CN114940955B - Mycoplasma gallisepticum attenuated vaccine strain and application thereof - Google Patents
Mycoplasma gallisepticum attenuated vaccine strain and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms and immunity, in particular to a mycoplasma gallisepticum attenuated vaccine strain and application thereof, wherein the collection number of the mycoplasma gallisepticum attenuated vaccine strain is CGMCC No.45082. The mycoplasma gallisepticum attenuated strain is a separated natural attenuated strain, has the advantages of safe use, no return of virulence of chicken, high growth titer and the like, and can obtain good immune protection effect by using eye-drop, nose-drop and aerosol immunization modes; and the chicken feed additive is convenient to use clinically and can be applied to chickens of various varieties and various ages.
Description
Technical Field
The invention relates to the technical field of microorganisms and immunity, in particular to a mycoplasma gallisepticum vaccine strain.
Background
Mycoplasma gallisepticum is a common, multiple infectious disease in chronic respiratory disease in chickens. The disease is often mixed with colibacillosis, newcastle disease, infectious bronchitis and the like, and causes great loss to chicken industry.
Mycoplasma gallisepticum infects chickens of various varieties and days old, so that the survival rate of commercial chickens is low, the growth is slow, the laying rate of the laying hens is reduced, and the laying peak period is delayed. Due to vertical infection, the mortality rate of the chicks is often increased and the contaminated area is enlarged. Infected chickens had poor appetite, reduced weight, emaciation, coughing, sneezing, wheezing with accompanying tracheal tube, swelling of the eyelids, and exudates from the nasal cavity and infraorbital sinus. Dissecting infected chickens, finding respiratory tract infection, inflammation on the inner wall of the trachea, visible mucus or catarrhal exudates, and finding that infected chickens suffer from air sac inflammation, air sac wall thickening and turbidity, and heavy people have more yellow secretion, and the infected chickens are in a block shape or cheese shape. The use cost of the medicine after the chicken flock is increased, the treatment effect of the medicine is not ideal enough, the medicine is easy to relapse after stopping the medicine, and the disease is difficult to eradicate once the disease is popular in chicken farms. Therefore, comprehensive measures such as seed source purification, full-in and full-out, vaccine prevention, strict environmental control, fine feeding management and the like are adopted for preventing, controlling and purifying mycoplasma gallisepticum, wherein the vaccine prevention plays a key role in controlling mycoplasma gallisepticum.
The clinical common mycoplasma gallisepticum vaccine is mainly divided into two types, namely an inactivated vaccine and a weak vaccine, wherein the inactivated vaccine can reduce the vertical transmission of mycoplasma gallisepticum, reduce toxin expelling, enhance humoral immunity and relieve infection symptoms, but can not thoroughly remove wild toxin in chicken and prevent wild toxin invasion. The attenuated vaccine can make organism produce local mucosa immunity, and its application is extensive, mainly includes F strain, 6/85 strain and Ts-11 strain.
The F strain vaccine has stronger capability than 6/85 and TS-11 vaccine strains to replace the virulent strain and can replace the mycoplasma gallisepticum wild strain in commercial egg-laying areas of various ages, however, when the farm in the area stops being immunized, the F strain continues to circulate among chicken flocks and cannot remove mycoplasma gallisepticum.
The strain Ts-11 can replace the strain F, but needs dry ice or liquid nitrogen for transportation and preservation, and is inconvenient to transport and use.
6/85 strains are derived from the United states, are suitable for chicken immunization of 6 weeks old and above by aerosol immunization, and have to be subjected to aerosol immunization for at least two times to achieve the best effect, thereby achieving the effect and being not easy for clinical application. In addition, TS-11 strain and 6/85 strain mycoplasma gallisepticum attenuated vaccine have the defects of weak pertinence, high cost and the like.
Therefore, development of a mycoplasma gallisepticum attenuated vaccine strain with good safety and strong immunogenicity becomes one of the technical problems to be solved in the field.
Disclosure of Invention
The invention aims to overcome the defects of the existing attenuated vaccine and provide a mycoplasma gallisepticum attenuated vaccine strain with convenient clinical use, good safety and strong immunogenicity and application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a mycoplasma gallisepticum attenuated vaccine strain is a mycoplasma gallisepticum DL attenuated strain, and the microorganism preservation number is CGMCC No.45082.
The mycoplasma gallisepticum attenuated vaccine strain can be used for preparing medicines for preventing and treating mycoplasma gallisepticum infection diseases.
The mycoplasma gallisepticum attenuated vaccine strain can be used for preparing mycoplasma gallisepticum vaccines.
The mycoplasma gallisepticum attenuated vaccine strain can also be used for preparing reagents for diagnosing or detecting diseases caused by mycoplasma gallisepticum.
A mycoplasma gallisepticum attenuated vaccine comprises the mycoplasma gallisepticum attenuated vaccine strain.
Wherein, the composition also comprises pharmaceutically acceptable auxiliary materials or adjuvants.
Compared with the prior art, the invention has the beneficial effects that:
the mycoplasma gallisepticum DL attenuated strain has the advantages of safe use, no return of virulence to the chicken body, stable genetic performance, high growth titer, good immunogenicity, convenient clinical use and applicability to chickens of various varieties and ages.
Compared with attenuated vaccine strains (F strain, 6/85 strain and Ts-11 strain) applied clinically in China, the mycoplasma gallisepticum DL attenuated strain is based on local clinical separation of natural attenuated vaccine strains, and has strong pertinency and better protection effect; and the mycoplasma gallisepticum DL attenuated strain is weaker than F strain widely applied in the market, has good safety, can be used as an alternative excellent strain for replacing F strain, can be freeze-dried and stored, and is convenient to transport.
In addition, the mycoplasma gallisepticum DL attenuated strain is very convenient to use, and can be used for eye drop, nose drop or aerosol immunization.
Drawings
FIG. 1 is an electrophoretically identified map of PCR amplification of Mycoplasma gallisepticum DL-virulent strain. M: DL2000Marker,1: MG positive control, 2: mycoplasma gallisepticum DL attenuated strain, 3: negative control, 4: blank control.
FIG. 2 is a colony morphology diagram of Mycoplasma gallisepticum DL-attenuated strain on a solid medium.
FIGS. 3 to 6 are graphs showing the co-linear comparison results of the Mycoplasma gallisepticum DL-attenuated strain and other strain genomes (TS-11 strain, R strain, 685 strain and F strain).
Information on preservation of microorganisms
The mycoplasma gallisepticum DL attenuated strain is preserved in China general microbiological culture Collection center, and the preservation address is: the institute of microbiology, national academy of sciences of China, national academy of sciences, north Chen West Lu 1, chaoyang, beijing, and the preservation agency is abbreviated: CGMCC, the preservation date is 2022, 02 and 17 days, the biological preservation number is CGMCC No.45082, and the name is: mycoplasma gallisepticum DL attenuated strain.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 isolation and identification of Mycoplasma gallisepticum (DL strain) natural attenuated strain
Pathogen separation: collecting infraorbital fluid, trachea and balloon separated mycoplasma from chicken in chicken farm of Dalian city, liaoning, culturing in shaking incubator at 37 deg.c, transplanting the liquid yellowing patient into fresh CM2 liquid culture medium in 10% proportion, and culturing to obtain the final product.
Identification medium identification culture: inoculating the isolated strain into eosin-Mei blue culture medium, maiconk culture medium, fresh blood culture medium, chocolate culture medium, and CM improvement 2 Culturing in liquid culture medium, isolating strain only in modified CM 2 The growth on the liquid culture medium is not carried out on the rest culture medium.
Simultaneously, the harvested culture fluid is coated on the modified CM 2 The solid medium was subjected to 3 clone cultures, and the bacterial liquid obtained by the final clone purification was designated as DL isolate, and PCR identification, colony morphology identification and color change unit (Colour Change Unit, CCU) measurement were performed.
PCR identification method: the primer is designed by taking a conservation region of a mycoplasma gallisepticum adhesion protein (PvpA) coding gene as a template, and PCR amplification is carried out. Wherein the primer sequences are as follows (as shown in SEQ ID No: 1-2):
PvpA-P1:ACTTACAGATTACAGGAGCAG
PvpA-P2: ATTTCCGTTGGTTGTTGACT, amplified fragment 321bp.
The PCR results showed that the size of the PvpA gene fragment amplified using the isolate genome as a template was identical to that of the positive control (using mycoplasma gallisepticum F strain genome as a template), as shown in FIG. 1.
Colony morphology observation: the harvested culture fluid is coated on a mycoplasma solid culture medium, and after being placed in a 5% carbon dioxide incubator at 37 ℃ for 3-5 days, the culture fluid is observed under a low power microscope to form a fried egg-like colony, as shown in figure 2.
Color Change Unit (CCU) assay: placing small test tubes on a test tube rack, and filling 1.8mL of mycoplasma culture medium into each test tube; adding 0.2ml of bacterial liquid to be detected into a first test tube, shaking and uniformly mixing, transferring 0.2ml into a 2 nd tube culture medium, sequentially carrying out 10-time serial dilution to 10-11, and taking a 12 th test tube as a contrast, and carrying out three parallels; marking the dilution of each tube, and placing the tube into a constant temperature incubator at 37 DEG CCulturing was performed for 7 days, and the growth of each tube was recorded daily. The CCU was highest at the dilution gradient at day 7, where no color change occurred. CCU assays showed that the titer of the macrodesmosome isolate reached 10 at day 5 10 CCU/ml, has a stable growth rate and a very high culture titer.
The Mycoplasma gallisepticum DL strain was subjected to whole genome sequencing and subjected to collinearity comparison with the genomes of other Mycoplasma gallisepticum strains (TS-11 strain, R strain, 685 strain and F strain), and the results show that the results are shown in FIG. 3-FIG. 6. Compared with the F strain, the DL strain has poor collinearity and has large-scale gene rearrangement phenomena such as gene inversion, insertion, deletion and the like; compared with the 685 strain and the R strain, the overall collinearity of the DL strain is general, and has the phenomenon of gene rearrangement such as multiple deletions, inversion and the like; the overall collinearity of the DL strain and the strain TS-11 with weak toxicity is good, and the gene rearrangement phenomena such as gene deletion, inversion and the like exist.
EXAMPLE 2 Mycoplasma gallisepticum DL strain virulence determination
The 40 SPF chickens with the age of 35 days are divided into A, B, C, D groups, 10 and A, B, C groups are evenly injected into eyes, nose and chest to attack 1 ml/each, and the toxin attack amount is 10 9 And a CCU. Wherein, the A group attacks the mycoplasma gallisepticum DL strain, the B group attacks the mycoplasma gallisepticum F36 strain, the C group attacks the mycoplasma gallisepticum R strain, the D group attacks normal saline (blank control group). After challenge, all test chickens were dissected 21 days under observation, and balloon lesions were observed and scored.
The toxicity measurement result shows that the group A and the group D have no difference, and the air bags are clean, transparent and thin; group B had 2/10 chicken air pockets with slightly increased thickness and slight turbidity; and 8/10 of the balloon lesions in the group C are judged to be more than or equal to 2. As can be seen from the toxicity measurement results, the group A mycoplasma gallisepticum DL strain has very weak toxicity, and the balloon lesions are uniformly lower than those of the F36 strain.
The air bag protection rate calculation method and the formula are as follows:
the protection rate was calculated by the air sac lesion procedure, and the air sac lesion degree of each side of the test chicken was classified into five grades:
0 minutes-normal air bag, clean, transparent and thin;
score 1-balloon with slight thickening and slight cloudiness, few gray or yellow exudate spots in the local area;
2 minutes-a portion of the balloon area had visible gray and yellow exudates with moderate balloon turbidity;
3 minutes-large air bags are full of yellow cheese-like exudate;
4 minutes-the entire balloon was full of yellow cheesy exudate and the balloon lost its elasticity.
The calculation formula of the air bag protection rate comprises the following steps:
protection ratio = [ (control chicken air sac injury score/only-test chicken air sac injury score/only)/control chicken air sac injury score/only ] ×100%
TABLE 1 determination of MGDL strain virulence, balloon lesion sharing Condition
EXAMPLE 3 chicken body passage Return test
Test of passage stability of mycoplasma gallisepticum DL strain: application improvement CM 2 Inoculating the solid mycoplasma gallisepticum DL strain into the liquid culture medium according to the proportion of 10% for 3 times, culturing in a constant-temperature shaking incubator at 37 ℃, and when the pH of the culture solution is reduced by more than 0.5, transferring the next generation, and then transferring to 60 generations (DL strain/DLF 60), sampling every 5 generations to determine the culture titer of the culture solution of each generation. The result shows that the mycoplasma gallisepticum DL strain is passaged for 60 generations, the character is stable, and the culture titer of the culture solution after passaging is stable at 10 every 5 generations 9 Above CCU/ml, no change occurs.
Chicken body passage back strength test: fresh culture solutions of mycoplasma gallisepticum DL strain and passage 60 (DLF 60) were injected into 10-day-old SPF chickens by eye, nose and chest, respectively, at 1 ml/day, and samples were aseptically taken from the air sac and trachea of vaccinated chickens for mycoplasma isolation on day 20. The first generation of returned isolates were continued to be injected at 1 ml/spot, nasal and chest, followed by mycoplasma isolation, and so on to the tenth generation. The respiratory reaction and the anatomical observation of the chicken after inoculation were observed for each generation, and the culture of the DL strain without feedback was used as a control. The test result shows that the DL strain/DLF 60 culture of each generation of 10 generations of the returned SPF chickens is inoculated to the chickens, the respiratory reaction of the inoculated chickens is not caused, the air sac of all inoculated chickens after dissection has no lesion, and the result of the DL strain of the non-returned SPF chickens is completely consistent. The result shows that the mycoplasma gallisepticum DL strain/DLF 60 can not return virulence after passing back through chicken bodies for 10 generations, and is safe to chickens and has no pathogenicity.
Example 4 determination of the different immunization System immune Effect of Mycoplasma gallisepticum (DL strain) live attenuated vaccine
After freeze-drying of mycoplasma gallisepticum DL strain and passage 60 (DLF 60), the total dose of the strain was 10 8 The content of CCU/ml was respectively spotted on eyes, dropped on nose, and air-mist immunized with SPF chickens of about 10 days old, and at 35 days post-inoculation, the same number of SPF chickens were inoculated in 500 to 600ml of crude culture (10 8-9 CCU/ml) aerosol challenge R strain virulence, anatomical observations at 14 days. According to the air bag protection rate calculation method and formula described in the embodiment 2, the immunogenicity results show that the mycoplasma gallisepticum DL strain and passage 60 (DLF 60) can produce better immune protection effects when the eye is dropped, the nose is dropped and the aerosol is immunized.
TABLE 2 determination of the effects of different immunization formats on DL strains
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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Claims (5)
1. A mycoplasma gallisepticum attenuated vaccine strain has a microorganism preservation number of CGMCC No.45082.
2. Use of a mycoplasma gallisepticum attenuated vaccine strain according to claim 1 in the manufacture of a medicament for the prevention and treatment of mycoplasma gallisepticum infection diseases.
3. Use of a mycoplasma gallisepticum attenuated vaccine strain according to claim 1 in the preparation of a mycoplasma gallisepticum vaccine.
4. A mycoplasma gallisepticum attenuated vaccine, characterized in that: a mycoplasma gallisepticum attenuated vaccine strain comprising the mycoplasma gallisepticum attenuated vaccine strain of claim 1.
5. The mycoplasma gallisepticum attenuated vaccine of claim 4, wherein: comprises pharmaceutically acceptable auxiliary materials.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1244581A (en) * | 1999-06-25 | 2000-02-16 | 江苏省农业科学院畜牧兽医研究所 | Cloned weakening strain of chicken virus mycoplasma |
| CN101896195A (en) * | 2007-09-11 | 2010-11-24 | 惠氏有限责任公司 | Attenuated mycoplasma gallisepticum strains |
| CN102985530A (en) * | 2010-05-19 | 2013-03-20 | 澳大利亚生物资源公司 | Method involving attenuated mycoplasma |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1244581A (en) * | 1999-06-25 | 2000-02-16 | 江苏省农业科学院畜牧兽医研究所 | Cloned weakening strain of chicken virus mycoplasma |
| CN101896195A (en) * | 2007-09-11 | 2010-11-24 | 惠氏有限责任公司 | Attenuated mycoplasma gallisepticum strains |
| RU2010108641A (en) * | 2007-09-11 | 2011-10-20 | ВАЙЕТ ЭлЭлСи (US) | ATTENUATED STRAINS of Mycoplasma gallisepticum |
| CN102985530A (en) * | 2010-05-19 | 2013-03-20 | 澳大利亚生物资源公司 | Method involving attenuated mycoplasma |
Non-Patent Citations (1)
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| 鸡毒支原体F弱毒疫苗株细胞免疫特性研究;林伯全等;《中国动物传染病学报》;第22卷(第3期);第78-81页 * |
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