Mycoplasma cynos, culture method and application thereof

Abstract

The application provides a canine mycoplasma, a culture method and application thereof. A new canine mycoplasma is isolated, and the canine mycoplasma is named QAU241017.16 and preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 23, 2025, with a preservation number of CGMCC No. 46643. The isolated strain has 8 amino acid site mutations on a sialidase gene, and the animal experiment proves that the virulence is significantly enhanced. The inactivated vaccine prepared from the canine mycoplasma has good immunogenicity and safety, and can effectively stimulate the body to produce antibodies against the canine mycoplasma. The inactivated vaccine prepared based on the strain can induce a neutralizing antibody titer of 1:128. The application can provide a new scheme for the prevention and control of the canine mycoplasma. The canine mycoplasma isolation and culture method provided by the application is lower in cost, more efficient, and can realize rapid proliferation of the mycoplasma, so that a higher viable bacterial titer is achieved. Technical support is provided for the isolation and 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: Use a concentration of 10 9 CCU / mL mycoplasma bacterial suspension was inoculated into liquid culture medium, sealed, and placed in a shaking incubator at 37℃ and 160 rpm in the dark.

[0019] Preparation of mycoplasma solid culture medium: 27.52g of mycoplasma broth medium was dissolved in 800ml of distilled water with 5.6g of agar powder. After sterilization and cooling to 55℃-60℃, 20% (v / v) of newborn calf serum was added, and the pH was adjusted to 7.6-7.8.

[0020] Culture conditions: Use a concentration of 10... 9 CCU / mL mycoplasma bacterial suspension was inoculated onto a solid culture medium and incubated upright in an incubator at 37℃ and 5% CO2 concentration for 24 hours, then inverted for 3-4 days. During the incubation period, the humidity of the culture environment should be maintained above 95%.

[0021] (2) Sample collection: Collect nasal and oral swabs from dogs with respiratory infections;

[0022] (3) Isolation and purification of mycoplasma: After filtering the positive sample through a 0.22 μm filter membrane, it was inoculated into the mycoplasma liquid culture medium prepared in step (1) and cultured in a shaking incubator at 37°C. When the culture medium turned clear orange-yellow, it was detected and passaged. The bacterial culture passaged to the 3rd generation was cultured to 10 9 CCU / mL, 1mL is spread evenly on the mycoplasma solid culture medium prepared in step (1), and incubated upright in an incubator at 37℃ and CO2 concentration of 5% for 24h, and then inverted for 3-4d. The solid culture medium turns from red to yellow. Observe the "fried egg" mycoplasma colonies under a microscope; pick a single colony into mycoplasma liquid culture medium and repeat the purification 3 times to obtain pure culture.

[0023] The novel canine mycoplasma isolation and culture method provided by this invention improves isolation efficiency and viable cell titer (CCU / mL) through the following optimization of culture medium and culture conditions:

[0024] Liquid culture was carried out in a shaking incubator in the dark. The dark culture avoided the adverse effects of ultraviolet light on the growth and metabolism of Mycoplasma canis, ensuring the normal growth of the bacteria and increasing the viable titer of the bacteria.

[0025] Solid culture was prepared using agar powder with a mass fraction of 0.7%. A tray of autoclaved pure water was placed in the incubator to maintain the humidity of the culture environment at above 95%. The agar concentration was verified by comparative experiments to be the optimal concentration for bacterial growth. The precise humidity control created 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 this invention are as follows: The canine mycoplasma isolation and culture method provided by this invention is lower in cost and more efficient than traditional culture methods, and can achieve rapid proliferation of mycoplasma, reaching a higher viable titer. This invention successfully obtained a canine mycoplasma isolate with a specific mutation in its virulence gene using an improved culture medium and method. This isolate has an 8-amino acid mutation in the sialidase gene, and animal experiments have confirmed its significantly enhanced virulence. Furthermore, this invention found that the inactivated vaccine prepared using this canine mycoplasma has good immunogenicity and safety, and can effectively stimulate the body to produce antibodies against canine mycoplasma. The inactivated vaccine prepared based on this strain can induce a neutralizing antibody titer of 1:128. This invention can provide a new solution for the prevention and control of canine mycoplasma. Attached Figure Description

[0027] Figure 1 Comparison of viable bacterial titers under different culture conditions in this invention. 1: Light-protected group; 2: Light-free group; A: Growth of Mycoplasma canis in liquid culture medium; B: Effect of CCU assay on Mycoplasma canis growth.

[0028] Figure 2 : Colony morphology of the canine mycoplasma isolate QAU241017.16 of this invention. A: Colony of the isolate; B: Microscopic examination of the colony of the isolate (40X).

[0029] Figure 3 Electron micrographs of the canine mycoplasma isolate QAU241017.16 of this invention. A (×60,000); B (×50,000).

[0030] Figure 4 Genetic evolutionary analysis diagram of the 16S gene sequence of the canine mycoplasma isolate QAU241017.16 of this invention. ● in the diagram represents strain QAU241017.16 of this invention; M. felis (feline mycoplasma); M. gallinarum (chicken mycoplasma); M. bovis (bovine mycoplasma).

[0031] Figure 5 Growth curve of the canine mycoplasma isolate QAU241017.16 of this invention.

[0032] Figure 6 The results of amino acid mutation site comparison of the sialidase gene of the canine mycoplasma isolate QAU241017.16 in this invention.

[0033] Figure 7Example 7 of this invention shows the changes in inflammatory markers in each group of dogs. A: Changes in C-reactive protein in the animal challenge test; B: Changes in leukocytes in the animal challenge test. In the figures, test group 1 is the test group infected with isolate QAU241017.16; test group 2 is the test group infected with isolate QAU240523.3.

[0034] Figure 8 Example 7 of this invention: Graph showing the change in viral shedding of canine pathogens in each group. In the graph, test group 1: infected with isolated strain QAU241017.16; test group 2: infected with isolated strain QAU240523.3. Detailed Implementation

[0035] During the research on the isolation and culture of canine mycoplasma, the applicant discovered that existing commercially available mycoplasma culture media cannot fully meet the nutritional requirements for mycoplasma growth. To address this issue, this invention, based on the growth and culture characteristics of mycoplasma, developed a novel improved culture medium and a highly efficient culture technique, and successfully isolated a strain of canine mycoplasma. Furthermore, an inactivated vaccine with good immunogenicity and safety was prepared, which can effectively stimulate the body to produce specific antibodies against canine mycoplasma.

[0036] The present invention will be further described below with reference to embodiments and accompanying drawings, but in no way should the present invention be limited.

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

[0038] Preparation of liquid culture medium: Weigh 27.52g of mycoplasma broth culture medium, dissolve it in 800ml of distilled water, autoclave at 121℃ for 15 minutes, cool to room temperature, add 10% to 20% (v / v) of newborn calf serum, and adjust the pH of the culture medium to 7.6-7.8 using pH buffer.

[0039] Optimization of liquid culture conditions: A concentration of 10... 9 400 μL of CCU / mL mycoplasma bacterial suspension was evenly inoculated into two 10 mL centrifuge tubes containing 4800 μL of the above liquid culture medium. The tubes were sealed with sealing film and placed in a shaking incubator at 37℃ and 160 rpm. One tube was incubated in the dark, while the other was incubated in the light-free environment. CCU levels were measured every 6 hours to observe the growth status and plot growth curves. The results are as follows: Figure 1 As shown: the viable bacterial titer in the light-protected group reached 10 after 24 hours of incubation. 9 CCU / mL was significantly higher than that of the non-light-protected group (10). 5 The CCU / mL test confirmed that light-protected conditions are a key factor in increasing the titer.

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

[0041] Screening of agar concentrations in solid culture media: Solid culture media with different agar concentrations were prepared by taking 0.5%, 0.7%, and 0.9% agar powder. These media were autoclaved at 121℃ for 15 minutes. When the temperature dropped to 50-60℃, 20% (v / v) newborn calf serum was added to adjust the pH to 7.6-7.8. Agar concentrations of 10... 9 500 μL of CCU / mL mycoplasma liquid culture was spread on a self-made solid culture medium and incubated upright in an incubator at 37℃ and 5% CO2 concentration for 24 hours. Then it was inverted and incubated for 3-4 days. During the incubation period, a tray of pure water sterilized by high pressure steam was placed in the incubator to maintain the humidity of the culture environment above 95%.

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

[0043] Based on the screening results, the final formula was established: 27.52g of mycoplasma broth culture medium was added to 5.6g of agar powder and dissolved in 800ml of distilled water, sterilized at 121℃ for 15 minutes, cooled to 55℃ and then 20% (v / v) of newborn calf serum was added.

[0044] Example 3: Isolation and Identification of Mycoplasma canis isolates

[0045] Sample collection: In 2023-2024, 390 oral and nasal swabs were collected from dogs suspected of having respiratory diseases in Qingdao Animal Hospital and kennels.

[0046] Isolation and purification of mycoplasma: Positive samples were filtered through a 0.22 μm filter membrane and then inoculated into a modified mycoplasma liquid culture medium, and cultured in a shaking incubator at 37°C. When the culture medium turned clear orange-yellow, quantitative real-time PCR was performed, and the samples were passaged. The culture cultured to the third generation was cultured for 10... 9 CCU / mL, 1 mL was evenly spread on a self-made mycoplasma solid culture medium, and incubated upright in an incubator at 37℃ and 5% CO2 concentration for 24 hours. Then it was inverted and incubated for 3–4 days. The solid culture medium changed from red to yellow. The "fried egg"-shaped mycoplasma colonies were observed under a microscope. Figure 2 As shown. Single colonies were picked and placed into mycoplasma liquid culture medium, and purified three times to obtain pure cultures.

[0047] Electron microscopy observations Figure 3 As shown: Isolates cultured to the third generation were used, with a bacterial concentration of 1×10⁻⁶. 9CCU / mL, after staining with phosphotungstic acid, can be observed under a transmission electron microscope to be circular with a diameter of 80nm-100nm.

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

[0049] Mycoplasma 16S gene amplification and sequencing analysis: Nucleic acid was extracted from the isolates using a DNA / RNA extraction kit, and PCR amplification was performed using bacterial 16S rDNA primers. The primer sequences are as follows:

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

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

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

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

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

[0055]

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

[0057] After the culture medium of the isolated strains turned from red to yellow, it was transferred to new liquid culture medium at a ratio of 5% and incubated in a shaking incubator at 37°C. A portion of the bacterial culture was taken every 12 hours to measure the color change units (CCU). The results are as follows: Figure 5 The results showed that the isolates were in the logarithmic growth phase within 24 hours of culture, remained stable from 24 to 36 hours, and then entered the death phase.

[0058] Example 6: Analysis of amino acid variation sites of the virulence gene sialidase in Mycoplasma canis isolate QAU241017.16

[0059] Sialidase protein amino acid sequence analysis: The Sialidase protein gene sequence of reference strains of Mycoplasma canis (CP0144281.1, JQ177148.1, JQ177149.1, OP354259.1) was obtained from the NCBI database. Amino acid sequence alignment analysis of the local isolate QAU241017.16 and the wild-type strain QAU240523.3 using MegAlign software showed that isolate QAU241017.16 had 8 unique mutation sites in the Sialidase protein: K34N, D95N, D96N, N136D, K212E, A224S, A293S, and A916V. This indicates that the isolate QAU241017.16 of this invention has undergone adaptive evolution under host immune pressure or environmental changes, which leads to changes in antigenic epitopes and allows the pathogen to escape the host's immune memory.

[0060]

[0061] The amino acid mutation site comparison results of the sialidase gene in the canine mycoplasma isolate QAU241017.16 are as follows: Figure 6 As shown.

[0062] Example 7: Pathogenicity verification of the canine mycoplasma mutant strain QAU241017.16

[0063] Nine healthy 3-month-old beagle dogs were randomly divided into experimental group 1 (n=3), experimental group 2 (n=3), and control group (n=3). The isolated mutant strain QAU241017.16 and wild-type strain QAU240523.3 were cultured to 1.0 × 10⁻⁶. 11 Dogs were injected intratracheally with 2 ml of mycoplasma culture medium (CCU / ml) into experimental groups 1 and 2, respectively, while the control group received 2 ml of mycoplasma liquid culture medium intratracheally. After infection, the three groups of dogs were isolated and observed for 14 days. Clinical manifestations were observed and recorded daily. Blood samples were collected every 3 days for complete blood count and C-reactive protein (CRP) determination. Nasopharyngeal and ocular swabs were collected every 3 days for DNA extraction and qPCR detection of canine mycoplasma pathogen shedding.

[0064] Table 1 Primer and probe sequences for qPCR detection of Mycoplasma canis

[0065] The results show that: Figure 7 The experimental group 1 infected with isolate QAU241017.16 showed higher C-reactive protein (CRP) and white blood cell (WBC) levels than the experimental group 2 and control group infected with isolate QAU240523.3 3–9 days post-infection. Figure 8 As shown, the viral shedding of Mycoplasma canis in experimental group 1 infected with strain QAU241017.16 was higher than that in experimental group 2 and the control group infected with strain QAU240523.3.

[0066] Example 8: Preparation of inactivated canine mycoplasma vaccine

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

[0068] Inactivation treatment: Add formaldehyde solution to the mycoplasma bacterial culture to make the final formaldehyde concentration 0.3%, and place it in a shaking incubator at 37℃ and 160rpm for 24 hours to inactivate the bacteria.

[0069] Preparation of inactivated vaccine: Inactivate mycoplasma bacterial suspension (1.0 × 10⁻⁶) 9 The inactivated vaccine was prepared by mixing CCU / mL with water-based adjuvant at a 1:1 volume ratio.

[0070] Example 9: Asepticity test of canine mycoplasma inactivated vaccine

[0071] Sterility test: The inactivated vaccine was spread onto TSA containing 5% serum and incubated upside down at 37°C for 48 hours. No other bacteria grew.

[0072] Example 10: Safety testing of inactivated canine mycoplasma vaccine

[0073] Safety test: Ten 6-week-old Kunming mice were used, with half in the control group and half in the experimental group. The experimental group was injected subcutaneously with 0.2 mL of vaccine, while the control group was injected subcutaneously with an equal volume of mycoplasma liquid culture medium. After 14 days of observation, all mice in the experimental group were healthy and met the safety test requirements.

[0074] Example 11: Test for formaldehyde residue in inactivated canine mycoplasma vaccine

[0075] Formaldehyde residue test: The formaldehyde residue was determined according to the appendix of the current edition of the Chinese Veterinary Pharmacopoeia. The formaldehyde residue was 0.09%, which meets the safety standards.

[0076] Example 12: Immunogenicity test of inactivated canine mycoplasma vaccine

[0077] Immunization test: Six healthy New Zealand white rabbits aged 7-8 days were randomly divided into two groups of three each. Rabbits in the vaccine group received a subcutaneous injection of 1 mL of inactivated vaccine in their necks, while rabbits in the control group received a subcutaneous injection of 1 mL of mycoplasma liquid culture medium containing 0.3% formaldehyde in their necks. A booster immunization was administered 7 days after the first immunization, 14 days after the second immunization, and 14 days after the third immunization.

[0078] Antibody titer detection: Blood was collected from the marginal ear vein every week, and the serum was serially diluted 2-fold to 10 gradients. The antibody titer of Mycoplasma canis in the serum of each group of rabbits was detected by neutralization test.

[0079] Neutralization test method: The mycoplasma isolate was cultured to 10... 9CCU / mL, take a 96-well cell culture plate, add 50 μL PBS to each well, add 50 μL of canine serum to the first well, and then perform serial dilutions (1:2 to 1:1024), with 3 replicates for each dilution, and set up a positive control (containing bacterial culture) and a negative control (sterile culture). Add an equal volume of mycoplasma bacterial culture to each well, gently shake to mix, and incubate at 37°C and 5% CO2 for 1 hour. Transfer 100 μL of the reaction solution from each well to a 2 mL centrifuge tube containing 900 μL of mycoplasma liquid culture medium, and incubate in a shaking incubator. Observe the color change of the culture medium every 3 hours, and continue monitoring until the positive control group turns completely yellow. The highest serum dilution that completely inhibits mycoplasma growth is taken as the neutralizing antibody titer. The judgment criterion is: if the culture medium remains red at a certain dilution, it is considered effective neutralization; otherwise, it is inactive. The results showed that the highest antibody titer in the vaccine group was 1:128, while the antibody titer in the control group was ≤1:2.

[0080] Table 2 Changes in antibody titers after immunization

[0081] The above embodiments demonstrate that the canine mycoplasma isolation and culture method of the present invention can achieve rapid proliferation of mycoplasma and reach a higher viable titer than traditional culture methods. The present invention successfully isolated a mutant isolate QAU241017.16 using an improved culture medium and culture method. This isolate has a mutation at 8 amino acid sites in the sialidase gene, and animal experiments have confirmed that its virulence is significantly enhanced. The inactivated vaccine prepared based on this 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 method for preparing a canine mycoplasma inactivated vaccine formulation, characterized in that, Includes the following steps: Strain culture: The canine mycoplasma isolate QAU241017.16 with preservation number CGMCC No. 46643 was inoculated into mycoplasma liquid culture medium and cultured at 37℃ with shaking in the dark until the logarithmic growth phase to obtain a high concentration of mycoplasma bacterial solution. Inactivation treatment: Add formaldehyde solution to the mycoplasma bacterial culture to make the final formaldehyde concentration 0.3%, and place it in a shaking incubator at 37℃ and 160 rpm for 24 hours to inactivate it; Preparation of inactivated vaccine: The inactivated mycoplasma bacterial solution is mixed with water-based adjuvant at a volume ratio of 1:1 to prepare an inactivated vaccine.

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