Streptococcus suis strains and multivalent vaccine for preventing streptococcus suis disease and use thereof

Abstract

The present application relates to the animal medical technology field, especially to the Streptococcus suis strain and the vaccine thereof for prevention and treatment, and provides Streptococcus suis type 2, type 3 and type 9 strains screened and obtained, and a trivalent inactivated vaccine prepared by mixing the three strains, which can safely and effectively prevent and protect against the attack of the three pathogenic bacteria of Streptococcus suis.

Description

Technical Field

[0001] This invention relates to the field of animal biopharmaceutical technology, and more specifically, to strains of Streptococcus suis, multivalent vaccines for the prevention and treatment of streptococcal disease in suis, and their applications. Background Technology

[0002] Streptococcus suis is an important zoonotic pathogen and a major cause of streptococcal disease in pigs. Infected pigs typically present with septicemia, meningitis, pneumonia, arthritis, and lymph node abscesses. In humans, it can cause meningitis and toxic shock-like syndrome, leading to death in severe cases. The large number of serotypes and wide prevalence of Streptococcus suis pose challenges to disease control. Based on differences in cell wall capsular polysaccharides (CSP), Streptococcus suis can be classified into 35 serotypes (types 1-34 and serotypes 1 / 2). Most pathogenic serotypes are types 1-9, with types 2 (serotypes 1 / 2), 7, and 9 currently exhibiting the highest virulence. Clinically, serotype 2 has the highest prevalence and pathogenicity. In recent years, serotypes 3 and 9 have also shown high prevalence and some virulence in pig farms in my country. Currently, antibiotics are the main treatment for streptococcal disease in pigs. Streptococcus suis is relatively sensitive to β-lactam antibiotics. However, due to the ease with which Streptococcus suis develops drug resistance and the national restrictions on antibiotic use in the breeding process, the use of vaccines for prevention of this disease has become a trend.

[0003] Currently, commercially available vaccines against streptococcal disease in pigs mainly include inactivated vaccines, live vaccines, and subunit vaccines. Live vaccines are primarily developed using the ST171 strain. The gene sequence of the ST171 attenuated strain of Streptococcus suis has been published (GenBank accession number CP002904). Because it is a group C streptococcus, the resulting vaccine does not provide ideal protection against Streptococcus suis type 2 strains, and this vaccine is now rarely used. In 2022, Wuhan Keqian Biotechnology evaluated the protective effect of the widely used inactivated vaccine (trivalent streptococcal disease vaccine SEZ+SS2+SS7) against Streptococcus suis types 3 and 9. The experimental results showed that this inactivated vaccine does not provide protection against virulent Streptococcus suis types 3 and 9. Commercially available subunit vaccines are primarily bivalent subunit vaccines against Streptococcus suis infection and Haemophilus parasuis infection. These include the HP0197 and HP1036 immune proteins against Streptococcus suis, as disclosed in ZL201010148662.5, mainly used to prevent infection with Streptococcus suis types 2 and 7. Therefore, there is currently a lack of commercially available vaccines against Streptococcus suis types 3 and 9. Thus, the development of trivalent inactivated vaccines against Streptococcus suis types 2, 3, and 9 is of significant practical importance.

[0004] Currently, the pathogenic mechanisms of virulence genes in *Streptococcus suis* are not fully understood, but it is generally believed that the strength of pathogenicity is largely correlated with the distribution of virulence genes in the strain. Investigations have found that the main virulence gene phenotype of most *Streptococcus suis* serotype 2 strains isolated in my country is sly+ / fbps+ / mrp+ / epf+, while the distribution of virulence genes in *Streptococcus suis* serotype 9 remains unclear.

[0005] In 2018, Jiangsu Veterinary Research Institute isolated Streptococcus suis SS2 and SS9. The virulence gene phenotypes of the SS2 and SS9 isolates were sly+ / fbps+ / orf2+ / mrp+ / sao+ / gdh+ / epf+ and sly- / fbps+ / orf2+ / mrp- / sao- / gdh+ / epf-, respectively. Moreover, all SS2 isolates were ST7, which is a relatively common sequence type isolated from diseased pigs in China.

[0006] In 2022, the College of Animal Science and Technology of Anhui Agricultural University isolated six strains of Streptococcus suis serotype 9 (SS9), named A to F. Isolates A, D, E, and F were all ST1330. Among them, isolates A and D had the same virulence genotype: gapdh+ / sly+ / fbps+ / orf2- / mrp+ / epf-. Isolates E and F also had the same virulence genotype: gapdh+ / sly+ / fbps+ / orf2+ / mrp+ / epf-. Isolates B and C were ST243, and their virulence genotypes were both gapdh+ / sly- / fbps+ / orf2- / mrp+ / epf-. ST243 was considered to be the ST type of SS9 with high virulence potential.

[0007] In 2022, Wuhan Keqian Biotechnology isolated two highly virulent strains of Streptococcus suis serotype 9, namely SS9-20 and SS9-22. Virulence gene amplification of strain SS9-22 revealed its genotype to be gapdh+ / sly+ / fbps+ / orf2+ / mrp- / 89K- / gdh+ / epf+, with an LD50 of 3.2 × 10⁷ CFU in mice. Wuhan Keqian Biotechnology also isolated a highly virulent strain of Streptococcus suis serotype 3, KQ3-1, with the genotype gapdh+ / sly+ / fbps- / orf2- / mrp- / 89K- / gdh+ / epf-, also with an LD50 of 5.2 × 10⁷ CFU in mice.

[0008] Wenyang Dong et al. from the College of Veterinary Medicine at Nanjing Agricultural University studied the relationship between the ST type and virulence of 30 SS9 isolates (24 Chinese isolates, 5 Vietnamese isolates, and 1 Danish isolate). They found that the strains were divided into two clusters (I and II). The genotype of cluster I was gapdh+ / sly- / fbps+ / orf2+ / mrp+ / epf-; the genotype of cluster II was gapdh+ / sly- / fbps+ / orf2- / mrp+ / epf-. ST 243 can be considered as a high-virulence ST in SS9. ST 243 is a strain in cluster I and has an LD50 of 2.5 × 10⁸ CFU.

[0009] Therefore, developing a trivalent vaccine that can simultaneously prevent diseases caused by three serotypes of Streptococcus suis: serotypes 2, 3, and 9 is an urgent problem to be solved in this field. Summary of the Invention

[0010] Therefore, the technical problem to be solved by the present invention is to provide a Streptococcus suis strain, a multivalent vaccine for the prevention and treatment of streptococcal disease in suis, and its application. The vaccine obtained by inactivating this strain has a good immune effect against Streptococcus suis.

[0011] The Streptococcus suis strain 9 with accession number CCTCC NO: M 2023031 is named Streptococcus suisSS1696.

[0012] The Streptococcus suis type 2 strain with accession number CCTCC NO: M 2023032 is named Streptococcus suisSS1803.

[0013] The Streptococcus suis strain 3 with accession number CCTCC NO: M 2023033 is named Streptococcus suisSS1803024.

[0014] The streptococcus suis strain provided by this invention was isolated from the brain, lungs, trachea, heart blood, and joint fluid of sick pigs exhibiting respiratory distress, septicemia, arthritis, and neurological symptoms, as well as dead pigs. The isolated strain can safely induce animal immunity and produce good protective effects.

[0015] The vaccine for preventing streptococcal disease in swine provided by the present invention is prepared from inactivated streptococcal type 2 strains, streptococcal type 3 strains, streptococcal type 9 strains and excipients.

[0016] Furthermore, the excipients include an adjuvant, the adjuvant being Summit Poly Solution, and the volume ratio of the inactivated strain mixture to the adjuvant is 4:1.

[0017] Furthermore, the concentration of inactivated Streptococcus suis type 2 strain in the vaccine was 1×10⁻⁶. 8 The concentration of Streptococcus suis type 3 strain was 7.5 × 10 CFU / mL. 8 The concentration of Streptococcus suis strain 9 (CFU / mL) was 1.5 × 10⁻⁶. 8 CFU / mL.

[0018] Furthermore, the excipients also include a diluent comprising 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, and a pH value of 7.4.

[0019] The method for preparing a trivalent inactivated vaccine provided by the present invention includes inactivating three serotypes of the bacterial strain, mixing them with a buffer solution and an adjuvant, and preparing the vaccine.

[0020] Furthermore, the concentration and preparation of inactivated Streptococcus suis trivalent antigen includes the following steps:

[0021] S1. Take the colonies of the activated strain and add them to THB liquid medium. Stir at 37°C and 180 r / min for 12 hours. Then transfer them to new THB liquid medium at a ratio of 1% (V / V). Place the medium on a shaker at 37°C and culture at 180 r / min until the late logarithmic phase to obtain the bacterial culture.

[0022] S2. Add 0.3%~0.4% formaldehyde solution (V / V) to the bacterial solution obtained in step S1, and place it in a shaker at 37°C and 180r / min to fully inactivate the bacteria.

[0023] S3. Centrifuge the inactivated bacterial solution at 8000 r / min for 10 min to obtain bacterial cells. Wash with PBS to remove formaldehyde solution. Adjust the ratio of the bacterial solution to the adjuvant to obtain the target immunization dose of vaccine.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. This invention provides three strains of Streptococcus suis: SS1803 (type 2), S1803024 (type 3), and SS1696 (type 9) obtained through screening. These three strains are highly pathogenic and have good immunogenicity, providing materials for basic and applied research on Streptococcus suis.

[0026] 2. The present invention also provides a trivalent inactivated vaccine of Streptococcus suis prepared based on the above three pathogenic bacteria. After mice are immunized with the vaccine, high levels of antibodies can be induced. The trivalent vaccine can protect mice safely and effectively against the attack of Streptococcus suis strain SS1803, Streptococcus suis strain SS1803024, and Streptococcus suis strain SS1696. Attached Figure Description

[0027] Figure 1 Figure A shows the PCR identification results of the strains; Figure B shows the identification results of Streptococcus suis serotype 2, 1 is SS1803 strain, 2 is positive control SC19, and 3 is negative control; Figure C shows the identification results of Streptococcus suis serotype 3, 1 is SS1803024 strain, and 2 is negative control; Figure C shows the identification results of Streptococcus suis serotype 9, 1 is SS1696 strain, and 2 is negative control.

[0028] Figure 2 Gram staining results for Streptococcus suis;

[0029] Figure 3 The growth curve of Streptococcus suis;

[0030] Figure 4 The results of virulence gene identification for three strains are shown in lane 1. gapdh Gene, 2 sly Genes, 3 fbps Genes, 4 orf2 Genes, 5 mrp Genes, 6 89K Genes, 7 gdh Gene;

[0031] Figure 5 The results are from the detection of specific antibodies in mouse serum; Figure 6 The survival curves of mice after challenge are shown in Figure 1. A represents the survival curve of mice after challenge with SS1803 strain, B represents the survival curve of mice after challenge with SS1803024 strain, and C represents the survival curve of mice after challenge with SS1696 strain. TIV: trivalent inactivated vaccine immunization group. Detailed Implementation

[0032] The present invention will now be described in detail with reference to specific embodiments. All experimental materials used in the present invention are commercially available products.

[0033] Example 1: Isolation of strains

[0034] 1. Pathological materials

[0035] Collect brain, lung, trachea, heart blood, and joint fluid from sick pigs exhibiting respiratory distress, septicemia, arthritis, and neurological symptoms, as well as from dead pigs in pig farms.

[0036] 2. Isolation and culture of bacteria

[0037] Under aseptic conditions, suspected streptococcal disease samples were evenly spread onto TSA solid medium containing 10% fetal bovine serum. After incubation at 37°C for 24 hours, suspected single streptococcal colonies were selected for PCR identification and Gram staining. Gram-positive, chain-like single colonies were selected and streaked onto TSA plates for subculturing. The purified strains were preserved in 50% glycerol and stored at -80°C for later use.

[0038] 3. Serological typing

[0039] PCR detection of target fragments for Streptococcus suis serotypes 2, 3, and 9: cps2I , cps3L and cps9J Primer sequences were designed based on the gene fragments. The sizes of the target fragments after amplification of each serotype of Streptococcus suis were 363bp, 210bp, and 409bp, as shown in Table 1. The serotypes were identified using these primer sequences.

[0040] Table 1 PCR Primers

[0041]

[0042] The PCR reaction system consisted of: 2 μL template, 12.5 μL 2×TaqMixture, 1 μL upstream primer, 1 μL downstream primer, and 8.5 μL pure water.

[0043] The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, and 30 cycles followed by a final extension at 72℃ for 10 min.

[0044] The PCR reaction products were photographed after electrophoresis on a 1.0% agarose gel, and the results are as follows. Figure 1Three strains of Streptococcus suis with different serotypes were discovered. The strain that tested positive for Streptococcus suis serotype 2 was named *Streptococcus suis* SS1803, accession number: CCTCC NO: M 2023032, accession date: January 5, 2023, depositary address: China Center for Type Culture Collection (Wuhan University, Wuhan, China), abbreviated as SS1803 strain; the strain that tested positive for Streptococcus suis serotype 3 was named *Streptococcus suis* SS1803024, accession number: CCTCC NO: M 2023033, accession date: January 5, 2023, depositary address: China Center for Type Culture Collection (Wuhan University, Wuhan, China), abbreviated as SS1803024 strain; the strain that tested positive for Streptococcus suis serotype 9 was named *Streptococcus suis* SS1696, accession number: CCTCC NO: M 2023031, deposited on January 5, 2023, deposited at China Center for Type Culture Collection (Wuhan University, Wuhan, China), abbreviated as SS1696.

[0045] like Figure 2 As shown, all three strains are Gram-positive bacteria and exist in both long and short chain forms. However, the chain lengths of the three strains are different. The chain of strain SS1803 is the longest; the chain of strain SS1803024 is the shortest, and it exists mostly as a single bacterium or a short chain; the chain length of strain SS1696 is between the two of the former two, with both long and short chains distributed.

[0046] 4. Preservation of microbial strains

[0047] The correctly identified Streptococcus suis was added to skim milk powder and freeze-dried for preservation.

[0048] 5. Growth curve determination

[0049] Single colonies of three strains (containing 100 mL / L newborn calf serum) were picked from a TSA plate (after subculturing and purification) in a sterile operating table and inoculated into THB liquid medium. The culture was incubated overnight at 37°C with a shaker at 180 rpm. The next day, the colonies were transferred to fresh THB liquid medium at a 1% (v / v) ratio and incubated again at 37°C with a shaker at 180 rpm. The culture tubes were removed at 0h, 2h, 4h, 6h, 8h, 10h, and 12h to measure the OD of the bacterial culture. 600nm The values ​​were calculated by repeating the process three times per tube, the average value was taken, and a growth curve was plotted. The results are as follows: Figure 3 As shown, all three strains entered the logarithmic growth phase in about 2-4 hours after being transferred to fresh culture medium.

[0050] 6. Virulence gene identification

[0051] Based on Table 2, select the main virulence factor of Streptococcus suis, hemolysin (… sly ), lysozyme-releasing protein ( mrp ), fibronectin ( fbps ), glyceraldehyde-3-phosphate dehydrogenase ( gapdh ), virulence-related factors ( orf2 ), glutamate dehydrogenase ( gdh )and 89k Using the virulence island as the identification gene, primers corresponding to 7 were designed (Table 2). The DNA of the three highly virulent strains obtained after screening was used as templates for PCR amplification to determine the virulence genotypes of SS1803, SS1803024 and SS1696.

[0052] The results are as follows Figure 4 The three strains had different virulence genotypes. Using SS1803 as a template, gapdh -F / R、 orf2 -F / R、 gdh -F / R、 fbps -F / R、 sly -F / R are the nucleotide sequences amplified by the primers, as shown in SEQ ID No. 01~05 respectively;

[0053] Using SS1803024 as a template, gapdh -F / R、 orf2 -F / R、 gdh -F / R、 fbps -F / R、 sly The nucleotide sequences amplified by the primers -F / R are shown in SEQ ID No. 06~10 respectively;

[0054] Using SS1696 as a template, gapdh -F / R、 orf2 -F / R、 gdh -F / R、 fbps -F / R、 sly The nucleotide sequences amplified by the primers -F / R are shown in SEQ ID No. 11~14 respectively;

[0055] Nucleotide sequences that were not amplified using SS1803, SS1803024, and SS1696 strains as templates and mrp-F / R and 89K-F / R primers were analyzed.

[0056] Table 2 PCR Primers

[0057]

[0058] The virulence genotype of strain SS1803 is: gapdh + / sly + / fbps + / orf2 + / mrp - / 89K - / gdh +, which is different from the virulence gene of the Streptococcus suis SS2 strain isolated by the Jiangsu Provincial Veterinary Research Institute;

[0059] The virulence genotype of strain SS1803024 is 3. gapdh + / sly + / fbps + / orf2 + / mrp - / 89K - / gdh +, which is different from the virulence gene of the highly virulent strain KQ3-1 of Streptococcus suis serological type 3 isolated by Wuhan Keqian Biotechnology;

[0060] The virulence genotype of strain SS1696 is... gapdh + / sly - / fbps + / orf2 + / mrp - / 89K - / gdh The virulence gene of Streptococcus suis serotype 9 isolated from the College of Animal Science and Technology of Anhui Agricultural University, the SS9-22 strain isolated from Wuhan Keqian Biotechnology, the virulence gene of Streptococcus suis serotype 9 isolated from Jiangsu Veterinary Research Institute, and the virulence gene of Streptococcus suis serotype 9 strain ST243 are all different from those of Streptococcus suis serotype 9 strain.

[0061] 7. Mouse pathogenicity test

[0062] One hundred and twenty-eight 6-7 week old female Balb / c mice were randomly divided into 16 groups of eight. SS1803 was administered at 2 × 10⁻⁶ doses from the following five different doses. 8 CFU / each, 1×10 8 CFU / each, 5 x 10 7 CFU / each, 2.5×10 7 CFU / each, and 1.3×10 7 CFU / unit; SS1803024 was challenged with the following 5 doses at 3×10⁻⁶ doses. 8 CFU / each, 1.5×10 8 CFU / each, 7.5×10 7 CFU / each, 3.8×10 7 CFU / only 1.9×10 7 CFU / animal; SS1696 was challenged with the following 5 doses at a rate of 1×10⁻⁶. 8 CFU / each, 5 x 10 7 CFU / each, 2.5×10 7CFU / each, 1.3×10 7 CFU / only 6.5×10 6 CFU / animal was used to challenge the virus, while a control group (0 CFU / animal) was set up and injected with an equal volume of physiological saline. All animals were challenged by intraperitoneal injection. After challenge, the animals were observed for 7 days and the mortality rate was recorded.

[0063] LD is calculated based on the improved Koch method. 50 The calculation formula is: LD50 ＝ lg -1 [Xm] － i ( ∑ P －0.5 )] ( Xm —Logarithm of the maximum dose; i—Difference between the logarithmic doses of two adjacent groups; P—Morale of animals in each group; ∑ P—the sum of mortality rates for all groups of animals.

[0064] The LD50 of SS1803, SS1803024, and SS1696 strains were calculated using the improved Koch method. 50 The median lethal dose (LD50) was 3.2 × 10⁻⁶. 7 CFU, 8.9×10 7 CFU and 1.1×10 7 CFU.

[0065] Therefore, the SS1696 strain is more virulent than existing strains.

[0066] Example 2: Preparation of Streptococcus suis type 2, 3 and 9 antigens

[0067] 1. Vaccine preparation

[0068] The freeze-dried strains SS1803, SS1803024 and SS1696 were revived and passaged. Single colonies were picked from TSA plates and inoculated into THB liquid medium. The plates were placed in a shaker at 37°C and cultured at 180 rpm for 12 hours overnight. The next day, the culture was transferred to fresh THB liquid medium at a ratio of 1% (volume ratio) and placed in a shaker at 37°C and cultured at 180 rpm until the late logarithmic phase. A portion of the bacterial culture was then taken for viable cell counting.

[0069] Take qualified Streptococcus suis type 2, 3, and 9 bacterial suspensions respectively, add 0.3%~0.4% formaldehyde solution (V / V) to the total volume of the bacterial suspension, and place in a shaker at 37℃ and 180r / min for complete inactivation. Samples are then taken for inactivation and sterility tests; no bacterial growth was observed.

[0070] The bacterial culture was then centrifuged at 4°C and 8000 rpm for 10 minutes to obtain bacterial cells. The cells were washed five times with PBS to remove residual formaldehyde solution. The concentration of PBS was adjusted to the levels shown in Table 3 according to the experimentally determined immunization dose. The inactivated vaccine was then mixed with SummitPoly Solution adjuvant at the volume ratios shown in Table 3 to prepare the required inactivated vaccine. The specific vaccine content percentages are shown in Table 3 below.

[0071] The antigen content (CFU / ml) of the prepared vaccine for Streptococcus serotype 2 SS1803 strain is 1×10⁻⁶. 8 CFU / mL, Streptococcus serotype 3 SS1803024 strain was 7.5 × 10⁻⁶. 8 CFU / mL, Streptococcus 9 SS1696 strain was 1.5 × 10⁻⁶. 8 CFUmL.

[0072] Table 3. Composition of Trivalent Vaccine

[0073]

[0074] 2. Mouse immunization

[0075] Thirty-six 6-week-old female Balb / c mice were selected and divided into six groups of six. Three groups were vaccinated with a trivalent inactivated vaccine, while the remaining three groups served as a control group and were injected with PBS. All mice were administered a 200 μL intramuscular injection in the leg. A second immunization was given 14 days after the initial immunization. Clinical symptoms and mortality were observed after challenge, and the protection rates of different vaccines were calculated.

[0076] 3. Safety Evaluation

[0077] The mice were in good condition; the injection site swelled briefly before returning to normal, and no mice died.

[0078] 4. Determination of IgG antibody levels in mouse serum

[0079] The three whole-bacterial proteins were diluted to 10 μg / mL with antigen coating buffer, and 100 μL was coated into each well of an ELISA plate. Serum levels of specific antibodies were measured using indirect ELISA. Mouse post-immunization serum was diluted 160-fold as the primary antibody, and HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody. The plates were developed with TMB chromogenic buffer, and the OD values ​​were read using a microplate reader. 630nm The value.

[0080] The results are as follows Figure 5 After two immunizations, the mice were able to produce IgG antibodies against the corresponding three serotype antigens.

[0081] 5. Virus challenge experiment

[0082] Fourteen days after the second immunization, mice in the three immunized groups were challenged with Streptococcus suis type 2, Streptococcus suis type 3, and Streptococcus suis type 9, respectively. The challenge dose for each strain was 5 × 10⁻⁶ for strain SS1803: SS1803024. 7 CFU / animal, SS1803024 strain: 1.7 × 10 8 CFU / unit and SS1696 plants: 1.5 × 10 7 CFU / animal. The three control groups were challenged with the same dose of the virus. The challenge method was intraperitoneal challenge. Patients were observed for 7 days post-challenge, and mortality was recorded.

[0083] The results are as follows Figure 6 All mice in the control group died, while the survival rates of the immunized mice after challenge with Streptococcus suis type 2, Streptococcus suis type 3, and Streptococcus suis type 9 were 83.3%, 66.7%, and 66.7%, respectively.

[0084] Table 4 Survival rates of each group after virus challenge

[0085]

[0086] Therefore, the trivalent inactivated vaccine against Streptococcus suis serotypes 2+3+9 in the immunized group can effectively prevent infection with virulent strains of Streptococcus suis serotypes 2, 3 and 9.

Claims

1. A vaccine for preventing Streptococcus suis, characterized in that, It was prepared from inactivated Streptococcus suis type 2, Streptococcus suis type 3, and Streptococcus suis type 9 strains and excipients. The Streptococcus suis type 9 strain has the preservation number CCTCC NO: M2023031 and is named as follows: Streptococcus suis SS1696; the preservation number of the Streptococcus suis type 2 strain is CCTCCNO: M 2023032, named Streptococcus suis SS1803; the preservation number of the Streptococcus suis strain 3 is CCTCC NO: M 2023033, named Streptococcus suis SS1803024; The concentration of inactivated Streptococcus suis type 2 strain in the vaccine is 1×10⁻⁶. 8 The concentration of Streptococcus suis type 3 strain was 7.5 × 10 CFU / mL. 8 The concentration of Streptococcus suis strain 9 (CFU / mL) was 1.5 × 10⁻⁶. 8 CFU / mL.

2. The vaccine according to claim 1, characterized in that, The excipients include an adjuvant, wherein the adjuvant is SummitPoly Solution, and the volume ratio of the inactivated strain of claim 1 to the adjuvant is 4:

1.

3. The vaccine according to claim 2, characterized in that, The excipients also include a diluent comprising 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4 and 0.24 g / L KH2PO4, with a pH of 7.

4.

4. A method for preparing the vaccine according to any one of claims 1 to 3, characterized in that, The method includes inactivating Streptococcus suis type 2, Streptococcus suis type 3 and Streptococcus suis type 9 strains as described in claim 1, and then mixing them with adjuvants and diluents to prepare a vaccine.

5. The preparation method according to claim 4, characterized in that, Includes the following steps: S1. Add the colonies of the activated strain to THB liquid medium, stir at 37°C and 180 r / min, and incubate for 12 hours. Then, transfer the colonies to new THB liquid medium at a ratio of 1% (V / V), and place the medium on a shaker at 37°C and incubate at 180 r / min until the late logarithmic phase to obtain the bacterial culture. S2. Add 0.3% to 0.4% formaldehyde solution (V / V) to the bacterial solution obtained in step S1, and place it in a shaker at 37°C and 180 r / min to fully inactivate the bacteria. S3. Centrifuge the inactivated bacterial solution at 8000 r / min for 10 min to obtain bacterial cells. Wash with PBS to remove formaldehyde solution, and then mix with adjuvant to adjust the ratio to obtain the target immunization dose of vaccine.

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