Chicken new tributary method glandular quintuplet vaccine and preparation method thereof

Abstract

The invention belongs to the technical field of poultry vaccines, and particularly relates to a chicken new tributary method adeno-quintuplet vaccine and a preparation method thereof.The chicken new tributary method adeno-quintuplet vaccine comprises chicken Newcastle disease venom, infectious bronchitis venom, avian influenza venom, infectious bursal disease virus liquid and fowl adenovirus venom, and the preparation method of the Newcastle disease venom comprises the steps that firstly, frozen MDCK suspension cells are provided; the frozen MDCK suspension cells are recovered at the temperature of 40-45 DEG C; 2, transferring the resuscitated MDCK suspension cells to a serum-free culture medium; according to the invention, the inactivated vaccine is prepared from the Newcastle disease venom, the infectious bronchitis venom, the avian influenza venom, the infectious bursal disease virus liquid and the fowl adenovirus venom by adopting a superstrong concentration process and a high-quality adjuvant, so that after animals are immunized, antibodies are generated quickly, the titer is high, the protection period is long, and the occurrence and spreading of epidemic diseases are prevented; and people can conveniently put multi-combined vaccines to cope with continuously changing epidemic disease prevention and control situations.

Description

Technical Field

[0001] This invention belongs to the field of poultry vaccine technology, specifically relating to a chicken neomycobacterial bronchitis-associated adenovirus pentavalent vaccine and its preparation method. Background Technology

[0002] Newcastle disease (ND) is a highly contagious disease caused by a paramyxovirus, also known as Asian fowl plague or pseudofowl plague. During its course, it often presents with septicemia, and the main clinical features include respiratory distress, diarrhea, neurological disorders, and hemorrhage of mucous membranes and serous membranes in affected chickens.

[0003] Infectious bronchitis (IB) is an acute, highly contagious respiratory disease in chickens caused by a virus belonging to the genus Coronavirus of the family Coronaviridae. Its clinical symptoms mainly include difficulty breathing, rales, coughing, open-mouth breathing, and sneezing. The mortality rate is generally low, but it can infect chickens of all ages and sexes.

[0004] Avian influenza (AI) is an acute, highly contagious disease caused by the avian influenza virus. It is prevalent in animals and humans and has a high mortality rate. It occurs widely around the world and poses a threat to humans and the poultry industry.

[0005] Infectious bursal disease (IBD) is a specific disease caused by the infectious bursal disease virus (IBDV) of the family DiRNAviridae, which damages the bursa of Fabricius in young chickens. It is characterized by diarrhea and exhaustion in affected chickens, with the bursa of Fabricius initially showing significant inflammation and enlargement, followed by atrophy, ultimately leading to death.

[0006] Avian adenovirus, this disease is caused by infection with serotype 8b of group I avian adenovirus. Epidemiological surveys have found that the disease has a high incidence rate in chicken flocks in my country, especially causing severe damage to chicks. It mainly occurs in chicks, and infected chicks exhibit stunted growth, ruffled feathers, and squatting.

[0007] Newcastle disease and avian influenza are two common and serious diseases that pose a significant threat to poultry. At the same time, infectious bronchitis, infectious bursal disease, and avian adenovirus infections also cause serious losses to the poultry industry. In order to reduce costs and avoid injection stress on chickens, farms urgently need safe, efficient, and easy-to-administer multivalent vaccines to cope with the ever-changing disease control situation. Therefore, it is necessary to research and develop a pentavalent inactivated vaccine for Newcastle disease, infectious bronchitis, avian influenza, infectious bursal disease, and avian adenovirus to make up for the shortcomings of existing vaccines.

[0008] Summary of the invention.

[0009] To address the problems mentioned in the background section, this invention provides a pentavalent vaccine for avian neomycosis and bronchitis and its preparation method, which features rapid antibody production, high potency, long-lasting protection, and prevention of disease occurrence and spread.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a five-in-one vaccine for chickens containing Newcastle disease, infectious bronchitis, avian influenza, infectious bursal disease virus, and avian adenovirus, and a method for preparing the same, comprising Newcastle disease virus solution, infectious bronchitis virus solution, avian influenza virus solution, infectious bursal disease virus solution, and avian adenovirus virus solution, wherein the Newcastle disease virus solution is prepared by:

[0011] 1. Provide frozen MDCK suspension cells and thaw the frozen MDCK suspension cells at 40-45°C; 2. Transfer the resuscitated MDCK suspension cells to serum-free culture medium and culture them at 45-50°C, 1-8% CO2, and 120-150 rpm for 2-3 days, then passage them 3-4 times. 3. Centrifuge MDCK cell suspension at a density of 3.5×106 cells / mL to 4.5×106 cells / mL. Resuspend the cell pellet in a pre-set amount of serum-free medium by pipetting. Then add 1–5 g / L of plant peptone, 1–5 g / L of SE50, 1–5 g / L of Yeast Extract, 1–10 g / L of glucose, 1–8 mg / L of TPCK, 1–5 mg / L of dexamethasone, and 2–5 mg / L of edaradoxone. Mix well to obtain Newcastle disease culture medium. IV. Inoculate the chicken Newcastle disease virus into the chicken Newcastle disease culture medium and incubate in a carbon dioxide incubator for 48-96 hours. Measure the virus titer during the incubation process. Harvest the virus solution when the virus titer is ≥8log2.

[0012] Preferably, the method for preparing the infectious bronchitis venom is as follows: 1. Inoculate chicken embryos with the virus strain at a reproduction temperature of 35℃. Candle the eggs twice a day and collect allantoic fluid from chicken embryos that die within 24-60 hours. For live embryos that are 60 hours old, incubate them overnight at 2℃ before collecting the allantoic fluid. When collecting the allantoic fluid, discard any chickens that died within 24 hours. Test the collected viral allantoic fluid. Only those with negative bacterial tests and a viral titer ≥10 EIDs0 / 0.1mL can be used as part of the antigen solution for vaccine preparation. 2. The virus solution was concentrated by zinc ester precipitation, purified by Sepharose 4FF gel chromatography column, and then inactivated by BPL for 24 hours. The inactivated virus solution was then subjected to a 37°C water bath for 4 hours to obtain the infectious bronchitis virus solution.

[0013] Preferably, the avian influenza vaccine is prepared in the following manner: 1. Centrifuge MDCK cell suspension at a density of 3.5×106 cells / mL to 4.5×106 cells / mL. Resuspend the cell pellet in serum-free medium by pipetting until homogeneous. Then add 1–5 g / L plant peptone, 1–5 g / L Yeast Extract, 1–10 g / L glucose, 3–8 mg / L TPCK, and 1–6 g / L HyPep1510, and mix well to obtain avian influenza culture medium. 2. Inoculate the avian influenza virus into the avian influenza culture medium and incubate it in a carbon dioxide incubator for 36-72 hours. During the incubation process, the virus titer is measured. When the virus titer is ≥8log2, the virus liquid is harvested and passaged to obtain avian influenza virus liquid.

[0014] Preferably, the preparation of the infectious bursal virus fluid is as follows: 1. Take 9-10 day old SPF chicken embryos, prepare chicken embryo fibroblasts, culture at 37°C, and after the cells grow into a monolayer, digest them with 0.2% trypsin solution, add twice the original volume of DMEM containing 10% newborn bovine serum, mix well, and then transfer the original volume of cell solution to another cell culture flask for culture until a monolayer is formed, then it is ready for use. 2. Discard the cell culture medium, take the production seed virus BJQ902 strain, dilute it appropriately with sterile physiological saline to 10⁻³, inoculate a monolayer of chicken embryo fibroblasts at 1 / 10 of the culture medium volume, incubate at 37°C for 1 hour, add the culture medium, and add 1% of the volume of 10% reduced glutathione solution, mix gently, and incubate at 37°C. 3. 24 hours after virus inoculation, add 0.2% of the total volume of 0.5 mol / L NaHCO3 to the culture medium every 6 hours, and culture for 3 to 4 days. Harvest the virus solution when 80% of the cells show specific lesions.

[0015] Preferably, the preparation of the avian adenovirus venom is as follows: 1. Culture LMH cells in square flasks using DMEM / F12 medium supplemented with 7% fetal bovine serum at 37°C. Passage is performed every 3 days at a ratio of 1:5. 2. Prepare avian adenovirus antigen solution. Expand the cell line to a 10-layer cell factory by passing the cells through each generation according to the passage ratio. 2. When the cell density is 100%, inoculate with avian adenovirus and replace with DMEM / F12 medium containing 2% fetal bovine serum for maintenance culture; when the cell pathogenesis is about 90% after 72 hours of culture, harvest the virus solution and store it at -15°C.

[0016] Preferred vaccine preparation method: I. Oil phase preparation: Take 95 parts of mineral oil and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80°C, then add 5 parts of Span 80, and maintain the temperature at 130°C for 60 minutes. After cooling, the oil phase preparation is complete. II. Preparation of aqueous phase: Take 100ml of each of the inactivated Newcastle disease virus, infectious bronchitis virus, avian influenza virus, infectious bursal disease virus, and avian adenovirus virus and mix them to obtain the antigen solution. Add 5 parts of sterile Tween-800ml and shake well to completely dissolve and mix thoroughly to prepare the aqueous phase. III. Emulsification preparation: Take 2 parts of the oil phase and put them into an emulsification tank. Add 1 part of the water phase and stir at 2500 r / min for 40 to 60 minutes to complete the emulsification preparation and obtain the pentavalent vaccine.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses Newcastle disease virus, infectious bronchitis virus, avian influenza virus, infectious bursal disease virus, and avian adenovirus virus to prepare an inactivated vaccine using a super-concentration process and high-quality adjuvants. After immunization, animals develop antibodies quickly, have high potency, and a long protection period, preventing the occurrence and spread of diseases. It also facilitates the administration of multivalent vaccines to cope with the ever-changing disease prevention and control situation. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 This invention provides the following technical solution: a five-in-one vaccine for chickens containing Newcastle disease, infectious bronchitis, avian influenza, infectious bursal disease virus, and avian adenovirus, and its preparation method includes Newcastle disease virus solution, infectious bronchitis virus solution, avian influenza virus solution, infectious bursal disease virus solution, and avian adenovirus virus solution; the preparation method of Newcastle disease virus solution is as follows: 1. Provide frozen MDCK suspension cells and thaw the frozen MDCK suspension cells at 40-45°C; 2. Transfer the resuscitated MDCK suspension cells to serum-free culture medium and culture them at 45-50°C, 1-8% CO2, and 120-150 rpm for 2-3 days, then passage them 3-4 times. 3. Centrifuge MDCK cell suspension at a density of 3.5×106 cells / mL to 4.5×106 cells / mL. Resuspend the cell pellet in a pre-set amount of serum-free medium by pipetting. Then add 1–5 g / L of plant peptone, 1–5 g / L of SE50, 1–5 g / L of Yeast Extract, 1–10 g / L of glucose, 1–8 mg / L of TPCK, 1–5 mg / L of dexamethasone, and 2–5 mg / L of edaradoxone. Mix well to obtain Newcastle disease culture medium. IV. Inoculate the chicken Newcastle disease virus into the chicken Newcastle disease culture medium and incubate in a carbon dioxide incubator for 48-96 hours. Measure the virus titer during the incubation process. Harvest the virus solution when the virus titer is ≥8log2.

[0020] Specifically, the preparation method of infectious bronchitis virus: 1. Inoculate chicken embryos with the virus strain at a reproduction temperature of 35℃. Candle the eggs twice a day and collect allantoic fluid from chicken embryos that die within 24-60 hours. For live embryos that are 60 hours old, incubate them overnight at 2℃ before collecting the allantoic fluid. When collecting the allantoic fluid, discard any chickens that died within 24 hours. Test the collected viral allantoic fluid. Only those with negative bacterial tests and a viral titer ≥10 EIDs0 / 0.1mL can be used as part of the antigen solution for vaccine preparation. 2. The virus solution was concentrated by zinc ester precipitation, purified by Sepharose 4FF gel chromatography column, and then inactivated by BPL for 24 hours. The inactivated virus solution was then subjected to a 37°C water bath for 4 hours to obtain the infectious bronchitis virus solution.

[0021] Specifically, the methods for preparing avian influenza vaccines: 1. Centrifuge MDCK cell suspension at a density of 3.5×106 cells / mL to 4.5×106 cells / mL. Resuspend the cell pellet in serum-free medium by pipetting until homogeneous. Then add 1–5 g / L plant peptone, 1–5 g / L Yeast Extract, 1–10 g / L glucose, 3–8 mg / L TPCK, and 1–6 g / L HyPep1510, and mix well to obtain avian influenza culture medium. 2. Inoculate the avian influenza virus into the avian influenza culture medium and incubate it in a carbon dioxide incubator for 36-72 hours. During the incubation process, the virus titer is measured. When the virus titer is ≥8log2, the virus liquid is harvested and passaged to obtain avian influenza virus liquid.

[0022] Specifically, the preparation of infectious bursal virus fluid: 1. Take 9-10 day old SPF chicken embryos, prepare chicken embryo fibroblasts, culture at 37°C, and after the cells grow into a monolayer, digest them with 0.2% trypsin solution, add twice the original volume of DMEM containing 10% newborn bovine serum, mix well, and then transfer the original volume of cell solution to another cell culture flask for culture until a monolayer is formed, then it is ready for use. 2. Discard the cell culture medium, take the production seed virus BJQ902 strain, dilute it appropriately with sterile physiological saline to 10⁻³, inoculate a monolayer of chicken embryo fibroblasts at 1 / 10 of the culture medium volume, incubate at 37°C for 1 hour, add the culture medium, and add 1% of the volume of 10% reduced glutathione solution, mix gently, and incubate at 37°C. 3. 24 hours after virus inoculation, add 0.2% of the total volume of 0.5 mol / L NaHCO3 to the culture medium every 6 hours, and culture for 3 to 4 days. Harvest the virus solution when 80% of the cells show specific lesions.

[0023] Specifically, the preparation of avian adenovirus venom: 1. Culture LMH cells in square flasks using DMEM / F12 medium supplemented with 7% fetal bovine serum at 37°C. Passage is performed every 3 days at a ratio of 1:5. 2. Prepare avian adenovirus antigen solution. Expand the cell line to a 10-layer cell factory by passing the cells through each generation according to the passage ratio. 2. When the cell density is 100%, inoculate with avian adenovirus and replace with DMEM / F12 medium containing 2% fetal bovine serum for maintenance culture; when the cell pathogenesis is about 90% after 72 hours of culture, harvest the virus solution and store it at -15°C.

[0024] Specifically, the vaccine preparation method: I. Oil phase preparation: Take 95 parts of mineral oil and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80°C, then add 5 parts of Span 80, and maintain the temperature at 130°C for 60 minutes. After cooling, the oil phase preparation is complete. II. Preparation of aqueous phase: Take 100ml of each of the inactivated Newcastle disease virus, infectious bronchitis virus, avian influenza virus, infectious bursal disease virus, and avian adenovirus virus and mix them to obtain the antigen solution. Add 5 parts of sterile Tween-800ml and shake well to completely dissolve and mix thoroughly to prepare the aqueous phase. III. Emulsification preparation: Take 2 parts of the oil phase and put them into an emulsification tank. Add 1 part of the water phase and stir at 2500 r / min for 40 to 60 minutes to complete the emulsification preparation and obtain the pentavalent vaccine.

[0025] Safety testing: Forty 10-day-old SPF chickens were used. Ten of them received an intramuscular injection of 1.0 ml of the vaccine in their breast, ten received an intravenous drip of 1 ml of the vaccine in their eyes, ten received a subcutaneous injection of 1 ml of the vaccine in their neck, and the last ten served as a control group without injection. All chickens were raised separately under the same conditions and observed for 14 consecutive days. Feed intake, water intake, and body weight of the SPF chickens before and 14 days after immunization were recorded. Dissection was performed to observe local lesions at the injection site and the absorption of the vaccine. No obvious adverse reactions were observed at the injection site or throughout the body in the 30 injected SPF chickens. There was no significant change in weight gain between the vaccine injection group and the control group. Dissection showed that the vaccine was well absorbed at the injection site, with no swelling or inflammation. All ten chickens in the control group died.

[0026] The pentavalent vaccine prepared using the method of this invention has good stability, can extend its shelf life, and has good immunization effect.

[0027] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pentavalent vaccine for chickens containing Newcastle disease, infectious bronchitis, avian influenza, infectious bursal disease virus, and avian adenovirus, and its preparation method thereof, comprising Newcastle disease virus, infectious bronchitis virus, avian influenza virus, infectious bursal disease virus, and avian adenovirus virus, characterized in that: The method for preparing Newcastle disease virus fluid:

1. Provide frozen MDCK suspension cells and thaw the frozen MDCK suspension cells at 40-45°C; 2. Transfer the resuscitated MDCK suspension cells to serum-free culture medium and culture them at 45-50°C, 1-8% CO2, and 120-150 rpm for 2-3 days, then passage them 3-4 times.

3. Centrifuge MDCK cell suspension at a density of 3.5×106 cells / mL to 4.5×106 cells / mL. Resuspend the cell pellet in a pre-set amount of serum-free medium by pipetting. Then add 1–5 g / L of plant peptone, 1–5 g / L of SE50, 1–5 g / L of Yeast Extract, 1–10 g / L of glucose, 1–8 mg / L of TPCK, 1–5 mg / L of dexamethasone, and 2–5 mg / L of edaradoxone. Mix well to obtain Newcastle disease culture medium. IV. Inoculate the chicken Newcastle disease virus into the chicken Newcastle disease culture medium and incubate in a carbon dioxide incubator for 48-96 hours. Measure the virus titer during the incubation process. Harvest the virus solution when the virus titer is ≥8log2.

2. The chicken neomycosis-associated bronchitis pentavalent vaccine and its preparation method according to claim 1, characterized in that: The method for preparing the infectious bronchitis venom:

1. Inoculate chicken embryos with the virus strain at a reproduction temperature of 35℃. Candle the eggs twice a day and collect allantoic fluid from chicken embryos that die within 24-60 hours. For live embryos that are 60 hours old, incubate them overnight at 2℃ before collecting the allantoic fluid. When collecting the allantoic fluid, discard any chickens that died within 24 hours. Test the collected viral allantoic fluid. Only those with negative bacterial tests and a viral titer ≥10 EIDs0 / 0.1mL can be used as part of the antigen solution for vaccine preparation.

2. The virus solution was concentrated by zinc ester precipitation, purified by Sepharose 4FF gel chromatography column, and then inactivated by BPL for 24 hours. The inactivated virus solution was then subjected to a 37°C water bath for 4 hours to obtain the infectious bronchitis virus solution.

3. The chicken neomycosis-associated bronchitis pentavalent vaccine and its preparation method according to claim 1, characterized in that: The method for preparing the avian influenza vaccine:

1. Centrifuge MDCK cell suspension at a density of 3.5×106 cells / mL to 4.5×106 cells / mL. Resuspend the cell pellet in serum-free medium by pipetting until homogeneous. Then add 1–5 g / L plant peptone, 1–5 g / L Yeast Extract, 1–10 g / L glucose, 3–8 mg / L TPCK, and 1–6 g / L HyPep1510, and mix well to obtain avian influenza culture medium.

2. Inoculate the avian influenza virus into the avian influenza culture medium and incubate it in a carbon dioxide incubator for 36-72 hours. During the incubation process, the virus titer is measured. When the virus titer is ≥8log2, the virus liquid is harvested and passaged to obtain avian influenza virus liquid.

4. The chicken neomycosis-associated bronchitis pentavalent vaccine and its preparation method according to claim 1, characterized in that: Preparation of the infectious bursal virus fluid:

1. Take 9-10 day old SPF chicken embryos, prepare chicken embryo fibroblasts, culture at 37°C, and after the cells grow into a monolayer, digest them with 0.2% trypsin solution, add twice the original volume of DMEM containing 10% newborn bovine serum, mix well, and then transfer the original volume of cell solution to another cell culture flask for culture until a monolayer is formed, then it is ready for use.

2. Discard the cell culture medium, take the production seed virus BJQ902 strain, dilute it appropriately with sterile physiological saline to 10⁻³, inoculate a monolayer of chicken embryo fibroblasts at 1 / 10 of the culture medium volume, incubate at 37°C for 1 hour, add the culture medium, and add 1% of the volume of 10% reduced glutathione solution, mix gently, and incubate at 37°C.

3. 24 hours after virus inoculation, add 0.2% of the total volume of 0.5 mol / L NaHCO3 to the culture medium every 6 hours, and culture for 3 to 4 days. Harvest the virus solution when 80% of the cells show specific lesions.

5. The chicken neomycosis-associated bronchitis pentavalent vaccine and its preparation method according to claim 1, characterized in that: Preparation of the avian adenovirus venom:

1. Culture LMH cells in square flasks using DMEM / F12 medium supplemented with 7% fetal bovine serum at 37°C. Passage is performed every 3 days at a ratio of 1:

5.

2. Prepare avian adenovirus antigen solution. Expand the cell line to a 10-layer cell factory by passing the cells through each generation according to the passage ratio.

2. When the cell density is 100%, inoculate with avian adenovirus and replace with DMEM / F12 medium containing 2% fetal bovine serum for maintenance culture; when the cell pathogenesis is about 90% after 72 hours of culture, harvest the virus solution and store it at -15°C.

6. The chicken neomycosis-associated bronchitis pentavalent vaccine and its preparation method according to claim 1, characterized in that: Vaccine preparation methods: I. Oil phase preparation: Take 95 parts of mineral oil and 1 part of aluminum stearate, mix them evenly in an oil phase preparation tank and heat to 80°C, then add 5 parts of Span 80, and maintain the temperature at 130°C for 60 minutes. After cooling, the oil phase preparation is complete. II. Preparation of aqueous phase: Take 100ml of each of the inactivated Newcastle disease virus, infectious bronchitis virus, avian influenza virus, infectious bursal disease virus, and avian adenovirus virus and mix them to obtain the antigen solution. Add 5 parts of sterile Tween-800ml and shake well to completely dissolve and mix thoroughly to prepare the aqueous phase. III. Emulsification preparation: Take 2 parts of the oil phase and put them into an emulsification tank. Add 1 part of the water phase and stir at 2500 r / min for 40 to 60 minutes to complete the emulsification preparation and obtain the pentavalent vaccine.