Poultry yellow glandular triple vaccine and preparation method thereof

Abstract

The invention relates to the field of preparation of poultry vaccines, in particular to a triple vaccine for avian yellow glands and a preparation method thereof, and the triple vaccine for avian yellow glands comprises 90-95 parts of mineral oil; 5 to 20 parts of a surfactant; 2-8 parts of a complexing agent; 1-10 parts of a divalent inorganic salt; 30-50 parts of a first virus liquid containing inactivated avian influenza virus; 30-40 parts of a second virus liquid containing inactivated fowl adenovirus; 40-60 parts of a third virus liquid containing inactivated chicken flavivirus; 5 to 10 parts of span-80; 5 to 10 parts of tween-80; and 1-3 parts of aluminum stearate. The vaccine prepared by the invention does not generate any adverse reaction on chickens, is high in experimental safety, and can induce high-level neutralizing antibodies in immunized chickens, which indicates that the immunized chickens can effectively resist virus attacks of chicken flavivirus, avian influenza and fowl adenovirus.

Description

Technical Field

[0001] The invention relates to the field of poultry vaccine preparation, and in particular to a poultry flu-yellow-gland triple vaccine and a preparation method thereof. Background Art

[0002] Avian Flavivirus is a flavivirus that can cause decreased egg production, decreased feed intake, growth retardation and death in poultry. Many domestic scholars have reported that the virus is pathogenic to ducks, geese, chickens and other poultry. As an emerging disease, the disease can cause a significant decrease in egg production or even complete egg production in breeding (eating) poultry, and a large number of commercial meat poultry die, causing significant economic losses to poultry, especially duck and goose farming. Gene sequence analysis results show that Avian Flavivirus is most closely related to Tembusu virus of the Ntaya virus group of the Flavivirus genus.

[0003] Avian influenza is caused by influenza A virus. It not only brings huge economic losses to animal husbandry, but also seriously threatens human health and life. At present, H5N1 subtype avian influenza virus has infected people in many countries. The World Health Organization has confirmed that avian influenza virus can be transmitted from person to person under certain conditions. Therefore, the development of new, efficient and safe avian influenza vaccines has become an important task for virologists. At present, the disease is mainly prevented by vaccination. Conventional inactivated vaccines have poor immune effects on the disease, while attenuated vaccines have potential biosafety issues. Therefore, it is imperative to develop new, efficient, cheap and safe vaccines. With the rapid development of molecular biology technology, scholars are constantly conducting research on new genetic engineering influenza vaccines.

[0004] Through the epidemiological survey of group I avian adenovirus, the disease has a high incidence rate in chickens in my country. It can be transmitted through both horizontal and vertical pathways, and the trend is increasing year by year. The host range of the disease is also getting wider and wider. White-feathered broilers, broiler breeders, laying hens, and yellow-feathered chickens can all be infected and fall ill. Especially after 2010, the incidence has shown an increasing trend and is prevalent throughout the country. Many group I avian adenoviruses can replicate in healthy poultry, and the symptoms are very mild or no symptoms of infection are shown. However, group I type 4 avian adenovirus is an exception. It can directly cause the disease in chickens. The main lesions are pericardial effusion and enlargement of the liver and kidneys, and the trend is increasing year by year, which brings serious harm to the chicken farming industry. At present, most of the composite vaccines used on the market are new flu vaccines, which do not contain flavivirus. However, this is relatively limited in terms of disease prevention. In addition, the abuse of antibiotics has led to the emergence of drug-resistant strains, making drug treatment ineffective and wasteful; the residues of antibiotics also make meat animals face the problem of not being able to pass when exported. Summary of the invention

[0005] Based on this, the present invention proposes a avian flu virus, avian influenza and avian adenovirus triple vaccine and a preparation method thereof, which can reduce the incidence of avian flu virus, avian influenza and avian adenovirus and improve the survival efficiency of chickens and eggs.

[0006] According to one aspect of the present invention, there is provided an avian influenza virus triple vaccine, comprising:

[0007] Mineral oil, 90-95 parts;

[0008] Surfactant, 5-20 parts;

[0009] Complexing agent, 2-8 parts;

[0010] Divalent inorganic salt, 1-10 parts;

[0011] 30-50 copies of the first virus solution containing inactivated avian influenza virus;

[0012] A second virus solution containing inactivated avian adenovirus, 30-40 portions;

[0013] A third virus solution containing inactivated chicken yellow virus, 40-60 portions;

[0014] Siben-80, 5-10 copies;

[0015] Tween-80, 5-10 parts;

[0016] 1-3 parts of aluminum stearate.

[0017] Furthermore, the divalent inorganic salt is selected from one of manganese gluconate, calcium gluconate, calcium aspartate, zinc gluconate, ferrous gluconate and calcium chloride.

[0018] Furthermore, the complexing agent is selected from one of ethylenediaminetetraacetic acid, sodium gluconate, potassium gluconate and sodium polyacrylate.

[0019] Further, the surfactant is an ethoxylated sugar ester.

[0020] According to a second aspect of the present invention, there is provided a method for preparing an avian influenza-yellow gland triple vaccine, comprising:

[0021] preparing and harvesting a first virus liquid containing inactivated avian influenza virus;

[0022] preparing and harvesting a second virus fluid containing inactivated avian adenovirus;

[0023] preparing and harvesting a third virus solution containing inactivated chicken yellow virus;

[0024] Take mineral oil and aluminum stearate, mix them evenly in an oil phase preparation tank and heat them to 80°C, then add SIBEN 80 and maintain the temperature at 115°C for 40 minutes. After cooling, the oil phase preparation is completed.

[0025] Mixing the first virus liquid, the second virus liquid and the third virus liquid to obtain a mixed virus liquid;

[0026] Take the mixed virus solution, 5 parts of sterilized Tween 80, and the divalent inorganic salt solution and mix them thoroughly to complete the preparation of the aqueous phase;

[0027] Take the oil phase and put it into an emulsification tank, add the water phase, where the volume of the oil phase to the water phase is 2:1, add the chelating agent and surfactant, and then stir at 3500r / min for 30-40 minutes to complete the vaccine preparation.

[0028] Furthermore, the separation process of the chicken yellow virus in the third virus liquid includes:

[0029] Virus isolation and culture: Aseptically collect the hemorrhagic follicles from laying hens showing abnormal egg production, homogenize according to the conventional method, freeze and thaw repeatedly for 4 times, centrifuge at 12,000 r / min for 10 min at 4°C, aspirate the supernatant under aseptic conditions, and freeze and store for later use;

[0030] Pathogenic avian embryo isolation and culture: the strain in the supernatant is continuously propagated through SPF chicken embryos for 4 generations, and the obtained SPF chicken embryo adapted strain is the chicken yellow virus strain;

[0031] The chicken yellow virus strain was diluted with sterile liquid at 1:10, inoculated into SPF chicken embryos via the allantoic cavity, cultured at 37°C, dead embryos within 24 hours were discarded, and when the embryos were dying, they were placed in a 4°C refrigerator for more than 4 hours, and the allantoic fluid of the dead embryos was harvested and frozen at -20°C;

[0032] The chicken yellow virus in the dead embryo allantoic fluid is detected. When the ELD50 of the chicken yellow virus is ≥10-4.0 / 0.1ml, it can be used for virus inactivation.

[0033] Furthermore, the preparation and harvesting of the third virus liquid containing inactivated chicken yellow virus comprises: inoculating the chicken yellow virus strain into SPF chicken embryos, collecting the allantoic fluid of SPF chicken embryos that died after inoculation, adding formaldehyde with a final concentration of 0.1%, inactivating at 37° C. for 24 hours, and concentrating by appropriate ultrafiltration as the third virus liquid containing inactivated chicken yellow virus, and storing at 37° C. for standby use.

[0034] Further, preparing and harvesting the first virus liquid containing inactivated avian influenza virus includes:

[0035] Dilute the virus seed with sterile PBS and inoculate SPF chicken embryos into the allantoic cavity, 0.1 ml per embryo, and continue incubation at 36-37°C. Harvest the embryo fluid of infected chickens that are not dead 72 hours after inoculation and place in a sterile container;

[0036] Mixing chicken embryo fluid that has been tested for sterility and has an agglutination titer of not less than 1:512 to a 1% chicken red blood cell suspension, and quantitatively packaging to obtain a first virus fluid containing avian influenza virus;

[0037] The avian influenza virus in the first virus liquid is inactivated.

[0038] Furthermore, inactivating the avian influenza virus in the first virus liquid includes: introducing the first virus liquid into an inactivation tank, adding a 10% formaldehyde solution in a metered manner, mixing thoroughly, the final concentration of the formaldehyde solution is 0.2%, inactivating at 37° C. for 16 hours, and obtaining a first virus liquid containing inactivated avian influenza virus.

[0039] Further, preparing and harvesting the second virus liquid containing inactivated avian adenovirus comprises:

[0040] Avian adenovirus was inoculated into chicken liver cells at a 1% dose;

[0041] Culture in a 37°C, 5% CO2 incubator, and harvest the virus solution when more than 80% of the cells show CPE;

[0042] The harvested venom is concentrated 2 to 3 times at 2 to 8°C using an ultrafiltration concentrator to obtain a concentrated solution;

[0043] The concentrated solution was introduced into an inactivation tank, 10% formaldehyde solution was added in a metered amount, and the mixture was fully mixed. The final concentration of the formaldehyde solution was 0.2%, and the mixture was inactivated at 37° C. for 16 hours to obtain a second virus solution containing inactivated avian adenovirus.

[0044] It can be seen from the above technical scheme that the avian influenza yellow gland triple vaccine and the preparation method thereof provided by the present invention have the following beneficial effects:

[0045] The present invention proposes an inactivated vaccine against chicken yellow virus, avian influenza and avian adenovirus. The research and preparation of the vaccine solves the defect that the disease cannot be effectively prevented in my country at present, and can effectively solve the high incidence of chicken yellow virus, avian influenza and avian adenovirus, which causes the loss of chicken egg production. The inactivated vaccine is prepared by using chicken yellow virus, which has a simple process and convenient operation, and the prepared vaccine has good antigenicity and high safety, has no adverse effect on the external environment, and is easy to pass the safety evaluation.

[0046] The vaccine prepared by the present invention will not produce any adverse reaction to chickens, has high experimental safety, and can induce high levels of neutralizing antibodies in the bodies of immunized chickens, indicating that the immunized chickens can effectively resist viral attacks of chicken yellow virus, avian influenza and avian adenovirus.

[0047] The vaccine of the present invention has high titer, good immunogenicity and can resist the attack of virus isolates of avian adenovirus disease in various places. The vaccine prepared by the present invention has good safety, and no local and systemic adverse reactions caused by the vaccine occur. In the shelf life test, the data of properties, safety tests and efficacy tests were analyzed. The results showed that there was no significant difference between the triple vaccine and the single vaccine of similar products, and all were stable and effective; the efficacy test results showed that the antibodies of the triple vaccine and other two-in-one vaccines or three single vaccines were maintained at a high level, and the antibodies were produced faster than similar products, while the antibodies of the control group were negative.

[0048] The invention has the advantages that the diseases caused by chicken yellow virus, avian influenza and fowl adenovirus can be treated at the same time, a synergistic immune effect can be produced by the combination induction of multiple immunogens, a specific immune effect is produced, the frequency of injection is reduced, the situation that at least two injections are needed to prevent the above diseases is changed, the immunization cost is reduced, the stress response of animals is reduced, and the triple inactivated vaccine has high titer content, convenient and fast immunization, avoids the adverse reaction of multiple vaccinations, and reduces the breeding cost. Compared with the current vaccine, more serotypes can be immunized with a single injection, so as to achieve the purpose of "one injection to prevent multiple diseases", and it is easy to use, reduces the stress of pigs, and there is no hidden danger of spreading poison, which is safe and reliable.

[0049] The present invention adopts a variety of adjuvants, including oily adjuvants and aqueous adjuvants, to reduce the side effects of redness, swelling and fever at the immunization site after immunization, will not affect the spirit and feeding conditions of the immunized poultry, and effectively improves the immune effect and production performance of the immunized poultry. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0051] At present, most of the compound vaccines used in the market are new flu vaccines, which do not contain flavivirus. However, this has limited effect in disease prevention. Moreover, due to the abuse of antibiotics, drug-resistant strains have been produced, making drug treatment ineffective and wasteful; the residue of antibiotics also makes it difficult for meat animals to pass when exported. Therefore, it is urgent to invent a new triple inactivated vaccine that can prevent diseases caused by avian influenza, avian flavivirus and avian adenovirus at the same time to solve the above problems.

[0052] In view of some existing problems, the present invention provides an avian influenza virus triple vaccine, comprising:

[0053] Mineral oil, 90-95 parts;

[0054] Surfactant, 5-20 parts;

[0055] Complexing agent, 2-8 parts;

[0056] Divalent inorganic salt, 1-10 parts;

[0057] 30-50 copies of the first virus solution containing inactivated avian influenza virus;

[0058] A second virus solution containing inactivated avian adenovirus, 30-40 portions;

[0059] A third virus solution containing inactivated chicken yellow virus, 40-60 portions;

[0060] Siben-80, 5-10 copies;

[0061] Tween-80, 5-10 parts;

[0062] 1-3 parts of aluminum stearate.

[0063] The present invention proposes an inactivated vaccine against chicken yellow virus, avian influenza and avian adenovirus. The research and preparation of the vaccine solves the defect that the disease cannot be effectively prevented in my country at present, and can effectively solve the high incidence of chicken yellow virus, avian influenza and avian adenovirus, which causes the loss of chicken egg production. The inactivated vaccine is prepared by using chicken yellow virus, which has a simple process and convenient operation, and the prepared vaccine has good antigenicity and high safety, has no adverse effect on the external environment, and is easy to pass the safety evaluation.

[0064] The vaccine prepared by the present invention will not produce any adverse reaction to chickens, has high experimental safety, and can induce high levels of neutralizing antibodies in the bodies of immunized chickens, indicating that the immunized chickens can effectively resist viral attacks of chicken yellow virus, avian influenza and avian adenovirus.

[0065] The vaccine of the present invention has high titer, good immunogenicity and can resist the attack of virus isolates of avian adenovirus disease in various places. The vaccine prepared by the present invention has good safety, and no local and systemic adverse reactions caused by the vaccine occur. In the shelf life test, the data of properties, safety tests and efficacy tests were analyzed. The results showed that there was no significant difference between the triple vaccine and the single vaccine of similar products, and all were stable and effective; the efficacy test results showed that the antibodies of the triple vaccine and other two-in-one vaccines or three single vaccines were maintained at a high level, and the antibodies were produced faster than similar products, while the antibodies of the control group were negative.

[0066] The invention has the advantages that the diseases caused by chicken yellow virus, avian influenza and fowl adenovirus can be treated at the same time, a synergistic immune effect can be produced by the combination induction of multiple immunogens, a specific immune effect is produced, the frequency of injection is reduced, the situation that at least two injections are needed to prevent the above diseases is changed, the immunization cost is reduced, the stress response of animals is reduced, and the triple inactivated vaccine has high titer content, convenient and fast immunization, avoids the adverse reaction of multiple vaccinations, and reduces the breeding cost. Compared with the current vaccine, more serotypes can be immunized with a single injection, so as to achieve the purpose of "one injection to prevent multiple diseases", and it is easy to use, reduces the stress of pigs, and there is no hidden danger of spreading poison, which is safe and reliable.

[0067] The present invention adopts a variety of adjuvants, including oily adjuvants and aqueous adjuvants, to reduce the side effects of redness, swelling and fever at the immunization site after immunization, will not affect the spirit and feeding conditions of the immunized poultry, and effectively improves the immune effect and production performance of the immunized poultry.

[0068] Furthermore, the divalent inorganic salt is selected from one of manganese gluconate, calcium gluconate, calcium aspartate, zinc gluconate, ferrous gluconate and calcium chloride.

[0069] Furthermore, the complexing agent is selected from one of ethylenediaminetetraacetic acid, sodium gluconate, potassium gluconate and sodium polyacrylate.

[0070] Further, the surfactant is an ethoxylated sugar ester.

[0071] According to a second aspect of the present invention, there is provided a method for preparing an avian influenza-yellow gland triple vaccine, comprising:

[0072] preparing and harvesting a first virus liquid containing inactivated avian influenza virus;

[0073] preparing and harvesting a second virus fluid containing inactivated avian adenovirus;

[0074] preparing and harvesting a third virus solution containing inactivated chicken yellow virus;

[0075] Take mineral oil and aluminum stearate, mix them evenly in an oil phase preparation tank and heat them to 80°C, then add SIBEN 80 and maintain the temperature at 115°C for 40 minutes. After cooling, the oil phase preparation is completed.

[0076] mixing the first virus liquid, the second virus liquid and the third virus liquid to obtain a mixed virus liquid;

[0077] Take the mixed virus solution, 5 parts of sterilized Tween 80, and the divalent inorganic salt solution and mix them thoroughly to complete the preparation of the aqueous phase;

[0078] Take the oil phase and put it into an emulsification tank, add the water phase, where the volume of the oil phase to the water phase is 2:1, add the chelating agent and surfactant, and then stir at 3500r / min for 30-40 minutes to complete the vaccine preparation.

[0079] The present invention proposes an inactivated vaccine against chicken yellow virus, avian influenza and avian adenovirus. The research and preparation of the vaccine solves the defect that the disease cannot be effectively prevented in my country at present, and can effectively solve the high incidence of chicken yellow virus, avian influenza and avian adenovirus, which causes the loss of chicken egg production. The inactivated vaccine is prepared by using chicken yellow virus, which has a simple process and convenient operation, and the prepared vaccine has good antigenicity and high safety, has no adverse effect on the external environment, and is easy to pass the safety evaluation.

[0080] The vaccine prepared by the present invention will not produce any adverse reaction to chickens, has high experimental safety, and can induce high levels of neutralizing antibodies in the bodies of immunized chickens, indicating that the immunized chickens can effectively resist viral attacks of chicken yellow virus, avian influenza and avian adenovirus.

[0081] The vaccine of the present invention has high titer, good immunogenicity and can resist the attack of virus isolates of avian adenovirus disease in various places. The vaccine prepared by the present invention has good safety, and no local and systemic adverse reactions caused by the vaccine occur. In the shelf life test, the data of properties, safety tests and efficacy tests were analyzed. The results showed that there was no significant difference between the triple vaccine and the single vaccine of similar products, and all were stable and effective; the efficacy test results showed that the antibodies of the triple vaccine and other two-in-one vaccines or three single vaccines were maintained at a high level, and the antibodies were produced faster than similar products, while the antibodies of the control group were negative.

[0082] The invention has the advantages that the diseases caused by chicken yellow virus, avian influenza and fowl adenovirus can be treated at the same time, a synergistic immune effect can be produced by the combination induction of multiple immunogens, a specific immune effect is produced, the frequency of injection is reduced, the situation that at least two injections are needed to prevent the above diseases is changed, the immunization cost is reduced, the stress response of animals is reduced, and the triple inactivated vaccine has high titer content, convenient and fast immunization, avoids the adverse reaction of multiple vaccinations, and reduces the breeding cost. Compared with the current vaccine, more serotypes can be immunized with a single injection, so as to achieve the purpose of "one injection to prevent multiple diseases", and it is easy to use, reduces the stress of pigs, and there is no hidden danger of spreading poison, which is safe and reliable.

[0083] The present invention adopts a variety of adjuvants, including oily adjuvants and aqueous adjuvants, to reduce the side effects of redness, swelling and fever at the immunization site after immunization, will not affect the spirit and feeding conditions of the immunized poultry, and effectively improves the immune effect and production performance of the immunized poultry.

[0084] Furthermore, the separation process of the chicken yellow virus in the third virus liquid includes:

[0085] Virus isolation and culture: Aseptically collect the hemorrhagic follicles from laying hens showing abnormal egg production, homogenize according to the conventional method, freeze and thaw repeatedly for 4 times, centrifuge at 12,000 r / min for 10 min at 4°C, aspirate the supernatant under aseptic conditions, and freeze and store for later use;

[0086] Isolation and culture of pathogenic avian embryos: The strain in the supernatant is propagated through SPF chicken embryos for four generations, and the obtained SPF chicken embryo-adapted strain is the chicken yellow virus strain;

[0087] The chicken yellow virus strain was diluted with sterile liquid at 1:10, inoculated into SPF chicken embryos through the allantoic cavity, cultured at 37°C, and dead embryos within 24 hours were discarded. When the embryos were dying, they were placed in a 4°C refrigerator for more than 4 hours, and the allantoic fluid of the dead embryos was harvested and frozen at -20°C.

[0088] Detection of chicken yellow virus in the allantoic fluid of dead embryos, when the ELD50 of the chicken yellow virus ≥

[0089] 10-4.0 / 0.1ml, it can be used for virus inactivation.

[0090] Furthermore, the preparation and harvesting of the third virus liquid containing the inactivated chicken yellow virus comprises: inoculating the chicken yellow virus strain into SPF chicken embryos, collecting the allantoic fluid of SPF chicken embryos that died after being inoculated with the virus, adding formaldehyde with a final concentration of 0.1%, inactivating the fluid at 37° C. for 24 hours, and concentrating the fluid by appropriate ultrafiltration as the third virus liquid containing the inactivated chicken yellow virus, and storing the fluid at 37° C. for standby use.

[0091] Further, preparing and harvesting the first virus liquid containing inactivated avian influenza virus includes:

[0092] Dilute the virus seed with sterile PBS and inoculate SPF chicken embryos into the allantoic cavity, 0.1 ml per embryo, and continue incubation at 36-37°C. Harvest the embryo fluid of infected chickens that are not dead 72 hours after inoculation and place in a sterile container;

[0093] Mixing chicken embryo fluid that has been tested for sterility and has an agglutination titer of not less than 1:512 to a 1% chicken red blood cell suspension, and quantitatively packaging to obtain a first virus fluid containing avian influenza virus;

[0094] The avian influenza virus in the first virus liquid is inactivated.

[0095] Furthermore, inactivating the avian influenza virus in the first virus liquid includes: introducing the first virus liquid into an inactivation tank, adding a 10% formaldehyde solution, mixing thoroughly, the final concentration of the formaldehyde solution is 0.2%, inactivating at 37° C. for 16 hours, and obtaining a first virus liquid containing inactivated avian influenza virus.

[0096] Further, preparing and harvesting the second virus liquid containing inactivated avian adenovirus comprises:

[0097] Avian adenovirus was inoculated into chicken liver cells at a 1% dose;

[0098] Culture in a 37°C, 5% CO2 incubator, and harvest the virus solution when more than 80% of the cells show CPE;

[0099] The harvested venom is concentrated 2 to 3 times at 2 to 8°C using an ultrafiltration concentrator to obtain a concentrated solution;

[0100] The concentrated solution was introduced into an inactivation tank, 10% formaldehyde solution was added in a metered amount, and the mixture was fully mixed. The final concentration of the formaldehyde solution was 0.2%, and the mixture was inactivated at 37° C. for 16 hours to obtain a second virus solution containing inactivated avian adenovirus.

[0101] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0102] Example 1: Preparation of samples.

[0103] Dilute the virus seed with sterile PBS and inoculate SPF chicken embryos into the allantoic cavity, 0.1 ml per embryo, and continue incubation at 36-37°C. Harvest the embryo fluid of infected chickens that are not dead 72 hours after inoculation and place in a sterile container;

[0104] Mixing chicken embryo fluid that has been tested for sterility and has an agglutination titer of not less than 1:512 to a 1% chicken red blood cell suspension, and quantitatively packaging to obtain a first virus fluid containing avian influenza virus;

[0105] The first virus liquid was introduced into an inactivation tank, 10% formaldehyde solution was added in a metered amount, and the mixture was fully mixed. The final concentration of the formaldehyde solution was 0.2%, and the mixture was inactivated at 37° C. for 16 hours to obtain the first virus liquid containing inactivated avian influenza virus.

[0106] Avian adenovirus was inoculated into chicken liver cells at a 1% dose;

[0107] The virus solution is harvested when more than 80% of the cells show CPE in an incubator at 37° C.; the harvested venom is concentrated 2 to 3 times by an ultrafiltration concentrator at 2 to 8° C. to obtain a concentrated solution; the concentrated solution is introduced into an inactivation tank, 10% formaldehyde solution is added by metering, and the solution is fully mixed to a final concentration of 0.2%; the solution is inactivated at 37° C. for 16 hours to obtain a second virus solution containing inactivated avian adenovirus.

[0108] The hemorrhagic follicles were collected aseptically from laying hens with abnormal egg production, homogenized according to conventional methods, and repeatedly frozen and thawed for 4 times. The follicles were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was aspirated aseptically and frozen for later use.

[0109] The strain in the supernatant was propagated through SPF chicken embryos for four consecutive generations, and the SPF chicken embryo-adapted strain obtained was the chicken yellow virus strain.

[0110] The chicken yellow virus strain is inoculated into SPF chicken embryos, and the allantoic fluid of SPF chicken embryos that died after infection is collected as the third virus fluid containing inactivated chicken yellow virus.

[0111] Take mineral oil and aluminum stearate, mix them evenly in an oil phase preparation tank and heat them to 80°C, then add SIBEN 80 and maintain the temperature at 115°C for 40 minutes. After cooling, the oil phase preparation is completed.

[0112] mixing the first virus liquid, the second virus liquid and the third virus liquid to obtain a mixed virus liquid;

[0113] Take the mixed virus solution, 5 parts of sterilized Tween 80, and the divalent inorganic salt solution and mix them thoroughly to complete the preparation of the aqueous phase;

[0114] Take the oil phase and put it into an emulsification tank, add the water phase, where the volume of the oil phase to the water phase is 2:1, add the chelating agent and surfactant, and then stir at 3500r / min for 30-40 minutes to complete the vaccine preparation.

[0115] Sample 1:

[0116] The triple inactivated vaccine was prepared by the above method, except that 95 parts of mineral oil, 5 parts of ethoxylated sugar ester, 2 parts of sodium polyacrylate, 1 part of manganese gluconate, 30 parts of a first virus solution containing inactivated avian influenza virus, 30 parts of a second virus solution containing inactivated avian adenovirus, 40 parts of a third virus solution containing inactivated chicken yellow virus, 5 parts of Siben-80, 5 parts of Tween-80, and 1 part of aluminum stearate were used as sample 1.

[0117] Sample 2:

[0118] The above method is used to prepare a two-unit inactivated vaccine, which is different from the above method in that 92 parts of mineral oil, 10 parts of ethoxylated sugar ester, 4 parts of potassium gluconate, 5 parts of calcium gluconate, 35 parts of a first virus solution containing inactivated avian influenza virus, 35 parts of a second virus solution containing inactivated avian adenovirus, 44 parts of a third virus solution containing inactivated chicken yellow virus, 70 parts of Siben-80, 6 parts of Tween-80, and 2 parts of aluminum stearate are used as sample 2.

[0119] Sample 3:

[0120] The above method is used to prepare a dual inactivated vaccine, which is different from the above method in that the following are used: 90 parts of mineral oil; 514 parts of ethoxylated sugar ester; 6 parts of sodium gluconate; 5 parts of calcium aspartate; 45 parts of a first virus solution containing inactivated avian influenza virus; 38 parts of a second virus solution containing inactivated avian adenovirus; 52 parts of a third virus solution containing inactivated chicken yellow virus; 8 parts of Siben-80; 7 parts of Tween-80; and 3 parts of aluminum stearate. This is used as sample 3.

[0121] Sample 4:

[0122] The above method is used to prepare a two-unit inactivated vaccine, which is different from the above method in that 90 parts of mineral oil, 20 parts of ethoxylated sugar ester, 8 parts of ethylenediaminetetraacetic acid, 10 parts of zinc gluconate, 50 parts of a first virus solution containing inactivated avian influenza virus, 40 parts of a second virus solution containing inactivated avian adenovirus, 60 parts of a third virus solution containing inactivated chicken yellow virus, 10 parts of Siben-80, 10 parts of Tween-80, and 3 parts of aluminum stearate are used as sample 4.

[0123] Control group 1:

[0124] A combined vaccine for avian influenza and avian adenovirus is commercially available.

[0125] Control group 2:

[0126] Commercially available avian flavivirus vaccines.

[0127] Blank control group:

[0128] Normal saline solution.

[0129] Embodiment 1:

[0130] To measure the compatibility of the vaccine provided by the present invention with in ovo inoculation of chicken embryos, 0.1 ml of each of the formulations of samples 1-4 of the present invention and the control group was injected into the amniotic cavity of 18-day fertilized eggs using an Intelliject syringe (Avitech) and a 17.5 cm, 18-gauge needle.

[0131] Toxicity was measured as the number of chicks hatched after injection compared to the number of chicks hatched after injection with 0.1 ml of saline (PBS). The results are shown in Table 1.

[0132] Table 1: Hatching rate after vaccination

[0133]

[0134]

[0135] As shown in Table 1, the observation results after the vaccine was inoculated into chicken embryos showed that the vaccine provided by the present invention had good biocompatibility. The vaccine prepared by the present invention did not produce any adverse reactions to chickens, had high experimental safety, and could induce high levels of neutralizing antibodies in the immunized chickens, indicating that the immunized chickens could effectively resist the viral attacks of chicken yellow virus, avian influenza and avian adenovirus.

[0136] 2. Vaccine immune efficacy determination:

[0137] 14 days after the chicks hatched, they were challenged with 2.0×106 CFU of avian yellow virus and their survival rates were measured. The results are shown in Table 2.

[0138] Table 2: Animal experiment results (%)

[0139] Porcine circovirus type 2 Attack rate Incidence Deaths Protection rate Experimental Group 1 5 1 1 0.8 Experimental Group 2 5 1 0 0.8 Experimental Group 3 5 0 0 1 Experimental Group 4 5 0 0 1 Control group 1 5 5 5 0 Control group 2 5 2 1 0.6 Blank control group 5 5 5 0

[0140] The present invention proposes an inactivated vaccine against chicken yellow virus, avian influenza and avian adenovirus. The research and preparation of the vaccine solves the defect that the disease cannot be effectively prevented in my country at present, and can effectively solve the high incidence of chicken yellow virus, avian influenza and avian adenovirus, and the loss of sudden drop in egg production caused by chickens. The inactivated vaccine is prepared by using chicken yellow virus, with simple process and convenient operation, and the prepared vaccine has good antigenicity, high safety and no adverse effect on the external environment.

[0141] The vaccine of the present invention has high titer, good immunogenicity and can resist the attack of virus isolated from various places of avian adenovirus disease. The vaccine prepared by the present invention has good safety, and no local and systemic adverse reactions caused by the vaccine occur. In the shelf life test, the data of the properties, safety test and efficacy test were analyzed. The results showed that the triple vaccine had no significant difference compared with the single vaccine of similar products, and all were stable and effective; the efficacy test results proved that the antibodies of the triple vaccine and other two-in-one vaccines or three single vaccines were maintained at a high level, and the antibodies were produced faster than similar products.

[0142] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A poultry flu vaccine, characterized in that: include: Mineral oil, 90-95 parts; Surfactant, 5-20 parts; Complexing agent, 2-8 parts; Divalent inorganic salt, 1-10 parts; 30-50 copies of the first virus solution containing inactivated avian influenza virus; A second virus solution containing inactivated avian adenovirus, 30-40 portions; A third virus solution containing inactivated chicken yellow virus, 40-60 portions; Siben-80, 5-10 copies; Tween-80, 5-10 parts; 1-3 parts of aluminum stearate.

2. The triple vaccine according to claim 1, characterized in that The divalent inorganic salt is selected from one of manganese gluconate, calcium gluconate, calcium aspartate, zinc gluconate, ferrous gluconate and calcium chloride.

3. The triple vaccine according to claim 1, characterized in that The complexing agent is selected from one of ethylenediaminetetraacetic acid, sodium gluconate, potassium gluconate and sodium polyacrylate.

4. The triple vaccine according to claim 1, characterized in that The surfactant is an ethoxylated sugar ester.

5. A method for preparing a triple vaccine for avian influenza, yellow glands and swine fever, characterized in that: include: preparing and harvesting a first virus liquid containing inactivated avian influenza virus; preparing and harvesting a second virus fluid containing inactivated avian adenovirus; preparing and harvesting a third virus solution containing inactivated chicken yellow virus; Take mineral oil and aluminum stearate, mix them evenly in an oil phase preparation tank and heat them to 80°C, then add SIBEN 80 and maintain the temperature at 115°C for 40 minutes. After cooling, the oil phase preparation is completed. Mixing the first virus liquid, the second virus liquid and the third virus liquid to obtain a mixed virus liquid; Take the mixed virus solution, 5 parts of sterilized Tween 80, and the divalent inorganic salt solution and mix them thoroughly to complete the preparation of the aqueous phase; Take the oil phase and put it into an emulsification tank, add the water phase, where the volume of the oil phase to the water phase is 2:1, add the chelating agent and surfactant, and then stir at 3500r / min for 30-40 minutes to complete the vaccine preparation.

6. The triple vaccine according to claim 5, characterized in that The separation process of the chicken yellow virus in the third virus liquid comprises: Virus isolation and culture: Aseptically collect the hemorrhagic follicles from laying hens showing abnormal egg production, homogenize according to the conventional method, freeze and thaw repeatedly for 4 times, centrifuge at 12,000 r / min for 10 min at 4°C, aspirate the supernatant under aseptic conditions, and freeze and store for later use; Pathogenic avian embryo isolation and culture: the strain in the supernatant is continuously propagated through SPF chicken embryos for 4 generations, and the obtained SPF chicken embryo adapted strain is the chicken yellow virus strain; The chicken yellow virus strain was diluted with sterile liquid at 1:10, inoculated into SPF chicken embryos via the allantoic cavity, cultured at 37°C, dead embryos within 24 hours were discarded, and when the embryos were dying, they were placed in a 4°C refrigerator for more than 4 hours, and the allantoic fluid of the dead embryos was harvested and frozen at -20°C; Detect the chicken yellow virus in the dead embryo allantoic fluid, when the ELD50 of the chicken yellow virus is ≥ 10-4.0 / 0.1ml, it can be used for virus inactivation.

7. The triple vaccine according to claim 6, characterized in that The preparation and harvesting of the third virus liquid containing the inactivated chicken yellow virus comprises: inoculating the chicken yellow virus strain into SPF chicken embryos, collecting the allantoic fluid of the SPF chicken embryos that died after the infection, adding formaldehyde with a final concentration of 0.1%, inactivating the fluid at 37°C for 24h, and using the third virus liquid containing the inactivated chicken yellow virus after appropriate ultrafiltration and concentration, and storing the fluid at 37°C for standby use.

8. The triple vaccine according to claim 5, characterized in that: The preparation and harvesting of the first virus solution containing inactivated avian influenza virus comprises: Dilute the virus seed with sterile PBS and inoculate SPF chicken embryos into the allantoic cavity, 0.1 ml per embryo, and continue incubation at 36-37°C. Harvest the embryo fluid of infected chickens that are not dead 72 hours after inoculation and place in a sterile container; Mixing chicken embryo fluid that has been tested for sterility and has an agglutination titer of not less than 1:512 to a 1% chicken red blood cell suspension, and quantitatively packaging to obtain a first virus fluid containing avian influenza virus; The avian influenza virus in the first virus liquid is inactivated.

9. The triple vaccine according to claim 8, characterized in that Inactivating the avian influenza virus in the first virus liquid includes: introducing the first virus liquid into an inactivation tank, adding 10% formaldehyde solution by metering, fully mixing, the final concentration of the formaldehyde solution is 0.2%, inactivating at 37° C. for 16 hours, and obtaining a first virus liquid containing inactivated avian influenza virus.

10. The triple vaccine according to claim 5, characterized in that The preparation and harvest of a second virus fluid containing inactivated avian adenovirus comprises: Avian adenovirus was inoculated into chicken liver cells at a 1% dose; Culture in a 37°C, 5% CO2 incubator, and harvest the virus solution when more than 80% of the cells show CPE; The harvested venom is concentrated 2 to 3 times at 2 to 8°C using an ultrafiltration concentrator to obtain a concentrated solution; The concentrated solution was introduced into an inactivation tank, 10% formaldehyde solution was added in a metered amount, and the mixture was fully mixed. The final concentration of the formaldehyde solution was 0.2%, and the mixture was inactivated at 37° C. for 16 hours to obtain a second virus solution containing inactivated avian adenovirus.

Images