Medical Mycology 2002, 40, 439–442
Accepted 27 December 2001; Final revision received 12 February 2002
Case Report
Molecular identi®cation of Trichophyton rubrum isolate
from a dog by chitin synthase 1 (CHS1) gene analysis
R. KANO*, A. HIRAI*, M. YOSHIIKEy, M. NAGATA**, Y. NAKAMURAz, S. WATANABEz & A. HASEGAWA*
*Department of Pathobiology, Nihon University School of Veterinary Medicine, 1866, Fujisawa, Kanagawa, 252-8510, Japan;
yYoshiike Animal Hospital, 3-25-101, Mistusawa Shimocho, Kanagawaku, Yokohama, Kanagawa, 221-0852, Japan; **Animal
Dermatology Center, ASC, 1-3-2, Higashicho, Jindaiji, Chofushi, Tokyo, 182-0012, Japan; zDepartment of Dermatology, Teikyo
University School of Medicine, 11-1, Kaga-2, Itabashiku, Tokyo, 173-8605, Japan.
A nonsporulating isolate from a dog with dermatophytosis was identiŽed as
Trichophyton rubrum by molecular analysis. The nucleotide sequence analysis of the
chitin synthase 1 (CHS1) gene from the isolate indicated more than 99% sequence
similarity with other human and canine isolates of T. rubrum. The molecular typing
suggested that isolates of T. rubrum from human and canine sources were genetically
identical.
Keywords
Trichophyton rubrum, dog, chitin synthase 1 gene, phylogeny
Introduction
Material and Methods
The isolates of Trichophyton species from human and
animal dermatophytoses have been identiŽed by morphological and biochemical analyses as well as mating
experiments. However, sterile isolates are difŽcult to
identify phenotypically. Furthermore, teleomorphs are
not formed by nearly the half of the recognized
dermatophyte species, including Trichophyton rubrum.
In our previous study, the chitin synthase 1 (CHS1)
gene of several dermatophyte species was sequenced and
analyzed, revealing the pattern of phylogenetic relationships within this group of organisms [1, 2]. We suggested
that sequence analysis of CHS1 gene should also be very
useful for molecular typing of dermatophyte species.
In the present study, a sterile isolate from a dog with
dermatophytosis was identiŽ ed as T. rubrum by molecular analysis. The CHS1 gene sequence was compared
to those of several other dermatophytes, including three
Arthroderma species with Trichophyton mentagrophyteslike anamorphs, T. violaceum, and Žve T. rubrum
isolates from human infections as well as four from
canine infections.
Patient
ÓÓ
Correspondence: R. Kano, Department of Pathobiology, Nihon
University School of Veterinary Medicine, 1866, Kameino,
Fujisawa Kanagawa, 252-8510, Japan. Tel.: ‡81-466-84-3649;
Fax: ‡81-466-84-3649; E-mail: [email protected]
2002
2002 ISHAM
ISHAM, Medical Mycology, 40, 439–442
A 9-year-old spayed crossbreed dog weighing 25.7 kg
was presented to the Yoshiike Animal Hospital with
eruption and pyoderma on the back and neck (Fig. 1).
The skin biopsy specimen revealed hyphae in and around
the hair Žlaments (Fig. 2). From skin scrapings, white
downy-to-uffy colonies were isolated on Sabouraud’s
dextrose agar [3] (Fig. 3). Griseofulvin was administered
at 25 mg kg¡1 given orally twice a day. After three
Fig. 1 Eruption and pyoderma on the abdomen, back and neck of
the canine patient.
440
Kano et al.
Fig. 2 Skin biopsy was showing hyphae inside and outside the hair
shafts (PAS stain).
months of treatment, the skin lesions were cured and the
fungus could not be cultured from the dog skin.
Mycological examination
The fungal colonies developed were morphologically
examined after subculturing on diluted Sabouraud
o
dextrose agar and on cornmeal agar at 24 C for 2 weeks
[3].
The colony of the clinical isolate was white and downy
to uffy, with a dark brown pigment on diluted
Sabouraud’s dextrose agar and red pigment on cornmeal
agar. Microscopic examination revealed only branched
hyphae without macroconidia or microconidia.
Molecular analysis
The CHS1 sequence from the clinical isolate was
investigated to determine its similarity to other dermatophyte sequences. Strains examined are listed in Table
1. About 10 mg of mycelial sample were lysed with 1 mg
of zymolyase-100T (Takara, Kyoto, Japan) per ml in a
lysis buffer containing 0.1 mM EDTA, 1% sodium
dodecyl sulfate (SDS), 10 mM Tris hydrochloride (pH
o
8.0) and 0.3% 2-mercaptoethanol at 37 C for 3 hrs. High
molecular weight DNAs were obtained from these
mycelial samples by phenol and chloroform extraction,
and then precipitated with ethanol. The DNA samples
were dissolved in TE buffer (10 mM Tris-hydrochloride,
pH 8.0 and 1 mM EDTA) and were used for polymerase
chain reaction (PCR) ampliŽcation. PCR was performed
in a reaction mixture (20 ml) containing 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2, 0.001%
gelatin, 200 mM each deoxynucleoside triphosphate, 1.0
unit of Taq polymerase (Takara) and 0.5 mg of each a
pair of primers. The sequences of the degenerate primers
for CHS1 as given in our based on the previous study
[1,2]: primer 1, 50 -CTG AAG CTT ACT(ACG) ATG
TAT(C) AAT(C) GAG(A) GAT(C)-30 and primer 2, 50 GTT CTC GAG (C)TTT (A)GTA (C)TTC (A)GAA
(A)GTT (T)CTG-30 . AmpliŽcation was carried out for
o
35 cycles of template denaturation (1 min, at 94 C),
o
primer annealing (2 min, at 50 C) and polymerization
o
(2 min, at 72 C). The PCR products were electrophoresed through 2% agarose gel and then stained with
ethidium bromide. The PCR products were gel-puriŽ ed
and cloned into a pCRII vector (Invitrogen, California,
USA). The plasmid DNAs of more than three clones
corresponding to each test species were extracted with
the QIAGEN plasmid kit (QIAGEN, California, USA)
and sequenced by the dideoxy chain termination method
using an ABI PRISM 310 Genetic Analyzer (PerkinElmer, California, USA).
To compare the CHS1 gene sequences obtained, we
used Clustal W multiple sequence alignment programs
[4] and the TREEVIEW program for display of
phylogenetic relationships [5]. Bootstrap analysis was
performed as described by Felsenstein [6] on 1000
random samples taken from a multiple alignment;
analysis was done using the Clustal W programs.
Results
Fig. 3 White, downy-to-uffy colonies of the nonsporulating
clinical isolate as seen on Sabouraud’s dextrose agar.
AmpliŽcation of the clinical isolate’s DNA with degenerate CHS1 primers yielded fragments of about 620bp,
consistent with the sizes previously reported for CHS1
gene fragments in dermatophytes [1,2]. The sequence of
the test isolate had more than 85% similarity with the
sequences of the other dermatophytes examined. The
sequence similarity between the clinical isolate and
known T. rubrum isolates exceeded 99%.
As the clinical isolate’s CHS1 sequence was essentially
identical to that of T. rubrum and distinct from those of
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2002 ISHAM, Medical Mycology, 40, 439–442
T. rubrum from a dog
Table 1
441
Species and strains used in this study
Special (Mating type)
Strain
origin
Arthroderma benhamiae (‡)
A. simii (‡)
A. vanbreuseghemii (‡)
VUT-77011
VUT-77009
VUT-77007
Trichophyton rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. violaceum
T. violaceum
T. violaceum
VUT-97014
VUT-97015
VUT-97016
VUT-97021
VUT-97020
VUT-97022
VUT-010001
VUT-98011
VUT-98012
VUT-98013
(IAM 12704 ˆ RV 26678)
(CBS 448.65)
(CBS 646.73 ˆ RV 27960)
(human)
(human)
(human)
(6-year-old female Dachshund)
(3-year-old female Yorkshire Terrier)
(11-year-old male Yorkshire Terrier)
(dog)
(human)
(human)
(human)
VUT: School of Veterinary Medicine, University ofTokyo
IAM: Institute of Applied Microbiology, University of Tokyo
RV: Institute de Medecine Tropicale, Antwerp, Belgium; CBS: Centraalbureau voor Schimmelcultures, Baarn, Netherlands.
Ó
Fig. 4 A tree showing relationships among CHS1 gene fragments of the nonsporulating clinical isolate and Žve Trichophyton species.
Numbers on branches were determined by bootstrap analysis indicating the percentage of 1000 repeat subsamples yielding consistent
monophyletic grouping. (T. mentagrophytes) indicates anamorphs.
2002 ISHAM, Medical Mycology, 40, 439–442
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Kano et al.
the other dermatophytes studied (Fig. 4), the isolate was
identiŽed as T. rubrum.
Discussion
T. rubrum is a cosmopolitan, anthropophilic species and
is the most frequent etiologic agent of human dermatophytosis throughout the world [3]. Although anthropophilic, it is also infrequently found in dermatological
disorders of dogs [7]. At least few cases of canine
ringworm due to T. rubrum have been reported from
Japan [7–9] and four strains were obtained from such
cases for the present study. These isolates might be
conjectured to represent a zooanthroponotic pathogen
genetically distinct from anthropophilic T. rubrum.
Yamada et al. [7] stated that studies were required to
clarify the role of T. rubrum as an anthropozoonotic and
zooanthroponotic pathogen.
Molecular comparisons have been applied to clinical
isolates of T. rubrum from humans but not to those from
dogs [10, 11]. The identity of the dog and human T.
rubrum CHS1 sequences is consistent with the Žnding of
identical ribosomal internal transcribed spacer 1 sequences in other studies of T. rubrum isolates [12],
suggesting that T. rubrum isolates are virtually genetically identical regardless of the speciŽc situation from
which they are isolated.
Kusida and Watanabe reported canine cases of T.
rubrum dermatophytosis that were probably transmitted
from the owner to the dog [8, 9]. In some other cases of
canine T. rubrum, however, could the source of infection
not be established [7]. The transmission of this infection
from an affected animal to a human is certainly possible;
therefore, care should be taken whenever infected
animals are handled, especially when they may be
infected with anthropophilic dermatophytes such as T.
rubrum [3].
References
1 Kano R, Nakamura Y, Watari T, et al. Molecular analysis of
chitin synthase 1 (CHS1) gene sequences of Trichophyton
mentagrophytes complex and T. rubrum. Curr Microbiol 1998;
37: 236–239.
2 Kano R, Okabayashi K, Nakamura Y, et al. Differences among
chitin synthase 1 gene sequences in Trichophyton rubrum and T.
violaceum. Med Mycol 2000; 38: 4750.
3 Kwon-Chung KJ and Bennett EJ, Dermatophytoses. In: Medical
Mycology. Philadelphia: Lea & Febiger; 1992: 136137 and 816–
826.
4 Thompson JD, Higgins DG, Gibson TJ, CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-speciŽc gap
penalties and weight matrix choice. Nucl Acid Res 1994; 22:
4673–4680.
5 Page RDM TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 1996; 12:
357–358.
6 Felsenstein J. ConŽdence limits on phylogenies: an approach
using the bootstrap. Evolution 1985; 39: 783–791.
7 Yamada C, Hasegawa A, Ono K, et al. Trichophyton rubrum
infection in a dog. Jpn J Med Mycol 1991; 32: 67–71.
8 Kushida T, Watanabe S. Canine ringworm caused by Trichophyton rubrum; probable transmission from man to animal.
Sabouraudia 1975; 13: 30–32.
9 Kushida T. An additional case of canine dermatophytosis
caused by Trichophyton rubrum. Nippon Juigaku Zasshi 1979;
41: 77–81.
10 Jackson CJ, Barton RC, Kelly SL, Evans EG. Strain identiŽcation of Trichophyton rubrum by speciŽc ampliŽcation of
subrepeat elements in the ribosomal DNA nontranscribed
spacer. J Clin Microbiol 2000; 38: 4527–4534.
11 Summerbell RC, Haugland RA, Li A, Gupta AK. rRNA gene
internal transcribed spacer 1 and 2 sequences of asexual,
anthropophilic dermatophytes related to Trichophyton rubrum.
J Clin Microbiol 1999; 37: 4005–4011.
12 Makimura K, Mochizuki T, Hasegawa A, Uchida K, Saito H,
Yamaguchi H. Phylogenetic classiŽcation of Trichophyton
mentagrophytes complex strains based on DNA sequences of
nuclear ribosomal internal transcribed spacer 1 regions. J Clin
Microbiol 1998; 36: 2629–2633.
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2002 ISHAM, Medical Mycology, 40, 439–442