Mycoplasma bovis JY-22002-4 and application thereof in construction of animal model

Abstract

The invention relates to the technical field of microorganisms, in particular to Mycoplasma bovis JY-22002-4 and application of the Mycoplasma bovis JY-22002-4 to construction of an animal model. The mycoplasma bovis JY-22002-4 is obtained through clinical separation, compared with conventional mycoplasma bovis, the mycoplasma bovis JY-22002-4 is easier to culture, when the mycoplasma bovis JY-22002-4 is used for constructing a mycoplasma bovis infected rabbit animal model, the obtained model animal has typical symptoms of mycoplasma bovis infection such as pulmonary abrupt change, and the mycoplasma bovis JY-22002-4 can be used for evaluating the effect of a mycoplasma bovis vaccine. The positive significance is realized on further in-depth research of the mycoplasma bovis.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a bovine mycoplasma ( Mycoplasma bovis )JY-22002-4 and its application in constructing animal models. Background Art

[0002] Mycoplasma bovis Mycoplasma bovis , M.bovis) is an important pathogenic mycoplasma that harms the cattle industry. In addition to causing pneumonia and mastitis, it also causes arthritis, keratoconjunctivitis, otitis, genital tract inflammation, abortion and infertility. The pathogen is mainly characterized by necrotizing pneumonia, with an incidence rate of 50% to 100% and a mortality rate of up to 10% to 50%.

[0003] The current research on M.bovis is progressing slowly, mainly due to the lack of suitable animal models for basic research and development of biological products. Therefore, the establishment of an M.bovis animal model is urgent. At present, there are reports on bovine mycoplasma guinea pig animal models in China, but guinea pigs have disadvantages such as high feeding accuracy, high mortality, and difficulty in blood collection; Lei Yuanyuan et al. conducted a test on bovine mycoplasma infected rabbits, but the infection model only showed symptoms such as pulmonary edema, pulmonary hemorrhage, and pleural effusion, but did not show symptoms of typical pulmonary consolidation of bovine mycoplasma disease, and could not accurately determine the results of the attack, and no relevant research for vaccine evaluation was carried out; Shi Xiaona et al. conducted a test on bovine mycoplasma infected rabbits. Although the pre-attack test showed symptoms such as lung tissue hyperplasia and hardening of the texture, when the attack frequency was changed to 3 days, the symptoms of typical pulmonary consolidation of bovine mycoplasma disease did not appear.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a bovine Mycoplasma ( Mycoplasma bovis )JY-22002-4 and its application in constructing animal models.

[0006] Specifically, the technical solution provided by the present invention is as follows: In a first aspect, the present invention provides a bovine Mycoplasma ( Mycoplasma bovis )JY-22002-4, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC), located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 46211.

[0007] The bovine mycoplasma JY-22002-4 described in the present invention is obtained by clinical isolation and is easier to culture than conventional bovine mycoplasma. It is used to construct a bovine mycoplasma infection rabbit animal model. The obtained model animal has typical symptoms of bovine mycoplasma infection such as pulmonary consolidation, and can be used for the effect evaluation of bovine mycoplasma vaccine.

[0008] In a second aspect, the present invention provides a method for culturing the bovine Mycoplasma JY-22002-4, wherein the bovine Mycoplasma JY-22002-4 is inoculated into a PPLO liquid culture medium containing 4-6% horse serum for culturing.

[0009] The cultivation of mycoplasma of different species requires a nutrient-rich culture medium, usually requiring the addition of at least 10% animal serum. When the bovine mycoplasma JY-22002-4 provided by the present invention is inoculated and cultured, the amount of serum added is only 4-6%, and the titer of the obtained bacterial liquid can still meet the production requirements, indicating that the bovine mycoplasma JY-22002-4 provided by the present invention is easier to culture, and the culture method proposed based on the bovine mycoplasma JY-22002-4 of the present invention can save production costs for large-scale production in the later stage.

[0010] In a third aspect, the present invention provides a bovine Mycoplasma culture, wherein the bovine Mycoplasma culture contains the bovine Mycoplasma JY-22002-4; or, the bovine Mycoplasma culture is obtained by culturing the bovine Mycoplasma JY-22002-4 according to claim 1.

[0011] The present invention does not particularly limit the specific culture method, and any culture method conventionally applicable to the growth and reproduction of the bovine Mycoplasma JY-22002-4 described in the present invention can be used.

[0012] In a fourth aspect, the present invention provides a bovine mycoplasma vaccine, wherein the bovine mycoplasma vaccine comprises the bovine mycoplasma JY-22002-4; or, the bovine mycoplasma vaccine is prepared from the bovine mycoplasma JY-22002-4.

[0013] The bovine mycoplasma JY-22002-4 of the present invention does not cause serious lesions in healthy negative animals at a toxin dose of 0.5-1 ml / animal. Therefore, the bovine mycoplasma JY-22002-4 of the present invention can be made into a bovine mycoplasma vaccine product.

[0014] In a fifth aspect, the present invention provides the use of the bovine Mycoplasma JY-22002-4 in constructing an animal model.

[0015] Preferably, the animal model is a rabbit animal model infected with Mycoplasma bovis, and / or the animal model has typical symptoms of pulmonary consolidation.

[0016] In a sixth aspect, the present invention provides a method for constructing a rabbit animal model of Mycoplasma bovis infection, comprising the following steps: S1. Inoculate bovine Mycoplasma JY-22002-4 in a PPLO liquid culture medium containing 4-6% horse serum for cultivation, collect the cells after the cultivation, and resuspend the collected cells to obtain a bovine Mycoplasma resuspension; S2. Use the bovine mycoplasma heavy suspension to perform virus challenge inoculation on healthy negative rabbits.

[0017] Preferably, the concentration of Mycoplasma bovis in the Mycoplasma bovis resuspension in step S1 is 1.0-3.0×10 11 CFU / piece.

[0018] Preferably, the weight of the healthy negative rabbit in step S2 is 2.0-3.0 kg.

[0019] Preferably, the challenge vaccination is performed continuously 2-4 times at a frequency of once per day.

[0020] Preferably, the dose of each challenge inoculation is 2 ml / each.

[0021] Preferably, the feeding continues after the challenge, and the ambient temperature of the feeding is 8-12°C; and / or the feeding time is ≥10 days.

[0022] In a more specific embodiment provided by the present invention, according to the typical symptoms of "pulmonary consolidation" of mycoplasma lesions, the method for determining the animal model of bovine mycoplasma infection in rabbits is preferably: the healthy negative rabbits are selected from Inner Mongolia large-eared white rabbits weighing 2.0-3.0 kg; the ambient temperature of the breeding is 10°C; the inoculation method is nasal inoculation or thoracic injection; the challenge dose is 2.0×10 11 CFU / individual×3 days; the date of sacrifice was 15 days after infection.

[0023] In a seventh aspect, the present invention also provides an application of the method for constructing the animal model of Mycoplasma bovis infected rabbits in the evaluation of the effect of Mycoplasma bovis vaccines. It has been verified that the method for constructing the animal model of Mycoplasma bovis infected rabbits and the animal model constructed by the present invention can be used for the evaluation of the effect of Mycoplasma bovis vaccines.

[0024] Description of biological deposit: Mycoplasma bovis Mycoplasma bovis )JY-22002-4, deposited on November 12, 2024 at the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), classified and named Mycoplasma bovis, with the deposit number: CGMCC No. 46211.

[0025] Beneficial effects: The present invention provides a bovine mycoplasma JY-22002-4 and an application thereof in constructing an animal model. The bovine mycoplasma JY-22002-4 of the present invention is obtained by clinical isolation, is easier to culture than conventional bovine mycoplasma, is used to construct a bovine mycoplasma infection rabbit animal model, and the obtained model animal has typical symptoms of bovine mycoplasma infection such as pulmonary consolidation, can be used for effect evaluation of bovine mycoplasma vaccine, and has positive significance for further in-depth research on bovine mycoplasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0027] Figure 1 These are the results of lung lesions in the rabbits challenged with Mycoplasma pneumonia in Example 1 of the present invention, where 1-10: typical symptoms of "pulmonary consolidation" of Mycoplasma pneumonia; 11: normal lung tissue.

[0028] Figure 2 The results of amplification using the specific primers for Mycoplasma bovis in Example 1 of the present invention, wherein M: DL2000bp DNA Marker; N: negative control; O: positive control; and 1 to 10 diseased lung tissues.

[0029] Figure 3 This is the "fried egg-like" colony of Mycoplasma bovis isolated and cultured in Example 1 of the present invention.

[0030] Figure 4 These are the results of lung lesions in rabbits immunized with poison in Example 2 of the present invention, where 1: 2.0 ml / rabbit immunized with poison group; 2: 1.0 ml / rabbit immunized with poison group; 3: 0.5 ml / rabbit immunized with poison group; 4: immunized with poison control group. DETAILED DESCRIPTION

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0032] Example 1 Establishment of a rabbit animal model of Mycoplasma bovis infection.

[0033] 1.1 Preparation of culture medium Liquid culture medium: PPLO broth powder 21g, glucose (containing 1 crystal water) 5.0g, 10ml of 10x concentrated MEM medium, 5.0g of yeast extract powder, 100ml of horse serum, 1% phenol red solution. Mix and dissolve the above ingredients, adjust the pH to 7.6~7.8 with 1.0mol / L sodium hydroxide solution, add water for injection to 1000ml, and filter to sterilize.

[0034] Solid culture medium: Add 15 g agar powder to the liquid culture medium, sterilize at 121℃ for 15 minutes, and then pour the complete culture medium into the plate when the temperature drops to 55℃.

[0035] (II) Preparation of the challenge solution The clinically isolated Mycoplasma bovis strain JY 22002-4 was inoculated into liquid culture medium containing 10% horse serum and 5% horse serum at a ratio of 10%, cultured at 37°C, and the bacterial solution was collected after 48 hours of culture; centrifuged at 8000rpm for 30 minutes at 4°C, the collected bacterial solution was resuspended in PPLO culture medium, 10μL of the suspension was aspirated and CFU counted on a solid culture plate of Mycoplasma bovis, and the colony count was performed after 48-72 hours of culture at 37°C and 5% CO2. The Mycoplasma bovis suspension of the required concentration was prepared according to the colony count results.

[0036] Results: The clinically isolated Mycoplasma bovis strain JY 22002-4 was inoculated into liquid culture medium containing 10% horse serum and 5% horse serum at a ratio of 10%. The titer of Mycoplasma bovis liquid could reach 1×10 9 CFU / ml. The cultivation of mycoplasma of different species requires nutrient-rich culture medium, which must be supplemented with at least 10% animal serum; when the strain was inoculated with only 5% serum added, the culture titer of the bacterial liquid could still meet the demand, indicating that the isolate is easy to culture and can save production costs for later large-scale vaccine production.

[0037] 3. Virus challenge test (1) Effects of experimental animal weight and feeding environment on challenge results: Select Inner Mongolian large-eared white rabbits weighing about 1.0-2.0 kg, 2.1-3.0 kg, and 3.1-4.0 kg, 4 of each size, a total of 12, and place them in an environment temperature of 10°C and 25°C for breeding (two large-eared white rabbits of different sizes were placed in different environments). Adjust the titer of the harvested bacterial solution to 1.0×10 11 CFU / ml, 2ml / rabbit was inoculated intranasally, and autopsies were performed 15 days after infection to observe lung lesions.

[0038] Results: The incidence of large-eared white rabbits raised at an ambient temperature of 10℃: local large-eared white rabbits of different weights showed different degrees of lung atrophy, pale lung color, and mild lung consolidation. The incidence of large-eared white rabbits raised at an ambient temperature of 25℃: local large-eared white rabbits of 1.0-2.0kg and 2.1-3.0kg showed mild lung atrophy and pale lung color; local large-eared white rabbits of 3.1-4.0kg showed no obvious symptoms. Therefore, the selection of local large-eared white rabbits below 3.0kg and the raising environment of 10℃ are conducive to the establishment of the animal challenge model of bovine Mycoplasma.

[0039] (2) The impact of different challenge methods and different challenge doses on the results: Twenty-one Inner Mongolian white rabbits weighing about 2.0-3.0 kg were selected and placed in different feeding rooms. The room temperature was adjusted to 10°C and randomly divided into 6 challenge groups and 2 control groups. Three groups were inoculated with intranasal drops at concentrations of 2.0×10 9 CFU / only (group A), 2.0×10 10 CFU / unit (Group B), 2.0×10 11 CFU / head (Group C) was challenged with a suspension of Mycoplasma bovis for 3 consecutive days with an inoculation volume of 2 ml; three groups were intrathoracically inoculated with concentrations of 2.0×10 9 CFU / unit (Group D), 2.0×10 10 CFU / only (Group E), 2.0×10 11 CFU / head (Group F) of bovine Mycoplasma suspension; the nasal inoculation volume of control group 1 was 2 ml of culture medium; the thoracic inoculation volume of control group 2 was 2 ml of culture medium. Autopsy was performed 15 days after infection to observe lung lesions.

[0040] PCR identification of lung lesion tissue: Take the lung tissue of the experimental rabbit, wipe the lung surface with 75% medical alcohol, select the junction of normal and diseased lung tissues aseptically, add culture medium for grinding, centrifuge at 2000rpm after grinding, and aspirate the supernatant, and perform PCR identification with bovine Mycoplasma specific primers.

[0041] Isolation and culture of Mycoplasma pneumoniae: Aseptically select tissue at the junction of normal and diseased lung tissue, dip the inner side of the tissue with an inoculation loop, smear it on solid culture medium, invert it at 37℃, 5% CO2, and culture for 3 to 4 days.

[0042] Results: Clinical symptoms showed that most rabbits in groups B, C, E, and F showed symptoms of poor appetite, depression, and dyspnea on the third day after the challenge; rabbits in the control group, A group, and D group were in normal mental state during the experiment. Lung observation showed that there was no lesion in the lungs of the control group, A group, and D group; pleural effusion, lung atrophy, and lung consolidation, typical symptoms of mycoplasma disease, appeared in the lungs of groups B, C, E, and F, and the degree of lesions became more severe with the increase of the challenge dose. The lesion tissue was separated and identified as positive for bovine Mycoplasma, as shown in Table 1.

[0043] .

[0044] Photos of lung tissue lesions in infected rabbits are shown in Figure 1 . Figure 1 In the figure, 1-2 show the lung tissues of 2 diseased rabbits in group B; 3-5 show the lung tissues of 3 diseased rabbits in group C; 6-7 show the lung tissues of 2 diseased rabbits in group E; 8-10 show the lung tissues of 3 diseased rabbits in group F; and 11 shows the normal lung tissue.

[0045] The results of PCR identification of diseased lung tissue are shown in Figure 2 . Figure 2 In the figure, M indicates DL2000bp DNA Marker; 1-2 indicate lung tissues of 2 diseased rabbits in group B; 3-5 indicate lung tissues of 3 diseased rabbits in group C; 6-7 indicate lung tissues of 2 diseased rabbits in group E; 8-10 indicate lung tissues of 3 diseased rabbits in group F; N indicates negative control; O indicates positive control.

[0046] See the photo of the "fried egg-like" colonies of Mycoplasma bovis Figure 3 .

[0047] Example 2 Vaccine efficacy testing.

[0048] Vaccine preparation: Inoculate the bovine Mycoplasma JY 22002-4 strain into the culture medium at a ratio of 10% for expansion and culture, place at 37°C, inactivate the culture after harvesting, add adjuvant after inactivation and mix to obtain the bovine Mycoplasma inactivated vaccine. The minimum immunization dose of the vaccine is 2ml according to the animal efficacy test and evaluation.

[0049] Immunization: 16 Inner Mongolian white rabbits weighing about 1.5-2.0 kg were selected and randomly divided into 3 immunization groups and a challenge control group, a total of 4 groups. The prepared bovine mycoplasma inactivated vaccine was used to immunize rabbits, with the immunization doses of 2 ml / rabbit (Group A), 1 ml / rabbit (Group B), and 0.5 ml / rabbit (Group C). A second immunization was performed 14 days after the first immunization, with the same immunization dose as the first immunization.

[0050] Challenge: 14 days after the second immunization, challenge with the virus according to Example 1 was performed, and an autopsy was performed 15 days after the challenge to observe the lung lesions and perform PCR identification on the lesion tissues.

[0051] Results: The challenge control group and immune group C showed typical mycoplasma lesions of pulmonary consolidation, which were specifically identified as bovine Mycoplasma by PCR; while the immune groups A and B did not show bovine Mycoplasma lesions, see Table 2.

[0052] .

[0053] Vaccine efficacy test results are available at Figure 4 . Figure 4 In the figure, 1 shows no lung lesions in group A; 2 shows no lung lesions in group B; 3 shows lung consolidation in group C; 4 shows lung consolidation in the control group.

[0054] The above results show that the bovine Mycoplasma infection rabbit animal model constructed by the present invention can be used for the evaluation of bovine Mycoplasma vaccines. When 1.5-2.0 kg rabbits are inoculated with 1 / 2 of the animal immunization dose (the animal immunization dose is 2 ml / rabbit), the immune protection results of the vaccine can indicate the immune efficacy results of the animal (can resist the attack of bovine Mycoplasma bacterial liquid).

[0055] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Mycoplasma bovis ( Mycoplasma bovis )JY-22002-4, its deposit number is CGMCC No. 46211.

2. The method for culturing Mycoplasma bovis JY-22002-4 according to claim 1, characterized in that: The bovine Mycoplasma JY-22002-4 was inoculated into a PPLO liquid culture medium containing 4-6% horse serum and cultured.

3. A culture of Mycoplasma bovis, characterized in that The Mycoplasma bovis culture contains the Mycoplasma bovis JY-22002-4 described in claim 1; or, the Mycoplasma bovis culture is obtained by culturing the Mycoplasma bovis JY-22002-4 described in claim 1.

4. A bovine mycoplasma vaccine, characterized in that: The bovine mycoplasma vaccine comprises the bovine mycoplasma JY-22002-4 described in claim 1; or, the bovine mycoplasma vaccine is prepared from the bovine mycoplasma JY-22002-4 described in claim 1.

5. Use of the bovine Mycoplasma JY-22002-4 according to claim 1 in constructing an animal model.

6. The use according to claim 5, characterized in that: The animal model is a rabbit animal model infected with Mycoplasma bovis, and / or the animal model has typical symptoms of pulmonary consolidation.

7. A method for constructing a rabbit animal model infected with Mycoplasma bovis, characterized in that: The steps include: S1. Inoculate bovine Mycoplasma JY-22002-4 in a PPLO liquid culture medium containing 4-6% horse serum for cultivation, collect the cells after the cultivation, and resuspend the collected cells to obtain a bovine Mycoplasma resuspension; S2. Use the bovine mycoplasma heavy suspension to perform virus challenge inoculation on healthy negative rabbits.

8. The method for constructing a rabbit animal model of Mycoplasma bovis infection according to claim 7, characterized in that: The concentration of Mycoplasma bovis in the Mycoplasma bovis resuspension in step S1 is 1.0-3.0×10 11 CFU / piece.

9. The method for constructing a rabbit animal model of Mycoplasma bovis infection according to claim 7, characterized in that: The weight of the healthy negative rabbits in step S2 is 2.0-3.0 kg; the challenge vaccination is performed 2-4 times continuously at a frequency of once a day, and the dose of each challenge vaccination is 2 ml / rabbit; the rabbits are continued to be fed after the challenge, and the ambient temperature of the feeding is 8-12° C.; the feeding time is ≥10 days.

10. Use of the method for constructing a bovine Mycoplasma infection rabbit animal model according to any one of claims 7 to 9 in evaluating the efficacy of a bovine Mycoplasma vaccine.

Images