Epidemiological Investigation and Pathogenicity of Streptococcus suis in Eastern China

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Currently, limited studies have investigated S. suis infections in major pig farms. This study investigated the serotypes, virulence genes, and pathogenicity of the isolates in 89 pig farms across 12 regions from 2022 to 2024. Results: The overall infection and isolation rates were 59.59% and 16.1%, respectively. The infection rate was the highest in Guangdong (72.41%) and the lowest in Hubei (43.75%). Suckling piglets, nursery pigs, fattening pigs, and pregnant sows were susceptible to S. suis infection with infection rates being as high as 60%. The infection rates in spring, summer, autumn, and winter were 70.72%, 60.67%, 40.62%, and 68.97%, respectively. Serotype analysis of 137 isolates revealed increased serotype diversity in coastal provinces, especially in Guangdong, Jiangsu, and Shandong. Serotype 1 was detected in Liaoning. The most prevalent serotype was NT (21.01%), especially in Anhui, Guangxi, and Guangdong, followed by Serotype 2 (20.29%) and Serotype 7 (18.12%). Virulence gene analysis revealed that the occurrence of gdh , gapdh , and orf2 (>89%) was high, whereas that of 89k and epf was low (≤ 28.47%). Serotypes 1 and 7 frequently harbored mrp and gdh but often lacked 89k and epf . Serotypes 2 and NT harbored all tested genes with low 89k occurrence rates. The occurrence rates of sly and epf (≤43.75%) were low in serotype 9. Animal challenge experiments demonstrated that Serotype 2 induced acute death in Landrace pigs aged 42 days with a mortality rate of 100%. In contrast, Serotype 7 was associated with low mortality rates (28.57%) and induced mild pathological symptoms, including pneumonia and pericarditis, and yellow effusion in the thoracic cavity. Conclusion： This study provides useful insights for the prevention and control of S. suis infection in pig farms in China. Streptococcus suis epidemiological investigation serotype virulence gene pathogenicit Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background S. suis , a Gram-positive bacterium that typically appears in pairs or short chains, can infect various animals, including pigs[ 1 ], horses[ 2 ], dogs, and cats[ 3 ], as well as humans[ 4 ]. In pigs, S. suis infections causes septicemia, pneumonia, arthritis, and endocarditis. Meanwhile, S. suis infection leads to symptoms, such as septicemia, skin ulcers, and meningitis[ 5 ]. S. suis was initially classified into 35 serotypes based on thein capsular antigens[ 6 ]. However, advances in molecular techniques have refined the classification system. Currently, S. suis is divided into 29 classical serotypes[ 7 ]. The serotype distribution of Streptococcus suis exhibits marked regional variation. Serotype 2, the most virulent global strain, accounts for 74.7% of human infections and dominates swine populations in Asia (44.2%) and North America (24.3%), while serotype 9 prevails in Europe (61%)[ 8 ]. Notably, South Korea shows an anomalous predominance of serotypes 3/4, and European countries such as Spain and the Netherlands display evolving serotype patterns (9/2/7)[ 9 ]. Alarmingly, nearly half of regions reporting human cases (e.g., Japan, Cambodia) lack synchronized swine surveillance data, critically hindering pathogen research and control[ 10 ]. Establishing an interdisciplinary monitoring network is urgently needed to address this zoonotic threat. The pathogenicity of S. suis is closely related to its virulence factors. The main virulence genes of S. suis include mrp , epf , sly, orf2 , fbps , gdh gapdh , and 89k , which are involved in the pathogenic process. Among the virulence genes, mrp , epf , and sly are considered the key virulence factors of S. suis [ 11 , 12 ]. In Eurasian strains, these genes are positively correlated with pathogenicity. The frequent phenotypes of diseased and healthy pigs are mrp ⁺ epf ⁺ sly ⁺and mrp ⁻ epf ⁻ sly ⁻, respectively[ 13 ]. However, some highly virulent strains from Canada do not express mrp , epf , and sly [ 14 ]. Additionally, avirulent strains harboring all these three genes have not been identified[ 15 ]. sly , which encodes a cytotoxic hemolysin, may enhance pathogenicity by modulating complement deposition and promoting the penetration of S. suis into deeper tissues[ 16 ]. epf severs as a phenotypic marker of virulence[ 17 ]. Additionally, differential orf2 sequences between highly virulent and weakly virulent strains may be associated with changes in strain pathogenicity[ 18 ]. fbps encodes a fibronectin/fibrinogen-binding protein that enables the bacteria to adhere and invade host cells[ 19 , 20 ]. Although gdh and gapdh encode metabolic enzymes, they may indirectly influence pathogenicity by regulating virulence or participating in host-pathogen interactions[ 21 , 22 ]. The 89k pathogenicity island is closely associated with highly virulent strains, indicating its importance in pathogenic mechanisms[ 23 ]. S. suis is a major pathogen affecting pigs worldwide. In China, S. suis drew significant attention in China after a human infection outbreak in Sichuan Province in 2005[ 24 ]. In the context of China's pig industry, there is a notable scarcity of reports concerning both the epidemiological data of S. suis infections and the genotypes associated with virulence-related factors. This study conducted a nationwide epidemiological investigation of Streptococcus suis infections from 2022 to 2024, encompassing 89 swine farms spanning 12 Chinese provinces. Our comprehensive analysis included serotyping identification, virulence gene profiling, and assessment of the pathogenic potential of predominant serotypes. These findings provide critical insights for developing evidence-based, targeted prevention and control strategies against S. suis infections in China's swine industry. Methods Sample Collection This study collected 1,428 samples from over 89 large-scale pig farms (approximately 5 million pigs) across 12 provinces in China (Anhui, Guangdong, Guangxi, Hainan, Hubei, Hunan, Jiangsu, Jiangxi, Liaoning, Shandong, Shanxi, and Shaanxi) between 2022 and 2024. The samples included nasopharyngeal swabs, pleural effusion, joint fluid from the legs, lungs, brain tissue, and vaginal pus from diseased piglets, nursery pigs, fattening pigs, and breeding pigs suspected with S. suis infections. These pigs exhibited symptoms, such as fever, swollen joints, and emaciation. Post-mortem examinations revealed septicemia, polyserositis, pneumonia, pericarditis, and hemorrhaging in multiple organs. Bacterial Isolation and Identification The samples were inoculated on tryptic soy agar (TSA, Difco Laboratories, Detroit, MI, USA) plates containing 5% bovine serum and incubated aerobically at 37°C for 48 h. The colonies were selected and cultured further in Todd-Hewitt broth (THB) with shaking at 37°C for 16 to 18 h. The broth cultures were subjected to polymerase chain reaction (PCR) to identify S. suis through the amplification of gdh . Positive strains were streaked onto THB plates for colony purification. Purified strains were heated in boiling water for 10 min and centrifuged at 13,000 g for 10 min. The supernatant was stored at − 20°C until use. Serotyping Referring to the methods of predecessors[ 6 , 25 – 27 ]. The serotype of isolated S. suis strains was determined using primers listed in Table 1 (synthesized by Sangon Biotech (Shanghai) Co., Ltd.). PCR amplification was performed using 2× Taq Quick-Load Master Mix (CW Biotech, Beijing, China) under the following conditions: 95°C for 5 min (initial denaturation), followed by 35 cycles of 95°C for 1 min (denaturation), 56°C for 1 min (annealing), and 72°C for 1 min (annealing), and 72°C for 5 min (final extension). Each sample was analyzed in triplicate. The amplicons were analyzed using 2% agarose gel electrophoresis. Virulence Gene Detection Referring to the methods of predecessors[ 26 , 28 , 29 ].Virulence genes in isolated S. suis strains were identified using primers listed in Table 2 (also synthesized by Sangon Biotech (Shanghai) Co., Ltd.). The detection method was based on previously reported PCR protocols targeting the following genes: gdh , fbps , sly , orf2 , mrp , 89k , gapdh , and epf . Animal Pathogenicity Experiment The pathogenicity of isolates SS2-1 (serotype 2) and SS7-1 (serotype 7) was evaluated in 24 healthy Landrace pigs aged 42 days (purchased from a pig farm in Qingyuan City, Guangdong Province). These pigs tested negative for S. suis and other exogenous pathogens, including classical swine fever (CSF), African swine fever (ASF), porcine reproductive and respiratory syndrome (PRRS), and porcine circovirus (PCV). The pigs were randomly divided into the following three groups (seven pigs per group): SS2, SS7, and control groups. All pigs had free access to water and food. Pigs in the SS2 and SS7 groups were intraperitoneally injected with 2 mL of 1.0 × 10⁶ CFU of SS2-1 and SS7-1, respectively, Meanwhile, pigs in the control group were injected with an equal volume of sterile phosphate-buffered saline (PBS). Clinical signs and mortality were recorded daily for 14 days post-infection. Dead pigs were immediately necropsied to observe pathological changes, and the lung tissues were collected for hematoxylin and eosin (H&E) staining analysis. The experiments were performed according to the Guidelines for Experimental Animals and was approved by the Ethics Committee of South China Agricultural University. At the end of the experiment, surviving pigs were euthanized to ensure animal welfare. Data Analysis The analysis and mapping of S. suis infection rates, as well as serotype identification, were performed using Office 2021 software. The correlation analysis between serotypes and virulence genes of the isolated strains, along with the generation of related charts, was performed using the online tool https://www.chiplot.online/ . The survival rate analysis and chart creation for animal experiments were conducted using GraphPad Prism 8 software. Results Detection and Infection Rate Analysis of Suspected S. suis Clinical Samples To investigate the epidemiological characteristics of S. suis in major pig-farming regions of China, 1,428 suspected infection samples were collected from 89 pig farms across 12 provinces and tested using PCR. The distribution of the 12 provinces and the number of collected samples and positive test samples are shown in Fig. 1 . The overall infection and isolation rates were 59.59%(851/1728) and 16.1% (137/851), respectively. The infection rates in different regions were as follows: (Fig. 2 A): Guangdong: 72.41%(63/87); Jiangxi: 65.88%(56/85); Guangxi: 65.73%(94/143); Jiangsu: 64.71%(77/119); Shandong: 64.29%(63/98); Shaanxi: 63.92%(186/291); Anhui: 62.79%(81/129); Liaoning: 61.54%(16/26); Hainan: 60.29%(41/68); Shanxi: 57.98%(69/119); Hunan: 44.72%(110/246); Hubei: 43.75%(7/16). Additionally, the infection rates in suckling piglets (aged 0–21 days), nursery pigs (aged 21–70 days), growing-finishing pigs (aged 70 days to 6 months, with a weight of approximately 100–120 kilograms), and pregnant sows (sows in the gestation period) were 57.27%(252/440), 61.21%(579/946), 50%(5/10), and 43.75%(14/32) respectively(Fig. 2 B). Next, the prevalence of S. suis infections in spring, summer, autumn, and winter was examined. The infection rates in spring(February - April), summer (May to July), autumn (August to October), and winter (From November to January of the following year) were 70.72%(215/304), 60.67%(344/567), 40.62%(132/325), and 68.97%(160/232), respectively (Fig. 2 C). Serotyping of S. suis This study systematically analyzed the serotype distribution characteristics and regional differences of Streptococcus suis(Fig. 3 ). In the eastern coastal provinces of China, there is a significant diversity of serotypes: In Guangdong, 9 serotypes including 2, 5, 7, 8, 9, 16, 33, and Serotype NT were detected; In Jiangsu Province, 8 serotypes including 2, 3, 4, 5, 7, 9, 18, and NT were found; In Zhejiang, 7 serotypes including 2, 3, 4, 5, 7, 9, and Serotype NT were detected. The prevalence of Serotype 2 was high in Guangdong (11 cases), Guangxi (7 cases), and Anhui (6 cases). In contrast, 1–3 serotypes were detected in inland provinces, such as Hubei, Hunan, and Shaanxi. In Liaoning Province, only Serotype 1 (2 cases) was identified. This distribution pattern may be influenced by sample representation, geographic barriers, or ecological niche competition among dominant serotypes. Further analysis of the 137 isolates revealed that Serotype 2 exhibited the highest occurrence rate (n = 42; 30.66%), followed by Serotype 7 (n = 29; 21.17%), Serotype 9 (n = 15; 10.95%), Serotype 1 (n = 12; 8.76%), Serotype NT (n = 10; 7.30%), Serotype 3 (n = 9; 6.57%), Serotype 4 (n = 8; 5.84%), Serotype 5 (n = 5; 4.38%). serotypes 33 (n = 2; 1.46%); serotype 16 (n = 2; 1.46%); Serotype 8 (n = 1; 1.46%), and Serotype 8 (n = 1; 1.46%)(Fig. 4 A and 4 B). Detection of Virulence Genes in S. suis This study analyzed the association between eight virulence genes ( gdh , fbps , sly , orf2 , mrp , 89k , gapdh , and epf ) and serotypes in 137 S. suis isolates. The occurrence rates of gdh , fbps , sly , orf2 , mrp , 89k , gapdh , and epf were 100% (137/137), 63.5% (87/137), 55.47% (76/137), 98.54% (135/137), 100% (137/137), 2.92% (4/137), 94.89% (130/137), and 56.93% (78/137), respectively(Fig. 5 A). All 12 isolates of Serotype 1 harbored mrp . Additionally, the other tested genes, except for 89k , were detected in all Serotype 1 isolates. The isolates of Serotypes 2 and NT harbored all tested genes. However, in serotype 2 and NT, the incidence of 89k is 4.76% (95% CI: 13.2%-15.79%) and 10% (95% CI: 17.9%-40.42%) respectively. Serotypes 4 and 7 did not harbor 89k and epf . The occurrence rates of gdh and orf2 in Serotypes 4 and 7 were 100% and ≥ 87%. The occurrence rates of epf in Serotypes 5 and 7 were 20% (95% CI: 36.2%-62.45%) and 0%(95% CI: 0-11.7%), respectively. The frequency of occurrence of most virulence factors, except for sly , 89k , and epf , were high in Serotype 7 with that of gdh and orf2 reaching 100%. Serotype 9 did not harbor 89k . The occurrence rates of sly , epf , and mrp in Serotype 9 were ≤ 43.75% with epf exhibiting a low occurrence rate (12.50%, 95% CI: 35-36.02%). However, the occurrence rates of gdh , fbps , gapdh , and orf2 in Serotype NT were in the range of 87.5%-100%. Additionally, all Serotypes 8, 16, 18, and 33 harbored fbps , orf2 , mrp , and gapdh . However, further validation is necessary as the sample size for these isolates was small (< 3 isolates)(Fig. 5 B). Animal Experiment To investigate the pathogenicity of the prevalent S. suis serotypes 2 and 7, strains SS2-1 and SS7-1 were selected for animal experimentation using 42-day-old, healthy Landrace pigs confirmed to be free of S. suis and other exogenous pathogens (CSF, ASF, PRRS, and PCV). The experimental results demonstrated that pigs in both the SS2-1 and SS7-1 challenge groups exhibited clinical signs such as lethargy, huddling, and a marked reduction or complete loss of appetite within 12 hours post-infection(Fig. 7 D and 7 G). Notably, all pigs in the SS2-1 group succumbed to acute infection by the fourth day, resulting in a mortality rate of 100%. In the SS7-1 group, no acute deaths occurred, but two pigs died on the 10th day, resulting in a mortality rate of 28.57% (Fig. 6 ). The surviving pigs were emaciated and exhibited slow growth, whereas no significant changes were detected in the control group(Fig. 7 A). Necropsy was performed on pigs that died after infection. The results revealed the following: In the SS2-1 group, the deceased pigs exhibited typical acute fibrinous myocarditis, with yellow-brown flocculent exudates accumulating in the pericardial cavity(Fig. 7 E). The lungs exhibited diffuse congestion and edema, characterized by dark red congested areas along the margins of the lung lobes(Fig. 7 F). In the SS7-1 group, pathological changes in the deceased pigs were similar to those observed in the SS2-1 group but were comparatively less severe. Principal findings included surface cardiac hemorrhage and enlargement, myocardial hemorrhage, and mild fibrinous myocarditis(Fig. 7 H). The lungs were enlarged and hemorrhagic, and a small quantity of serous or mildly fibrinous exudate was present within the thoracic cavity(Fig. 7 I). In contrast, no significant pathological alterations were observed in the control group during necropsy(Fig. 7 B and 7 C). Histopathological examination revealed that in both the SS2-1 and SS7-1 groups, myocardial cells exhibited disorganization, increased infiltration of inflammatory cells, interstitial edema, and erythrocyte exudation(Fig. 8 B and 8 C). The alveolar structures were either disrupted or altered, with eosinophilic exudates and scattered erythrocyte leakage present within the alveolar cavities. Hemorrhage and enlargement were also prominent in the lung interstitium(Fig. 8 E and 8 E). In contrast, no significant pathological alterations were observed in the control group(Fig. 8 A and 8 G). Discussion S. suis is one of the most threatening bacterial infectious diseases in the global pig farming industry. Its typical clinical symptoms include purulent meningitis, septicemia, and polyarthritis, which imposes substantial economic burdens on global swine production systems annually [ 30 , 31 ]. Epidemiological investigation shows that in several regions including Europe, North America, and Australia, the infection rate of Streptococcus suis has exceeded 90%[ 32 ].In China, it is reported that the carrier rate of this pathogen in large-scale pig farms exceeds 40%[ 33 ]This phenomenon not only poses a serious biosecurity threat to the health of pigs, but also has a significant impact on the global agricultural economy This study, based on cross-regional epidemiological surveys, found that the overall detection rate of S. suis in large-scale pig farms across 12 provinces in China reached 59.59%, markedly higher than the previou sly reported prevalence of 40.8% [ 34 ].Importantly, the findings also revealed the pathogen’s full-cycle infection capability, with positive cases identified across all age groups of pigs, underscoring its persistent and pervasive threat within pig farming systems. The infection rates in suckling piglets (< 21 days old) and nursery pigs (21–70 days old) were 57.4% and 61.21%, respectively, which is consistent with previous reports[ 35 ]. Previous studies have indicated that Streptococcus suis infections can occur year-round, with higher isolation rates observed during summer months, likely due to increased humidity during this season[ 36 ]. However, this study found that infection rates were significantly higher in winter and spring compared to summer and autumn, a finding that may be associated with regional factors. In recent years, Chinese researchers have identified several novel variants, including serotypes 21/29, NCL21–NCL26, and Chz, expanding the known diversity of S. suis and underscoring the evolving complexity of its epidemiological landscape[ 37 , 38 ]. Although the prevalence of S. suis serotypes and genotypes varies across geographic regions and time periods, serotype 2 remains one of the most prevalent serotypes globally. Moreover, The distribution of S. suis serotypes shows significant regional differences: In the Spain, serotypes 2 (21.7%), 1 (21.3%), and 9 (19.3%) dominate[ 39 ], whereas in North America, serotypes 2 (24.3%) and 3 (21%) prevail. In Asia, serotype 2 accounts for the highest proportion (44.2%), with a detection rate of 34.08% in Jiangxi Province, China, whereas serotypes 3 and 4 account for 12.4% and 5.6%, respectively[ 40 ]. Similarly, this investigation revealed that the distribution of S. suis serotypes in China displays pronounced regional characteristics. For instance, multiple serotypes were found to coexist within individual regions along the eastern coast, such as Guangdong, Jiangsu, and Shandong. Notably, serotype NT was highly prevalent in Anhui, Guangxi, and Guangdong, accounting for 65.5% of all NT isolates—a pattern seldom reported in existing literature. On a global scale, the predominant S. suis serotypes isolated from clinical pig cases, in descending order of frequency, are serotypes 2, 9, 3, 1/2, and 7, with approximately 15% classified as NT[ 41 ]. In this study, serotypes NT and 2 were dominant, followed by serotype 7, which is partially consistent with global trends. However, the higher proportion of NT (21.01%) suggests that regional characteristics or differences in detection methods may influence serotype distribution patterns. The lungs serve as the core infection site (33.33–100%), with serotypes 2, 7, 9, and NT exhibiting multi-organ invasion capabilities, indicating strong tissue invasiveness, which exacerbates disease complexity and control challenges. The virulence of S. suis is closely associated with its serotypes, with notable variations in virulence factors across different serotypes. To date, over 100 virulence-associated components have been identified, including hemolysins, adhesins, and proteases, which collectively contribute to the pathogen's differential pathogenicity[ 42 ]. Among these, the hemolysin sly (57 kDa) is recognized as a pivotal virulence factor, capable of disrupting the blood-brain barrier, inhibiting complement-mediated bactericidal activity, and triggering robust inflammatory responses[ 43 ]. In serotype 2 strains, sly -positive isolates have been significantly correlated with heightened pathogenic potential. Yin et al. reported the 89K pathogenicity island in serotype 2 S. suis , a genomic element strongly linked to the severe outbreaks in Jiangsu and Sichuan and regarded as a major determinant of virulence[ 44 ]. However, in the present study, the detection rate of the 89K pathogenicity island in serotype 2 strains was only 7.14% (2/28), and intriguingly, the gene was also found in serotype 16 and NT strains, suggesting a broader distribution and variability than previously reported. Mrp , epf , fbps, and sly are recognized as key pathogenic marker genes of S. suis serotype 2[ 45 ]. Previous reports have indicated universal presence of the sly gene across isolates, whereas the carriage rates of mrp and epf were notably lower, at 33% and 4% respectively, which contrasts markedly with clinical isolates from North America and Europe, where the carriage rates of mrp and epf can reach 92% and 31%, respectively[ 11 , 46 ]. In the present study, however, the carriage rates of sly , mrp , and epf in serotype 2 isolates were found to be 92.85% (26/28), 96.43% (27/28), and 78.57% (22/28), respectively—levels comparable to those observed in clinical isolates from North America and Europe. Additionally, the mrp gene was highly prevalent in serotypes 1, 3, 4, 5, 7, 8, 9, 16, 18, 33, and NT, indicating that mrp might be a key virulence factor of porcine-derived S. suis . The gdh of S. suis is a specific protein that can serve as a marker antigen for detection. About 305 porcine serum samples were found to show a gdh seropositivity rate fo 73.1%, suggesting its potential application in diagnosing S. suis infections[ 47 ]. This study further confirmed this finding: among 137 S. suis isolates, the carriage rate of the gdh gene was 96.35% (132/137). Except for serotype NT, where the carriage rate was 82.76%, all other serotypes carried the gdh gene at 100%. These findings indicate that the gdh gene is also a key virulence factor of porcine-derived S. suis , and studies on gdh gene deletion provide important insights for vaccine development. gapdh is an important virulence factor closely linked to the bacterial adhesion process. Studies have demonstrated that deletion of the gapdh gene significantly impairs the bacterium's ability to adhere to host cells[ 48 ]. Research further indicates that the gapdh gene is widely distributed among various streptococcal species, including serotypes 2, 7, and 9 of S. suis . Only a few nonpathogenic strains lack this gene, underscoring its universality and importance[ 49 ]. This study also supports this view, with the carriage rate of the gapdh gene being 89.78% (123/137). Serotypes 1, 2, 3, 7, 8, 16, 18, and 33 carried it at 100%, whereas serotypes 4, 5, 9, and NT had carriage rates as high as 87.5%. Thus, regardless of whether the strain is low or highly virulent, this gene is universally present. The orf2 gene is closely related to the virulence of Streptococcus suis, and it is present in at least 78.3% of Streptococcus suis isolates[ 50 ]. This study revealed that the carriage rate of orf2 in 137 isolates was as high as 93%, and orf2 is widely present in different serotypes, suggesting its universality. However, its function and impact on virulence need to be comprehensively evaluated in combination with other factors. Currently, limited research has been conducted on the pathogenicity of S. suis serotypes 2 and 7 in pigs, with the majority of existing studies based on mouse models. Evidence indicates that serotype 2 is the most virulent, capable of inducing acute mortality in pigs through septicemia and polyserositis[ 51 ]. The present study corroborates this finding, which may be attributed to the high-frequency carriage of key virulence genes, including gdh , fbp s, sly , orf2 , mrp , and gapdh . In challenge experiments with serotype 7 in 42-day-old Landrace pigs, although no acute deaths were observed, two pigs died on the 10th day postinfection, both of which presented with leg arthritis and typical polyserositis lesions. This result is consistent with the experimental findings of Boetner AG et al. who used serotype 7 to infect 7-day-old piglets, indicating that serotype 7 has some pathogenicity in piglets of this age group[ 52 ]. Additionally, 42-day-old Landrace pigs infected with serotypes 2 and 7 both presented obvious pneumonia and pericarditis symptoms, suggesting that the lungs and heart may be the primary target organs of these two serotypes. In summary, this study revealed that the overall infection rate is markedly higher than previously reported, with pigs of all age groups demonstrating susceptibility and no evident seasonal variation. Serotypes NT and 2 emerged as the predominant strains, followed by serotype 7. While this distribution trend is partially consistent with global patterns, the elevated prevalence of serotype NT indicates that regional factors or methodological differences in detection may contribute to variations in serotype distribution. Notably, the carriage rates of virulence genes varied significantly across serotypes, with gdh , fbps , sly , orf2 , mrp , and gapdh being widely detected, whereas 89k and epf were found at lower frequencies. Moreover, both serotypes 2 and 7 can cause clinical symptoms similar to those of S. suis disease, but serotype 2 is significantly more pathogenic than is serotype 7. This study provides important scientific evidence for formulating precise prevention and control strategies. Conclusion In this study, we investigated the prevalence of S. suis in 89 large-scale pig farms in 12 provinces in the western region of our country, and analyzed the serotypes and the presence of virulence genes of the isolates as well as the pathogenicity of serotypes 2 and 7. The results of this paper provide important baseline information on the serotype characteristics and virulence genes of Streptococcus suis and the pathogenicity of epidemic strains in China, which is of great significance for understanding its epidemiological characteristics and the development of vaccines used to prevent Streptococcus suis infection in pigs. Declarations Author contributions DY and JX conceived and designed the experiments; JX, DY, MH，and JZ performed the experiments; JX and DY analyzed the data and wrote the manuscript. DY and XH were responsible for manuscript review and editing, supervision, resource provision, and funding acquisition. All authors reviewed and endorsed the final version for submission. Funding This research was supported by the Yunfu Innovation Team Project (CYRC202301) and the Research and Application of Epidemiology and Prevention Technology for Important Bacterial Diseases in Swine Respiratory System (WS-GG-JKYZ-202309-002). Availability of data and materials Reasonable requests for the datasets utilized or examined in this study can be directed to the corresponding author Ethics approval and consent to participate We confirm that for the purchased pigs, informed consent for the use of the animals in this study has been obtained from the owner of a certain pig farm in Qingyuan City, Guangdong Province. Animal infection experiments were conducted according to the Guidelines for Experimental Animals established by the Ministry of Science and Technology of China (Beijing) and were supervised and approved by the National Animal Ethics and Use Committee. The study was approved by the South China Agricultural University (Approval No.: SYXK-2019–0136). Competing interests The authors state that they have no conflicts of interest. Author details 1 College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou, 510642, China. 2 Guangdong Enterprise Key Laboratory for Animal Health and Environmental Control, Wen's Foodstuff Group Co. Ltd, Yunfu, 527439, China. References Mi K, Li M, Sun L, et al. Determination of Susceptibility Breakpoint for Cefquinome against Streptococcus suis in Pigs[J]. Antibiot (Basel). 2021;10(8). 10.3390/antibiotics10080958 . Devriese LA, Haesebrouck F. Streptococcus suis infections in horses and cats[J]. Vet Rec. 1992;130(17):380. 10.1136/vr.130.17.380 . Salasia SI, Lammler C, Devriese LA. Serotypes and putative virulence markers of Streptococcus suis isolates from cats and dogs[J]. Res Vet Sci. 1994;57(2):259–61. 10.1016/0034-5288(94)90070-1 . 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Petrocchi-Rilo M, Martinez-Martinez S, Aguaron-Turrientes A, et al. Anatomical Site, Typing, Virulence Gene Profiling, Antimicrobial Susceptibility and Resistance Genes of Streptococcus suis Isolates Recovered from Pigs in Spain[J]. Antibiot (Basel). 2021;10(6). 10.3390/antibiotics10060707 . Gottschalk M, Lebrun A, Wisselink H, et al. Production of virulence-related proteins by Canadian strains of Streptococcus suis capsular type 2[J]. Can J Vet Res. 1998;62(1):75–9. Vecht U, Wisselink HJ, van Dijk JE, et al. Virulence of Streptococcus suis type 2 strains in newborn germfree pigs depends on phenotype[J]. Infect Immun. 1992;60(2):550–6. 10.1128/iai.60.2.550-556.1992 . King SJ, Heath PJ, Luque I, et al. Distribution and genetic diversity of suilysin in Streptococcus suis isolated from different diseases of pigs and characterization of the genetic basis of suilysin absence[J]. Infect Immun. 2001;69(12):7572–82. 10.1128/IAI.69.12.7572-7582.2001 . Wisselink HJ, Reek FH, Vecht U, et al. Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR[J]. Vet Microbiol. 1999;67(2):143–57. 10.1016/s0378-1135(99)00036-x . de Greeff A, Buys H, Wells JM, et al. A naturally occurring nucleotide polymorphism in the orf2/folc promoter is associated with Streptococcus suis virulence[J]. BMC Microbiol. 2014;14:264. 10.1186/s12866-014-0264-9 . Musyoki AM, Shi Z, Xuan C, et al. Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis[J]. Proc Natl Acad Sci U S A. 2016;113(48):13869–74. 10.1073/pnas.1608406113 . Esgleas M, Lacouture S, Gottschalk M. Streptococcus suis serotype 2 binding to extracellular matrix proteins[J]. FEMS Microbiol Lett. 2005;244(1):33–40. 10.1016/j.femsle.2005.01.017 . Du B, Ji W, An H, et al. Functional analysis of c-di-AMP phosphodiesterase, GdpP, in Streptococcus suis serotype 2[J]. Microbiol Res. 2014;169(9–10):749–58. 10.1016/j.micres.2014.01.002 . Chittick L, Okwumabua O. Loss of expression of the glutamate dehydrogenase (gdh) of Streptococcus suis serotype 2 compromises growth and pathogenicity[J]. Microb Pathog. 2024;188:106565. 10.1016/j.micpath.2024.106565 . Chen C, Tang J, Dong W, et al. A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates[J]. PLoS ONE. 2007;2(3):e315. 10.1371/journal.pone.0000315 . Yu H, Jing H, Chen Z, et al. Human Streptococcus suis outbreak, Sichuan, China[J]. Emerg Infect Dis. 2006;12(6):914–20. 10.3201/eid1206.051194 . Kerdsin A, Akeda Y, Hatrongjit R, et al. Streptococcus suis serotyping by a new multiplex PCR[J]. J Med Microbiol. 2014;63(Pt 6):824–30. 10.1099/jmm.0.069757-0 . Kerdsin A, Dejsirilert S, Akeda Y, et al. Fifteen Streptococcus suis serotypes identified by multiplex PCR[J]. J Med Microbiol. 2012;61(Pt 12):1669–72. 10.1099/jmm.0.048587-0 . Smith HE, Veenbergen V, van der Velde J, et al. The cps genes of Streptococcus suis serotypes 1, 2, and 9: development of rapid serotype-specific PCR assays[J]. J Clin Microbiol. 1999;37(10):3146–52. 10.1128/JCM.37.10.3146-3152.1999 . Ju A, Wang C, Zheng F, Pan X, Dong Y, Ge J, Lu J Study on molecular epidemiology of major pathgenic Streptococcus suis serotypes in middle part of Jiangsu province[J]. Chin Epidemiol, Kerdsin A, Dejsirilert S, Akeda Y et al. Fifteen Streptococcus suis serotypes identified by multiplex PCR[J]. J Med Microbiol, 2012,61(Pt 12):1669–1672. Pan J, Zhang H, Hen B, Zhou M, Wang Z, Xu G, Isolation. Identification and Pathogenicity of Streptococcus suis Type 2[J]. China J Veterinary Drug. 2020;54:14–9. 10.11751/ISSN.1002 -1280.2020.08.03 . Susilawathi NM, Tarini N, Fatmawati N, et al. Streptococcus suis-Associated Meningitis, Bali, Indonesia, 2014–2017[J]. Emerg Infect Dis. 2019;25(12):2235–42. 10.3201/eid2512.181709 . Dutkiewicz J, Zajac V, Sroka J, et al. Streptococcus suis: a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II - Pathogenesis[J]. Ann Agric Environ Med. 2018;25(1):186–203. 10.26444/aaem/85651 . Lv R, Zhang W, Sun Z, et al. Current prevalence and therapeutic strategies for porcine Streptococcus suis in China[J]. Appl Environ Microbiol. 2025;91(3):e216024. 10.1128/aem.02160-24 . Segura M, Aragon V, Brockmeier SL et al. Update on Streptococcus suis Research and Prevention in the Era of Antimicrobial Restriction: 4th International Workshop on S. suis[J]. Pathogens, 2020,9(5). 10.3390/pathogens9050374 . DOI: 10.1016/j.onehlt.2023.100513. Liu P, Zhang Y, Tang H, et al. Prevalence of Streptococcus suis in pigs in China during 2000–2021: A systematic review and meta-analysis[J]. One Health. 2023;16:100513. 10.1016/j.onehlt.2023.100513 . Correa-Fiz F, Neila-Ibanez C, Lopez-Soria S, et al. Feed additives for the control of post-weaning Streptococcus suis disease and the effect on the faecal and nasal microbiota[J]. Sci Rep. 2020;10(1):20354. 10.1038/s41598-020-77313-6 . Zhang B, Ku X, Yu X, et al. Prevalence and antimicrobial susceptibilities of bacterial pathogens in Chinese pig farms from 2013 to 2017[J]. Sci Rep. 2019;9(1):9908. 10.1038/s41598-019-45482-8 . Huang J, Liu X, Chen H, et al. Identification of six novel capsular polysaccharide loci (NCL) from Streptococcus suis multidrug resistant non-typeable strains and the pathogenic characteristic of strains carrying new NCLs[J]. Transbound Emerg Dis. 2019;66(2):995–1003. 10.1111/tbed.13123 . Pan Z, Ma J, Dong W, et al. Novel variant serotype of streptococcus suis isolated from piglets with meningitis[J]. Appl Environ Microbiol. 2015;81(3):976–85. 10.1128/AEM.02962-14 . Petrocchi-Rilo M, Martinez-Martinez S, Aguaron-Turrientes A, et al. Anatomical Site, Typing, Virulence Gene Profiling, Antimicrobial Susceptibility and Resistance Genes of Streptococcus suis Isolates Recovered from Pigs in Spain[J]. Antibiot (Basel). 2021;10(6). 10.3390/antibiotics10060707 . Tan MF, Tan J, Zeng YB, et al. Antimicrobial resistance phenotypes and genotypes of Streptococcus suis isolated from clinically healthy pigs from 2017 to 2019 in Jiangxi Province, China[J]. J Appl Microbiol. 2021;130(3):797–806. 10.1111/jam.14831 . Goyette-Desjardins G, Auger JP, Xu J, et al. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing[J]. Emerg Microbes Infect. 2014;3(6):e45. 10.1038/emi.2014.45 . Roodsant TJ, Van Der Putten B, Tamminga SM, et al. Identification of Streptococcus suis putative zoonotic virulence factors: A systematic review and genomic meta-analysis[J]. Virulence. 2021;12(1):2787–97. 10.1080/21505594.2021.1985760 . Lin L, Xu L, Lv W, et al. An NLRP3 inflammasome-triggered cytokine storm contributes to Streptococcal toxic shock-like syndrome (STSLS)[J]. PLoS Pathog. 2019;15(6):e1007795. 10.1371/journal.ppat.1007795 . Yin S, Li M, Rao X, et al. Subtilisin-like protease-1 secreted through type IV secretion system contributes to high virulence of Streptococcus suis 2[J]. Sci Rep. 2016;6:27369. 10.1038/srep27369 . Dong W, Ma J, Zhu Y, et al. Virulence genotyping and population analysis of Streptococcus suis serotype 2 isolates from China[J]. Infect Genet Evol. 2015;36:483–9. 10.1016/j.meegid.2015.08.021 . Fittipaldi N, Fuller TE, Teel JF, et al. Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States[J]. Vet Microbiol. 2009;139(3–4):310–7. 10.1016/j.vetmic.2009.06.024 . Xia XJ, Wang L, Shen ZQ, et al. Development of an Indirect Dot-PPA-ELISA using glutamate dehydrogenase as a diagnostic antigen for the rapid and specific detection of Streptococcus suis and its application to clinical specimens[J]. Antonie Van Leeuwenhoek. 2017;110(4):585–92. 10.1007/s10482-016-0825-z . Brassard J, Gottschalk M, Quessy S. Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphate dehydrogenase and its involvement as an adhesin[J]. Vet Microbiol. 2004;102(1–2):87–94. 10.1016/j.vetmic.2004.05.008 . Wang Z, Guo M, Kong L, et al. TLR4 Agonist Combined with Trivalent Protein JointS of Streptococcus suis Provides Immunological Protection in Animals[J]. Vaccines (Basel). 2021;9(2). 10.3390/vaccines9020184 . Zhao X, Han S, Zhang F, et al. Identification and characterization of Streptococcus suis strains isolated from eastern China Swine Farms, 2021–2023[J]. Sci Rep. 2025;15(1):5677. 10.1038/s41598-025-90308-5 . Lun ZR, Wang QP, Chen XG, et al. Streptococcus suis: an emerging zoonotic pathogen[J]. Lancet Infect Dis. 2007;7(3):201–9. 10.1016/S1473-3099(07)70001-4 . Boetner AG, Binder M, Bille-Hansen V. Streptococcus suis infections in Danish pigs and experimental infection with Streptococcus suis serotype 7[J]. Acta Pathol Microbiol Immunol Scand B. 1987;95(4):233–9. 10.1111/j.1699-0463.1987.tb03118.x . Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6553183","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":457324467,"identity":"4e12d8a3-6da9-43e3-b54c-69796a72f98f","order_by":0,"name":"Jingyu Xu","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jingyu","middleName":"","lastName":"Xu","suffix":""},{"id":457324468,"identity":"4d5210d4-da32-4b93-9dd1-62d49d03a3e7","order_by":1,"name":"Meiling Hu","email":"","orcid":"","institution":"Wen's Foodstuff Group Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Meiling","middleName":"","lastName":"Hu","suffix":""},{"id":457324469,"identity":"e6066a30-b105-4d52-a8cf-a09e2c1f6460","order_by":2,"name":"Jinmei Zhu","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jinmei","middleName":"","lastName":"Zhu","suffix":""},{"id":457324470,"identity":"11f4e80b-a409-453a-868b-61a014d446a3","order_by":3,"name":"Xianhui Huang","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xianhui","middleName":"","lastName":"Huang","suffix":""},{"id":457324472,"identity":"6b222c61-5371-4db5-a883-7a704f0fdaa2","order_by":4,"name":"Dehong Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYLACHoYDDPzMzIcfkKZFsp0tzYA0LQbneRQkiFJtcPzs4RdvKu7IGR/mYTBgqLGJJqzlTF6a5Zwzz4zNDvMeeMBwLC23gaCWAzlmxrxthxO3HeZLMGBsOEyElvNvgFr+HU7c3MxjIEGclhs5xo95Gw4nbmAmVovkjTdmjHOOHTaWOAwM5ARi/MJ3Psf4w5uaw3L8/YcPP/hQY0NYi8IBBjZEdCQQUg4C8g0MzB+IUTgKRsEoGAUjGAAAq/BFBqFWwxYAAAAASUVORK5CYII=","orcid":"","institution":"South China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Dehong","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-04-29 06:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6553183/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6553183/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83023702,"identity":"22213996-8a2b-45a1-af1d-67a9b2d4b0ef","added_by":"auto","created_at":"2025-05-19 07:59:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4280384,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic distribution, sample size, and number of positive samples of suspected \u003cem\u003eStreptococcus suis\u003c/em\u003e from pigs in 12 regions of China.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/dd8745858ad9a4ee0a8788d5.png"},{"id":83022393,"identity":"33e1873c-21ee-4490-9980-2923db40fef5","added_by":"auto","created_at":"2025-05-19 07:51:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4509411,"visible":true,"origin":"","legend":"\u003cp\u003ePrevalence of \u003cem\u003eS. suis\u003c/em\u003e in different regions of China. A. Infection rates of \u003cem\u003eS. suis\u003c/em\u003e in 12 regions of China; B. Distribution of \u003cem\u003eS. suis \u003c/em\u003einfections across different growth stages of pigs; C. Seasonal variations in\u003cem\u003e S. suis\u003c/em\u003einfection rates.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/f439e147265d1ee4d3a73fa7.png"},{"id":83022416,"identity":"14ddac47-5ee5-412e-a3bb-c869e9bf702f","added_by":"auto","created_at":"2025-05-19 07:51:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2286895,"visible":true,"origin":"","legend":"\u003cp\u003eSerotype distribution characteristics of 137 \u003cem\u003eS. suis\u003c/em\u003e isolates collected from 12 regions of China. NT indicates non-typeable strains.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/72c05e1e985a5ad74e8b7663.png"},{"id":83022401,"identity":"5d8fffad-e2d5-4096-bbf9-2e512a87ead3","added_by":"auto","created_at":"2025-05-19 07:51:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3841508,"visible":true,"origin":"","legend":"\u003cp\u003eSerotype distribution patterns of 137 \u003cem\u003eS. suis \u003c/em\u003eisolates. A. Number of isolates corresponding to each serotype; B. Detailed breakdown of isolates by serotype.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/6747e5d0ac4aa8e009e8a55e.png"},{"id":83023707,"identity":"fff9bbf0-6f32-4b7d-8219-01f57a90cd8d","added_by":"auto","created_at":"2025-05-19 07:59:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8577949,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between serotypes and virulence genes in 137 \u003cem\u003eS. suis\u003c/em\u003e isolates. A. Positive counts of different virulence genes detected in the 137 isolates; B. Heatmap showing the relationships between different serotypes and virulence genes among the isolates.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/88bd2d22b4ae96966f1f1703.png"},{"id":83023701,"identity":"a1764a1b-bd82-4ff8-97cd-fa520ffc592b","added_by":"auto","created_at":"2025-05-19 07:59:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":459617,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curves of 42-day-old Landrace pigs after artificial infection with \u003cem\u003eS. suis.\u003c/em\u003eThe survival rates for the SS-2 and SS-7 challenge groups were 0% and 71.42%, respectively, whereas no deaths occurred in the control group.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/d95d9710b5b10b3c79325fb7.png"},{"id":83022412,"identity":"dbfc6e45-74a4-45f0-88b1-42acdfa3ffa5","added_by":"auto","created_at":"2025-05-19 07:51:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":32487733,"visible":true,"origin":"","legend":"\u003cp\u003eClinical manifestations and post-mortem lesions in 42-day-old Landrace pigs after artificial infection. A-C show the clinical status, heart, and lung lesions of pigs in the control group; D-F depict the clinical status, heart, and lung lesions in the SS2-challenged group; G-I illustrate the clinical status, heart, and lung lesions in the SS7-challenged group.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/bc2292f5cfa8cb341c15554c.png"},{"id":83022428,"identity":"b0316eea-41cd-4d14-bf95-2b9c02e9eaf5","added_by":"auto","created_at":"2025-05-19 07:51:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":14389714,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological changes in the heart and lungs of 42-day-old Landrace pigs after artificial infection. A-C represent histopathological changes in the lungs of the control group, SS2-challenged group, and SS7-challenged group, respectively; D-F show histopathological changes in the hearts of the control group, SS2-challenged group, and SS7-challenged group, respectively.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/d2e1434e62c144bb3e0aea40.png"},{"id":89026664,"identity":"79e010de-4c96-4dfe-a0ef-a3b44c9835d0","added_by":"auto","created_at":"2025-08-14 00:02:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":64745527,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/2ed32cbe-ff97-4edb-ac4f-4d3520b3105e.pdf"},{"id":83023706,"identity":"832ba82f-d694-4ed6-901a-8379d129d135","added_by":"auto","created_at":"2025-05-19 07:59:17","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13882,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/59e253efdb5209db29e1c627.xlsx"},{"id":83022415,"identity":"b7310dc0-cebd-4f92-a06a-d39b50b12ea5","added_by":"auto","created_at":"2025-05-19 07:51:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11773,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6553183/v1/3209d6a63f0291d6ea9b6a70.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epidemiological Investigation and Pathogenicity of Streptococcus suis in Eastern China","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eS. suis\u003c/em\u003e, a Gram-positive bacterium that typically appears in pairs or short chains, can infect various animals, including pigs[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], horses[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], dogs, and cats[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], as well as humans[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In pigs, \u003cem\u003eS. suis\u003c/em\u003e infections causes septicemia, pneumonia, arthritis, and endocarditis. Meanwhile, \u003cem\u003eS. suis\u003c/em\u003e infection leads to symptoms, such as septicemia, skin ulcers, and meningitis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eS. suis\u003c/em\u003e was initially classified into 35 serotypes based on thein capsular antigens[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, advances in molecular techniques have refined the classification system. Currently, \u003cem\u003eS. suis\u003c/em\u003e is divided into 29 classical serotypes[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The serotype distribution of Streptococcus suis exhibits marked regional variation. Serotype 2, the most virulent global strain, accounts for 74.7% of human infections and dominates swine populations in Asia (44.2%) and North America (24.3%), while serotype 9 prevails in Europe (61%)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Notably, South Korea shows an anomalous predominance of serotypes 3/4, and European countries such as Spain and the Netherlands display evolving serotype patterns (9/2/7)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Alarmingly, nearly half of regions reporting human cases (e.g., Japan, Cambodia) lack synchronized swine surveillance data, critically hindering pathogen research and control[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Establishing an interdisciplinary monitoring network is urgently needed to address this zoonotic threat.\u003c/p\u003e \u003cp\u003eThe pathogenicity of \u003cem\u003eS. suis\u003c/em\u003e is closely related to its virulence factors. The main virulence genes of \u003cem\u003eS. suis\u003c/em\u003e include \u003cem\u003emrp\u003c/em\u003e, \u003cem\u003eepf\u003c/em\u003e, sly, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003egdh gapdh\u003c/em\u003e, and \u003cem\u003e89k\u003c/em\u003e, which are involved in the pathogenic process. Among the virulence genes, \u003cem\u003emrp\u003c/em\u003e, \u003cem\u003eepf\u003c/em\u003e, and \u003cem\u003esly\u003c/em\u003e are considered the key virulence factors of \u003cem\u003eS. suis\u003c/em\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In Eurasian strains, these genes are positively correlated with pathogenicity. The frequent phenotypes of diseased and healthy pigs are \u003cem\u003emrp\u003c/em\u003e⁺\u003cem\u003eepf\u003c/em\u003e⁺\u003cem\u003esly\u003c/em\u003e⁺and \u003cem\u003emrp\u003c/em\u003e⁻\u003cem\u003eepf\u003c/em\u003e⁻\u003cem\u003esly\u003c/em\u003e⁻, respectively[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, some highly virulent strains from Canada do not express \u003cem\u003emrp\u003c/em\u003e, \u003cem\u003eepf\u003c/em\u003e, and \u003cem\u003esly\u003c/em\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, avirulent strains harboring all these three genes have not been identified[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003esly\u003c/em\u003e, which encodes a cytotoxic hemolysin, may enhance pathogenicity by modulating complement deposition and promoting the penetration of \u003cem\u003eS. suis\u003c/em\u003e into deeper tissues[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eepf\u003c/em\u003e severs as a phenotypic marker of virulence[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, differential \u003cem\u003eorf2\u003c/em\u003e sequences between highly virulent and weakly virulent strains may be associated with changes in strain pathogenicity[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003efbps\u003c/em\u003e encodes a fibronectin/fibrinogen-binding protein that enables the bacteria to adhere and invade host cells[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although \u003cem\u003egdh\u003c/em\u003e and \u003cem\u003egapdh\u003c/em\u003e encode metabolic enzymes, they may indirectly influence pathogenicity by regulating virulence or participating in host-pathogen interactions[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The \u003cem\u003e89k\u003c/em\u003e pathogenicity island is closely associated with highly virulent strains, indicating its importance in pathogenic mechanisms[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eS. suis\u003c/em\u003e is a major pathogen affecting pigs worldwide. In China, \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eS. suis\u003c/span\u003e drew significant attention in China after a human infection outbreak in Sichuan Province in 2005[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the context of China's pig industry, there is a notable scarcity of reports concerning both the epidemiological data of \u003cem\u003eS. suis\u003c/em\u003e infections and the genotypes associated with virulence-related factors. This study conducted a nationwide epidemiological investigation of Streptococcus suis infections from 2022 to 2024, encompassing 89 swine farms spanning 12 Chinese provinces. Our comprehensive analysis included serotyping identification, virulence gene profiling, and assessment of the pathogenic potential of predominant serotypes. These findings provide critical insights for developing evidence-based, targeted prevention and control strategies against S. suis infections in China's swine industry.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection\u003c/h2\u003e \u003cp\u003eThis study collected 1,428 samples from over 89 large-scale pig farms (approximately 5\u0026nbsp;million pigs) across 12 provinces in China (Anhui, Guangdong, Guangxi, Hainan, Hubei, Hunan, Jiangsu, Jiangxi, Liaoning, Shandong, Shanxi, and Shaanxi) between 2022 and 2024. The samples included nasopharyngeal swabs, pleural effusion, joint fluid from the legs, lungs, brain tissue, and vaginal pus from diseased piglets, nursery pigs, fattening pigs, and breeding pigs suspected with \u003cem\u003eS. suis\u003c/em\u003e infections. These pigs exhibited symptoms, such as fever, swollen joints, and emaciation. Post-mortem examinations revealed septicemia, polyserositis, pneumonia, pericarditis, and hemorrhaging in multiple organs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial Isolation and Identification\u003c/h3\u003e\n\u003cp\u003eThe samples were inoculated on tryptic soy agar (TSA, Difco Laboratories, Detroit, MI, USA) plates containing 5% bovine serum and incubated aerobically at 37\u0026deg;C for 48 h. The colonies were selected and cultured further in Todd-Hewitt broth (THB) with shaking at 37\u0026deg;C for 16 to 18 h. The broth cultures were subjected to polymerase chain reaction (PCR) to identify \u003cem\u003eS. suis\u003c/em\u003e through the amplification of \u003cem\u003egdh\u003c/em\u003e. Positive strains were streaked onto THB plates for colony purification. Purified strains were heated in boiling water for 10 min and centrifuged at 13,000 \u003cem\u003eg\u003c/em\u003e for 10 min. The supernatant was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e\n\u003ch3\u003eSerotyping\u003c/h3\u003e\n\u003cp\u003eReferring to the methods of predecessors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The serotype of isolated \u003cem\u003eS. suis\u003c/em\u003e strains was determined using primers listed in Table\u0026nbsp;1 (synthesized by Sangon Biotech (Shanghai) Co., Ltd.). PCR amplification was performed using 2\u0026times; Taq Quick-Load Master Mix (CW Biotech, Beijing, China) under the following conditions: 95\u0026deg;C for 5 min (initial denaturation), followed by 35 cycles of 95\u0026deg;C for 1 min (denaturation), 56\u0026deg;C for 1 min (annealing), and 72\u0026deg;C for 1 min (annealing), and 72\u0026deg;C for 5 min (final extension). Each sample was analyzed in triplicate. The amplicons were analyzed using 2% agarose gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003eVirulence Gene Detection\u003c/h3\u003e\n\u003cp\u003eReferring to the methods of predecessors[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].Virulence genes in isolated \u003cem\u003eS. suis\u003c/em\u003e strains were identified using primers listed in Table\u0026nbsp;2 (also synthesized by Sangon Biotech (Shanghai) Co., Ltd.). The detection method was based on previously reported PCR protocols targeting the following genes: \u003cem\u003egdh\u003c/em\u003e, \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003esly\u003c/em\u003e, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, \u003cem\u003e89k\u003c/em\u003e, \u003cem\u003egapdh\u003c/em\u003e, and \u003cem\u003eepf\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eAnimal Pathogenicity Experiment\u003c/h3\u003e\n\u003cp\u003eThe pathogenicity of isolates SS2-1 (serotype 2) and SS7-1 (serotype 7) was evaluated in 24 healthy Landrace pigs aged 42 days (purchased from a pig farm in Qingyuan City, Guangdong Province). These pigs tested negative for \u003cem\u003eS. suis\u003c/em\u003e and other exogenous pathogens, including classical swine fever (CSF), African swine fever (ASF), porcine reproductive and respiratory syndrome (PRRS), and porcine circovirus (PCV). The pigs were randomly divided into the following three groups (seven pigs per group): SS2, SS7, and control groups. All pigs had free access to water and food.\u003c/p\u003e \u003cp\u003ePigs in the SS2 and SS7 groups were intraperitoneally injected with 2 mL of 1.0 \u0026times; 10⁶ CFU of SS2-1 and SS7-1, respectively, Meanwhile, pigs in the control group were injected with an equal volume of sterile phosphate-buffered saline (PBS). Clinical signs and mortality were recorded daily for 14 days post-infection. Dead pigs were immediately necropsied to observe pathological changes, and the lung tissues were collected for hematoxylin and eosin (H\u0026amp;E) staining analysis. The experiments were performed according to the Guidelines for Experimental Animals and was approved by the Ethics Committee of South China Agricultural University. At the end of the experiment, surviving pigs were euthanized to ensure animal welfare.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eThe analysis and mapping of \u003cem\u003eS. suis\u003c/em\u003e infection rates, as well as serotype identification, were performed using Office 2021 software. The correlation analysis between serotypes and virulence genes of the isolated strains, along with the generation of related charts, was performed using the online tool \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The survival rate analysis and chart creation for animal experiments were conducted using GraphPad Prism 8 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetection and Infection Rate Analysis of Suspected S. suis Clinical Samples\u003c/h2\u003e \u003cp\u003eTo investigate the epidemiological characteristics of \u003cem\u003eS. suis\u003c/em\u003e in major pig-farming regions of China, 1,428 suspected infection samples were collected from 89 pig farms across 12 provinces and tested using PCR. The distribution of the 12 provinces and the number of collected samples and positive test samples are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe overall infection and isolation rates were 59.59%(851/1728) and 16.1% (137/851), respectively. The infection rates in different regions were as follows: (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA): Guangdong: 72.41%(63/87); Jiangxi: 65.88%(56/85); Guangxi: 65.73%(94/143); Jiangsu: 64.71%(77/119); Shandong: 64.29%(63/98); Shaanxi: 63.92%(186/291); Anhui: 62.79%(81/129); Liaoning: 61.54%(16/26); Hainan: 60.29%(41/68); Shanxi: 57.98%(69/119); Hunan: 44.72%(110/246); Hubei: 43.75%(7/16). Additionally, the infection rates in suckling piglets (aged 0\u0026ndash;21 days), nursery pigs (aged 21\u0026ndash;70 days), growing-finishing pigs (aged 70 days to 6 months, with a weight of approximately 100\u0026ndash;120 kilograms), and pregnant sows (sows in the gestation period) were 57.27%(252/440), 61.21%(579/946), 50%(5/10), and 43.75%(14/32) respectively(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, the prevalence of \u003cem\u003eS. suis\u003c/em\u003e infections in spring, summer, autumn, and winter was examined. The infection rates in spring(February - April), summer (May to July), autumn (August to October), and winter (From November to January of the following year) were 70.72%(215/304), 60.67%(344/567), 40.62%(132/325), and 68.97%(160/232), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSerotyping of\u003c/b\u003e \u003cb\u003eS. suis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study systematically analyzed the serotype distribution characteristics and regional differences of Streptococcus suis(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the eastern coastal provinces of China, there is a significant diversity of serotypes: In Guangdong, 9 serotypes including 2, 5, 7, 8, 9, 16, 33, and Serotype NT were detected; In Jiangsu Province, 8 serotypes including 2, 3, 4, 5, 7, 9, 18, and NT were found; In Zhejiang, 7 serotypes including 2, 3, 4, 5, 7, 9, and Serotype NT were detected. The prevalence of Serotype 2 was high in Guangdong (11 cases), Guangxi (7 cases), and Anhui (6 cases). In contrast, 1\u0026ndash;3 serotypes were detected in inland provinces, such as Hubei, Hunan, and Shaanxi. In Liaoning Province, only Serotype 1 (2 cases) was identified. This distribution pattern may be influenced by sample representation, geographic barriers, or ecological niche competition among dominant serotypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of the 137 isolates revealed that Serotype 2 exhibited the highest occurrence rate (n\u0026thinsp;=\u0026thinsp;42; 30.66%), followed by Serotype 7 (n\u0026thinsp;=\u0026thinsp;29; 21.17%), Serotype 9 (n\u0026thinsp;=\u0026thinsp;15; 10.95%), Serotype 1 (n\u0026thinsp;=\u0026thinsp;12; 8.76%), Serotype NT (n\u0026thinsp;=\u0026thinsp;10; 7.30%), Serotype 3 (n\u0026thinsp;=\u0026thinsp;9; 6.57%), Serotype 4 (n\u0026thinsp;=\u0026thinsp;8; 5.84%), Serotype 5 (n\u0026thinsp;=\u0026thinsp;5; 4.38%). serotypes 33 (n\u0026thinsp;=\u0026thinsp;2; 1.46%); serotype 16 (n\u0026thinsp;=\u0026thinsp;2; 1.46%); Serotype 8 (n\u0026thinsp;=\u0026thinsp;1; 1.46%), and Serotype 8 (n\u0026thinsp;=\u0026thinsp;1; 1.46%)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of Virulence Genes in\u003c/b\u003e \u003cb\u003eS. suis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study analyzed the association between eight virulence genes (\u003cem\u003egdh\u003c/em\u003e, \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003esly\u003c/em\u003e, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, \u003cem\u003e89k\u003c/em\u003e, \u003cem\u003egapdh\u003c/em\u003e, and \u003cem\u003eepf\u003c/em\u003e) and serotypes in 137 \u003cem\u003eS. suis\u003c/em\u003e isolates. The occurrence rates of \u003cem\u003egdh\u003c/em\u003e, \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003esly\u003c/em\u003e, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, \u003cem\u003e89k\u003c/em\u003e, \u003cem\u003egapdh\u003c/em\u003e, and \u003cem\u003eepf\u003c/em\u003e were 100% (137/137), 63.5% (87/137), 55.47% (76/137), 98.54% (135/137), 100% (137/137), 2.92% (4/137), 94.89% (130/137), and 56.93% (78/137), respectively(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). All 12 isolates of Serotype 1 harbored \u003cem\u003emrp\u003c/em\u003e. Additionally, the other tested genes, except for \u003cem\u003e89k\u003c/em\u003e, were detected in all Serotype 1 isolates. The isolates of Serotypes 2 and NT harbored all tested genes. However, in serotype 2 and NT, the incidence of 89k is 4.76% (95% CI: 13.2%-15.79%) and 10% (95% CI: 17.9%-40.42%) respectively. Serotypes 4 and 7 did not harbor \u003cem\u003e89k\u003c/em\u003e and \u003cem\u003eepf\u003c/em\u003e. The occurrence rates of \u003cem\u003egdh\u003c/em\u003e and \u003cem\u003eorf2\u003c/em\u003e in Serotypes 4 and 7 were 100% and \u0026ge;\u0026thinsp;87%. The occurrence rates of \u003cem\u003eepf\u003c/em\u003e in Serotypes 5 and 7 were 20% (95% CI: 36.2%-62.45%) and 0%(95% CI: 0-11.7%), respectively. The frequency of occurrence of most virulence factors, except for \u003cem\u003esly\u003c/em\u003e, \u003cem\u003e89k\u003c/em\u003e, and \u003cem\u003eepf\u003c/em\u003e, were high in Serotype 7 with that of \u003cem\u003egdh\u003c/em\u003e and \u003cem\u003eorf2\u003c/em\u003e reaching 100%. Serotype 9 did not harbor \u003cem\u003e89k\u003c/em\u003e. The occurrence rates of \u003cem\u003esly\u003c/em\u003e, \u003cem\u003eepf\u003c/em\u003e, and \u003cem\u003emrp\u003c/em\u003e in Serotype 9 were \u0026le;\u0026thinsp;43.75% with \u003cem\u003eepf\u003c/em\u003e exhibiting a low occurrence rate (12.50%, 95% CI: 35-36.02%). However, the occurrence rates of \u003cem\u003egdh\u003c/em\u003e, \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003egapdh\u003c/em\u003e, and \u003cem\u003eorf2\u003c/em\u003e in Serotype NT were in the range of 87.5%-100%. Additionally, all Serotypes 8, 16, 18, and 33 harbored \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, and \u003cem\u003egapdh\u003c/em\u003e. However, further validation is necessary as the sample size for these isolates was small (\u0026lt;\u0026thinsp;3 isolates)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Experiment\u003c/h2\u003e \u003cp\u003eTo investigate the pathogenicity of the prevalent \u003cem\u003eS. suis\u003c/em\u003e serotypes 2 and 7, strains SS2-1 and SS7-1 were selected for animal experimentation using 42-day-old, healthy Landrace pigs confirmed to be free of \u003cem\u003eS. suis\u003c/em\u003e and other exogenous pathogens (CSF, ASF, PRRS, and PCV). The experimental results demonstrated that pigs in both the SS2-1 and SS7-1 challenge groups exhibited clinical signs such as lethargy, huddling, and a marked reduction or complete loss of appetite within 12 hours post-infection(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Notably, all pigs in the SS2-1 group succumbed to acute infection by the fourth day, resulting in a mortality rate of 100%. In the SS7-1 group, no acute deaths occurred, but two pigs died on the 10th day, resulting in a mortality rate of 28.57% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The surviving pigs were emaciated and exhibited slow growth, whereas no significant changes were detected in the control group(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNecropsy was performed on pigs that died after infection. The results revealed the following: In the SS2-1 group, the deceased pigs exhibited typical acute fibrinous myocarditis, with yellow-brown flocculent exudates accumulating in the pericardial cavity(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The lungs exhibited diffuse congestion and edema, characterized by dark red congested areas along the margins of the lung lobes(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). In the SS7-1 group, pathological changes in the deceased pigs were similar to those observed in the SS2-1 group but were comparatively less severe. Principal findings included surface cardiac hemorrhage and enlargement, myocardial hemorrhage, and mild fibrinous myocarditis(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). The lungs were enlarged and hemorrhagic, and a small quantity of serous or mildly fibrinous exudate was present within the thoracic cavity(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). In contrast, no significant pathological alterations were observed in the control group during necropsy(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eHistopathological examination revealed that in both the SS2-1 and SS7-1 groups, myocardial cells exhibited disorganization, increased infiltration of inflammatory cells, interstitial edema, and erythrocyte exudation(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The alveolar structures were either disrupted or altered, with eosinophilic exudates and scattered erythrocyte leakage present within the alveolar cavities. Hemorrhage and enlargement were also prominent in the lung interstitium(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). In contrast, no significant pathological alterations were observed in the control group(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eS. suis\u003c/em\u003e is one of the most threatening bacterial infectious diseases in the global pig farming industry. Its typical clinical symptoms include purulent meningitis, septicemia, and polyarthritis, which imposes substantial economic burdens on global swine production systems annually [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Epidemiological investigation shows that in several regions including Europe, North America, and Australia, the infection rate of Streptococcus suis has exceeded 90%[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].In China, it is reported that the carrier rate of this pathogen in large-scale pig farms exceeds 40%[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]This phenomenon not only poses a serious biosecurity threat to the health of pigs, but also has a significant impact on the global agricultural economy This study, based on cross-regional epidemiological surveys, found that the overall detection rate of \u003cem\u003eS. suis\u003c/em\u003e in large-scale pig farms across 12 provinces in China reached 59.59%, markedly higher than the previou\u003cem\u003esly\u003c/em\u003e reported prevalence of 40.8% [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].Importantly, the findings also revealed the pathogen\u0026rsquo;s full-cycle infection capability, with positive cases identified across all age groups of pigs, underscoring its persistent and pervasive threat within pig farming systems. The infection rates in suckling piglets (\u0026lt;\u0026thinsp;21 days old) and nursery pigs (21\u0026ndash;70 days old) were 57.4% and 61.21%, respectively, which is consistent with previous reports[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have indicated that Streptococcus suis infections can occur year-round, with higher isolation rates observed during summer months, likely due to increased humidity during this season[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, this study found that infection rates were significantly higher in winter and spring compared to summer and autumn, a finding that may be associated with regional factors.\u003c/p\u003e \u003cp\u003eIn recent years, Chinese researchers have identified several novel variants, including serotypes 21/29, NCL21\u0026ndash;NCL26, and Chz, expanding the known diversity of \u003cem\u003eS. suis\u003c/em\u003e and underscoring the evolving complexity of its epidemiological landscape[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Although the prevalence of \u003cem\u003eS. suis\u003c/em\u003e serotypes and genotypes varies across geographic regions and time periods, serotype 2 remains one of the most prevalent serotypes globally. Moreover, The distribution of S. suis serotypes shows significant regional differences: In the Spain, serotypes 2 (21.7%), 1 (21.3%), and 9 (19.3%) dominate[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], whereas in North America, serotypes 2 (24.3%) and 3 (21%) prevail. In Asia, serotype 2 accounts for the highest proportion (44.2%), with a detection rate of 34.08% in Jiangxi Province, China, whereas serotypes 3 and 4 account for 12.4% and 5.6%, respectively[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Similarly, this investigation revealed that the distribution of S. suis serotypes in China displays pronounced regional characteristics. For instance, multiple serotypes were found to coexist within individual regions along the eastern coast, such as Guangdong, Jiangsu, and Shandong. Notably, serotype \u003cem\u003eNT\u003c/em\u003e was highly prevalent in Anhui, Guangxi, and Guangdong, accounting for 65.5% of all NT isolates\u0026mdash;a pattern seldom reported in existing literature. On a global scale, the predominant \u003cem\u003eS. suis\u003c/em\u003e serotypes isolated from clinical pig cases, in descending order of frequency, are serotypes 2, 9, 3, 1/2, and 7, with approximately 15% classified as NT[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, serotypes NT and 2 were dominant, followed by serotype 7, which is partially consistent with global trends. However, the higher proportion of NT (21.01%) suggests that regional characteristics or differences in detection methods may influence serotype distribution patterns. The lungs serve as the core infection site (33.33\u0026ndash;100%), with serotypes 2, 7, 9, and NT exhibiting multi-organ invasion capabilities, indicating strong tissue invasiveness, which exacerbates disease complexity and control challenges.\u003c/p\u003e \u003cp\u003eThe virulence of \u003cem\u003eS. suis\u003c/em\u003e is closely associated with its serotypes, with notable variations in virulence factors across different serotypes. To date, over 100 virulence-associated components have been identified, including hemolysins, adhesins, and proteases, which collectively contribute to the pathogen's differential pathogenicity[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Among these, the hemolysin \u003cem\u003esly\u003c/em\u003e (57 kDa) is recognized as a pivotal virulence factor, capable of disrupting the blood-brain barrier, inhibiting complement-mediated bactericidal activity, and triggering robust inflammatory responses[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In serotype 2 strains, \u003cem\u003esly\u003c/em\u003e-positive isolates have been significantly correlated with heightened pathogenic potential. Yin et al. reported the \u003cem\u003e89K\u003c/em\u003e pathogenicity island in serotype 2 \u003cem\u003eS. suis\u003c/em\u003e, a genomic element strongly linked to the severe outbreaks in Jiangsu and Sichuan and regarded as a major determinant of virulence[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, in the present study, the detection rate of the \u003cem\u003e89K\u003c/em\u003e pathogenicity island in serotype 2 strains was only 7.14% (2/28), and intriguingly, the gene was also found in serotype 16 and NT strains, suggesting a broader distribution and variability than previously reported.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMrp\u003c/em\u003e, \u003cem\u003eepf\u003c/em\u003e, fbps, and \u003cem\u003esly\u003c/em\u003e are recognized as key pathogenic marker genes of \u003cem\u003eS. suis\u003c/em\u003e serotype 2[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Previous reports have indicated universal presence of the sly gene across isolates, whereas the carriage rates of mrp and \u003cem\u003eepf\u003c/em\u003e were notably lower, at 33% and 4% respectively, which contrasts markedly with clinical isolates from North America and Europe, where the carriage rates of mrp and epf can reach 92% and 31%, respectively[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In the present study, however, the carriage rates of \u003cem\u003esly\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, and \u003cem\u003eepf\u003c/em\u003e in serotype 2 isolates were found to be 92.85% (26/28), 96.43% (27/28), and 78.57% (22/28), respectively\u0026mdash;levels comparable to those observed in clinical isolates from North America and Europe. Additionally, the mrp gene was highly prevalent in serotypes 1, 3, 4, 5, 7, 8, 9, 16, 18, 33, and NT, indicating that mrp might be a key virulence factor of porcine-derived \u003cem\u003eS. suis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003egdh\u003c/em\u003e of \u003cem\u003eS. suis\u003c/em\u003e is a specific protein that can serve as a marker antigen for detection. About 305 porcine serum samples were found to show a \u003cem\u003egdh\u003c/em\u003e seropositivity rate fo 73.1%, suggesting its potential application in diagnosing S. suis infections[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This study further confirmed this finding: among 137 \u003cem\u003eS. suis\u003c/em\u003e isolates, the carriage rate of the \u003cem\u003egdh\u003c/em\u003e gene was 96.35% (132/137). Except for serotype NT, where the carriage rate was 82.76%, all other serotypes carried the gdh gene at 100%. These findings indicate that the gdh gene is also a key virulence factor of porcine-derived \u003cem\u003eS. suis\u003c/em\u003e, and studies on gdh gene deletion provide important insights for vaccine development.\u003c/p\u003e \u003cp\u003e \u003cem\u003egapdh\u003c/em\u003e is an important virulence factor closely linked to the bacterial adhesion process. Studies have demonstrated that deletion of the \u003cem\u003egapdh\u003c/em\u003e gene significantly impairs the bacterium's ability to adhere to host cells[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Research further indicates that the gapdh gene is widely distributed among various streptococcal species, including serotypes 2, 7, and 9 of \u003cem\u003eS. suis\u003c/em\u003e. Only a few nonpathogenic strains lack this gene, underscoring its universality and importance[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This study also supports this view, with the carriage rate of the gapdh gene being 89.78% (123/137). Serotypes 1, 2, 3, 7, 8, 16, 18, and 33 carried it at 100%, whereas serotypes 4, 5, 9, and NT had carriage rates as high as 87.5%. Thus, regardless of whether the strain is low or highly virulent, this gene is universally present. The orf2 gene is closely related to the virulence of Streptococcus suis, and it is present in at least 78.3% of Streptococcus suis isolates[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This study revealed that the carriage rate of \u003cem\u003eorf2\u003c/em\u003e in 137 isolates was as high as 93%, and orf2 is widely present in different serotypes, suggesting its universality. However, its function and impact on virulence need to be comprehensively evaluated in combination with other factors.\u003c/p\u003e \u003cp\u003eCurrently, limited research has been conducted on the pathogenicity of \u003cem\u003eS. suis\u003c/em\u003e serotypes 2 and 7 in pigs, with the majority of existing studies based on mouse models. Evidence indicates that serotype 2 is the most virulent, capable of inducing acute mortality in pigs through septicemia and polyserositis[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The present study corroborates this finding, which may be attributed to the high-frequency carriage of key virulence genes, including \u003cem\u003egdh\u003c/em\u003e, \u003cem\u003efbp\u003c/em\u003es, \u003cem\u003esly\u003c/em\u003e, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, and \u003cem\u003egapdh\u003c/em\u003e. In challenge experiments with serotype 7 in 42-day-old Landrace pigs, although no acute deaths were observed, two pigs died on the 10th day postinfection, both of which presented with leg arthritis and typical polyserositis lesions. This result is consistent with the experimental findings of Boetner AG et al. who used serotype 7 to infect 7-day-old piglets, indicating that serotype 7 has some pathogenicity in piglets of this age group[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Additionally, 42-day-old Landrace pigs infected with serotypes 2 and 7 both presented obvious pneumonia and pericarditis symptoms, suggesting that the lungs and heart may be the primary target organs of these two serotypes.\u003c/p\u003e \u003cp\u003eIn summary, this study revealed that the overall infection rate is markedly higher than previously reported, with pigs of all age groups demonstrating susceptibility and no evident seasonal variation. Serotypes NT and 2 emerged as the predominant strains, followed by serotype 7. While this distribution trend is partially consistent with global patterns, the elevated prevalence of serotype NT indicates that regional factors or methodological differences in detection may contribute to variations in serotype distribution. Notably, the carriage rates of virulence genes varied significantly across serotypes, with \u003cem\u003egdh\u003c/em\u003e, \u003cem\u003efbps\u003c/em\u003e, \u003cem\u003esly\u003c/em\u003e, \u003cem\u003eorf2\u003c/em\u003e, \u003cem\u003emrp\u003c/em\u003e, and \u003cem\u003egapdh\u003c/em\u003e being widely detected, whereas \u003cem\u003e89k\u003c/em\u003e and \u003cem\u003eepf\u003c/em\u003e were found at lower frequencies. Moreover, both serotypes 2 and 7 can cause clinical symptoms similar to those of \u003cem\u003eS. suis\u003c/em\u003e disease, but serotype 2 is significantly more pathogenic than is serotype 7. This study provides important scientific evidence for formulating precise prevention and control strategies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we investigated the prevalence of \u003cem\u003eS. suis\u003c/em\u003e in 89 large-scale pig farms in 12 provinces in the western region of our country, and analyzed the serotypes and the presence of virulence genes of the isolates as well as the pathogenicity of serotypes 2 and 7. The results of this paper provide important baseline information on the serotype characteristics and virulence genes of Streptococcus suis and the pathogenicity of epidemic strains in China, which is of great significance for understanding its epidemiological characteristics and the development of vaccines used to prevent Streptococcus suis infection in pigs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eDY and JX conceived and designed the experiments; JX, DY, MH，and JZ performed the experiments; JX and DY analyzed the data and wrote the manuscript. DY and XH were responsible for manuscript review and editing, supervision, resource provision, and funding acquisition. All authors reviewed and endorsed the final version for submission.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Yunfu Innovation Team Project (CYRC202301) and the Research and Application of Epidemiology and Prevention Technology for Important Bacterial Diseases in Swine Respiratory System (WS-GG-JKYZ-202309-002).\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003cbr\u003eReasonable requests for the datasets utilized or examined in this study can be directed to the corresponding author\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eWe confirm that for the purchased pigs, informed consent for the use of the animals in this study has been obtained from the owner of a certain pig farm in Qingyuan City, Guangdong Province. Animal infection experiments were conducted according to the Guidelines for Experimental Animals established by the Ministry of Science and Technology of China (Beijing) and were supervised and approved by the National Animal Ethics and Use Committee. The study was approved by the South China Agricultural University (Approval No.: SYXK-2019–0136).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors state that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eAuthor details\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003eCollege of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou, 510642, China. \u003cstrong\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003eGuangdong Enterprise Key Laboratory for Animal Health and Environmental Control, Wen's Foodstuff Group Co. 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Acta Pathol Microbiol Immunol Scand B. 1987;95(4):233\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1699-0463.1987.tb03118.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1699-0463.1987.tb03118.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Streptococcus suis, epidemiological investigation, serotype, virulence gene, pathogenicit","lastPublishedDoi":"10.21203/rs.3.rs-6553183/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6553183/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eStreptococcus suis\u003c/em\u003e,\u003cem\u003e \u003c/em\u003ea zoonotic Gram-positive bacterium, is the etiological factor for septicemia and pneumonia in humans and pigs, posing a global public health threat. Currently, limited studies have investigated \u003cem\u003eS. suis \u003c/em\u003einfections in major pig farms. This study investigated the serotypes, virulence genes, and pathogenicity of the isolates in 89 pig farms across 12 regions from 2022 to 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe overall infection and isolation rates were 59.59% and 16.1%, respectively. The infection rate was the highest in Guangdong (72.41%) and the lowest in Hubei (43.75%). Suckling piglets, nursery pigs, fattening pigs, and pregnant sows were susceptible to \u003cem\u003eS. suis \u003c/em\u003einfection with infection rates being as high as 60%. The infection rates in spring, summer, autumn, and winter were 70.72%, 60.67%, 40.62%, and 68.97%, respectively. Serotype analysis of 137 isolates revealed increased serotype diversity in coastal provinces, especially in Guangdong, Jiangsu, and Shandong. Serotype 1 was detected in Liaoning. The most prevalent serotype was NT (21.01%), especially in Anhui, Guangxi, and Guangdong, followed by Serotype 2 (20.29%) and Serotype 7 (18.12%). Virulence gene analysis revealed that the occurrence of \u003cem\u003egdh\u003c/em\u003e, \u003cem\u003egapdh\u003c/em\u003e, and \u003cem\u003eorf2 \u003c/em\u003e(\u0026gt;89%) was high, whereas that of \u003cem\u003e89k\u003c/em\u003e and \u003cem\u003eepf \u003c/em\u003ewas low (≤ 28.47%). Serotypes 1 and 7 frequently harbored mrp and gdh but often lacked \u003cem\u003e89k \u003c/em\u003eand \u003cem\u003eepf\u003c/em\u003e. Serotypes 2 and NT harbored all tested genes with low \u003cem\u003e89k\u003c/em\u003e occurrence rates. The occurrence rates of \u003cem\u003esly\u003c/em\u003e and \u003cem\u003eepf \u003c/em\u003e(≤43.75%) were low in serotype 9. Animal challenge experiments demonstrated that Serotype 2 induced acute death in Landrace pigs aged 42 days with a mortality rate of 100%. In contrast, Serotype 7 was associated with low mortality rates (28.57%) and induced mild pathological symptoms, including pneumonia and pericarditis, and yellow effusion in the thoracic cavity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion：\u003c/strong\u003eThis study provides useful insights for the prevention and control of \u003cem\u003eS. suis \u003c/em\u003einfection in pig farms in China.\u003c/p\u003e","manuscriptTitle":"Epidemiological Investigation and Pathogenicity of Streptococcus suis in Eastern China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 07:51:11","doi":"10.21203/rs.3.rs-6553183/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"923bc5f3-aa8d-48ec-bfa1-03d8b8ad0a4e","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-13T23:53:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-19 07:51:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6553183","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6553183","identity":"rs-6553183","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}