CN108465107B - Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine - Google Patents
Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine Download PDFInfo
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Abstract
The invention provides a Muscovy duck type 2 adenovirus and Muscovy duck parvovirus disease bivalent inactivated vaccine, wherein antigens are inactivated duck type 2 adenovirus and Muscovy duck parvovirus; the preservation number of the Muscovy duck parvovirus is CCTCC NO of V201812; the preservation number of the duck type 2 adenovirus is CCTCC No: v201633. The duck type 2 adenovirus and Muscovy duck parvovirus disease bivalent inactivated vaccine prepared by the invention has good safety, and no local and systemic adverse reaction caused by the vaccine occurs. After analysis of property, safety test and efficacy test data in the retention period test, all indexes are stable and effective; the immune effect of the vaccine is evaluated by a serology method and an immune challenge method, and the result shows that the bivalent inactivated vaccine prepared by the invention can provide effective immune protection for duck groups and has good commercial development prospect.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to preparation and application of a duck type 2 adenovirus and Muscovy duck parvovirus disease bivalent inactivated vaccine.
Background
Adenoviruses are common infectious pathogens of poultry. Avian adenoviruses are divided into three groups: the group I is avian adenovirus separated from respiratory tract infection of chicken, turkey, goose and duck, has common group specific antigen, and can be divided into A, B, C, D, E5 kinds and 12 serotypes; group II comprises hemorrhagic enteritis virus of turkey, marmot virus of pheasant and splenopathy virus of chicken; group III is an adenovirus isolated from the egg laying syndrome of chickens and ducks. Duck type 2 adenovirus belongs to avian adenovirus I, is isolated from diseased Muscovy ducks by France Hungary people in 1977, and infected ducks mainly show symptoms of mental depression, diarrhea, emaciation and the like. Mainly has the transmission mode decision, namely horizontal transmission and vertical transmission, and the virus is difficult to eliminate once being carried into the duck group.
Muscovy duck parvovirus is an acute infectious disease caused by Duck Parvovirus (DPV), mainly attacks young Muscovy ducks of 1-3 weeks old, and has the main clinical symptoms of dyspnea (asthma), diarrhea, pancreatic necrosis, exudative enteritis and the like, and the morbidity and mortality are high. The duck is resistant to growth retardation and becomes a stiff duck, and the economic value of the duck is lost. The disease is distributed worldwide, and brings huge economic loss to Muscovy duck breeding industry.
Epidemiological investigation on the two diseases shows that the two diseases have increasing tendency in Muscovy duck group in China. Based on the fact that the immune effect of the commercial inactivated vaccines for preventing the two diseases at home and abroad is not good, the disease phenomenon or even death of individual duck farms still occurs, the disease phenomenon or even death of the individual duck farms may be related to the variation of viruses, and the vaccine needs to be prepared by screening the variant virus strains. In addition, multiple needles and multiple doses of a single seedling bring unnecessary stress reaction to Muscovy duck groups. The development of safe and effective bivalent inactivated vaccines is urgent, and the problem must be overcome by innovative technology.
Disclosure of Invention
The invention aims to provide a Muscovy duck type 2 adenovirus and Muscovy duck parvovirus disease bivalent inactivated vaccine which can effectively prevent duck type 2 adenovirus and Muscovy duck parvovirus disease.
The invention provides a Muscovy duck type 2 adenovirus and Muscovy duck parvovirus bivalent vaccine, wherein antigens are inactivated duck type 2 adenovirus and Muscovy duck parvovirus; the Muscovy duck parvovirus is a Muscovy duck parvovirus GDL strain (MDPV-GDL), is preserved in a China center for type culture Collection (address: Wuhan university in Wuhan, China) in 2018, 4 months and 20 days, and has the preservation number of CCTCC NO: V201812.
Preferably, the duck type 2 adenovirus is inactivated GD strain virus; the used duck type 2 adenovirus GD strain is preserved in the China center for type culture Collection of the university of Wuhan in 2016, 6 months and 5 days, and the preservation number is CCTCC No: v201633.
The vaccine is an inactivated vaccine.
In the inactivated vaccine, the duck type 2 adenovirus and Muscovy duck parvovirus are inactivated by formaldehyde, and the virus content of the duck type 2 adenovirus in the vaccine is more than or equal to 106.5TCID500.1ml, the virus content of Muscovy duck parvovirus is more than or equal to 105.2ELD50/0.2ml。
Adding formaldehyde with the final concentration of 0.2% into GD strain virus liquid, stirring and inactivating for 16 hours at 37 ℃, adding formaldehyde with the final concentration of 0.2% into MDPV-GDL strain virus liquid, stirring and inactivating for 16 hours at 37 ℃, mixing two antigens (GD strain and MDPV-GDL strain) according to the volume ratio of 0.6: 1-1: 1 after inactivation inspection, adding Tween-80 to prepare a water phase, and mixing and emulsifying the dissolved water phase and a white oil adjuvant 1: 2 to obtain the duck type 2 adenovirus and Muscovy duck parvovirus disease bivalent inactivated vaccine.
The duck type 2 adenovirus and Muscovy duck parvovirus disease bivalent inactivated vaccine prepared by the invention has good safety, and no local and systemic adverse reaction caused by the vaccine occurs. After analysis of property, safety test and efficacy test data in the retention period test, all indexes are stable and effective; the immune effect of the vaccine is evaluated by a serology method and an immune challenge method, and the result shows that the bivalent inactivated vaccine prepared by the invention can provide effective immune protection for duck groups and has good commercial development prospect.
Drawings
FIG. 1 shows the PCR detection result of Muscovy duck parvovirus MDPV-GDL strain, wherein 1 is the PCR amplification result, M is 2000DNAmarker, and 2 is the negative control;
FIG. 2 is a dendrogram of Muscovy duck parvovirus MDPV-GDL strain;
FIG. 3 shows a homology comparison of Muscovy duck parvovirus MDPV-GDL strain genes;
Detailed Description
The present inventors screened a strain of duck adenovirus type 2 GD and a strain of Muscovy duck parvovirus MDPV-GDL, both of which are variant strains, and thus contributed to the present invention. The present invention will be described in detail with reference to examples.
Example 1 isolation and identification of strains
1.1 isolation and identification of GD Strain
1.1.1 isolation of viruses of GD Strain in 2015, the inventors succeeded in isolating a Duck type 2 adenovirus from Muscovy Duck group in a certain duck farm in Guangdong. Grinding the tissues of the liver, spleen, kidney and the like of the diseased Muscovy duck, homogenizing with sterile PBS, repeatedly freezing and thawing for 3 times, centrifuging at 6000r/min for 10 minutes, taking the supernatant, adding streptomycin, standing overnight at 4 ℃, filtering, performing sterile inspection, and storing for later use. Inoculating the filtered solution containing 1% of virus into Muscovy duck embryo fibroblasts, culturing in a serum-free M199 culture solution, performing static culture at 37 ℃ for 60-78 hours to obtain obvious cytopathic effect, and collecting virus solution.
1.1.2 etiological identification the collected virus liquid is purified and processed with chloroform treatment, hemagglutination identification, physicochemical property test, virus content determination, immunogenicity, specificity and purity etc. virus property analysis and detection. The results show that: the virus cannot agglutinate red blood cells of SPF chickens and ducks, and cannot agglutinate the red blood cells even if the concentration of the red blood cells is changed. The virus has no envelope, is resistant to ether and chloroform, and is acid resistant. After treatment with sodium hydroxide (pH 10) and temperature (50 ℃, 1 hour), cells were seeded and free of cytopathic effects, and negative for PCR detection. The nucleic acid type of the separated strain is DNA, and the virus is not alkali-resistant and can be inactivated at 60 ℃ for 1 hour. The virus content of the strain is 107.50TCID500.1 ml. The neutralization group neutralization index of the virus and the GD strain positive serum is far higher than that of the epidemic strain positive serum, which indicates that the virus and the GD strain serumSpecific neutralization reactions occurred and there were differences in neutralization titers. The virus has no contamination of bacteria, mycoplasma and exogenous virus.
1.1.3 molecular biology identification the isolated viruses were subjected to PCR detection, gene sequencing, homology analysis. Through PCR detection and Hexon gene sequencing, the isolated strain Hexon gene belongs to the same branch with the group I avian adenovirus through NCBI BLAST comparison analysis, and has 99.02 percent and 99.70 percent of homology with nucleotide and amino acid of GR strain (accession number: NC-024486.1) in GenBank database respectively. The GD strain and GR strain differ in six amino acid positions encoded by Hexon. The part of nucleotide homology of GD strain and I group avian adenovirus in GenBank database is 31% -88%.
1.2 isolation and identification of MDPV-GDL Strain
1.2.1 epidemiological survey in 2017 for 4 months, a large area of disease with soft feet, asthma and diarrhea as symptoms appears in a Muscovy duck group of 11 days old in Muscovy duck farm in Guangdong. The Muscovy duck farm immunizes commercial goose plague and Muscovy duck parvovirus bivalent inactivated vaccine at 1 day of age, and young Muscovy ducks at the early stage of 8 days of age are mainly manifested by mental retardation, inappetence, weakness of feet, no desire to walk and mouth opening for breathing; diarrhea of varying degrees, yellow-green or white watery stool, and sticking around the anus. After 3 days, the large batch died. The dead duck is dissected to find diseases with the characteristics of liver, pancreas, kidney, spleen and other organs, and the Muscovy duck parvovirus disease is preliminarily diagnosed through clinical investigation and laboratory detection.
1.2.2 Virus is separated and 50-100 g of diseased tissues of liver, spleen, pancreas, intestinal tract and the like of the Muscovy duck is taken, the tissues are ground by a mortar, liquid nitrogen is continuously added in the process until the tissues are ground into powder (no obvious visible particles), the powder is homogenized with sterile physiological saline according to the proportion of 1:5(w/v), the homogenate is repeatedly frozen and thawed for 3 times and then is centrifuged at 6000r/min for 10 minutes, the supernatant is taken and added with 10000IU/ml of each of penicillin and streptomycin, the mixture is kept overnight at 4 ℃, and the mixture is filtered by a 0.22 mu m micropore filter and is preserved for later use after being qualified in sterile inspection.
1.2.3 identification of viruses isolated viruses were subjected to culture characterization, physical and chemical characterization, assay of viral content, viral purity, specificity, and immunizationAnd (3) comprehensively identifying immunogenicity and storage life tests and the like. The result shows that after 12-day-old Muscovy duck embryos are inoculated for 120 hours, the head, the neck and chorioallantoic membranes of the embryos are obviously edematous through the autopsy, the whole body is bleeding, the change characteristics are obvious, and the culture characteristics are stable. The virus has no hemagglutination activity, and can not agglutinate red blood cells of SPF chicken and duck, and can not agglutinate red blood cells even if the concentration of the red blood cells is changed. It is insensitive to chloroform, ether and pancreatin; it has resistance to pH 3.0, pH 5.0, and heat (60 deg.C, 1 hr), and is sensitive to ultraviolet radiation. The virus content of the strain used for preparing the vaccine is 106.5ELD50/0.1ml，
The virus strain screened by the invention is used as positive serum prepared by antigen, and the neutralizing capacity of the virus screened by the invention is obviously better than that of the positive serum prepared by epidemic strain; and the neutralizing effect on the strain is obviously different compared with that of an epidemic strain. The virus has no contamination of bacteria, mycoplasma and exogenous virus. The strain is prepared into an inactivated vaccine to immunize young Muscovy ducks and adult female Muscovy ducks, the young Muscovy ducks of 1 day old are immunized, the antibody can reach more than 90% of positive rate within 2 weeks, and the immunization period can reach 5 months; the age of Muscovy ducks on slaughtering days is reached or exceeded; the offspring generated by immunizing adult female muscovy duck at 2 months can generate complete virus counteracting protection, which indicates that the virus strain is an ideal antigen.
1.2.4 PCR detection results of Muscovy duck parvovirus MDPV-GDL strains through the sequence of gosling plague gene published by NCBI (structural gene VP) with the total length of 2199bp and coding 732 amino acids, a pair of primers is designed to cover the total length of VP gene, and an upstream primer P1: 5'-ATGGAGAATGAACAATAAAGGTGAT-3', downstream primer P2: 5'-ACCTGGTACCAATCAGCCTATCTT-3' are provided. The allantoic fluid of Muscovy duck embryo virus is taken, virus DNA is extracted by using a tissue cell genome DNA rapid extraction kit, PCR detection is carried out, and a DNA band appears at about 2232bp by 1% agarose gel electrophoresis (figure 1).
1.2.5 sequencing result of Muscovy Duck parvovirus MDPV-GDL strain sequencing and splicing the amplified fragment of the virus VP gene, and comparing and analyzing the published gene sequence through NCBI, wherein the sequence homology of the VP gene of the MDPV-GDL strain and the published VP gene of the MDPV is between 87.7% and 99.7%. As can be seen by gene evolutionary tree analysis, the MDPV-GDL strain has a close relationship with SAAS-SHNH and a far relationship with other strains, and belongs to a new epidemic strain. The gene evolution tree is shown in FIGS. 2 and 3.
1.2.6 the MDPV-GDL strain and the epidemic strain screened by the invention are subjected to subculture and purification to prepare inactivated vaccines, the rabbits are respectively immunized, the immunization dose is 1.0 ml/rabbit, the immunization is performed once every 2 weeks for 3 times, the blood is collected 2 weeks after the three-immunization, the serum is separated, and the specificity test is performed after the inactivation at 56 ℃ for 1 hour.
Diluting MDPV-GDL strain virus liquid by 50 times, mixing with positive serum of the strain and epidemic strain in equal volume, neutralizing at room temperature for 1 hr, inoculating Muscovy duck embryo of 12 days old, 0.2 ml/piece, and observing for 168 hr. The result shows that the Muscovy duck embryo in the serum neutralization group of Muscovy duck parvovirus of the virus is completely alive, the Muscovy duck embryo in the positive serum neutralization group of other epidemic strains is only 60-80% of the whole virus embryos in the serum neutralization group, and the Muscovy duck embryo in the virus group without serum neutralization is completely dead, which indicates that the virus and the Muscovy duck parvovirus serum have specific neutralization reaction. The virus strain screened by the invention is used as positive serum prepared by antigen, and the neutralizing capacity of the virus screened by the invention is obviously better than that of the positive serum prepared by epidemic strain; and the neutralizing effect on the strain is obviously different compared with that of an epidemic strain.
The results show that the two strains obtained by the invention are new variant strains, thereby reducing the immune effect of the existing vaccine.
Example 2 vaccine manufacture and semi-finished product testing
2.1 preparation of seed Virus
2.1.1 preparation of poison seed of GD Strain, selecting Muscovy duck embryo fibroblast grown in monolayer, discarding original culture solution, adding 1% poison seed (3 rd generation of GD Strain) serum-free M199 maintaining solution with final concentration, standing and culturing at 37 deg.C for 60-78 hr to obtain obvious cytopathy, and harvesting when the cytopathy reaches more than 80%. The method is used for continuously transmitting 10 generations which are respectively marked as C3-C10 generations. The virus content was determined for each passage. The same generation is tested to be sterile and the virus content is more than or equal to 106.0TCID500.1ml of virus solution, quantitatively packaging, freeze-drying and preservingAnd (4) storing.
2.1.2 preparation of seed of MDPV-GDL strains allantoic fluid (3 rd generation of MDPV-GDL strain original seed) is diluted 100 times with sterilized normal saline, 10 Muscovy duck embryos of 12 days old are inoculated via allantoic cavity, each embryo is 0.2ml, incubation is continued at 36-38 ℃, and duck embryos of 48-144 hours after inoculation are selected. Allantoic fluid was collected and centrifuged, and the supernatant was collected for the next generation. The method is used for continuously transmitting 10 generations which are respectively marked as C3-C10 generations. The virus content was determined for each passage. The same generation is tested to be sterile and the virus content is more than or equal to 105.2ELD500.2ml of virus liquid, quantitatively subpackaging, freeze-drying and storing.
2.2 preparation of antigens
2.2.1 preparation of GD Strain antigens
2.2.1.1 preparation of duck embryo fibroblast and GD strain virus liquid.
2.2.1.2 preparation of cells Muscovy Duck embryo of 11-13 days old is digested with 0.25% pancreatin, and cultured with a proper amount of M199 culture solution containing 10% serum.
2.2.1.3 inoculating virus, selecting full monolayer Muscovy duck embryo fibroblast, discarding original culture solution, adding 1% virus seed maintaining solution, and culturing at 37 deg.C.
2.2.1.4 after harvesting and inoculation, the cytopathic conditions were recorded by observing 2 times daily. Harvesting when cytopathic effect reaches more than 80%, freeze-thawing for 2 times, and storing at-20 deg.C before inactivating the harvested cytotoxicity.
2.2.2 preparation of MDPV-GDL Strain antigen
Diluting the virus seeds by 100 times with sterile normal saline, inoculating 12-day-old susceptible duck embryos into an allantoic cavity, incubating at 37 ℃, collecting allantoic fluid and embryo body tissues of the duck embryos within 48-120 hours, homogenizing, repeatedly freezing and thawing for 3 times, centrifuging, mixing supernatant in a sterilized container, and storing at 2-8 ℃.
2.3 measurement of Virus content
2.3.1 measurement of Virus content of GD Strain the virus seed was serially diluted 10-fold with a cell maintenance solution, and 10 cells were taken﹣6、10﹣7、10﹣83 dilutions were inoculated into duck embryo fibroblasts grown in monolayer (96 wells)Cell plates) were seeded at 6 wells per dilution, 0.1ml per well. Setting virus positive control hole and cell negative control hole at the same time, culturing at 37 deg.C, and 5% CO2Culturing in incubator and observing for 168 hr, judging the CPE as infection, and calculating TCID50。
2.3.2 measurement of MDPV-GDL Strain Virus content MDPV-GDL Strain Virus solution was serially diluted 10 times with sterilized physiological saline, 10 times each﹣5、10﹣6、10﹣7And 3 dilutions, inoculating 12-day-old Muscovy duck embryos with 0.2ml of each embryo through allantoic cavities, and simultaneously inoculating 5 inoculated embryos with physiological saline in a contrast manner with 0.2ml of each embryo. Incubation was continued at 37 ℃ and observed for 168 hours. The death of duck embryo after 48 hours and the occurrence of allantoic membrane edema and thickening, and the hemorrhagic lesions of the whole body such as head, neck and back are judged as infection, and the ELD of MDPV-GDL strain is respectively calculated50。
2.4 inactivation two antigens (GD strain and MDPV-GDL strain) are mixed according to the volume ratio of 0.6: 1-1: 1, and are respectively led into an inactivation tank, 10% of formaldehyde solution is metered and added, a stirrer is started to stir, so that the two antigens are fully mixed, and the final concentration of formaldehyde is 0.2%. The formaldehyde solution is added and then introduced into another inactivation tank to avoid that viruses adhered near the tank opening can not contact the inactivator. Inactivating at 37 ℃ for 16 hours (starting to time when the temperature in the tank reaches 37 ℃, and starting a stirrer to stir continuously), taking out, and storing at 2-8 ℃ for no more than 1 month.
2.5 inspection of semi-finished products
2.5.1 sterile examination the inactivated virus solution is taken, examined according to the appendix of the current pharmacopoeia of Chinese beasts, and is required to grow aseptically.
2.5.2 inactivation assay
2.5.2.1 GD strain inactivation test comprises diluting inactivated GD strain virus solution 10 times, inoculating full monolayer Muscovy duck embryo fibroblast (24-well cell plate) with 4 wells (0.2 ml per well), and supplementing maintenance solution to 2.0 ml; meanwhile, the virus solution which is not inoculated and inactivated is used as a negative control, the virus solution which is not inoculated and inactivated is used as a positive control, the temperature is 37 ℃, and the CO content is 5 percent2The incubator was incubated and observed for 168 hours. The culture is harvested, frozen and thawed repeatedly, and then the first generation is passed through in a blind mode, and the culture is continued for 120 hours. Observe whether CPE is present.
2.5.2.2 MDPV-GDL strain inactivation test 10 Muscovy duck embryos of 12 days old are inoculated with virus liquid of the inactivated MDPV-GDL strain through allantoic cavities, each embryo is 0.1ml, and the embryos are placed at 37 ℃ for continuous incubation and continuous observation for 168 hours.
2.6 preparation of oil emulsion inactivated vaccine
2.6.1 oil phase preparation:
taking 94 parts of mineral oil and 2 parts of aluminum stearate (g), stirring while adding until the mineral oil is completely transparent, adding 6 parts of Span-80 (Span-80) (ml), fully mixing, and sterilizing by high-pressure steam at 121 ℃ for later use.
2.6.2 aqueous phase preparation:
mixing virus liquid qualified by inactivation inspection, adding 96 parts (ml) of the virus liquid into 4 parts (ml) of sterilized Tween-80 (Tween-80), and fully shaking until the Tween-80 is completely dissolved.
2.6.3 emulsification
And (3) introducing 2 parts of the oil phase into an emulsification tank, starting a motor to stir at a low speed, slowly adding 1 part of the water phase, and emulsifying for 30 minutes at 1000r/min to obtain the oil emulsion inactivated vaccine.
And 2.6.4, subpackaging quantitatively, sealing by covering, sticking a label, and storing at 2-8 ℃.
Example 3 Final vaccine testing
3.1 Properties
The appearance was a milky white emulsion.
The dosage form is water-in-oil type. A clean pipette is taken to suck a small amount of vaccine and drip the vaccine into cold water, and the vaccine should not spread except the 1 st drop.
The stable vaccine is sucked by 10ml and added into a centrifuge tube, and centrifuged for 15 minutes at 3000r/min, and the water separated out from the tube bottom is not more than 0.5ml correspondingly.
The viscosity is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the viscosity is in accordance with the regulations.
3.2 the filling quantity is checked according to the appendix of the current Chinese animal pharmacopoeia, and the filling quantity is in accordance with the regulations.
3.3 sterility test the sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the sterility growth is required.
3.4 safety inspection to ensure the safety of the vaccine, 10 Muscovy ducks of 1 day old are immunized subcutaneously, 1.0ml of vaccine is injected into each neck part subcutaneously in points, the growth and development are normal after 21 days of observation, the mental state is good, the vaccine is well absorbed at the injection part of the autopsy, and inflammatory reactions such as red swelling, tissue necrosis and the like do not exist.
3.5 efficacy test
3.5.1 serological method 30 Muscovy ducks of 1 day old, 10 neck subcutaneous immunizations, 10 breast muscle immunizations, 20 total, each 0.2ml, and another 10 immunization controls. And (3) taking blood of each duck 21-28 days after immunization, separating serum, and detecting Muscovy duck serum of an immune group and a control group by using an agar immunodiffusion test, wherein the GD strain immune group is at least 8 positive, and the control group is all negative. The MDPV-GDL strain immune group should be at least 10 positive, and the control group should be all negative.
Serum results: the GD strain is positive to more than 8/10 antibodies in agar diffusion test through neck subcutaneous and chest muscle immunization for more than 21 days, and all the control group ducks are negative; the MDPV-GDL strain is positive for the Qiongzhan antibody more than 14 days through neck subcutaneous and chest muscle immunity, and all ducks in a control group are negative; the tests all accord with the efficiency detection standard. The immune period can reach more than 5 months; the age of the Muscovy ducks in slaughtering days is reached or exceeded. See table 1 for details.
Table 1: antibody levels of young Muscovy ducks at different times after immunization of bigeminal vaccine
3.5.2 Muscovy duck source epidemic strain vaccine immunization and virus challenge protection effects of the invention 60 Muscovy ducks of 1 day age are selected, a duck type 2 adenovirus epidemic strain and a Muscovy duck parvovirus epidemic strain with higher neutralization potency and virus content are respectively selected to prepare the bivalent inactivated vaccine, and the preparation method is the same as that of the invention. The neck of each seedling is immunized subcutaneously by 10, 40, 0.2ml each, and another 20 immunization controls. Challenge tests were performed 14 days after immunization. Intramuscular injection of GD strain virus solution (about 10) into young Muscovy ducks of all immune groups and control groups6.0TCID500.1ml) and virus solution of MDPV-GDL strain (about 10% in5.2ELD500.2ml), 0.2ml each. After the toxin is attacked, continuously observing for 14 days, every dayThe clinical manifestations of the ducks were observed and the morbidity and mortality were recorded.
Vaccine immunization and GD strain and MDPV-GDL strain challenge protection results: the 14-day protection number of the vaccine is 100 percent after immunization, while the 14-day protection number of the epidemic strain is lower than 80 percent after immunization; in a control group, the strains for preparing the vaccine are used for resisting the virus and all the diseases are caused within 14 days, namely the disease rate is 100 percent, and the death rate is more than 90 percent. The vaccine prepared by the invention has the best immune effect. The data are detailed in table 2 below.
Table 2: immune and virus challenge protection result of 1-day-old muscovy duck epidemic strain vaccine
3.5.3 relationship between maternal antibody of offspring and protection against toxic pathogen 30 adult healthy Muscovy ducks immunized subcutaneously in each neck at 0.5ml, and 30 non-immunized ducks as control.
Eggs laid 2 months after immunization are hatched to produce Muscovy ducks, 20 Muscovy ducks in the immune group are taken, 20 Muscovy ducks in the control group are taken, and the eggs are raised for challenge test. The immune group and the control group of Muscovy ducks are respectively injected with GD strain virus solution (about 10 percent)6.0TCID500.1ml), MDPV-GDL strain virus liquid (about 10% in content)5.2ELD500.1ml) and 0.2ml of epidemic strain virus liquid each. After the challenge, the clinical manifestations of the ducks are observed every day, the morbidity and the mortality are recorded, and the observation is continuously carried out for 14 days. And (4) effective inspection standard: GD strain immune duck should be at least 8 normal, and control group should be at least 8 diseased. The MDPV-GDL strain immune ducks should be at least 8 normal ducks and the control group should have at least 8 diseased ducks.
The results of the offspring maternal antibody and challenge protection: the agar-agar antibodies of offspring produced by 2 months old adult female muscovy ducks immunized with the bigeminy seedlings are 100% positive when the offspring are 7 days old. All controls were negative. The offspring 7-day old Muscovy duck attacks to prepare a strain and an epidemic strain for the vaccine, wherein the epidemic strain is selected from 4 representative strains. After continuously observing for 14 days, the protection number of the immune group reaches 90 percent; the control group uses the vaccine strain to fight against the virus within 14 days and the mortality rate reaches more than 90 percent. The protection number of the control group in 14 days after the control group is challenged with the epidemic strains is lower than 20 percent. The cross protection rate is high. The vaccine prepared by the invention can effectively prevent the infection of duck type 2 adenovirus and Muscovy duck parvovirus which are popular in various places at present, and has good immune effect. The specific data are shown in tables 3 and 4 below.
Table 3: maternal antibody and toxicity counteracting protection test of offspring 7-day old muscovy duck
Table 4: cross protection test of maternal antibody and epidemic strain of offspring 7-day-old muscovy duck
In conclusion, the vaccine prepared from the GD strain and the MDPV-GDL strain screened by the invention is a bivalent inactivated vaccine with ideal immune effect, and can effectively prevent the group I duck adenovirus 2 disease and Muscovy duck parvovirus disease.
The above embodiments are preferred embodiments of the present invention, and any other changes, modifications, substitutions, combinations, and equivalents which do not depart from the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A goose parvovirus and duck 2 adenovirus bivalent inactivated vaccine is characterized in that the antigens of the bivalent inactivated vaccine are duck 2 adenovirus and goose parvovirus, wherein the preservation number of the goose parvovirus is CCTCC NO: V201811; the duck type 2 adenovirus is GD strain virus; the preservation number is CCTCC No: v201633.
2. The bivalent vaccine as claimed in claim 1, wherein the inactivation of duck adenovirus type 2 and muscovy duck derived goose parvovirus is carried out by formaldehyde inactivation.
3. The bivalent vaccine according to claim 1, wherein the virus content of duck type 2 adenovirus in the vaccine is 10 or more6.5TCID500.1ml, the virus content of Muscovy duck source goose parvovirus is more than or equal to 105.0ELD50/0.2ml。
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Inventor after: Wang Xiangqin Inventor after: Wang Danna Inventor after: Shan Xueqiang Inventor after: Sun Nana Inventor after: Lin Wenchao Inventor after: Wang Xuebo Inventor after: Li Chaoyang Inventor after: Liu Yang Inventor before: Wang Xiangqin Inventor before: Wang Danna Inventor before: Liu Yang Inventor before: Sun Nana Inventor before: Lin Wenchao Inventor before: Wang Xuebo Inventor before: Li Chaoyang |
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Denomination of invention: A combined inactivated vaccine of duck type 2 adenovirus and Muscovy Duck Parvovirus Disease Effective date of registration: 20211222 Granted publication date: 20200221 Pledgee: Shandong Zhucheng rural commercial bank Limited by Share Ltd. Pledgor: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Registration number: Y2021980015708 |
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Denomination of invention: A Dual Inactivated vaccine of Duck Adenovirus Type 2 and Muscovy Duck Parvovirus Effective date of registration: 20230608 Granted publication date: 20200221 Pledgee: Shandong Zhucheng rural commercial bank Limited by Share Ltd. Pledgor: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Registration number: Y2023980043376 |
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