtainable databases.22 There 10 days. Susceptibility was screened by means of disc are no reports on identification of reptile-associated fungi diffusion testing. Fungal colonies were first diluted in using MALDI-TOF MS. In order to facilitate and enhance Nutrient Broth with Glucose (Oxoid), then the fluid was the practicability of fungal species identification in veteri- distributed on SAB-CHL/GEN (Oxoid) and incubated at Downloaded from https://academic.oup.com/mmy/article-abstract/56/5/610/4372450 by Georgetown University Library user on 04 June 2018 612 Medical Mycology, 2018, Vol. 56, No. 5 35◦C for 120 h. Utilized test discs included voriconazole sity of Leipzig, Leipzig, Germany). Cultures were incubated 1 µg (CT1807B; Oxoid), fluconazole 25 µg (CT1806B; for 10 days, followed by inoculation of samples in two Oxoid), itraconazole 8 µg (81812 N; Neo-SensitabsTM, fluid-culture mediums, Brain-Heart-Infusion broth (BHI-B, Rosco Diagnostica A/S, Taastrup, Denmark), nystatin 100 Sifin, Berlin, Germany) and Sabouraud broth (SAB-B, Sifin), international units (CT0073B; Oxoid), amphotericin B which were then incubated for one to two days at room 20 µg (051916048; LiofilchemR , Roseto degli Abruzzi, temperature. Ten microliters