IMMUNOLOGY
MEDICAL
MICROBIOLOGY
ELSEVIER
FEMS
Immunology
and Medical
Microbiology
AND
13 ( 1996) I47- 154
Karyotyping by PFGE of clinical isolates of Sporothrix schenckii
Takeshi Tateishi a-* , Somay Yamagata Murayama b, Fujio Otsuka a,
Hideyo Y amaguchi b~c
Received
30 August
1995: revised 26 October
1995; accepted 27 October
1995
Abstract
From October1991to December1992we hadeight patientswith sporotrichosis
at TsukubaUniversity Hospitalin Japan.
With 8 strainsisolated from thesepatients,PFGE(pulsed-fieldgel electrophoresis)
analyseswere carriedout to examine
whetherthe karyotypeof S. schenckiiis distinguished
by our methodandwhetherthismolecularapproachis a usefulmeans
of biotypingof S. schenckiistrains.Chromosomes
wereseparated
by contour-clamped
homogeneous
electricfield (CHEF)
gel electrophoresis.
The strainshadsix to eight chromosomes
and a total genomesizewasapprox.28 Mbp. Although these
karyotypesof all the isolateslookedcloselysimilarto eachother, they weregroupedinto threetypes.
Key~rords:
Sporothrh
schewkii:
Sporotrichosis:
Pulsed-field
gel electrophoresia:
1. Introduction
Sporotrichosis is a subacute or chronic disease
causedby Spomthrix schenckii, a pathogenic fungus
classified as a member of the deuteromycetes.Infection is usually limited to the skin, subcutaneous
tissue. and lymphatic glands. In most caseslesions
are indolent nodules that often suppurate, ulcerate,
and drain [l]. Sporotrichosis is worldwide in distribution. In Japan approximately 150 casesare recorded
every year. There is regional difference in incidence
of this fungal infection; it is particularly high in the
Kanto district, which includes Ibaraki prefecture, and
in the Kyushu district (Fig. 1) [2].
_ Corresponding
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Microbiological
Karyotype
S. schenckii is dimorphic; at room temperature it
grows as a mycelial form on Sabouraud’s glucose
agar or other conventional media, while in infected
tissueyeast form cells are predominant. Such yeastform cells of this fungus also develop in cultures
grown at 35-37°C on blood agar containing cysteine
or in brain heart infusion broth. In nature S. schenckii
exists as a saprophyte and is found in soil and
various plant materials [3]. Ajello and Kaplan [4]
described a new variety of S. schenckii termed S.
schenckii var. luriei
isolated from a patient with
sporotrichosis,and Mackinnon et al. [5] and Dixon et
al. [6] reported environmental isolates which were
closely similar to S. schenckii in mycological properties. In recent years, molecular biological techniques
have been applied to epidemiological and taxonomic
study of several pathogenic fungi, particularly CanSocieties.
All rights reserved
148
[lo]. Karyotyping has been also done of a variety of
pathogenic fungi, including C. al&cans, C. glabrutu
[ 1 I], C. tropicalis [ 121, Cyptococcus
neoformans
[ 131, Aspergillus
nidulans [ 141, A. niger [I 51,
Malassezia pachydermatis [ 161 and Coccidioides immitis [17]. However, there is no information available on the karyotype of S. schenckii.
In this paper. we analyzed the electrophoretic
karyotype of S. schenckii isolated from patients with
sporotrichosis in the Tsukuba University Hospital, in
order to know, firstly, if the electrophoretic karyotype is really distinguished by our PFGE method,
secondly, if this molecular method is useful in strain
typing of S. schenckii.
2. Materials
and methods
2.1. Strains
0
20klll
Fig. I. Geographic
distribution
of aporotrichosis
patients from
whom testing strains of S. schenckii
were isolated. Symbol: 0,
habitat of each patient.
didu albicans. Molecular biological methods have
been used to explore the phylogenic relationship of
S. schenckii with some other fungi. as well as its
strain typing. Berbee and Taylor demonstrated the
phylogenic relationship between S. schenckii and
Ophiostoma stenoceras by comparing the DNA sequence of 18s ribosomal RNA gene (18s rDNA) [7].
Based on restriction profiles (restriction fragment
length polymorphisms
(RFLPs)) of mitochondrial
DNA, Suzuki et al. grouped S. schenckii into 11
types [8]. RFLP analysis of whole cell DNA was
carried out by Cooper et al. with clinical and environmental isolates of Sporothrix spp. collected in the
United States [9].
More recently, pulsed-field gel electrophoresis
(PFGE) has become useful for separating large DNA
molecules over IO kbp can be favorably resolved
[lo]. The size and number of chromosomal DNAs
can be separated by PFGE to specify the karyotype.
Karyotyping has been successfully done with various
organisms including mammals, plants and bacteria
All strains of S. schenckii used in this study were
isolated from patients diagnosed with sporotrichosis
and were identified on the basis of cultural and
morphological properties at the Department of Dermatology, University of Tsukuba, School of Medicine
between October 199 1 and December 1992. The
strains isolated from patients 1. 2, 3, 4, 5, 6, 7 and 8
were designated as strain nos. 1-8, respectively.
2.2. Preparation
of intact chromosomul DNA
Yeast-form cells of S. schenckii were grown at
33.5-345°C
in brain heart infusion broth for 7 days
with gentle shaking, harvested by centrifugation
( 15,000 X g ) and washed twice with Tris-EDTA (TE)
buffer (10 mM Tris-HCl, 50 mM EDTA, pH 7.6).
We used two methods for preparation of intact chromosomal DNA: an agarose-spheroplast method and a
liquid-spheroplast
method. In the former, washed
cells were suspended in TE buffer containing 1
mg/ml of Zymolyase 20T (Seikagaku Co., Tokyo),
mixed with low melting point agarose (Bio-Rad) to a
final concentration of 0.75%, and the cell concentration adjusted to 5 X 10’ cells/ml. The mixture was
poured into plug molds and cooled at 4°C for 15 min
to solidify the agarose. The agarose plugs were
pushed into LET buffer (500 mM EDTA, IO mM
Tris-HCl, 1% 2-mercaptoethanol. pH 7.6) and incu-
bated at 37°C for 18 h. Then they were transferred to
ESP solution (500 mM EDTA, 1% sodium lauroyl
sarcosinate, 1 mg/ml Proteinase K) and incubated at
50°C for 48 h. After the incubation, the agarose
plugs were washed at 50°C twice with TE buffer
containing 1 mM phenylmethylsulfonyl
fluoride
(PMSF) and another two times with PMSF-free TE
buffer, then stored at 4°C until use.
In the liquid-spheroplast
method, washed cells
were suspended in 1 ml of lysis solution (1 M KCl,
10 mM Tris-HCl.
pH 7.51, to which 50 ~1 of
/3-mercaptoethanol and Zymolyase 20T (1 mg/ml
cell solution) were added and were incubated at 37°C
for 3 h to induce spheroplasts. The resultant spheroplasts were harvested by centrifugation (10,000 X g),
resuspended in 0.67 ml of lysis solution, and mixed
with 0.37 ml of 2% low melting point agarose
containing 50 mM EDTA. Final concentrations of
agarose and fungal cells were the same as those
using the agarose-spheroplast method. The mixture
of agarose and fungal cells was solidified at 4°C for
15 min in a plug mold. The agarose plugs were
incubated in ESP solution and the subsequently procedure followed that in the agarose-spheroplast
method.
2.3. Electrophoresis
CHEF (contour-clamped
homogeneous electric
field gel electrophoresis) was performed using the
CHEF DRII, DRIB or Mapper (Bio-Rad) apparatus.
DNAs of Saccharomyces cerecisiae YNN295 strain
(Bio-Rad) and Schizosaccharomyces
pombe strain
972h- (Bio-Rad) were used as the size markers. The
composition of buffer, concentration of agarose
(pulsed-field grade agarose, Bio-Rad), voltage, switch
time, run time and angle of electrophoresis were
selected according to the size of DNA, as described
in the legends to figures. After a run, the DNA was
visualized with ethidium bromide and photographed
under ultraviolet light using instant film (FP3000B,
Fuji).
3. Results
3.1. Clinical
tien ts
characteristics
of sporotrichosis
pa-
From November 1991 to December 1992 we isolated each S. schenckii strain from 8 patients with
Ml2345678
+-
460 kbp
Fig. 2. CHEF electrophoresis
of 8 S. schenckii strains. The electrophoresis
was performed
by CHEF DRII apparatus using the following
conditions:
I .O% agarose. included angle of 120” and switch time of 60 s for I5 h and then 90 s for 9 h at 14°C in 0.5 X TBE buffer (40 mM
Tris, 45 mM borate. I .O mM EDTA. pH 8.3). Lane M: S. cere, isine YNN795
strain.
150
Ml2345678
Fig. 3. CHEF electrophoresis
of 8 S. s&w&i
strains. The electrophoresis
was performed
V/cm,
120” included angle. 120 s switch interval. 24 h run time followed by 240 s switch
14°C. Lane M: S. cerec~i.siw YNN295
strain.
MlM21
2345
by CHEF DRIII apparatus, 1.0% agarose, 2
interval. 24 h run time in 0.5 X TBE buffer at
6 78
c- 3900 kbp
+ 3400 kbp
t-3000
kbp
Fig. 3. CHEF electrophoresis
of 8 S. schenckii strains, The electrophoresis
was performed
by CHEF DRIII apparatus, 0.8% agarose, 2
V/cm,
106” included angle, 500 s switch interval, 36 h run time followed by 1200 s switch interval, 36 h run time in 1.0 X TAE (40 mM
Tris, 50 mM acetate. 2.0 mM EDTA. pH 8.0) buffer at 14°C. Lane Ml: S. c’erel isine YNN295
strain; lane M2: S. pombe 972hstrain.
sporotrichosis. The clinical form in all patients, was
either lymphocutaneous or fixed cutaneous. It is
noted that 7 of the 8 patients were farmers, supporting the general notion that the soil is an important
reservoir of S. schenckii.
3.2. Karyotypes
of S.
schenckii
Both the agarose-spheroplastmethod and the liquid-spheroplast method were used to prepare DNA
samplesas stated. The two methods gave virtually
the same results, although clearer patterns were obtained with samplesprepared by the former means
(data not shown), probably becauseDNA was more
easily degraded during the preparation of spheroplasts in fluid than in solid agar.
The results of electrophoresis patterns obtained
with DNA prepared by the agarose-spheroplast
method are shown in Figs. 2-5. Four different analytical experimental conditions were used to separate
DNAs with a wide range of molecular weight from
460 kbp to 6200 kbp. Under the initial conditions.
three chromosomal DNA bands were resolved by
CHEF analysis (Fig. 2). In this gel the smallest
chromosomalband was detected in strain nos. 1. 2.
MlM21
4, 6, and 7 and estimatedto be 460 kbp in molecular
mass. In second condition both switching time and
running time were prolonged (Fig. 31, since prolongation of switching time and lowering agaroseconcentration are reported to be effective in separating
high molecular mass DNAs from each other [I 81.
The smallestband was seenin strain nos. 1, 2, 4: 6.
7 and 8 and estimated to be 1100 kbp. To further
separate the larger band(s), the following changes
were madein analytical conditions: useof 1.OX TAE
buffer in place of 0.5 X TBE buffer; decrease in
included angle; prolongation of switching time; and
decrease in the agarose concentration. It is also
known that Tris-acetate buffer can speed up migration and that the decreaseof the included angle can
increase the running velocity [ 181.Under the modified analytical conditions, the two large bandsshown
in Fig. 3 were separatedinto 6 bands(Fig. 4). three
smaller bands were observed in all strains and estimated to be 3900, 3400 and 3000 kbp in molecular
size. The switch time was further increasedto 1800 s
to analyze the largest three bands. As shown in Fig.
5, they were clearly separablefrom each other in all
strainsand estimatedto be 6200, 5600 and 4600 kbp.
However, there remainsthe possibility that the largest
2345678
+
-
6200 kbp
5600 kbp
f-
4600 kbp
Fig. 5. CHEF electrophoresis
of 8 S. schmckii
strains. The electrophoresis
was performed
by CHEF Mapper apparatus. 0.8% agarose. 2
V/cm, 106” included angle, 1800 s witch interval, 72 h run time in 1 .O X TAE buffer at 14°C. Lane MI: S. cerrc~isitrr YNN295 strain; lane
M2: S. pomhe
972h- strain.
152
T. Tat&hi
et al. / FEMS
Immunology
md
Microhiologv
13 ClYY61
147-134
4. Discussion
W
6200
5600
4600
3900
-------m
3400
3000
--m-
---m
-w-----m
1100
460
Strain
Medical
No.
Fig. 6. Summarized
illustrated.
1
2345678
data of electrophoretic
analysis
schematically
band may exceed 6200 kbp becauseno size marker
larger than 5700 kbp which is equivalent to the
largest chromosome of S. pombe was available.
These higher molecular bands from 3 to 7 Mbp were
smudging because of the limitation of our PFGE
apparatus.The intensities of the bandsestimated460
kbp, I 100 kbp and 4600 kbp were very weak compared with others. We do not know whether they
reflected the ploidy or the chromosomelength polymorphism. But all chromosome bands revealed in
Fig. 6 could be detected reproducibly.
Summarized data on electrophoretic analysis are
schematically illustrated in Fig. 6. Total genomesize
was calculated to be approx. 28 Mbp. The number of
chromosomesvaried from six to eight among the
different strains. Three different karyotypes were
recognized: the first type (strain nos. I, 2, 4, 7 and 8)
was characterized by existence of all eight chromosomal bands, the second type (strain nos. 3 and 5)
was lacking in both 460 kbp and I 100 kbp bands,
and the third type (strain no. 6) was lacking in only
the 460 kbp band.
The conventional taxonomical classification typing of fungi is usually made on the basis of their
morphological. biochemical and/or serological
properties. With S. schenckii, there is broad variety
in the productivity of pigment, the utility of sugar(s),
morphological characteristics. suchas conidial states,
growth temperature [19]. and the conditions of conversion to yeast form among different strains. No
serological type or physiological type of this fungal
specieshas been clearly defined. Although a limited
number of papers dealing with serological [20] and
biochemical [ 191typing of S. schenckii have been
published, no definite serotype or biotype of this
fungus has yet been accepted.
Various molecular biological techniques have recently been introduced to medical mycology for
identification and typing of pathogenic fungi [21].
PFGE has now proved useful as one meansof this
molecular typing. In the present study, the karyotypes of eight S. schenckii strains isolated from
patients with sporotrichosis who lived within a radius of 20 km of each other in Ibaraki prefecture, a
part of the Kanto district in Japan (Fig. I) were
determined to be engagedin farming or dairy cattle.
BecauseS. scherzckii inhabits the soil and plants [3],
it is highly probable that patients became infected
with this fungus while they were in the field and in
contact with soil within the restricted area. Our
finding showed that the S. schenckii strainshave 6 to
8 chromosomesand it is interesting to note that their
detected karyotypes were relatively well conserved.
Although the karyotypic patterns of the 8 strains
tested looked similar, they could be classified into
three groups depending on the presenceor absence
of 460 kbp and/or 1100 kbp chromosomes.
As shown in the present study, PFGE karyotype
of S. schenckii isolates do not appear to be related
with the clinical forms of sporotrichosisthey caused.
Nishikawa et al. 1201reported that although there are
antigenic varieties among different S. schenckii
strains, they did not find any relationship between
antigenic pattern and isolation sources,clinical forms
of the disease, or the morphology of yeast-form
cells. We were also unable to correlate the karyotype
of S. schenckii strains with any of their cultural or
morphological properties, including the morphology
of growing colonies on conventional agar media or
microconidia produced. However, it is of note that
all of the 8 strains tested in this study converted into
yeast form cell at about 34°C and none of them was
capable of growing at a temperature of 35°C or
above. It is generally accepted that optimal temperature for mycelial to yeast-form conversion of S.
s&r&i
is usually around 37°C [3]. Dixon et al.
reported that only 29 of 69 environmental Sporothrix
isolates but all of 21 clinical isolatesof S. schenckii
grew at 37°C and converted into yeast form [6].
Karyotyping studies with those S. schenckii strains
in which mycelial to yeast-form conversion occurs at
37°C are needed.It remainsto be answered whether
or not a karyotype relates to certain phenotypes of S.
schenckii. Working on C. nlbicans strains, Mahrous
et al. attempted to find a relationship between karyotype and phenotype (morphology, API profile.
serotype. production of proteinase or virulence to
mice) but failed [22]. Increased numbers of phenotypic characters will be neededfor such studieswith
C. albicnns and S. schenckii.
In the present study, we succeededin distinguishing the karyotype of S. schenckii
by our PFGE
method and demonstratedthat the PFGE karyotypes
of different S. schenckii isolates show substantial
variations in two small DNA bands. In connection
with this, those S. schmckii
strains which share a
common karyotype could be further classified into
subgroupswhen they are subjected to Southern hybridization analysis [23], or polymerase chain reaction (PCR) fingerprinting analysis [24]. These additional molecular biological techniques. when used in
combination with PFGE, will give us more precise
information on molecular types of S. whenckii
and
probably some other pathogenic fungi. Strain variation. in terms of karyotype. is well known for C.
albicms
[25] and C. mwfortnans
[26]. The present
study in which only 8 strainsof S. schenckii isolated
from patients inhabiting in such a small and restricted area provides evidence that karyotypes of
this speciesin restricted area shows a similarity. To
know the occurrence of the variation in that of S.
schenckii,
such as C. albicuns or C. neof~~rmuns,
further studieson karyotyping of S. schenckii strains
collected in different areas in Japan and foreign
countries should be explore. Takeda et al. reported
the results of their molecular epidemiological study
carried out with S. schenckii strains isolated from
sporotrichosis patients in Japan using the RFLP of
fungal mitochondrial DNA [27]. They commented
that the RFLP pattern tends to relate to the area
where the strain was isolated, but that the group
pattern of RFLP differed dependingon the restriction
enzymes used: this presumably reflects the existence
of many repetitive sequencesin the mitochondrial
genes. along with the occurrence of high rate of
point mutation 1281.
Electrophoretic karyotyping is the first step for
the physical map. The genetic knowledge of S.
schenckii is very poor. Only the sequenceof the 18s
rRNA gene and chitin synthetasegeneswere known.
Therefore our karyotype of this specieswill be helpful to analyze its genetic information.
References
111Rippon.
J.W.
(1988)
Sporotrichosis.
In:
Medical
Mycology:
the Pathogenic
Actinomycetes.
Third
edition.
pp.
Company.
121Fukushiro.
chosis
257.
[31 Miyaji.
WonsiewicL
Philadelphia.
R. (1984)
Epidemiology
and ecology
in Japan.
Zentralbl.
Bakteriol.
Mikrobiol.
22X-233.
M.
and
Yamaguchi,
H.
(Ed.),
Fungi and the Pathogenic
325-352,
W.B.
Saunders
(1988)
of sporotriHyg. [a]
Sporotrichosis.
In:
Balows.
A. Hausler,
W.J. Jr. Ohashi,
M. and Turano,
A.
(Eds.).
Laboratory
Diagnosis
of Infectious
Diseases.
Principle and Practice.
pp. 706-714.
Springer-Verlag,
New York.
[U Ajello.
L. and Kaplan.
W. (1969)
A new variant
of S/xvwrhriv
Mykoaen
12. 633-644.
J.E.. Conti.
D.I.. Geruele.
E.. Civila,
E. and Luz.
151 Mackinnon.
Sd. (I 969) Isolation
of Sporot/tri.u
sc~hewkii
from nature and
considerations
on it> pathogenicity
and ecology.
Sabouraudia
.scherlckii.
7. 38-15.
I61 Dixon.
D.M..
Salkin.
J.H., Kemna.
characterization
environmental
demx
[71 Berbee.
gene
of sporotrichosla.
J. Clin.
M.L.
and Taylor,
J.W.
sequence
Spororhri.i-
87-9
.schwLii
character\
in genu\
C.J.,
DNA
sporotrichosis
1101Watson,
R.A.,
F.B.
whmckii
with
Hurd,
N.J.,
Haines,
(1991)
Isolation
from clinical
the largest
U.S.
Microbial.
(1992)
18s
place
the
Ophiostoma.
29. I lO6riboaomal
human
Exp.
and
and
epiI1 13.
RNA
pathogen
Mycol.
16,
I.
K.. Kawasaki.
[81 Suzuki.
of restriction
profiles
sckewkii
and related
[91 Cooper,
(1992)
1.F.. Duncan.
M.E.
and Colea.
of Sporothri.v
sources
associated
J.D..
Breslin.
typing
epidemic.
Gilman.
M. and Ishiraki.
H. (198X)
Analysis
of mitochondrial
DNA from Sporothri.u
fungi.
Mycopathologia
103, l17- I5 I.
of
B.J.. Dixon.
D.M.
isolates
associated
and Salkin.
with the
J. Clin.
Microbial.
M., Witkowski,
30, 1631-1635.
J. and Zoller.
1.F.
1988
M.
(1992) In: Recombinant
DNA. Second edition, pp. 583-602.
Scientific American Books. New York.
[I I] Magee. B.B. and Magee, P.T. (1987) Electrophoretic
karyotypes and chromosome
numbers in Cc&i&l
species. J. Gen.
Microbial.
133, 425-430.
[l2] Doebbeling,
B.N.. Lehmann.
P.F.. Hollis, R.J.. Wu. L.C.,
Widmer. A.F.. Vors, A. and Pfaller. M.A. (1993) Compari\on of pulsed-field
gel electrophoresis
with isoenzyme profiles as a typing system for Cc&i&
tiwpicrrlis. Clin. Infect.
Dis. 16, 377-383.
[I31 Perfect, J.R.. Magee, B.B. and Magee, P.T. (1989) Separation of chromosomea
of Cryptococcus
neoformnns
by pulsed
field gel electrophoreais.
Infect. Immun. 57, 2624-2627.
[ I41 Brody, H. and Carbon, J. (1989) Electrophoretic
karyotype
of
A.vprrgi/lus
t~idulcrns.
Proc. Natl. Acad. Sci. USA 86, 62606263.
[IS]
Debets, A.J.. Holub. E.F., Swart. K., Broek, Hvd. and Bos,
C.J. (I 990) An electrophoretic
karyotype
of A.cpurgillm
niger. Mol. Gen. Genet. 224, Xx-268.
[ 161 Kiuchi, A.. Taharaguchi.
S.. Hanazawa. R., Hara. M.. Ikeda,
T. and Tabuchi.
K. (1992) Chromosome-sized
DNA
of
Mnlnsse:in
p”“l7~demrrttis
by pulsed-field
gel electrophoresis. J. Vet. Med. Sci. 54. 1219-1220.
[ I71 Pan. S. and Cole. G.T. (1992). Electrophoretic
karyotypes
of
clinical isolates of Corcidioidr,
irwnitis.
Infect. Immun. 60,
Serologic
differences
in strains of Sporothrh
schenckii.
Sabouraudla
13, 285-290.
[?I] Maslow,
J.N., Mulligan.
M.E. and Arbeit.
R.D. (1993)
Molecular
epidemiology:
Application
of contemporary
techniques to the typing of microorganisms.
CID 17, l53- 162.
[22] Mahrous.
M., Lott, T.J.. Meyer.
S.A., Sawant, A.D. and
Ahearn. D.G. (1988) Electrophoretic
karyotyping
of typical
and atypical Cundidn
rrlhicclns. J. Clin. Microbial.
28, 876881.
[23] Mahrous.
M.. Sawant, A.D.. Pruitt. W.R., Lott, T., Meyer,
S.A. and Ahearn. D.G. (1992) DNA relatedness. karyotyping
and gene probing of Cmdido
tropiudis.
Cmdida
albicms
and its synonyms
Cmdidtr
strlktfoidea
and Candida
cltrussenii.
Eur. J. Epidemiol. 8, 444-45 I.
[a] Meyer, W., Mitchell. T.G.. Freedman, E.Z. and Vilgalys, R.
(1993) Hybridization
probes for conventional
DNA fingerprinting
used as single primers in the polymerase
chain
reaction to distinguish
strains of C~ptococtw
rze&nntms.
J. Clin. Microbial.
31. 2274-2280.
[25] Rustchenko-Bulgac.
E.P. and Howard, D.H. (1993) Multiple
chromosomal
and phenotypic
changes in spontaneous
mutants of Cmdidtr
czlhictrn.!. J. Gen. Microbial.
139. I l951207.
[Xi]
4872-4880.
[ 181 Birren, B. and Lai, E. (1993). In: Pulsed Field Gel Electrophoreris:
A Practical Guide. pp. 107- 140, Academic Press,
California.
[ 191 Taylor. J.J. (I 970) A comparison of some Ceratocystis specie\
with Sporothvi.t
.stiwrc~X-ii. Mycopathol.
Mycol.
Appl. 47.
233-240.
[20] Nishikawa.
T., Harada.
T.. Harada,
S. and Hatano.
H. (197.5)
Perfect, J.R.. Ketabchi. N., Cox, G.M.. Ingram. C.W. and
Beiser. C.L. (1993) Karyotypin g of Ciytocom~s
neqfirm(my as an epidemiological
tool. J. Clin. Microhiol.
31,
3305-3309.
Takeda, Y., Kawasaki, M. and Ishizaki. H. (1991) Phylogeny
and molecular epidemiology
of Sporothrir.
.rchunckii
in Japan.
Mycopathologia
116. 9-14.
[28] Watson. J.D.. Gilman.
M.. Witkowski.
J. and Zoller. M.
(1992) In: Recombinant
DNA. Second edition, pp. 433-45 1,
Scientific American Books. NY.
[27]