Investigation on clinical healthy swine carrier status of Streptococcus suis in Hebei Province of China

A total of 600 samples of nose swabs collected from Hebei Province, China, were examined by polymerase chain reaction (PCR) for the presence of Streptococcus suis in healthy swine and the serotype were identified. Results showed that 148 strains (24.67%) of 600 tested were positive with S. suis, including 27 strains (18.24%) were identified to be type 7, 24 strains (16.22%) were type 2 and 20 strains (13.51%) were type 9. But serotypes of other 75 strains (50.68%) were undetermined. To our knowledge, this is the first epidemiological investigation of S. suis in healthy swine from Hebei Province of China.


INTRODUCTION
Streptococcus suis is an important pathogenic bacteria hazard in modern swine industry.It can be divided into 35 serotypes (type 1 to 34 and 1/2) according to the differences in antigenic properties of polysaccharide capsular.S. suis serotype 2 is considered to be the most widely popular and highest pathogenicity isolated in both swine and humans, which is also an important zoonotic pathogen.It reported that slaughterhouse employees can be infected with S. suis and to be considered as an occupational disease (He et al., 2000;Hu et al., 2000).In recent years, S. suis outbreaks in many countries from Europe, Americas and Asian.It can not only cause huge losses to the world's swine industry, but also endangers with public health and safety (Staats et al., 1997;Touil et al., 1998;Torremorell et al., 1998).S. suis serotype 2 (SS2) was discovered firstly in Guangdong Province, China, in 1990.There were two large outbreaks of SS2, people had been found infected with SS2 died in Jiang su, Si chuan Province, in 1998 (Shen et al., 2000;Liu et al., 2005).In addition to SS2, SS1, SS7, SS9, etc are

Primers
Five pairs of primers were designed according to the reference (Okwumabua et al., 2003;Smith et al., 1999;Wang et al., 2009) respectively and synthesized in Sangon Biotech (Shanghai) Co., LTD.The details of the primers were listed in Table 1.

Sample collection
600 nasal swabs were collected from healthy pigs (different growth stage) on farms of Hebei Province in China, such as Shijiazhuang, Xingtai, Zhangjiakou, Cangzhou, Tangshan and Qinhuangdao.Samples were collected and processed to refrigerated storage.

Bacteria culture and identification
100 μl samples of nasal swab were inoculated into 2 ml of Streptococcus liquid selection medium (containing 15 μg/ml polymyxin B, 30 μg/ml nalidixic acid and 0.2 g/ml crystal purple) for 18 to 24 h at 37°C, S. suis was observed in Gram's method by a microscope.

Preparation of the templates
1000 μl Gram-positive Streptococcus culture liquid were centrifuged at 10000r / min for 1 min, supernatant was discarded, resuspended with 200 μl ddH2O and then boiled for 10 min, after cooling, centrifuged at 7000r / min for 5 min, supernatant were stored at -20°C.DNA was extracted using Bacteria genomic DNA extraction kit.

PCR identification of S. suis
Firstly, GDH sequence of S. suis was applied to identify the strain.
Samples were also identified by PCR based on the S. suis serotype 1,2,7,9.The final PCR volume was 25 μl, the reaction components are as follows: 10×PCR bufferr (Mg 2+ Plus) 2.5 μl, dNTPs Mixture (2.5 mM) 2.0 μl, upstream and downstream primer 1.0 μl, Ex Taq DNA polymerase 0.2 μl (5 U), DNA template 2.5 μl, added ddH2O to 25 μl.PCRs consisted of 30 cycles of denaturation for 5 min at 95°C, then 95°C denaturation 15S (gdh and cps1I) or 94°C for 30 s (Cps2J, Cps7H, Cps9H), annealing at 55°C for 45 s, and extension for 30 s at 72°C.A final extension was performed for 10 min at 72°C.PCR reaction condition of the rest genes are the same as except for annealing temperature.Simultaneously, negative control was designed.The PCR products were detected by electrophoresis in 1.2% agarose gel.

Isolation and identification of S. suis
The strain was identified as S. suis in morphology.S. suis is Grampositive cocci in pairs or short chains of broth cultures by microscopy.Bacterial liquid rules on blood agar plates, at 37°C for 18 ~ 24 h, 3～6 colonies each plate with α hemolytic, smooth, moist, white translucent, diameter 1 ~2 mm were picked selectively, and inoculated into 2 ml 5% bovine serum of THB, suspected of S. suis were stored at 4°C.Finally, PCR methods established above were used to identified the isolated bacteria.

Serum agglutination test
Each drop of diagnose serum antibodies known and bacilli were mixed in the slide, a few minutes after, it was identified as positive when there was emergence of visible agglutination.Also set up normal saline as control.PCR identification as S. suis serotype 2 were to have further identification with 1/2 Standard antiserum, PCR identification as S. suis serotype 1, and S. suis serotype 14 and 1/2 standard anti-serum were used for further identification, respectively.

S. suis carrying in different regions
PCR analysis showed that 148 samples in 600 nasal   swabs were positive for S. suis, the positive rate was 24.67%, results were shown in Table 2. Zhangjiakou, Cangzhou positive rates were 11 and 17%, significantly lower than other regions, the difference was significant (p<0.01),Qinhuangdao, Tangshan, Shijiazhuang positive samples were high, were 30, 32 and 34%, significantly higher than other regions, which showed that the S. suis infection had some regional differences (Table 2).The electrophoresis graphs of gdh, cps2J, cps1I, cps7H and

Major pathogenic S. suis serotype carrying cases in different regions
PCR test results showed that each serotype carrying case as follows: SS7 were the highest (4.5%), followed by SS2 up to 4%; SS9 of 3.3%; SS1 was the least, only 0.33%.The positive rate of SS7 in Shijiazhuang or Qinhuangdao was significantly (p<0.01)higher than other areas; SS1 positive rate was lower, in addition to one was detected in Qinhuangdao, Cangzhou, other regions were not detected positive.The positive rate of SS9 in Qinhuangdao was significantly higher than other regions (p <0.01) (Table 3).

S. suis serotype mixed infections in different parts
Statistics of nasal swab test results showed that the same one sample could be infected with two or more different serotypes of Streptococcus suis.But the samples were limited，the proportion in the sample was less than 5%.The samples of infected with both SS7 and SS9 sample were the most, up to 1.67% of the total sample, both SS2 and SS7 were the second, accounting for 1%, both SS2 and SS9 infection accounted for 0.67%.There were also infected with SS2, SS7 and SS9 or more serotypes, but the proportion was very small, only 0.5%.(Table 4).

Serum agglutination test
Results for 30 isolates with a slide serum agglutination test showed that 11 S. suis serotype 2 bacilli only had antiserum agglutination with S. suis type 2, not with S. suis 1/2 type, so it was judged as S. suis type 2. S. suis serotype 7 (11) and type 9 (8) bacilli had specific agglutination with S. suis serotype 7, type 9 antiserum respectively.All results were consistent with the PCR analysis results.

DISCUSSION
The S. suis detection rate is different in the normal swine herds in different regions of China.Study found that SS7 had the highest detection rate, 4.5%, followed by SS2, SS9, and the detection rates were 4 and 3.3% respectively, these results suggested SS2, SS7 and SS9 were the most important popular serotypes in the Hebei region of China, and SS2 was zoonotic disease.More attention should be paid to this disease.SS7 was the highest detection strain.The authors found the pig cases infected with SS7 in Hebei, therefore, it needs to conduct deeper research on SS7 and strengthen prevention and control.SS1 carrier rate was relatively low (0.33%).There were multiple S. suis serotypes in the same nasal swabs by PCR, but it was small rate related to the carrier rate of each serotypes, its epidemiological significance remains to be studied.The study also found that, samples collected from Zhangjiakou, Cangzhou was significantly lower than other places of the province, can be inferred there are some regional differences in SS infection or the incidence of different farms will be different.
PCR is the most commonly method for the detection of S. suis.But PCR cannot distinguish between S. suis type 1 and type 14, S. suis 1/2 and type 1, type 2. To make test results more accurate, standard antiserum of S. suis type 2, type 7, type 9, 14 and 1/2 prepared by our laboratory were used to have a re-examination for this epidemiological survey.S. suis type 1 and 1/2 were not isolated in this study, it may be relative to the two serotypes little in Hebei Province.

Table 2 .
Results of S. suis positive rates.

Table 3 .
Major pathogenic S. suis serotype carrying cases in different regions.