Introduction

Trichophyton equinum is a zoophilic dermatophyte that is frequently isolated from horse dermatophytosis and rare infections in humans [1,2,3]. Morphological and physiological characteristics of this fungus have been previously described, as well as molecular analyses were also performed for the identification [1,2,3,4]. The related species, T. tonsurans, is an anthropophilic dermatophyte, which is frequently reported as a source of human dermatophytosis throughout the world [4]. Since approximately 2000, cases of this dermatophytosis in Japan have significantly increased among contact sports participants [5], indicating that this dermatophytosis represents an emerging fungal disease epidemic in this country [5]. While T. tonsurans and T. equinum have been treated as different species [4], they are indistinguishable from each other when tested by standard morphological and physiological tests [6]. However, de Hoog et al., sequenced for internal transcribed spacer (ITS) region, D1–D2 region of large subunit (LSU) of ribosomal DNA and partial β-tubulin genes of both species. They reported that T. equinum could as yet not be distinguished from T. tonsurans by the molecular typing [4]. Kandemir et al. reported that 40 isolates (26 of T. equinum and 14 of T. tonsurans) harbored a “T” single nucleotide polymorphism (T-SNP) at position 18 in the internal transcribed spacer (ITS) region. All strains with an ITS region C-SNP at this position were human isolates of T. tonsurans [7]. The authors concluded that the evolution of T. tonsurans and T. equinum must be relatively recent and the speciation process might not yet be complete [7]. Therefore, the taxonomy of both dermatophytes is complicated. Furthermore, since horse dermatophytosis due to T. equinum is one of the important diseases in the field of veterinary dermatology, the classification of this fungus is important in the veterinary medicine. In the present study, molecular and physiological testing were performed on T. equinum isolated from dermatophytoses of Japanese racehorses to assess genotype and phenotype patterns of these strains. To our knowledge, this is the first report employing genotyping and phenotyping analyses of T. equinum in Asia to understand the evolution of this species.

Materials and Methods

Strains

Seven T. equinum clinical isolates were obtained from seven horse cases in Ibaraki, Japan, in 2019 (Table 1). In all cases, dermatophytoses were diagnosed by detecting arthroconidia around the affected hairs by skin scraping examination.

Table 1 Strains and identifications of results of this study
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ITS Region Sequences and β-tubulin (BT2) Sequence Identification

Genomic DNA was isolated as reported previously [8]. Molecular characteristics of the strains were also identified by sequence analysis of the ITS region. The ITS region of isolates was amplified using universal fungal primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATAGC-3′) [9].

To sequence BT2 of isolates, primers were prepared based on the T. equinum isolate NBRC 31,610 beta-tubulin gene, partial sequence (GenBank accession no. JF731092). The primers TeTUB-S (5′- GCCCGAAAAACACGACACGG-3′; position 60–79) and TeTUB-R (5′- GCTCACCAGAAAGCAGCACC-3′; position 320–339) were used.

PCR was carried out in a total reaction volume of 30 µl amplification mixture [10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 200 mM of each deoxynucleotide triphosphate, 1.0 U of PrimeSTAR HS DNA Polymeras (Takara, Kyoto, Japan), and 50 µM of each primer]. PCR amplification was performed using the following conditions: 30 cycles of denaturation for 30 s at 95 °C, primer annealing for 30 s at 55 °C, and extension for 1 min at 72 °C. Resulting amplified DNA fragments were electrophoresed on a 2% (w/v) agarose gel with 1 × TAE buffer and visualized by ethidium bromide staining. (Fig. 1).

Fig. 1
figure 1

Colony and microscopic morphologies of the strain NUBS20001. a, Photo showing the flat and radial grooves in the center, brown to yellow in color, with a villous surface b, Microscopic photo showing numerous subspherical to pyriform microconidia were present. Macroconidia were abundant and cigar- to club-shaped and were smooth and thin-walled

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DNA bands, approximately 550-bp for ITS and 280-bp for BT2 were excised from the gel, purified using the ExoSAP-IT® kit (USB Corporation, Cleveland, OH, USA), and sequenced on the ABI PRISM 3130 DNA Analyzer (Thermo Fisher Scientific, Inc., Tokyo, Japan).

Comparative sequence analyses were carried out using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website.

PCR Analysis of Alpha-box (MAT1-1) and HMG-box (MAT1-2) Genes Specific to the Genomic DNA of Isolates

Primers TmMATa1S and TmMATa1R amplify a 471-bp fragment of the T. mentagrophytes alpha-box (MAT1-1) gene fragment. Primers TmHMG1S and TmHMG1R amplify a 524-bp fragment of the T. mentagrophytes HMG-box (MAT1-2) gene fragment [8].

Genomic DNA (100 ng) from the clinical isolates was amplified by PCR in a total reaction volume of 30 µl amplification mixture containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 200 mM of each deoxynucleoside triphosphate, 1.0 unit Taq polymerase (Takara), and 50 nM of each primer. Amplification was carried out over 35 cycles consisting of template denaturation for 1 min at 94 °C, primer annealing for 1 min at 55 °C, and polymerization for 2 min at 72 °C. Resultant PCR products were detected by electrophoresis on 2% agarose gels followed by staining with ethidium bromide and visualization under UV light.

Physiological Investigation for Nicotinic Acid Requirement

Trichophyton Agar 1, 4 and 5 (Thermo Fisher Scientific, Waltham, MA, USA) were used to investigate nicotinic acid requirement [1,2,3, 10].

Results

ITS Identity and Comparison of SNPs in the ITS Region and BT2

Comparative nucleotide sequence analysis using the BLAST algorithm on the NCBI website showed that ITS sequences amplified from all isolates were 100% identical to T. equinum reference strains (GenBank accession nos. MH862816.1 and KT155693.1). Additionally, the homology of region sequences of all isolates was also 99% identical to T. tonsurans reference strains (GenBank accession nos. NR_144891.1 and MH865912.1).

ITS sequences amplified among the isolates were 100% identical and harbored a T-SNP at position 18 (Table 1).

Comparative nucleotide sequence analysis using the BLAST algorithm on the NCBI website showed that BT2 sequences amplified from all isolates were 100% identical to T. equinum reference strains (GenBank accession no. JF731092). Additionally, the homology of region sequences of all isolates were also 99% identical to T. tonsurans reference strains (GenBank accession nos. MN714012).

The BT2 sequences amplified among the isolates were 100% identical and harbored a A-SNP at position 176 as in T. equinum BT2 [Trichophyton equinum isolate NBRC 31,610 beta-tubulin gene, partial sequence (GenBank accession no. JF731092)] (Table 1). The G-SNP exists at the same position in T. tonsurans BT2 [Trichophyton tonsurans isolate NBRC 5928 beta-tubulin gene, partial sequence (GenBank accession no. JF731073)] (Table 1).

PCR Detection of MAT1-2

The MAT1-2 allele was detected by PCR in all seven isolates. Conversely, the MAT1-1 allele was not detected in any isolate (Table 1).

Nicotinic Acid Requirement

All horse isolates grew only on Trichophyton Agar 5 and did not grow on Trichophyton Agar 1 and 4 (Table 1).

Disscussion

In this report, molecular and physiological testing were performed on T. equinum isolated from dermatophytoses of Japanese racehorses. All strains were physiologically (nicotinic acid requirement) and genetically (harbored a T-SNP at position 18 in the ITS region) close to the European type of T. equinum. However, the nucleotide sequence similarity of the ITS region was 100% among the examined isolates, suggesting that Japanese T. equinum is a clonal population with T. equinum reference strains.

PCR analysis of MAT1-2 and MAT1-1 genes indicated that the MAT1-2 allele was detected by PCR in all seven isolates. Kandemir et al. also reported that all clinical isolates of T. equinum were possessed the MAT1-2 allele and not the MAT1-1 allele [7]. In our previous study, PCR analysis confirmed that all 60 T. tonsurans strains in Japan contained the MAT1-1 allele, while none contained the MAT1-2 allele [11]. Moreover, T. equinum and T. tonsurans showed a SNP difference in the BT2 that were A-SNP at position 176 in T. equinum and G-SNP at the same position in T. tonsurans, respectively.

All horse isolates grew only on Trichophyton Agar 5 and did not grow on Trichophyton Agar 1 and 4 (Table 1). Trichophyton agar 5, indicating that nicotinic acid is required [1,2,3, 10]. T. tonsurans can grow on Trichophyton Agar 1 and 4 [1,2,3, 10].

From the above results, it was reconfirmed that T. equinum and T. tonsurans isolated in Japan are molecularly and phylogenetically different. The origin of Japanese racehorses was the thoroughbred, which was imported from the European region after nineteenth century. Japanese T. equinum might have been imported together with horses at that time and has evolved a monophyletic.