CN111110839A - Goose astrovirus bivalent inactivated vaccine for preventing gosling gout - Google Patents
Goose astrovirus bivalent inactivated vaccine for preventing gosling gout Download PDFInfo
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Abstract
The invention provides a goose astrovirus bivalent inactivated vaccine, which comprises inactivated antigens of two virus strains and a vaccine adjuvant, wherein the antigens are a novel goose astrovirus GD strain and a goose astrovirus variant SC-3 strain with the preservation number of CCTCC NO: V201976. The bivalent inactivated vaccine provided by the invention can play a good role in preventing the current gosling gout disease prevalent in China, thereby reducing the loss of the breeding industry.
Description
Technical Field
The invention belongs to the technical field of preparation of poultry vaccines, and particularly relates to a goose astrovirus bivalent inactivated vaccine for preventing gosling gout.
Background
Since 2015, the infectious disease mainly occurs in goslings of 5-15 days old, the mortality rate is 20-30%, the feeding condition is poor and reaches up to 40%, the death rate is higher when the incidence days are smaller, the death rate is higher, the goslings are basically not dead after 15 days old, the dead goslings mainly show serious urate deposition of organs and joints of the whole body, the over-resistant goslings are prone to cause low feed conversion rate due to digestive absorption disorder, the organism is poor in development, the growth of large groups is uneven, the production performance of goose groups is seriously influenced, and the serious loss is caused to the goose industry of China.
The pathogen separation and identification confirm that the gosling gout is mainly caused by goose astrovirus of two genotypes. At present, China has no vaccine aiming at the goose astrovirus, so the screening of the goose astrovirus and the preparation of the vaccine have great significance for the prevention and control of the gosling gout disease.
Disclosure of Invention
The invention provides a goose astrovirus bivalent inactivated vaccine, thereby filling the blank that no goose astrovirus vaccine exists in China.
The invention provides a goose astrovirus bivalent inactivated vaccine, which comprises inactivated antigens of two virus strains and a vaccine adjuvant, wherein the antigens are a goose astrovirus GD strain (goose astrovirus, purchased from China veterinary medicine inspection institute) and a goose astrovirus FLX variant virus SC-3 strain with the preservation number of CCTCC NO: V201976;
wherein the virus content of the two virus strains is not less than 2 × 106.0TCID50/0.1ml；
Wherein the virus strain is inactivated using formaldehyde solution.
The vaccine adjuvant is an oil phase adjuvant, and one of the oil phase adjuvant comprises 94 parts of animal white oil, 1 part of aluminum stearate and 5 parts of span-80.
The preparation method of the bivalent inactivated vaccine comprises the following steps:
1) virus propagation and harvesting: performing virus propagation on GD strains on LMH cells, and performing virus propagation on SC-3 strains on chorioallantoic membranes of goose embryos to obtain virus liquid;
2) and (3) concentrating the virus: concentrating the harvested 2 virus solutions by 2 times by using an ultrafiltration concentrator at the temperature of 2-8 ℃, and preparing the concentrated virus solutions for inactivation;
3) virus inactivation: introducing the concentrated virus solution into an inactivation tank, metering and adding 10% of formaldehyde solution, and fully mixing, wherein the final concentration of the formaldehyde solution is 0.1%; inactivating the mixture in a shaker at 37 ℃ for 20 hours by shaking at 200rpm, taking out the mixture, and storing the mixture at 2-8 ℃;
4) preparing an oil phase: taking 95 parts of white oil for livestock and 1 part of aluminum stearate, placing the white oil and the aluminum stearate in an oil phase preparation tank, heating the mixture to 80 ℃, then adding span-805 parts of span-30 min until the temperature reaches 115 ℃, and cooling the mixture for later use;
5) preparing an aqueous phase, mixing 96 parts of antigen liquid of the inactivated goose astrovirus GD strain and the SC-3 strain in the step 3) with 4 parts of sterilized Tween-80 respectively, and stirring in an aqueous phase preparation tank until the Tween-80 is completely dissolved;
6) emulsification: placing 2 parts of oil phase in a high speed shearing machine, starting a motor to rotate slowly and stir, simultaneously slowly adding 1 part of water phase, and emulsifying at 10000r/min to prepare the vaccine.
The bivalent inactivated vaccine provided by the invention can play a good role in preventing the current gosling gout disease prevalent in China, thereby reducing the loss of the breeding industry.
Drawings
FIG. 1: anatomy figures after different modes of toxicity attack;
FIG. 2: cloaca swab viral gene RT-PCR detection map of different challenge group geese, 11 detected by random sampling of each group, M lane: marker, "N" represents a negative control;
FIG. 3: RT-PCR detection images of virus genes of goose cloaca swabs after challenge of different immunization groups, wherein lanes 1-20 respectively represent numbers of geese, and positive bands are subjected to gel cutting and then are subjected to sequencing identification to obtain goose astrovirus sequences; lane M: marker; the toxin expelling conditions of 2, 4, 6, 8, 10 and 12 days after toxin attack are consistent with 14 days after toxin attack;
FIG. 4: and (3) a post-challenge anatomical map of young geese hatched by hatching eggs laid by the immune breeding geese and the non-immune breeding geese.
Detailed Description
The present invention will be described in detail with reference to examples.
Example 1:
1. isolation and identification of viral strains
The invention uses 2 strains of goose astrovirus, wherein the strain of the goose astrovirus FLX variant virus SC-3 is preserved in China center for type culture Collection of Wuhan university in Wuhan, Wuhan university in 2019, 10 and 29 months, and the preservation number is CCTCC NO: V201976;
the goose astrovirus FLX mutant virus SC-3 strain is separated from a 5-15-day-old disease and dead gosling sample which is inspected from Guangdong, Sichuan, Hebei, Shandong, Anhui, Liaoning and the like in the period of 10 months to 5 months in 2019, and the separated strain has strong pathogenicity on goose embryos, so that the diseased goose has clinical manifestations different from those of classical strains and mainly shows the serious urate deposition phenomenon of organs of the whole body, which shows that the biological characteristics of the goose astrovirus separated by the invention are obviously changed compared with the classical strains, particularly the pathogenicity on the goose embryos and the clinical symptoms caused by the goose.
The genome total length of the goose astrovirus FLX variant virus SC-3 strain is 7288bp, the genome total length of the classical goose astrovirus is 7299bp (sequence number: KY271027), the whole genomes of the two strains of viruses, ORF1a gene, ORF1b gene and ORF2 gene sequences all have base mutation or deletion with different degrees, wherein the ORF1a gene is highly conserved, the ORF1b gene is relatively conserved, but the capsid protein ORF2 gene (4869-one 6992) coding the virus protective antigen has larger change and generates larger base mutation or deletion, thereby causing the change of translated amino acid.
Neutralization experiments show that SC-3 strain positive serum and YB-1 strain positive serum have strong self-neutralization capability but poor neutralization capability to each other, the antigen correlation R values of the SC-3 strain positive serum and the YB-1 strain positive serum are only 0.22 (Table 4), and the SC-3 strain positive serum and the YB-1 strain positive serum belong to different serotypes.
Another strain of the goose astrovirus is purchased from China institute of veterinary medicine, and for convenience of description, the goose astrovirus purchased from China institute of veterinary medicine is named as goose astrovirus GD strain in the specification.
2. Animal regression test
The goose astrovirus (novel goose astrovirus GD strain, goose astrovirus FLX variant virus SC-3 strain) used by the invention is respectively subjected to virus propagation (the propagation method is shown in vaccine preparation 1.1 virus propagation and harvest) on LMH cells and goose embryos, and the obtained virus liquid is used for animal regressionTest comprises dividing healthy 120 Yangzhou white gooses of 1 day old into 4 groups and 30 white gooses, feeding in animal isolator, adding feed and drinking water daily, and respectively attacking with novel goose astrovirus GD strain, goose astrovirus FLX variant virus SC-3 strain and novel goose astrovirus GD strain + goose astrovirus FLX variant virus SC-3 strain with attacking dose GD of 106.0TCID500.1ml, SC-3 is 106.0ELD500.1ml, simultaneously setting a blank control group without counteracting the toxin, observing the mental state, ingestion and morbidity of the goose group every day, collecting the cloaca swab of each goose on the 2 nd, 4 th, 6 th, 8 th, 10 th, 12 th and 14 th days after counteracting the toxin, detecting the toxin expelling condition by using an RT-PCR method, and recording test data.
Table 1: test record table after gosling attacking poison
Animal regression test results show that single goose astrovirus GD strain or SC-3 strain is used for attacking toxin to cause animal diarrhea, but the single goose astrovirus GD strain or SC-3 strain cannot reproduce the gosling gout disease consistent with clinical morbidity, the goose astrovirus GD strain and SC-3 strain are used for combined attacking toxin to successfully reproduce the typical goose gout disease consistent with clinical morbidity (figure 1), and the clinical manifestations and toxin expelling conditions of an attacking goose are shown in tables 1 and 2, so that the two strains of viruses causing the gosling gout disease are determined to be the goose astrovirus GD strain and the goose astrovirus FLX variant virus SC-3 strain according to the definition of the Koehler's law.
Example 2: preparation and detection of vaccines
1. Preparation of vaccines
1.1 Virus propagation and harvesting
Performing virus propagation on the goose astrovirus GD strain and the goose astrovirus FLX variant virus SC-3 strain on LMH cells and goose embryos respectively to obtain pure virus liquid with high sufficient titer.
Goose astrovirus GD strain: subculturing LMH cells in a T25 cell bottle, pouring cell culture solution in the bottle when the cell density reaches about 95%, washing the cells for three times by using serum-free DMEM medium, inoculating viruses according to 1% dose, adsorbing the viruses for 1h at 37 ℃, slightly swinging the cell bottle every 10min so as to completely adsorb the viruses, pouring the virus receiving solution after the adsorption is finished, adding the DMEM medium containing 1% fetal calf serum to continuously culture and observe the viruses, collecting the viruses when the cytopathic effect reaches about 80% after 96h, putting the cell bottle into an ultra-low temperature refrigerator with the temperature of-80 ℃, repeatedly freezing and thawing for three times so as to completely release the viruses from the cells, centrifuging the cells for 30min at 5000rpm in a centrifuge with the temperature of 4 ℃, and carefully transferring supernatant into a sterile bottle.
Goose astrovirus FLX mutant virus SC-3 strain: diluting the virus stock solution by 100 times, inoculating goose embryos of 10 days old, 0.2 ml/piece, culturing in an incubator at 37 ℃ by means of chorioallantoic membrane, irradiating the embryos 2 times per day, discarding the dead goose embryos within 48 hours, recording, and cooling the goose embryos for 24 hours at 2-8 ℃ after 5 days. Taking out the cooled goose embryo, harvesting the allantoic membrane and allantoic fluid of goose embryo villi, sucking the allantoic fluid, putting the embryo fluid into a 1L sterile bottle, peeling off the allantoic membrane of the goose embryo villi with scissors and forceps, fully grinding (adding quartz sand) in a mortar, transferring the grinding fluid into the sterile bottle containing the embryo fluid, repeatedly freezing and thawing for three times in a refrigerator at-80 ℃, centrifuging for 30min at 5000rpm in a centrifuge at 4 ℃, and carefully transferring the supernatant into the sterile bottle. The two virus solutions are subjected to sterility test according to the current Chinese veterinary pharmacopoeia. The numbers of GD strain and SC-3 strain are marked, and the GD strain and SC-3 strain are stored at 4 ℃ for later use.
1.2 concentration of the virus: and (3) concentrating the harvested 2 virus solutions by using an ultrafiltration concentrator at the temperature of 2-8 ℃, concentrating by 2 times, reserving samples, carrying out semi-finished product inspection, and inactivating the rest virus solutions.
1.3 inactivating the virus, namely introducing the goose star-shaped virus liquid into an inactivation tank, metering and adding 10% of formaldehyde solution, and fully mixing, wherein the final concentration of the formaldehyde solution is 0.1%. And inactivating the mixture for 20 hours by shaking at 200rpm in a shaker at 37 ℃, taking out the mixture, and storing the mixture at 2-8 ℃.
1.4 inspection of semi-finished products
(1) And (4) performing sterile inspection according to the method in the appendix of the current Chinese veterinary pharmacopoeia, and determining that the prepared virus solution has no bacterial infection.
(2) Virus content determination harvested virus fluid (sampled before inactivation) is serially diluted 10 times with sterilized normal saline, and taken10-4、10-5、10-6、10-74 dilutions and the diluted GD samples were applied to prepared 96 well cell plates that had grown into LMH cell monolayers (typically 24 h), 6 wells per dilution, 100. mu.l/well. Meanwhile, 6 normal cell control wells were set. The loading sequence should be based on the loading of normal cell control before the high dilution to low dilution into the cell plate. The cell culture plate was placed at 37 ℃ and 5% CO2Adsorbing for 1 hr in incubator, adding 1% fetal calf serum DMEM cell culture solution 100 μ l, standing at 37 deg.C and 5% CO2Culturing in an incubator, continuously observing until 7 th day, and judging the result. The pathological changes of the cells in each 96-well cell culture plate were recorded and the TCID was calculated50The virus content is not less than 2X 106.0TCID500.1 ml. The harvested virus liquid (sampled before inactivation) SC-3 strain is serially diluted by 10 times with sterilized normal saline, 10 are taken-4、10-5、10-6、10-74 dilutions, inoculating 10-day-old goose embryos with each embryo of 0.1ml through chorioallantoic membrane, and simultaneously inoculating 5 control embryos of physiological saline of 0.1 ml. Incubation was continued at 37 ℃ with embryo irradiation 2 times a day for 144 h. Observing and recording death condition of each embryo, and calculating ELD50Determining the virus content not less than 2 × 106.0ELD50/0.1ml。
(3) And (3) preparing 6 bottles of LMH cells by inactivation test, inoculating 0.2ml of inactivated virus solution GD strain into each bottle, observing the cells for 2 times every day, continuously observing for 6 days, collecting cell solution, conducting blind transmission for 2 generations according to the same method, observing and recording the pathological change condition of the cells, and completely inactivating the cells and preventing the pathological change of the cells. (ii) a Taking 6 goose embryos of 10 days old, inoculating 0.2ml of inactivated virus liquid into each chorioallantoic membrane, observing for 7 days after each embryo is inoculated for 2 times per day, collecting embryo liquid and chorioallantoic membrane, conducting blind transmission for 2 generations according to the method, observing and recording the death condition of the goose embryos, and finally, completely inactivating the goose embryos without dead embryos.
1.5 preparation of inactivated vaccine: and (3) preparing the vaccine by using the semi-finished antigen after passing the inspection (the liquid components in the following preparation are calculated according to the volume ratio).
(1) Preparing an oil phase, namely putting 95 parts of white oil for livestock and 1 part of aluminum stearate in an oil phase preparation tank, heating to 80 ℃, then adding span-805 parts until the temperature reaches 115 ℃, maintaining for 30min, and cooling for later use.
(2) And (3) preparing an aqueous phase, mixing 96 parts of inactivated antigen liquid of the goose astrovirus GD strain and the SC-3 strain with the sterilized tween-804 parts respectively, and stirring in an aqueous phase preparation tank until the tween-80 is completely dissolved.
(3) Emulsifying 2 parts of oil phase in a high-speed shearing machine, starting a motor to rotate slowly and stirring, and slowly adding 1 part of water phase at 10000r/min for emulsifying for 5 minutes. After emulsification, 10ml of the mixture is taken out and centrifuged at 3000r/min for 15 minutes, and the water phase separated out from the bottom of the tube is not more than 0.5 ml.
(4) Preparing individual oil seedlings of the goose astrovirus GD strain and the SC-3 strain prepared by the steps according to the ratio of 1:1, and mixing and emulsifying for a plurality of times in a small amount in the emulsifying process so as to ensure that emulsion droplets of the two antigens are uniformly distributed.
1.6 vaccine product testing
1.6.1 Properties
Appearance: the prepared vaccine is milk white emulsion without impurities.
The preparation formulation is as follows: is of water-in-oil type. A clean suction tube is taken, a small amount of vaccine is sucked and dripped into cold water, and the vaccine does not diffuse except for the 1 st drop.
Stability: 10ml of the vaccine is sucked and added into a centrifuge tube, and the centrifuge tube is centrifuged for 15 minutes at 3000r/min, and the water phase separated out from the tube bottom is not more than 0.5 ml.
Viscosity: the detection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the corresponding regulations are met.
1.6.2 sterility test: according to the appendix of the existing Chinese animal pharmacopoeia, the result conforms to the regulations.
1.6.3 safety inspection: no local and systemic adverse reactions caused by the vaccine occur. Respectively injecting 1.0ml of bivalent vaccine subcutaneously into the neck of 10 SPF chickens of 7 days old and 10 goslings of 7 days old, respectively, simultaneously respectively setting 5 controls, feeding under the same conditions, continuously observing for 14 days, and recording the ingestion, drinking water and clinical conditions of the test chickens and the goslings. Any local and systemic adverse reactions caused by the vaccine should not occur.
The results show that the chickens and geese in the test group and the control group develop normally and are in good mental state, and the vaccine at the injection part is well absorbed by the autopsy test group without inflammatory reactions such as red swelling, tissue necrosis and the like. The trial vaccine is safe and harmless and has no influence on the growth of animals.
1.6.4 efficacy test
10 SPF chickens of 21 days old are injected with goose astrovirus vaccine 0.3 ml/chicken subcutaneously in each neck, 5 other chickens of the same day old are taken as control, and after 21 days of immunization, blood is collected and serum is separated, and antibodies are respectively measured.
The antibody titer of the immune group is not less than 6.0log2VN antibodies. The control groups should be negative.
2. Virus neutralization assay
The inactivated vaccines are prepared by carrying out subculture and purification on the goose astrovirus GD strains and the SC-3 strains according to the method, 21-day-old SPF (specific pathogen free) chickens are immunized respectively, the immunization dose is 0.5 ml/chicken, the immunization is carried out once every 2 weeks for 3 times, and blood sampling and serum separation are carried out 2 weeks after three-time immunization. Adopting a fixed virus dilution serum method to perform cross neutralization titer determination on positive serum of GD strains and SC-3 strains, which comprises the following steps: respectively diluting GD strain virus liquid and SC-3 strain virus liquid to 200TCID50/0.1ml、200ELD500.1ml, mixing with the dilution positive serum of GD strain and SC-3 strain one by one in equal volume, shaking and neutralizing at 37 ℃ for 1 hour, inoculating 5 holes of LMH cells, 0.2 ml/hole or 5 goose embryos of 10 days old, 0.2 ml/egg to each neutralization group, observing once every 24 hours, discarding the dead goose embryos within 24 hours, and continuously observing for 7 days. The results showed that there was no neutralization between the serum-viruses of the goose astrovirus GD strain and SC-3 strain, and the R value of the antigen correlation between the two strains was 0 (table 2). This shows that there is no cross-neutralization ability between the novel goose astrovirus GD strain and the goose astrovirus FLX variant virus SC-3 strain, and the antibodies of the novel goose astrovirus GD strain and the goose astrovirus FLX variant virus SC-3 strain can only generate good neutralization ability for the virus, but have no neutralization ability for non-virus. From serological results, the goose astrovirus GD strain and the SC-3 strain are two viruses without serological relationship.
Table 2: serum cross-neutralization test results of goose astrovirus GD strains and SC-3 strains
Note: denotes a virus; serum is
3 challenge protection test
Culturing GD strain and SC-3 strain of goose astrovirus on LMH cell and 10-day-old goose embryo, respectively, and performing TCID50、ELD50The antigen content of each strain is 10 respectively-6.0TCID50/0.1ml、10-6.0TCID50/0.1ml, after harvesting the antigen, concentrating by an ultrafiltration concentrator for 2 times, inactivating the antigen by formaldehyde with the final concentration of 1.0 per mill at 37 ℃ for 20 hours, and mixing the components according to the following oil phase: preparing bivalent inactivated vaccines of the goose astrovirus GD strain, the goose astrovirus SC-3 strain and the goose astrovirus GD strain plus the SC-3 strain according to the proportion of 2:1 of the water phase. 100 1-day-old goslings were divided into 5 groups (Table 3) and 20 goslings/group, the immunized group goslings were injected with bivalent inactivated vaccines of goose astrovirus GD, goose astrovirus SC-3, goose astrovirus GD + SC-3, and 0.5 ml/gosling, and the control group goslings were injected with physiological saline, 0.5 ml/gosling, respectively.
Combined challenge with goose astrovirus GD strain and SC-3 strain (1: 1) at a dose of 1 ml/strain (GD 10) 14 days after immunization- 7.0TCID50，SC-3＝10-7.0ELD50) And a blank control group goose is arranged, so that the immunity and the toxicity are not attacked. Observing the mental state, ingestion and morbidity of the goose flocks every day, collecting the cloaca swab of each goose on the 2 nd, 4 th, 6 th, 8 th, 10 th, 12 th and 14 th days after the challenge, detecting the detoxification condition by using an RT-PCR method, and recording test data.
Table 3: test grouping design table
Table 4: toxin expelling condition table after toxin expelling by using goose astrovirus GD strain and SC-3 strain in different immunization groups
The result of the challenge protection test shows that the immune goose astrovirus GD strain and SC-3 strain bivalent inactivated vaccine of the gosling can provide complete protection for combined challenge of the goose astrovirus GD strain and the SC-3 strain, the gosling in an immune group does not expel toxin, does not show any clinical symptoms and does not die, and the goslings in the immune goose astrovirus GD strain vaccine group, the goose astrovirus SC-3 strain vaccine group and a challenge control group all have toxin expelling phenomenon (figure 3, enumerates the toxin expelling detection on 14 days after challenge, and the objective goose is sent to the Shanghai biological engineering company Limited after being subjected to glue cutting for sequencing and identification to be correct), and the gosling is listened, white excrement discharging and extremely thin and later stage goose emaciation, wherein 2 goslings in the immune goose astrovirus GD strain vaccine group die 12 days (28 days old) after challenge, and have no typical gout by autopsy, and can be related to the day age (15 days old) of the challenge goose, as the age of the goose in days is increased, the resistance of the goose to the virus is enhanced, and all goslings in the blank control group are normal. The result shows that the goose astrovirus GD strain and SC-3 strain bivalent inactivated vaccine can prevent the gosling from expelling toxin and can well prevent diseases caused by the goose astrovirus.
4. Immunization of breeding goose vaccine and challenge protection test of gosling
The gosling gout is caused by goose astrovirus, but mainly occurs at 5-15 days of age, the disease rarely occurs after 20 days of age, even if the disease occurs, no obvious death exists, and the clinical symptoms are slight. Therefore, clinically, the gosling gout disease is mainly prevented and controlled in the gosling stage, but in the 1-day-old vaccine immunization, the goose astrovirus antibodies in the gosling body can play a protective role at least after 14 days, and the immune blank period before 14 days is most prone to the gosling gout disease. Therefore, the goose astrovirus GD strain + SC-3 strain bivalent inactivated vaccine is mainly applied to breeding geese, the breeding geese immunize the vaccine, and after hatching eggs are hatched out of shells, the gosling can safely survive a disease high-incidence period of 5-15 days old under the protection of maternal antibodies as long as enough maternal antibodies are available in the hatching eggs. Therefore, it is necessary to immunize the inactivated vaccine on breeding geese, collect hatching eggs of the geese, and verify the effectiveness of the vaccine by using young geese hatched out of shells to perform challenge protection testProperty of (2)。
The Yangzhou white goose parents are randomly divided into two groups (A group) for health (laboratory detection without goose astrovirus infection)Immunizing goose astrovirus GD strain and SC-3 strain at 150-day age and 180-day age respectively, injecting the bivalent inactivated vaccine for 1.0 ml/time, immunizing the other group (B group) of geese without the goose astrovirus vaccine, collecting 500 hatching eggs produced by the two groups of breeding geese after 210-day age, hatching the hatching eggs in an incubator, immediately transferring the hatching eggs to an isolator for feeding, and using goose astrovirus GD strain and SC-3 strain for 1:1 mixed virus challenge with the virus challenge dose of 1ml (GD is 10 ═ for-7.0TCID50/0.1ml，SC-3＝10-7.0ELD500.1ml), hatching 100 hatching eggs laid by the group B breeding geese out of the shell and setting as a blank control (group C), and not attacking poison. Observing the mental state, ingestion and morbidity of the goose flocks every day, collecting the cloaca swab of each goose on the 2 nd, 4 th, 6 th, 8 th, 10 th, 12 th and 14 th days after the challenge, detecting the detoxification condition by using an RT-PCR method, and recording test data.
After the challenge, the commercial gosling generation A grows normally and is not abnormal; group B commercial goslings begin to suffer from listlessness, wilting and anorexia on the 3 rd day after the detoxification, do not want to walk in a squatting state, begin to die on the 4 th day, continue until the 10 th day, the dead goslings undergo severe visceral gout by caesarean examination, and in some cases, the goslings also suffer from arthrolithia, the death rate is 138/500-27.6%, the enduring goslings have poor growth and development, are emaciated and are ragged; the blank control group gosling was all normal without any abnormality (see fig. 4). The goose astrovirus GD strain and SC-3 strain bivalent inactivated vaccine can provide complete protection effect on the commercial generation gosling, and under the condition that the commercial generation gosling is not immunized by the vaccine, the gosling can safely pass through the immunization blank period of 1-14 days old through the effect of the maternal antibody of the breeding goose. Therefore, the bivalent inactivated vaccine of the goose astrovirus GD strain and the SC-3 strain is popularized and immunized on breeding geese, can well prevent and control gout of goslings popular in China at present, is the first vaccine for the gout disease caused by the goose astrovirus in China, and has great significance for the goose industry in China.
Claims (6)
1. The bivalent inactivated vaccine of the goose astrovirus is characterized by comprising inactivated antigens of two virus strains and a vaccine adjuvant, wherein the antigens are a GD strain of the goose astrovirus and an FLX variant virus SC-3 strain of the goose astrovirus with a preservation number of CCTCC NO: V201976.
2. The bivalent inactivated vaccine according to claim 1, wherein the virus strain has a virus content of not less than 2 x 106.0TCID50/0.1ml。
3. The bivalent inactivated vaccine according to claim 1 or 2, wherein the virus strain is inactivated using a formaldehyde solution.
4. The bivalent inactivated vaccine according to claim 1, wherein the vaccine adjuvant is an oil phase adjuvant.
5. The bivalent inactivated vaccine according to claim 4, wherein the oil phase adjuvant comprises 94 parts of white animal oil, 1 part of aluminum stearate and 5 parts of span-80.
6. The bivalent inactivated vaccine according to claim 5, characterized in that the preparation method of the bivalent inactivated vaccine is as follows:
1) virus propagation and harvesting: performing virus propagation on GD strains on LMH cells, and performing virus propagation on SC-3 strains on chorioallantoic membranes of goose embryos to obtain virus liquid;
2) and (3) concentrating the virus: concentrating the harvested 2 virus solutions by 2 times by using an ultrafiltration concentrator at the temperature of 2-8 ℃, and preparing the concentrated virus solutions for inactivation;
3) virus inactivation: introducing the concentrated virus solution into an inactivation tank, metering and adding 10% of formaldehyde solution, and fully mixing, wherein the final concentration of the formaldehyde solution is 0.1%; inactivating the mixture in a shaker at 37 ℃ for 20 hours by shaking at 200rpm, taking out the mixture, and storing the mixture at 2-8 ℃;
4) preparing an oil phase: taking 95 parts of white oil for livestock and 1 part of aluminum stearate, placing the white oil and the aluminum stearate in an oil phase preparation tank, heating the mixture to 80 ℃, then adding span-805 parts of span-30 min until the temperature reaches 115 ℃, and cooling the mixture for later use;
5) preparing an aqueous phase, mixing 96 parts of antigen liquid of the inactivated goose astrovirus GD strain and the inactivated goose astrovirus variant virus SC-3 strain in the step 3) with 4 parts of tween-80 after sterilization respectively, and stirring in an aqueous phase preparation tank until the tween-80 is completely dissolved;
6) emulsification: placing 2 parts of oil phase in a high speed shearing machine, starting a motor to rotate slowly and stir, simultaneously slowly adding 1 part of water phase, and emulsifying at 10000r/min to prepare the vaccine.
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| CN111905100A (en) * | 2020-08-17 | 2020-11-10 | 山东信得科技股份有限公司 | Goose astrovirus bivalent inactivated vaccine and yolk antibody and preparation method thereof |
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