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REVIEW PAPER
Methods for the detection and characterizationof Streptococcus suis: from conventional bacterial culturemethods to immunosensors
Xiaojing Xia . Xin Wang . Xiaobing Wei . Jinqing Jiang . Jianhe Hu
Received: 10 March 2018 / Accepted: 14 June 2018 / Published online: 23 June 2018
� Springer International Publishing AG, part of Springer Nature 2018
Abstract One of the most important zoonotic
pathogens worldwide, Streptococcus suis is a swine
pathogen that is responsible for meningitis, toxic
shock and even death in humans. S. suis infection
develops rapidly with nonspecific clinical symptoms
in the early stages and a high fatality rate. Recently,
much attention has been paid to the high prevalence of
S. suis as well as the increasing incidence and its
epidemic characteristics. As laboratory-acquired
infections of S. suis can occur and it is dangerous to
public health security, timely and early diagnosis has
become key to controlling S. suis prevalence. Here, the
techniques that have been used for the detection,
typing and characterization of S. suis are reviewed and
the prospects for future detection methods for this
bacterium are also discussed.
Keywords Streptococcus suis � Detection �Immunological � PCR � Immunosensor
Introduction
Streptococcus suis, an oval or olive-shaped Gram-
positive coccus that can occur singly or in pairs, is
widely found in nature and in porcine tonsils and
tracheal secretions. The bacterium is an important
zoonotic pathogen (Feng et al. 2014). S. suis is highly
resistant and capable of surviving for extended periods
in faeces, dust and water. As flies can carry the
bacterium, which remain infectious for more than
5 days, flies are considered important vectors for this
infection. Serotypes 20, 22, 26 and 33 were removed
from the S. suis taxon based on DNA–DNA homology
and sodA and recN phylogenies and serotypes 32 andXin Wang is the co-first author.
X. Xia � X. Wei � J. Jiang (&) � J. HuCollege of Animal Science and Veterinary Medicine,
Henan Institute of Science and Technology, No. 90,
Hualan Street, Xinxiang 453003, Henan, People’s
Republic of China
e-mail: [email protected]
X. Xia � J. Hu (&)
Postdoctoral Research Base, Henan Institute of Science
and Technology, No. 90, Hualan Street,
Xinxiang 453003, Henan, People’s Republic of China
e-mail: [email protected];
X. Xia
Post-doctoral Research Station, Henan Agriculture
University, Zhengzhou 450002, People’s Republic of
China
X. Wang
College of Agriculture and Forestry Science, Linyi
University, Linyi 276005, People’s Republic of China
123
Antonie van Leeuwenhoek (2018) 111:2233–2247
https://doi.org/10.1007/s10482-018-1116-7(0123456789().,-volV)(0123456789().,-volV)
34 were removed from the S. suis taxon based on
genetic analysis, respectively (Hill et al. 2005; Tien
et al. 2013). Hence, there are currently 29 remaining
true S. suis serotypes, of which 1, 2, 1/2, 7, 9, and 14
are pathogenic, with serotype 2 (SS2) being the most
virulent and the most widely distributed. Experimental
studies have confirmed that S. suis capsular polysac-
charide (CPS), extracellular factor (EF), muramidase-
released protein (MRP), hemolysin, adhesin, fibro-
nectin binding protein (fbpS) and glutamate dehydro-
genase play important roles in the pathogenesis of this
pathogen (Segura et al. 2017). In the pig industry, sales
and meat-processing workers are susceptible to the
disease; indeed, after contact with the infected pigs,
the bacterium can penetrate the damaged skin, mucous
membranes or the digestive tract and cause human
Streptococcus toxic shock syndrome (STSS) and
streptococcal meningitis syndrome (SMS) (Mohapatra
et al. 2015). Both STSS and SMS are characterized by
acute onset, rapid progression, and high mortality.
Despite lifetime treatment, some patients may develop
permanent deafness and other sequelae.
Human S. suis infection is a new disease that poses a
significant public health threat to the life and health of
humankind. It also has a serious negative impact on the
social order and economic development. Of note, two
large-scale outbreaks of lethal SS2 infection with a
hallmark of streptococcal toxic shock-like syndrome
(STSLS) occurred in China in 1998 and 2005, respec-
tively, raising grave concerns for public health (Yu et al.
2006; Tang et al. 2006; Ye et al. 2006). Hence, accurate
and early detection is critical for controlling S. suis
infection. Scientists have long been committed to
establishing a highly sensitive and rapid diagnostic ap-
proach for S. suis. However, conventional isolation,
culture and biochemical identification are time-con-
suming and have low-sensitivity (Xia et al. 2017a, b). In
recent years, the development of molecular biology
techniques have opened up new possibilities for S. suis
detection. In particular, the rapidly developed methods
of colloidal gold immunochromatography and
immunosensors have the advantages of being simple,
quick, specific and sensitive, and having achieved rapid
development (Wang et al. 2013; Ju et al. 2010). Figure 1
provides a comparison of conventional bacterial culture
methods and culture-independent detection methods
(Wang and Salazar 2016). This article reviews the
published literature on the methods employed for the
detection, typing and characterization of S. suis with
particular emphasis on developments in immunological
and nucleic-acid based detection methods for the
detection of this organism.
Conventional bacterial culture methods
Bacteria are isolated from the typical diseased organs
of affected animals, and preliminary identification of
S. suis can be achieved by bacterial morphology as
well as culture and biochemical characteristics
(Table 1). S. suis is an aerobic or facultative
anaerobe with high nutritional requirements, show-
ing poor growth on ordinary medium, but the ability
to grow well in anaerobic broth. The typical S. suis
colony on a blood plate is alpha-hemolytic, needle-
tip-sized, round, dewdrop, and translucent. Gram
staining reveals a single or double arrangement of
Gram-positive cocci with a few short chains (No-
moto et al. 2015). Rosendal et al. have recommended
trypticase soy agar containing gentamicin, crystal
violet, nalidixic acid and 5% defibrinated bovine
blood for culturing S. suis (Rosendal et al. 1986). In
addition, Kataoka et al. isolated S. suis using a
selective medium that consisted of Todd-Hewitt
broth, Bacto-agar, defibrinated sheep blood, crystal
violet, colistin and nalidixic acid (Kataoka et al.
1991). A commercial product, the API 20 Strep
identification system can be used for the identifica-
tion of S. suis at the species level (Haleis et al.
2009). Overall, the results of biochemical tests for
different types of S. suis vary greatly, and the
morphological, cultural and biochemical reactions
and phenotypic characteristics of bacteria are diffi-
cult to type, requiring other test methods for accurate
typing.
Immunological-based methods
Immunoassay techniques exploit the highly specific
binding that occurs between antigens and antibodies
and facilitate quantitative or qualitative detection
based on the specific reactions caused by this binding.
Modern immunological techniques have enabled
highly sensitive and rapid diagnostic detection and
have been developed into a variety of immunoassay
methods by introducing enzymatically catalyzed
reactions, fluorescent or isotopic labeling as a specific
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2234 Antonie van Leeuwenhoek (2018) 111:2233–2247
measure of antigen–antibody binding, such
approaches have been developed into a variety of
immunoassay methods (Table 1).
Enzyme linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is
based on immunoassay techniques that utilize
enzyme-catalyzed reactions to enhance the sensitivity
of specific antigen–antibody reactions (Schalhorn and
Wilmanns 1980). ELISA is widely used for the
detection of a variety of pathogenic microorganisms
and is considered to be one of the most successful
detection technologies in the past few decades.
ELISAs were first used by Vecht et al. to detect SS2
pathogenic strains and non-pathogenic strains. The
results for the proteins MRP and Epf were almost
identical to those of western blotting, indicating that
the established assays were not only simple but also
rapid and reliable for identifying SS2 strains (Vecht
et al. 1993). Campo et al. detected the antibody against
SS2 with ELISA using capsule polysaccharides as the
diagnostic antigen (CPS–ELISA) and compared the
results with ELISA using the bacterium as the antigen
(WCA–ELISA). The results showed that the speci-
ficity of WCA–ELISA was very low when detecting
other serotypes using rabbit antiserum due to a cross-
reaction because of common antigens. In contrast,
Fig. 1 Comparison of dection methods
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Antonie van Leeuwenhoek (2018) 111:2233–2247 2235
Table 1 Approaches for detection, identification and typing of Streptococcus suis
Different approaches Description References
Conventional bacterial culture methods
Selective medium-based
cultivation
Trypticase soy agar, 5% defibrinated bovine blood,
crystal violet, nalidixic acid and gentamicin;
Rosendal et al. (1986)
Todd-Hewitt, bacto-agar, defibrinated sheep blood,
nalidixic acid, colistin and crystal violet
Kataoka et al. (1991)
API 20 Strep identification
system
Identification of Streptococcus suis at species level Haleis et al. (2009)
Immunological-based methods
Enzyme linked immunosorbent
assay (ELISA)
Capsule polysaccharide as diagnostic antigen (CPS–
ELISA)
Del et al. (1996)
Whole-bacterium as diagnostic antigen (PPA–
EILSA)
Sun et al. (2008)
Dot–ELISA Anti-MRP and anti-EF antibody as capture antibody Oo et al. (2001)
Sao-M as diagnostic antigen Xia et al. (2017a, b)
Colloidal gold-based
immunochromatographic
assay (GICA)
Capsule polysaccharide as diagnostic antigen Yang et al. (2007)
Anti-SS2 antibody as capture antibody Ju et al. (2010)
Polyclonal antibodies (pAbs) against S. suis as
capture antibody
Nakayama et al. (2014)
Immunouorescence methods FITC labeled anti-SS2 antibody as capture antibody Zhu et al. (2010)
CdSe/ZnS quantum dot fluorescent probe based on
the SS2 of MRP antibody
Wu et al. (2009)
SERS Surface enhanced Raman scattering (SERS) with
MRP protein as capture antigen
Chen et al. (2012)
Nucleic acid-based detection methods
General PCR sly as target gene Okwumabua et al. (1999)
93 nucleic acid probes specific to genes in the cps
locus
Wang et al. (2012)
recN as target gene Ishida et al. (2014)
Multiplex PCR cps, epf, mrp, sly and arcA as target gene Silva et al. (2006)
cps as target gene Kerdsin et al. (2014)
Major clonal complexes (CCs) as target gene Hatrongjit et al.(2016)
FQ-PCR sodA Tang et al. (2012)
cps2J Sun et al. (2008) and Bonifait et al.
(2014)
Fibronectin binding protein (fbpS) Srinivasan et al. (2016)
16S rRNA Su et al. (2008)
cps9H Dekker et al. (2016)
PFGE Pulsed-field gel electrophoresis Schwartz and Cantor (1984), Berthelot-
Herault et al. (2002) Marois et al.
(2007)
RFLP Restriction fragment length polymorphism Mogollon et al. (1990), Amass et al.
(1997)
MLST Multilocus Sequence typing King et al (2002), Princivalli et al.
(2009), Dong et al. (2017), Zheng et al.
(2018)
RAPD Random amplified polymorphic DNA Vecht et al. (1991), Gottschalk et al.
(1998), Martinez et al. (2002), (2003)
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2236 Antonie van Leeuwenhoek (2018) 111:2233–2247
standardized CPS–ELISA significantly reduced the
cross-reaction when using 0.1 mg antigen per pore
(Del et al. 1996). To enlarge the detection range to
simultaneously detect samples from different sources,
such as guinea pigs, rabbits, and pig serum, Xia et al.
established PPA–ELISA by utilizing enzyme-labeled
streptavidin (SPA) to replace the secondary antibody,
avoiding the requirement of a variety of secondary
antibodies (Xia et al. 2017a, b). Although specificity is
one of the main advantages of ELISA, many secreted
proteins of S. suis share high homology with those of
other bacteria, which increases the possibility of false-
positive results.
Dot enzyme-linked immunosorbent assay (dot–
ELISA), also known as dot-immunobinding, is an
immunoassay technique that employs a cellulose
membrane as the carrier. Dot–ELISA is simple and
fast in operation, and the results are readily
observed, without the need for any special equip-
ment. This approach is thus suitable for the
massive and on-site diagnosis of S. suis infection.
As an example, Oo et al. purified S. suis virulence-
associated proteins MRP and EF and prepared their
antibodies against these proteins. Dot–ELISA and
the indirect ELISA methods were established using
these antibodies and used to detect 17 strains of S.
suis from swine and 2 strains of S. suis from hu-
mans; MRP and EF positivity rates were 61% (11/
18) (Oo and Lu 2001). Xia et al. also established
dot–PPA–ELISA using glutamate dehydrogenase as
a diagnostic antigen, and the results of an assay of
160 samples well coincided with those of conven-
tional plate ELISA (Xia et al. 2017a, b).
Colloidal gold-based immunochromatographic
assay (GICA)
Colloidal gold-based immunochromatographic assay
(GICA) is an in vitro diagnostic technology that
combines colloidal gold labeling, immunoassay, chro-
matography, monoclonal antibody technology and
new material technology. GICA is convenient with
definitive results, without complicated operation or
techniques and special equipment, and it has become a
new direction in the field of clinical and quarantine
diagnosis. Yang et al. employed colloidal gold-
labelled staphylococcal protein A (SPA) as a probe
and purified SS2 CPS and healthy pig IgG as a
detection line reagent and contrast reagent, respec-
tively, to develop a rapid detection strip for SS2. The
test results for 14 serum samples from pigs that
survived challenge with SS2 and 24 hyperimmune
serum samples raised against SS2 showed a 100%
correlation between conventional ELISA and
immunochromatographic results (Yang et al. 2007).
In addition, Ju et al. used the citrate reduction method
to prepare colloidal gold particle-labeled SS2 poly-
clonal antibody to establish an immunochromato-
graphic test for SS2 detection. The results showed that
the optimum antibody labeling amount per ml of
colloidal gold was 22 lg mL-1, and that the optimal
coating antibody concentration was 2 mg mL-1. The
lower limit of detection of the colloidal gold
immunochromatographic test strip was
106 CFU mL-1, and the detection time was
5–15 min. Moreover, there was no cross-reaction of
the antibodies with other related pathogens and 15
serotypes of S. suis, indicating that the method is
simple in operation with high sensitivity and strong
Table 1 continued
Different approaches Description References
Ribotyping Ribotyping Okwumabua et al. (1995), Smith et al.
(1997), Staats et al. (1998), Vanier
et al. (2009)
LAMP Loop-mediated isothermal amplification Zhu et al. (2010), Huy et al. (2012),
Zhang et al. (2013), Arai et al. (2015)
DNA microarray DNA microarray Zheng et al. (2008)
Nano material based immunosensors
Electrochemiluminescence
immunosensor
It is based on the strategy of enhancing reacted
efficiency of co-reactants
Wang et al. (2013, 2014)
Amperometric immunosensor It is based on functionalized nanoparticles Zhu et al. (2013)
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Antonie van Leeuwenhoek (2018) 111:2233–2247 2237
specificity and can be used for rapid early screening
and detection of S. suis (Ju et al. 2010). Nakayama
et al. developed a rapid diagnosis kit that detects S.
suis antigens in urine using a colloidal gold-based
immunochromatographic stripe (ICS) test, which
enables the quantitative detection of S. suis antigens.
The ICS sensitivity is such that 1.0 9 104 CFU of the
streptococci and 0.05 mg of the CPS can be detected.
No cross-reactivity was observed with Streptococcus
agalactiae, Streptococcus pneumoniae, Escherichia
coli, Enterococcus faecalis, Pseudomonas aerugi-
nosa, Staphylococcus aureus, or Klebsiella pneumo-
niae (Nakayama et al. 2014).
Immunofluorescence methods
Although immune enzyme technology has good sensi-
tivity, its relatively complicated procedure limits further
development. However, as immunofluorescence meth-
ods developed on the basis of immunoassay techniques
involve simpler procedures and have high sensitivity and
specificity, they are considered among the most promis-
ing pathogen analysis methods. In addition, the develop-
ment of nanotechnology has brought new opportunities
for fluorescence analysis. For example, recently devel-
oped nanomaterials, such as fluorescent quantum dots,
have the excellent properties of high fluorescence
quantum yields, broad excitation spectral ranges, narrow
emission spectrum ranges, strong bleaching resistance
and good space compatibility.Wu et al. developedCdSe/
ZnS quantum dots with mercaptoacetic acid modified to
high luminous efficiency as well as a preparation of an
anti-MRP antibody (MRPAb) CdSe/ZnS quantum dot
fluorescent probe based on the S. suis type 2 virus-
induced factor-related, an important force of MRP
antibody to develop a new method for detecting MRP
antigen (MRPAg). The linear detection range of this
method was 5.0 9 10-8–1.5 9 10-6 mol/L, with a
detection limit of 1.9 9 10-8 mol/L, which providing a
new method for S. suis detection (Wu et al. 2009).
Surface-enhanced Raman scattering (SERS)
With the rapid development of modern nanotechnology,
new diagnostic techniques and analytical methods have
been emerging for S. suis. Recently, Chen et al. reported
an immunoassay based on surface-enhanced Raman
scattering (SERS) spectroscopy that was developed to
detect SS2 anti-MRP antibodies by utilizing thorny gold
nanoparticles (tAuNPs) as SERS substrates. Initially,
multi-branching tAuNPs were produced by seed-medi-
ated growth methods in the absence of surfactant and
template, facilitating the covalent attachment of p-
mercaptobenzoic acid (pMBA) to tAuNP via S–Au
linkage. The obtained immuneSERS tag,which affords a
strong Raman signal, enabled indirect detection of SS2
anti-MRP antibodies, with the sandwich assay being
performed at a highly sensitive level. The Raman
intensity at 1588 cm-1 was proportional to the logarithm
of the concentration of the anti-MRP antibody in the
range of 0.01–100 ng mL-1, with a detection limit of
0.1 pg mL-1. In addition, the results of the proposed
SERS method for anti-MRP antibody detection in
porcine serum samples were consistent with the results
of the ELISA, indicating that there is great potential for
clinical application in diagnostic immunoassays (Chen
et al. 2012).
Immunomagnetic separation (IMS)
IMS is a technique that utilizes the specific reaction of
antigen and antibody and the magnetic response of
magnetic beads for separation and enrichment. It has
the characteristics of strong specificity, high sensitiv-
ity and fast separation speed. It can also eliminate
matrix interference and enrich target detection objects
from complex samples (Fedio et al. 2011; Safarik et al.
1995; Gottschalk et al. 1999). This method was first
used for the selective isolation of S. suis serotypes 2
and 1/2 from tonsils of carrier animals in 1999.
Superparamagnetic polystyrene beads were coated
with either a purified monoclonal antibody (MAb)
directed to a capsular sialic acid-containing epitope or
purified rabbit immunoglobulin G, both specific for S.
suis serotypes 2 and 1/2. Results showed that this
method can be used to isolate a specific serotype from
carrier pigs, which low-pathogenic serotypes and non-
typable strains compete for the same target site in the
tonsils, with a detection limit of 101 CFU/0.1 g of
tonsil (Gottschalk et al. 1999).
Nucleic acid-based detection methods
With the development and application of new tech-
nologies, research has switched from routine etiolog-
ical identification to molecular aspects. There are a
number of diagnostic tests for S. suis, and these
123
2238 Antonie van Leeuwenhoek (2018) 111:2233–2247
technologies can both detect the bacterium and even
distinguish between different serotypes of S. suis
(Table 1).
Polymerase chain reaction (PCR)-based detection
Polymerase chain reaction (PCR) has high sensitivity,
specificity, good repeatability and easy operation. It
can provide rapid and accurate etiological diagnosis in
a short amount of time. In recent years, PCR has been
widely applied for S. suis detection, with remarkable
progress.
General PCR
Okwumabua et al. designed primers based on the
suilysin (sly) gene sequences of type 2 strains to
establish PCR, but the PCR results showed that this
approach could not detect all serotypes or pathogenic
strains (Okwumabua et al. 1999). In a study by Wang
et al. 2–6 serotype–specific genes of each of eight
serotypes (3, 4, 5, 8, 10, 19, 23, and 25) were identified
by cross-hybridization with 93 nucleic acid probes
specific to sequences in the cps locus, and these
authors further developed serotype–specific PCR
assays for rapid and sensitive detection of the eight
serotypes of SS (Wang et al. 2012). Since 2005,
serotypes 20, 22, 26, 32, 33, and 34 have been
successively removed from the S. suis taxon (Hill et al.
2005; Tien et al. 2013). In a recent study, Ishida et al.
designed a PCR method using the recombination/
repair protein (recN) gene of S. suis. Its specificity was
confirmed by comparison with other PCR methods for
S. suis. In addition, the recN PCR limits of detection
for all reference S. suis strains were similar, indicating
that recN PCR can provided reliable results for
different bacterial strains and isolates (Ishida et al.
2014).
Multiplex PCR (m-PCR)
Multiplex PCR (m-PCR), also known as multiplex
primer PCR, is a PCR amplification technique devel-
oped based on conventional PCR. Multiplex PCR
employs multiple pairs of specific primers to simul-
taneously amplify different DNA fragments in a PCR
system, greatly improving the detection efficiency and
saving manpower, materials and financial resources
for detection. This allows for rapidly determining
multiple pathogens or different bacterial serotypes at
the same time. Based on virulence-related genes such
as EPF, MRP and sly, a multiplex PCR that distin-
guishes at least 6 MRP variants was developed (Silva
et al. 2006). In 2012, Kerdsin et al. proposed multiplex
PCR assays using serotype-specific cps genes, which
can distinguish among 15 serotypes of S. suis isolates
from humans and pigs. Subsequently, these research-
ers developed an expanded multiplex PCR assay, that
was able to detect all serotypes of S. suis in four
reactions (Kerdsin et al. 2014). Recently, the major
clonal complexes (CC) method was applied to devel-
oped a multiplex PCR assay to detect S. suis strains
relevant to human infection (Hatrongjit et al. 2016).
Fluorescence quantitative real-time polymerase chain
reaction (FQ-PCR)
Fluorescence quantitative real-time-PCR (FQ-PCR)
has the characteristics not only of high conventional
PCR amplification efficiency as well as high probe
specificity, high sensitivity and high precision of
spectral technology. FQ-PCR has been widely used in
the detection of pathogenic microorganisms (Tang
et al. 2012); indeed, FQ-PCR can be employed to solve
the ‘‘window period’’ problem of immunological
detection and to determine whether the infection is
latent or subclinical. In addition, FQ-PCR can distin-
guish between current and previous infection, an
aspect that antibody detection fails to do. For instance,
Sun et al. established a SYBR Green influorescence
real-time quantitative PCR detection method for SS2
using cps2J (in the capsule antigen-encoding gene
cluster) as the target gene, and real-time quantitative
detection of the target bacteria was realized through
establishment of a standard curve. The method can
accurately reflect the intensity of infection or pollu-
tion, to a large extent avoiding false-positive results,
and further improve the detection of S. suis (Sun et al.
2008). Nga et al. developed a real-time PCR assay for
the specific detection of S. suis serotypes 2 and 1/2 for
cps2J (Nga et al. 2011). Given the pathogenic potential
of several serotypes (Gustavsson and Rasmussen,
2014), the ability to detect all known serotypes is
highly desirable. In 2016, Srinivasan et al. used Primer
Express 3.0 to develop degenerate oligonucleotide
primers and probes for S. suis targeting the fbpS gene.
The primers and fluorescent dye-labeled probe were
designed by aligning multiple fbpS gene sequences
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Antonie van Leeuwenhoek (2018) 111:2233–2247 2239
from different serotypes available in GenBank and
partial fbpS genes sequenced from all known ser-
otypes. The assay could detect all 35 recognized
serotypes (1–34 and 1/2), with the same sensitivity
(\ 10 copies/assay) as the assay reported by Srini-
vasan et al. (Srinivasan et al. 2016). Other FQ-PCR
methods for S. suis detection and quantification in pig,
human and environmental samples included targeting
the 16S rRNA gene (Su et al. 2008), the serotypes 2
(and 1/2)-specific cps2J gene (Bonifait et al. 2014),
and the cps9H gene (Dekker et al. 2016).
Loop-mediated isothermal amplification (LAMP)
Loop-mediated isothermal amplification (LAMP) is
a nucleic acid amplification technology developed
by Japanese scholar Notomi that was developed in
2000. It has caught the research community’s
attention because it is specific, sensitive, simple,
and rapid and there is no need for expensive
equipment. Huy et al. designed LAMP primers for
the 16S RNAs of four meningitis bacteria (S.
aureus, S. pneumoniae, S. suis, and S. agalactiae),
whereby infection by one of the four can be ruled
out if there is no amplification. A positive result is
followed by enzyme digestion with restriction
enzyme Dde I and Hae III and agarose gel elec-
trophoresis is used to analyze the products, and the
bacteria can be determined by the sizes of the
fragments (Huy et al. 2012). Zhu et al. designed 4
primers according to the gene sequence of the 89K
virulence island of the SS2 China isolate
(05ZYH33) and applied LAMP to detect 21 strains
of SS2, 18 strains of other Streptococcus, 9 strains
of Staphylococcus, 5 other strains of other species
and 49 unknown samples. The findings showed that
LAMP only displayed a positive result for the SS2
endemic strain containing the 89K virulence island,
whereas other strains were negative, indicating that
this method is specific. A negative result was
obtained by application of LAMP for practical
examination of the 89K pathogenicity island gene
in 49 different source samples, 32 of which were
nasopharyngeal swabs from patients with unknown
fever and 15 from normal emergency pig tonsil
throat swab samples; one blood sample from two 2
cases of suspected SS2 was also negative, in
agreement with the results of common PCR/quan-
titative PCR (Zhu et al. 2010). In 2013, Zhang et al.
designed a LAMP method with primers targeting the
recN gene for detecting SS2 (Zhang et al. 2013).
Furthermore, Arai et al. developed a novel LAMP
method (designated LAMPSS) targeting the recN to
assess S. suis in raw pork meat. This method could
detect all serotypes of S. suis, except for those
taxonomically removed from authentic S. suis, i.e.,
serotypes 20, 22, 26, 32, 33, and 34 (Arai et al.
2015).
DNA microarray
Microarrays are small devices that consist of short,
single-stranded DNA oligonucleotide probes attached
to slides or chips (McLoughlin 2011). The probe on
the device is typically a short 25–80 bp sequence that
is complementary to the gene or genomic tag of a
different target pathogen (Severgnini et al. 2011). In
such an analysis, DNA (or RNA) from the target
organism is extracted and labeled with a fluorescent
dye and denatured to produce single-stranded mole-
cules that bind to the corresponding complementary
probes on the array. When double-stranded DNA is
formed, a fluorescent signal is emitted, and the
intensity is proportional to the concentration of the
target DNA sequence (Lauri andMariani 2009).When
a large number of nucleic acid probes are affixed, a
sample can be analyzed in a high-throughput, multi-
target gene detection manner. With the large number
of pathogenic microorganisms being sequenced, DNA
microarrays are being widely applied for the rapid
detection of pathogenicmicroorganisms. For example,
to identify the major causative serotypes, Zheng et al.
designed oligonucleotide probes for the conserved
regions of S. suis cps1 (SS1 and SS14), cps2 (SS2 and
SS1/2) and cps9 according to the related sequences of
S. suis in GenBank, though the strain-specific probes
designed for different strains did not achieve the
expected test results (Zheng et al. 2008). In addition,
there are several patents related to the detection of S.
suis using microarray technology. As a new method of
detecting S. suis strains, pathogenic serotypes and
virulence factors, the chip system has good specificity
and sensitivity and is of great value for the high-
throughput identification of S. suis strains and their
virulence.
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Typing-oriented analyses
Typing-oriented analyses, such as pulsed-field gel
electrophoresis (PFGE) and restriction fragment
length polymorphism (RFLP), are a subgroup of
nucleic acid-based detection methods. In general,
PFGE, random amplified polymorphic DNA (RAPD)
and RFLP can offer clues about genomic differences
between different strains/serotypes, and multilocus
sequence typing (MLST) can directly capture the
nucleotide sequence deviation used for typing pur-
poses (Feng et al. 2014).
Pulsed field gel electrophoresis (PFGE)
PFGE is achieved by digesting the bacterial genome
and then separating the fragments by agarose gel
electrophoresis, with continual change in direction of
the electric field to obtain a DNA pattern of the
polymorphism on the gel. PFGE is known as the gold
standard of bacterial molecular biology typing
because of its good repeatability and strong resolving
power. Schwartz et al. first reported the successful
isolation of yeast chromosomes by PFGE (Schwartz
and Cantor, 1984). Furthermore, PFGE is one of the
most effective molecular typing methods for assessing
complex genetic differences between different S. suis
strains. Compared with traditional serotyping meth-
ods, PFGE is more accurate and suitable for applied
and epidemiological studies. In 2002, Berthelot-
Herault et al. used PFGE to characterize 123 isolates
of S. suis isolates derived from French pigs and from
different countries with 2, 2, 3, 7, and 9 serum
samples. A total of 74 PFGE types were divided into 3
groups (A, B, C), with a homology of 60% and a
further 8 (a, b, c, d, e, f, g, h) subtypes (Princivalli et al.
2009; Berthelot-Herault et al. 2002). In a study of S.
suis obtained from tonsils, Marois et al. found that by
using using the capsular polysaccharide antigen
method, 58% of strains were not screened for
serotypes, suggesting that capsule antigen typing is
not sufficient for distinguishing these samples (Marois
et al. 2007). However, PFGE genotyping can identify
the genetic diversity of S. suis and distinguish
pathogenic and non-pathogenic strains from pig and
human isolates, which may be a very important
method in epidemiological investigations of this
pathogen.
Restriction fragment length polymorphism (RFLP)
RFLP technique originates from the natural variation
in biological genomic DNA. Grodjicker et al. first
proposed RFLP in 1974, and as the first generation of
molecular genetic markers, it greatly promoted the
study of human DNA polymorphism, with wide use in
the diagnosis of human genetic diseases as well as in
animal genetics. Mogollond et al. first used RFLP to
study S. suis and found that when digested with Hae
III, 23 serotypes produced easily distinguishable
patterns, whereas serotypes 9, 11, 12 and 16 were
resistant to Hae III digestion. A large difference
between the 9 and 16 patterns obtained with Hind III
was observed. Through the study of 110 strains, a
variety of discoveries with regard to DNA fingerprint-
ing and with clinical relevance were obtained, indi-
cating that DNA polymorphism technology can be
used for epidemiological investigation (Mogollon
et al. 1990). For instance, the use of RFLP technology
by Amass et al. demonstrated that the infection of
piglets was caused by vertical transmission. RFLP
analysis of S. suis type 5 isolates from 3 sows of the
same herd and from the piglets produced showed that
the isolates from piglets and their corresponding sows
had the same DNA fingerprinting but that there was a
large difference between the DNA profiles of samples
without maternal relationship (Amass et al. 1997). The
advantage of RFLP technology is that it is efficient and
reliable and can detect a large number of restriction
fragments in a single reaction. Nonetheless, the RFLP
technique is cumbersome, and the polymorphisms
obtained are not as clear compared with PFGE.
Multilocus sequence typing (MLST)
As a high-resolution and high-accuracy identification
method, MLST has been successfully applied for the
identification of several bacteria (Subaaharan et al.
2010; Lott et al. 2010). In 2002, King et al. first used
the MLST method to amplify and sequence the four
housekeeping genes of 294 S. suis strains in the UK
and obtained 92 sequence types (STs), with the highest
frequency found for ST1, which was reported 141
times in 6 countries. In addition, ST1 is often found in
meningitis, arthritis and sepsis samples, whereas ST27
and ST87 are mainly observed in lung samples and in
samples from farms with a good clinical background
(King et al. 2002). When studying strains isolated in
123
Antonie van Leeuwenhoek (2018) 111:2233–2247 2241
Italy from 2003 to 2007, Princivalli et al. found that
ST1 dominated the past few years and most of strains
carried the MRP, EF and suilysin genes (Princivalli
et al. 2009). Dong compared 30 strains of S. suis
serotype 9 isolates, including 24 strains of from China
between 2004 and 2013, 5 strains of clinical isolates
from Vietnam and a serotype reference from Den-
mark, by MLST analysis to exploit the genetic
relationships among those isolates. The phylogenetic
tree based on the MLST data divides the isolates into
two clades (I and II), which are in good accordance
with a virulence genotyping analysis detecting 23
virulence-related genes. Interestingly, these Asia
strains were shown to be highly heterogeneous, with
16 of 17 STs being described for the first time (Dong
et al. 2017). Additionally, Zheng et al. reported that
most Spanish strains were either ST123 or ST125,
whereas a high number of different STs were detected
amongst Canadian strains based on MLST analysis
(Zheng et al. 2018).
The random amplified polymorphic DNA (RAPD)
RAPD amplifies the genomic DNA of a strain using
random primers to obtain a DNA polymorphism map.
Chatellier et al.’s RAPD analysis of 88 strains of S suis
isolates from pigs and humans using three pairs of
random primers showed the presence of 5 spectral
types in a group with five phenotypes, MRP?EF?-
SLY?, MRP?EF?SLY-, MRP?EF-SLY-, MRP--
EF-SLY?, MRP-EF-SLY?, with excellent
phenotype correlation. Eight percent of North Amer-
ican isolates were MRP?EF?, whereas 55% of
European isolates were MRP?EF?, consistent with
the previously reported virulence factors of MRP and
EF in European isolates (Vecht et al. 1991). In
addition, 22% of North American isolates were
Sly?, as were 66% of European isolates, which is
consistent with reports that the virulence factors EF,
MRP, and Sly are not commonly found in North
America (Gottschalk et al. 1998). In 2002, the
polymorphisms of SS2 in S. suis isolates from
slaughterhouses were analyzed by the RAPD method
and the diversity of serum 1/2-type strains was found
to be lower than that of serotype 2 strains (Martinez
et al. 2002). Martinez et al. assessed the genetic
diversity of an S. suis serotype 2 isolated from healthy
pigs using RAPD. According to the results, RAPD
revealed not only the prevalence of S. suis but also the
source and the transmission route of infection in pigs
(Martinez et al. 2003). Compared with other molecular
bio-typing techniques, RAPD technology is simple
and low cost, and can random evaluation can be
performed on the entire bacterial genome; however, its
repeatability is poor, and standardization is difficult.
Ribotyping
Ribotyping, a method developed on the basis of
Southern blotting and RFLP, was the first molecular
fingerprinting method used for bacterial typing. The
bacterial 16S rRNA has the characteristics of inter-
group specificity, and its sequence is highly conserved,
which can be helpful for diagnosis and epidemiolog-
ical investigations of bacteria. In 1995, the ribotyping
technique was first applied to the polymorphism
analysis of 54 S. suis strains, including 35 serotype
strains; genetic heterogeneity was found, with virulent
strains and attenuated strains being distinguished
based on certain special bands (Okwumabua et al.
1995). Analysis of the relationships between ribotypes
and the virulence of different strains revealed that
compared to moderate strains and attenuated strains,
most virulent strains (5/7) were significantly associ-
ated with ribotyping B. The ribotypes of avirulent and
moderately virulent strains showed greater hetero-
geneity (Staats et al. 1998). In a study of the same and
different serotypes, Vanier et al. found that this
method can distinguish between the genes of type 2
pathogenic strains and non-pathogenic strains. The
results showed that the ribotyping technique can not
only successfully distinguish the strains that cannot be
detected by serological and biochemical tests and can
also determine the virulence of S. suis (Vanier et al.
2009). Smith utilized ribotyping to classify 42 S. suis
strains of 5 serotypes and found that these strains had
different pathogenicity from pigs and expressed
different virulence factors (MRP and EF) (Smith
et al. 1997).
With the development of microbial genomics and
bioinformatics, the latest advances in whole genome
sequencing (WGS) technology now allow the rapid
and relatively inexpensive sequencing of hundreds of
bacterial genomes. The WGS method has replaced all
these techniques, such as PFGE, RFLP, MLST, RAPD
and ribotyping. The arrival of WGS is revolutionizing
microbiological typing in human and veterinary
medicine and strengthening public health goals such
123
2242 Antonie van Leeuwenhoek (2018) 111:2233–2247
as disease surveillance, epidemiological investigation,
and infection control (Koser et al. 2012; Athey et al.
2014).
Nano material-based immunosensors
In 1975, Janata reported that immune electrodes can
be regarded as a prototype of immunosensors (Moss
et al. 1975), and Henry first introduced the concept of
immunosensors in 1990 (Henry, 1990). An
immunosensor is a type of biosensor designed based
on the specific binding and chemical changes of
organisms and is mainly composed of receptors,
transducers and amplifiers. Because the result needs
to be converted to an output signal by the transducer,
often depends on the accuracy and the stability of the
transducers used, such that the type of transducer
appears to be particularly important to the sensing
system. The technique is based on the transducer’s
special status in the sensor. The types of immunosen-
sors are generally divided according to the different
transducers, thus far into the following categories:
electrochemical immunosensors, mass detection
immunosensors, optical immunosensors and calorime-
try sensors. Compared to conventional culture meth-
ods, an advantage of immunosensors is that the
antigen–antibody-specific binding determines its sen-
sitivity without interference or decrease in the detec-
tion limit (Kwon et al. 2006). Moreover, the detection
time is short, usually only a few minutes or tens of
minutes, and the cost is low (Hansen et al. 2006). In
recent years, research into novel nano-biomaterials
and nanocomposites has attracted much attention. Due
to their structure, strong adsorption capacity, good
directional ability, biological compatibility, trapping
and binding ability of biological molecules, and
molecular biological advantages, immunosensors
have been widely used for pathogen detection and
analysis (Li et al. 2016; Jin 2014; Skrabalak et al.
2008) (Table 1).
Some nanomaterials have properties similar to
those of biological enzymes. For example, hollow Pt–
Pd nanomaterials (HPtPd) not only have a large
specific surface area, good biocompatibility and high
catalytic capacity, but they also catalyze the decom-
position of H2O2 to produce O2 as a horseradish
peroxidase (HRP) mimic enzyme. Accordingly, Wang
et al. constructed ultra-sensitive enhanced
chemiluminescence (ECL) immunosensors in which
HPtPd combined with glucose oxidase (GOD) com-
prises a double-enzyme system. In the presence of
glucose, GOD produces H2O2 from this substrate, and
HPtPd acts as an HRP mimetic enzyme to decompose
the H2O2 to O2, which acts as a co-reactant for S2O82-
and effectively amplifies the ECL signal and signif-
icantly increases sensitivity. The electrochemical
luminescence intensity of the ECL immunosensors
was linearly correlated with the logarithm of the SS2
concentration in the range of 0.0001–100 ng/mL, with
a detection limit of 33 fg/mL (Wang et al. 2013).
Composite nanomaterials combine nanomaterials
with different properties, resulting in more features.
Wang et al. generated L-cysteine (L-Cys)-linked
fullerene (C60) functionalized hollow palladium
nanocage (PdNCs) nanocomposites (C60-L-Cys-
PdNCs) and used them for GOD immobilization and
ECL signal electrocatalytic amplification. Similar to
the strategy above, GOD immobilized onto C60-L-
Cys-PdNCs produces H2O2 from glucose, and PdNCs
decomposes H2O2 to produce O2, enhancing the
S2O82- ECL signal. These reserachers constructed a
sandwich-type ECL immunosensor with a wide linear
detection range of 0.1 pg mL-1–100 ng mL-1 and a
relatively low detection limit of 33.3 fg mL-1,
enabling the sensitive detection of the SS2 antigen
(Wang et al. 2014).
Simultaneous multi-analyte immunoassays
(SMIAs) are a more attractive method of analysis
than are traditional single-analyte immunoassays, with
the advantages of smaller sample sizes, lower cost per
test, and improved productivity efficiency. Zhu et al.
reported a standard sandwich-type immunosensor for
the multiplex detection of alpha-fetoprotein (AFP),
carcinoembryonic (CEA) and SS2 using protein A
(PA) adsorbed onto Nafion-modified electrodes for
primary antibody (anti-CEA, anti-AFP and anti-SS2)
immobilization and antibody-functionalized graphene
sheets (GSs), containing abundant gold nanoparticles
(AuNPs) and carboxyl groups for target labeling. The
detection limits were as follows: 5.4 pg mL-1 (AFP),
2.8 pg mL-1 (CEA) and 4.2 pg mL-1 (SS2) (Zhu
et al. 2013).
123
Antonie van Leeuwenhoek (2018) 111:2233–2247 2243
Summary and prospects
S. suis is a common opportunistic pathogen in swine
herds that usually colonizes the upper respiratory tract
of the aniamls, especially the tonsils and nasal
passages, the genital tract or the digestive tract and
in severe cases can cause pneumonia, meningitis,
septicemia, and arthritis (Nakayama et al. 2014; Haleis
et al. 2009; Wertheim et al. 2009). Human infection
can also occur, manifesting as meningitis, sepsis,
arthritis, pneumonia, endocarditis, endophthalmitis
and peritonitis, with other serious symptoms, and
can even cause death. As the threat to food safety,
livestock production safety and related industries is
enormous (Huong et al. 2014; Choi et al. 2012;
Gottschalk et al. 2010; Lutticken et al. 1986), accurate
and rapid detection of S. suis is vital for the early
diagnosis and treatment of infection. Serological
techniques remain the most basic diagnostic methods
for this disease, though the cost of commercial
diagnostic sera is quite high. Therefore, rapid diagno-
sis is challenging. With further research on the
molecular biology of Streptococcus, PCR technology
is playing an increasingly important role in the
diagnosis and typing of these bacteria. However, due
to its limitations, such as the need for professional and
technical personnel and expensive equipment, the
promotion of grass-roots technology has encountered
some difficulties. Based on the combination of anti-
gen–antibody-specific reactions and signal amplifica-
tion of nanomaterials, immunosensors have the
advantages of high detection selectivity and sensitiv-
ity, and they are small in size, easy to operate and
readily automated. Therefore, there is a wide range of
applications for S. suis. Despite the many methods for
identifying S. suis, the approaches have limitations.
Therefore, regarding the diagnosis of S. suis, we
should adhere to the principle of a combination of
various methods, accelerate research into standard
diagnostic methods, and lay a solid foundation for the
rapid diagnosis, prevention and control of swine
streptococcal disease.
Acknowledgements This review was funded by the National
Key Research and Development Program of China (No.
2016YFD0500708-4), the National Natural Science
Foundation of China (Nos. 31702263, 31672559), the China
Postdoctoral Science Foundation (No. 2017M622346), and the
Excellent Youth Foundation of He’nan Scientific Committee
(2017JQ0005).
Conflict of interest The authors declare that they have no
conflict of interest.
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