Mycoplasma caninum as well as culture method and application thereof

Abstract

The invention provides a mycoplasma caninum, a culture method and application thereof. According to the invention, a new strain of mycoplasma canis is obtained through separation, the mycoplasma canis is named as QAU241017.16, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 23, 2025, and the preservation number is CGMCC No. 46643. The isolates have eight amino acid site mutations on the sialidase gene, and animal experiments prove that the toxicity of the isolates is remarkably enhanced. The inactivated vaccine prepared from the mycoplasma canine has good immunogenicity and safety, and can effectively stimulate an organism to generate an antibody aiming at the mycoplasma canine. The inactivated vaccine prepared on the basis of the strain can induce the neutralizing antibody titer to be 1: 128. The invention can provide a new scheme for prevention and control of canine mycoplasma. Compared with a traditional culture method, the canine mycoplasma separation culture method provided by the invention is lower in cost, more efficient and capable of realizing rapid proliferation of mycoplasma and achieving higher viable bacterium titer. And a technical support is provided for isolated culture of the canine mycoplasma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a Mycoplasma canis, a culture method and application thereof, in particular to a Mycoplasma canis (Mycoplasma canis, M. canis) isolated strain QAU241017.16 with a specific mutation in a sialidase gene, a high-efficiency separation and culture method of the strain, pathogenicity analysis and an inactivated vaccine prepared based on the strain. BACKGROUND

[0002] Mycoplasma canis is a kind of prokaryotic cell type microorganism, and is one of the important pathogens of canine respiratory disease complex (CRDC). The pathogen is transmitted by aerosol and can cause acute bronchitis, bronchiolitis and interstitial pneumonia and other respiratory diseases. The incidence rate can reach 70% to 90% in high-density breeding environments such as dog houses. Mycoplasma canis often forms a synergistic infection with canine distemper virus, adenovirus and bronchial septic Bordetella, etc., leading to prolonged course and complicated treatment. At present, the prevention and treatment of Mycoplasma canis mainly relies on antibiotics, but the abuse of antibiotics may lead to the emergence of drug resistance, thereby affecting the treatment effect. Therefore, it is of great practical significance to develop a safe and effective vaccine for Mycoplasma canis.

[0003] In the field of pathogen research, the separation and culture of Mycoplasma canis still faces technical bottlenecks. The strain needs to be cultured in a Mycoplasma culture medium containing 10% to 20% horse serum at 5% CO2 and 37℃ for 5 to 7 days to form a typical "scrambled egg-like" colony. The separation and identification of Mycoplasma canis are time-consuming and difficult. Since the titer of Mycoplasma in the primary culture is usually less than 10 4 CCU / mL, and is easily contaminated by symbiotic bacteria such as L-form bacteria, the positive detection rate is less than 55%, and the probability of successfully isolating Mycoplasma canis is very low. In addition, Mycoplasma canis has high nutritional requirements, grows slowly and has a low titer, so the number of strains available for vaccine production is extremely limited. Therefore, it is of great significance to develop a low-cost and high-efficiency culture method for Mycoplasma canis to improve the separation efficiency of Mycoplasma canis and promote vaccine preparation. SUMMARY

[0004] The purpose of the present application is to provide a Mycoplasma canis with a specific mutation in a virulence gene, a high-efficiency separation and culture method thereof and an inactivated vaccine with good immunogenicity and safety prepared based on the strain.

[0005] The present application provides a Mycoplasma canis, which is named QAU241017.16 and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 23, 2025, with a preservation number of CGMCC No. 46643.

[0006] The DNA sequence of the Mycoplasma cynos QAU241017.16S is shown as SEQ ID No. 1.

[0007] The sialidase of the Mycoplasma cynos QAU241017.16 has eight amino acid mutations of K34N, D95N, D96N, N136D, K212E, A224S, A293S, and A916V, and the amino acid sequence of the sialidase is shown as SEQ ID No. 2. It is verified by animal challenge test that the mutant strain can cause C-reactive protein, white blood cell inflammation index, and respiratory tract detoxification amount to be higher than the wild strain QAU240523.3, confirming that the virulence is enhanced.

[0008] The application also provides application of the Mycoplasma cynos in preparation of a vaccine for preventing bronchitis and pneumonia caused by Mycoplasma cynos.

[0009] The application also provides a Mycoplasma cynos inactivated vaccine preparation, which comprises an immunologically effective amount of inactivated Mycoplasma cynos QAU241017.16 and a vaccine adjuvant.

[0010] The vaccine preparation contains at least 1×10 9 CCU / mL of bacteria per dose.

[0011] The preparation method of the Mycoplasma cynos inactivated vaccine preparation comprises the following steps:

[0012] Strain culture: inoculate the Mycoplasma cynos isolated strain QAU241017.16 into a Mycoplasma liquid culture medium, and cultivate at 37℃ under oscillation and light avoidance to the logarithmic growth phase to obtain a high-concentration Mycoplasma bacterial solution;

[0013] Inactivation treatment: add a formaldehyde solution to the Mycoplasma bacterial solution to make the final concentration of formaldehyde 0.3%, and place in a 37℃, 160rpm oscillation incubator for inactivation for 24 hours;

[0014] Inactivated vaccine preparation: mix the inactivated Mycoplasma bacterial solution with the water-for-injection adjuvant at a volume ratio of 1:1 to prepare the inactivated vaccine.

[0015] The application also provides a Mycoplasma cynos isolation and culture method, comprising the following steps:

[0016] (1) Optimization of Mycoplasma cynos culture medium and culture conditions:

[0017] Preparation of the Mycoplasma liquid culture medium: take 27.52g of Mycoplasma broth culture medium, dissolve in 800ml of distilled water, sterilize and cool to room temperature, then add 10%-20% volume fraction of newborn calf serum, and adjust the pH value of the culture medium to 7.6-7.8;

[0018] Liquid culture conditions: take the concentration of 10 9 CCU / mL mycoplasma bacteria liquid is inoculated in the liquid culture medium, and is cultured in the shaking incubator at 37 DEG C and 160 rpm in the dark;

[0019] Mycoplasma solid culture medium preparation: mycoplasma broth medium 27.52 g is added to agar powder 5.6 g, dissolved in 800 ml distilled water, sterilized and cooled to 55-60 DEG C, then 20% volume fraction of newborn calf serum is added, and the pH value is adjusted to 7.6-7.8;

[0020] Culture conditions: the concentration of 10 9 CCU / mL mycoplasma bacteria liquid is inoculated on the solid culture medium, and is cultured in the incubator at 37 DEG C and 5% CO2 concentration for 24 h, and then is cultured for 3-4 d, and the humidity of the culture environment needs to be maintained above 95%;

[0021] (2) sample collection: collecting the oral and nasal swabs of the dogs infected with respiratory diseases;

[0022] (3) isolation and purification of mycoplasma: the positive sample is filtered through a 0.22 mu m filter membrane, inoculated in the mycoplasma liquid culture medium prepared in step (1), cultured in a shaking incubator at 37 DEG C, and detected and subcultured when the culture medium becomes clear orange; the bacteria liquid subcultured to the 3rd generation is cultured to 10 9 CCU / mL, 1 mL is inoculated on the mycoplasma solid culture medium prepared in step (1), cultured in the incubator at 37 DEG C and 5% CO2 concentration for 24 h, and then cultured for 3-4 d, and the solid culture medium changes from red to yellow, which is observed under a microscope; a single colony is picked to the mycoplasma liquid culture medium, and repeated purification is carried out for 3 times to obtain pure culture.

[0023] The new mycoplasma canis isolation and culture method provided by the application improves the isolation efficiency and viable bacterial titer (CCU / mL) by optimizing the culture medium and culture adjustment:

[0024] The liquid culture is cultured in the shaking incubator in the dark, which avoids the adverse effects of ultraviolet rays on the growth and metabolism of mycoplasma canis, ensures the normal growth of the bacteria, and improves the viable bacterial titer of the bacteria.

[0025] The solid culture adopts agar powder with a mass fraction of 0.7% to prepare the solid culture medium, and a plate of pure water sterilized by high pressure is placed in the incubator to maintain the humidity of the culture environment above 95%, the agar concentration is verified as the most suitable concentration for the growth of bacteria through comparative experiments, and the precise humidity control creates a suitable environment for the growth of mycoplasma canis, further improving the growth effect and culture success rate of mycoplasma canis on the solid culture medium.

[0026] The beneficial effects of the present application are that the Mycoplasma cynos isolation and culture method provided by the present application is lower in cost and more efficient than the traditional culture method, and can achieve rapid proliferation of Mycoplasma and reach a higher viable bacterial titer. The present application successfully obtains a Mycoplasma cynos isolate with specific mutations in virulence genes by using an improved culture medium and culture method. The isolate has 8 amino acid site mutations in the sialidase gene, and animal experiments confirm that its virulence is significantly enhanced. Further, the present application finds that the inactivated vaccine prepared from the Mycoplasma cynos has good immunogenicity and safety, and can effectively stimulate the body to produce antibodies against Mycoplasma cynos. The inactivated vaccine prepared based on the strain can induce a neutralizing antibody titer of 1:128, and the present application can provide a new solution for the prevention and control of Mycoplasma cynos. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : Comparison chart of viable bacterial titers under different culture conditions of the present application. 1: light-avoiding group; 2: non-light-avoiding group; A: growth chart of Mycoplasma cynos in liquid culture medium; B: influence test chart of CCU determination of Mycoplasma cynos growth.

[0028] Figure 2 : Colony morphology chart of Mycoplasma cynos isolate QAU241017.16 of the present application. A: isolate colony; B: isolate colony microscopic examination (40X).

[0029] Figure 3 : Electron microscope observation chart of Mycoplasma cynos isolate QAU241017.16 of the present application. A (×60,000); B (×50,000).

[0030] Figure 4 : Genetic evolution analysis chart of 16S gene sequence of Mycoplasma cynos isolate QAU241017.16 of the present application. In the chart, ● represents the strain QAU241017.16 of the present application; M.felis cat Mycoplasma; M.gallinarum. chicken Mycoplasma; M.bovis. bovine Mycoplasma.

[0031] Figure 5 : Growth curve chart of Mycoplasma cynos isolate QAU241017.16 of the present application.

[0032] Figure 6 : Comparison result of sialidase gene amino acid mutation sites of Mycoplasma cynos isolate QAU241017.16 of the present application.

[0033] Figure 7: Figure of changes of inflammation indexes of each group of dogs in Example 7 of the present application. Wherein A: figure of changes of C-reactive protein in animal challenge test; B: figure of changes of white blood cells in animal challenge test. In the figure, test group 1: test group infected by isolated strain QAU241017.16; test group 2: test group infected by isolated strain QAU240523.3;

[0034] Figure 8 : Figure of changes of pathogen detoxification amount of each group of dogs in Example 7 of the present application. In the figure, test group 1: infection group of isolated strain QAU241017.16; test group 2: infection group of isolated strain QAU240523.3. DETAILED DESCRIPTION

[0035] In the research process of Mycoplasma canis isolation and culture, the present applicant found that the existing Mycoplasma culture medium on the market cannot completely meet the nutritional conditions required for the growth of Mycoplasma. In view of this problem, the present application develops a new type of improved culture medium and efficient culture technology from the growth and culture characteristics of Mycoplasma, and successfully isolates a strain of Mycoplasma canis. Further, an inactivated vaccine with good immunogenicity and safety is prepared, which can effectively stimulate the body to produce specific antibodies against Mycoplasma canis.

[0036] The present application will be further described below in conjunction with examples and drawings, but in no way limits the present application.

[0037] Example 1: Optimization of liquid culture medium and culture conditions of Mycoplasma canis

[0038] Preparation of liquid culture medium: 27.52 g of Mycoplasma broth medium was dissolved in 800 ml of distilled water, and sterilized at 121℃ for 15 minutes. After cooling to room temperature, 10% to 20% volume fraction of newborn calf serum was added, and the pH value of the culture medium was adjusted to 7.6-7.8 by pH buffer.

[0039] Optimization of liquid culture conditions: 400 μL of Mycoplasma bacteria solution with a concentration of 10 9 CCU / mL was inoculated into two 10 mL centrifuge tubes containing 4800 μL of the above liquid culture medium, sealed with a sealing film, and placed in a shaking incubator at 37℃ and 160 rpm. One was cultured in the dark, and the other was not cultured in the dark. The CCU was measured every 6 h, the growth state was observed, and the growth curve was drawn, and the results are shown in Figure 1 : The viable bacteria titer of the dark culture group reached 10 9 CCU / mL after 24 h of culture, which was significantly higher than 10 5 CCU / mL of the non-dark culture group, confirming that the dark condition is a key factor for improving the titer.

[0040] Example 2: Optimization of solid culture medium and culture conditions of Mycoplasma canis

[0041] Agar concentration screening of solid medium: 0.5%, 0.7%, and 0.9% agar powder were used to prepare solid medium with different concentrations of agar, which was autoclaved at 121°C for 15 minutes. When the temperature decreased to 50-60°C, 20% newborn calf serum was added, and the pH value was adjusted to 7.6-7.8. 10 9 CCU / mL of mycoplasma liquid was plated on the self-made solid medium and incubated upright in a 37°C incubator with 5% CO2 for 24 hours, followed by inverted incubation for 3-4 days. During the incubation period, a plate of distilled water sterilized by high-pressure steam was placed in the incubator to maintain the humidity above 95%.

[0042] The test results showed that after 4 days, no colonies grew on the 0.5% solid medium, 33±5 colonies grew on the 0.9% solid medium, and 59±5 colonies grew on the 0.7% solid medium. Therefore, the optimal growth concentration of agar was 0.7%.

[0043] Based on the screening results, the final formula was established: 27.52g of mycoplasma broth medium was added with 5.6g of agar powder dissolved in 800ml of distilled water, autoclaved at 121°C for 15 minutes, and cooled to 55°C before adding 20% newborn calf serum.

[0044] Example 3: Isolation and identification of canine mycoplasma isolates

[0045] Sample collection: In 2023-2024, 390 nasal swabs from dogs suspected of respiratory disease infection were collected from Qingdao Animal Hospital and Dog Farm.

[0046] Isolation and purification of mycoplasma: Positive samples were filtered through a 0.22 μm filter and inoculated into modified mycoplasma liquid medium, then incubated in a 37°C shaking incubator. When the medium turned clear orange, fluorescence quantitative PCR detection was performed and the culture was passaged. The 3rd passage of the culture was incubated to 10 9 CCU / mL, 1mL was plated on the self-made mycoplasma solid medium and incubated upright in a 37°C incubator with 5% CO2 for 24 hours, followed by inverted incubation for 3-4 days. The solid medium turned from red to yellow, and it was observed under a microscope to observe the "scrambled egg-like" mycoplasma colonies, as shown in Figure 2 The single colonies were picked into mycoplasma liquid medium and purified for 3 times to obtain pure culture.

[0047] Electron microscopy observation as shown in Figure 3 The third passage of the isolate was taken, and the concentration of the bacterial solution was 1×10 9CCU / mL, which were observed as round shape with 80-100 nm in diameter under transmission electron microscope after staining with phosphotungstic acid.

[0048] Example 4: Genetic evolution analysis of Mycoplasma canis isolate QAU241017.16

[0049] Mycoplasma 16S gene amplification and sequencing analysis: The nucleic acid of the isolate was extracted using a DNA / RNA extraction kit, and PCR amplification was performed using bacterial 16S rDNA primers, the primer sequences were as follows:

[0050] F, 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 3);

[0051] R, 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID No. 4).

[0052] Reaction system: 2x Phanta Max Master Mix (Dye Plus) 12.5 μL; upstream primer 1.0 μL; downstream primer 1.0 μL; template 2 μL; ddH2O 8.5 μL.

[0053] Reaction conditions: pre-denaturation 98℃ 3min; 98℃ 10s, 55℃ annealing 15s, 72℃ extension 20s, 34 cycles; 72℃ final extension 2min.

[0054] The sequencing results of the isolated strain were compared in GenBank by BLAST. The results showed that the 16S rDNA gene sequence of the QAU241017.16 isolate had the highest homology with the sequence of M. canis published in NCBI. The phylogenetic tree was constructed using MEGA 7, as shown in Figure 4 Figure 1, showing that the isolate was located in the same branch as M. canis.

[0055]

[0056] Example 5: Growth curve determination of Mycoplasma cynos isolate QAU241017.16

[0057] After the culture solution of the isolate to be separated turned from red to yellow, it was transferred to new liquid culture medium at a proportion of 5%, and cultured in a 37°C shaking incubator. Every 12 h, a portion of the bacterial solution was taken to determine the color change unit (CCU). The results are shown in Table 2. Figure 5 It is shown that the isolate is in the logarithmic growth phase within 24 h of culture, remains stable from 24 to 36 h, and then enters the decline phase.

[0058] Example 6: Analysis of sialidase amino acid mutation sites of Mycoplasma cynos isolate QAU241017.16

[0059] Sialidase (Sialidase protein) amino acid sequence analysis: The Sialidase protein gene sequences of Mycoplasma cynos reference strains (CP0144281.1, JQ177148.1, JQ177149.1, OP354259.1) were obtained through the NCBI database, and the MegAlign software was used to perform amino acid sequence alignment analysis on the local isolate QAU241017.16 and the wild strain QAU240523.3. It is shown that the isolate QAU241017.16 identifies 8 unique mutation sites in the Sialidase protein: K34N, D95N, D96N, N136D, K212E, A224S, A293S, and A916V, indicating that the isolate QAU241017.16 of the present application has adaptive evolution under host immune pressure or environmental changes, which further leads to changes in antigenic epitopes, so that the pathogen escapes the host immune memory.

[0060]

[0061] The sialidase gene amino acid mutation site alignment results of the Mycoplasma canis isolate QAU241017.16 are shown in Table 2. Figure 6

[0062] Example 7: Pathogenicity verification of Mycoplasma canis mutant strain QAU241017.16

[0063] 9 three-month-old healthy Beagle dogs were randomly divided into test group 1 (3 dogs), test group 2 (3 dogs), and a control group (3 dogs). The mutant isolate QAU241017.16 and the wild strain QAU240523.3 were both cultured to 1.0 x 10 11 CCU / ml, and were injected into the trachea in a route of 2 ml for each of the test group 1 and the test group 2, and 2 ml of Mycoplasma liquid medium was injected into the trachea of the control group. After infection, the three groups of test dogs were isolated and fed, and were observed for 14 days. The clinical manifestations of the test dogs were observed and recorded every day, blood was collected every 3 days for determination of blood routine and C-reactive protein indicators, and eye and nose pharynx swabs were collected every 3 days for DNA extraction, and qPCR was performed to detect the pathogen shedding amount of Mycoplasma canis.

[0064] Table 1: Mycoplasma canis qPCR detection primer probe sequence

[0065] The results show that, as shown in Table 3, the C-reactive protein (CRP) and white blood cell (WBC) indicators of the test group 1 infected with the isolate QAU241017.16 were higher than those of the test group 2 infected with the isolate QAU240523.3 and the control group from 3 to 9 days after infection. As shown in Table 4, the pathogen shedding amount of Mycoplasma canis of the test group 1 infected with the isolate QAU241017.16 was higher than that of the test group 2 infected with the isolate QAU240523.3 and the control group. Figure 7 Figure 8

[0066] Example 8: Preparation of Mycoplasma canis inactivated vaccine

[0067] Strain culture: The Mycoplasma canis isolate QAU241017.16 was inoculated into Mycoplasma liquid medium and cultured at 37°C with shaking to the logarithmic growth phase to obtain a high-concentration Mycoplasma bacterial solution.

[0068] Inactivation treatment: Formaldehyde solution was added to the Mycoplasma bacterial solution to a final concentration of 0.3%, and the solution was placed in a 37°C, 160 rpm shaking incubator for inactivation for 24 hours.

[0069] Inactivated vaccine preparation: The inactivated Mycoplasma bacterial solution (1.0 x 10 9 CCU / mL) was mixed with the water-based adjuvant at a volume ratio of 1:1 to prepare the inactivated vaccine.​​​

[0070] Example 9: Sterility test of Mycoplasma canis inactivated vaccine

[0071] Sterility test: The inactivated vaccine was coated into TSA containing 5% serum, and incubated at 37°C for 48h, and no bacteria grew.

[0072] Example 10: Safety test of Mycoplasma canis inactivated vaccine

[0073] Safety test: 10 six-week-old Kunming mice were prepared, half of them were in the control group and the other half were in the test group. The mice in the test group were injected subcutaneously with 0.2 mL vaccine, and the mice in the control group were injected subcutaneously with the same volume of Mycoplasma liquid medium. After 14 days of observation, all the mice in the test group were alive, which met the safety test requirements.

[0074] Example 11: Formaldehyde residue test of Mycoplasma canis inactivated vaccine

[0075] Formaldehyde residue test: According to the current edition of Chinese Veterinary Pharmacopoeia Appendix, the formaldehyde residue was 0.09%, which met the safety standards.

[0076] Example 12: Immunogenicity test of Mycoplasma canis inactivated vaccine

[0077] Immunization test: 6 healthy 7-8-day-old New Zealand white rabbits were randomly divided into 2 groups, 3 rabbits in each group. The rabbits in the vaccine group were injected subcutaneously with 1 mL inactivated vaccine in the neck, and the rabbits in the control group were injected subcutaneously with 1 mL Mycoplasma liquid medium containing 0.3% formaldehyde in the neck. Seven days after immunization, the same route was used for booster immunization once, and 14 days after the second immunization, the same route was used for booster immunization once, and 14 days after the third immunization, the same route was used for booster immunization once.

[0078] Antibody titer detection: every 1 week, blood was collected from the marginal vein, and the serum was diluted 10 times in 2 times gradient, and the antibody titer of Mycoplasma canis in the serum of rabbits in each group was detected by neutralization test method.

[0079] Neutralization test method: Mycoplasma isolated bacteria were cultured to 10 9CCU / mL, 96-well cell culture plates were taken, 50 μL PBS was added to each well, 50 μL of the canine serum to be tested was added to the first well, and then serial dilution (1:2 to 1:1024) was performed in turn, 3 repeated wells were set for each dilution, and positive control (containing bacterial liquid) and negative control (without sterile liquid) were set. Add an equal volume of mycoplasma bacteria liquid to each well, mix gently, and then place in an incubator at 37°C and 5% CO2 concentration for 1 hour. Transfer 100 μL of reaction solution from each well to a 2 mL centrifuge tube containing 900 μL of mycoplasma liquid medium, and place in a shaking incubator. Observe the color change of the medium every 3 hours, and continue to monitor until the positive control group is completely yellow. The highest serum dilution that completely inhibits the growth of mycoplasma is the neutralizing antibody titer. The judgment standard is: if the medium of a certain dilution remains red, it is determined to be effective neutralization, otherwise it is inactive. The results show that the antibody titer of the vaccine group of rabbits is 1:128, and the antibody titer of the control group of rabbits is ≤1:2.

[0080] Table 2 Change of antibody titer after immunization with vaccine

[0081] The above examples show that the mycoplasma canella isolation and culture method of the present application can achieve rapid proliferation of mycoplasma and achieve a higher viable bacterial titer than the traditional culture method; the improved culture medium and culture method are used to successfully isolate a mutant isolate QAU241017.16, which has 8 amino acid site mutations in the sialidase gene, and animal experiments confirm that its virulence is significantly enhanced; the inactivated vaccine prepared based on the strain can induce a neutralizing antibody titer of 1:128, providing a new solution for the prevention and control of canine mycoplasma.

Claims

1. A Mycoplasma canis strain, characterized in that: Mycoplasma canis QAU241017.16 was deposited in the General Microbiology Center of China General Culture Collection Administration CGMCC on July 23, 2025, with the deposit number CGMCC No. 46643.

2. The Mycoplasma canis according to claim 1, characterized in that The 16S DNA sequence of the Mycoplasma canis QAU241017.16 is shown in SEQ ID No.

1.

3. The Mycoplasma canis according to claim 1, characterized in that The Mycoplasma canis QAU241017.16 sialidase has eight amino acid mutations: K34N, D95N, D96N, N136D, K212E, A224S, A293S, and A916V. The amino acid sequence of the sialidase is shown in SEQ ID No.

2.

4. Use of the Mycoplasma canis according to claim 1 in the preparation of a vaccine for preventing bronchitis and pneumonia caused by Mycoplasma canis.

5. A canine Mycoplasma inactivated vaccine preparation, characterized in that: The preparation comprises an immunologically effective amount of the inactivated Mycoplasma canis according to claim 1 and a vaccine adjuvant.

6. An inactivated Mycoplasma canis vaccine preparation according to claim 5, characterized in that: Each dose contains at least 1×109 CCU / mL of bacteria.

7. A method for preparing an inactivated Mycoplasma canis vaccine preparation, characterized in that: The following steps are involved: Strain culture: Mycoplasma canis isolate QAU241017.16 was inoculated into mycoplasma liquid culture medium and cultured at 37°C in the dark with shaking until the logarithmic growth phase to obtain a high-concentration mycoplasma culture solution. Inactivation treatment: add formaldehyde solution to the mycoplasma solution to make the final formaldehyde concentration 0.3%, and place it in a shaking incubator at 37°C and 160 rpm for 24 hours; Preparation of inactivated vaccine: Mix the inactivated mycoplasma solution with the water adjuvant in a 1:1 volume ratio to prepare the inactivated vaccine.

8. A method for isolating and culturing Mycoplasma canis, characterized in that: The steps include: (1) Optimization of culture medium and culture conditions for Mycoplasma canis: Preparation of Mycoplasma liquid culture medium: Dissolve 27.52 g of Mycoplasma broth in 800 ml of distilled water, sterilize and cool to room temperature, then add 10% to 20% by volume of newborn calf serum and adjust the pH of the culture medium to 7.6-7.

8. Liquid culture conditions: take the concentration of 10 9 CCU / mL of mycoplasma bacteria were inoculated into liquid culture medium, sealed, and placed in a shaking incubator at 37°C and 160 rpm in the dark for incubation; Preparation of mycoplasma solid culture medium: Add 5.6 g agar powder to 27.52 g of mycoplasma broth and dissolve it in 800 ml of distilled water. Sterilize and cool to 55-60°C. Then add 20% by volume of newborn calf serum and adjust the pH to 7.6-7.

8. Culture conditions: The concentration was 10 9 CCU / mL of mycoplasma liquid was inoculated onto solid culture medium and cultured upright for 24 hours in an incubator at 37°C and 5% CO2 concentration, then inverted for 3-4 days. The humidity of the culture environment should be maintained above 95% during the culture period. (2) Sample collection: Collect oral and nasal swabs from dogs infected with respiratory diseases; (3) Isolation and purification of mycoplasma: After filtering the positive sample through a 0.22 μm filter membrane, inoculate it into the mycoplasma liquid culture medium prepared in step (1) and culture it in a shaking incubator at 37°C. When the culture medium turns clear orange, perform the test and pass it on to the next generation. Culture the culture medium of the third generation to 10 9 1 mL of CCU / mL was spread on the mycoplasma solid culture medium prepared in step (1), and cultured upright in an incubator at a temperature of 37°C and a CO2 concentration of 5% for 24 h, and then cultured in an inverted manner for 3 to 4 days. When the solid culture medium turns from red to yellow, the "fried egg-like" mycoplasma colonies are observed under a microscope; a single colony was picked and transferred to the mycoplasma liquid culture medium, and purified three times to obtain a pure culture.

9. A liquid culture medium for Mycoplasma canis, characterized in that The liquid culture medium is prepared by the following method: 27.52 g of mycoplasma broth culture medium is dissolved in 800 ml of distilled water, sterilized and cooled to room temperature, and then 10% to 20% volume fraction of newborn calf serum is added, and the pH value of the culture medium is adjusted to 7.6-7.

8.

10. A solid culture medium for Mycoplasma canis, characterized in that: The solid culture medium is prepared by the following method: 27.52 g of mycoplasma broth culture medium is added with 5.6 g of agar powder and dissolved in 800 ml of distilled water, sterilized and cooled to 55° C.-60° C., and then 20% volume fraction of newborn calf serum is added, and the pH value is adjusted to 7.6-7.8.

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