Sheep mycoplasma tissue poison as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a sheep mycoplasma tissue toxin as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing a sheep mycoplasma positive disease material with a mycoplasma culture medium, and grinding to prepare a toxic substance counteracting emulsion; the toxin counteracting emulsion is inoculated to healthy sheep, after the body temperature of the healthy sheep rises to 41 DEG C or above, dissecting and killing are conducted within 3-5 days, lesion tissue is taken and mixed with a mycoplasma culture medium, then grinding is conducted, and then the lesion tissue is mixed with a freeze-drying protective additive and freeze-dried. The prepared sheep mycoplasma tissue poison has the advantages of being high in toxicity, stable in toxicity, easy to operate and the like, can be used for evaluating the immune protection capacity of sheep mycoplasma vaccines, has good practical application value, and lays a foundation for related research of sheep mycoplasma diseases.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a mycoplasma ovis tissue toxin and a preparation method and application thereof. Background Art

[0002] Mycoplasma ovis pneumonia, also known as ovine contagious pleuropneumonia, is a chronic respiratory infectious disease of sheep and goats caused by Mycoplasma ovipneumoniae (Mo). Its main clinical features are coughing, wheezing, progressive weight loss and proliferative inflammation of the lung interstitium. It is widely prevalent throughout the world. Both goats and sheep are susceptible to the disease, causing significant losses to the sheep industry.

[0003] Mycoplasma ovis has high nutritional requirements and is difficult to culture, and its virulence is generally weak, which has led to slow research progress on Mycoplasma ovis. At present, there have been reports on the research of Mycoplasma ovis vaccines, but the efficacy test and evaluation generally adopts the method of bacterial liquid challenge. The culture time of Mycoplasma ovis generally takes 3 to 5 days, and the CCU test results need to wait for 5 to 7 days. At the same time, the titer of Mycoplasma ovis bacterial liquid will decrease with the extension of storage time. Therefore, it is impossible to accurately control the titer of the challenge bacterial liquid, which leads to the instability of the efficacy test challenge results. The Mycoplasma ovis tissue toxin prepared by Mao Li et al. can be used for efficacy test and evaluation, but there are disadvantages that the dose used for the challenge of a single test sheep is large and the number of test toxins prepared in a single batch cannot guarantee the efficacy test and evaluation of the vaccine multiple times. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention is proposed.

[0005] First, the present invention provides a freeze-drying protective agent, which is composed of the following substances: Water, trehalose, mannitol, bovine serum albumin and gelatin.

[0006] The lyophilization protectant of the present invention is suitable for freeze-drying and storing Mycoplasma ovis tissue toxin, and can ensure that the Mycoplasma ovis tissue toxin still has high toxicity after long-term freeze-drying and storage, and has good physical properties during the long-term freeze-drying and storage process.

[0007] Preferably, in the lyoprotectant, the concentration of trehalose is 0.05-0.1 g / mL, the concentration of mannitol is 0.025-0.05 g / mL, the concentration of bovine serum albumin is 0.025-0.05 g / mL, and the concentration of gelatin is 0.04-0.1 g / mL.

[0008] Furthermore, the present invention provides a method for preparing Mycoplasma ovis tissue toxin, comprising: (1) Mixing the Mycoplasma ovis positive diseased material with the Mycoplasma culture medium and grinding them to prepare a challenge emulsion; (2) The challenge emulsion is inoculated into healthy sheep, and when the body temperature of the healthy sheep rises to above 41°C, the sheep are killed within 3 to 5 days, and the diseased tissue is mixed with a mycoplasma culture medium and then ground to obtain an emulsion, and then the emulsion is mixed with the lyophilization protectant according to claim 1 or 2 and freeze-dried to obtain the sheep mycoplasma tissue toxin.

[0009] The present invention finds that after the body temperature of healthy sheep rises to above 41°C after inoculation with the challenge emulsion, the Mycoplasma ovis tissue prepared from the diseased tissue taken within 3 to 5 days has high virulence and stable challenge results, and the Mycoplasma ovis positive diseased materials from different sources are used to prepare the challenge emulsion for verification, and consistent excellent results are achieved.

[0010] Preferably, the Mycoplasma ovis positive material is the tissue at the junction of healthy sheep lung tissue and diseased sheep lung tissue.

[0011] Preferably, the mycoplasma culture medium is a mycoplasma liquid culture medium in the veterinary pharmacopoeia.

[0012] Preferably, the healthy sheep are healthy lambs of 3 to 4 months old.

[0013] Preferably, the inoculation method is tracheal injection, and each healthy sheep is inoculated with 4-6 mL of the challenge emulsion, and each 1 mL of the challenge emulsion contains at least 0.1 g of the Mycoplasma ovis positive material.

[0014] Preferably, the emulsion is mixed with the lyoprotectant according to claim 1 or 2 in a volume ratio of 1:1.

[0015] Furthermore, the present invention provides Mycoplasma ovis tissue toxin prepared according to any of the above embodiments.

[0016] Furthermore, the present invention also provides the use of the above-mentioned Mycoplasma ovis tissue virus in the efficacy test of Mycoplasma ovis vaccine.

[0017] Furthermore, the present invention also provides the use of the above-mentioned Mycoplasma ovis tissue toxin in the preparation of reagents or kits; the reagents or kits are used to test the efficacy of Mycoplasma ovis vaccines.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The sheep mycoplasma tissue toxin prepared by the present invention has the advantages of high virulence, stable virulence, easy operation, etc., can be used to evaluate the immune protection of sheep mycoplasma vaccines, has good practical application value, and lays a foundation for related research on sheep mycoplasma disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a photo of the lungs of the lambs challenged with the virus of the present invention showing typical symptoms of mycoplasma pneumonia.

[0020] Figure 2 This is a photo of the lungs of the lambs challenged with poison according to the present invention showing a normal state. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0023] Example 1 This embodiment provides a lyophilization protective agent, and the preparation steps are as follows: Weigh trehalose, mannitol, bovine serum albumin, and gelatin and dissolve them in sterile water to a final concentration of 0.05 g / mL for trehalose, 0.05 g / mL for mannitol, 0.025 g / mL for bovine serum albumin, and 0.05 g / mL for gelatin. Sterilize at 116°C for 15 minutes before use.

[0024] Example 2 This example provides a lyoprotectant, which is different from Example 1 in that the final concentration of trehalose is 0.05 g / mL, the final concentration of mannitol is 0.025 g / mL, the final concentration of bovine serum albumin is 0.05 g / mL, and the final concentration of gelatin is 0.1 g / mL.

[0025] Example 3 This example provides a lyoprotectant, which is different from Example 1 only in that the final concentration of trehalose is 0.1 g / mL, the final concentration of mannitol is 0.025 g / mL, the final concentration of bovine serum albumin is 0.025 g / mL, and the final concentration of gelatin is 0.04 g / mL.

[0026] Example 4 This embodiment provides a method for preparing Mycoplasma ovis tissue toxin, the steps comprising: 1. Acquisition of diseased lung tissue Dorper × Han sheep with suspected ovine mycoplasma pneumonia characteristics in a sheep farm in Bayannur City, Inner Mongolia were screened, and the lungs of sick sheep with typical ovine mycoplasma pneumonia characteristics were aseptically collected. The tissue at the junction of healthy lung tissue and diseased lung tissue was cut, and the mycoplasma liquid culture medium recorded in the veterinary pharmacopoeia was added and ground to obtain a lung tissue emulsion.

[0027] 2. Isolation and identification of diseased lung tissue (1) Take part of the lung tissue emulsion and use a nucleic acid extraction kit to extract nucleic acid. After full pathogen identification, the results showed that ovine mycoplasma was positive and exogenous pathogens were negative.

[0028] (2) Use a disposable inoculation loop to dip the lung tissue emulsion, streak it on the mycoplasma solid plate, place it in an incubator at 37°C and 5% CO2 and invert it for culture. After 6-7 days of culture, place the solid culture medium under a microscope and observe it. You can see that the colonies are of a single morphology and there are no "umbilicus" or "mulberry-shaped" colonies. Select the colonies and inoculate them into mycoplasma liquid culture medium. After they turn yellow, harvest the culture for characteristic identification. The culture is Gram-negative; Wright staining microscopy shows that its morphology is polymorphic, such as ring-shaped and spherical microorganisms; the culture ferments glucose culture medium to produce acid; the culture grows well on mycoplasma liquid culture medium and is slightly turbid.

[0029] 3. Lung tissue challenge test Eight healthy lambs aged 3 to 4 months were selected and divided into two groups; the first group was the challenge group, with a total of 6 lambs, and the challenge method was tracheal injection. The challenge dose was 4 mL of lung tissue emulsion, containing 0.4 g of lung tissue; the second group was the control group, with a total of 2 lambs, and 4 mL of mycoplasma liquid culture medium was injected into the trachea.

[0030] Three to four days after infection, the sheep in the infection group developed symptoms such as coughing, runny nose, and fever. They were killed after their body temperature rose to above 41°C and remained there for several days. The control group had normal body temperature and no respiratory symptoms.

[0031] 4. Preparation of Mycoplasma ovis tissue toxin The sheep in the challenge group were killed after the body temperature was raised to 41°C for 3-5 days (Group 1), 7-9 days (Group 2), and 10-12 days (Group 3). After killing, the lungs showed typical mycoplasma lesions of "pulmonary consolidation", and the lesions were mainly concentrated in the apical lobe, cardiac lobe, etc. After the lungs were collected aseptically, the surface was washed with physiological saline containing 2000 units of penicillin per milliliter, and the lung tissue blocks at the junction of the characteristic lesions of mycoplasma disease and the healthy lung were cut out aseptically, and the small bronchi were removed. After weighing, the mycoplasma liquid culture medium was added and stirred, and then ground with a grinder on an ice bath to make an emulsion, with 0.1g of lung tissue in each mL of emulsion.

[0032] The emulsions prepared at different dissection times were mixed with the lyophilization protective agents in Examples 1 to 3 at a volume ratio of 1:1, respectively, and packaged and freeze-dried. The freeze-dried tissue toxicity appearance, residual moisture, vacuum degree and other freeze-dried physical properties were tested to be qualified and stored at -70°C.

[0033] Test Example 1 The toxicity of the freeze-dried Mycoplasma ovis tissue toxin prepared in Example 4 was determined.

[0034] Twelve healthy lambs aged 3 to 4 months were selected and divided into 6 groups, with 2 lambs in each group. The freeze-dried sheep mycoplasma tissue toxins prepared by different slaughter times and different freeze-dried protective agents were diluted with 4 mL of normal saline, and the toxin challenge experiment was carried out according to the grouping in Table 1. The control group was prepared by mixing the mycoplasma liquid culture medium and the freeze-dried protective agent in a volume ratio of 1:1, freeze-drying, and then diluting with 4 mL of normal saline. The results are shown in Table 1.

[0035] Table 1

[0036] The results showed that lambs in groups 1 to 3 developed symptoms such as coughing, runny nose, and elevated body temperature 3 to 5 days after infection. After being killed, the lungs showed typical symptoms of mycoplasma pneumonia ( Figure 1 ); Lambs in Groups 4, 5, and 6 showed no adverse clinical symptoms, and their lungs were normal after being killed on the 20th day after the challenge ( Figure 2 ).

[0037] Test Example 2 The freeze-dried Mycoplasma ovis tissue toxin (the freeze-dried protective agent of Example 1 + the diseased tissue emulsion taken 3 to 5 days after freezing for 12 months) was diluted with normal saline to different titers and injected into the trachea of ​​healthy lambs aged 3 to 4 months. The lambs were killed 20 days after the challenge, and the lung lesions were observed and scored according to the 28-point scoring method.

[0038] The results are shown in Table 2. The results show that when the challenge dose is 0.0005g / lamb, the challenge lamb can still get sick, with symptoms such as fever, runny nose, and lung consolidation, and the freeze-dried tissue toxin MID is determined to be 0.0005g / lamb. It is proved that the sheep mycoplasma tissue toxin prepared by the present invention has strong virulence, and one test toxin can meet the needs of multiple vaccine efficacy tests and evaluations. In addition, the present invention also uses sheep mycoplasma positive disease materials from other different individual sources to prepare the challenge emulsion for verification, and all have achieved consistent excellent results.

[0039] Table 2

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A freeze-drying protective agent, characterized in that It is composed of the following substances: Water, trehalose, mannitol, bovine serum albumin and gelatin.

2. The lyoprotectant according to claim 1, characterized in that In the lyophilization protective agent, the concentration of trehalose is 0.05-0.1 g / mL, the concentration of mannitol is 0.025-0.05 g / mL, the concentration of bovine serum albumin is 0.025-0.05 g / mL, and the concentration of gelatin is 0.04-0.1 g / mL.

3. A method for preparing Mycoplasma ovis tissue toxin, characterized in that: include: (1) Mixing the Mycoplasma ovis positive diseased material with the Mycoplasma culture medium and grinding them to prepare a challenge emulsion; (2) The challenge emulsion is inoculated into healthy sheep, and when the body temperature of the healthy sheep rises to above 41°C, the sheep are killed within 3 to 5 days, and the diseased tissue is mixed with a mycoplasma culture medium and then ground to obtain an emulsion, and then the emulsion is mixed with the lyophilization protectant according to claim 1 or 2 and freeze-dried to obtain the sheep mycoplasma tissue toxin.

4. The preparation method according to claim 3, characterized in that: The Mycoplasma ovis positive specimen is the junction tissue between healthy sheep lung tissue and diseased sheep lung tissue.

5. The preparation method according to claim 3, characterized in that: The mycoplasma culture medium is a mycoplasma liquid culture medium in the veterinary pharmacopoeia.

6. The preparation method according to claim 3, characterized in that: The healthy sheep are healthy lambs aged 3 to 4 months.

7. The preparation method according to claim 3, characterized in that: The inoculation method is tracheal injection, and each healthy sheep is inoculated with 4-6 mL of the challenge emulsion, and each 1 mL of the challenge emulsion contains at least 0.1 g of the Mycoplasma ovis positive disease material.

8. The preparation method according to claim 3, characterized in that: The emulsion is mixed with the lyophilization protectant according to claim 1 or 2 in a volume ratio of 1:

1.

9. A Mycoplasma ovis tissue toxin, characterized in that: It is prepared by the preparation method described in any one of claims 3 to 8.

10. Use of the Mycoplasma ovis tissue toxin according to claim 9 in at least one of the following aspects: (1) Application in the efficacy test of ovine mycoplasma vaccine; (2) Preparing a reagent or a kit; the reagent or the kit is used to test the efficacy of the ovine Mycoplasma vaccine.

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