A streptococcus suis disease-porcine contagious pleuropneumonia-blue ear disease triple vaccine and a preparation method thereof

Abstract

The application discloses a Streptococcus suis disease-porcine contagious pleuropneumonia-blue ear disease triple vaccine and a preparation method thereof. The triple vaccine comprises a Streptococcus suis and a porcine contagious pleuropneumonia double inactivated vaccine, and a blue ear live vaccine. The Streptococcus suis, the porcine contagious pleuropneumonia double inactivated vaccine and the blue ear live vaccine are combined to form a combined vaccine, and the Streptococcus suis, the porcine contagious pleuropneumonia double inactivated vaccine and the blue ear live vaccine have a synergistic immune function, so that the immune effect of the combined vaccine can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of multivalent inactivated vaccine technology, specifically relating to a triple vaccine for swine streptococcal disease, swine infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS) and its preparation method. Background Technology

[0002] Streptococcal disease in pigs is an infectious disease caused by Streptococcus suis (SS). Infected pigs exhibit meningitis, arthritis, and septicemia as the main clinical symptoms. Streptococcus suis not only causes significant economic losses to the global pig farming industry but is also a zoonotic pathogen, seriously threatening public health security.

[0003] Porcine contagious pleuropneumonia is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae (APP). It is mainly characterized by pulmonary hemorrhage, necrosis and fibrinous exudation. It is highly contagious and often mixed with other pathogens. It can infect pigs of any age and has caused huge economic losses to the pig industry in countries around the world.

[0004] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Its main characteristics include reproductive disorders in pregnant sows such as abortion, stillbirth, mummified fetuses, and weak piglets, as well as high mortality rates in piglets. Highly pathogenic PRRSV can cause 100% morbidity and over 50% mortality in piglets, and over 30% abortion rate in sows. This virus causes immunosuppression in pigs and makes them susceptible to secondary infections with other pathogens. Current control measures primarily involve immunization and disease eradication, and an increasing number of pig farms are using vaccines for prevention.

[0005] Currently, the market only offers bivalent inactivated vaccines for swine streptococcal disease, swine contagious pleuropneumonia, highly pathogenic swine reproductive and respiratory syndrome (PRRS) live vaccines, PRRS live vaccines, and PRRS inactivated vaccines. There is no trivalent vaccine that can simultaneously prevent all three diseases. Simultaneous prevention of all three diseases requires separate vaccination with each of these vaccines, which can cause repeated stress in pigs during vaccination, potentially affecting the vaccine's effectiveness.

[0006] Therefore, developing a method for preparing a combined inactivated vaccine and a live PRRSV vaccine that provides multiple protections with a single injection, reduces stress in pigs, and has good immunization effects, and can simultaneously prevent streptococcal disease, infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS), is of great practical significance. Summary of the Invention

[0007] To address the aforementioned shortcomings in the existing technology, this invention provides a triple vaccine for swine streptococcal disease, swine infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS) and its preparation method. This invention aims to provide a combined vaccine that offers multiple protections with a single injection, reduces stress response in pigs, and enhances immune efficacy.

[0008] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0009] A triple vaccine for swine streptococcal disease, swine infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS), comprising a bivalent inactivated vaccine for streptococcal disease and swine infectious pleuropneumonia, and a live PRRS vaccine.

[0010] Furthermore, the live bacteria count of the inactivated swine streptococcus vaccine before inactivation is not less than 1–3 × 10⁻⁶. 9 CFU / dose; the live bacteria count of the porcine infectious pleuropneumonia inactivated vaccine before inactivation is not less than 1–3 × 10⁻⁶. 9 CFU / dose; the viral load of each dose of the porcine reproductive and respiratory syndrome (PRRS) live vaccine is not less than 10 CFU / dose. 5 TCID 50 .

[0011] Furthermore, the live bacteria count of the inactivated swine streptococcus vaccine before inactivation is not less than 1×10⁻⁶. 9 CFU / dose; the live bacteria count of the porcine infectious pleuropneumonia inactivated vaccine before inactivation is not less than 1×10⁻⁶. 9 CFU / dose; each dose of the live PRRSV vaccine contains no less than 10 CFU / dose. 5 TCID 50 .

[0012] Furthermore, the trivalent vaccine also includes pharmaceutically acceptable adjuvants.

[0013] The above-mentioned method for the triple vaccine against swine streptococcal disease, swine infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS) involves diluting one dose of live PRRS vaccine with one dose of inactivated swine streptococcal and swine infectious pleuropneumonia bivalent vaccine.

[0014] Furthermore, the preparation process of the bivalent inactivated vaccine against Streptococcus suis and porcine contagious pleuropneumonia is as follows:

[0015] (1) Preparation of Streptococcus suis antigen

[0016] S1. Preparation of primary production seeds

[0017] Streptococcus suis strain ZY-2 was inoculated into a solid culture medium and cultured at 36.5–37.5°C for 20–24 hours. Then it was inoculated into a solid agar slant and cultured at 36.5–37.5°C for another 18–24 hours to obtain the first-grade production seed.

[0018] S2, Preparation of secondary production seeds

[0019] The primary production seeds are inoculated into liquid culture medium at an inoculation rate of 1% to 2%, and cultured in a shaker at 36.5 to 37.5°C for 50 to 60 r / min for 10 to 12 hours to obtain secondary production seeds;

[0020] S3, bacterial culture

[0021] Inoculate the secondary production seed culture solution into the liquid culture medium at an inoculation rate of 2% to 5%, and incubate at 36.5 to 37.5°C for 10 to 12 hours;

[0022] S4, Inactivation

[0023] At 37±0.2℃, while stirring, formaldehyde solution accounting for 0.3% of the total volume of the bacterial solution was added, and the inactivation treatment was carried out for 48 hours to obtain the inactivated solution.

[0024] S5, Concentrated Formaldehyde Removal

[0025] Centrifuge the inactivation solution obtained in step S4, wash and filter it with PBS several times, concentrate and resuspend it to obtain streptococcal antigen.

[0026] (2) Preparation of porcine infectious pleuropneumonia antigen

[0027] S1. Preparation of primary production seeds

[0028] The SC strain of Actinobacillus pleuropneumoniae was inoculated into a solid culture medium and cultured at 36.5–37.5°C for 24 hours. Then it was inoculated into a solid agar slant and cultured at 36.5–37.5°C for 16–18 hours to obtain the first-grade production seed.

[0029] S2, Preparation of secondary production seeds

[0030] The primary production seeds were inoculated into liquid culture medium at an inoculation rate of 1% to 2% and cultured at 36.5 to 37.5°C for 16 to 18 hours to obtain secondary production seeds.

[0031] S3, bacterial culture

[0032] Inoculate the secondary production seed culture solution into the liquid culture medium at an inoculation rate of 2% to 3% and incubate at 36.5 to 37.5°C for 16 to 18 hours;

[0033] S4, Inactivation

[0034] At 37±0.2℃, while stirring, formaldehyde solution accounting for 0.3% of the total volume of the bacterial solution was added, and the inactivation treatment was carried out for 48 hours to obtain the inactivated solution.

[0035] S5, Concentrated Formaldehyde Removal

[0036] Centrifuge the inactivated solution obtained in step S4, wash and filter it with PBS several times, concentrate and resuspend it to obtain porcine infectious pleuropneumonia antigen.

[0037] (3) Preparation of a bivalent inactivated vaccine against Streptococcus suis and porcine infectious pleuropneumonia.

[0038] After mixing equal amounts of Streptococcus suis antigen and Actinobacillus pleuropneumoniae antigen, aluminum hydroxide glue is added and the mixture is stirred thoroughly to obtain a bivalent inactivated vaccine against Streptococcus suis and infectious pleuropneumoniae.

[0039] Furthermore, in step (3), the weight percentage of aluminum hydroxide gel is 5% of the mixed antigen.

[0040] The beneficial effects of this invention are:

[0041] 1. The present invention obtains bacterial antigens by first inactivating, then concentrating, and repeatedly washing, filtering and resuspending, which can effectively remove formaldehyde from inactivated vaccines to avoid the impact of formaldehyde on live porcine reproductive and respiratory syndrome (PRRS) vaccines.

[0042] 2. A combined vaccine is formed by using a bivalent inactivated vaccine of Streptococcus suis and infectious pleuropneumonia and a live PRRSV vaccine. The bivalent inactivated vaccine of Streptococcus suis and infectious pleuropneumonia and the live PRRSV vaccine have synergistic immune function, which can effectively improve the immune effect of the combined vaccine.

[0043] 3. Combination vaccines can simplify the vaccination process, save manpower and resources, reduce the number of vaccinations, reduce the chance of adverse reactions, and improve the immunization effect. Detailed Implementation

[0044] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0045] Example 1: Preparation of Combined Vaccine

[0046] 1. Preparation of Streptococcus suis antigen

[0047] (1) Preparation of primary production seed: Unseal the freeze-dried strain ZY-2 of Streptococcus suis, inoculate it into 10% sheep blood Martin agar plates, and incubate at 36.5-37.5℃ for 20-24 hours. Take 10 typical colonies of uniform size and inoculate them into 10% sheep blood Martin agar slants. Incubate at 36.5-37.5℃ for 18-24 hours. These are used as primary seeds and stored at 2-8℃ for no more than 30 days. The number of generations should not exceed 4.

[0048] (2) Preparation of secondary production seeds: Wash the primary seeds off the Martin agar slant with streptococcal culture medium, and inoculate them into pig streptococcal culture medium containing 4% bovine serum and 0.2% glucose at an inoculation rate of 1% to 2%. Incubate at 36.5 to 37.5°C on a shaker at 50 to 60 r / min for 10 to 12 hours to obtain secondary seeds. Store at 2 to 8°C for no more than 4 days.

[0049] (3) Bacterial culture: The secondary seed bacterial culture was inoculated into a Streptococcus suis culture medium containing 4% bovine serum and 0.2% glucose at an inoculation rate of 2% to 5%, and cultured at 36.5 to 37.5°C for 10 to 12 hours to obtain the bacterial culture.

[0050] (4) Inactivation: The bacterial solution obtained in step (3) is subjected to inactivation treatment for 48 hours at 37±0.2℃ with the addition of 0.3% of the total bacterial solution in formaldehyde solution while stirring, to obtain the inactivation solution.

[0051] (5) Concentration and formaldehyde removal: Centrifuge the inactivation solution obtained in step (4), wash and filter with PBS several times, and then concentrate and resuspend. This yields the Streptococcus suis antigen.

[0052] 2. Preparation of Actinobacillus pleuropneumoniae antigen

[0053] (1) Preparation of primary production seed: Take the freeze-dried strain of Actinobacillus pleuropneumoniae SC and inoculate it into a TSA plate containing 0.1% NAD and 4% newborn calf serum. Incubate at 36.5-37.5℃ for 24 hours. Select 10 typical colonies of uniform size and inoculate them into several TSA slant plates containing 0.1% NAD and 4% newborn calf serum. Incubate at 36.5-37.5℃ for 16-18 hours. This is the primary seed. It should not exceed 7 days and should not be passaged more than 3 times.

[0054] (2) Preparation of secondary production seeds: Wash the primary seeds off the TSA slant with TSB, and inoculate them at an inoculation rate of 1% to 2% into trypsin soybean broth (TSB) medium containing 0.1% NAD and 4% newborn calf serum. Incubate at 36.5 to 37.5°C for 16 to 18 hours as secondary seeds. The inoculation period should not exceed 7 days.

[0055] (3) Culture of bacterial suspension: Inoculate the secondary seed bacterial suspension with 0.1% NAD and 4% newborn calf serum at an inoculation rate of 2% to 3% and culture at 36.5 to 37.5°C for 16 to 18 hours to obtain bacterial suspension.

[0056] (4) Inactivation: The bacterial solution obtained in step (3) is subjected to inactivation treatment for 24 hours at 37±0.2℃ with 0.3% of the total bacterial solution in formaldehyde solution while stirring, to obtain the inactivation solution.

[0057] (5) Concentration and formaldehyde removal: Centrifuge the inactivated solution obtained in step (4), wash and filter with PBS several times, then concentrate and resuspend. Porcine Actinobacillus pleuropneumoniae antigen is obtained.

[0058] 3. Preparation of a bivalent inactivated vaccine against Streptococcus suis and infectious pleuropneumoniae.

[0059] After mixing equal amounts of Streptococcus suis antigen and Actinobacillus pleuropneumoniae antigen, 5% aluminum hydroxide gel by their respective weights is added, and the mixture is stirred thoroughly to obtain a bivalent inactivated vaccine against Streptococcus suis and infectious pleuropneumoniae.

[0060] 4. Preparation of combination vaccines

[0061] (1) Live PRRSV vaccine: Highly pathogenic porcine reproductive and respiratory syndrome live vaccine, produced by our company (batch number: 2022005, virus content per dose is 10). 6.6 TCID 50 ).

[0062] (2) Dilute one dose of live porcine reproductive and respiratory syndrome (PRRS) vaccine with one dose of inactivated Streptococcus suis and infectious pleuropneumonia (IPP) bivalent vaccine; wherein the number of live Streptococcus suis bacteria before inactivation in the inactivated Streptococcus suis and IPP bivalent vaccine shall not be less than 1.0 × 10⁻⁶. 9 CFU / dose, the viable count of Actinobacillus pleuropneumoniae before inactivation is not less than 1.0 × 10⁻⁶. 9 CFU / dose, highly pathogenic porcine reproductive and respiratory syndrome live vaccine, with a viral load of not less than 10 CFU / dose. 5.0 TCID 50 .

[0063] Example 2: Safety and efficacy trial of the combination vaccine

[0064] The combined vaccines were prepared according to the proportions shown in Table 1, and were used as controls with a bivalent inactivated vaccine against Streptococcus suis, a bivalent inactivated vaccine against infectious pleuropneumonia, and a live vaccine against highly pathogenic porcine reproductive and respiratory syndrome, respectively, and subsequent experiments were conducted.

[0065] Table 1 Combination Vaccines

[0066]

[0067] 1. Safety inspection

[0068] Twenty-five healthy, susceptible pigs aged 28–35 days were divided into five groups of five each. Each group received four doses of the combination vaccine per pig. During the 14-day observation period following vaccination, all pigs exhibited normal clinical signs, including mental state, respiration, and appetite. No inflammation, swelling, or lumps were observed at the injection sites upon palpation, and all pigs remained healthy. The results indicate that the safety of the combination vaccine was not affected by different proportions (see Table 2).

[0069] Table 2. Safety test results of vaccines with different proportions.

[0070] 1 5 / 5 Healthy Living 2 5 / 5 Healthy Living 3 5 / 5 Healthy Living Swine streptococcus and infectious pleuropneumonia bivalent inactivated vaccine 5 / 5 Healthy Living Highly pathogenic porcine reproductive and respiratory syndrome live vaccine 5 / 5 Healthy Living

[0071] 2. Validity test

[0072] (1) Streptococcus suis

[0073] Thirty healthy, susceptible pigs aged 28–35 days were divided into six groups of five each. One group served as the control group, while the immunized groups were each vaccinated with one dose of the combined vaccine. These pigs were then fed together with the five control pigs. Twenty-one days after immunization, the pigs were challenged with 2.0 mL of Streptococcus suis ZY-2 strain. After 14 days of observation following the challenge, 5 / 5 of the pigs in the highly pathogenic porcine reproductive and respiratory syndrome live vaccine group and the control group died, while 5 / 5 of the remaining four groups survived (see Table 3). This demonstrates that different proportions of the combined vaccine did not affect its effectiveness against Streptococcus suis.

[0074] (2) Actinobacillus pleuropneumoniae in pigs

[0075] Thirty healthy, susceptible pigs aged 28–35 days were divided into six groups of five each. One group served as the control group, while the immunized groups were each vaccinated with one dose of the combined vaccine. These pigs were then fed together with the five control pigs. Twenty-one days after immunization, the pigs were challenged with 2.0 mL of Actinobacillus pleuropneumoniae SC strain. After 14 days of observation following the challenge, 5 out of 5 pigs in the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) live vaccine group and the control group developed the disease, while 5 out of 5 pigs in the other four groups remained healthy (see Table 3). This demonstrates that different proportions of the combined vaccine did not affect the effectiveness against Actinobacillus pleuropneumoniae.

[0076] Table 3. Results of the efficacy of different ratios of combined vaccines against viral challenge.

[0077]

[0078] (3) Highly pathogenic porcine reproductive and respiratory syndrome virus

[0079] Take one dose of the combination vaccine from each group, and serially dilute it 10-fold with cell maintenance medium. -3 10 -4 10 -5 10 -6 10 -7 Five dilutions were seeded onto 96-well cell culture plates containing a monolayer of well-grown Marc-145 cells, with 8 wells seeded per titer. The plates were incubated at 37°C with 5% CO2 for 5–7 days. Cytopathic effects were observed, and TCID was calculated. 50 / Toufen.

[0080] The results showed that no PRRSV virus was detected in the bivalent inactivated vaccine group containing Streptococcus suis and contagious pleuropneumoniae, while the virus content of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) was higher than 10 in the other four vaccine groups. 5.0 TCID 50 / dosage. Different ratios of combined vaccines did not affect the efficacy of the batch of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) live vaccine. The viral load of PRRS in combined vaccine 3 was lower than that in the live PRRS vaccine; therefore, subsequent trials were conducted only on the ratios of combined vaccine 1 and combined vaccine 2.

[0081] Table 4. Results of the efficacy of different ratios of combination vaccines against PRRSV.

[0082]

[0083] Example 3: Shelf life test of combined vaccines

[0084] Considering that the combination vaccine needs to be kept at room temperature for a certain period of time during the vaccination process in pig farms, the combination vaccine was prepared according to the ratio of combination vaccine 1 and combination vaccine 2 in Table 1 of Example 2. At the same time, the storage period test was carried out with different storage times using streptococcus suis, infectious pleuropneumonia bivalent inactivated vaccine and highly pathogenic porcine reproductive and respiratory syndrome live vaccine as controls (storage time is shown in Table 5).

[0085] 1. Safety inspection

[0086] For each storage period: 20 healthy, susceptible pigs aged 28–35 days were divided into 4 groups of 5 pigs each. Each group was vaccinated with 4 doses of the combination vaccine per pig and observed for 14 days. During the 14-day observation period after vaccination, all pigs showed normal clinical behavior, and no abnormal changes such as inflammation, swelling, or lumps were observed upon palpation of the injection site. All pigs were healthy and alive (Table 5). This demonstrates that the safety of the combination vaccine with different proportions was not affected after storage for different periods.

[0087] Table 5. Vaccine safety test results after different ratio combinations were placed for different times.

[0088]

[0089] 2. Validity test

[0090] (1) Streptococcus suis

[0091] For each storage period: 25 healthy, susceptible pigs aged 28–35 days were divided into 5 groups of 5 pigs each, with one group serving as the control group. Each immunized pig in the immunization group received one dose of the combined vaccine. These pigs, along with the 10 control pigs, were fed together. 21 days after immunization, pigs were challenged with 2.0 mL of Streptococcus suis ZY-2 strain. They were observed for 14 days post-challenge. In the group challenged with the combined vaccines at different storage times, 5 / 5 of the pigs in the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) live vaccine group and the control group died. In contrast, 5 / 5 of the pigs in the combined vaccine 1 and combined vaccine 2 groups survived (Table 6-10). This indicates that storage time did not affect the efficacy of the combined vaccines against Streptococcus suis.

[0092] (2) Actinobacillus pleuropneumoniae in pigs

[0093] For each storage period: 25 healthy, susceptible pigs aged 28–35 days were divided into 5 groups of 5 pigs each, with one group serving as a control group. Each immunized group received one dose of the combined vaccine. These pigs, along with the 10 control pigs, were fed together. 21 days after immunization, pigs were challenged with 2.0 mL of *Actinobacillus pleuropneumoniae* SC strain. They were observed for 14 days post-challenge. In the pigs challenged with *Streptococcus suis* using the combined vaccines stored for different durations, 5 / 5 of the pigs in the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) live vaccine group and the control group died. In contrast, 5 / 5 of the pigs in the combined vaccine 1 and combined vaccine 2 groups stored for different durations survived (Table 6-10). This indicates that storage time did not affect the effectiveness of the combined vaccines against *Actinobacillus pleuropneumoniae*.

[0094] Table 6. Results of the efficacy of different ratio combination vaccines against viral challenge after 0 hours.

[0095]

[0096] Table 7. Results of the effectiveness of different ratio combination vaccines against viral challenge 1 hour after placement.

[0097]

[0098]

[0099] Table 8. Results of the efficacy of different ratio combination vaccines against viral challenge after 2 hours.

[0100]

[0101]

[0102] Table 9. Results of the efficacy of different ratio combination vaccines against viral challenge 4 hours after administration.

[0103]

[0104] Table 10 Results of the efficacy of different ratio combination vaccines against challenge after 8 hours.

[0105]

[0106]

[0107] (3) Highly pathogenic porcine reproductive and respiratory syndrome virus

[0108] For each storage period, take one dose of the combination vaccine from each group, and serially dilute one dose of vaccine 10-fold with cell maintenance medium. Take 10 doses of the combination vaccine. -3 10 -4 10 -5 10 -6 10 -7 Five dilutions were seeded onto 96-well cell culture plates containing a monolayer of well-grown Marc-145 cells, with 8 wells seeded per titer. The plates were incubated at 37°C with 5% CO2 for 5–7 days. Cytopathic effects were observed, and TCID was calculated. 50 / Toufen.

[0109] The results showed that no highly pathogenic porcine reproductive and respiratory syndrome (PRRS) virus was detected in the bivalent inactivated vaccine group containing Streptococcus suis and contagious pleuropneumoniae. However, the other four vaccine groups showed PRRS virus levels exceeding 10 after different storage times. 5.0 TCID 50 / Head portion (Table 11).

[0110] As the storage time increased, the viral load of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) live vaccine decreased. However, when the storage time was the same, the viral load of the PRRS live vaccines in combination vaccines 1 and 2 was higher than that in combination vaccines 2. This indicates that the combination of the bivalent inactivated vaccine against streptococcus suis and contagious pleuropneumonia with the PRRS live vaccine can delay the decrease in the viral load of the PRRS live vaccine and effectively improve the immunization effect of the combination vaccine.

[0111] Table 11 Results of the efficacy of different ratio combination vaccines against highly pathogenic porcine reproductive and respiratory syndrome.

[0112]

[0113]

[0114] Both combination vaccines 1 and 2 showed that the decrease in viral load of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) live vaccine was delayed after 8 hours of storage without affecting the efficacy against Streptococcus suis and Actinobacillus pleuropneumoniae. Considering the proportion of live vaccines (Streptococcus suis, pleuropneumoniae bivalent inactivated vaccine, and highly pathogenic PRRS live vaccine) in combination vaccines 1 and 2, the final preparation method for the combination vaccines was determined as follows: one dose of highly pathogenic PRRS live vaccine was diluted with one dose of Streptococcus suis and pleuropneumoniae bivalent inactivated vaccine.

[0115] Example 4: Shelf life test of live combination vaccine for porcine reproductive and respiratory syndrome.

[0116] 1. Preparation of combination vaccines

[0117] (1) Live PRRSV vaccine: Live vaccine for porcine reproductive and respiratory syndrome, produced by our company (batch number: 2022005, virus content per dose is 10). 5.6 TCID 50 ).

[0118] (2) Dilute one dose of live porcine reproductive and respiratory syndrome (PRRS) vaccine with one dose of the bivalent inactivated Streptococcus suis and infectious pleuropneumoniae vaccine prepared in Example 1; wherein the number of live Streptococcus suis bacteria in the bivalent inactivated Streptococcus suis and infectious pleuropneumoniae vaccine before inactivation is not less than 1.0 × 10⁻⁶. 9 CFU / dose, the viable count of Actinobacillus pleuropneumoniae before inactivation is not less than 1.0 × 10⁻⁶. 9 CFU / dose, highly pathogenic porcine reproductive and respiratory syndrome live vaccine, with a viral load of not less than 10 CFU / dose. 5.0 TCID 50 .

[0119] 2. Safety Inspection

[0120] For each storage period: 15 healthy, susceptible pigs aged 28–35 days were divided into 3 groups of 5 pigs each. Each group was vaccinated with 4 doses of the combination vaccine per pig and observed for 14 days. During the 14-day observation period after vaccination, all pigs showed normal clinical behavior, and no abnormal changes such as inflammation, swelling, or lumps were observed upon palpation of the injection site. All pigs were healthy and alive (Table 12). This demonstrates that the safety of the combination vaccine was not affected by different proportions after storage for different periods.

[0121] Table 12. Results of vaccine safety testing after different storage times for the combined vaccine.

[0122]

[0123] 3. Validity test

[0124] (1) Streptococcus suis

[0125] For each storage period: 20 healthy, susceptible pigs aged 28–35 days were divided into 4 groups of 5 pigs each, with one group serving as a control group. Each immunized pig in the immunization group received one dose of the combined vaccine. These pigs, along with the 10 control pigs, were fed together. 21 days after immunization, pigs were challenged with 2.0 mL of Streptococcus suis ZY-2 strain. They were observed for 14 days post-challenge. Of the pigs challenged with the combined vaccine at different storage times, 5 / 5 died in the live vaccine group and the control group, while 5 / 5 survived in the combined vaccine group (Tables 13-17). Storage time did not affect the efficacy of the combined vaccine against Streptococcus suis.

[0126] (2) Actinobacillus pleuropneumoniae in pigs

[0127] For each storage period: 20 healthy, susceptible pigs aged 28–35 days were divided into 4 groups of 5 pigs each, with one group serving as a control group. Each immunized pig in the immunization group received one dose of the combined vaccine. These pigs, along with the 10 control pigs, were fed together. 21 days after immunization, the pigs were challenged with 2.0 mL of *Actinobacillus pleuropneumoniae* SC strain. They were observed for 14 days post-challenge. Of the pigs challenged with *Actinobacillus pleuropneumoniae*, 5 / 5 in the live vaccine group and the control group developed the disease, while 5 / 5 in the combined vaccine group remained viable (Tables 13-17). This indicates that storage time did not affect the effectiveness of the combined vaccine against *Actinobacillus pleuropneumoniae*.

[0128] Table 13 Results of the efficacy of the combined vaccine against challenge after 0 hours.

[0129]

[0130] Table 14 Results of the efficacy of the combined vaccine against challenge 1 hour after placement.

[0131]

[0132] Table 15 Results of the efficacy of the combined vaccine against challenge 2 hours after placement.

[0133]

[0134]

[0135] Table 16 Results of the efficacy of the combination vaccine against challenge 4 hours after administration.

[0136]

[0137] Table 17 Results of the efficacy of the combined vaccine against challenge 8 hours after administration.

[0138]

[0139] (3) Porcine reproductive and respiratory syndrome virus

[0140] For each storage period, take one dose of the combination vaccine from each group, and serially dilute one dose of vaccine 10-fold with cell maintenance medium. Take 10 doses of the combination vaccine. -3 10 -4 10 -5 10 -6 10 -7 Five dilutions were seeded onto 96-well cell culture plates containing a monolayer of well-grown Marc-145 cells, with 8 wells seeded per titer. The plates were incubated at 37°C with 5% CO2 for 5–7 days. Cytopathic effects were observed, and TCID was calculated. 50 / Toufen.

[0141] The results showed that no porcine reproductive and respiratory syndrome (PRRS) virus was detected in the combined inactivated Streptococcus suis and contagious pleuropneumoniae vaccine group, while the PRRS virus content of the other vaccines was higher than 10 after different storage times. 5.0 TCID 50 / Toufen.

[0142] As the storage time increased, the viral load of the live porcine reproductive and respiratory syndrome (PRRS) vaccine decreased. Under the same storage time, the viral load of the combined vaccine was higher than that of the live porcine PRRS vaccine. This indicates that the combination of the bivalent inactivated vaccine against streptococcus and contagious pleuropneumonia with the live porcine PRRS vaccine can delay the decrease in the viral load of the live porcine PRRS vaccine and effectively improve the immunization effect of the combined vaccine.

[0143] Table 18 Results of the efficacy of combined vaccines against porcine reproductive and respiratory syndrome.

[0144]

[0145] In summary, the combination vaccine is prepared by diluting one dose of swine streptococcus and infectious pleuropneumonia bivalent inactivated vaccine with one dose of porcine reproductive and respiratory syndrome (PRRS) live vaccine. It can be used as a live vaccine for highly pathogenic porcine reproductive and respiratory syndrome (PRRS).

Claims

1. A triple vaccine for swine streptococcal disease, swine infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS), characterized in that, The trivalent vaccine is made by diluting one dose of Streptococcus suis and a bivalent inactivated vaccine for porcine infectious pleuropneumonia with one or two doses of live porcine reproductive and respiratory syndrome (PRRS) vaccine. The inactivated streptococcal vaccine of pigs has a live bacteria count of not less than 1×10⁻⁶ before inactivation. 9 CFU / dose; the live bacteria count of the porcine infectious pleuropneumonia inactivated vaccine before inactivation is not less than 1×10⁻⁶. 9 CFU / dose; the viral load of each dose of the porcine reproductive and respiratory syndrome (PRRS) live vaccine is not less than 10 CFU / dose. 5 TCID 50 .

2. The triple vaccine for swine streptococcal disease, swine infectious pleuropneumonia, and porcine reproductive and respiratory syndrome (PRRS) according to claim 1, characterized in that, The trivalent vaccine also includes a pharmaceutically acceptable adjuvant.

3. A method for preparing the porcine streptococcal disease-porcine infectious pleuropneumonia-PRRS trivalent vaccine as described in claim 1 or 2, characterized in that, It is made by diluting one or two doses of live porcine reproductive and respiratory syndrome (PRRS) vaccine with a bivalent inactivated vaccine of Streptococcus suis and porcine infectious pleuropneumonia.

4. The method according to claim 3, characterized in that, The preparation process of the bivalent inactivated vaccine against Streptococcus suis and porcine contagious pleuropneumonia is as follows: (1) Preparation of Streptococcus suis antigen S1. Preparation of primary production seeds Streptococcus suis strain ZY-2 was inoculated into a solid culture medium and cultured at 36.5-37.5℃ for 20-24 hours. Then it was inoculated into a solid agar slant and cultured at 36.5-37.5℃ for another 18-24 hours to obtain the first-grade production seed. S2, Preparation of secondary production seeds The primary production seeds are inoculated into liquid culture medium at an inoculation rate of 1% to 2%, and cultured in a shaker at 36.5 to 37.5°C for 50 to 60 r / min for 10 to 12 hours to obtain the secondary production seeds; S3, bacterial culture Inoculate the secondary production seed culture solution into the liquid culture medium at an inoculation rate of 2% to 5%, and incubate at 36.5 to 37.5°C for 10 to 12 hours; S4. Inactivate the cultured bacterial solution with formaldehyde, and then remove the formaldehyde to obtain the streptococcal antigen. (2) Preparation of porcine infectious pleuropneumonia antigen S1. Preparation of primary production seeds The SC strain of Actinobacillus pleuropneumoniae was inoculated into a solid culture medium and cultured at 36.5-37.5℃ for 24 hours. Then it was inoculated into a solid agar slant and cultured at 36.5-37.5℃ for 16-18 hours to obtain the first-grade production seed. S2, Preparation of secondary production seeds The primary production seeds were inoculated into liquid culture medium at an inoculation rate of 1% to 2% and cultured at 36.5 to 37.5°C for 16 to 18 hours to obtain secondary production seeds. S3, bacterial culture Inoculate the secondary production seed culture solution into the liquid culture medium at an inoculation rate of 2% to 3%, and incubate at 36.5 to 37.5°C for 16 to 18 hours; S4. Inactivate the cultured bacterial solution with formaldehyde, and then remove the formaldehyde to obtain the Actinobacillus pleuropneumoniae antigen. (3) Preparation of a bivalent inactivated vaccine against Streptococcus suis and porcine infectious pleuropneumonia. After mixing equal amounts of Streptococcus suis antigen and Actinobacillus pleuropneumoniae antigen, aluminum hydroxide glue is added and the mixture is stirred thoroughly to obtain a bivalent inactivated vaccine against Streptococcus suis and infectious pleuropneumoniae.

5. The method according to claim 4, characterized in that, In step (3), the weight percentage of aluminum hydroxide gel is 5% of the mixed antigen.

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