Mycoplasma synoviae of chickens, the resulting live vaccine, and its preparation method and application

Abstract

The present invention provides a Mycoplasma synoviae of chicken, a resulting live vaccine, a preparation method thereof and an application, belonging to the technical field of veterinary vaccines. The Mycoplasma synoviae of chicken is the Mycoplasma synoviae MS 7 strain, which was deposited at the China Center for Type Culture Collection on July 10, 2024, and its deposit number is CCTCC M 20241533. Preparing a live vaccine from the Mycoplasma synoviae MS 7 strain of chicken has a lower cost, is more suitable for industrial production, is convenient for storage and transportation, has a simple usage method, and is conducive to large-scale popularization and use. Compared with the attenuated vaccine prepared by fully emulsifying the existing MSHLB-200 strain and white oil for injection and the commercial vaccine MS-H, it is convenient for storage and transportation and has an ideal immune effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary vaccines, and particularly relates to Mycoplasma synoviae of chickens, a resulting live vaccine, a preparation method thereof, and an application thereof. Background Art

[0002] Mycoplasma synoviae (hereinafter referred to as "MS") can cause subclinical respiratory diseases in various poultry such as chickens, swelling of foot pads or joints, a decrease in egg production rate, eggshell quality, and feed-to-meat ratio, and has the ability of horizontal transmission and vertical transmission. Horizontal transmission is mainly through the respiratory tract. Latently infected chickens, diseased chickens, and their secretions and excreta are the main sources of infection of this disease; vertical transmission is through eggs or semen. Chicks hatched from infected eggs carry MS, and through horizontal transmission, more chickens will be infected. 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 at present, and it has seriously affected the economic benefits of the poultry industry in China.

[0003] The prevalence and infection rate of MS in chicken flocks in China are high, including various breeds such as broilers, laying hens, breeding chickens, and local yellow chickens in China. Moreover, the high-density intensive farming mode is also conducive to the horizontal transmission of MS; in addition, breeding chickens infected with MS are also prone to vertical transmission, which all increases the prevention and control difficulty of MS. At home and abroad, the prevention and control of MS disease are still in the exploratory stage. At present, the drug resistance of MS to common bacterial antibiotics is increasing year by year, and it is becoming increasingly difficult to solely rely on antibiotics to prevent and control MS infection in commercial broilers and commercial laying hens. Vaccines play a key role in the prevention and control of livestock and poultry infectious diseases. At present, only the inactivated Mycoplasma synoviae vaccine (YBF-MS1 strain) independently developed by Qingdao Yibang Bioengineering Co., Ltd. and the live Mycoplasma synoviae vaccine (MS-H strain) developed by Australian BioResources Company have obtained domestic registration and are permitted for sale. The inactivated vaccine can provide good humoral immunity, but it cannot block infection. Therefore, it is not sufficient to completely control the prevalence of this disease; studies at home and abroad on the live Mycoplasma synoviae vaccine (MS-H strain) show that when used by eye drop in MS-negative chickens, it can prevent and reduce clinical symptoms, reduce the degree of MS infection and horizontal transmission. However, this vaccine is expensive and can only be stored below -70°C, and the storage cost is extremely high, which is not conducive to large-scale promotion and use in the front line of clinical practice. Therefore, there is an urgent need to develop a commercial live vaccine that is safe and effective, convenient for storage and transportation, and can be widely promoted and used on a large scale. Summary of the Invention

[0004] The present invention provides a Mycoplasma synoviae of chickens, a resulting live vaccine, a preparation method thereof, and an application thereof. Preparing a live vaccine from the Mycoplasma synoviae MS 7 strain has a lower cost, is more suitable for industrial production, is convenient for storage and transportation, has a simple usage method, is conducive to large-scale promotion and use, and is convenient for storage and transportation and has an ideal immune effect compared with the attenuated vaccine prepared by fully emulsifying the existing MSHLB-200 strain and white oil for injection and the commercial vaccine MS-H.

[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 freeze-drying 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 1Growth of MSHLB-95, MSHLB-120, MSHLB-160, and MSHLB-200 on solid plates. Among them, A: MSHLB-95 group; B: MSHLB-120 group; C: MSHLB-160 group; D: MSHLB-200 group;

[0025] Figure 2 Partial PCR amplification. Among them, "+": MS positive control; "-": negative control; 1: MS strain 6; 2: MS strain 7; 3: MS strain 8; 4: MS strain 9;

[0026] Figure 3 Growth of MS strain 1, MS strain 7, and MS strain 20 in liquid medium;

[0027] Figure 4 Embryo infection of some infected group chicken embryos. Among them, A: MS strain 1; B: MS strain 7; C: PBS control; Specific implementation mode

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

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

[0030] Take MSHLB-95, MSHLB-120, MSHLB-160, and MSHLB-200 stored at -80°C after laboratory isolation and purification, numbered H95, H120, H160, and H200 respectively (Inoculate the MSHLB-95 strain into the modified Frey's liquid medium at a ratio of 10%, place it in an oscillator at 37°C for continuous subculture in vitro. Among them, MSHLB-95 is derived from the passage of MSHLB-1, and MSHLB-1 is the strain (Mycoplasma synoviae HLB-1) preserved in the laboratory of Ningxia University on March 3, 2017, recorded in CN117946928A). Resuscitate it on the modified Frey's solid plate, and select 5 monoclonal colonies with the same size and a "fried egg shape" (as Figure 1 shown), with typical mycoplasma morphology (The H95 monoclonal colonies are respectively denoted as MS1 to MS5; the H120 monoclonal colonies are respectively denoted as MS6 to MS10; the H160 monoclonal colonies are respectively denoted as MS11 to MS15; the H200 monoclonal colonies are respectively denoted as MS16 to MS20), and harvest them when the pH of the modified Frey's liquid medium reaches 6.8 - 6.9.

[0031] By the following viable count method (serially dilute the bacterial solution 10-fold with modified Frey's liquid medium to 10 -10 , respectively take 1.0 ml from 10 -7 , 10 -8 , 10 -9 , 10 -10 tubes and inoculate 1.0 ml into sterile test tubes containing 1.0 ml of medium. Inoculate 6 small tubes for each dilution. Additionally, set up 2 sterile test tubes containing 2.0 ml of liquid medium as controls. Incubate at 37°C with shaking at 100 r / min for 7 days. Count the number of tubes with color change (the medium color changes from red to yellow) at each dilution. Calculate the median color-changing unit (CCU 50 ) to measure the growth curve, and extract the DNA of the harvested monoclonal bacterial solution. Refer to the conserved sequence of the nanA gene in the GenBank database MS (accession number: AE017245), and use primer 6.0 software to design a pair of specific primers:

[0032] F: 5’-GGCTTATCGCAGGTAATCAC-3’;

[0033] R: 5’-TTAACTTCGGAGACGACTGC-3’;

[0034] The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After synthesis, the two primers were dissolved in ddH 2 O to a primer concentration of 20 pmol / μl and stored at -20°C for later use. Use the synthesized MS-specific primers to amplify the nanA-specific gene sequence for PCR-specific amplification. Use the specific PCR reaction system of a PCR amplifier for amplification. The amplification conditions are: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 56°C for 46 s; extension at 72°C for 30 s, for a total of 29 cycles; extension at 72°C for 5 min. The amplified product was detected by 1% agarose gel electrophoresis to identify the size of the amplified fragment. The size of the target fragment amplified by the primers is 483 bp, and this is used for PCR identification.

[0035] The results showed that for the in vitro morphological observations of different passages, the modified Frey's solid medium in the H95, H120, H160, and H200 experimental groups was cultured in a 37°C incubator containing 5% CO 2 for 3 days. After microscopic examination, typical "fried egg-like" colonies were observed. The DNA of the harvested monoclonal bacterial solution was extracted, and after PCR amplification, the Mycoplasma synoviae target fragment with a fragment length of 483 bp could be obtained, as Figure 2As shown, it meets the expected results, and it is proved at both the morphological and molecular levels that the isolated monoclonal strain is Mycoplasma synoviae of chickens.

[0036] Strains MS1 - MS5 (H95 passage) reached the optimal growth state at 16 - 24 hours (the growth of some strains is as shown Figure 3 ), and the viable cell count was 10 8.0～9.0 CCU 50 / ml; Strains MS6 - MS10 (H120 passage) reached the optimal growth state at 16 - 24 hours, and the viable cell count was 10 8.5～9.5 CCU 50 / ml; Strains MS11 - MS15 (H160 passage) reached the optimal growth state at 12 - 18 hours, and the viable cell count was 10 8.5～9.5 CCU 50 / ml; Strains MS16 - MS20 (H200 passage) reached the optimal growth state at 6 - 12 hours, and the viable cell count was 10 8.8～9.5 CCU 50 / ml, among which the viable cell counts of MS7, MS15, and MS16 were the highest, all being 10 9.5 CCU 50 / ml.

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

[0038]

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

[0040]

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

[0042]

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

[0044]

[0045] Example 2 Chicken embryo infection test of Mycoplasma synoviae of chickens with different monoclonal strains of different passages

[0046] 1. Experimental strains and embryos

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

[0048] 2. Mycoplasma synoviae chicken embryo infection test with different passages and different monoclonal strains

[0049] 420 7-day-old SPF chicken embryos were selected and randomly divided into 22 groups, with 20 embryos in each infected group, 10 embryos in the PBS control group, and 10 embryos in the blank control group. Among them, groups MS1 to MS20 were inoculated with H95, H120, H160 and H200 monoclonal bacterial solutions through the yolk sac cavity, 0.1 ml per embryo, and the viable bacteria count inoculated was 10 8.0 CCU 50 / ml. The 6th group was inoculated with the same dose of PBS as the blank control group. They were cultured at 37.5 - 38.0 °C, observed daily, the chicken embryos that died within 24 hours were discarded, the yolk fluid of the chicken embryos was collected for strain pathogen identification, and the death situation of the chicken embryos 14 days after inoculation was recorded.

[0050] 3. Autopsy observation of chicken embryos with different passages and different monoclonal strains

[0051] After inoculation, in the dead chicken embryos of each infected group, it could be observed that the chicken embryo blood vessels adhered to the wall or the blood vessels were blurred. Autopsy showed that the embryo bodies of the chicken embryos in each test group had varying degrees of hyperemia and hemorrhage, while there was no hyperemia and hemorrhage in the PBS group. Among them, the states of hyperemia and hemorrhage of the embryo bodies of the chicken embryos in MS1 - MS5 (H95 group), MS6, MS8 - MS10 (H120 group), MS11, MS15 (H160 group), MS18 (H200 group) were relatively serious, and there was almost no hyperemia and hemorrhage in the embryo bodies of the chicken embryos in MS7 (H120 group), MS12 - MS14 (H160 group), MS16, MS17, MS19, MS20 (H200 group), nor were there obvious pathological changes, as Figure 4 shown.

[0052] 4. Death situation of chicken embryos infected with different passages and different monoclonal strains

[0053] The results showed that for MS1 - MS5 (60 - 75%), MS6 - MS10 (10 - 60%, with a 10% mortality rate of chicken embryos for MS7), MS11 - MS15 (10 - 20%), and MS16 - MS20 (10 - 20%), the mortality rate of chicken embryos generally decreased gradually with the increase in the passage number. However, it was found that the virulence of the MS7 monoclonal strain picked from the H120 group was significantly lower than that of other monoclonal strains of the same passage. The mortality rate of chicken embryos in the PBS group and the blank group was 0%. At the same time, the yolk sac fluid isolated from each group was aspirated into liquid medium and solid medium for culture respectively. Twenty samples were isolated from each infected group. The liquid medium of each sample turned yellow, and "fried egg-like" colonies were observed in the solid medium. PCR identification showed 20 / 20 positive. Ten samples were isolated from the PBS group and the blank group respectively, but the liquid medium of each sample did not turn yellow, and no "fried egg-like" colonies were observed in their solid medium. The positive rate of PCR identification was 0 / 10.

[0054] Table 5 Results of Mycoplasma synoviae chicken embryo infection with different monoclonal strains at different passages

[0055]

[0056] Note: " / " indicates not applicable.

[0057] From the above experiments, the following conclusions can be drawn: The pathogenicity of the monoclonal MS7 strain at the H120 passage, the monoclonal MS13 strain at the H160 passage, and the monoclonal MS16 and MS17 strains at the H200 passage is the weakest. In view of the results of different monoclonal cultures at different passages in the early stage, the monoclonal MS7 strain at the H120 passage and the monoclonal MS16 strain at the H200 passage can be used as candidate strains for the live attenuated MS vaccine.

[0058] Example 3 Production of live Mycoplasma synoviae vaccine

[0059] 1. Preparation of production seeds

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

[0061] Sodium chloride 5.0 g, potassium chloride 0.4 g, magnesium sulfate (with 7 crystal waters) 0.2 g, disodium hydrogen phosphate (with 12 crystal waters) 1.6 g, anhydrous potassium dihydrogen phosphate 0.2 g, glucose (with 1 crystal water) 10.0 g, lactalbumin hydrolyzate 5.0 g, yeast extract powder 5.0 g, 1% coenzyme I solution 10 ml, 1% L-cysteine solution 10 ml, 2% arginine solution 20 ml, porcine serum 100 ml, 1% phenol red solution 1.0 ml. Add water for injection to make up to 1000 ml. Mix and dissolve the above components, adjust the pH value to 7.6 - 7.8 with 1.0 mol / ml sodium hydroxide, filter through bacteria, and store at 2 - 8°C.

[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 sub-packaging into 3 ml vials, the lyophilization conditions are set as follows: (The pre-freezing temperature is -40°C, and the pre-freezing time is 4 - 5 hours (this step is used to maintain biological activity, prepare for lyophilization, improve lyophilization efficiency, avoid product volume expansion, and create conditions for long-term storage of the vaccine); the sublimation temperature is -10 to -12°C, and the sublimation time is 24 - 26 hours (this step is used to remove moisture, maintain vaccine stability, prevent microbial growth, extend the vaccine shelf life, reduce volume and weight, maintain physical form, prepare for analysis, which is the most important step, directly affecting the quality and lyophilization effect of the vaccine, ensuring the original biological activity and immunogenicity after lyophilization); the analysis temperature is 27°C, maintained for 12 hours (this step is used to remove bound water, improve stability, prevent aggregation during rehydration, extend the shelf life, ensure vaccine uniformity, prepare for sealing and storage, ensure the long-term stability and effectiveness of the lyophilized vaccine, and provide convenience for the final use of the vaccine).) Immediately carry out freeze-vacuum drying, capping, and labeling.

[0071] 3. Test Results

[0072] 3.1 Appearance

[0073] Light yellow spongy loose mass, easily detached from the vial wall, and quickly dissolved after adding diluent.

[0074] 3.2 Purity Test, Residual Moisture, Vacuum Degree

[0075] Tested according to the current "Chinese Veterinary Pharmacopoeia", all are qualified.

[0076] 3.3 Viable Bacteria Count After restoring the vaccine to its original volume, perform CCU 50 determination. The viable bacteria count of the freeze-dried vaccine of MS 7 strain is 10 9.3 CCU 50 / ml, and the viable bacteria count of the freeze-dried vaccine of MS16 strain is 10 8.8 CCU 50 / ml.

[0077] 4. Vaccine Safety Test Comparison

[0078] Dilute the vaccine with sterilized normal saline. Use 80 21-day-old SPF chickens, 20 in each group. Inoculate 0.03 ml of MS 7 strain, MS16 strain and commercial vaccine MS-H by eye drop and 0.3 ml of the attenuated vaccine prepared with MSHLB-200 strain and white oil for injection by nasal drip. Observe continuously for 14 days. Except for the immunized chickens in the H200 group showing open-mouth breathing 2 days after immunization and starting to recover 3 days later, the autopsy results were normal. However, the nasal drip immunization dose in the H200 group was larger, the immunization time was longer, and the clinical symptom of open-mouth breathing appeared 2 days after immunization and gradually recovered 3 days later.

[0079] Table 6 Comparison Results of Vaccine Safety Tests

[0080]

[0081]

[0082] 5. Vaccine potency test comparison

[0083] Dilute the vaccine with sterilized normal saline. Use 90 SPF chickens at 28 days of age and randomly divide them into 5 groups, with 20 chickens in each group. Instill 0.03 ml (10 6.5 CCU 50 / bird) of MS 7 strain, MS16 strain, and commercial vaccine MS-H into the eyes; instill 0.3 ml of the attenuated vaccine prepared from MSHLB-200 strain and white oil for injection into the nasal cavity of 20 chickens; another 10 chickens are not inoculated as a control. 35 days after inoculation, each chicken is challenged with a virulent strain of Mycoplasma synoviae of chickens. Observe for 14 days. After 14 days, dissect and observe the air sac lesions and score them. A score of 2 or above is judged as diseased, and a score below 2 is judged as protected. 8 / 10 of the control chickens were diseased, 16 / 20 of the chickens immunized with MS 7 strain were protected, 14 / 20 of the chickens immunized with MS16 strain were protected, 14 / 20 of the chickens immunized with MS-H strain were protected, and 13 / 20 of the chickens immunized with the MSHLB-200 attenuated vaccine were protected.

[0084] Table 7 Comparison results of vaccine potency test

[0085]

[0086] Note: " / " indicates not applicable.

[0087] The results show that the freeze-drying loss of the vaccine prepared from MS 7 strain is much lower than that of the vaccine prepared from MS16 strain, which is more conducive to large-scale production in the future. The results of other test items all meet the requirements. In addition, the results of the vaccine immune potency show that the vaccine prepared from MS 7 strain is superior to the vaccines prepared from MS16 strain, commercial vaccine MS-H, and the attenuated vaccine prepared by fully emulsifying MSHLB-200 strain and white oil for injection. Therefore, MS 7 strain is selected as the vaccine strain for the future. The Mycoplasma synoviae MS 7 strain of chickens was deposited in the China Center for Type Culture Collection on July 10, 2024, and its deposit number is CCTCC M 20241533.

[0088] Test the vaccine stored at 2 - 8 °C for one year. The results show that the results of the properties, residual moisture, vacuum degree, purity test, viable count, safety test, and potency test are basically the same as those of the previous research. Therefore, after the vaccine is prepared from MS 7 strain, it can be stored at 2 - 8 °C for at least one year.

[0089] Table 8 Test results of the live vaccine of Mycoplasma synoviae of chickens MS 7 strain stored at 2 - 8 °C 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. A live vaccine of Mycoplasma synoviae, characterized in that: The method is prepared by fully mixing a Mycoplasma gallinarum bacterial liquid and a lyophilization protective agent in a volume ratio of 3:1 and then freeze-drying, wherein the Mycoplasma gallinarum 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 CCTCC M 20241533; 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.

2. The Mycoplasma gallinarum live vaccine according to claim 1, characterized in that: The freeze-drying protective agent is a gelatin composite stabilizer.

3. The Mycoplasma gallinarum live vaccine according to claim 1, 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.

4. The Mycoplasma gallinarum live vaccine according to claim 1, 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.

5. The Mycoplasma gallinarum live vaccine according to any one of claims 1 to 4, 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%.

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