A streptococcus suis type 9 vaccine strain, vaccine and application thereof

Abstract

This invention discloses a Streptococcus suis serotype 9 vaccine strain, the vaccine, and its application. The Streptococcus suis serotype 9 vaccine strain is deposited at the China Center for Type Culture Collection (CCTCC) and named Streptococcus suis SS9-CZ, with a deposit date of November 3, 2023, and accession number CCTCC NO: M20232125. Compared with existing technologies, the SS9-CZ strain of this invention was isolated from the lung tissue of clinically ill pigs, serotyped as Streptococcus suis serotype 9, grows well in THB medium, exhibits strong pathogenicity in piglets, and possesses good immunogenicity, making it a good Streptococcus suis serotype 9 vaccine strain. Immunization of piglets with the vaccine prepared using the SS9-CZ strain effectively resists attack by virulent Streptococcus suis strains, demonstrating good immunogenicity and providing technical support for the effective prevention of streptococcal disease in pigs.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to a Streptococcus suis type 9 vaccine strain, the vaccine, and its application. Background Technology

[0002] Streptococcal disease in pigs is a common bacterial infectious disease reported in various pig-farming regions. Clinical manifestations mainly include meningitis, septicemia, and arthritis. The primary pathogen is *Streptococcus suis* (SS), which can infect not only pigs but also humans, posing a significant threat to public health. *Streptococcus suis* is a capsule-bearing Gram-positive coccus, classified into 33 serotypes (serotypes 1–31, 33, and 1 / 2) based on differences in capsular polysaccharide antigens. In recent years, the clinical infection rate of *Streptococcus suis* serotype 9 (SS9) has been continuously increasing in pig farms in China, Australia, Belgium, the Netherlands, and Germany, becoming the main pathogenic strain for streptococcal disease in pig farms in China and globally. Han Rui et al. collected samples from eight large-scale pig farms in Nanchang, Fuzhou, and Jiujiang cities in Jiangxi Province from 2018 to 2021, isolating 10 pathogenic *Streptococcus suis* strains, seven of which were SS9. Huang Yuan et al. isolated three strains of *Streptococcus suis* from samples taken from a slaughterhouse in Dongguan in 2021, two of which were *Streptococcus suis* serotype 9. Ma Tingting et al. isolated 201 strains of *Streptococcus suis* from Napo County, Daxin County, Dongxing City, Jingxi City, Baise City, and Yulin City in Guangxi Province in 2017, 15 of which were *Streptococcus suis* serotype 9. Peng Zeren et al. isolated 60 strains of *Streptococcus suis* from Guangxi Province in 2021-2022, 7 of which were *Streptococcus suis* serotype 9; they also isolated 41 strains of *Streptococcus suis* from Sichuan Province, 5 of which were *Streptococcus suis* serotype 9. Our team isolated a total of 283 strains of *Streptococcus suis* from large-scale pig farms in East my country from 2020-2022, 91 of which were *Streptococcus suis* serotype 9. Vaccination is the main means of preventing and controlling streptococcal disease in pigs. Currently, there are no vaccines for streptococcus 9 (SS9) in China or abroad, and the development of an SS9 vaccine is urgent. Summary of the Invention

[0003] Purpose of the invention: To address the above-mentioned technical problems, this invention provides a Streptococcus suis type 9 vaccine strain, the vaccine, and its application. The vaccine strain has good growth performance and immunogenicity. After immunization with the vaccine made from it, it can effectively resist the attack of highly virulent strains of Streptococcus suis and has good immunoprotective efficacy.

[0004] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0005] In a first aspect, the present invention provides a Streptococcus suis type 9 vaccine strain, which is deposited at the China Center for Type Culture Collection (CCTCC) and named Streptococcus suis SS9-CZ, with a deposit date of November 3, 2023 and accession number CCTCC NO: M20232125.

[0006] Secondly, the present invention provides a Streptococcus suis type 9 vaccine, the vaccine containing an inactivated strain of Streptococcus suis SS9-CZ.

[0007] As one implementation, the vaccine contains an inactivated strain of Streptococcus suis SS9-CZ and an aqueous adjuvant.

[0008] Thirdly, the present invention provides a method for preparing the above-mentioned Streptococcus suis type 9 vaccine, comprising mixing an inactivated Streptococcus suis SS9-CZ bacterial suspension with an aqueous adjuvant, stirring evenly, to obtain the vaccine.

[0009] Fourthly, the present invention provides the aforementioned Streptococcus suis type 9 vaccine strain and its application in the preparation of a drug for preventing animal infection with Streptococcus suis.

[0010] Furthermore, the present invention provides the aforementioned Streptococcus suis type 9 vaccine strain and its application in the preparation of a drug for preventing pig infection with Streptococcus suis.

[0011] Preferably, the aforementioned Streptococcus suis includes Streptococcus suis type 9 or Streptococcus suis type 2, and more preferably, highly virulent strains of the aforementioned bacteria.

[0012] Fifthly, the present invention provides the aforementioned Streptococcus suis type 9 vaccine strain and its application in the preparation of drugs for preventing Streptococcus suis-related diseases.

[0013] Furthermore, the diseases associated with Streptococcus suis include meningitis, septicemia, and arthritis.

[0014] Beneficial effects: Compared with existing technologies, the SS9-CZ strain of this invention, isolated from the lung tissue of clinically ill pigs, was serotyped as Streptococcus suis type 9. It grows well in THB medium, exhibits strong pathogenicity in piglets, and demonstrates good immunogenicity, making it a good vaccine strain for Streptococcus suis type 9. Immunization of piglets with the vaccine prepared using the SS9-CZ strain effectively resists attacks from highly virulent Streptococcus suis strains, demonstrating good immunogenicity and providing technical support for the effective prevention of streptococcal disease in pigs. Attached Figure Description

[0015] Figure 1 Gram staining and microscopic examination of Streptococcus suis type 9 vaccine strain SS9-CZ;

[0016] Figure 2 Growth curve determination of Streptococcus suis type 9 vaccine strain SS9-CZ;

[0017] Figure 3 Piglets exhibited visceral pathological changes such as pulmonary congestion and pericardial effusion after being challenged with the SS9-CZ strain of Streptococcus suis vaccine. Detailed Implementation

[0018] To make the present invention easier to understand, specific examples are provided below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Specific experimental methods not mentioned in the following embodiments are generally performed according to conventional experimental methods.

[0019] Healthy weaned piglets aged 3-4 weeks were purchased from a pig farm in Jiangsu Province. The piglets tested negative for both Streptococcus suis antigen and antibody. THB culture medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; 2×Taq PCR Mix and 2000bp DNA Marker were purchased from Nanjing Novizan Biotechnology Co., Ltd.; all other reagents were domestically produced analytical grade.

[0020] Example 1: Isolation and Identification of Streptococcus suis Type 9 Vaccine Strain

[0021] 1. Isolation of Streptococcus suis type 9 vaccine strain SS9-CZ

[0022] Pathological samples were collected from the lungs of acutely deceased pigs at a pig farm in Jiangsu Province. After aseptic treatment of the collected lung tissue, the lung tissue was cut open, and a small amount of alveolar fluid was collected using an inoculation loop and streaked onto THB agar plates. The plates were incubated overnight at 37°C. Small, round, smooth, moist colonies with regular edges (grayish-white) were picked and inoculated onto THB liquid culture medium. Gram-stained microscopic examination of the liquid culture revealed Gram-positive, short-chain, blue-purple cocci under an oil immersion optical microscope (see...). Figure 1 Following the methods described in the references, PCR identification confirmed that the clinically isolated strain was Streptococcus suis type 9. Based on its serotype and collection location, it was named strain SS9-CZ.

[0023] The above-mentioned strain was deposited at the China Center for Type Culture Collection (CCTCC) and named Streptococcus suis SS9-CZ. The deposit date was November 3, 2023, the accession number was CCTCC NO: M20232125, and the deposit address was Wuhan University, Wuhan, China.

[0024] 2. Growth curve determination of Streptococcus suis type 9 vaccine strain SS9-CZ

[0025] Using an inoculation loop, collect the first-generation bacterial suspension of Streptococcus suis type 9 vaccine strain SS9-CZ, streak it onto THB solid medium, and incubate overnight at 37°C to obtain second-generation single colonies. Pick three second-generation single colonies and inoculate them into 5 mL of THB liquid medium, incubate overnight at 37°C with a shaker at 180 rpm to obtain the third-generation bacterial suspension. Take 1 mL of the third-generation bacterial suspension and inoculate it into an Erlenmeyer flask containing 100 mL of THB liquid medium, incubate at 37°C with a shaker at 180 rpm. Take bacterial suspension samples hourly to determine OD. 600nm The values ​​were used to plot a growth curve. The results are as follows: Figure 2 As shown, the OD of SS9-CZ strain after inoculation with THB liquid medium... 600nm The culture time to reach the peak value was 8 hours, and the viable count of SS9-CZ strain at the growth plateau was 4.2 × 10⁻⁶. 9 CFU / mL.

[0026] 3. Virulence detection of Streptococcus suis type 9 vaccine strain SS9-CZ

[0027] Thirty healthy piglets aged 7-8 weeks were randomly divided into 6 groups of 5 piglets each. Groups 1-5 were each injected intravenously via the ear vein with 1 mL of Streptococcus suis serotype 9 bacterial suspension, with a viable bacterial count of 4 × 10⁻⁶ respectively. 6 CFU / mL, 2×10 7 CFU / mL, 1×10 8 CFU / mL, 5×10 8 CFU / mL, 2.5×10 9 CFU / mL. Group 6 was the blank control group, without challenge. Piglets were observed for 14 consecutive days after challenge to assess the pathogenicity of the Streptococcus suis type 9 vaccine strain SS9-CZ to piglets. Results showed that after challenging 7-8 week old piglets with the Streptococcus suis type 9 vaccine strain SS9-CZ via the ear vein, piglets in groups 1-5 exhibited clinical symptoms such as lethargy, reduced feed and water intake, and lameness after challenge with different doses of the Streptococcus suis type 9 vaccine strain SS9-CZ. Necropsy performed 14 days after challenge revealed significant pulmonary congestion, pericardial effusion, and other visceral pathological changes in piglets in groups 1-5. Figure 3 Group 5 (2.5×10) 9 Of the five experimental piglets tested (CFU / mL), three developed severe clinical symptoms on day 4 post-challenge and died on day 5. The other two piglets in group 5 also developed significant clinical symptoms on day 4 post-challenge, which persisted until day 10 post-challenge. These results indicate that the Streptococcus suis type 9 vaccine strain SS9-CZ isolated in this invention has strong virulence.

[0028] Example 2: Immunogenicity detection of Streptococcus suis type 9 vaccine strain SS9-CZ

[0029] Streptococcus suis type 9 vaccine strain SS9-CZ was taken, revived, and a bacterial suspension was prepared. After inactivation, it was mixed with a water-soluble adjuvant at a ratio of 9:1 (V:V) to prepare an inactivated vaccine (containing 1×10⁻⁶ Streptococcus suis type 9 vaccine strain SS9-CZ). 9 Thirty healthy, susceptible piglets aged 2-3 weeks were randomly divided into four groups. Groups 1 and 2, each with 10 piglets, received a 2 mL intramuscular injection of inactivated Streptococcus suis SS9-CZ vaccine (CFU / mL) in the neck. Groups 3 and 4, each with 5 piglets, received a non-immune challenge control group via intramuscular injection of saline in the neck. Three weeks after the initial immunization, a second immunization was administered via intramuscular injection on the opposite side of the neck with the same dose. Two weeks after the second immunization, challenge was initiated. Each piglet in groups 1 and 3 received a 1 mL intravenous injection of Streptococcus suis SS9-CZ bacterial suspension (containing 2.5 × 10⁻⁶ live bacteria) via the marginal ear vein. 9 CFU); In groups 2 and 4, each piglet was injected intravenously via the ear vein with 1 mL of highly virulent Streptococcus suis type 2 ZY05719 (SS2-ZY05719) solution (containing 2 × 10⁻⁶ live bacteria). 7 (CFU). After challenge, the piglets were observed for 14 days. Results are shown in Table 1. In the non-immune challenge control groups (groups 3 and 4), 100% of the experimental piglets developed the disease. The SS9-CZ vaccine strain used to immunize piglets showed 100% immunoprotective efficacy against challenges from homologous virulent strains (group 1, SS9-CZ challenge); and 80% immunoprotective efficacy against challenges from heterologous virulent strains of Streptococcus suis type 2 (group 2, SS2-ZY05719 challenge). This indicates that the SS9-CZ Streptococcus suis type 9 vaccine strain screened in this invention has good immunogenicity.

[0030] Table 1. Immunogenicity detection of Streptococcus suis strain SS9-CZ (Type 9 vaccine).

[0031]

[0032]

[0033] The above embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Although the invention has been described in detail with reference to the embodiments, those skilled in the art should understand that various combinations, modifications, or equivalent substitutions of the technical solutions of the invention do not depart from the spirit and scope of the invention and should be covered within the scope of the claims of the invention.

Claims

1. A Streptococcus suis type 9 vaccine strain, characterized in that, The Streptococcus suis type 9 vaccine strain is deposited at the China Center for Type Culture Collection (CCTCC) and named Streptococcus suis SS9-CZ. The deposit date is November 3, 2023, and the accession number is CCTCC NO: M20232125.

2. A Streptococcus suis type 9 vaccine, characterized in that, The vaccine contains the inactivated strain of Streptococcus suis SS9-CZ as described in claim 1.

3. The Streptococcus suis type 9 vaccine according to claim 2, characterized in that, The vaccine contains an inactivated strain of Streptococcus suis SS9-CZ as described in claim 1 and an aqueous adjuvant.

4. The method for preparing the Streptococcus suis type 9 vaccine according to claim 3, characterized in that, The method involves mixing an inactivated Streptococcus suis SS9-CZ bacterial suspension with an aqueous adjuvant and stirring until homogeneous to obtain the vaccine.

5. The use of the Streptococcus suis type 9 vaccine strain according to claim 1, or the Streptococcus suis type 9 vaccine according to claim 2 or 3, in the preparation of a drug for preventing pig infection with Streptococcus suis.

6. The use of the Streptococcus suis type 9 vaccine strain according to claim 1, or the Streptococcus suis type 9 vaccine according to claim 2 or 3, in the preparation of a medicament for the prevention of Streptococcus suis-related diseases, characterized in that... The diseases associated with Streptococcus suis are meningitis, sepsis, or arthritis.

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