CN117085120A - Newcastle disease and avian influenza H9 subtype bivalent inactivated oral immunogen composition as well as preparation method and application thereof - Google Patents
Newcastle disease and avian influenza H9 subtype bivalent inactivated oral immunogen composition as well as preparation method and application thereof Download PDFInfo
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- CN117085120A CN117085120A CN202311064567.0A CN202311064567A CN117085120A CN 117085120 A CN117085120 A CN 117085120A CN 202311064567 A CN202311064567 A CN 202311064567A CN 117085120 A CN117085120 A CN 117085120A
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Classifications
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- A61P31/12—Antivirals
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- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a bigeminal inactivated oral immunogen composition for preventing newcastle disease and avian influenza H9 subtype, and a preparation method and application thereof; comprises the following components in volume ratio (2-4): 1 and a water adjuvant complex; wherein the inactivated antigen comprises at least one inactivated newcastle disease whole virus antigen and at least one inactivated avian influenza H9 subtype whole virus antigen; the water adjuvant compound is water-soluble adjuvant; the water-soluble auxiliary agent comprises the following raw materials in parts by weight: 4-8 parts of poloxamer polyether, 1-2 parts of glucan and 0.2-0.5 part of astragalus polysaccharide. The chicken newcastle disease and avian influenza (H9 subtype) bigeminal inactivated oral immunogen composition can be used for orally taking or drinking water to immunize chickens to maintain high-level serum antibodies (which can be equivalent to injection vaccines), can be used for immunization at any day age, has no inoculation stress, and solves the problem that an inactivated vaccine is difficult to obtain the high-level serum antibodies through oral administration.
Description
Technical Field
The invention relates to the field of veterinary medicines, in particular to a dual inactivated oral immunogen composition for newcastle disease and avian influenza H9 subtype, a preparation method and application thereof, and an oral immunogen composition comprising the same, and a preparation method and application thereof.
Background
Newcastle disease virus (Newcastledisease virus, NDV) is also known as asian fowl plague virus, pseudo fowl plague virus or avian pneumoencephalitis virus. Newcastle disease virus is a ssRNA virus, enveloped. Viral particles are polymorphic, and have circular, oval, and long rod shapes. Mature virions have a diameter of 100 to 400nm. The virus is mainly harmful to chickens, turkeys and turkeys, and can be rapidly transmitted in the affected chickens, and the virulent strain can destroy the chickens entirely. The attenuated strain only causes respiratory tract infection and egg production reduction of chicken flocks, but can be recovered rapidly. The immune control of newcastle disease should take preventive inoculation as a main measure.
Avian Influenza (AI) is an infection or disease syndrome in an Avian or animal caused by Influenza virus type a of the genus Influenza virus of the family orthomyxoviridae. Avian influenza can be classified into highly pathogenic avian influenza (Highly pathogenic avian influenza, HPAI) and less pathogenic avian influenza (Lowly pathogenic avian influenza, LPAI) according to pathogenicity of pathogens.
The H9N2 subtype avian influenza virus belongs to low pathogenicity avian influenza virus, can cause respiratory tract symptoms of laying hens to occur, the laying rate to decrease, broiler chickens to cause respiratory tract symptoms, and the constitution of infected chickens to decrease, and secondary bacterial diseases to cause the increase of death rate. Researchers in 1966 first isolated from turkeys to H9N2 subtype avian influenza virus and then subsequently isolated from chickens, pheasants, ducks and quails. Domestic Guo Yuanji is equal to that of being separated from the drinking water of ducks in 1980 for the first time, and after 1994, a large amount of viruses are separated from chicken flocks, and the viruses become one of important epidemic diseases which threaten the healthy development of the poultry industry in China.
Diphtheria Toxin (DT) is an exotoxin produced by Corynebacterium diphtheriae infected with the beta phage genome, and has strong cytotoxicity. Cross-reactive substance 197 (ccross-reacting material 197, CRM 197) is a DT non-toxic mutant. The human immune protein carrier which is often used as a safe and effective human immune protein carrier is widely applied to the field of biological medicine. CRM197 as a neutralizing antibody capable of remarkably improving the immunogenicity of target viral proteins and improving the high titer of experimental animals is widely applied to the development fields of hepatitis E, zika virus vaccines and avian influenza fusion expression vaccines. CRM197 inhibits the growth of epidermal cytokines, thereby preventing the growth of epidermal cytokines and achieving the purpose of treating tumors. CRM197 as a widely applied protein molecule not only can improve the immunogenicity of target proteins in the fields of virus protein vaccines, bacterial polysaccharide conjugate vaccines and the like, but also can be used as a molecular protein of an immune adjuvant to improve the cellular immunity and humoral immunity level of organisms, and has obvious effect on tumor treatment.
Vaccines for preventing newcastle disease and avian influenza on the market today fall into two major categories, live and inactivated, such as:
1) Live vaccines, usually attenuated live viruses, are used for nasal drip and water immunization, but lack of high serum antibodies to inactivated vaccines, and the chicken flock only gets partial protection rate after immunization, which aggravates the respiratory tract reaction of the chicken flock. The main reason is that excessive attenuation can lead to insufficient efficacy; insufficient attenuation and the possibility of pathogenicity.
2) Inactivated vaccines, generally prepared using oil adjuvants, are used to vaccinate chicken flocks by the neck subcutaneous or chest intramuscular route. In order to reduce the injection stress and the injection cost, the breast intramuscular injection of small day old (1-7 days old) or adult chickens (60-110 days old) is adopted as much as possible, and the vaccination is carried out at other days old, so that the immunization blank period is enlarged.
In addition, although the inactivated vaccine is safer, the inactivated vaccine has short immunization period because of the inactivated virus, and needs multiple injections, thereby greatly increasing the workload of the farm owner.
Theoretically, it is required that the antigenicity of the vaccine strain and the epidemic pathogen be matched to each other in order to obtain the best protective effect. There is a need to develop vaccines suitable for multiple routes of vaccinations of animals, which reduce stress, increase the age-of-day period of use, and increase the population protection rate in the future.
Therefore, the preparation of the inactivated oral vaccine of the newcastle disease and avian influenza H9 type is tried, and the inactivated oral vaccine has great practical significance and wide market development scenes, but has the following difficulties: 1) The adjuvant adopted for inactivating viruses is usually an oil adjuvant which is insoluble in the environment in the organism in water phase, and the viscosity of the oil adjuvant is high, generally 80-100 cp, and the oil adjuvant has no flowing effect and is difficult to be used for oral administration, so that a new adjuvant needs to be developed; 2) Since inactivated viruses do not continue to multiply, it is also a difficult task to study how to maintain high serum levels of antibodies after oral administration.
Disclosure of Invention
Object of the Invention
The invention aims to provide a bigeminal inactivated oral immunogen composition for preventing newcastle disease and avian influenza H9 subtype, and a preparation method and application thereof.
The adjuvant compound adopted by the invention takes macromolecule F127, chitosan, astragalus polysaccharide and CRM197 protein molecules as main components, and has the main functions of improving the cellular immunity and humoral immunity of organisms under the action of plant extract astragalus polysaccharide through the crosslinking action of the macromolecule F127 and the chitosan. The CRM197 protein molecule fully activates phagocytic function of intestinal epithelial cells and activates humoral immune response, thereby playing an immune effect in cooperation with antigen molecules. The water adjuvant can meet the application requirement of oral administration, and meanwhile, oral administration immunity mainly utilizes the immune response of intestinal mucosa immune system of the organism. Therefore, the bivalent oral immunogen composition for preventing newcastle disease and avian influenza (H9 subtype) can immunize chickens to maintain high-level serum antibodies (which can be equivalent to injection vaccines), can immunize chickens at any age of day, has no inoculation stress, and solves the problem that inactivated vaccines are difficult to obtain high-level serum antibodies through oral administration. The application of the chicken newcastle disease and avian influenza bigeminal inactivated oral immunogen composition has great practical significance and wide market development prospect.
Solution scheme
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the invention provides a bivalent inactivated oral immunogenic composition for preventing newcastle disease and avian influenza H9 subtype, comprising the following components in volume ratio (2-4): 1 and a water adjuvant complex;
the inactivated antigens comprise at least one inactivated newcastle disease whole virus antigen and at least one inactivated avian influenza H9 subtype whole virus antigen. Optionally, the newcastle disease holoviral antigen is newcastle disease La Sota strain holoviral antigen, the avian influenza H9 subtype holoviral antigen is inactivated avian influenza H9 subtype WD strain holoviral antigen, and the virus content in the inactivated antigen is not less than 10 9.0 EID 50 0.1ml, optionally with a virus content of 10 9.3 ～10 9.5 EID 50 /0.1ml。
The water adjuvant compound is a water-soluble adjuvant and comprises a water-soluble auxiliary agent and diphtheria toxin mutant CRM197 protein;
the water-soluble auxiliary agent comprises the following raw materials in parts by weight: 4-8 parts of poloxamer polyether, 1-2 parts of glucan and 0.2-0.5 part of astragalus polysaccharide;
the concentration of diphtheria toxin mutant CRM197 protein in the adjuvant complex is 100-400 mg/L.
Further, the water-soluble auxiliary agent comprises the following raw materials in parts by weight: 8 parts of poloxamer polyether, 2 parts of glucan and 0.5 part of astragalus polysaccharide;
optionally, the water-soluble auxiliary agent also comprises physiological saline, and optionally, the physiological saline is supplemented to 100 parts by weight;
optionally, the poloxamer polyether is F127;
optionally, the astragalus polysaccharide is not less than 150mg per 1g of astragalus polysaccharide calculated by glucose.
Further, the weight ratio of the inactivated antigen to the adjuvant complex is (2-4): 1, alternatively 2:1 or 4:1.
further, in the inactivated antigen, the virus volume content ratio of the newcastle disease holoviral antigen to the avian influenza H9 subtype holoviral antigen is (0.5-2): 1, alternatively 1:1.
further, it is in the form of a liquid aqueous formulation;
in a second aspect, there is provided a method for preparing the bivalent oral immunogenic composition according to the first aspect, mixing the inactivated antigen and the water adjuvant complex, mixing in a stirring tank at 20-25 ℃ and 100-200 r/min, stirring for 4-6 hours, and adjusting the pH to 6.5-7.5, thereby obtaining the inactivated oral immunogenic composition.
Further, the method for judging the high-titer inactivated antigen comprises the following steps: the mortality rate of more than 60% of the virus inoculated chick embryos after rejuvenation is considered to be qualified.
Further, the preparation method of the inactivated antigen comprises the following steps:
a) Inoculating chicken newcastle disease La Sota strain virus into small day-old SPF chicken, taking viscera tissues for grinding 6 days after inoculation to obtain tissue virus solution, diluting 100-1000 times, inoculating small day-old non-immune chicken embryo, incubating for 72-96 hours, and harvesting to treat diseasesToxin content 10 9.3 ～10 9.5 EID 50 /0.1ml。
B) Inoculating avian influenza H9 subtype WD strain virus to SPF chicken of small day old, taking viscera tissue, grinding to obtain tissue virus solution, diluting 100-1000 times, inoculating non-immune chicken embryo of small day old, incubating for 72-96 hr, harvesting, and virus content of 10 9.3 ～10 9.5 EID 50 /0.1ml。
The preparation of A) and B) is not sequential.
In a third aspect, there is provided a use of the bivalent oral immunogenic composition according to the first aspect or the bivalent oral immunogenic composition prepared by the preparation method according to the second aspect for preparing a medicament for preventing diseases caused by newcastle disease and/or avian influenza H9 subtype virus infection.
Further, the method of administering the oral immunogenic composition comprises: the chickens orally take the oral immunogenic composition daily for three consecutive days when immunized for the first time; performing a second immunization 12-15 days after the first immunization, orally administering the oral immunogenic composition daily for three consecutive days; optionally, the oral immunogenic composition has a daily oral dose of 0.3 to 0.5 ml/plume;
or when the serum HI antibody of the newcastle disease is not higher than 8log2 and the HI antibody of the avian influenza H9 subtype is not higher than 8log2, the chicken is immunized by drinking water, and the oral immunogenic composition is contained in water after three days of continuous drinking, wherein the daily oral dosage is 0.2-0.5 ml/feather.
In a fifth aspect, a method for evaluating the immune effect of a dual inactivated oral immunogenic composition of newcastle disease and avian influenza H9 subtype is provided, wherein at least two oral immunizations are performed on animals without specific pathogens or at least one drinking immunization is performed on laying hens in the egg producing period, the antigen-specific antibody level of the immunized animals is tested and/or the protection rate of the vaccine is determined by a challenge test, and when the antibody level and/or the protection rate of the vaccine is not lower than a predetermined value, the inactivated oral immunogenic composition for animals and/or the immunization method thereof are considered to be qualified, wherein:
at least two oral immunizations of animals without specific pathogens were: the SPF chickens orally ingest the oral immunogenic composition daily for three consecutive days upon first immunization; performing a second immunization 12-15 days after the first immunization, orally administering the oral immunogenic composition daily for three consecutive days; the daily oral dosage of the oral immunogenic composition is 0.3-0.5 ml/feather;
at least one drinking water immunization of the laying hens in the egg producing period (optionally with the age of 180 days to 450 days) is as follows: when the serum HI antibody of the newcastle disease is not higher than 8log2 and the HI antibody of the avian influenza H9 subtype is not higher than 8log2, the chicken is immunized by drinking water, and the oral immunogenic composition is contained in water after three days of continuous drinking, wherein the daily oral dosage is 0.2-0.5 ml/feather.
Further, the judging method includes:
after 21-28 days of primary immunization of SPF chickens, the Newcastle disease and the avian influenza H9 subtype all generate high-titer hemagglutination inhibition antibodies, and the average value is not lower than 1:256; the protection rate of the vaccine is 100% by the attack of the newcastle disease strong virus and/or the avian influenza H9 subtype strong virus as a virus attack test; when the chicken newcastle disease and the avian influenza H9 subtype serum hemagglutination inhibition antibody level is not lower than 1:64 or 9log2 when the chicken newcastle disease and the avian influenza H9 subtype serum hemagglutination inhibition antibody level are immunized for 2 months for the first time, and the oral immunogen composition and the immunization method thereof are considered to be qualified; and/or the number of the groups of groups,
the egg laying period laying hen (180-450 days old) generates high-titer hemagglutination inhibition antibodies after immunization for 21-28 days, and the newcastle disease and the avian influenza H9 subtype all generate high-titer hemagglutination inhibition antibodies, and the level of the hemagglutination inhibition antibodies is not lower than 10log2 by improving at least 2 titers. And when the level of the serum hemagglutination inhibition antibodies of the newcastle disease and the avian influenza H9 subtype is not lower than 9log2 when the oral immunogen composition is immunized for 2 months for the first time, judging that the immunization method of the oral immunogen composition is qualified.
Alternatively, the dual inactivated oral immunogenic composition is selected from the group consisting of the dual inactivated oral immunogenic composition of newcastle disease and avian influenza H9 subtype according to the first aspect and the dual inactivated oral immunogenic composition prepared by the preparation method according to the second aspect
Advantageous effects
The diphtheria toxin mutant CRM197 added in the invention can be matched with other substances in an adjuvant, and after being crosslinked with an antigen, the diphtheria toxin mutant CRM197 increases the phagocytic efficiency from intestinal tract to the antigen, promotes the secretion of plasma cells and generates serum antibodies.
The avian influenza H9 subtype WD strain and the newcastle disease La Sota strain provide necessary conditional control for obtaining high-titer antigens by subsequent inoculation of non-immune chick embryos.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
The present invention will be described in detail below.
The reagents, enzymes, media and other chemical materials used in the examples below were all commercially available.
Some commonly used biological materials, such as competent cells, vectors, etc., are also commercially available products.
Some synthetic biomaterials, such as diphtheria toxin mutant CRM197 sequences, require artificial synthesis, all delegated to the synthesis company.
Example 1: preparation and examination of diphtheria toxin mutant CRM197 and Water adjuvant Complex
1.1 expression and examination of diphtheria toxin mutant CRM197
1.1.1 vector construction and transformation construction of an expression vector of a diphtheria toxin mutant CRM197, selecting a partial sequence of an R region of a diphtheria toxin mutant CRM197 complete sequence for expression, constructing a pET-28a expression vector with 123 amino acids, transforming competent cells of escherichia coli BL21 (DE 3), screening positive clones (amp+ resistance) by using a culture dish, and selecting a single colony for verification expression.
The amino acid sequence of the R region part of the CRM197 complete sequence is shown in SEQ:1 is shown as follows:
RTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSL
encoding SEQ id no: 1 is shown in SEQ:2 is shown as follows:
AGAACTGGTTTCCAAGGTGAATCTGGTCACGACATCAAGATCACTGCTGAAAACACTCCATTGCCAATCGCTGGTGTTTTGTTGCCAACTATCCCAGGTAAGTTGGACGTTAACAAGTCTAAGACTCACATCTCTGTTAACGGTAGAAAGATCAGAATGAGATGTAGAGCTATCGACGGTGACGTTACTTTCTGTAGACCAAAGTCTCCAGTTTACGTTGGTAACGGTGTTCACGCTAACTTGCACGTTGCTTTCCACAGATCTTCTTCTGAAAAGATCCACTCTAACGAAATCTCTTCTGACTCTATCGGTGTTTTGGGTTACCAAAAGACTGTTGACCACACTAAGGTTAACTCTAAGTTGTCTCTT
1.1.2 picking colony, placing the colony into 5ml LB (containing amp+) liquid culture medium, shake culturing for 4-5 h at 37 ℃, adding 1.6ul 1M IPTG to induce for 4-5 h to detect protein expression quantity, preserving strain with high expression quantity (1 ml culture solution is added with equal volume of 50% glycerol, preserving strain below-80 ℃), and expanding induced expression at the same time.
1.1.3 transferring the high-expression bacteria into Amp+ resistant liquid LB with final concentration of 100ug/ml, and culturing in 1L triangular flask with 500ml LB liquid culture medium at 37 ℃ at 200rpm/min until OD600 is between 0.6 and 0.8. Adding 200ul of IPTG with the concentration of 1M into the culture, inducing for 4-5 h at 37 ℃,
1.1.4 taking out 500ml of bacterial liquid after induction, centrifuging at 1200rpm for 15-20 min, discarding supernatant, adding 10 times of sterilized normal saline according to the weight of bacterial mud, and homogenizing at 500-800 bar pressure for three times under high pressure. And (3) centrifuging at 1200rpm for 15-20 min, taking the supernatant to obtain a protein solution, and carrying out SDS-PAGE and protein content inspection.
1.1.5 samples are added with 5x protein loading buffer solution, boiled in boiling water for 10min, 5ul of loading gel is taken, SDS-PAGE is observed after dyeing and decoloration, and obvious target bands appear near 40 kDa; the content of the detected protein is more than 500 mg/L.
1.2 preparation of Water adjuvant Complex
1.2.1 taking 8% F127 (final concentration after dissolving in physiological saline), 2% glucan (final concentration after dissolving in physiological saline), 0.5% astragalus polysaccharide (content 20%) (final concentration after dissolving in physiological saline), dissolving the above raw materials in water for injection fully in sequence, adjusting pH to 6.5-7.5, and sterilizing at 116 ℃ for 20min under high pressure. After the temperature is reduced to 20-25 ℃, 1.1 expression purified diphtheria toxin mutant CRM197 is added into the mixture, so that the concentration of the diphtheria toxin mutant CRM197 is 400mg/L, the pH value is adjusted to 6.5-7.5, and the mixture is stirred for 4-6 hours at 2-8 ℃ and 500r/min, thus obtaining the water adjuvant compound. Lot number ZJ 001.
1.2.2 taking 4% F127 (final concentration after dissolving in physiological saline), 1% glucan (final concentration after dissolving in physiological saline), 0.2% astragalus polysaccharide (content 20%) (final concentration after dissolving in physiological saline), dissolving the above raw materials in water for injection fully in sequence, adjusting pH to 6.5-7.5, and sterilizing at 116 ℃ for 20min under high pressure. After the temperature is reduced to 20-25 ℃, 1.1 expression purified diphtheria toxin mutant CRM197 is added into the mixture, so that the concentration of the diphtheria toxin mutant CRM197 is 100mg/L, the pH value is adjusted to 6.5-7.5, and the mixture is stirred for 4-6 hours at 2-8 ℃ and 500r/min, thus obtaining the water adjuvant compound. Lot number ZJ002 lot.
1.2.3 simultaneously water adjuvant complexes were prepared without expression of purified diphtheria toxin mutant CRM 197. Taking 8% of F127,2% of glucan and 0.5% of astragalus polysaccharide (content of 20%) (final concentration after dissolving in normal saline), dissolving the raw materials in water for injection fully, adjusting pH to 6.5-7.5, and sterilizing at 116 ℃ for 20min under high pressure. And cooling to 20-25 deg.c. Lot number ZJ003 lot.
The water adjuvant compound ZJ001 batch, ZJ002 batch and ZJ003 batch are tested, and the results show that the pH value is between 6.9 and 7.1, the sterile test is qualified, the stability is qualified, and the injection safety of SPF chickens and mice is qualified. The results are shown in Table 1.
Table 1 water adjuvant complex assay
EXAMPLE 2 preparation of high titer antigen
2.1 preparation method of newcastle disease La Sota strain rejuvenation and high-titer antigen
2.1.1 rejuvenation of Newcastle disease Virus La Sota strainThe preserved freeze-dried strain La Sota is added with 2ml of sterilized normal saline, diluted by 10 times, inoculated with 10 SPF chickens of 7 days old, injected into the wings by intravenous injection of 0.2ml and injected into the chest by intramuscular injection of 0.5ml, the inoculated chickens are sacrificed 3 days after inoculation, tissues such as liver, intestinal tract, lung and the like are taken, and the tissues of 10 chickens are mixed to be used as one sample. Grinding by adding sterilized normal saline according to w/v of 1:5. Freezing and thawing the grinding fluid at-80 ℃ for 3 times, centrifuging for 20min at 12000r/min, and taking the supernatant as seed for producing the newcastle disease virus La Sota strain.
2.1.2 examination of seed productionAnd (3) carrying out sterile inspection and exogenous virus inspection on the seeds produced by the Newcastle disease virus La Sota strain according to the annex of the current Chinese veterinary pharmacopoeia. The result shows that the seed produced by the chicken newcastle disease virus La Sota strain grows aseptically without mycoplasma. After seed virus stock solution produced by Newcastle disease virus La Sota strain and positive serum for resisting Newcastle disease are neutralized, after chick embryo is inoculated by allantoic cavity and chorioallantoic membrane, noDeath of chick embryo, no pathological changes of chick embryo, negative chick embryo allantoic fluid HA; inoculating chicken embryo fibroblasts, culturing for 5 days, and having no cytopathy and no adsorption phenomenon to 1% chicken erythrocytes; inoculating chicken embryo fibroblasts, culturing for 5 days, and then carrying out passage for 2 generations, taking cell liquids of the first generation, the second generation and the third generation of freeze thawing, and detecting chicken leukemia pathogen by using an ELISA method, wherein the third generation is negative; chicken embryo fibroblasts are inoculated, cultured for 5 days, reticuloendotheliosis virus fluorescent monoclonal antibody is stained, and no specific fluorescence appears. The result shows that the seed produced by the chicken newcastle disease virus La Sota strain is pure and has no foreign virus pollution.
2.1.3 production of high titer antigensThe chicken newcastle disease virus La Sota strain production seeds are inoculated with 10-11 days old non-immunized chick embryos (HI antibody of newcastle disease egg yolk antibody is lower than 1:4) according to the allantoic cavity route after 100-1000 times dilution, the chick embryos are harvested after incubation for 72-96 hours, and the death rate of the chick embryos is kept above 60 percent and is judged to be qualified for inoculation. In the method, the classical La Sota strain is rejuvenated in SPF chickens of small day age, and the death rate of the rejuvenated virus inoculated chick embryo is more than 60 percent, which is the key process step for obtaining high-titer antigen.
2.1.4 harvesting and detection of antigen
(1) Chick embryo antigen ND001 batch prepared according to 2.1.3 method has hemagglutination titer of 1:2048 and virus content of 10 9.5 EID 50 /0.1ml；
(2) The hemagglutination titer of the chick embryo antigen ND002 batch prepared according to the method of 2.1.3 is 1:2048, and the virus content is 10 9.3 EID 50 /0.1ml。
2.2 preparation method of avian influenza (H9 subtype) WD strain rejuvenation and high-titer antigen
2.2.1 rejuvenation of avian influenza (H9 subtype) WD strainsThe preserved freeze-dried strain WD strain is added with 2ml of sterilized normal saline, diluted by 10 times, inoculated with 10 SPF chickens of 7 days old, injected into the wings of 0.2ml, injected into the chest for intramuscular injection of 0.5ml, the inoculated chickens are sacrificed 3 days after inoculation, tissues such as liver, intestinal tract, lung and the like are taken, and the tissues of 10 chickens are mixed to be used as one sample. Adding sterilized normal saline according to w/v of 1:5, grinding. Freezing and thawing the grinding fluid at-80 ℃ for 3 times, centrifuging for 20min at 12000r/min, and taking the supernatant as an avian influenza (H9 subtype) WD strain to produce seeds.
2.2.2 inspection of seed productionThe production seeds of the avian influenza (H9 subtype) WD strain are subjected to sterile inspection and exogenous virus inspection according to the annex of the current Chinese veterinary pharmacopoeia. The results show that the avian influenza (H9 subtype) WD strain produces seed sterile growth without mycoplasma growth. After the positive serum of the seed virus stock solution produced by the avian influenza (H9 subtype) WD strain and the avian influenza (H9 subtype) is neutralized, the allantoic cavity and chorioallantoic membrane are inoculated with chick embryos, so that no dead chick embryos are produced, no lesions are produced on the chick embryos, and the chick embryo allantoic fluid is HA negative; inoculating chicken embryo fibroblasts, culturing for 5 days, and having no cytopathy and no adsorption phenomenon to 1% chicken erythrocytes; inoculating chicken embryo fibroblasts, culturing for 5 days, and then carrying out passage for 2 generations, taking cell liquids of the first generation, the second generation and the third generation of freeze thawing, and detecting chicken leukemia pathogen by using an ELISA method, wherein the third generation is negative; chicken embryo fibroblasts are inoculated, cultured for 5 days, reticuloendotheliosis virus fluorescent monoclonal antibody is stained, and no specific fluorescence appears. The result shows that the avian influenza (H9 subtype) WD strain has pure seed and no foreign virus pollution.
2.2.3 production of high titer antigensThe production seeds of the avian influenza (H9 subtype) WD strain are inoculated with 10-11 days old non-immunized chick embryos (HI antibody of avian influenza (H9 subtype) egg yolk antibody is lower than 1:4) according to the allantoic cavity route after 100-1000 times dilution, the chick embryos are harvested after incubation for 72-96 hours, and the death rate of the chick embryos is maintained above 60 percent and is judged to be qualified after inoculation. In the method, the SPF chicken with the small day age is rejuvenated by classical avian influenza (H9 subtype) WD strain, and the death rate of more than 60% of virus inoculated chick embryo after rejuvenation is a key process step for obtaining high-titer antigen.
2.2.4 harvesting and detection of antigen
(1) Chick embryo antigen AI001 batch prepared according to 2.2.3 method has hemagglutination potency at 1:2048, virus content 10 9.3 EID 50 /0.1ml；
(2) The hemagglutination titer of the chick embryo antigen AI002 batch prepared according to the method of 2.2.3 was 1:2048, virus content 10 9.5 EID 50 /0.1ml。
2.3 inactivation of antigen the qualified newcastle disease virus liquid and the avian influenza H9 subtype virus liquid are respectively added into 10% formaldehyde solution, so that the final concentration of the formaldehyde solution is 0.2%, and the mixture is stirred while adding so as to be fully mixed. The virus solution of the La Sota strain of the newcastle disease virus is inactivated at 37 ℃ for 16 hours, and the virus solution of the H9 subtype of avian influenza is inactivated at 37 ℃ for 24 hours. And (3) performing sterile inspection and inactivation inspection, and preserving the inactivated virus liquid at 2-8 ℃ for no more than 2 months.
Example 3 screening of a Combined inactivated oral immunogenic composition for New castle disease and avian influenza (subtype H9)
3.1 preparation of immunogenic composition inactivated antigen of Newcastle disease La Sota strain (virus content 10) 9.3 EID 50 0.1ml, ND002 batch), avian influenza (H9 subtype) WD strain inactivated antigen (virus content 10) 9.3 EID 50 0.1ml, AI001 batch) 1:1, respectively preparing an oral immunogenic composition by mixing the mixed antigen with ZJ001 batch, ZJ002 batch and ZJ003 batch according to the following steps: the adjuvant is 2:1, preparing vaccine, wherein the batch number is 202201 batches of chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition, 202202 batches of chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition and 202203 batches of chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition. Simultaneously, antigen is set: physiological saline is 2:1 preparing a control in proportion, and 202204 batches of a dual inactivated oral immunogen composition for newcastle disease and avian influenza (H9 subtype); setting an antigen: physiological saline (100 mg/L added to express diphtheria toxin mutant CRM197 protein) was 2:1 control, chicken newcastle disease and avian influenza (H9 subtype) bivalent inactivated oral immunogenic composition 202205 batches were prepared.
3.2.2 oral vaccine Effect test Each group of vaccine was orally immunized against 21 day old SPF chickens at 0.5 ml/day, at 3 days following continuous oral immunization, and at 3 days following 14 days, the "3+3" regimen; each group of 10 chickens, 5 control groups, 45 total.
3.3.3 determination of application Effect oral test chickens were subjected to 14 days after the first dripping and 14 days after the second oral administration, serum was collected respectively, serum newcastle disease and avian influenza (H9 subtype) hemagglutination inhibition antibodies were detected (detection method is hemagglutination inhibition test method), and antibody production conditions of each immune group were counted.
The results show that the effect of expressing diphtheria toxin mutant CRM197 protein on inducing high-level serum antibodies by using an oral vaccine is 4.2log2, the effect of using the hemagglutination inhibition antibodies of newcastle disease is 4.5log2, and the effect of using the hemagglutination inhibition antibodies of avian influenza (H9 subtype) is obviously higher than that of a control group (2.4 log2 and 2.3log 2) added with normal saline only. The results also indicate that the diphtheria toxin mutant CRM197 protein and water-soluble adjuvant synergistically contribute more significantly to the pro-antibody production of oral vaccines. Oral vaccines for avian influenza (H9 subtype) containing 100-400 mg/L diphtheria toxin mutant CRM197 protein can induce high levels of antibody to 8log 2-9 log2 (1:256-1:512).
TABLE 2 serum New castle disease and avian influenza (H9 subtype) antibody results with different concentrations of CRM197 protein in water adjuvant complexes
Example 4 preparation of a Combined inactivated oral vaccine against newcastle disease and avian influenza (H9 subtype) of the present invention
4.1 selection of antigen and adjuvantTaking antigen of Newcastle disease La Sota strain (virus content 10) 9.3 EID 50 0.1ml, ND002 batch), avian influenza (H9 subtype) WD strain antigen (virus content 10) 9.3 EID 50 0.1ml, AI001 batch) 1:1, adjuvant selection ZJ001 batch (400 mg/L diphtheria toxin mutant CRM 197); the volume ratio of the antigen to the water adjuvant compound is 4:1 and 2:1, preparing vaccine in a proportion, wherein the batch number is 202206 batches and 202207 batches; taking antigen of Newcastle disease La Sota strain (virus content 10) 9.5 EID 50 0.1ml, ND001 batch), avian influenza (H9 subtype) WD strain antigen (virus content 10) 9.5 EID 50 0.1ml, AI002 batch) 1:1, adjuvant selection ZJ002 lot (containing 100mg/L diphtheria toxin mutant CRM 197); the volume ratio of the antigen to the water adjuvant compound is 4:1 and 2:1, lot 202208, 202209The method comprises the steps of carrying out a first treatment on the surface of the Vaccine lot numbers and compositions are prepared as shown in table 2.
4.2 verification of the application Effect of the proportioningEach group of vaccine is orally immunized to SPF chickens of 28 days old according to 0.5 ml/day, and continuously orally immunized for 3 days after 14 days, namely a 3+3 scheme; each group of 10 chickens, 5 control groups, 45 total.
3.3 determination of application EffectSerum was collected from the orally administered test chickens 14 days after the first oral drip and 14 days after the second oral administration, respectively, and serum newcastle disease and avian influenza (H9 subtype) hemagglutination-inhibiting antibodies were detected. Antibody production was counted for each immune group.
The results show that when the water adjuvant complex of the invention is used for preparing a chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition, the virus content of the inactivated antigen is 10 9.3 ～10 9.5 EID 50 When the ratio of the inactivated antigen to the water adjuvant complex is 2:1-4:1, the high-level antibody can be induced to be generated, and the ratio of the inactivated antigen to the water adjuvant complex is 8log 2-9 log2 (1:256-1:512).
TABLE 3 results of New castle disease and avian influenza (H9 subtype) antibodies with different ratios of Water adjuvant complexes to inactivated antigen
The main advantages of this solution are: can continuously stimulate intestinal mucosa immune system during the first oral immunization, and form immune memory after the second continuous oral immunization, thereby promoting the organism to produce high-potency serum antibody.
Example 5 safety verification of the newcastle disease and avian influenza (H9 subtype) bivalent inactivated oral vaccine of the present invention
50 SPF chickens (21 days old) were taken, and 10 animals each had 0.6ml of vaccine injected subcutaneously into the neck of each of the 202208 and 202209 animals (as described in example 4) by using a combination of newcastle disease and avian influenza (H9 subtype) to inactivate oral immunogenic composition, and each had 2.0ml of vaccine injected orally, and no local and systemic adverse effects caused by the vaccine were observed for 14 days. The results are shown in Table 4.
TABLE 4 results of safety tests of different approaches for Newcastle disease and avian influenza (H9 subtype) bivalent inactivated oral immunogen compositions
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EXAMPLE 6 formulation of optimal immunization doses for the Bie-inactivated oral immunogenic composition of New castle disease and avian influenza (subtype H9) of the invention
In order to determine the optimal immunization dose of the chicken newcastle disease and avian influenza (H9 subtype) bivalent inactivated oral immunogen composition, according to the research results of the chicken newcastle disease inactivated oral vaccine and the avian influenza (H9 subtype) inactivated oral inactivated vaccine, a '3+3' scheme is adopted, and different oral doses are designed for effect verification:
6.1 application scheme of Newcastle disease and avian influenza (H9 subtype) bivalent inactivated oral vaccineNewcastle disease, avian influenza (H9 subtype) inactivated oral immunogenic composition 202207 (as described in example 4) was orally immunized with 0.3 ml/day, 0.5 ml/day, 0.8 ml/day, 28 day old SPF chickens, 3 consecutive oral immunizations each followed by 3 consecutive oral immunizations each, and 3 consecutive oral immunizations each after 14 days, i.e. "3+3" regimen, control group oral saline; each group of 20 chickens and 10 controls. Wherein the number of newcastle disease parts and avian influenza parts is 5, and the total number is 70.
6.2 determination of application EffectCollecting serum from the orally administered test chicken 14 days after the first dripping and 14 days after the second oral administration, respectively, and detecting serum chicken newcastle disease hemagglutination inhibition antibody and avian influenza (H9 subtype) hemagglutination inhibition antibody; after the blood sampling is completed 14 days after the second drip, toxicity attack experiments are carried out: wherein each chicken of the Newcastle disease part is intramuscular injected with 0.5ml (containing 10) of chicken Newcastle disease virulent BJ strain (CVCC AV1611 strain purchased from Chinese veterinary drug administration) 5.0 ELD 50 ) The observation was carried out for 14 days. Statistics of immune groupsProtection rate; avian influenza part Each chicken wing is injected with 0.2ml (containing 10) of avian influenza (H9 subtype) WD strain by intravenous injection 7.5 EID 50 ) Observing for 5 days, collecting throat swab and cloaca swab, inoculating SPF chick embryo of 9-11 days old, counting virus separation rate of immune group and control group, and judging the protection of immune group.
TABLE 5 optimal applied dose validation results for Newcastle disease and avian influenza (H9 subtype) bivalent inactivated oral immunogen compositions
6.3 conclusionThrough continuous 3 days of oral immunization, the rising serum antibody of 0.8ml dose reaches 5.1-5.3 log2 higher; and then through the second drip immunization, the serum antibody level of more than 8.0log2 is reached in the dosage of 0.3ml, 0.5ml and 0.8ml for 3 days continuously, and the toxicity attack protection rate newcastle disease part and the avian influenza (H9 subtype) are both 10/10. Therefore, consider the validation effect of drip immunization: the 3+3 scheme is adopted, the dosage is 0.3-0.8 ml, which is oral dosage, and the recommended dosage is 0.3-05 ml/dose.
Namely: the chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition is subjected to drop immunization for three consecutive days, 0.3-0.5 ml/dose, and after 14 days, the chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition is subjected to second drop immunization for three consecutive days, and 0.3-0.5 ml/dose. The serum antibody level of newcastle disease and avian influenza is not lower than 1:256 (8.0 log 2) 14 days after the second drip immunization, and the toxicity attack protection rate is 10/10.
Example 7 comparison of the efficacy of the Bie-inactivated oral immunogenic composition of New castle disease and avian influenza (subtype H9) with commercial vaccine
7.1 Newcastle disease and avian influenza (subtype H9) bigeminal inactivated oral administrationImmunogenic compositionsImmunization protocolThe chicken newcastle disease and avian influenza (H9 subtype) are combined and inactivated into an oral immunogen composition 202208 batch according to 0.5 ml/daySPF chickens of 28 days of age were orally immunized for 3 consecutive days, and further orally immunized for 3 days after 14 days, and were performed according to the "3+3" regimen. Simultaneously injecting and inoculating commercial vaccine seedlings (purchased from Beijing Xinde organisms) of chicken newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine as an injection group, wherein the injection group is 0.5 ml/chicken newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine; commercial vaccine (La Sota strain) of chicken newcastle disease (source of Beijing China Xingyang biotechnology Co., ltd.) is orally taken as a live vaccine drip immune group, and physiological saline is orally taken as a control group. Each group of 20 chickens, 10 control groups, and 70 chickens.
7.2 determination of the immune EffectThe immunogen composition 202208 batches of drip test chickens, commercial vaccine injection chickens and live vaccine drip test chickens (only newcastle disease part) respectively collect serum 21 days (7 days after drip secondary immunization), 28 days, 42 days, 49 days and 60 days after the primary immunization, and detect the serum newcastle disease hemagglutination inhibition antibodies and the avian influenza (H9 subtype) hemagglutination inhibition antibodies; and after 60 days of blood sampling after immunization, carrying out a toxicity attack experiment: wherein each chicken of the Newcastle disease part is intramuscular injected with 0.5ml (containing 10) of chicken Newcastle disease virulent BJ strain (CVCC AV1611 strain purchased from Chinese veterinary drug administration) 5.0 ELD 50 ) The observation was carried out for 14 days. Counting the protection rate of an immune group; avian influenza part Each chicken wing is injected with 0.2ml (containing 10) of avian influenza (H9 subtype) WD strain by intravenous injection 7.5 EID 50 ) Observing for 5 days, collecting throat swab and cloaca swab, inoculating SPF chick embryo of 9-11 days old, counting virus separation rate of immune group and control group, and judging the protection of immune group.
TABLE 6 comparison of the immunopotency of Newcastle disease and avian influenza (H9 subtype) bivalent oral immunogen compositions
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7.3 conclusion
7.3.1 drip immunization is carried out according to the scheme of '3+3', the chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition drip immunization and commercial vaccine injection immunization are carried out, the two groups of inoculation routes are 21-42 days after immunization, serum antibodies are not greatly different, the serum antibody level is above 1:256, the live vaccine drip immunization newcastle disease antibodies are lower, and the number of the live vaccine drip immunization newcastle disease antibodies is 5.0log 2-6.0 log2. After the immunization duration is prolonged, the newcastle disease serum antibody of 60 days after the immunization of the immunogen composition by drip mouth immunization is more than 6.0log2, the avian influenza serum antibody is more than 7.0log2, and the toxicity attack protection rate of the newcastle disease and the avian influenza part is 10/10.
7.3.2 drop immunization, wherein the peak period of the serum antibody is 21-42 days after immunization, and the serum antibody level is above 1:256; the duration is 60 days (more than 6.0log 2) after immunization, the newcastle disease serum antibody is more than 6.0log2, and the avian influenza serum antibody is more than 7.0log 2. The method is fully applicable to commercial broilers (about 42 days old in the field) with shorter feeding period, and is also applicable to the antibody maintenance in the egg laying period and the immunization period of 2 months of commercial broilers.
The oral vaccine of the invention can reach high-level serum antibody equivalent to injection immunity, and overcomes the problem of low serum antibody level of the inactivated virus oral vaccine.
Example 8 application of the Newcastle disease and avian influenza (H9 subtype) bigeminal inactivated oral immunogen composition of the present invention to commercial laying hens
8.1, carrying out drinking water immunization on 180-day-old commercial laying hens according to 0.2 ml/day in 202206 batches of dual inactivated oral immunogen composition for newcastle disease and avian influenza (H9 subtype), and 18000 continuous 3 days; meanwhile, 200 control groups which only drink water and are not immunized are arranged; meanwhile, 200 injection groups for injecting chicken newcastle disease and bird flu (H9 subtype) bivalent inactivated vaccine commercial vaccine are arranged, and the volume of the vaccine is 0.2 ml/vaccine.
8.2, carrying out drinking water immunization on 450-day-old commercial laying hens according to 0.5 ml/day in 202207 batches of dual inactivated oral immunogen composition for newcastle disease and avian influenza (H9 subtype), 24000 eggs for 3 continuous days; meanwhile, 200 control groups which only drink water and are not immunized are arranged; meanwhile, 200 injection groups for injecting chicken newcastle disease and bird flu (H9 subtype) bivalent inactivated vaccine commercial vaccine are arranged, and the volume of the vaccine is 0.5 ml/vaccine.
8.3 judgment of immune EffectFixing deviceThe immunogen composition is used for respectively collecting serum 21 days, 42 days and 63 days after drinking water immunization, and detecting serum newcastle disease hemagglutination inhibition antibodies and avian influenza (H9 subtype) hemagglutination inhibition antibodies.
TABLE 7 results of the efficacy of a Newcastle disease and avian influenza (H9 subtype) bivalent oral immunogen composition on commercial laying hens (180 days old)
Table 8 results of the efficacy of the Newcastle disease and avian influenza (H9 subtype) bivalent oral immunogen composition on commercial laying hens (450 days old)
8.3 conclusion
8.3.1 drinking water immunization is carried out according to the scheme of 'continuous drinking water for 3 days', the chicken newcastle disease and avian influenza (H9 subtype) dual inactivated oral immunogen composition is used for drinking water immunization and commercial vaccine injection immunization, the two groups of inoculation ways are 21-60 days after immunization, the difference between the chicken newcastle disease and the avian influenza (H9 subtype) serum antibodies is small, and the serum antibody level is above 9log2 (1:512).
8.3.2 in commercial laying hens in the presence of maternal antibodies at antibody levels below 8log2, a double inactivated oral immunogenic composition of newcastle disease and avian influenza (subtype H9) was immunized against drinking water: according to the scheme of 'continuous drinking water immunization for 3 days', the serum antibodies of newcastle disease and avian influenza (H9 subtype) are not greatly different from 60 days after drinking water immunization, and the serum antibody level is more than 9log2 (1:512).
8.3.3 the immunogenic composition of the invention in the presence of maternal antibodies, the drinking water immunization is a booster immunization, which can reduce the drinking water dosage by 3 times and also achieve high levels of serum antibodies comparable to the injection immunization.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A bivalent inactivated oral immunogenic composition for preventing newcastle disease and avian influenza H9 subtype, which is characterized by comprising the following components in volume ratio (2-4): 1 and a water adjuvant complex;
the inactivated antigen comprises at least one inactivated newcastle disease whole virus antigen and at least one inactivated avian influenza H9 subtype whole virus antigen;
the water adjuvant compound is a water-soluble adjuvant and comprises a water-soluble auxiliary agent and diphtheria toxin mutant CRM197 protein;
the water-soluble auxiliary agent comprises the following raw materials in parts by weight: 4-8 parts of poloxamer polyether, 1-2 parts of glucan and 0.2-0.5 part of astragalus polysaccharide;
the concentration of diphtheria toxin mutant CRM197 protein in the adjuvant complex is 100-400 mg/L.
2. The dual inactivated oral immunogenic composition according to claim 1, wherein the water soluble adjuvant comprises the following raw materials in parts by weight: 8 parts of poloxamer polyether, 2 parts of glucan and 0.5 part of astragalus polysaccharide;
optionally, the water-soluble auxiliary agent also comprises physiological saline, and optionally, the physiological saline is supplemented to 100 parts by weight;
optionally, the poloxamer polyether is F127;
optionally, the astragalus polysaccharide is not less than 150mg per 1g of astragalus polysaccharide calculated by glucose.
3. The dual inactivated oral immunogenic composition according to claim 1 or 2, wherein the volume ratio of inactivated antigen to water adjuvant complex is (2-4): 1, alternatively 2:1 or 4:1, a step of;
and/or, in the inactivated antigen, the virus volume content ratio of the newcastle disease holoviral antigen to the avian influenza H9 subtype holoviral antigen is (0.5-2): 1, alternatively 1:1,
and/or, the virus content in the inactivated antigen is not less than 10 9.0 EID 50 0.1ml, optionally with a virus content of 10 9.3 ～10 9.5 EID 50 /0.1ml。
4. A dual inactivated oral immunogenic composition according to any one of claims 1 to 3, which is in the form of a liquid aqueous formulation.
5. A process for the preparation of a dual inactivated oral immunogenic composition according to any one of claims 1 to 4, wherein the inactivated antigen is mixed with the water adjuvant complex, mixed in a stirred tank at 20-25 ℃, 100-200 r/min, stirred for 4-6 hours, and pH adjusted to 6.5-7.5 to obtain the dual inactivated oral immunogenic composition.
6. The method of claim 5, wherein the method of preparing the inactivated antigen comprises:
a) Inoculating chicken newcastle disease La Sota strain virus into small day-old SPF chicken, taking viscera tissue for grinding 6 days after inoculation to obtain tissue virus solution, diluting 100-1000 times, inoculating small day-old non-immune chicken embryo, incubating for 72-96 hours, harvesting, and obtaining the virus content of 10% 9.3 ～10 9.5 EID 50 /0.1ml；
B) Inoculating avian influenza H9 subtype WD strain virus to SPF chicken of small day old, taking viscera tissue, grinding to obtain tissue virus solution, diluting 100-1000 times, inoculating non-immune chicken embryo of small day old, incubating for 72-96 hr, harvesting, and virus content of 10 9.3 ～10 9.5 EID 50 /0.1ml。
7. Use of a dual inactivated oral immunogenic composition of newcastle disease and avian influenza H9 subtype according to any one of claims 1 to 4 or a dual inactivated oral immunogenic composition prepared by the preparation method according to any one of claims 5 to 6 for the preparation of an oral immunogenic composition for preventing diseases caused by newcastle disease and/or avian influenza H9 subtype virus infection.
8. Use of a dual inactivated oral immunogenic composition according to claim 7, wherein the method of administration of the oral immunogenic composition comprises: the chickens orally take the oral immunogenic composition daily for three consecutive days when immunized for the first time; performing a second immunization 12-15 days after the first immunization, orally administering the oral immunogenic composition daily for three consecutive days; optionally, the oral immunogenic composition has a daily oral dose of 0.3 to 0.5 ml/plume;
or when the serum HI antibody of the newcastle disease is not higher than 8log2 and the HI antibody of the avian influenza H9 subtype is not higher than 8log2, the commercial laying hen is immunized by drinking water for three days, the oral immunogen composition is contained in water, the daily oral dosage is 0.2-0.5 ml/feather, and optionally, the day-old of the commercial laying hen is 180-450 days old.
9. A method for evaluating the immune effect of a combination of a newcastle disease and an avian influenza H9 subtype, characterized in that an animal without specific pathogens is subjected to at least two oral immunizations or a laying hen in the laying period is subjected to at least one drinking immunization, the antigen-specific antibodies of the immunized animal are tested and/or the protection rate of the vaccine is determined by a challenge test, and when the antibody level and/or the protection rate of the vaccine are not lower than a predetermined value, the veterinary combination of the inactivated oral immunogen and/or the immunization method thereof is considered to be qualified, wherein:
at least two oral immunizations of animals without specific pathogens were: the SPF chickens orally ingest the oral immunogenic composition daily for three consecutive days upon first immunization; performing a second immunization 12-15 days after the first immunization, orally administering the oral immunogenic composition daily for three consecutive days; the daily oral dosage of the oral immunogenic composition is 0.3-0.5 ml/feather;
at least one drinking immunization of the laying hens in the egg producing period is as follows: when the serum HI antibody of the newcastle disease is not higher than 8log2 and the HI antibody of the avian influenza H9 subtype is not higher than 8log2, the chicken is immunized by drinking water, and the oral immunogenic composition is contained in water after three days of continuous drinking, wherein the daily oral dosage is 0.2-0.5 ml/feather.
10. The method for evaluating an immune effect according to claim 9, wherein the determination method comprises:
after 21-28 days of primary immunization of SPF chickens, the newcastle disease and the avian influenza generate high-titer hemagglutination inhibition antibodies, and the average value is not lower than 1:256; the protection rate of the vaccine is 100% by the attack of the newcastle disease strong virus and/or the bird flu strong virus as a virus attack test; when the chicken newcastle disease and the avian influenza H9 subtype serum hemagglutination inhibition antibody level is not lower than 1:64 or 9log2 when the chicken newcastle disease and the avian influenza H9 subtype serum hemagglutination inhibition antibody level are immunized for 2 months for the first time, and the oral immunogen composition and the immunization method thereof are considered to be qualified; and/or the number of the groups of groups,
the egg laying period laying hen generates high titer hemagglutination inhibition antibodies after 21-28 days after immunization, and the level of the hemagglutination inhibition antibodies is not lower than 10log2 when the newcastle disease and the avian influenza H9 subtype are both raised by at least 2 titers; when the level of the serum hemagglutination inhibition antibodies of the newcastle disease and the avian influenza H9 subtype is not lower than 9log2 when the oral immunogen composition is immunized for 2 months for the first time, judging that the immunization method of the oral immunogen composition is qualified; optionally, the day-old of the commercial laying period laying hens is 180-450 days old;
alternatively, the dual inactivated oral immunogenic composition is selected from the group consisting of the dual inactivated oral immunogenic composition of newcastle disease and avian influenza H9 subtype according to any one of claims 1 to 4 or the dual inactivated oral immunogenic composition prepared by the preparation method according to any one of claims 5 to 6.
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