Methods for the detection and characterization of ... · and nucleic-acid based detection methods...

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];

[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

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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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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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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

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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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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


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


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


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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