Culture method and application of mycoplasma synoviae
By using serum-free LMH cell line culture medium and optimized culture conditions, the problems of long cycle and low protein yield of traditional culture media were solved, achieving efficient Mycoplasma synovitis culture and efficient vaccine preparation, which significantly improved the immune protection rate.
Patent Information
- Application Number
- CN202511404455.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2026-01-13
AI Technical Summary
The traditional culture method for Mycoplasma synoviae in chickens in the present technology relies on modified Frey medium containing serum, which results in long culture cycles, complex serum composition, large batch-to-batch variability, low protein yield, and the protection rate of traditional vaccines is less than 50%.
A highly effective Mycoplasma synovitis vaccine was prepared by using serum-free LMH cell line culture medium supplemented with key factors such as cholesterol, coenzyme I, and vitamin B12, optimizing the inoculation amount and culture conditions, and combining concentration and inactivation processes.
High-density proliferation of Mycoplasma synoviae was achieved, with a protein yield of 800 mg/L and a titer of 2.84 × 10¹⁰ CCU₅₀/mL. The prepared vaccine had an immunoprotective rate of ≥ 90%, significantly improving the prevention and control of Mycoplasma synoviae disease in chickens.
Smart Images
Figure BDA0005619719070000031 
Figure BDA0005619719070000041 
Figure BDA0005619719070000042
Abstract
Description
Technical Field
[0001] This invention relates to the field of veterinary biological products, specifically to a method for culturing and applying Mycoplasma synoviae. Background Technology
[0002] Mycoplasma synoviae (MS) is a highly pathogenic microorganism that is mainly transmitted through the respiratory tract and hatching eggs. Infected poultry exhibit obvious joint and footpad swelling, tenosynovitis, and synovitis, leading to lameness, stunted growth, decreased egg production performance, and increased mortality, causing significant economic losses to the poultry industry, especially in laying hens and breeding chickens.
[0003] Traditional methods for culturing Mycoplasma synoviae rely on modified Frey medium containing serum, which results in long culture periods, complex serum composition, significant batch-to-batch variability, and low protein yield. Furthermore, according to statistics from the World Organisation for Animal Health (OIE), in 2024, traditional vaccines provided less than 50% protection against MS strains. Therefore, developing a serum-free, efficient, stable, and immunogenic culture method is crucial for improving the quality of Mycoplasma synoviae vaccines. Summary of the Invention
[0004] The purpose of this invention is to provide a method for culturing Mycoplasma synovitis.
[0005] The purpose of this invention is to provide an application of Mycoplasma synovitis.
[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:
[0007] A method for culturing Mycoplasma synovitis, the method comprising:
[0008] S1. Prepare serum-free culture medium solution;
[0009] S2. Inoculate LMH cells and adjust cell density;
[0010] S3. Inoculate with the revived Mycoplasma synoviae culture for further culture;
[0011] S4. After culturing, concentrate and inactivate the enzyme.
[0012] Furthermore, the serum-free culture medium described in step S1 contains LMF-SFMII basal medium, cholesterol, coenzyme I, and vitamins.
[0013] Furthermore, the LMH cell density in step S2 is 1.0 × 10⁻⁶. 6 cells / mL.
[0014] Furthermore, the concentration of the Mycoplasma synoviae bacterial solution introduced in step S3 is 10%.
[0015] Furthermore, in step S1, the serum-free culture medium contains 25 mg / L cholesterol, 50 mg / L nicotinamide adenine dinucleotide, and 2 μg / L vitamins.
[0016] Furthermore, the culture conditions after inoculating the Mycoplasma synoviae culture in step S3 are as follows: culture temperature 37℃±0.5, pH 7.2±0.05, DO 10±5%, and stirring speed 50-150rpm.
[0017] Furthermore, the titer of Mycoplasma synovitis can reach 2.84 × 10⁻⁶. 10 CCU 50 / mL.
[0018] Application of a culture method for Mycoplasma synovitis in the preparation of Mycoplasma synovitis vaccines.
[0019] Compared to existing technologies, the beneficial effects of this invention are:
[0020] This invention is the first to propose using the LMH cell line to culture Mycoplasma synoviae in chickens. Compared with the traditional Frey medium culture, it does not require the addition of serum and the protein yield reaches 800 mg / L.
[0021] Furthermore, this invention also adds a certain proportion of key factors such as cholesterol, coenzyme I, and vitamin B12 to the culture medium components, achieving high-density proliferation of Mycoplasma synoviae with a titer reaching 2 × 10⁻⁶. 10 CCU 50 / mL.
[0022] Cholesterol accounts for 36% of the mycoplasma membrane composition, and sufficient cholesterol is necessary to maintain membrane fluidity and structural integrity. Coenzyme I participates in energy (ATP) production and redox reactions. Mycoplasma lacks a complete tricarboxylic acid cycle and relies on coenzyme I to maintain its metabolism.
[0023] By optimizing the inoculation amount, culture conditions and inactivation process, the final inactivated vaccine can significantly improve the immune protection rate and is suitable for the prevention and control of Mycoplasma synoviae disease in poultry. The prepared Mycoplasma synoviae vaccine for chickens has a challenge protection rate of ≥90%. Detailed Implementation
[0024] The following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.
[0025] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods.
[0026] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
[0027] Example 1: Optimization experiments of different cell lines
[0028] 1.1 Experimental Materials: LMH cells, DF-1 cells, and MDCC cells were obtained from commercially available cell lines; LMF-SFMII medium, Frey medium, etc., were all commercially available culture media; Mycoplasma synoviae MS strain RPMSA1301 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20232235, located at Wuhan University, China.
[0029] 1.2 Bacterial Strain Resuscitation: Lyophilized Mycoplasma synoviae strain RPMSA1301 was revived using Frey medium at a 1:10 ratio and statically incubated at 37℃ for 24-36 hours. The culture was harvested when the culture turned orange-yellow and the pH dropped to 6.9±0.1. The culture that passed purity testing was used as the revived strain, and samples were taken for CCU analysis. 50 .
[0030] 1.3 Preparation of Primary Seeds: The revived seed solution was inoculated into Frey medium at a ratio of 1:10. The medium was incubated at 37℃ with 5% CO2 and shaken at 100±10 rpm for 24-36 hours. The culture was harvested when the color turned orange-yellow and the pH dropped to 6.9±0.1. The culture that passed purity testing was used as primary seed. Samples were taken for CCU testing. 50 It is used for subsequent cell inoculation.
[0031] 1.4 LMH, DF-1, and MDCC cell lines were passaged into nine 125 mL Erlenmeyer flasks containing 20 mL of cell culture medium each using LMF-SFMII. Three parallel culture flasks were set up for each experimental group. The cell culture flasks were placed at 37℃ and 5% CO2 with shaking at 120±10 rpm for 48 h. Cells were cultured until the density reached 1-3 × 10⁻⁶ cells / mL. 6 When the cell density reaches 1.0 × 10⁶ cells / mL and the viability is ≥ 90%, the cells are resuspended in fresh serum-free medium and the cell density is adjusted to 1.0 × 10⁶ cells / mL. 1.3 primary seed cultures are used for inoculation, with the amount of Mycoplasma synoviae being 1%. The culture is carried out at 37℃ and 100 rpm with shaking.
[0032] 1.5 Viable Bacterial Count (Titer): The *Mycoplasma synoviae* cell lines cultured above were diluted 10-fold to 10⁻¹¹, with five replicates for each dilution. The cells were then incubated statically at 37°C. Results were evaluated after two weeks. The number of tubes showing discoloration (pH ≥ 0.5) at each dilution was counted, and the half-maximum number of color-changing units (CCU) was calculated using the Reed and Muench methods. 50 The half-maximum color change endpoint was calculated using the Reed-Muench method ( / mL).
[0033] 1.6 Concentration and inactivation of culture: First, centrifuge at low speed (2000 rpm, 10 min) to remove LMH cells, then centrifuge at 9000 rpm / min for 60 min, resuspend in PBS solution to 1 / 10 of the original volume, and inactivate the 10-fold concentrated culture with 10% formaldehyde solution at 37℃ and 50 rpm / min for 24 h. The final formaldehyde concentration is 0.2%. The inactivated antigen is then sonicated under the following conditions: sonication power 300 W, sonication for 3 s, interval for 5 s, total duration 10 min, and protein concentration is measured.
[0034] 1.7 Protein concentration determination: The total protein concentration of the cultures after sonication was determined using a commercially available total protein concentration assay kit (BCA kit).
[0035] Table 1: Results of Mycoplasma synoviae cultured in different cell cultures
[0036] Cell types Incubation time (h) Protein concentration (mg / L) <![CDATA[CCU 50 (×10 8 / mL)]]> LMH 24 98 1.2 DF-1 24 86 0.7 MDCC 24 67 0.1
[0037] Table 1 shows the results: using LMH suspension cells, DF-1 suspension cells, and MDCC suspension cells, with a culture time of 24 h, the titers were 1.2 × 10⁻⁶ and 1.2 × 10⁻⁶, respectively. 8 0.7×10 8 0.1×10 8 CCU 50 / mL, the protein concentrations at 10-fold concentrations were 98, 86, and 67 mg / L, respectively. Compared with the three cell lines LMH, DF-1, and MDCC, LMH cells showed the best fit for MS, with titers 71.4% higher than DF-1 and 11 times higher than MDCC.
[0038] Example 2: Optimization experiment under different inoculation conditions
[0039] LMH cells were passaged into three 125 mL Erlenmeyer flasks containing 20 mL of cell culture medium using LMF-SFMII medium. The flasks were then incubated at 37°C with 5% CO2 and shaking at 120 ± 10 rpm for 48 h, until the cell density reached 1-3 × 10⁶ cells / mL. 6Cells were collected at concentrations of 1.0 × 10⁶ cells / mL with a viability ≥ 90%. Cells were resuspended in fresh serum-free medium and the cell density was adjusted to 1.0 × 10⁶ cells / mL. The concentrations of *Mycoplasma synoviae* inoculated were 1%, 5%, and 10%, respectively, and then cultured at 37°C with shaking at 100 rpm. CCU levels at different time points were then measured. 50 And protein content (mg / L).
[0040] Table 2. Culture results at different inoculum sizes
[0041]
[0042]
[0043] Table 2 shows the results: when the inoculum size was 1%, 5%, and 10%, and the culture time was 24 hours, the titers were 0.6 × 10⁻⁶ and 0.6 × 10⁻⁶, respectively. 8 2.2×10 8 4.6×10 8 CCU 50 / mL, the protein concentrations at 10x concentrations are 110, 136, and 215 mg / L, respectively, with an optimal inoculum size of 10%.
[0044] Example 3: Optimization Experiment of Different Culture Medium Components
[0045] 3.1. Optimization Methods
[0046] Serum-free medium LMF-SFMII was used as the basal medium, supplemented with seven target substances: cholesterol, L-arginine, L-cysteine, vitamin B12, insulin, transferrin, and nicotinamide adenine dinucleotide (coenzyme I). The levels are shown in Table 3. Factors significantly affecting mycoplasma growth were rapidly screened using effect values and P-values calculated via Minitab 17.
[0047] Table 3 Experimental Design Scheme
[0048] Serial Number factor Low level (-1) High level (+1) A L-arginine 100mg / L 500mg / L B cholesterol 5mg / L 50mg / L C L-cysteine 50mg / L 200mg / L D Coenzyme I 1ng / L 50ng / L E Vitamin B12 0.1 μg / L 10 μg / L F transferrin 5mg / L 50mg / L G Coenzyme I 1mg / L 50mg / L
[0049] 3.2 Experimental Results
[0050] Based on Tables 4-5, cholesterol, coenzyme I, and vitamin B12 were identified as significant influencing factors. Mycoplasma synovitis was cultured in LMH cells selected in Examples 1 and 2 under optimized culture conditions. The LMF medium was supplemented according to the dosages listed in Table 6, and the titers were measured. The results showed that the highest titer reached was 2.84 × 10⁻⁶ after adding 25 mg / L cholesterol, 50 mg / L coenzyme I, and 2 μg / L vitamin B12. 10 CCU 50 / mL.
[0051] Table 4. Culture results with different culture medium components
[0052]
[0053]
[0054] Table 5 Coefficient Table
[0055] Serial Number factor effect coefficient T-value p-value Significance A L-arginine -1.25 -0.625 -1.72 0.161 Not significant B cholesterol 2.517 1.258 3.46 0.026 Significant C L-cysteine -1.417 -0.708 -1.95 0.123 Not significant D Coenzyme I 1.183 0.592 1.63 0.179 Not significant E Vitamin B12 -2.65 -1.325 -3.64 0.022 Significant F transferrin 0.783 0.392 1.08 0.342 Not significant G Coenzyme I 2.183 1.092 3.00 0.040 Significant
[0056] Table 6 Comparison of culture media with different nutrient components
[0057]
[0058] Example 4: Large-scale culture experiment
[0059] 4.1 Experimental materials: LMH cells, mycoplasma standard strains, 10L bioreactor, CO2 incubator, etc., LMF-SFMII medium.
[0060] 4.2 Experimental Methods:
[0061] 4.2.1 The serum-free LMH cell culture medium for Mycoplasma synovitis was prepared according to the optimal additive composition ratio screened in the experimental results of Example 3. 25 mg / L cholesterol, 50 mg / L coenzyme I, and 2 μg / L vitamin B12 were weighed, completely dissolved, and diluted to 1 L of purified water. 1 L of the prepared solution was mixed with 4 L of serum-free culture medium solution, and the pH was adjusted to 7.2 ± 0.05 with 1 mol / L sodium hydroxide. The mixture was then filtered through a 0.22 μm filter for sterilization and stored at 2-8°C. No additional serum was added.
[0062] 4.2.2 Add 5L of optimized filter medium to 10L of the bioreactor, inoculate with LMH cells, and when the LMH cell density reaches the logarithmic growth phase, the cell density will be 4×10⁴ cells / year. 6 At a concentration of cells / mL, a 10% inoculum of *Mycoplasma synoviae* standard strain was inoculated. During cultivation, the culture temperature was strictly controlled at 37℃±0.5, pH at 7.2±0.05, and dissolved oxygen (DO) at 10±5% using the bioreactor control system. The stirring speed was linked to dissolved oxygen levels, with a stirring range between 50-150 rpm. Samples were periodically taken from the bioreactor to detect the CCU titer. 50 / mL, harvest the Mycoplasma synoviae culture medium when the cell viability drops to 50-60%.
[0063] 4.2.3 The above-mentioned cell culture of Mycoplasma synovitis was diluted 10-fold to 10⁻¹¹, with five replicates at each dilution, and cultured statically at 37°C. Results were evaluated after two weeks. The number of tubes showing discoloration (pH ≥ 0.5) at each dilution was counted, and the half-maximum number of color-changing units (CCU) was calculated using the Reed and Muench methods. 50 The half-value ( / mL) was calculated using the Reed-Muench method to determine the color change endpoint.
[0064] 4.2.4 Bacterial Culture Concentration and Inactivation: Cells were removed by centrifugation at 2000 rpm for 10 min, followed by centrifugation at 12000 rpm for 40 min. The culture was resuspended in sterile PBS to 1 / 10 of its original volume and inactivated with formaldehyde to a final concentration of 0.2%. The inactivated culture was then subjected to sonication at 300 W for 3 seconds, followed by a 5-second interval, for a total duration of 10 min.
[0065] 4.2.5 Protein concentration determination: The total protein concentration of the cultures after sonication was determined using a commercially available total protein concentration assay kit (BCA kit).
[0066] 4.3 Experimental Results: After 24 hours of incubation, the titer of Mycoplasma synoviae remained stable at 10. 10 CCU 50 The culture volume of Mycoplasma synoviae was increased to above 100 mL, enabling large-scale and efficient culture of Mycoplasma synoviae.
[0067] 4.4 Fermentation was conducted using four methods: Frey medium containing 15% serum, Frey medium containing 15% serum + additives (cholesterol, coenzyme I, vitamin B12), LMH cells + LMF-SFMII medium, and LMH cells + LMF-SFMII medium + additives (cholesterol, coenzyme I, vitamin B12). (The dosage of additives is shown in 4.2.1). The results are shown in Table 7. Viable cell count (CCU) 50 The values are 2.53 × 10⁻⁶ respectively. 9 1.64×10 9 6.47×10 8 and 2.2×10 10 The protein concentrations of the 10-fold concentrated cultures were 540, 460, 330, and 820 mg / L, respectively. This shows that using LMH cells and adding additives to serum-free culture medium for large-scale culture resulted in higher titers and protein concentrations of Mycoplasma synoviae, making it more suitable for large-scale culture.
[0068] Table 7. Results of culture on different culture media at different scales
[0069]
[0070]
[0071] Example 5: Vaccine preparation and immune challenge protection test
[0072] 5.1 Vaccine preparation: The inactivated culture of Mycoplasma synovitis (MS) was stored at -20℃ and tested for sterility before use. Montanide ISA 206VG (water-in-oil) adjuvant was used to emulsify the adjuvant with the antigen at a 1:1 ratio for vaccine preparation.
[0073] 5.2 Antibody Detection: A commercially available IDEXX ELSA detection kit was used. An S / P > 0.40 was considered positive. An indirect ELISA method was employed, using MS whole-cell antigen-coated 96-well plates. Procedure: Sample dilution (1:100) → Incubation at 37℃ for 1 h → HRP-labeled secondary antibody reaction → TMB color development → Reading at 450 nm. Data Recording: OD values were calculated at each time point. 450 Mean value, S / P value for each group.
[0074] 5.3 Vaccine efficacy assessment: 21-day-old SPF chickens were randomly immunized via subcutaneous injection in the neck with an inactivated vaccine prepared from serum-free LMH cell culture of Mycoplasma synoviae, 0.2 mL × 10 8 Each group consisted of 10 birds per CCU (Central Custodian Custodian Unit). An inactivated vaccine prepared from *Mycoplasma synoviae* cultured in Frey medium and a control group were also included as challenges. Blood samples were collected before vaccination and at 14, 21, and 28 days after immunization; serum was then separated for antibody detection.
[0075] Table 8. S / P values of each ELISA group after Mycoplasma synoviae immunization in chickens
[0076]
[0077] This invention uses serum-free LMH cell culture to prepare Mycoplasma synovitis vaccine. The results of the S / P values of the ELISA results after immunization, compared with those of vaccines prepared in conventional Frey medium and control groups, are shown in Table 8. The serum-free LMH cell-prepared Mycoplasma synovitis vaccine of this invention showed an S / P value of 0.45 14 days after immunization, indicating a significant antibody response (S / P > 0.4). After 28 days, the S / P value reached 1.0, indicating a strong response. The antibody level of the MS vaccine prepared using serum-free LMH cell culture was significantly higher than that of the conventional Frey medium control group (S / P value 0.38). The S / P values of the non-immunized groups were all less than 0.4, thus establishing a valid negative control.
[0078] 5.4 Vaccine Immunization Assessment and Challenge Method: 28 days after vaccination, the chickens in all three groups were placed in a 3m... 3 Inside the isolator, a fresh culture of Mycoplasma synoviae was injected intravenously at a dose of 0.2 × 10⁻⁶. 7CCU / feather, observed for 21 days after challenge, necropsy, disease status determined, and relative protection rate calculated.
[0079] Table 9 Results of Mycoplasma synoviae Immunogenicity Test
[0080]
[0081] As can be seen from the table, when chickens were immunized with the vaccine prepared using the culture method of this invention, the protection rate against Mycoplasma synoviae challenge was 90%, while the protection rate against challenge with the traditional Frey medium culture was 20%. All members of the challenge control group developed the disease. Compared with the vaccine prepared with the traditional Frey medium, the serum-free LMH cell culture Mycoplasma synoviae vaccine prepared by this invention has stronger protective efficacy.
[0082] The above embodiments are merely illustrative and not restrictive. The scope of the invention is defined by the claims. Those skilled in the art will understand that various modifications and variations can be made to the invention without departing from the spirit and scope of this application, and all such modifications and variations are within the scope of the invention.
Claims
1. A method for culturing Mycoplasma synoviae, characterized in that, The cultivation method includes: S1. Prepare serum-free culture medium solution; S2. Inoculate chicken liver cancer cell line LMH cells and adjust cell density; S3. Inoculate with the revived Mycoplasma synoviae culture for further culture; S4. After culturing, concentrate and inactivate the enzyme.
2. The method for culturing Mycoplasma synoviae according to claim 1, characterized in that, The serum-free culture medium described in step S1 contains LMF-SFMII basal medium, cholesterol, nicotinamide adenine dinucleotide, and vitamins.
3. The method for culturing Mycoplasma synoviae according to claim 1, characterized in that, The density of the chicken liver cancer cell line LMH in step S2 was 1.0 × 10⁻⁶ cells. 6 cells / mL.
4. The method for culturing Mycoplasma synoviae according to claim 1, characterized in that, The concentration of the Mycoplasma synoviae bacterial solution introduced in step S3 is 10%.
5. The method for culturing Mycoplasma synoviae according to claim 2, characterized in that, The serum-free culture medium in step S1 contains 25 mg / L cholesterol, 50 mg / L nicotinamide adenine dinucleotide, and 2 μg / L vitamins.
6. The method for culturing Mycoplasma synovitis according to claim 1, characterized in that, The culture conditions after inoculating the Mycoplasma synoviae culture in step S3 are as follows: culture temperature 37℃±0.5, pH 7.2±0.05, DO 10±5%, and stirring speed 50-150rpm.
7. The method for culturing Mycoplasma synoviae according to claim 1, characterized in that, The titer of Mycoplasma synovitis can reach 2.84 × 10⁻⁶. 10 CCU 50 / mL.
8. The application of the method for culturing Mycoplasma synovitis as described in claim 1 in the preparation of Mycoplasma synovitis vaccine.