Methods for the detection and characterization of Streptococcus suis: from conventional bacterial culture methods to immunosensors

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.

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 and 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 polysaccharide (CPS), extracellular factor (EF), muramidase-released protein (MRP), hemolysin, adhesin, fibronectin binding protein (fbpS) and glutamate dehydrogenase 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, respectively, 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 approach for S. suis. However, conventional isolation, culture and biochemical identification are time-consuming 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.

Fig. 1

Comparison of dection methods

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, showing 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 (Nomoto 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 identification 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 difficult to type, requiring other test methods for accurate typing.

Table 1 Approaches for detection, identification and typing of Streptococcus suis

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 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 specificity of WCA–ELISA was very low when detecting other serotypes using rabbit antiserum due to a cross-reaction because of common antigens. In contrast, 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 equipment. 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 humans; 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 conventional 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, chromatography, 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, respectively, 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 polyclonal antibody to establish an immunochromatographic test for SS2 detection. The results showed that the optimum antibody labeling amount per ml of colloidal gold was 22 μg 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 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 × 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 aeruginosa, Staphylococcus aureus, or Klebsiella pneumoniae (Nakayama et al. 2014).

Immunofluorescence methods

Although immune enzyme technology has good sensitivity, its relatively complicated procedure limits further development. However, as immunofluorescence methods developed on the basis of immunoassay techniques involve simpler procedures and have high sensitivity and specificity, they are considered among the most promising pathogen analysis methods. In addition, the development of nanotechnology has brought new opportunities for fluorescence analysis. For example, recently developed 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. developed CdSe/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 × 10⁻⁸–1.5 × 10⁻⁶ mol/L, with a detection limit of 1.9 × 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-mediated 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 immune SERS 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 sensitivity 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 technologies, research has switched from routine etiological identification to molecular aspects. There are a number of diagnostic tests for S. suis, and these 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 developed based on conventional PCR. Multiplex PCR employs multiple pairs of specific primers to simultaneously 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 distinguishes 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 researchers 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 developed 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 distinguish 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 pollution, 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 from different serotypes available in GenBank and partial fbpS genes sequenced from all known serotypes. 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 Srinivasan 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 electrophoresis 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/quantitative 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 molecules 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 and Mariani 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 pathogenic microorganisms. 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.

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 purposes (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 methods, PFGE is more accurate and suitable for applied and epidemiological studies. In 2002, Berthelot-Hérault 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 fingerprinting and with clinical relevance were obtained, indicating 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 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 Denmark, 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 American 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 epidemiological 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 associated with ribotyping B. The ribotypes of avirulent and moderately virulent strains showed greater heterogeneity (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 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 immunosensors are generally divided according to the different transducers, thus far into the following categories: electrochemical immunosensors, mass detection immunosensors, optical immunosensors and calorimetry sensors. Compared to conventional culture methods, an advantage of immunosensors is that the antigen–antibody-specific binding determines its sensitivity without interference or decrease in the detection 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 decomposition 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) comprises 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 significantly 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).

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 diagnosis 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 antigen–antibody-specific reactions and signal amplification of nanomaterials, immunosensors have the advantages of high detection selectivity and sensitivity, 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.