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Abstract

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
Thumbnail image of graphical abstractThumbnail image of graphical abstract

Background

Molecular analysis methods have led to many changes in the taxonomy of dermatophyte species.

Hypothesis/Objectives

We hypothesized that fungi displaying morphology consistent with a traditional identification of ‘Trichophyton mentagrophytes’ represent multiple species, consistent with the new taxonomy.

Methods

Fungal specimens (= 20) were collected directly from animals with dermatophytosis, were among those submitted for diagnostic analysis or were part of historical teaching collections. Primers that amplified a portion of the 28S ribosomal RNA gene and primers specific for a fragment from the internal transcribed spacer region were used for PCR amplification of genomic DNA. The DNA sequences from the amplified products were compared with databases to identify the isolates.

Results

Of the 80% (= 16) of the fungal isolates identified as Arthroderma benhamiae, eight were collected from dogs. One isolate was identified as Arthroderma vanbreuseghemii, two were Trichophyton erinacei and one was Nannizziopsis (Chrysosporium) guarroi, which was probably present as a saprophyte.

Conclusions and clinical importance

Frequent isolation of A. benhamiae from dogs suggests a greater host range for this fungus than reflected in the current literature. Our data also suggest the potential for geographical restriction of strain types within the species. Efforts to identify fungal isolates using molecular techniques create a better understanding of diversity and epidemiology of the dermatophytes.

Résumé

Contexte

Les méthodes d'analyse moléculaire ont menées à de nombreux changements de taxonomie des espèces de dermatophytes.

Hypothèses/Objectifs

Nous supposons que les champignons dont la morphologie est compatible avec l'identification traditionnelle de ‘Trichophyton mentagrophytes’ représentent de multiples espèces correspondant à la nouvelle taxonomie.

Méthodes

Les espèces de champignons (= 20) ont été isolées directement sur des animaux atteints de dermatophytose, parmi les prélèvements soumis pour analyse diagnostique ou faisaient partie de collections d'enseignement. Les amorces qui amplifiaient une portion de gène ribosomique 28S et les amorces spécifiques d'un fragment de région d’ITS ont été utilisées pour amplification par PCR. Les séquences d’ADN des produits amplifiés étaient comparées avec les bases de données pour identifier les isolats.

Résultats

Sur 80% (= 16) des souches fongiques identifiées en tant que Arthroderma benhamiae, huit étaient prélevées sur des chiens. Une souche était identifiée en tant que Arthroderma vanbreuseghemii, deux étaient Trichophyton erinacei et une était Nannizziopsis (Chrysosporium) guarroi, probalement un saprophyte.

Conclusions et importance clinique

L'isolement fréquent de A. benhamiae des chiens suggère un portage plus important pour ce champignon qu’évoqué dans la littérature actuelle. Nos données suggèrent également une potentielle restriction géographique des types de souches au sein des espèces. Les efforts d'identification des souches fongiques à l'aide de techniques moléculaires, permettent une meilleure compréhension de la diversité et de l’épidémiologie des dermatophytes.

Resumen

Introducción

el uso de métodos de análisis molecular han llevado a muchos cambios en la taxonomia de las especies de dermatofitos.

Hipótesis/Objetivos

nuestra hipótesis es que los hongos que presentan una morfología típica de Trichophyton mentagrophytes representan múltiples especies, en consonancia con la nueva taxonomia.

Métodos

se obtuvieron muestras de hongos (n = 20) directamente de animales con dermatofitosis entre muestras remitidas para análisis diagnostico o que formaban parte de colecciones históricas para enseñanza. Se utilizaron iniciadores que ampliaban una porción del gen RNA 28S ribosomal e iniciadores específicos de un fragmento de la región interna espaciadora transcrita para la amplificación mediante PCR del DNA genómico. Las secuencias de DNA de los productos amplificados se compararon con bases de datos para identificar los aislados.

Resultados

del 80% (n = 16) de los aislados identificados como Arthroderma benhamiae, ocho fueron obtenidos de perros. Un aislado fue identificado como Arthroderma vanbreuseghemii, dos fueron Trichphyton erinacei y uno fue Nannizziopsis (Chrysosporium) guarroi, el cual era posiblemente un saprofito.

Conclusiones e importancia clínica

el aislamiento frecuente de A. benhamiae de perros sugiere un rango de hospedadores más amplio para este hongo que lo que refleja la literatura científica actual. Nuestros datos también sugieren la posibilidad de una restricción geográfica de algunas cepas dentro de la especie. Los esfuerzos para identificar los aislados fúngicos utilizando técnicas moleculares crea un mejor conocimiento de la diversidad y epidemiología de los dermatofitos.

Zusammenfassung

Hintergrund

Molekulare Analysemethoden haben zu vielen Änderungen in der Taxonomie der Dermatophyten Spezies geführt.

Hypothese/Ziele

Wir stellen die Hypothese auf, dass Fungi, die eine Morphologie aufweisen, die traditionell zur Identifizierung als Trichophyton mentagrophytes führt, multiple Spezies repräsentieren, die mit der neuen Taxonomie übereinstimmen.

Methoden

Es wurden Pilzelemente (= 20) direkt von Tieren mit einer Dermatophytose gesammelt und zur diagnostischen Analyse eingeschickt. Andere Proben kamen aus einer historischen Kollektion, die zu Schulungszwecken verwendet wurde. Zur PCR Amplifizierung der genomischen DNA wurden Primer verwendet, die einen Teil des 28S ribosomalen RNA Gens amplifizierten, sowie Primer, die spezifisch für ein Fragment der inneren transkribierten Spacer Region waren. Um die Isolate zu identifizieren wurden DNA Sequenzen der amplifizierten Produkte mit denen in Datenbanken verglichen.

Ergebnisse

Von den 80% (= 16) der Pilzisolate, die als Arthroderma benhamiae identifiziert wurden, wurden acht von Hunden genommen. Eines der Isolate wurde als Arthroderma vanbreuseghemii identifiziert, zwei waren Trichophyton erinacei und eines war Nannizziopsis (Chrysosporium) guarroi; letzterer kam eventuell als Saprophyt vor.

Schlussfolgerungen und klinische Bedeutung

Eine häufige Isolierung von A. benhamiae von Hunden weist auf eine größere Breite an Wirten hin, als in der gängigen Literatur reflektiert wird. Unsere Daten weisen auch auf die Möglichkeit einer geografischen Einschränkung von Stammtypen innerhalb einer Spezies hin. Versuche, Pilzisolate mittels molekularen Techniken zu identifizieren führen zu einem besseren Verständnis der Diversität und der Epidemiologie der Dermatophyten.


Introduction

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal species classified as dermatophytes are the cause of dermatophytosis, more commonly known as ringworm. Dermatophytes can be highly contagious, with spread between humans, between animals, or zoonotic transfer between animals and humans. Isolation and culture of the dermatophyte associated with a clinical condition is a prerequisite for its identification. Identification is often pursued with the intent of understanding the source of the infection and to prevent its spread. Identification historically, and even currently, is commonly accomplished by evaluation of colony and cellular morphology of the dermatophyte isolate.

Molecular methods for dermatophyte identification have been introduced in the recent past and provide the opportunity for a more detailed taxonomy of dermatophyte species. This area of study is dynamic, with species names in seemingly continuous flux as new insights are gained into the diversity of dermatophyte species. Gräser et al.[1] reviewed these concepts and proposed a polyphasic approach to dermatophyte taxonomy. They highlighted the idea that isolates traditionally identified as ‘Trichophyton mentagrophytes’ represent a diverse taxonomic group, i.e. a species complex. The goal of this work was to use molecular identification methods on a collection of fungi that were isolated from clinical cases of dermatophytosis in animal species. We hypothesized that the isolates that fit the traditional description of ‘T. mentagrophytes’ represent a diverse range of fungal taxa.

Materials and methods

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates (n = 20; Table 1) with colony and microscopic morphology that could fit within the broad designation of ‘T. mentagrophytes’ were obtained from the Veterinary Teaching Hospital, the Veterinary Diagnostic Laboratory and historical teaching collections at the College of Veterinary Medicine, University of Illinois at Urbana-Champaign. Medical records were available for 13 of the isolates; these were collected between 2006 and 2011. Teaching collection isolates dated back as far as 1989.

Table 1. Summary of fungal isolates analysed in this study
Morphological identificationMolecular identificationSource of isolate
CatDogChinchillaHedgehogUnknown
Trichophyton mentagrophytes (n = 20) 19217
 Arthroderma benhamiae (n=16)181 6
 Arthroderma vanbreuseghemii (n=1)  1  
 Trichophyton erinacei (n=2)   11
 Nannizziopsis (Chrysosporium) guarroi (n=1) 1   

Genomic DNA was extracted from cultures grown on Sabouraud agar plates. Mycelium was homogenized by vortexing in the presence of glass beads, similar to the method of Ninet et al.[2] The homogenate was extracted with phenol–chloroform–isoamyl alcohol, precipitated with ethanol and further purified using a QIAamp DNA mini kit according to the manufacturer's instructions (Qiagen).

Genomic DNA from each fungal isolate was amplified by PCR using primers specific to the internal transcribed spacer (ITS) region of the nuclear-encoded ribosomal RNA genes (rDNA; primers 18SF1 and 58SR1),[3] as well as primers specific for the 28S rRNA-encoding gene (primers LSU1 and LSU2).[2, 4]

The PCR products were purified and their DNA sequences determined by the staff at the Keck Functional Genomics Laboratory of the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Multiple approaches were used to identify the isolates, based on their DNA sequences. BLAST searches (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) were completed. Although this approach was helpful, misleading results may be obtained because many sequences have been submitted to public databases without rigorous identification of the fungal isolate. To minimize this difficulty, ITS sequences were matched against the ITS rDNA barcode database (http://www.cbs.knaw.nl/dermatophytes/BioloMICS.aspx). This database contains sequences from fungal isolates that have been rigorously identified using methods such as mating to tester strains to define species.[5] As our identification results became more refined, we compared our data with reference sequences from type strains or carefully validated isolates,[1, 4, 5] to lend additional confidence to our conclusions. Genus and species names used here followed conventions described by Gräser et al.[1]

Results

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Table 1 shows the results for molecular characterization of the 20 fungal isolates. Sixteen of the strains (3208, 3209, 3216, 3220, 3329, 3350, 3355, 3356, 3360, 3361, 3368, 3369, 3370, 3371, 3376 and 3378) were identified as Arthroderma benhamiae. The ITS amplicon sequences (335 bp) from all 16 isolates were identical and a 100% match to the corresponding region from the A. benhamiae type strain sequence (GenBank accession number AB088677). The 28S sequences (313 bp) from all 16 isolates were identical to each other, but showed one mismatch to the reference sequence from a carefully characterized tester strain (accession number GU646875);[4] our isolates were polymorphic at position 293 in this sequence, showing A or G (single nucleotide code R), compared with G in the reference sequence. Collectively, these results provided confidence that the 16 isolates were correctly identified as A. benhamiae.

One of the fungal isolates (3210; from a chinchilla) was identified as Arthroderma vanbreuseghemii (Table 1). The ITS sequence for this isolate (343 bp) matched 100% in the overlapping region with the GenBank sequence AF170452. This sequence was derived from a fungal strain that was also isolated from a chinchilla (IHEM 10162).[5] Strain IHEM 10162 provided fertile mating products with A. vanbreuseghemii tester strains.[5] The 28S sequence for strain 3210 (313 bp) was a 100% match with the corresponding sequence from strain CBS428.63, a strain recognized as A. vanbreuseghemii (Trichophyton interdigitale).[1]

Two of the isolates (3211 and 3362) matched sequences from Trichophyton erinacei strains. The ITS sequences (335 bp) from these strains were identical to each other. Searching the ITS barcode database showed 100% identity between our sequence and T. erinacei, which accounted for the top 10 matches in the list. Comparison of our sequence with GenBank accession number Z97997, which was cited as representative of the species,[1] further supported our conclusions. Owing to ambiguities in the large public databases, searches with the 28S sequences from these isolates were not productive.

BLAST searches with the ITS (301 bp) and 28S (314 bp) sequences from isolate 3353 (collected from a dog) matched 100% with sequences from Chrysosporium guarroi strain CCFVB CH10 (GenBank accession numbers FJ839684 and EU018451, respectively). Also apparent on the search results were numerous sequences from additional isolates of the same species, now renamed as Nannizziopsis guarroi.[6] Re-examination of the colony morphology for strain 3353 indicated its similarity to published N. guarroi images.[6]

Discussion

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results showed the utility of DNA sequence analysis to identify and differentiate between isolates that fit into the traditional category of T. mentagrophytes. Considerable effort has been dedicated to understanding the taxonomy of this group of fungi in the recent past.[1, 4, 5] Results here also display the complexities and dynamic nature of taxonomy for this group of fungi. In the current literature, manuscripts with the newest taxonomic designations are found side by side with those that use historical terminology. Conclusions regarding fungal diversity within a species and the epidemiology of the corresponding disease will gain clarity as the nomenclature becomes consistent.

The association between fungus and host species supported some previously reported relationships, as well as revealing some new trends. Consistent with our findings, A. vanbreuseghemii has been isolated from chinchillas,[5] and T. erinacei has been isolated from hedgehogs.[7] Isolation of Nannizziopsis (Chrysosporium) guarroi from a dog may be an incidental finding, because Chrysosporium spp. may be saprophytic and unrelated to the dog's clinical presentation. Nannizziopsis guarroi is an emerging pathogen of reptiles.[6] Chrysosporium spp. are known to produce a colour change on dermatophyte test medium, perhaps prompting preservation of the isolate from the primary culture.[8]

Results for the A. benhamiae isolates were the most novel. Current literature emphasizes the connection between A. benhamiae isolates and guinea pigs.[9] This correlation emerged from a survey of 274 pets in Switzerland in 2006–2007. The study noted isolation of A. vanbreuseghemii from dogs and cats, but A. benhamiae only from guinea pigs. In our study, 80% of the fungal isolates were A. benhamiae (Table 1), with 80% of strains of known origin collected from dogs. All of these dogs had a clinical presentation consistent with a diagnosis of dermatophytosis.

Arthroderma benhamiae taxonomy is gaining research attention. Symoens et al.[4] completed additional taxonomic analysis of this fungal species, focusing on DNA sequence comparisons and mating interactions for strains that originated in Switzerland. Their work identified two groups among the Swiss A. benhamiae strains, i.e. group I (white colony type) and group II (yellow colony type). Our A. benhamiae sequences matched the ITS (AB088677) and 28S (GU646875) reference sequences for group II (yellow); however, our isolates have many, varied colony phenotypes, each of which more closely resembles the white type described for group I. Symoens et al.[4] showed that this combination of sequence data and colony phenotype is typified by the most ‘classic’ mating tester strains for A. benhamiae, some of which date back to description of the A. benhamiae species in 1967.[10] It is possible that A. benhamiae strains demonstrate geographical restriction and that our species are typical of a North American isolate. Our frequent isolation of these strains suggests that they are common and readily collected from dogs. Further analysis of these specimens will provide additional insight into the growing picture of strain diversity and epidemiology of A. benhamiae.

Acknowledgements

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Karen Campbell, Carol Maddox, Pat Hoien-Dalen, and Sandy Grable for making fungal isolates available for this study. We thank Sherry Qiu, a participant in the University Laboratory High School Summer Research Experience, for her enthusiasm and contributions to the project. We thank the staff at the Keck Functional Genomics Laboratory of the Roy J. Carver Biotechnology Center (UIUC) for generating the DNA sequence data.

References

  1. Top of page
  2. RésuméResumenZusammenfassung
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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    Makimura K, Tamura Y, Mochizuki T et al. Phylogenetic classification and species identification of dermatophyte strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol 1999; 37: 920924.
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    Carroll HF. Evaluation of dermatophyte test medium for diagnosis of dermatophytosis. J Am Vet Med Assoc 1974; 165: 192195.
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    Drouot S, Mignon B, Fratti M et al. Pets as the main source of two zoonotic species of the Trichophyton mentagrophytes complex in Switzerland, Arthroderma vanbreuseghemii and Arthroderma benhamiae. Vet Dermatol 2009; 20: 1318.
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    Ajello L, Cheng S-L. The perfect state of Trichophyton mentagrophytes. Sabouraudia 1967; 5: 230234.