Chicken new branch method glandular slip quintuplet vaccine and preparation method thereof

Abstract

The invention belongs to the technical field of research and development of quintuplet vaccines, and particularly relates to a chicken Newcastle disease virus strain LaSota, a chicken Newcastle disease virus strain H120, a chicken Newcastle disease virus strain N49, a chicken Newcastle disease virus strain FADV-4 and a chicken Newcastle disease virus strain HN01. The chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain FADV-4 and the chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain FADV-4 and the chicken Newcastle disease virus strain HN01 are combined. The chicken Newcastle disease virus LaSota strain is proliferated in chicken embryo fibroblasts or chicken kidney cells, and the chicken Newcastle disease virus LaSota strain and the preparation method thereof have the beneficial effects that the chicken Newcastle disease virus LaSota strain and the preparation method thereof can be used for preparing the chicken Newcastle disease virus LaSota strain. The preparation method comprises the following steps: selecting an inactivated newcastle disease virus LaSota strain, an inactivated bronchitis H120 strain, an inactivated bursal disease N49 strain, an inactivated fowl adenovirus FADV-4 strain and an inactivated mycoplasma synovium HN01 strain; it can be guaranteed that a follow-up vaccine has the advantages of being low in toxicity, high in biological safety, high in cross protection capacity, high in immune response speed, long in antibody protection period, high in cellular immune level and high in virus culture content.

Description

Technical Field

[0001] This invention belongs to the field of pentavalent vaccine research and development technology, specifically relating to a pentavalent vaccine for chicken new bronchitis and adenovirus syringovirus and its preparation method. Background Technology

[0002] In modern chicken farming, the types of infectious diseases are increasing and their occurrence is becoming more frequent. Practice has proven that immunization is the most effective method for preventing infectious diseases. Immunization refers to the artificial inoculation of chicken flocks with vaccines, thereby stimulating the flock to develop specific resistance to certain pathogenic microorganisms and transforming susceptible animals into non-susceptible animals. In epidemic areas or areas with potential risks of certain infectious diseases, planned immunization of healthy chicken flocks is one of the important measures for preventing and controlling the occurrence of chicken infectious diseases. However, chickens have many infectious diseases, and ordinary vaccines can only treat a limited number of infectious diseases. Therefore, there is a need for a highly effective, safe, protective, and long-lasting pentavalent vaccine that can also address chicken new bronchitis, mycoplasma, and adenovirus infections. Summary of the Invention

[0003] To address the problems mentioned in the background section, this invention provides a pentavalent vaccine for avian mycoplasma and bronchopneumoniae virus (MPV) and its preparation method, which is characterized by high efficiency, safety, strong protective efficacy, long-lasting protection, and the ability to eliminate MPV.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a pentavalent vaccine for Newcastle disease, bronchitis, adenovirus, and synovitis in chickens, wherein the pentavalent vaccine is composed of inactivated Newcastle disease virus LaSota strain, inactivated bronchitis virus H120 strain, inactivated infectious bursal disease virus N49 strain, inactivated avian adenovirus FADV-4 strain, and inactivated mycoplasma synovitis virus HN01 strain.

[0005] Preferably, the Newcastle disease virus LaSota strain proliferates in chicken embryo fibroblasts or chicken kidney cells; the bronchitis H120 strain proliferates in chicken embryo fibroblasts; the infectious bursal disease N49 strain proliferates in chicken embryo fibroblasts; the avian adenovirus FADV-4 strain proliferates in chicken embryo fibroblasts; and the synovial mycoplasma HN01 strain proliferates in chicken embryo fibroblasts. Preferred, a method for preparing a pentavalent vaccine for Newcastle disease, bronchitis, and synovitis in chickens includes the following steps: Step 1: Selecting strains, specifically selecting Newcastle disease virus LaSota strain, bronchitis H120 strain, infectious bursal disease N49 strain, avian adenovirus FADV-4 strain and synovitis mycoplasma HN01 strain. Step 2: Virus propagation. The selected Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis strain are placed with chicken embryo fibroblasts in a cell culture incubator at a temperature of 37 degrees Celsius, humidity of 95%, and carbon dioxide concentration of 5%. The propagation time is between 2 and 3 days. After propagation, the strains are inactivated by one of the following methods: high-pressure steam inactivation, formaldehyde fumigation inactivation, and ultraviolet irradiation inactivation. The propagated viruses are then purified by centrifugation or ultrafiltration. Step 3: Vaccine preparation. Inactivated Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, Mycoplasma synovitis strain, adjuvant, and stabilizer are placed inside a mixer. The mixer is used at room temperature to mix the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, Mycoplasma synovitis strain, adjuvant, and stabilizer. The adjuvant is either an aluminum salt adjuvant or a distillation adjuvant. The stabilizer is either an amino acid or a buffer salt. The mixing ratio is 1 unit of Newcastle disease virus strain : 1 unit of infectious bursal disease virus strain : 1 unit of avian adenovirus strain : 1 unit of Mycoplasma synovitis strain : 1 unit of Mycoplasma synovitis strain : 10 units of adjuvant : 10 units of stabilizer. Step 4: Safety Testing and Evaluation. The prepared vaccine is injected into a batch of animals, such as mice and chickens. The Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis virus strain are injected into the animals in sequence according to the time. At the same time, five control experiments are set up, and the five control experimental animals are injected with the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis virus strain respectively. The animals are observed for 20-35 days, and the vaccine effect is recorded and evaluated. Step 5: Packaging and Storage. After testing, the vaccines are packaged into appropriate containers, labeled, and heat-sealed. The ambient temperature during the packaging process is generally controlled between 23 and 26 degrees Celsius. When storing the vaccines, the temperature should be controlled between 2 and 8 degrees Celsius.

[0006] Preferably, the tools required for virus proliferation in step two include cell culture flasks, pipettes, cell culture media, cell culture incubators, and inverted microscopes.

[0007] Preferably, the preparation temperature of the vaccine in step three is maintained at room temperature, and the tools used are a mixer, a sterile container, and a sterile filter.

[0008] Preferably, the tools used in step four, safety testing and evaluation, include syringes, laboratory animals, and observation equipment.

[0009] Preferably, the tools used for packaging and storage in step five include packaging machines, aluminum foil bags, vacuum packaging materials, and labels.

[0010] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention, by selecting inactivated Newcastle disease virus LaSota strain, inactivated bronchitis virus H120 strain, inactivated infectious bursal disease virus N49 strain, inactivated avian adenovirus FADV-4 strain, and inactivated mycoplasma synovitis strain HN01 strain, can ensure that the subsequent vaccine has the advantages of low virulence, high biosafety, strong cross-protection, accelerated immune response, extended antibody protection period, improved cellular immunity level, and high virus culture content. 2. In this invention, by setting Newcastle disease virus LaSota strain, inactivated bronchitis H120 strain, inactivated infectious bursal disease N49 strain, inactivated avian adenovirus FADV-4 strain, and inactivated mycoplasma synovitis HN01 strain into chicken embryo fibroblasts for proliferation, it is possible to ensure both the proliferation effect and the proliferation efficiency. Detailed Implementation

[0011] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0012] Example 1 The present invention provides the following technical solution: a pentavalent vaccine for Newcastle disease, bronchitis, adenovirus and synovitis in chickens, wherein the pentavalent vaccine is composed of inactivated Newcastle disease virus LaSota strain, inactivated bronchitis virus H120 strain, inactivated infectious bursal disease virus N49 strain, inactivated avian adenovirus FADV-4 strain and inactivated mycoplasma synovitis virus HN01 strain.

[0013] Specifically, the Newcastle disease virus LaSota strain proliferates in chicken embryo fibroblasts or chicken kidney cells, the bronchitis H120 strain proliferates in chicken embryo fibroblasts, the infectious bursal disease N49 strain proliferates in chicken embryo fibroblasts, the avian adenovirus FADV-4 strain proliferates in chicken embryo fibroblasts, and the synovial mycoplasma HN01 strain proliferates in chicken embryo fibroblasts.

[0014] Specifically, a method for preparing a pentavalent vaccine for chickens with Newcastle disease, bronchitis, infectious bursal disease, and mycoplasma synovitis includes the following steps: Step 1: Selecting virus strains, specifically selecting the Newcastle disease virus (LaSota) strain, bronchitis virus (H120) strain, infectious bursal disease (N49) strain, avian adenovirus (FADV-4) strain, and mycoplasma synovitis strain; Step 2: Virus propagation, mixing the selected Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis strain with chicken embryo fibroblasts. The cells are placed in a cell culture incubator at a temperature of 37 degrees Celsius, a humidity of 95%, and a carbon dioxide concentration of 5%. The first-generation proliferation time is between 2 and 3 days. After the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis strain are proliferated, the strains are then inactivated by one of the following inactivation methods: high-pressure steam inactivation, formaldehyde fumigation inactivation, and ultraviolet irradiation inactivation. After that, the proliferated viruses are purified by centrifugation or ultrafiltration. Step 3: Vaccine preparation. Inactivated Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, Mycoplasma synovitis strain, adjuvant, and stabilizer are placed inside a mixer. The mixer is used at room temperature to mix the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, Mycoplasma synovitis strain, adjuvant, and stabilizer. The adjuvant is either an aluminum salt adjuvant or a distillation adjuvant. The stabilizer is either an amino acid or a buffer salt. The mixing ratio is 1 unit of Newcastle disease virus strain : 1 unit of infectious bursal disease virus strain : 1 unit of avian adenovirus strain : 1 unit of Mycoplasma synovitis strain : 1 unit of Mycoplasma synovitis strain : 10 units of adjuvant : 10 units of stabilizer. Step 4: Safety Testing and Evaluation. The prepared vaccine is injected into a batch of animals, such as mice and chickens. The Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis virus strain are injected into the animals in sequence according to the time. At the same time, five control experiments are set up, and the five control experimental animals are injected with the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis virus strain respectively. The animals are observed for 20-35 days, and the vaccine effect is recorded and evaluated. Step 5: Packaging and Storage. After testing, the vaccines are packaged into appropriate containers, labeled, and heat-sealed. The ambient temperature during the packaging process is generally controlled between 23 and 26 degrees Celsius. When storing the vaccines, the temperature should be controlled between 2 and 8 degrees Celsius.

[0015] Specifically, the tools required for virus proliferation in step two include cell culture flasks, pipettes, cell culture media, cell culture incubators, and inverted microscopes.

[0016] Specifically, in step three, the vaccine preparation temperature is maintained at room temperature, and the tools used are a mixer, a sterile container, and a sterile filter.

[0017] Specifically, the tools used in step four, safety testing and evaluation, include syringes, laboratory animals, and observation equipment.

[0018] Specifically, the tools used for packaging and storage in step five include packaging machines, aluminum foil bags, vacuum packaging materials, and labels.

[0019] The working principle and usage process of this invention: Step 1: Selecting strains, specifically Newcastle disease virus LaSota strain, bronchitis H120 strain, infectious bursal disease N49 strain, avian adenovirus FADV-4 strain and mycoplasma synovitis HN01 strain. Step 2: Virus propagation. The selected Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis strain are placed with chicken embryo fibroblasts in a cell culture incubator at a temperature of 37 degrees Celsius, humidity of 95%, and carbon dioxide concentration of 5%. The propagation time is between 2 and 3 days. After propagation, the strains are inactivated by one of the following methods: high-pressure steam inactivation, formaldehyde fumigation inactivation, and ultraviolet irradiation inactivation. The propagated viruses are then purified by centrifugation or ultrafiltration. Step 3: Vaccine preparation. Inactivated Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, Mycoplasma synovitis strain, adjuvant, and stabilizer are placed inside a mixer. The mixer is used at room temperature to mix the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, Mycoplasma synovitis strain, adjuvant, and stabilizer. The adjuvant is either an aluminum salt adjuvant or a distillation adjuvant. The stabilizer is either an amino acid or a buffer salt. The mixing ratio is 1 unit of Newcastle disease virus strain : 1 unit of infectious bursal disease virus strain : 1 unit of avian adenovirus strain : 1 unit of Mycoplasma synovitis strain : 1 unit of Mycoplasma synovitis strain : 10 units of adjuvant : 10 units of stabilizer. Step 4: Safety Testing and Evaluation. The prepared vaccine is injected into a batch of animals, such as mice and chickens. The Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis virus strain are injected into the animals in sequence according to the time. At the same time, five control experiments are set up, and the five control experimental animals are injected with the Newcastle disease virus strain, bronchitis virus strain, infectious bursal disease virus strain, avian adenovirus strain, and mycoplasma synovitis virus strain respectively. The animals are observed for 20-35 days, and the vaccine effect is recorded and evaluated. Step 5: Packaging and Storage. After testing, the vaccines are packaged into appropriate containers, labeled, and heat-sealed. The ambient temperature during the packaging process is generally controlled between 23 and 26 degrees Celsius. When storing the vaccines, the temperature should be controlled between 2 and 8 degrees Celsius.

[0020] 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 new branch of the gland of chickens five vaccine, characterized in that: The chicken new branch law gland slide five vaccine is composed of inactivated chicken newcastle disease virus LaSota strain, inactivated bronchitis H120 strain, inactivated bursa disease N49 strain, inactivated fowl adenovirus FADV-4 strain and inactivated synovial mycoplasma HN01 strain.

2. The new branch gland slide five-in-one vaccine for chicken according to claim 1, characterized in that: The chicken newcastle disease virus LaSota strain is proliferated in chicken embryo fibroblasts or chicken kidney cells, the bronchitis H120 strain is proliferated in chicken embryo fibroblasts, the bursa disease N49 strain is proliferated in chicken embryo fibroblasts, the fowl adenovirus FADV-4 strain is proliferated in chicken embryo fibroblasts, and the synovial mycoplasma HN01 strain is proliferated in chicken embryo fibroblasts.

3. A method for preparing a chicken new-branch glandular slip five-in-one vaccine according to any one of claims 1-2, comprising the following steps, characterized in that: Step one: selecting strains, respectively selecting chicken newcastle disease virus LaSota strain, bronchitis H120 strain, bursa disease N49 strain, fowl adenovirus FADV-4 strain and synovial mycoplasma HN01 strain; Step two: virus proliferation, placing the selected chicken newcastle disease virus strain, bronchitis strain, bursa disease strain, fowl adenovirus strain and synovial mycoplasma strain into a cell culture box with a temperature of 37 degrees, humidity of 95% and carbon dioxide concentration of 5%, and one generation of proliferation time is between 2-3 days, and after the proliferation of the chicken newcastle disease virus strain, the bronchitis strain, the bursa disease strain, the fowl adenovirus strain and the synovial mycoplasma strain, one of the inactivation methods of high-pressure steam inactivation, formaldehyde fumigation inactivation and ultraviolet irradiation inactivation is used for inactivation treatment, and then the proliferated virus is purified by centrifugation or ultrafiltration; Step three: vaccine preparation, placing the inactivated chicken newcastle disease virus strain, bronchitis strain, bursa disease strain, fowl adenovirus strain, synovial mycoplasma strain, adjuvant and stabilizer into a mixer, and the mixer mixes the chicken newcastle disease virus strain, bronchitis strain, bursa disease strain, fowl adenovirus strain, synovial mycoplasma strain, adjuvant and stabilizer at room temperature, the selection of adjuvant is one of aluminum salt adjuvant or emulsion adjuvant, the selection of stabilizer is one of amino acid or buffer salt, and the mixing ratio is 1 unit of newcastle disease strain: 1 unit of bursa disease strain: 1 unit of fowl adenovirus strain: 1 unit of synovial mycoplasma strain: 1 unit of synovial mycoplasma strain: 10 units of adjuvant: 10 units of stabilizer; Step four: safety detection and evaluation, injecting the prepared vaccine into a batch of animals, such as mice and chickens, and injecting the chicken newcastle disease virus strain, bronchitis strain, bursa disease strain, fowl adenovirus strain and synovial mycoplasma strain into the animals in sequence, setting up five groups of control experiments at the same time, and injecting the chicken newcastle disease virus strain, bronchitis strain, bursa disease strain, fowl adenovirus strain and synovial mycoplasma strain into the five groups of control experiment animals respectively, and observing for 20-35 days to record and evaluate the effect of the vaccine; Step five: packaging storage, detection of finished vaccine sub-packaged to the appropriate container, and the container is labeled and heat-sealed treatment, sub-packaging process ambient temperature control between approximately 23-26 degrees, the vaccine storage, the vaccine needs to be controlled at 2 degrees-8 degrees.

4. The process for the preparation of a new type of five-in-one vaccine against Gumboro disease in chickens according to claim 3, characterized by the fact that: The tools required for the virus propagation in step two are cell culture bottles, pipettes, cell culture medium, cell incubators and inverted microscopes, etc.

5. A process for the preparation of a new type of avian Pasteurella multocida vaccine according to claim 3, characterized in that: The preparation temperature of the vaccine preparation in step three is kept at room temperature, and the tools used are mixers, sterile containers and sterile filters.

6. A process for the preparation of a new type of avian Pasteurella multocida vaccine according to claim 1, characterized by: The tools used in the safety evaluation in step four are syringes, experimental animals and observation equipment, etc.

7. A process for the preparation of a new type of avian Pasteurella multocida vaccine according to claim 1, characterized by: The tools used in step five for packaging and storage are packaging machines, aluminum foil bags, vacuum packaging materials and labels, etc.