Chicken mycoplasma synoviae, live vaccine and preparation method and application thereof

Abstract

The invention provides chicken mycoplasma synoviae, an obtained live vaccine as well as a preparation method and application of the live vaccine, and belongs to the technical field of veterinary vaccines. The chicken mycoplasma synoviae is a chicken mycoplasma synoviae MS 7 strain, and is preserved in the China Center for Type Culture Collection on July 10, 2024, and the preservation number of the chicken mycoplasma synoviae is CCTCC M 20241533. The live vaccine prepared from the mycoplasma synoviae MS7 strain is lower in cost, more suitable for industrial production, convenient to store and transport, simple in use method and beneficial to large-scale popularization and application, and compared with an attenuated vaccine and a commercial vaccine MS-H prepared after the existing MSHLB-200 strain and white oil for injection are fully emulsified, the live vaccine is convenient to store and transport and ideal in immune effect.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to a Mycoplasma synoviae, a live vaccine obtained, and a preparation method and application thereof. Background Art

[0002] Mycoplasma synoviae (hereinafter referred to as "MS") can cause subclinical respiratory diseases in chickens and other poultry, swelling of foot pads or joints, decreased egg production, eggshell quality and feed-to-meat ratio, and has the ability to spread horizontally and vertically. Horizontal transmission is mainly through the respiratory tract, and latently infected chickens and sick chickens and their secretions and excrement are the main sources of infection of the disease; vertical transmission is through eggs or semen, and infected eggs hatch chicks carrying MS, which will cause more chickens to be infected through horizontal transmission. Once infected, they will carry the bacteria for life. This is one of the important reasons for the high incidence and wide distribution of MS, which has seriously affected the economic benefits of my country's poultry industry.

[0003] MS has a high prevalence in domestic chickens, including broilers, laying hens, breeders, and local yellow chickens. The high-density intensive breeding model is also conducive to the horizontal spread of MS. In addition, breeders infected with MS are prone to vertical transmission, which increases the difficulty of MS prevention and control. The prevention and control of MS disease at home and abroad is still in the exploratory stage. At present, the resistance of MS to commonly used bacterial antibiotics is increasing year by year. It is increasingly difficult to rely solely on antibiotics to prevent and control MS infection in commercial broilers and laying hens. Vaccines play a key role in the prevention and control of livestock and poultry infectious diseases. At present, only the chicken synoviae mycoplasma inactivated vaccine (YBF-MS1 strain) independently developed by Qingdao Yibang Bioengineering Co., Ltd. and the chicken synoviae mycoplasma live vaccine (MS-H strain) developed by Australian Bioresources have been registered in China and licensed for sale. Inactivated vaccines can provide good humoral immunity, but cannot block infection, so they are not enough to completely control the epidemic of the disease; domestic and foreign studies have shown that the live vaccine of Mycoplasma synoviae (MS-H strain) can prevent and alleviate clinical symptoms, reduce the degree of MS infection and horizontal transmission by eye drops in MS-negative chickens, but the vaccine is expensive and can only be stored below -70°C. The storage cost is extremely high, which is not conducive to large-scale promotion and use in the clinical front line. Therefore, it is urgent to develop a safe and effective commercial live vaccine that is easy to store and transport and can be promoted on a large scale. Summary of the invention

[0004] The invention provides a Mycoplasma gallinarum, a live vaccine obtained therefrom, and a preparation method and application thereof. The Mycoplasma gallinarum MS 7 strain is made into a live vaccine with lower cost, is more suitable for industrial production, is convenient for storage and transportation, has a simple use method, and is conducive to large-scale promotion and use. Compared with the existing attenuated vaccine prepared by fully emulsifying the MSHLB-200 strain and white oil for injection and the commercial vaccine MS-H, the live vaccine is convenient for storage and transportation and has an ideal immune effect.

[0005] In order to achieve the above-mentioned object, the present invention provides a Mycoplasma gallinarum, which is the Mycoplasma gallinarum MS 7 strain, which was deposited in the China Center for Type Culture Collection on July 10, 2024, and its deposit number is CCTCCM 20241533.

[0006] The present invention provides a use of the Mycoplasma gallinarum according to the above technical solution in the preparation of a live Mycoplasma gallinarum vaccine.

[0007] The present invention provides a live vaccine of Mycoplasma gallinarum, which is prepared by using the Mycoplasma gallinarum described in the above technical solution.

[0008] Preferably, the Mycoplasma gallinarum live vaccine is prepared by fully mixing the Mycoplasma gallinarum bacterial liquid with a lyophilization protective agent in a volume ratio of 3:1 and then freeze-drying.

[0009] Preferably, the lyophilization protective agent is a gelatin composite stabilizer, and the gelatin composite stabilizer consists of 2% gelatin and 10% sucrose in terms of mass percentage.

[0010] Preferably, the freeze-drying conditions meet the following procedures:

[0011] The pre-freezing temperature is -40°C and the pre-freezing time is 4 to 5 hours;

[0012] The sublimation temperature is -10 to -12°C and the sublimation time is 24 to 26 hours;

[0013] The desorption temperature was 27°C and maintained for 12 hours.

[0014] Preferably, the Mycoplasma gallinarum bacterial liquid is prepared by the following method:

[0015] The Mycoplasma synoviae MS 7 strain was inoculated into the modified Frey's liquid medium at 5% of the volume of the medium, and cultured at 37°C and 100 r / min until the pH value of the medium dropped to 6.7-6.9. After the microscopic examination was qualified, the strains were harvested and used as the first-level seeds. The method was then used to expand the culture step by step to the fourth-level seeds.

[0016] The culture medium was added at 60% to 80% of the volume of the fermentation tank, and the production seed liquid was inoculated at 5% of the volume of the culture medium. The culture was stirred at 37° C. and the bacterial liquid of Mycoplasma synoviae MS 7 strain was harvested when the pH value of the culture medium dropped to 6.7 to 6.9.

[0017] Preferably, the number of live bacteria of the MS 7 strain in the Mycoplasma synoviae live vaccine is 10 9.0 CCU 50 / ml or above.

[0018] As a preference, the Mycoplasma synoviae live vaccine is 6.5 CCU 50 / Feather concentration, the protection efficiency of 0.03ml immunization method through eye drops reached more than 75%.

[0019] Compared with the prior art, the advantages and positive effects of the present invention are:

[0020] 1. The present invention is to recover the MSHLB-95, MSHLB-120, MSHLB-160, and MSHLB-200 strains (numbered H95, H120, H160, and H200, respectively) separated and purified from the laboratory and stored at -80°C to a modified Frey solid plate, select monoclones with uniform size, "fried egg shape", and typical mycoplasma morphology in a modified Frey liquid medium, and measure the growth curves of different clones of different generations by the method of viable bacteria counting, and then preliminarily screen out clones with weaker pathogenicity by a chicken embryo infection test of Mycoplasma gallinarum as candidate strains for live vaccine of Mycoplasma gallinarum;

[0021] After the culture was expanded, freeze-drying protective agents were added and then freeze-dried. The properties, purity test, live bacteria count, safety test and efficacy test were carried out. It was found that the freeze-drying loss of MS 7 strain was small after freeze-drying, and it was safe for SPF chickens after eye drop immunization, and the immunization effect was ideal.

[0022] The MS 7 strain was made into a vaccine and stored at 2-8°C for one year. The vaccine was then verified to be qualified using the above method, indicating that the production process is reliable. At the same time, the test results confirmed that the live vaccine produced by this production process is low-cost, easy to use, safe, effective, and has stable and controllable quality.

[0023] 2. Although the attenuated vaccine prepared by fully emulsifying the MSHLB-200 strain obtained by in vitro passage of MSHLB-95 in CN117946928A with white oil for injection has a good immune effect, it is only a laboratory research product. Because it needs to add white oil for emulsification, it is inconvenient to use, has high production cost, and cannot be stored for a long time. Therefore, it cannot be used as a commercial vaccine for large-scale promotion and use. Compared with it, the live vaccine obtained in this application has lower cost, is more suitable for industrial production, is easy to store and transport, has a simple method of use, and has an ideal immune effect, which is conducive to large-scale promotion and use. Compared with the commercial vaccine MS-H, it still has the advantages of easy storage and transportation, and ideal immune effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The growth conditions of MSHLB-95, MSHLB-120, MSHLB-160 and MSHLB-200 on solid plates, where A: MSHLB-95 group; B: MSHLB-120 group; C: MSHLB-160 group; D: MSHLB-200 group;

[0025] Figure 2 Part of the PCR amplification results, where "+": MS positive control; "-": negative control; 1: MS 6 strains; 2: MS 7 strains; 3: MS 8 strains; 4: MS 9 strains;

[0026] Figure 3 The growth conditions of strains MS1, MS7, and MS20 in liquid culture medium;

[0027] Figure 4 The infection status of chicken embryos in some infection groups, A: MS1 strain; B: MS 7 strain; C: PBS control; DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 Screening of candidate strains of Mycoplasma synoviae live vaccine

[0030] MSHLB-95, MSHLB-120, MSHLB-160, and MSHLB-200 isolated and purified in the laboratory and stored at -80°C were numbered H95, H120, H160, and H200, respectively (MSHLB-95 strain was inoculated at a ratio of 10% into a modified Frey's liquid medium, cultured at 37°C with shaking, and continuously subcultured in vitro, wherein MSHLB-95 was subcultured from MSHLB-1, which was a (Mycoplasma synoviae HLB-1) strain deposited in the laboratory of Ningxia University on March 3, 2017, and recorded in CN117946928A), revived to a modified Frey's solid plate, and selected strains of uniform size and "fried egg shape" (such as Figure 1 ), 5 monoclones with typical mycoplasma morphology (H95 monoclones were recorded as MS1 to MS 5; H120 monoclones were recorded as MS 6 to MS10; H160 monoclones were recorded as MS11 to MS15; H200 monoclones were recorded as MS16 to MS20) were harvested when cultured in modified Frey's liquid medium to pH 6.8-6.9.

[0031] The viable bacteria were counted by the following method (the bacterial solution was serially diluted 10-fold to 10 using modified Frey's liquid medium). -10 , respectively from 10 -7 , 10 -8 , 10 -9 , 10 -10 Take 1.0 ml of each dilution and inoculate it into a sterile test tube containing 1.0 ml of culture medium. Inoculate 6 small tubes for each dilution. Set up 2 sterile test tubes containing 2.0 ml of liquid culture medium as a control. Incubate at 37°C and 100 r / min with shaking. Observe for 7 days. Count the number of small tubes of each dilution that change color (the color of the culture medium changes from red to yellow). Calculate the half color change unit (CCU) according to the Reed-Muench method. 50 ) was used to measure the growth curve, and DNA of the monoclonal bacterial solution was extracted and harvested. Referring to the conserved sequence of the nanA gene in the GenBank database MS (accession number: AE017245), a pair of specific primers were designed using primer 6.0 software:

[0032] F: 5'-GGCTTATCGCAGGTAATCAC-3';

[0033] R: 5'-TTAACTTCGGAGACGACTGC-3';

[0034] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The two synthesized primers were dissolved in ddH2O to a primer concentration of 20pmol / μl and stored at -20°C for future use. The synthesized MS-specific primers were used to amplify the nanA-specific gene sequence and perform PCR-specific amplification. The amplification was performed using the specific PCR reaction system of the PCR amplifier. The amplification conditions were: 94°C pre-denaturation for 3min; 94°C denaturation for 30s; 56°C annealing for 46s; 72°C extension for 30s, a total of 29 cycles; 72°C extension for 5min. The amplified product was detected by 1% agarose gel electrophoresis to identify the size of the amplified fragment. The target fragment size amplified by the primers was 483bp, which was used for PCR identification.

[0035] The results showed that the results of in vitro morphological observations of different generations showed that the modified Frey solid medium of the H95, H120, H160, and H200 test groups were cultured in a 37°C incubator containing 5% CO2 for 3 days. Typical "fried egg-like" colonies were observed under microscopic examination. The DNA of the monoclonal bacterial liquid was extracted and harvested, and the target fragment of Mycoplasma gallinarum with a length of 483 bp was obtained after PCR amplification. Figure 2 As shown, the results were consistent with the expected results, and both the morphological and molecular levels proved that the isolated monoclonal strain was Mycoplasma gallinarum.

[0036] The strains MS1 to MS 5 (H95 generation) reached their optimal growth state in 16 to 24 hours (the growth of some strains is shown in Figure 3 The viable count is 10 8.0～9.0 CCU 50 / ml; MS 6-MS10 (H120 generation) strains reach the best growth state in 16-24 hours, and the number of viable bacteria is 10 8.5～9.5 CCU 50 / ml; MS11-MS15 (H160 generation) strains reached the best growth state in 12-18 hours, and the number of viable bacteria was 10 8.5～9.5 CCU 50 / ml; MS16-MS20 (H200 generation) strains reached the best growth state in 6-12 hours, and the number of viable bacteria was 10 8.8～9.5 CCU 50 / ml, among which MS 7, MS15, and MS16 had the highest viable counts, all at 10 9.5 CCU 50 / ml.

[0037] Table 1 Growth of different monoclonal strains of MSHLB-95

[0038]

[0039] Table 2 Growth of different monoclonal strains of MSHLB-120

[0040]

[0041] Table 3 Growth of different monoclonal strains of MSHLB-160

[0042]

[0043] Table 4 Growth of different monoclonal strains of MSHLB-200

[0044]

[0045] Example 2 Chicken embryo infection test of Mycoplasma synoviae of different generations and different monoclonal strains

[0046] 1. Experimental strains and embryos

[0047] The experiment used monoclonal strains selected from MSHLB-95, MSHLB-120, MSHLB-160 and MSHLB-200 strains that were separated and purified and stored at -80°C. 420 SPF chicken embryo eggs were purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd.

[0048] 2. Chicken embryo infection test of Mycoplasma synoviae of different generations and different monoclonal strains

[0049] 420 7-day-old SPF chicken embryos were randomly divided into 22 groups, with 20 in each infection group, 10 in each PBS control group, and 10 in each blank control group. The MS1-MS20 groups were inoculated with 0.1 ml of H95, H120, H160, and H200 monoclonal bacterial solution through the yolk sac cavity, respectively, and the number of viable bacteria inoculated was 10 8.0 CCU 50 / ml. Group 6 was inoculated with the same dose of PBS as a blank control group. The cells were cultured at 37.5-38.0℃ and observed daily. Chicken embryos that died within 24 hours were discarded, and the chicken embryo yolk fluid was collected for strain pathogen identification. The death of chicken embryos 14 days after inoculation was recorded.

[0050] 3. Observation of chicken embryo autopsies of different monoclonal strains at different generations

[0051] After inoculation, it can be observed that the blood vessels of the dead chicken embryos in each infection group are attached to the wall, or the blood vessels are blurred. Autopsy showed that the chicken embryos in each test group showed different degrees of congestion and bleeding, while there was no congestion and bleeding in the PBS group. Among them, the chicken embryos of MS1~MS 5 (H95 group), MS 6, MS 8~MS10 (H120 group), MS11, MS15 (H160 group), and MS18 (H200 group) showed more serious congestion and bleeding. The chicken embryos of MS 7 (H120 group), MS12~MS 14 (H160 group), MS16, MS17, MS19, and MS20 (H200 group) groups showed almost no congestion and bleeding, and no obvious pathological changes, such as Figure 4 shown.

[0052] 4. Death of chicken embryos infected with different monoclonal strains of different generations

[0053] The results showed that the mortality rate of chicken embryos of MS1-MS 5 (60-75%), MS 6-MS10 (10-60%, of which the mortality rate of MS 7 chicken embryos was 10%), MS11-MS15 (10-20%), and MS16-MS20 (10-20%) generally decreased gradually with the increase of the number of generations, but it was found that the virulence of the MS 7 monoclonal strain selected by the H120 group was significantly lower than that of other monoclonal strains of the same generation. The mortality rate of chicken embryos of the PBS group and the blank group was 0%. At the same time, the yolk sac fluid separated from each group was sucked into the liquid culture medium and the solid culture medium for culture respectively. Each infection group was separated from 20 samples, and the liquid culture medium of each sample turned yellow, and "fried egg-like" colonies were observed from the solid culture medium, and PCR identification was 20 / 20 positive. The PBS group and the blank group were separated from 10 samples, but the liquid culture medium of each sample did not turn yellow, and "fried egg-like" colonies were not observed in its solid culture medium, and the PCR identification positive rate was 0 / 10.

[0054] Table 5 Results of chicken embryo infection with Mycoplasma synoviae from different generations and different monoclonal strains

[0055]

[0056] Note: “ / ” indicates not applicable.

[0057] The above experiments can conclude that the H120-generation monoclonal MS 7 strain, the H160-generation monoclonal MS13 strain, the H200-generation monoclonal MS16 and MS17 strains have the weakest pathogenicity. In view of the results of the previous monoclonal cultures of different generations, the H120-generation monoclonal MS 7 strain and the H200-generation monoclonal MS16 strain can be used as candidate strains for MS attenuated live vaccine.

[0058] Example 3 Production of Live Vaccine of Mycoplasma synoviae

[0059] 1. Seed preparation for production

[0060] 1.1 Preparation of modified Frey's liquid medium

[0061] 5.0g of sodium chloride, 0.4g of potassium chloride, 0.2g of magnesium sulfate (containing 7 crystal waters), 1.6g of disodium hydrogen phosphate (containing 12 crystal waters), 0.2g of anhydrous potassium dihydrogen phosphate, 10.0g of glucose (containing 1 crystal water), 5.0g of milk protein hydrolyzate, 5.0g of yeast extract powder, 10ml of 1% coenzyme I solution, 10ml of 1% L-cysteine ​​solution, 20ml of 2% arginine solution, 100ml of porcine serum, 1.0ml of 1% phenol red solution, add water for injection to make up to 1000ml, mix and dissolve the above ingredients, adjust the pH to 7.6-7.8 with 1.0mol / ml sodium hydroxide, filter and sterilize, and store at 2-8℃.

[0062] 1.2 Seed propagation and identification

[0063] The MS 7 and MS16 strains of Mycoplasma synoviae were reconstituted in a modified Frey's liquid medium, and the reconstituted seed solution was inoculated at 5% of the volume of the medium, and the culture was placed at 37°C and 100r / min with shaking. When the pH value of the modified Frey's liquid medium dropped to 6.7-6.9, the culture was stopped, and the seeds were harvested and used as first-level seeds after microscopic examination. This method was gradually expanded to fourth-level seeds.

[0064] 2. Preparation of bacterial solution for seedling production

[0065] 2.1 Bacterial liquid culture: Add 60% to 80% of the volume of the fermentation tank, inoculate 5% of the volume of the culture medium with the fourth-grade seed liquid, and culture at 37℃ with stirring. When the pH value of the culture medium drops to 6.7 to 6.9, harvest the bacterial liquid and take samples for live bacteria count and purity test. Store below -15℃ for no more than 14 days.

[0066] 2.2 Semi-finished product inspection

[0067] 2.2.1 Live bacteria count The number of live bacteria is 10 9.5 CCU 50 / ml.

[0068] 2.2.2 Purity test: Inoculate the harvested bacterial liquid into 2 tubes of TG tube, GA slant and modified Frey's liquid culture medium, 0.2 ml each, culture one tube at 35-37℃ and the other at 23-25℃. Observe for 5 days and all the results are pure.

[0069] 2.3 Preparation and Packaging The qualified Mycoplasma gallinarum bacterial solution is fully mixed with a lyophilization protective agent at a volume ratio of 3:1, wherein the lyophilization protective agent is a gelatin composite stabilizer, and the gelatin composite stabilizer is composed of 2% gelatin and 10% sucrose in terms of mass percentage.

[0070] 2.4 After freeze-drying, package at 3 ml / bottle and set the freeze-drying conditions as follows: (pre-freezing temperature is -40°C, pre-freezing time is 4 to 5 hours (this step is used to maintain biological activity, prepare for freeze-drying, improve freeze-drying efficiency, avoid product volume expansion, and create conditions for long-term storage of vaccines); sublimation temperature is -10 to -12°C, sublimation time is 24 to 26 hours (this step is used to remove moisture, maintain vaccine stability, prevent microbial growth, increase vaccine shelf life, reduce volume and weight, maintain physical form, prepare for analysis, is the most important step, directly affects the quality of the vaccine and freeze-drying effect, and ensures that the original biological activity and immunogenicity are maintained after freeze-drying); analysis temperature is 27°C, maintained for 12 hours (this step is used to remove bound water, improve stability, prevent aggregation during rehydration, extend shelf life, ensure vaccine uniformity, prepare for sealing and storage, ensure long-term stability and effectiveness of the vaccine after freeze-drying, and provide convenience for the final use of the vaccine) quickly perform freeze vacuum drying, capping and labeling.

[0071] 3. Test results

[0072] 3.1 Properties

[0073] Slightly yellow spongy loose lumps, easily separated from the bottle wall, and quickly dissolved after adding diluent.

[0074] 3.2 Purity test, residual moisture, vacuum degree

[0075] They were tested according to the current "Chinese Veterinary Pharmacopoeia" and all passed.

[0076] 3.3 Live bacteria count After the vaccine is restored to its original volume, CCU 50 The live bacterial count of MS 7 freeze-dried vaccine was 10 9.3 CCU 50 / ml, the live bacteria count of MS16 freeze-dried vaccine is 10 8.8 CCU 50 / ml.

[0077] 4. Comparison of vaccine safety tests

[0078] The vaccine was diluted with sterile saline and 80 SPF chickens aged 21 days were used, with 20 chickens in each group. They were inoculated with 0.03 ml of MS 7 strain, MS16 strain and commercial vaccine MS-H and 0.3 ml of attenuated vaccine prepared with MSHLB-200 strain and injectable white oil by intranasal drops and observed for 14 days. Chickens in group H200 showed open mouth breathing 2 days after immunization and began to recover after 3 days. The autopsy was normal. However, the intranasal immunization dose of group H200 was larger, the immunization time was longer, and the clinical symptoms of open mouth breathing appeared 2 days after immunization, and gradually recovered after 3 days.

[0079] Table 6 Comparison results of vaccine safety tests

[0080]

[0081]

[0082] 5. Vaccine efficacy test comparison

[0083] The vaccine was diluted with sterile saline and 90 SPF chickens aged 28 days were randomly divided into 5 groups. Each group was inoculated with 0.03 ml (10 6.5 CCU 50 / feather), 20 chickens were intranasally inoculated with 0.3ml of attenuated vaccine prepared with MSHLB-200 strain and white oil for injection; the other 10 chickens were not vaccinated as controls. 35 days after vaccination, each chicken was challenged with virulent Mycoplasma synoviae and observed for 14 days. After 14 days, the air sac lesions were observed and scored. 2 points or more were judged as disease, and less than 2 points were judged as protection. 8 / 10 control chickens were diseased, 16 / 20 chickens immunized with MS7 strain were protected, 14 / 20 chickens immunized with MS16 strain were protected, 14 / 20 chickens immunized with MS-H strain were protected, and 13 / 20 chickens immunized with MSHLB-200 attenuated vaccine were protected.

[0084] Table 7 Comparison results of vaccine efficacy test

[0085]

[0086] Note: “ / ” indicates not applicable.

[0087] The results showed that the freeze-drying loss of the vaccine prepared by the MS 7 strain was much lower than that of the vaccine prepared by the MS16 strain, which was more conducive to subsequent large-scale production. Other test items were in compliance with regulations: In addition, the results of vaccine immune efficacy showed that the vaccine prepared by the MS 7 strain was better than the attenuated vaccine prepared by fully emulsifying the commercial vaccine MS-H and MSHLB-200 strains with white oil for injection, so the MS 7 strain was selected as the vaccine strain in the future. The chicken synoviae Mycoplasma MS 7 strain was deposited in the China Center for Type Culture Collection on July 10, 2024, and its deposit number is CCTCC M 20241533.

[0088] The vaccine stored at 2-8°C for one year was tested, and the results showed that the results of properties, residual moisture, vacuum degree, purity test, live bacteria count, safety test, and efficacy test were basically consistent with previous studies. Therefore, after the MS 7 strain is produced, it can be stored at 2-8°C for at least one year.

[0089] Table 8 Test results of Mycoplasma synoviae live vaccine MS 7 strain stored at 2-8℃ for one year

[0090]

[0091] Compared with the inactivated vaccine, the present application prepares the live vaccine of the MS 7 strain of Mycoplasma synoviae, which can play a competitive exclusion role by occupying the position, induce local mucosal immunity, effectively prevent the spread of wild-type Mycoplasma synoviae, and after freeze-drying, it can be stored at 2-8°C for at least one year, which is convenient for cold chain transportation; compared with the attenuated vaccine prepared by fully emulsifying the existing MSHLB-200 strain with white oil for injection, it has lower cost, is more suitable for industrial production, is convenient for storage and transportation, and has a simple method of use, convenient operation, and ideal immune effect, which is conducive to large-scale promotion and use; compared with the commercial vaccine MS-H strain, it is convenient for storage and transportation (MS-H strain needs to be stored below -70°C), and has an ideal immune effect. As can be seen from the above, the live vaccine prepared by the MS 7 strain of Mycoplasma synoviae is low in cost, suitable for industrial production, convenient for storage and transportation, simple in method of use, safe, and has an ideal immune effect, which is conducive to large-scale promotion and use.

Claims

1. Mycoplasma gallinarum, characterized in that The gallinarum mycoplasma is the gallinarum mycoplasma MS 7 strain, which was deposited in the China Center for Type Culture Collection on July 10, 2024, and its deposit number is CCTCC M 20241533.

2. Use of the Mycoplasma gallinarum according to claim 1 in the preparation of a live Mycoplasma gallinarum vaccine.

3. A live vaccine of Mycoplasma synoviae, characterized in that: The method is prepared by using the Mycoplasma synoviae described in claim 1.

4. The Mycoplasma gallinarum live vaccine according to claim 3, characterized in that: The Mycoplasma gallinarum live vaccine is prepared by fully mixing the Mycoplasma gallinarum bacterial liquid with a lyophilization protective agent in a volume ratio of 3:1 and then freeze-drying.

5. The Mycoplasma gallinarum live vaccine according to claim 3, characterized in that: The freeze-drying protective agent is a gelatin composite stabilizer, and the gelatin composite stabilizer is composed of 2% gelatin and 10% sucrose in terms of mass percentage.

6. The Mycoplasma gallinarum live vaccine according to claim 3, characterized in that: The freeze-drying conditions meet the following procedures: The pre-freezing temperature is -40°C and the pre-freezing time is 4 to 5 hours; The sublimation temperature is -10 to -12°C and the sublimation time is 24 to 26 hours; The desorption temperature was 27°C and maintained for 12 hours.

7. The Mycoplasma gallinarum live vaccine according to claim 3, characterized in that: The Mycoplasma gallinarum bacterial liquid is prepared by the following method: The MS7 strain of Mycoplasma synoviae was inoculated into the modified Frey's liquid medium at 5% of the volume of the medium and cultured at 37°C and 100 rpm until the pH value of the medium dropped to 6.7-6.9, after passing the microscopic examination, they are harvested and used as first-grade seeds, and the culture is gradually expanded to fourth-grade seeds in this way; Add 60% to 80% of the fermentation tank volume into the culture medium, inoculate 5% of the culture medium volume with the fourth-grade seed solution, and culture at 37°C with stirring until the pH value of the culture medium drops to From 6.7 to 6.9, the bacterial liquid of Mycoplasma synoviae MS 7 strain was harvested.

8. The Mycoplasma gallinarum live vaccine according to any one of claims 3 to 7, characterized in that The number of live bacteria of the MS 7 strain in the Mycoplasma synoviae live vaccine is 10 9.0 CCU 50 / ml or above.

9. The Mycoplasma gallinarum live vaccine according to any one of claims 3 to 7, characterized in that The live vaccine of Mycoplasma synoviae is 6.5 CCU 50 / Feather concentration, the protection efficiency of 0.03ml immunization method through eye drops reached more than 75%.

Images