Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China
et al. (2013) Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern
China. PLoS ONE 8(7): e67755. doi:10.1371/journal.pone.0067755
Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China
Yongchun Yang 0
Yinglong Liu 0
Yunlei Ding 0
Li Yi 0
Zhe Ma 0
Hongjie Fan 0
Chengping Lu 0
Dongsheng Zhou, Beijing Institute of Microbiology and Epidemiology, China
0 College of Veterinary Medicine, Nanjing Agriculture University , Nanjing , China
One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae.
Streptococcus agalactiae, also referred to as group B
streptococcus (GBS), is one of the leading causes of bovine
mastitis, which has economically important implications for the
dairy cattle industry throughout the world . S. agalactiae is
also an important human pathogen that can induce invasive
infections in neonates, the elderly and pregnant women [2,3,4].
There is indirect evidence that S. agalactiae is transmitted
between cattle and humans . Clearly, control and prevention
of S. agalactiae mastitis will improve the quantity and quality of
milk production and have important significance for animal
welfare and public health. As we know, studies of the molecular
epidemiology of field S. agalactiae strains is vitally important in
implementing efficient management practices in dairy farms.
However, no information regarding the molecular
characterization of S. agalactiae strains occurring in farms in
China has previously been documented.
Multilocus sequence typing (MLST) and pulsed-field gel
electrophoresis (PFGE) typing are two genotype methods used
to characterize and distinguish specific clones among GBS
isolates . MLST is an unambiguous sequence-based and
reliable typing tool, allowing comparison of the gene distribution
of different isolates collected from all geographic areas and
further investigation of the population structure [6,7]. Five
hundred and eighty-three S. agalactiae sequence types (STs)
have been identified and made available on the MLST website
as of May 2012 (http://pubmlst.org/sagalactiae/), although
information about bovine strains is still limited.
Capsular serotyping is a classical method used for S.
agalactiae in epidemiological studies. To date, ten serotypes,
based on the S. agalactiae capsular polysaccharides (CPS),
have been identified, including Ia, Ib, II-VIII and a new serotype
IX . Capsular genotyping is considered more suitable for
epidemiological investigation because the serotypes can be
identified with or without the CPS expression.
The alpha-like protein (Alp) family play an important role in S.
agalactiae pathogenesis and are also vaccine candidates .
Six members of the Alp family have been extensively studied,
including Alpha-C, Rib, Alp1 (Epsilon), Alp2, Alp3 and Alp4
, they are encoded by genes bca, alp1 (Epsilon), alp2/3,
Rib, and alp4, respectively.
6 (1), 7 (25),
8 (5), 10 (3),
103 16 1 6 2 9 9 2 Ia (58) 57 2b NT (58) 14 (8),) 15
(7), 17 (1),
18 (5), 19 (3)
2 (1), 5 (1),
12 (2), 16
(6), 20 (1),
568 16 1 6 2 51 9 2 Ia (32) 31 2b NT (32) 21 (1), 23
(5), 24 (2),
25 (9), 26
(3), 27 (1)
a CC, clonal complex. b ST, sequence type. c The allelic profiles are presented in the following order: adhP, pheS, atr, glnA, sdhA, glcK, and tkt. d PI, pilus island. Minus in
this column indicates the absence of a PI-1 gene. e Alp, alpha-like protein. f NT, non-typeable.
Three types of pili of S. agalactiae have been identified and
designated as pilus island 1 (PI-1), PI-2a and PI-2b, of which
PI-2a and PI-2b are encoded by genes located in two distinct
loci in the same region of the genome, while the PI-1 gene is
located in a separate region .
The objectives of this study were to elucidate the
relationships between bovine S. agalactiae strains by
molecular characterization based on capsular genotyping, pilus
and Alp gene profiling and MLST and PFGE analyses.
Identification of S. agalactiae in dairy cows
Milk samples were collected from a total of 619 cows with
subclinical mastitis from 33 dairy farms located in six provinces
(Jiangsu, Anhui, Shandong, Zhegjiang, Jiangxi and Fujjian) and
one municipality (Shanghai). S. agalactiae was detected in milk
samples from 102 cows from 21 farms located at five provinces
(except Fujian province) and one municipality. In total, 102
bovine isolates (one isolate per cow) of S. agalactiae were
selected and analyzed (a complete list of all isolates is
provided in Table S1).
Capsular genotype and alpha-like protein
All 102 bovine S. agalactiae isolates belonged to capsular
genotype Ia and II (Table 1) according to the multiplex PCR
assay. Overall, type Ia was the most prevalent accounting for
approximately 89% (91/102) of isolates. Type II was
represented by 11% (11/102) of isolates. A large proportion
(93%; n = 95/102) of the bovine S. agalactiae isolates was
nontypeable (NT) to any of the detected Alp genes according to the
multiple PCR assay (Table 1). Only 5% (5/102) of Alp1
genepositive strains and 2% (2/102) Alp4 gene-positive strains were
characterized. The alpha-like protein genes associated with the
capsular genotype were also identified (Table 1): alp1 and alp4
associated with genotype II, NT associated with genotype II
and Ia (2=36.595, P < 0.001).
Each strain was detected in the three PCR assays for pilus
typing. PI-1 and PI-2a genes were absent in all of the
investigated bovine strains although the corresponding genes
were detected in the positive controls. All 102 bovine S.
agalactiae strains carried the PI-2b gene alone (Table 1).
MLST sequence types and PFGE analysis
All 102 isolates were characterized using MLST; one novel
allele (sdhA allele 51, GenBank accession number is
KF006268) and two new STs (ST 568 and ST570) were
identified. ST568 is a single-locus variant (SLV, in which one
allele differs from the ST) of ST103, ST570 is a double-locus
variant (DLV, in which two alleles differ from the ST) of ST64
and ST67 according to eBURST analysis. ST568 was assigned
based on the presence of a novel allele (sdhA allele 51) and
ST570 was assigned based on new combinations of allele
profiles. In general, all 102 bovine isolates were clustered in six
STs: ST103, ST568, ST67, ST301, ST313 and ST570, and
were grouped in three clonal complexes (CC): CC67, CC64
and CC103 by eBURST analysis (Figure 1). Figure 1 also
shows the close correlation between CC67 and CC64, which
are both subgroups of CC17. This was further supported by the
relationship between ST570 and ST67 (ST570 was a DLV of
ST67) as described previously. However, CC103 remained as
a distinct group.
The isolates were grouped in 27 PFGE types in this study, of
which 16 common (represented by dashed rectangles) and 11
unique types were identified, with a similarity between 43.9%
and 100% (Figure 2). Eight major PFGE types (7, 8, 14, 15, 16,
18, 23 and 25 shown in Figure 2) comprised close to 70%
(71/102) of all the isolates. A total of eight more minor types,
comprising only two or three isolates, corresponded to almost
20% (20/102) of all the isolates.
Table 1 shows the 11 capsular genotype II isolates that were
distributed among four STs (ST67, ST301, ST313, ST570) and
eight PFGE types. The 91 type Ia isolates belonged to three
STs (ST67, ST103, ST568) and 21 different PFGE types.
ST103 and novel sequence type ST568, both belonging to CC
103 (Figure 1), were the predominant sequence types. These
types comprised genotype Ia isolates associated with NT Alp
genes and represented 57% (58/102) and 31% (32/102) of the
isolates, respectively (Table 1). These isolates were clustered
into 20 types according to the PFGE analysis. ST67, ST313
(DLV of ST67) and ST301 (SLV of ST67), were grouped in
CC67 (Figure 1). ST67 represented 5% (5/102) of the isolates
assigned to capsular genotype II (4 isolates) and Ia (1 isolate)
associated with NT Alp genes, and were further clustered in
four PFGE types. ST313 and ST301 comprised a total of 5%
(5/102) of the isolates, which were assigned to capsular
genotype II carrying Alp1 genes and belonged to five PFGE
types. ST570, belonging to CC64, represented only two
isolates of serotype II carrying the Alp4 gene and clustered in
PFGE type 9 (Table 1).
The isolates clustered in the same ST were clearly divided
into several distinct PFGE types. For example, ST103 and 568,
were divided into nine and eleven different PFGE types,
respectively (Table 1 Figure 2). It was also were observed that
the isolates belonging to different capsular genotypes were
clustered in the same ST or PFGE type, while the same
capsular genotypes were divided into different ST or PFGE
types (Table 1 Figure 2).
Distribution of S. agalactiae between and within farms
The STs, molecular genotypes, Alp genes of S. agalactiae
among the 21 S. agalactiae positive farms are presented in
Figure 3. Seven different genotypes were found when
combining these three types. The predominant type, ST568, a
novel ST which is a SLV of ST103, combined with Ia and NT
and was found exclusively among isolates from farms located
in northern provinces (Jiangsu and Shandong) only. ST103, the
predicted founder of ST568, combined with Ia and NT, and with
few exceptions, was found exclusively among isolates in the
farms located in the southern provinces (including Shanghai,
Anhui and Zhejiang).
On the basis of the combination of genotypes described, the
isolates were characterized into 31 types when combined with
the PFGE genotypes (Figure 3). Comparison of S. agalactiae
isolates between herds revealed similar genotypes (10 types)
distributed in different herds, and herd specific types (21 types)
were also observed (Figure 3). Comparison of S. agalactiae
isolates from the same herd showed S. agalactiae isolates
displaying either different types (in 12 herds) or the same type
(in 9 herds) within a same herd. Clustering of multiple isolates
of the same type within an individual herd was also observed
on several farms.
In the present study, 21 of 33 dairy farms screened positive
for S. agalactiae, although control measures were managed in
these farms. It was reported that the herd level prevalence of
S. agalactiae increased steadily from 2000 to 2008 in
Denmark, even with systematic control measures in place,
which had previously been effective in preventing S. agalactiae
mastitis . The increasing prevalence may be due to an
increase infection rate that exceeds the capacity for
eradication. This demonstrates that S. agalactiae remains a
significant cause of mastitis in cattle herds, and more effective
management is required to control S. agalactiae mastitis.
The CPS of S. agalactiae are one of the most important
virulence factors and form the main components of multivalent
vaccines . Capsular genotyping by multiplex PCR revealed
that Ia was the most predominant capsular genotype in Eastern
China. Similar findings have been reported in Germany where
serotype Ia was shown to be prevalent in 19 of 79 bovine S.
agalactiae isolates . However, serotype III and serotypes V
and IV were found to be the most prevalent in Quebec
(Canada) and Norway, respectively [15,16]. The diversity in
serotype distribution of mastitis S. agalactiae might be the
result of divergent geographical regions, times, management
practices and breeds of cow. In the present study, all the
bovine S. agalactiae isolates from 21 farms of five provinces
and one municipality in Eastern China belonged to serotype Ia
The major surface-localized protein antigens of S. agalactiae
belong to the alpha-like protein (Alp) family of surface proteins.
It is reported that S. agalactiae strains usually carry at least
one of the alpha-like proteins, of which proteins Epsilon (Alp1),
Alp2, Alp3, Alp4, Alpha-C and Rib have been extensively
studied , although several bovine and human strains have
been reported to be negative for any of the six proteins
[16,17,18]. In this study, the multiple PCR assay for detecting
Alp genes showed that 92% of the tested bovine isolates were
non-typeable, possibly due to mismatch of the primers for
genes encoding alpha-like proteins in these isolates. Many
studies have previously shown the associations of Alp genes
and serotypes; for example, the Alpha-C protein gene with
serotypes Ia and II [17,19], the Epsilon/alp1 gene with serotype
Ia  and the Rib gene with serotype II . However, in this
study, different combinations of Alp genes and capsular
genotypes were found; the Epsilon/alp1and alp4 genes were
significantly associated with type II, and NT Alp genes with type
Ia and II. The novel combinations of Alp genes and serotypes
are suggested by the emergence of new S. agalactiae clones
S. agalactiae strains carry at least one of the three pilus
islands. Margarit et al.  demonstrated that all 289 human
strains they investigated carried either PI-2a (73% strains) or
PI-2b (27% strains), while PI-1 was missing in 28% of the
strains. Following analysis of 238 isolates, Srensen and
colleagues  found that all the isolates carried PI-2, but PI-1
was missing in 54% of the bovine isolates and in 24% of the
human isolates; therefore, it was concluded that PI-1 genes do
not exist in several lineages. In this study, all of the 102 bovine
S. agalactiae strains from Eastern China carried only PI-2b,
while PI-1 was absent from all of the strains. Since previous
studies have shown that pilus-based vaccines can be effective
in preventing infections caused by homologous challenge of S.
agalactiae , PI-2b proteins may be considered as potential
vaccine candidates for formulation of subunit vaccines against
bovine S. agalactiae mastitis.
Two novel sequence types, ST568 and ST570, were found in
this study, which was expected because the information in the
MLST database about the S. agalactiae isolates prevailing in
China is severely lacking. Thus, the submission of our data to
this database enriches the available data on bovine S.
agalactiae isolates. The novel sequence type ST568 and its
predicted founder ST103 were the predominant STs found in
this study. ST103 is occasionally obtained from dairy cows
according to previous reports . However, a recent study
shows ST103 to be a predominant ST in bovine strains from
Denmark  and here it is shown to be prevalent in Eastern
China, which contributes to a better understanding of the global
epidemiology of mastitis S. agalactiae isolates. The new
sequence type ST570, which is a SLV of ST 64, represented
only 2% of the isolates in this study. Conversely, its predicted
founder ST64 has been reported as a common ST among
bovine S. agalactiae strains . ST67, ST301 and ST313
were grouped in CC67, representing only a total of 12% of the
isolates in this study. However, ST67 has previously been
considered to be the most common ST among bovine isolates
. The occurrence of unique and identical STs identified
between Eastern China and other countries shows that the
types of ST and predominant STs differ in bovine isolates from
divergent geographical regions. However, limited clonal groups
appear in different region and countries according to MLST
analysis. In present study, CC67 and CC64 showed a high
degree clonal of relatedness, while CC103 remained as a
distinct group, suggesting the S. agalactiae strains from bovine
milk in Eastern China comprise two genetically distinct
In this study, identical capsular genotypes belonged to
different clonal groups and identical STs, PFGE types shared
by various capsular genotypes were observed. This switching
may be the result of horizontal transfer of capsular genes,
which is likely to be driven by the host immune response and
supported by the increased fitness acquired by isolates
showing specific phenotype-genotype combinations [6,24].
Similar sequence types prevalent on several different farms
were observed in this study. The novel ST568 is prevalent in
the north, while ST103, the predicted founder of ST568, is
predominant in the south of this region, thus indicating that the
prevailing S. agalactiae on these related farms are from the
same strain, and might be transmitted from farms in the south
to those in the north. It can be speculated that this occurs as a
result of the commercial movement of infected animals
between farms. Further analysis using PFGE demonstrated
that the isolates grouped in the same ST were divided into
several different PFGE types. The combination of genotypes
presented several phenomena: farm specific in several farms,
homology of the isolates among several farms, heterogeneity in
isolates within several individual herds and multiple cows
infected by a single strain on the same farms. S. agalactiae is a
well-known contagious mastitis pathogen, with transmission
occurring between cows within herds . This mode of
transmission is thought to explain how a single strain prevails
in the same herd in the present study and in previous reports
. Different PFGE types were found in isolates clustered in
the same STs, which may be accounted for by different
management practices between farms. Homology of isolates in
different farms and heterogeneity in isolates within individual
herds are reported for the first time in this study. As previously
speculated, the homology of isolates in different farms might
also be due to commercial movement of infected animals
between farms; however, it is not known why and how several
different genotypes of S. agalactiae strains were obtained from
the same herd, because few molecular epidemiological studies
of sources of infection or transmission routes have been
conducted in cattle. This heterogeneity might suggest the
emergence of several strains originating from several sources
of infection, with infected humans as the suspected infection
In conclusion, the results of the present study demonstrate
that the distribution of STs, capsular genotypes, and pili genes
among the dairy cattle in Eastern China were similar to
previously reports of bovine S. agalactiae strains; however
some geographic characteristics were revealed by the
emergence of unique Alp profiles and prevalent novel STs.
ST103 to be a predominant ST in bovine mastitis from Eastern
China, which contributes to a better understanding of the global
epidemiology of mastitis S. agalactiae isolates. The conserved
pilus type of S. agalactiae isolates provides positive information
for the development of vaccines against S. agalactiae. Further
investigation is necessary to establish the epidemiology of S.
agalactiae, and to determine how S. agalactiae can be best
controlled and prevented.
Materials and Methods
Milk samples were obtained with consent from animals with
subclinical mastitis under the ethical approval granted by
College of Veterinary Medicine, the Nanjing Agricultural
University Veterinary College. The protocol was permitted by
the owners of the dairy farms under investigation. All efforts
were made to minimize animal suffering.
S. agalactiae reference strains
Four reference strains of S. agalactiae: ATCC 13813
(serotype II), ATCC 12403 (Serotype III), ATCC BAA-611
(Serotype V) and A909 (Serotype Ia), used in this study were
obtained from the American Type Culture Collection (ATCC) as
Identification of S. agalactiae from milk samples
A total of 619 cows from 33 large-scale diary farms located in
Eastern China were selected to participate in this study
according to the willing of the farmers (information about the
farms were provided in Table S2). Holsteins dairy cows were
preferably kept on the 33 dairy farms with a mean yield at 305
d of 7,539 kg of milk. For each farm, milk somatic cell counts
(SCC) of each cow were routinely carried out twice per month
by electronic counting (Fossomatic 5000TM, Foss Electric,
Hillerd, Denmark). Subclinical mastitis is suspected when
SCC 300,000 cells/ml, with decreased milk production and no
inflammation of the udder. Milk samples were aseptically
collected from dairy cattle with subclinical mastitis in these
farms from January to December in 2011. In brief, each udder
of the subclinical mastitis cow was washed and dried with a
clean face towel. Each teat was disinfected with swabs soaked
in 70% ethyl alcohol. The first few streams were discarded
before milk samples were collected in 10-ml sterile plastic
tubes and numbered. Each teat was disinfected with 70% ethyl
alcohol after collection. The samples were taken to the
laboratory as soon as possible, or stored at -20C for later
For isolation of S. agalactiae, 20 l of milk was streaked on
5% sheep blood agar plates and incubated at 37C for 24 h. S.
agalactiae were identified by conventional methods  and
were further confirmed by PCR with 16S DNA species-specific
primers as described by Martinez et al. . A single isolate
from each sampled cow was selected and stored at -70C for
further experimental use.
Bacterial culture and DNA extraction
The isolates, including the reference strains, were cultured in
3 ml of Tryptic soya broth (TSB, MO BIO, Laboratories. Inc.)
overnight at 37C. The bacterial culture was centrifuged
(14,000 g for 5 min at room temperature) and the pellets were
harvested and resuspended in 200 l TE buffer (10 mM
TrisHCl, pH 8.0; 1 mM EDTA, pH 8.0) supplemented with 20 mg/ml
(final concentration) of lysozyme and incubated at 37C for 30
min. Genomic DNA was extracted using the Bacterial DNA
extraction kit following manufacturers specifications for
Grampositive bacteria (Omega Bio-Tek, USA). The extracted DNA
was used as the template for PCR.
The capsular genotype Ia, Ib, IIIX of S. agalactiae was
identified by a multiplex PCR assay as previously described
. The PCR system (total volume, 25 l) contained 50 ng
DNA template, 1 PCR buffer; 2 mmol/1 MgCl2, 200 mol/l
dNTPs (dATP, dTTP dCTP, and dGTP), 400 nmol/l primers
cpsI-Ia-6-7-F and cpsI-7-9-F, 250 nmol/l of each other primer
and 0.3 U of HotMaster Taq DNA Polymerase (Tiangen,
China). The PCR amplification conditions were as follows:
preheating at 95C for 5 min, followed by 15 cycles of 95C for
1 min, 54C for 1 min, and 72C for 2 min and then by an
additional 25 cycles of 95C for 1 min, 56C for 1 min, and
72C for 2 min with a final cycle of 72C for 10 min. Serotypes
of strains were identified by analyzing the unique banding
pattern following agarose gel (1.5% wt./vol.) electrophoresis.
Alpha-like protein (Alp) family
The alpha-like protein genes bca, alp1 (Epsilon), alp2/3, Rib,
and alp4 in the strains were detected by a simple multiplex
PCR assay described by Creti, et al. . In brief, the PCR
system (total volume, 25 l) contained the following: 50 ng
DNA template, 1 PCR buffer; 2 mmol/l MgCl2, 200 mol/l
dNTPs, 400 nmol/l of each of the five pairs of primers; 0.3 U of
HotMaster Taq DNA Polymerase (Tiangen, China). The
amplification conditions were as follows: preheating at 96C for
3 min, followed by 30 cycles of 95C for 1 min, 58C for 45 s,
and 72C for 45 s, with a final cycle of 72C for 10 min.
Amplification of the alpha-like protein genes was evaluated by
agarose gel (2% wt./vol.) electrophoresis of the PCR products.
Strains that tested negative for any of the Alp genes under
investigation were considered non-typeable (NT).
To identify the presence of pilus islands in the strains, three
PCR assays were used to detect the PI-1, PI-2a or PI-2b genes
as described previously . In brief, the PCR system (total
volume, 25 l) contained 10 ng DNA template, 1 PCR buffer;
2 mmol/l MgCl2, 200 mol/l dNTPs, 400 nmol/l of each of the
six primers; and 0.25 U of TaqDNA polymerase (Tiangen,
China). The amplification conditions were as follows:
preheating at 94C for 5 min, followed by 35 cycles of 94C for
45 s, 54C for 45 s, and 72C for 1 min and 30 s to 2 min
(according to the lengths of the amplicons) and concluding with
a cycle of 72C for 10 min. The identity of the PCR products
was analyzed by agarose gel (1% wt./vol.) electrophoresis and
deemed to be positive based on the expected size of the PCR
All the isolates were typed using multilocus sequence typing
(MLST) in this study. The seven housekeeping genes (adh,
pheS, atr, glnA, sdhA, glcK and tkt) were amplified by PCR and
internal fragments sequences were obtained as described
previously . For each isolate, the allele number and
sequence types (STs) were defined by analysis of the alleles
sequence in the MLST database (http://pubmlst.org/
sagalactiae/). The allele sequences or previously undescribed
ST were assigned new numbers and the data were deposited
in the MLST database. CC analysis was performed using the
entire S. agalactiae MLST database and eBURST program
DNA was extracted and digested with the SmaI restriction
enzyme (Takara, China.) as previously described [31,32]. The
PFGE program was performed according to Chen et al. . A
Salmonella serotype Braenderup H9812 DNA digested with
XbaI was used as the molecular size standard as
recommended in PulseNet . The S. agalactiae PFGE
patterns were analyzed with BioNumerics (Applied Maths
BVBA, Belgium) using an optimization setting of 0.00% and
band position tolerance of 1.5%. Cluster analysis was
performed using the Dice coefficient and UPGMA of the
digitalized PFGE patterns for the 102 S. agalactiae strains.
Genotypic related groups, characterized at 80% similarity or
above are represented by dashed rectangles in the
Categorical data were analyzed by using the Pearson
chisquared test. When data were insufficient for the test
demands, Fishers exact test and Likelihood-ratio tests were
used. P values of <0.05 were considered to indicate statistical
We wish to thank Huimin Qian (Jiangsu Provincial Center for
Disease Prevention and Control, China) for the helping in
PFGE analysis with BioNumerics software.
Conceived and designed the experiments: YY HF CL.
Performed the experiments: YY YL YD. Analyzed the data: YY
ZM HF. Contributed reagents/materials/analysis tools: YY LY.
Wrote the manuscript: YY HF.
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