CN114940955A - Chicken mycoplasma attenuated vaccine strain and application thereof - Google Patents
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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 chicken body virulence, 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 clinical use is convenient, and the chicken feed can be applied to various varieties of chickens with various ages in days.
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 and multiple infectious disease in chronic respiratory diseases of chickens. The disease is often mixed with colibacillosis, newcastle disease, infectious bronchitis and the like, and causes great loss to the chicken industry.
The mycoplasma gallisepticum infects chickens of various varieties and various ages in days, so that the survival rate of commercial chickens is low, the growth of the commercial chickens is slow, the laying rate of laying hens is reduced, and the egg laying peak period is delayed. Due to vertical infection, the death rate of the born chicks is often increased and the contaminated area is enlarged. Infected chicken does not eat enough food, lose weight, cough, sneeze, asthma accompanied by tracheal rales, swelling of eyelid, and exudation from nasal cavity and infraorbital sinus. Infected chicken is dissected, respiratory tract infection is found, inflammation exists on the inner wall of the trachea, mucus or catarrhal exudates can be seen, and infected chicken is found to have sacculitis, thickened and turbid air sac walls, and more yellow exudates are found in serious cases, and the infected chicken is in a block shape or a cheese shape. The use cost of the medicine is increased after the chicken flocks are attacked, the medicine treatment effect is not ideal enough, the disease is easy to relapse after the medicine is stopped, and the disease is difficult to eradicate once the disease is epidemic in chicken farms. Therefore, the prevention, control and purification of mycoplasma gallisepticum must adopt comprehensive measures of provenance purification, all-in-all-out, vaccine prevention, strict environmental control, fine feeding management and the like, wherein the vaccine prevention plays a key role in controlling mycoplasma gallisepticum.
The mycoplasma gallisepticum vaccine commonly used in clinic is mainly divided into inactivated vaccine and attenuated vaccine, wherein the inactivated vaccine can reduce vertical propagation of mycoplasma gallisepticum, reduce toxin expelling, enhance humoral immunity and relieve infection symptoms, but cannot completely eliminate wild virus in chicken and prevent invasion of the wild virus. The attenuated vaccine can make organism produce local mucosal immunity, and has wide application including 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 challenge strains, and can replace mycoplasma gallisepticum field strains in commercial egg-laying areas of various ages, however, when the immunity of farms in the areas is stopped, the F strain continues to circulate among chicken flocks, and the mycoplasma gallisepticum cannot be eliminated.
The Ts-11 strain can replace the F strain, but needs dry ice or liquid nitrogen for transportation and storage, and is inconvenient to transport and use.
6/85 strain is originated from America, is suitable for chicken of 6 weeks and above through aerosol immunization, and in order to achieve the best effect, at least two aerosol immunizations are needed to achieve the effect, so the clinical application is not easy. In addition, the attenuated vaccines of the TS-11 strain and the 6/85 strain of the mycoplasma gallisepticum have the defects of weak pertinence, high cost and the like.
Therefore, the development of the mycoplasma gallisepticum attenuated vaccine strain with good safety and strong immunogenicity becomes one of the technical problems to be solved urgently 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 purpose, the invention provides the following technical scheme:
a mycoplasma gallisepticum attenuated vaccine strain is mycoplasma gallisepticum DL attenuated strain with microorganism preservation number of 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 attenuated vaccine strain of mycoplasma gallisepticum can also be used for preparing a reagent for diagnosing or detecting diseases caused by mycoplasma gallisepticum.
The invention discloses a mycoplasma gallisepticum attenuated vaccine, which 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 toxicity when being transmitted back to chickens, stable genetic property, high growth titer, good immunogenicity, convenient clinical use and application to chickens of various varieties and various ages in days.
Compared with the attenuated vaccine strains (F strains, 6/85 strains and Ts-11 strains) clinically applied in China, the chicken mycoplasma DL attenuated strain is a natural attenuated vaccine strain isolated based on local clinic, and has strong pertinence and better protection effect; and the attenuated strain of mycoplasma gallisepticum DL has weaker toxicity than the F strain widely used in the market and good safety, the DL strain can be used as an alternative excellent strain for replacing the F strain, and the strain can be stored in a freeze-drying way and is convenient to transport.
In addition, the mycoplasma gallisepticum DL low virulent strain is very convenient to use and can be used for eye drop, nasal drop or aerosol immunization.
Drawings
FIG. 1 is the electrophoresis identification diagram of PCR amplification of Mycoplasma gallisepticum DL attenuated strain. M is DL2000Marker, 1 is MG positive control, 2 is mycoplasma gallisepticum DL low virulent strain, 3 is negative control, and 4 is blank control.
FIG. 2 is a colony morphology diagram of Mycoplasma gallisepticum DL attenuated strains on solid medium.
FIGS. 3 to 6 are graphs showing the results of the collinearity alignment of the Mycoplasma gallisepticum DL attenuated strain with the genomes of other strains (TS-11 strain, R strain, 685 strain and F strain).
Information on microbial deposits
The mycoplasma gallisepticum DL low virulent strain is preserved in the China general microbiological culture Collection center, and the preservation address is as follows: the microorganism research institute of academy of sciences of China, Xilu No. 1, Beijing, Chaoyang, and the Collection is abbreviated as: CGMCC with preservation date of 2022, 17 days 02 and month, biological preservation number of CGMCC No.45082, naming: mycoplasma gallisepticum DL attenuated strain.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1 isolation and identification of Natural attenuated strains of Mycoplasma gallisepticum (DL strain)
Pathogen isolation: collecting infraorbital fluid, trachea and air sac of suspected chicken suffering from chronic respiratory disease in a certain chicken farm in Dalian city of Liaoning, separating mycoplasma, culturing in a constant-temperature shaking incubator at 37 ℃, transplanting the liquid with yellowing into a fresh improved CM2 liquid culture medium according to a proportion of 10%, and subculturing when the culture solution becomes yellow.
Differential culture of a differential culture medium: inoculating the isolated strains to eosin methylene blue culture medium, Mackanka culture medium, fresh blood culture medium, chocolate culture medium, and modified CM 2 Culturing in liquid medium, and separating the strain only in the modified CM 2 The growth on the liquid medium, and the growth on the rest of the medium.
Simultaneously coating the harvested culture solution on the modified CM 2 The solid medium was subjected to cloning culture 3 times, and the bacterial solution obtained by the last cloning purification was designated as DL isolate, and subjected to PCR identification, colony morphology identification and Color Change Unit (CCU) assay.
The PCR identification method comprises the following steps: designing a primer by taking a conserved region of a mycoplasma gallisepticum adhesion protein (PvpA) encoding gene as a template, and carrying out PCR amplification. Wherein the primer sequences are as follows (shown as SEQ ID No: 1-2):
PvpA-P1:ACTTACAGATTACAGGAGCAG
PvpA-P2: ATTTCCGTTGGTTGTTGACT, amplified fragment 321 bp.
The PCR results showed that the size of the PvpA gene fragment amplified using the genome of the isolate as a template was identical to the size of the fragment amplified in the positive control (using the genome of Mycoplasma gallisepticum strain F as a template), as shown in FIG. 1.
And (3) observing colony morphology: the harvested culture solution is smeared on a mycoplasma solid culture medium, and after the culture solution is placed in a 5% carbon dioxide incubator at 37 ℃ for 3-5 days, a fried egg-like bacterial colony is observed under a low power microscope, as shown in figure 2.
Color Change Unit (CCU) measurement: 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 to-be-detected bacterial liquid into a first test tube, shaking and uniformly mixing, transferring 0.2ml of to-be-detected bacterial liquid into a culture medium of a 2 nd tube, sequentially carrying out 10-fold serial dilution to 10-11 in the way, and taking a 12 th test tube as a reference to make three parallel tests; marking the dilution of each tube, placing the tube into a constant-temperature incubator at 37 ℃ for culture for 7 days, and recording the growth condition of each tube every day. The highest CCU was the dilution gradient that did not change color any more on day 7. CCU determination shows that the titer of the Dalian lian isolate can reach 10 at the 5 th day 10 CCU/ml, stable growth rate and high culture titer.
The mycoplasma gallisepticum DL strain was subjected to whole genome sequencing and colinear alignment with the genomes of other mycoplasma gallisepticum strains (TS-11 strain, R strain, 685 strain and F strain), and the results were shown in fig. 3-6. Compared with the strain F, the strain DL has poor colinearity 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 DL strain has general colinearity and has gene rearrangement phenomena such as multiple deletions, inversions and the like; the DL strain and the TS-11 strain with weak toxicity have good overall collinearity and have gene rearrangement phenomena such as gene deletion, inversion and the like.
Example 2 Mycoplasma gallisepticum DL Strain virulence assay
Dividing 40 SPF chickens of 35 days old into A, B, C, D groups, each group comprises 10 SPF chickens, A, B, C groups each have 1ml of antidote for eye, nose and chest injection, and the antidote dosage is 10 9 The CCU. Wherein, group A challenge mycoplasma gallisepticum DL strain, group B challenge mycoplasma gallisepticum F36 strain, and group C challenge mycoplasma gallisepticum R strainGroup D, physiological saline (placebo). After challenge, all test chickens were dissected, observed for 21 days, observed for balloon lesions and scored.
The toxicity test result shows that the A group and the D group have no difference with the control group, and the air bags are clean, transparent and thin; 2/10 chickens in group B had slightly thickened and slightly turbid air sacs; in group C, 8/10 ballon lesions were judged to be 2 or more. As can be seen from the results of virulence determination, the group A mycoplasma gallisepticum DL strain has very weak virulence, and the air sac lesions are all lower than those of the F36 strain.
The air bag protection rate calculation method and the formula are as follows:
the protective rate is calculated by an air sac lesion program, and the degree of air sac lesions on each side of the test chicken is divided into five grades:
0 min-normal air bag, clean, transparent and thin;
score 1-balloon slightly thickened and slightly turbid, with a few blotches of grey or yellow exudate on the part;
score 2-some of the balloon area had visible gray and yellow exudates with moderate turbidity of the balloon;
3 minutes-a large number of air bags are full of yellow cheese-like exudates;
4 points-the whole bladder was covered with a yellow cheese-like exudate and the bladder lost elasticity.
The air bag protection rate calculation formula is as follows:
the protection rate is [ (control chicken air sac damage/one-test chicken air sac damage/one)/control chicken air sac damage/one ] × 100%
TABLE 1 virulence determination of MGDL strain, equipartition of balloon lesions
Example 3 hen body passage Return Strength test
Passage stability test of mycoplasma gallisepticum DL strain: application improvement CM 2 Inoculating Mycoplasma gallisepticum DL strain which is subjected to solid cloning for 3 times in a liquid culture medium according to a proportion of 10%, and culturing in a constant-temperature shaking incubator at 37 DEG CWhen the pH of the culture solution is reduced to more than 0.5, the culture solution can be passed through the next generation, and then the culture solution is passed through 60 generations (DL strain/DLF 60), and samples are taken every 5 generations to determine the culture titer of each generation of the culture solution. 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 every 5 generations after passage is stable at 10 9 Above CCU/ml, no change occurred.
Chicken passage strength-return test: fresh culture solutions of mycoplasma gallisepticum DL strain and 60 generations (DLF60) were injected into 5 SPF chickens (1 ml/chicken) 10 days old by eye dropping, nose dropping and chest cavity, and mycoplasma isolation was performed by sampling air sac and trachea of inoculated chickens aseptically on day 20. The first generation returned isolate was injected with 1ml per eye, nose and chest, and then the original separation was performed, and so on to the tenth generation. Respiratory tract reactions and dissections of the inoculated chickens were observed for each generation, and the DL strain culture which was not returned was used as a control. Test results show that the chicken inoculated with the DL strain/DLF 60 culture of each generation of 10 generations of returned SPF chicken do not cause respiratory reaction of the inoculated chicken, and air sacs of all inoculated chicken after being dissected are not diseased, and the results are completely consistent with those of DL strains of SPF chicken which are not returned. The result shows that the mycoplasma gallisepticum DL strain/DLF 60 has no virulence return to the 10 th generation after being returned by the chicken body, is safe to the chicken and has no pathogenicity.
Example 4 determination of the immunological Effect of Mycoplasma gallisepticum (DL strain) attenuated live vaccine in different immunization modes
Mycoplasma gallisepticum DL strain and passage 60 (DLF60) were lyophilized to 10 8 The SPF chickens with the content of CCU/ml are respectively inoculated by eye dropping, nose dropping and aerial fog immunization for about 10 days, and 500-600 ml of original culture (10 ml) is added with the same number of SPF chickens 35 days after inoculation 8-9 CCU/ml) aerosol challenge R strain is virulent, and results are observed in a dissecting mode at 14 days. According to the calculation method and the formula of the air bag protection rate in the embodiment 2, the immunogenicity result shows that the mycoplasma gallisepticum DL strain and the 60-generation passage (DLF60) eye drop, nasal drop and aerosol immunization can generate better immune protection effect.
TABLE 2 determination of the different immunization patterns of the DL strains
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Mtoyowa Biotechnology (Nanjing) Ltd
<120> mycoplasma gallisepticum attenuated vaccine strain and application thereof
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<213> Artificial Sequence (Artificial Sequence)
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Claims (6)
1. A mycoplasma gallisepticum attenuated vaccine strain with a microorganism preservation number of CGMCC No. 45082.
2. Use of the mycoplasma gallisepticum attenuated vaccine strain of claim 1 in the preparation of a medicament for the prevention and treatment of mycoplasma gallisepticum infection.
3. Use of the mycoplasma gallisepticum attenuated vaccine strain of claim 1 in the preparation of a mycoplasma gallisepticum vaccine.
4. The use of the mycoplasma gallisepticum attenuated vaccine strain of claim 1 in the preparation of a reagent for diagnosing or detecting a disease caused by mycoplasma gallisepticum.
5. An attenuated mycoplasma gallisepticum vaccine, comprising: comprising the attenuated mycoplasma gallisepticum vaccine strain of claim 1.
6. The mycoplasma gallisepticum attenuated vaccine of claim 5, wherein: comprises pharmaceutically acceptable auxiliary materials or adjuvants.
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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 | Methods relating to an 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 | Methods relating to an attenuated mycoplasma |
Non-Patent Citations (2)
| Title |
|---|
| ANNA KANCI CONDELLO等: "Mycoplasma gallisepticum strain ts-304 is a safe and effective live attenuated vaccine for use in chickens", 《VET MICROBIOL . 》, vol. 244, pages 1 - 8 * |
| 林伯全等: "鸡毒支原体F弱毒疫苗株细胞免疫特性研究", 《中国动物传染病学报》, vol. 22, no. 3, pages 78 - 81 * |
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