Rapid identication of Streptococcus nae by specic PCR

assay utilizing genetic markers in TS rDNA

S M Zhou
1
, Y Fan
1
, X Q Zhu
2
, M Q Xie
1
and A X Li
1
1 Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, School of
Life Sciences, Sun Yat-sen University, 135 Xingang West Road, Guangzhou 510275, China
2 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of
Agricultural Sciences, Lanzhou, China
Abstract
The 16S-23S intergenic spacers (ITS) of ribosomal
DNA from ten independent isolates of Streto.o..us
.n.ue and one reference strain ATCC29178 were
chain reaction (PCR) primer set for rapid and
S. .n.ue. This
S. .n.ue , but not from other
mized to allow detection of the organism from agar,
of the PCR assay was established by the detection of
DNA as low as 0.02 ng or as few as 10 CFU bac-
S. .n.ue.
1e,words. 16S-23S intergenic spacers (ITS), spe-
Streto.o..us
.n.ue.
ntroduction
Streto.o..us .n.ue is considered one of the most
distribution, capable of causing invasive disease in
environments (Shoemaker, Klesius & Evans 2001;
Colorni, Diamant, Eldar, Kvitt & Zlotkin 2002). It
world, including the commercially important spe-
drum and yellowtail (Kitao 1993; Shoemaker et uI.
2001). Streto.o..us .n.ue
American Tilapia Association as the most important
pathogen affecting the tilapia culture industry
(Bowser, Wooster, Getchell & Timmons 1998).
More importantly, S. .n.ue can also infect human
beings, and most of the infected people are
infection (Weinstein, Low, McGeer, Willey, Rose,
Coulter, Wyper, Borczyk, Lovgren & Facklam
1997; Lau, Woo, Tse, Leung, Wong & Yuen 2003;
Koh, Kurup & Chen 2004).
S. .n.ue include biochemical characterization, as
systems such as BioMerieux Vitek (Lauet uI. 2003;
Facklam, Elliott, Shewmaker & Reingold 2005),
ATB Expression system (Lau et uI. 2003) and
Microscan (Facklam et uI. 2005) were unable to
identify S. .n.ue because it was not in the corre-
sponding databases. In the study of Roach, Levett
& Lavoie (2006), only 19/25 of the S. .n.ue strains
plate panels and Microlog database. Moreover, the
species diversity of Streto.o..us
tion of S. .n.ue based only on phenotypic traits
(Mata, Gibello, Casamayor, Blanco, Dominguez &
Fernandez-Garayzabal 2004b). Therefore, misiden-
tional biochemical tests has frequently occurred,
JournaI of Fish Diseases 2011, 34, 265-271 doi:10.1111/j.1365-2761.2010.01233.x
Correspondence A X Li, Schoo| of Life Sciences, SunYat-sen
University, 135 Xinan West Poa, Haizhu District, Guanzhou
510275, Guanon Province, China (e-mai|. anxin_|i2002
yahoo.com.cn)
265
2011
Blackwell Publishing Ltd
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
sequenced, aligned and used to design a polymerase
especially in human clinical cases (Lau et uI. 2003;
Lau, Woo, Luk, Fung, Hui, Fong, Chow, Wong &
Yuen 2006).
As a useful alternative to phenotypic traits,
genetic techniques such as 16S rDNA sequence
analysis (Lau et uI.
(Berridge, Fuller, de Azavedo, Low, Bercovier &
Frelier 1998; Zlotkin, Hershko & Eldar 1998;
Mata, Blanco, Dominguez, Fernandez-Garayzabal
& Gibello 2004a) have been used to identify
bacterial pathogens. Although 16S rRNA gene
analysis is a reliable and practical approach to
identify S. .n.ue and other Streto.o..us species (Lau
et uI.
pathogen, especially when the latter is used in
epidemiologic studies associated with the outbreak
of S. .n.ue et uI. 1998;
Mata et uI. 2004a). The 16S rDNA, 16S-23S
rDNA intergenic spacer (ITS) region and lactate
oxidase gene (I.tO
sequences to identify S. .n.ue (Berridge et uI. 1998;
Zlotkin et uI. 1998; Mata et uI. 2004a). However,
S. .n.ue on the basis of its 16S rDNA
sequence, was not able to differentiate S. .n.ue from
Streto.o..us uguIu.t.ue in some cases because of
high genetic relatedness between them (Mata et uI.
2004a). Moreover, the primer set targeting the ITS
sequence designed by Berridge et uI. (1998) was
found ineffective in detecting S. .n.ue isolated in
South China or the reference strain ATCC29178
(unpublished data).
Therefore, the objective of this study was to
develop a new S. .n.ue
designing a new primer set targeting the ITS
sequence, which could be used to diagnose disease
Materials and methods
Bacterial strains and growth condition
Bacterial strains used in this study are listed in
Table 1, together with their sources and the media
used for their culture. The S. .n.ue reference strains
was obtained from the American Type Culture
Collection (ATCC29178) and the Belgian Coordi-
nated Collections of Microorganisms (LMG14520).
Other S. .n.ue strains were isolated from diseased
traditional biochemical characterization and spe-
All S. .n.ue strains were cultured in brain heart
infusion agar plates (BHIA; HuanKai ) supple-
mented with 1.0% NaCl and incubated at 27 C
for 24-48 h.
DNA amplihcation and sequencing
The template DNA extraction of S. .n.ue strains
used in this study was performed as described
previously (Zhou et uI. 2008). The conserved
from the S. .n.ue strains were reported previously
(Berridge et uI. 1998): primer A1 5-AG-
Table 1 Bacterial strains used to evaluate
the new primer set based on the ITS
sequence of Streptococcus iniae
Baotorial strains
Numbor
oxaminod Modia
a
Origin
b
Streptococcus iniae 28 BHA ATCC/LMG/SYSU
Streptococcus aa|actiae 4 BHA SYSU
Streptococcus ysa|actiae 4 BHA SYSU
Streptococcus parauberis 1 BHA MCCC
Lactococcus arveiae 1 BHA MCCC
Photobacterium amse|ae subsp.
piscicia
2 TSA SYSU
Nocaria serio|ae 3 EA SYSU
Lactococcus |actis 1 TSA ATCC
Staphy|ococcus aureus 1 TSA ATCC
Vibrio sp. 4 TCBSA SYSU
Aeromonas hyrophi|a 3 TSA ZJFF
ITS, 16S-23S intergenic spacers.
a
ATCC, American Type Culture Collection; LMG, Belgian Coordinated Collections of Micro-
organisms; SYSU, Sun Yat-Sen University; MCCC, Marine culture collection of China; ZJIFF,
Zhejiang Institute of Freshwater Fisheries.
b
BHIA, brain heart infusion agar; TSA, tryptic soy agar; EA, Eugon agar; TCBSA, thiosulphate
citrate bile salts sucrose agar.
266
2011
Blackwell Publishing Ltd
JournaI of Fish Diseases 2011, 34, 265-271 S M Zhou et al. 1dent.J.ut.on o[ Streptococcus iniae I, se..J. 1CR ussu,
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
TCGTAACAAGGTAAGCCG-3 and primer B1
5 CT/CA/GT/CTGCCAAGCATCCAC-T3. The
PCR mixture contained bacterial DNA, PCR buffer
(10 m Tris-HCl, pH 8.8, 50 m KCl, 2 m
MgCl
2
, 0.08% Nonidet P40), a 200 concen-
tration of each deoxynucleoside triphosphate,
0.1 of each primer and 1.0 U of Tu polymer-
ase (Fermentas ). The thermocycling parameter
used for this conserved primer set was 35 cycles of
94 C for 1 min, 55 C for 1 min and 72 C for
C for 5 min in
an automated thermal cycler (PTC-100; Bio-
Rad S. .n.ue
isolates in South China and the reference strain
from two national bacterial collections were gel-
)
and sequenced using the primer A1.
Design of primers for specihc amplihcation of
Stveptvcvccus 1u1ue ITS rDNA
A comparison of the sequences of S. .n.ue strains
and related species was made using the ITS
sequences obtained in this study and those available
in the GenBank database (Table 2). Based on the
ITS sequence of S. .n.ue strains obtained in this
study and homologous sequences from the Gen-
Bank database, a new primer set was designed to
amplify a S. .n.ue
ITS. The sequences of the primers were SP-1
(sequence positions 5 95-119 bp): 5-GAAA-
ATAGGAAAGAGACGCAGTGTC-3 and SP-2
(sequence positions 5 447-471 bp): 5-CCTTAT-
TTCCAGTCTTTCGACCTTC-3 (see GenBank
accession numbers GU330188). The primers were
synthesized by SBS Genetech
were performed in a 50 L reaction buffer con-
tained bacterial DNA, PCR buffer (10 m Tris-
HCl, pH 8.8, 50 m KCl, 2.5 m MgCl
2
, 0.08%
Nonidet P40), 200 concentration of each
deoxynucleoside triphosphate, 0.1 of each
primer and 1.0 U of Tu polymerase (Fermen-
tas ). The optimized PCR parameters used for this
C
for 1 min, 60 C for 1 min and 72 C for 1 min,
C for 10 min in an
automated thermal cycler (PTC-100; Bio-Rad ).
Specihcity and sensitivity of the PCR assay
using DNA extracted from all bacterial strains listed
genomic DNA isolated from these bacteria controls
(Table 1), PCR was performed to amplify the 16S
rDNA using the universal primer pA/pH designed
by Edwards, Rogall, Blocker, Emde & Bottger
(1989). Moreover, another 20 S. .n.ue strains
determine their ability to produce the appropriate
Table 2 ITS sequences from the
GenBank databases used to design
Streto.o..us .n.ue
Baotoria spooios Samplo oodo
a
Aooossion numbor
b
Streptococcus iniae ATCC29178/LMG14520 GU330188/GU330189
BCPC 14744 DQ204504
SO-2 GU330193
HD-1 GU330190
HD-2 GU330191
HD-3 GU330195
HD-4 GU330196
HD-5 GU330197
HD-6 GU330198
YJ-1 GU330192
YJ-2 GU330194
Streptococcus parauberis SAP 99 AF284577
Streptococcus aa|actiae ATCC27956 AY347540
Streptococcus ysa|actiae subsp.
ysa|actiae
NCTC4335 EU860341
Streptococcus phocae NCTC 12719 AF489596
Lactococcus arvieae L1-5 AF225968
Staphy|ococcus aureus E AF317718
ITS, 16S-23S intergenic spacers.
a
BCRC, Bioresources Collection and Research Center; NCTC, National Collection of Type
Cultures.
b
GU330188-GU330198 were sequenced in this study.
267
2011
Blackwell Publishing Ltd
JournaI of Fish Diseases 2011, 34, 265-271 S M Zhou et al. 1dent.J.ut.on o[ Streptococcus iniae I, se..J. 1CR ussu,
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
fragment at varying concentrations of genomic
template or bacterial cells. DNA concentrations of
100 ng, 10 ng, 1 ng, 0.5 ng, 0.2 ng, 0.1 ng, 50 pg,
20 pg and 10 pg were evaluated. Also, a single
colony of the strain ATCC29178 was picked from
an agar plate and serially tenfold diluted with sterile
phosphate-buffered saline to 10
8
. Each dilution
was then plated to BHIA to determine cells in each
dilution. These dilutions were used to determine a
minimum level of detection at optimal PCR
conditions.
Preparation of experimentally infected hsh
tissues
Twenty tilapia 25.0 2.0 g, which had been
S. .n.ue by
conventional microbiologic methods, were feed for
1 week and then challenged with S. .n.ue at the
dose of LD50 (4 10
7
CFU). Tissues of brain,
liver, spleen, kidney and muscle were collected from
The presence of S. .n.ue in these tissues was
determined by inoculating them onto BHIA plate
and then incubating at 27 C for 24 h. All
microbiologically positive tissues were investigated
by PCR. Samples of non-inoculated and microbi-
ologically negative healthy tilapia were used as
negative controls.
Organ samples of approximately 0.1-0.2 g each
were lysed by 200 L lysis buffer (20 m Tris-
HCl, 5 m EDTA Na
2
, 400 m NaCl, 1% SDS,
100 mg L
1
proteinase K, pH 8.0) at 55 C with
occasional inversion for 1 h. The resultant lysates
were extracted with phenol-chloroform-isoamylol
(25:24:1) and precipitated with ice-cold 95%
ethanol.
Results
ITS sequencing and analysis
The universal ITS primer set used in this study
yielded the expected 550-bp amplicon for all
ten S. .n.ue strains and the reference strains
ATCC29178 and LMG14520. The ITS sequences
determined in this study have been deposited in the
GenBank nucleotide sequence database, and
the GenBank accession numbers are listed in
Table 2.
Sequence comparison revealed that the
ITS sequences of all S. .n.ue isolates had
99.3% to 100% homology to the ITS sequence
of the reference S. .n.ue strains ATCC29178 or
LMG14520.
Specihcity and sensitivity of the primer set
Using the universal primer set, approximately
of the bacteria controls used in this study (Fig. 1a).
As expected, the new primer set SP-1 and SP-2
of 377 bp with S. .n.ue strains only (Fig. 1b), even
when the annealing temperature decreased to 55 C.
No amplicons were produced from the heteroge-
neous species S. uguIu.t.ue, S. d,sguIu.t.ue, S. uru-
uIer.s, 1u.to.o..us gurv.eue
pathogens or non-pathogenic bacteria listed in
Table 1. Sequencing of one representative amplicon
shown). Identical results were obtained when PCR
was performed with whole bacterial cells boiled for
10 min instead of extracted DNA. The sensitivity of
the PCR assay was established by the detection of
DNA as low as 0.02 ng and as few as 10 CFU
template DNA was obtained by the boiling method
which is quicker and easier than the common DNA
extraction method (Zhou et uI. 2008).

Figure 1 Agarose gel electrophoresis of 16S rDNA PCR

primer set (b). M represents DNA size marker. Lane S represents
strain ATCC 29178 (the reference strain), lanes 1-11 represent
Streto.o..us uguIu.t.ue, Streto.o..us d,sguIu.t.ue, Streto.o..us
uruuIer.s, 1u.to.o..us gurve.ue, 1otoIu.ter.um dumseIue subsp.
.s....du, Ao.urd.u ser.oIue, 1u.to.o..us Iu.t.s, Stu,Io.o..us
uureus, V.Ir.o ungu.IIurum, eromonus ,dro.Iu, and no-DNA
control, respectively. ITS, 16S-23S intergenic spacers.
268
2011
Blackwell Publishing Ltd
JournaI of Fish Diseases 2011, 34, 265-271 S M Zhou et al. 1dent.J.ut.on o[ Streptococcus iniae I, se..J. 1CR ussu,
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
PCR detection of Stveptvcvccus 1u1ue in
experimentally infected hsh
The results displayed that all microbiologically
positive tissues were PCR positive. The 377-bp
all infected tissues regardless of brain, liver, spleen,
kidney or muscle, whereas the tissues from non-
Discussion
Given the low intraspecies heterogeneity but sig-
ITS rDNA has been exploited for the development
bacteria including Ao.urd.u ser.oIue, Streto.o..us
o.ue (Kono, Ooyama, Chen & Sakai 2002;
Hassan, Vossen, Lammler, Siebert & Fernandez-
Garayzabal 2008) and S. .n.ue (Berridge et uI.
1998). These tools have revolutionized the identi-
problems remain relating to reproducibility, spec-
lems have led to the development of several
different primer sets that target different DNA
S. .n.ue.
Berridge et uI.
primer set that targeted the ITS region to identify
S. .n.ue. This primer set was claimed to be useful for
S. .n.ue isolates
s study that we
could refer to. Using this primer set, Roach et uI.
(2006) were consistently able to amplify amplicons
from all tested isolates including the reference strain
cons obtained by them were obscure. According to
et uI. 2006), it
was obviously shorter than the expected amplicon of
373 bp. Surprisingly, this primer set did not work
in our laboratory under generally accepted PCR
conditions. It was ineffective in detecting S. .n.ue
isolated in China or the reference strain
ATCC29178, even when the PCR parameters were
optimized or the primer set of Berridge was used
when synthesized by three different companies.
Given this situation, we sequenced the ITS
rDNA of nine Chinese S. .n.ue strains and one
reference strain from America (ATCC29178) and
Europe (LMG14520) separately in the present
study. Sequence alignment showed that the ITS
sequences were extremely conserved among differ-
ent S. .n.ue strains. The homology of the ITS DNA
sequences was 99.3-100%. However, sequence
alignment displayed that there were 18 nucleotide
positions where unmatched base pairs were detected
between the ITS sequences of the reference strain
ATCC29178 uploaded by Berridge et uI. (1998) to
GenBank (accession number AF048773.1) and
that obtained in this study. Importantly, seven base
pairs of the reverse primer of Berridge do not match
ITS sequences we obtained (marked by box in
Fig. 4). According to our results, we consider that
there are mistakes in Berridge s ITS sequence and
reverse primer set.
In addition to the ITS sequence, 16S rDNA and
the lactate oxidase gene (I.tO) have also been used
as genetic markers to identify S. .n.ue (Berridge

Figure 2 Sensitivity of the Streto.o..us .n.ue

(a) M represents DNA size marker. Lanes 1-8 represent S. .n.ue
DNA of 100 ng, 10 ng, 1 ng, 0.5 ng, 0.2 ng, 0.1 ng, 50 pg,
20 pg and 10 pg, respectively. (b) M represents DNA size
marker. Lanes 1-8 represent the number of bacteria used for the
10
6
, 8 10
5
, 8 10
4
, 8 10
3
, 788,
70, 10 and 1, respectively.
Figure 3
primer set. Lane M represents a DNA size marker; lane S
represents Streto.o..us .n.ue genomic DNA, positive control;
lanes 1, 3, 5, 7 and 9 represent the brain, liver, spleen, kidney
represent brain, liver, spleen, kidney and muscle of negative
269
2011
Blackwell Publishing Ltd
JournaI of Fish Diseases 2011, 34, 265-271 S M Zhou et al. 1dent.J.ut.on o[ Streptococcus iniae I, se..J. 1CR ussu,
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
et uI. 1998; Zlotkin et uI. 1998; Mata et uI. 2004a).
S. .n.ue based on the 16S rDNA
sequence was not able to differentiate S. .n.ue and
S. uguIu.t.ue because of high genetic relatedness
between them (Mata et uI. 2004a). The develop-
ment of the lactate oxidase gene (I.tO) PCR assay
by Mata et uI. (2004a) revealed that the primer pair
LOX-1/LOX-2 could be used successfully to aid in
S. .n.ue via the generation of a
Nawawi, Baiano, Kvennefors & Barnes (2009), a
novel 920-bp variant of the I.tO gene was found in
some of the Australian S. .n.ue isolates when using
the LOX-1/LOX-2 primer pair.
primer sets mentioned above, a new primer set
based on the ITS sequence was developed to rapidly
and accurately identify S. .n.ue in this study. This
new primer set could identify all the strains that
genotypes (Zhou et uI. 2008), as well as the
reference strain ATCC29178. Moreover, the new
primer set SP-1-SP-2 evaluated in the present study
S. .n.ue and
could differentiate S. .n.ue from other common
Streto.o..us S. uguIu.t.ue,
S. d,sguIu.t.ue, S. uruuIer.s and 1. gurv.eue. There
pathogens including 1otoIu.ter.um dumseIue
subsp. .s....du, A. ser.oIue, eromonus ,dro.Iu
and V.Ir.o sp. and non-pathogenic bacteria includ-
ing 1u.to.o..us Iu.t.s and Stu,Io.o..us uureus.
The new primer set SP-1-SP-2 is useful for the
S. .n.ue in pure culture from
colonies on agar plates or from broth. This assay is
easy to use because it works well under generally
accepted PCR conditions. Moreover, quick DNA
template preparation by boiling the bacterial cells
gave the same results as DNA prepared by phenol
extraction. Furthermore, our primer set is also useful
for detection of S. .n.ue in infected tissues of tilapia.
We could detect S. .n.ue in the brain, kidney, liver,
spleen and muscle which were freshly sampled or
preserved at 70 C using this new primer set.
In conclusion, the results of the present study
demonstrated that the PCR assay using this new
S. .n.ue strains in different clinical
situations.
Acknowledgement
The project was supported the Key Projects in
the National Science & Technology Pillar Pro-
gram in the Eleventh Five-year Plan Period
(2007BAD29B05).
References
Berridge B.R., Fuller J.D., de Azavedo J., Low D.E., Bercovier
Figure 4 The unmatched base pairs in the ITS sequence between the reference strain ATCC29178 of Streto.o..us .n.ue uploaded by
Berridge et uI. (1998) to GenBank (accession number AF048773.1) and a representative ITS sequence obtained in this study
(accession number GU330188). ITS, 16S-23S intergenic spacers.
270
2011
Blackwell Publishing Ltd
JournaI of Fish Diseases 2011, 34, 265-271 S M Zhou et al. 1dent.J.ut.on o[ Streptococcus iniae I, se..J. 1CR ussu,
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
oligonucleotide PCR primers for the Streto.o..us .n.ue
16S-23S ribosomal DNA intergenic spacer. ]ournuI o[ CI.n..uI
A..roI.oIog, 36, 2778-2781.
Bowser P.R., Wooster G.A., Getchell R.G. & Timmons M.B.
(1998) Streto.o..us .n.ue infection of tilapia Oreo.rom.s
n.Iot..us in a recirculation production facility. ]ournuI o[ te
WorId uu.uIture So..et, 29, 335-339.
Colorni A., Diamant A., Eldar A., Kvitt H. & Zlotkin A. (2002)
Streto.o..us .n.ue infections in Red Sea cage-cultured and wild
1.seuses o[ uut.. Orgun.sms 49, 165-170.
Edwards U., Rogall T., Blocker H., Emde M. & Bottger E.C.
(1989) Isolation and direct complete nucleotide determination
of entire genes. Characterization of a gene coding for 16S
ribosomal RNA. Au.Ie.. ..ds Reseur. 17, 7843-7853.
Facklam R., Elliott J., Shewmaker L. & Reingold A. (2005)
Streto.o..us .n.ue isolated from humans. ]ournuI o[ CI.n..uI
A..roI.oIog, 43, 933-937.
Hassan A.A., Vossen A., Lammler C., Siebert U. & Fernandez-
Garayza
sequences of 16S rDNA and 16S-23S rDNA intergenic spacer
Streto.o..us o.ue. A..roI.oIog..uI
Reseur. 163, 132-135.
Kitao T. (1993) Streptococcal infections. In: Bu.ter.uI 1.seuses
o[ 1.s (ed. by V. Inglis, R.J. Roberts & N.R. Bromage),
Koh T.H., Kurup A. & Chen J. (2004) Streto.o..us .n.ue discitis
in Singapore. 1merg.ng 1n[e.t.ous 1.seuses 10, 1694-1696.
Kono T., Ooyama T., Chen S.C. & Sakai M. (2002) Sequencing
of 16S-23S rRNA internal transcribed spacer and its appli-
Ao.urd.u ser.oIue by polymerase
chain reaction. uu.uIture Reseur. 33, 1195-1197.
Lau S.K., Woo P.C., Tse H., Leung K.W., Wong S.S. & Yuen
K.Y. (2003) Invasive Streto.o..us .n.ue infections outside
North America. ]ournuI o[ CI.n..uI A..roI.oIog, 41, 1004-
1009.
Lau S.K., Woo P.C., Luk W.K., Fung A.M., Hui W.T., Fong
A.H., Chow C.W., Wong S.S. & Yuen K.Y. (2006) Clinical
isolates of Streto.o..us .n.ue from Asia are more mucoid
and beta-hemolytic than those from North America. 1.ug-
nost.. A..roI.oIog, und 1n[e.t.ous 1.seuse 54, 177-181.
Mata A.I., Blanco M.M., Dominguez L., Fernandez-Garayzabal
J.F. & Gibello A. (2004a) Development of a PCR assay for
Streto.o..us .n.ue based on the lactate oxidase (I.tO) gene with
potential diagnostic value. Veter.nur, A..roI.oIog, 101, 109-
116.
Mata A.I., Gibello A., Casamayor A., Blanco M.M., Dominguez
L. & Fernandez-Garayzabal J.F. (2004b) Multiplex PCR assay
for detection of bacterial pathogens associated with warm-
water Streto.o..os.s I.ed und 1nv.ronmentuI
A..roI.oIog, 70, 3183-3187.
Nawawi R.A., Baiano J.C.F., Kvennefors E.C..E. & Barnes A.C.
(2009) Host-directed evolution of a novel lactate oxidase in
Streto.o..us .n.ue isolates from barramundi (1utes .uI.ur.[er).
I.ed und 1nv.ronmentuI A..roI.oIog, 75, 2908-2919.
Streto.o..us .n.ue
systems. ]ournuI o[ CI.n..uI A..roI.oIog, 27, 20-26.
Shoemaker C.A., Klesius P.H. & Evans J.J. (2001) Prevalence of
Streto.o..us .n.ue in tilapia, hybrid striped bass, and channel
mer..un ]ournuI o[ Veter.nur, Reseur. 62, 174-177.
Weinstein M., Low D., McGeer A., Willey B., Rose D.,
Coulter M., Wyper P., Borczyk A., Lovgren M. & Facklam R.
Streto.o..us
.n.ue. Aew 1ngIund ]ournuI o[ Aed...ne 337, 589-594.
Zhou S.M., Xie M.Q., Zhu X.Q., Ma Y., Tan Z.L. & Li A.X.
Streto-
.o..us .n.ue ]ournuI
o[ 1.s 1.seuses 31, 869-875.
Zlotkin A., Hershko H. & Eldar A. (1998) Possible transmission
of Streto.o..us .n.ue
I.ed und 1nv.ronmentuI A..roI.oIog, 64, 4065-4067.
Re.e.ved. !5 SetemIer 2u!u
Rev.s.on re.e.ved. !2 O.toIer 2u!u
..eted. !6 O.toIer 2u!u
271
2011
Blackwell Publishing Ltd
JournaI of Fish Diseases 2011, 34, 265-271 S M Zhou et al. 1dent.J.ut.on o[ Streptococcus iniae I, se..J. 1CR ussu,
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m
C
lic
k
t
o
b
u
y
N
O
W
!
P
D
F
-XChan
g
e
w
w
w
.d
o
cu-trac
k
.c
o
m