Inactivated vaccine for preventing mycoplasma gallisepticum and preparation method thereof
The killed Mycoplasma gallisepticum vaccine, prepared with specific strain CVCC3616 and adjuvants, effectively addresses the challenge of chicken mycoplasmosis by enhancing immune response and reducing disease incidence in poultry.
Patent Information
- Application Number
- CN202411854911.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-25
AI Technical Summary
Chicken mycoplasmosis, caused by Mycoplasma gallisepticum, leads to chronic respiratory disease in chickens, increasing mortality rates, reducing egg production, and prolonging growth periods, posing significant economic threats to poultry farming due to high infection rates and secondary diseases.
A killed vaccine for Mycoplasma gallisepticum, comprising a specific strain (CVCC3616) with an antigen content of at least 2×10^9 CCU/mL, prepared through a process involving cultivation, concentration, inactivation with formaldehyde, and emulsification with adjuvants, enhancing immune response.
The vaccine significantly boosts the immune system of chickens, effectively preventing Mycoplasma gallisepticum infection, demonstrated by improved protection rates in experimental trials.
Smart Images
Figure CN119679928A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to an inactivated vaccine for preventing Mycoplasma gallisepticum disease and a preparation method thereof. Background Art
[0002] Mycoplasma gallisepticum is a chronic respiratory disease (CRD) in chickens and turkeys caused by Mycoplasma gallisepticum (MG). Clinically, the main manifestations are coughing, runny nose, rales when breathing, and open mouth breathing in severe cases. With the increase in the scale of chicken farming, changes in feeding methods and increased feeding density, the incidence of the disease is getting higher and higher. According to statistics, after Mycoplasma gallisepticum infects chickens, the rate of weak chicks increases by about 10%, the egg production rate of laying hens decreases by 10-20%, the weight of broilers decreases by 38%, the market period is extended, the feed conversion rate is reduced, the immunity of the infected chickens is suppressed and causes secondary or concomitant diseases, which can indirectly cause a large amount of medical expenses. It is one of the important diseases that endanger the poultry industry.
[0003] In summary, it is necessary to research and develop inactivated vaccines to prevent Mycoplasma gallisepticum disease and improve the preventive effect of Mycoplasma gallisepticum disease. Summary of the invention
[0004] The purpose of the present invention is to provide an inactivated vaccine for preventing Mycoplasma gallisepticum disease and improve the preventive effect on Mycoplasma gallisepticum disease.
[0005] The technical solution adopted by the present invention is: an inactivated vaccine for preventing Mycoplasma gallisepticum disease, comprising an antigen and a vaccine adjuvant, wherein the antigen is an inactivated Mycoplasma gallisepticum CR strain purchased from the China Veterinary Drug Administration and numbered CVCC3616, and the antigen content in each milliliter of the inactivated vaccine is not less than 2×10 9 CCU / mL.
[0006] Another object of the present invention is to provide an inactivated vaccine for preventing Mycoplasma gallisepticum disease and a preparation method thereof, comprising the following steps:
[0007] Step S1, inoculating Mycoplasma gallisepticum into the finished culture medium at a ratio of 10-20%, and harvesting the bacterial liquid after shaking culture at 37° C. for 18-30 hours as the first generation seed liquid, and gradually expanding it to the content required for the vaccine;
[0008] Step S2, using ultrafiltration concentration method to concentrate the culture solution of Mycoplasma gallisepticum to a viable bacterial content of not less than 6×10 9 CCU / mL;
[0009] Step S3, adding 10% formaldehyde solution to the concentrated bacterial solution to a final concentration of 0.2%, inactivating the solution at 37° C. for 24 hours, and storing the inactivated bacterial solution at 2-8° C. to obtain a Mycoplasma gallisepticum antigen solution;
[0010] Step S4, taking 94 parts of white oil for injection and 2 parts of aluminum stearate and mixing them evenly in an oil phase preparation tank, then adding 6 parts of Tween-80, and sterilizing them by high pressure after mixing as the oil phase for later use; adding Tween-80 with a final concentration of 5% to the Mycoplasma gallisepticum antigen liquid that has passed the inspection, and mixing them well as the water phase for later use; emulsifying the oil phase and the water phase in a volume ratio of 2:1 to obtain the Mycoplasma gallisepticum inactivated vaccine.
[0011] Preferably, the preparation method of the finished culture medium described in step S1 is: take 27g of culture medium powder, add distilled water to 900mL, adjust the pH value to 7.8-8.0, filter and sterilize with a 0.22μm filter element, and store at 4°C for use; before use, add 100mL of serum, 1 / 1000 penicillin and 1 / 10000 coenzyme.
[0012] The advantages and positive effects of the present invention are:
[0013] The invention provides an inactivated vaccine for preventing Mycoplasma gallisepticum disease and a preparation method thereof. The invention can significantly improve the immune ability of chickens and enhance the preventive effect on Mycoplasma gallisepticum disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 It is a flow chart of the preparation method of the Mycoplasma gallisepticum vaccine of the present invention;
[0015] Figure 2 It is a flow chart of the efficacy test of the Mycoplasma gallisepticum vaccine in the present invention and a flow chart of establishing the immunization program. DETAILED DESCRIPTION
[0016] In order to further understand the content, features and effects of the present invention, the following embodiments are given to explain in detail.
[0017] The inactivated vaccine for preventing Mycoplasma gallisepticum disease of the present invention comprises an antigen and a vaccine adjuvant. The antigen is an inactivated Mycoplasma gallisepticum CR strain purchased from the China Veterinary Drug Administration and is numbered CVCC3616. The antigen content in each milliliter of the inactivated vaccine is not less than 2×10 9 CCU / mL.
[0018] See also Figure 1 The method for preparing the inactivated vaccine for preventing Mycoplasma gallisepticum disease of the present invention comprises the following steps:
[0019] Step S1, inoculating Mycoplasma gallisepticum into the finished culture medium at a ratio of 10-20%, and harvesting the bacterial liquid after shaking culture at 37° C. for 18-30 hours as the first generation seed liquid, and gradually expanding it to the content required for the vaccine;
[0020] In this step, the preparation method of the finished culture medium is as follows: take 27g of culture medium powder, add distilled water to 900mL, adjust the pH value to 7.8-8.0, filter and sterilize with a 0.22μm filter element, and store at 4°C for use; before use, add 100mL of serum, 1 / 1000 penicillin and 1 / 10000 coenzyme.
[0021] Step S2, using ultrafiltration concentration method to concentrate the culture solution of Mycoplasma gallisepticum to a viable bacterial content of not less than 6×10 9 CCU / mL;
[0022] Step S3, adding 10% formaldehyde solution to the concentrated bacterial solution to a final concentration of 0.2%, inactivating the solution at 37° C. for 24 hours, and storing the inactivated bacterial solution at 2-8° C. to obtain a Mycoplasma gallisepticum antigen solution;
[0023] Step S4, taking 94 parts of white oil for injection and 2 parts of aluminum stearate and mixing them evenly in an oil phase preparation tank, then adding 6 parts of Tween-80, and sterilizing them by high pressure after mixing as the oil phase for later use; adding Tween-80 with a final concentration of 5% to the Mycoplasma gallisepticum antigen liquid that has passed the inspection, and mixing them well as the water phase for later use; emulsifying the oil phase and the water phase in a volume ratio of 2:1 to obtain the Mycoplasma gallisepticum inactivated vaccine.
[0024] Specific examples are given below for illustration:
[0025] 1. Preparation of 1L finished culture medium
[0026] Take 27 g of culture medium powder, add distilled water to 900 mL, adjust the pH value to 7.8-8.0, filter and sterilize with a 0.22 μm filter element to obtain 1 L of liquid finished culture medium, and store at 4°C for later use.
[0027] Before use, add 100 mL of serum, 1 / 1000 penicillin and 1 / 10000 coenzyme.
[0028] 2. Preparation of bacterial solution for seedling production
[0029] 2.1 Primary seed propagation
[0030] Take 1 mL of the finished culture medium and inject it into the freeze-dried bottle to revive the strain, inoculate it into 10 mL of the finished culture medium at a ratio of 10%, culture it in a constant temperature incubator at 37°C for 18 to 24 hours, and harvest it when the pH value drops by 0.5 to 0.8 as the first-level seeds.
[0031] 2.2 Secondary seed propagation
[0032] Take the first-level seeds, inoculate them into 100 mL of finished culture medium at a ratio of 10% of the volume of the culture medium, culture for 18 to 24 hours, and harvest them when the pH value drops by 0.5 to 0.8 as second-level seeds.
[0033] 2.3 Preparation of bacterial solution for seedling production
[0034] The bacteria were propagated step by step according to the above method, and the inoculation amount of the strain was maintained at 10% in each step. The bacteria were harvested when the pH dropped from 7.5 to 6.7-7.0. The number of generations of the strain propagation did not exceed 10, and samples were taken from the last generation for viable bacteria titer determination.
[0035] 2.4 Concentration
[0036] The qualified Mycoplasma gallisepticum bacterial liquid was concentrated to 1 / 10 of the original volume using a tangential flow ultrafiltration concentration device with a molecular weight cutoff of 200 kDa.
[0037] After concentration, samples were taken for viable bacterial titer determination.
[0038] 2.5 Inactivation
[0039] A 10% formaldehyde solution was added to the concentrated bacterial solution to a final concentration of 0.2%, and the solution was inactivated at 37°C for 24 hours. The inactivated bacterial solution was stored at 2-8°C, and both the inactivation test and the sterility test were qualified.
[0040] The method for determining the viable bacterial titer is as follows:
[0041] Take samples from the harvested Mycoplasma gallisepticum antigen and perform 10-fold serial dilutions. Dispense the finished culture medium into sterile small white tubes with a specification of 4.5 mL / tube. Dispense into 18 small white tubes in total, select 10 tubes and mark them from 1 to 10, and select 4 tubes each and mark them as 9 and 10 as parallel controls.
[0042] Use a pipette to accurately draw 0.5 mL of the test sample, add it to tube 1, mix the bacterial solution evenly, dilute it to tube 10 in sequence according to the above method, mix all the small white tubes evenly, place them in a 37°C incubator, and culture them statically for 14 days. Observe the color change of the culture medium. The number of test tubes that change color to the highest dilution on the 14th day is used as the color change unit of the test bacterial solution, and CCU / mL is used to represent the viable bacterial concentration of mycoplasma.
[0043] The results showed that the concentration of live bacteria of Mycoplasma gallisepticum antigen before concentration was 1×10 9 CCU / mL, the concentration of live bacteria of Mycoplasma gallisepticum antigen after concentration is 8×10 9 CCU / mL.
[0044] The aforementioned sterility test method is:
[0045] Take the inactivated antigen liquid and test it according to the appendix of the current "Chinese Veterinary Pharmacopoeia". It should be free of sterile growth.
[0046] The method of the aforementioned inactivation test is:
[0047] The inactivated antigen was inoculated into the mycoplasma culture medium and cultured at 37°C for 7 days. If the culture medium did not change in color, the antigen was completely inactivated.
[0048] 3. Vaccine preparation
[0049] Take 94 parts of white oil for injection and 2 parts of aluminum stearate and mix them evenly in an oil phase preparation tank, then add 6 parts of Siben-80, mix and sterilize by high pressure as the oil phase for later use. Add Tween-80 with a final concentration of 5% to the qualified Mycoplasma gallisepticum antigen liquid and mix them well as the water phase for later use. Emulsify the oil phase and the water phase in a volume ratio of 2:1 to obtain the Mycoplasma gallisepticum inactivated vaccine.
[0050] 4. Vaccine testing
[0051] 4.1 Physical properties inspection
[0052] The inactivated vaccine is an oil-in-water type, and the appearance is a milky white uniform emulsion. Take a clean pipette, draw a small amount of vaccine and drop it into cold water. Except for the first drop, it does not spread. Draw 10mL of vaccine into a centrifuge tube, centrifuge at 3000r / min for 15min, and the water phase precipitated at the bottom of the tube does not exceed 0.5mL.
[0053] 4.2 Sterility test
[0054] Tested according to the appendix of the current "Chinese Veterinary Pharmacopoeia", all showed no sterile growth.
[0055] 4.3 Safety Inspection
[0056] Ten 40-day-old SPF chickens were subcutaneously injected with the vaccine at the back of the neck, 1.0 mL per chicken. After 14 days of observation, all the chickens were healthy and had no local or systemic adverse reactions such as loss of appetite, depression, local inflammation, etc.
[0057] 4.4 Effectiveness test
[0058] Ten 40-day-old SPF chickens were subcutaneously injected with the vaccine at the back of the neck, 0.5 mL per chicken. 28 days later, 0.2 mL of Mycoplasma gallisepticum R strain (5 × 10 live Mycoplasma gallisepticum per mL of bacterial solution) was injected into the air sac of 10 control chickens under the same conditions. 8 The patients were kept in CCU for 14 days and then autopsied to observe the air sac lesions and to calculate the lesion scores (see Note).
[0059] In the control group, at least 4 chickens should have lesions of 2 points or more on the air sac, and the average air sac protection rate of immunized chickens should be ≥60%.
[0060] Table 1 Test results of the efficacy of Mycoplasma gallisepticum inactivated vaccine
[0061]
[0062] The results showed that the protection rate of this experimental vaccine against Mycoplasma gallisepticum was 62.5%.
[0063] 5. The preventive effect of the combined use of SPF chicken Chinese patent medicine and inactivated Mycoplasma gallisepticum vaccine
[0064] 5.1 Usage of Chinese patent medicine
[0065] The main ingredient of the Chinese patent medicine is Codonopsis pilosula and Astragalus membranaceus extract. Every 500g of Chinese medicine powder is diluted with 1000kg of water. Each chicken is fed 100mL of Chinese medicine water every day and the water is continuously fed for seven days before immunization.
[0066] 5.2 Safety Inspection
[0067] Take 10 33-day-old SPF chickens, feed each chicken 100mL of Chinese medicine water every day, and continue to feed water for seven days before immunization. Seven days later, inject the vaccine subcutaneously at the back of the neck, 1.0mL per chicken, and observe for 14 days. All chickens are alive and well, without local or systemic adverse reactions such as loss of appetite, depression, local inflammation, etc.
[0068] 5.3 Effectiveness test
[0069] Take 20 33-day-old SPF chickens, of which 10 were fed 100 mL of Chinese medicine solution every day for seven days, and the other 10 were given normal water. Seven days later, the 20 chickens were injected subcutaneously with the vaccine at the back of the neck, 0.5 mL each. 28 days later, together with 10 control chickens under the same conditions, 0.2 mL of Mycoplasma gallisepticum R strain (5×10 live Mycoplasma gallisepticum per mL of bacterial solution) was injected into the air sac 8 The patients were kept in CCU for 14 days and then autopsied to observe the air sac lesions and to calculate the lesion scores (see Note).
[0070] In the control group, at least 4 chickens should have lesions of 2 points or more on the air sac, and the average air sac protection rate of immunized chickens should be ≥60%.
[0071] Table 2 Test results of the efficacy of SPF chicken Chinese medicine plus Mycoplasma gallisepticum inactivated vaccine
[0072]
[0073] The results showed that the protection rate of the Chinese medicine plus vaccine group against Mycoplasma gallisepticum immunization challenge was 84.6%, and the protection rate of the vaccine alone group against Mycoplasma gallisepticum immunization challenge was 61.54%, an increase of 23.1%.
[0074] 6. The preventive effect of the combined use of commercial chicken Chinese patent medicine and inactivated vaccine of Mycoplasma gallisepticum
[0075] 6.1 Safety Inspection
[0076] Take 10 commercial chickens aged 33 days, feed each chicken 100mL of Chinese medicine water every day, and continue to feed water for seven days before immunization. Seven days later, inject the vaccine subcutaneously at the back of the neck, 1.0mL per chicken, and observe for 14 days. All the chickens are alive and well, without local or systemic adverse reactions such as loss of appetite, depression, local inflammation, etc.
[0077] 6.2 Effectiveness test
[0078] Take 20 commercial chickens of 33 days old, of which 10 are fed with 100 mL of Chinese medicine solution every day for seven days, and the other 10 are given normal water. Seven days later, these 20 chickens are injected with vaccine subcutaneously at the back of the neck, 0.5 mL per chicken. 28 days later, together with 10 control chickens under the same conditions, 0.2 mL of Mycoplasma gallisepticum R strain (the number of live Mycoplasma gallisepticum bacteria per milliliter is 5×10 8 The patients were kept in CCU for 14 days and then autopsied to observe the air sac lesions and to calculate the lesion scores (see Note).
[0079] In the control group, at least 4 chickens should have lesions of 2 points or more on the air sac, and the average air sac protection rate of immunized chickens should be ≥60%.
[0080] Table 3 Test results of the efficacy of commercial chicken Chinese patent medicine plus Mycoplasma gallisepticum inactivated vaccine
[0081]
[0082] The results showed that the protection rate of the Chinese medicine plus vaccine group against Mycoplasma gallisepticum immunization challenge was 76.9%, and the protection rate of the vaccine alone group against Mycoplasma gallisepticum immunization challenge was 61.5%, an increase of 15.4%.
[0083]
Notes
[0084] 1.1 Scoring criteria for air sac lesions:
[0085] 0 points: The air sac is normal, clean, transparent and thin;
[0086] 1 point: The air sac is slightly thickened and slightly turbid, with a few gray or yellow exudate spots locally;
[0087] 2 points: gray and yellow exudates are visible in some air sac areas, accompanied by moderate thickening of the air sacs;
[0088] 3 points: most of the air sacs are covered with yellow cheesy exudate;
[0089] 4 points: The entire air sac is covered with yellow cheesy exudate and the air sac loses its elasticity.
[0090] 1.2 Protection rate calculation formula:
[0091] Average air sac protection rate = (average air sac lesion score of control chickens - average air sac lesion score of experimental chickens) / average air sac lesion score of control chickens × 100%.
Claims
1. An inactivated vaccine for preventing Mycoplasma gallisepticum disease, characterized in that: The invention comprises an antigen and a vaccine adjuvant. The antigen is an inactivated Mycoplasma gallisepticum CR strain purchased from the China Veterinary Drug Administration and numbered as CVCC3616. The antigen content in each milliliter of the inactivated vaccine is not less than 2×10 9 CCU / mL.
2. The method for preparing an inactivated vaccine for preventing Mycoplasma gallisepticum disease as claimed in claim 1, characterized in that: Includes the following step, Step S1, inoculating Mycoplasma gallisepticum into the finished culture medium at a ratio of 10-20%, and harvesting the bacterial liquid after shaking culture at 37° C. for 18-30 hours as the first generation seed liquid, and gradually expanding it to the content required for the vaccine; Step S2, using ultrafiltration concentration method to concentrate the culture solution of Mycoplasma gallisepticum to a viable bacterial content of not less than 6×10 9 CCU / mL; Step S3, adding 10% formaldehyde solution to the concentrated bacterial solution to a final concentration of 0.2%, inactivating the solution at 37° C. for 24 hours, and storing the inactivated bacterial solution at 2-8° C. to obtain a Mycoplasma gallisepticum antigen solution; Step S4, taking 94 parts of white oil for injection and 2 parts of aluminum stearate and mixing them evenly in an oil phase preparation tank, then adding 6 parts of Tween-80, and sterilizing them by high pressure after mixing as the oil phase for later use; adding Tween-80 with a final concentration of 5% to the Mycoplasma gallisepticum antigen liquid that has passed the inspection, and mixing them well as the water phase for later use; emulsifying the oil phase and the water phase in a volume ratio of 2:1 to obtain the Mycoplasma gallisepticum inactivated vaccine.
3. The method for preventing Mycoplasma gallisepticum disease according to claim 2, characterized in that: The preparation method of the finished culture medium described in step S1 is as follows: take 27g of culture medium powder, add distilled water to 900mL, adjust the pH value to 7.8-8.0, filter and sterilize with a 0.22μm filter element, and store at 4°C for use; before use, add 100mL of serum, 1 / 1000 penicillin and 1 / 10000 coenzyme.