CN111551747A - Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection - Google Patents
Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection Download PDFInfo
- Publication number
- CN111551747A CN111551747A CN202010410668.9A CN202010410668A CN111551747A CN 111551747 A CN111551747 A CN 111551747A CN 202010410668 A CN202010410668 A CN 202010410668A CN 111551747 A CN111551747 A CN 111551747A
- Authority
- CN
- China
- Prior art keywords
- virus
- serum
- porcine
- rabbit
- rabbits
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Endocrinology (AREA)
- Gastroenterology & Hepatology (AREA)
- Diabetes (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Rheumatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a method for detecting the effectiveness of porcine epikavirus inactivated vaccine by using rabbits based on antibody detection, belonging to the field of biological products for livestock. The invention comprises the steps of immunizing a plurality of healthy susceptible rabbits by using the porcine epinaesia virus inactivated vaccine; after 28 days of immunization, the neutralizing antibody titer of the seneca virus serum in all rabbit sera is detected; when the neutralizing antibody titer of the seneca virus serum in the serum of at least 4/5 rabbit individuals is more than or equal to 1:64, the protective efficacy of the inactivated vaccine for the porcine seneca virus is judged to be qualified. The method can effectively use rabbits to replace experimental pigs to establish a substitute method for the efficacy test of the inactivated vaccine against the Seneca virus disease, effectively reduces the cost, and has the advantages of simple and easy operation, safety and convenience.
Description
Technical Field
The invention belongs to the field of biological products for veterinary use, and particularly relates to a method for testing the efficacy of an inactivated vaccine against porcine epicaavirus disease by using rabbits based on antibody detection.
Background
Seneca Virus A (SVA), a virus that can cause porcine vesicular disease, has been a serious disease condition at home and abroad in recent years. The virus is RNA virus with single-stranded non-graded positive strand, belongs to the genus Senecavirus of the family of small RNA viruses, and clinical evidence shows that the virus can cause the infected pig to have vesicular lesion and death of newborn piglets after the pig is infected, and the symptoms are very similar to foot-and-mouth disease. The virus was first discovered in 1998, and gradually noticed by people after a large-scale outbreak in brazil in 2014. Outbreak epidemic situation occurs for the first time in a certain pig farm in Guangdong in 2015 for 3 months in China; subsequently, 6 different farms in Guangdong province were identified 6 new SVA isolates from 2015, 5 months to 2016, 1 months, which were divided into two different branches by gene-phylogenetic tree analysis; in 2016, there was another outbreak of SVA infection in Hubei province in China. Up to now, the cases of SVA infection in Guangdong, Fujian, Heilongjiang, Hubei and Henan provinces in China are reported, which causes serious economic loss in pig industry.
The prevention and treatment of the virus are mainly carried out, however, no commercial vaccine for the virus of the Sendai virus exists in China at present, and the test vaccine is in the research, development and inspection stage. The efficacy test of the epikavirus test vaccine is mainly carried out by pig bodies, the price is increased due to the influence of African swine fever, the buying and selling and transportation of pigs are limited, the experimental pigs are difficult to purchase and are not safe and reliable any more, and the research and development work of the inactivated vaccine of the epikavirus disease is directly hindered. Therefore, establishing an alternative method for testing the efficacy of the inactivated vaccine against the seneca virus is crucial to the development of the inactivated vaccine against the seneca virus, and can greatly save the research and development cost.
Disclosure of Invention
Aiming at one or more problems in the prior art, the invention provides a method for testing the effectiveness of inactivated vaccine against porcine epicaavirus disease by using rabbits based on antibody detection, which comprises the following steps:
1) immunizing a plurality of healthy susceptible rabbits by using the porcine epinakavirus inactivated vaccine;
2) after 28 days of immunization, the neutralizing antibody titer of the seneca virus serum in all rabbit sera is detected;
3) when the neutralizing antibody titer of the seneca virus serum in the serum of at least 4/5 rabbit individuals is more than or equal to 1:64, the protective efficacy of the inactivated vaccine for the porcine seneca virus is judged to be qualified.
In the method, the neutralizing antibody titer of the seneca virus serum in the serum of the healthy susceptible rabbit in the step 1) is less than 1: 2.
In the method, the dosage of the immunization in the step 1) is at least 1ml per mouse, and the route of the immunization adopts leg intramuscular injection.
Among the above methods, the method for detecting the titer of the serum neutralizing antibody in step 2) includes, but is not limited to, the fixed virus dilution serum method in the neutralization assay.
In the method, in the step 3), when the neutralizing antibody titer of the seneca virus serum in the serum of all the rabbit individuals is more than or equal to 1:64, the protective efficacy of the inactivated vaccine against the porcine seneca virus is judged to be qualified.
The method for testing the efficacy of the inactivated vaccine against the porcine epinakavirus disease based on the antibody detection provided by the technical scheme can effectively use rabbits to replace experimental pigs to establish a substitute method for testing the efficacy of the inactivated vaccine against the porcine epinakavirus disease, and eliminate the influence caused by swine infectious diseases such as African swine fever and the like; meanwhile, in the method provided by the invention, the rabbits are used as experimental animals, compared with experimental pigs, the raising cost and the purchasing cost are relatively low, the cost can be effectively reduced, and the operation is simple, easy, safe and convenient. The data show that the rabbit is used for replacing the experimental pig in the provided method, and the vaccine efficacy detection results of the rabbit and the experimental pig are consistent, so that the method for detecting the serum neutralizing antibody after the rabbit is immunized can be used for evaluating the immune efficacy of the inactivated vaccine against the Seneca virus instead of a pig immunization challenge method, and meanwhile, the method does not relate to a challenge protection test, and is high in biological safety and short in detection period. In addition, the method provided by the invention uses rabbits to replace experimental pigs, and when virus challenge verification is carried out after inactivated vaccine immunization, white or red bulges with millet sizes appear at the junction of noses, lips and noselips of the rabbits infected with SVA and having the disease with little or no hairs, so that the confusion of the water vacuole-like symptoms and the disease symptoms of foot-and-mouth disease and the like after the pigs are infected with SVA can be avoided, and the infection of the SVA of the pigs and the diseases of the foot-and-mouth disease and the like of the pigs are distinguished.
Detailed Description
The invention aims to establish a method for testing the efficacy of the porcine epinakavirus inactivated vaccine by replacing a pig with a rabbit, and provides a new vector and a replacement method for testing the efficacy of a novel epinakavirus vaccine.
The present invention will be described in detail with reference to the following specific embodiments.
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, which are helpful for understanding the invention, but should not be taken as limiting the content of the invention.
Example 1 preparation of inactivated Swine Selenecar Virus vaccine
In the embodiment, SVV/CH/ZZ/2016CGMCC No.14886(CN110551694A) is selected as a strain to prepare the seneca virus solution, and the vaccine in the following embodiments is obtained after inactivation and emulsification, and specifically comprises the following steps:
inoculating the Selenecar virus strain to a cell density of 3.0 × 106PK15-JY suspension cells (porcine kidney suspension cells, provided by Jinyubao Ling biopharmaceutical Co., Ltd.) with cell viability rate of more than 95 percent are placed at 37 ℃ in 5 percent of CO2After 3 days of culture in an incubator, repeatedly freezing and thawing for 3 times to obtain a virus culture solution, adding BEI (diethylene imine, Sigma) to enable the final concentration to be 0.003mol/L, inactivating at 26 ℃ for 48 hours, and adding a sodium thiosulfate solution with the concentration of 50% according to the content of 4% (V/V) to block inactivation to obtain an inactivated antigen. Mixing the inactivated antigen with 206 adjuvant (SEPPIC) according to the mass ratio of 1:1, emulsifying for 30 minutes at 30 ℃, subpackaging and storing at 2-8 ℃ for later use. Batches of 4 vaccines, numbered 2019002, 2019005, 2019006 and 2019007, were prepared in this manner and used in the following examples to evaluate the efficacy of the vaccines.
Example 2 preparation of Seneca Virus solution
This example selects SVV/CH/ZZ/2016CGMCC No.14886(CN110551694A) as the strain to prepare the Selcard virus solution for neutralizing and testing in the following examples, which specifically comprises the following steps:
the seneca virus strain was inoculated into a full monolayer of PK-15 cells (porcine kidney cells, gold)Yubaoling biopharmaceutical Co., Ltd.), CO at 37 deg.C2Culturing in incubator for 5 days, repeatedly freezing and thawing for 3 times to obtain virus culture solution, recording as F1 generation, and storing at-20 deg.C. Collecting harvested virus culture fluid F1 generation, inoculating full monolayer PK-15 cells according to content of 10% (V/V), and placing in CO at 37 deg.C2The cells were incubated in an incubator and observed for cytopathic effect (CPE) daily, and the virus broth was harvested 48 hours after inoculation and recorded as F2 generation. Passage was continued as described above until the appearance of typical lesions (manifested by cell rounding, aggregation, voiding, or even shedding), which occurred in this example when cultured for F3. Continuing to passage F7, collecting virus liquid as the most typical lowest passage of the lesion, and determining the titer of virus to 107.5TCID500.1 ml. The process is completed by national engineering laboratory of Jinyubingling biopharmaceutical GmbH.
Example 3 Immunity test of inactivated vaccine against porcine epikavirus disease
This example carried out a potency test on the inactivated porcine Seneca virus vaccine (batch No. 2019002) obtained in example 1 above, using experimental animals: 1.0-2.0 kg of 25 healthy susceptible rabbits, a neutralizing antibody of Seneca virus serum is less than 1:2, and the neutralizing antibody is purchased from Dalatel's permanent agriculture and animal husbandry development company, Ordos, Ordovicus; the neutralizing virus used was the in-stopper virus solution obtained in example 2 above. The specific operation method comprises the following steps:
3.1 Swine Securidaca virus inactivated vaccine immunization of rabbits
1) Grouping: randomly dividing healthy susceptible rabbits into 5 groups of 5 rabbits, wherein 4 groups are immune groups, and 1 group is reserved as a control group;
2) immunization: the immune group is injected with 0.25ml, 0.5ml, 1.0ml and 2.0ml of porcine epinakay virus inactivated vaccine respectively, leg muscle injection is adopted in the injection way, the control group is injected with normal saline with the same dosage by the same method, and all rabbits are fed in the same way under the same environment after being immunized;
3) antibody detection: on days 7, 14, 21 and 28 after immunization, all rabbits were bled, sera were separated at 3500rpm for 20min, and the neutralizing antibody titer of the rabbit senecavir sera of each group (i.e., the neutralizing antibody titer of the sera, the neutralizing virus used was the senecavir virus solution prepared in example 2) was measured by the fixed virus dilution sera method in the neutralization assay; the results of the measurements are shown in Table 1 below.
Table 1: rabbit Sessilekavirus serum neutralizing antibody test results (log2)
As shown in the above table 1, it can be seen that specific serum neutralizing antibodies can be generated after rabbits are inoculated with inactivated vaccines for the Seneca virus disease, and the antibody titer (i.e., the value of the serum neutralizing antibodies) continuously increases with the passage of time; wherein the antibody titer in the rabbit serum of the 2.0ml vaccine immunization group is highest, the antibody titer in the rabbit serum of the 1.0ml vaccine immunization group is slightly higher than that in the rabbit serum of the 0.5ml vaccine immunization group, the antibody titer in the rabbit serum of the above three groups after immunization at 28 days is higher than 1:128, and the antibody titer in the rabbit serum of the 0.25ml immunization group is lower than that in other immunization groups; the neutralizing antibody titer of the rabbit serum of the control group is less than 1: 2.
3.2 serum neutralizing antibody level correlation test with challenge protective Rate
1) The operation method comprises the following steps: collecting blood of each group of rabbits in 3.1, and collecting with a poison value of 107.5TCID50/0.1ml of Seneca virus solution (prepared in example 2) attacks all rabbits with a toxin attacking dose of 2.0 ml/rabbit, and the toxin attacking mode adopts a nasal drip approach, and the rabbits are observed once every morning and afternoon after toxin attacking for 15 days continuously;
2) and (3) judging standard: after the rabbit attacks poison, the junction of the nose, the lip and the nose lip appears white or red bulge with millet grain size at the hairless or hairless position, and the rabbit is judged to have a disease if the symptom lasts for 2-3 days; the asymptomatic one is judged as protection. The results of the rabbit immune challenge test are shown in table 2 below.
Table 2: rabbit immune toxicity challenge test result
As can be seen from the above table 2, when the titer of the serum neutralizing antibody is more than or equal to 1:64, the immune rabbit does not attack the toxin and does not attack the disease; after challenge, the protection rates of 2.0ml, 1.0ml and 0.5ml vaccine immunization groups of rabbits are 100%, the protection rate of 0.25ml immunization groups is 40%, wherein the two sera are used for protection after challenge with the rabbit with the antibody titer of more than or equal to 1:64, and the rabbits after challenge with the control group are all attacked (the protection rate is 0%).
From the above results, it is understood that the minimum immune dose of the inactivated seneca virus vaccine prepared in example 1 to rabbits is 0.5 ml/rabbit, and 100% immune protection to rabbits can be achieved. When the vaccine efficacy test is carried out, the dosage used can be 0.5ml of the minimum immune dose per tube, but in practical operation, the dosage 2 times of the minimum immune dose is often selected, namely the immune dose is set to be 1.0ml per tube, so that the immune efficacy of the vaccine can be judged more objectively; when the titer of neutralizing antibodies of the Seneca virus in the serum of the rabbits is more than or equal to 1:64, the attacking rabbits can not attack the diseases, the effective protection effect can be achieved, and the correlation between the antibody detection result and the attacking protection rate is good.
Example 4 comparison of the vaccine efficacy test method Using rabbits and Experimental pigs
This example carried out a potency test on the inactivated porcine Seneca virus vaccine obtained in example 1 above (lots 2019005, 2019006, 2019007) using experimental animals: healthy and susceptible pigs over 60 days old, the neutralizing antibody of Seneca virus serum is less than 1:2, and the pigs are purchased from a positive pig farm in the great call of Harvest; 1.0-2.0 kg of 20 healthy susceptible rabbits, wherein the neutralizing antibody of the Seneca virus serum is less than 1:2, and the virus is purchased from Dalatel's permanent agriculture and animal husbandry development company, Ordos, Ordovicus; the test virus and neutralizing virus used were the seneca virus solutions obtained in example 2 above. The specific operation method comprises the following steps:
4.1 vaccine efficacy test method Using rabbits
Selecting 20 healthy susceptible rabbits of 1.0-2.0 kg, randomly dividing the rabbits into 4 groups of 5 rabbits, wherein 3 groups of the rabbits are used for immunizing porcine epikavirus inactivated vaccines (corresponding to batch numbers 2019005, 2019006 and 2019007 respectively); taking 1 group as a negative control, performing immunization according to the method in the following table 3, collecting blood of all rabbits at 28 days after immunization, separating serum at 3500rpm for 20min, and detecting the neutralizing antibody titer of the rabbit Sessincard virus serum of each group by a fixed virus dilution serum method in a neutralization test method; the rabbit senecavir serum neutralizing antibody detection results are shown in table 4 below.
Table 3: method of vaccination
Table 4: rabbit Sessilekavirus serum neutralizing antibody test results (log2)
As can be seen from the data in Table 4 above, after 3 batches of inactivated vaccines for Secoid virus disease are used for immunizing various groups of immunized rabbits respectively, the rabbits can generate neutralizing antibodies specific to Secoid virus, the antibody titer is higher than 1:64 in 28 days after immunization, and the serum neutralizing antibody titer of the rabbits in the non-immunized groups is 0.
4.2 vaccine efficacy test method using experimental pigs
Selecting 20 healthy susceptible pigs over 60 days old, randomly dividing into 4 groups of 5 pigs each, wherein 3 groups of the healthy susceptible pigs are used for immunizing the inactivated vaccines of porcine epikavacirus (corresponding to the batch numbers of 2019005, 2019006 and 2019007 respectively); the other 1 group was a non-immunized group as a negative control, and the immunization was performed according to the method shown in table 5 below, and the immunization was performed 1 time at the same dose and the same inoculation method on the 21 st day after the immunization according to the method shown in table 5; the use poison price is 10 on the 14 th day after the second immunization7.5TCID50/0.1ml of the Seneca virus liquid attacks the immune pig, the dose of attacking the virus is 5.0 ml/head, the attacking method adopts a nose-dropping way, each nostril is 2.5ml, the virus is observed once every morning and afternoon every day after attacking, and even if the virus is connected with the nose-dropping way, the medicine is used for treating the pigThe observation was continued for 15 days. The results of the swine immune challenge test are shown in table 6 below.
Table 5: method of vaccination
Table 6: pig immunity challenge test result
As can be seen from the data in the table 6, after 3 batches of the porcine epinakalin inactivated vaccines are used for immunizing experimental pigs of each immunization group, the virus attack protection rate is 100 percent; the experimental pigs in the non-immunized group (4-1 group) all suffered from the disease after the challenge (the protection rate is 0%).
As can be seen from the above tables 4 and 6, when the immune potency of the inactivated vaccine against Seneca virus was examined by domestic rabbits, the detection of neutralizing antibodies in serum was carried out 28 days after immunization, and the antibody titer in 100% of the individual sera of the immunized rabbits inoculated with 3 batches of inactivated vaccine against Seneca virus from pigs was higher than 1: 64; the results of the immune challenge test on experimental pigs show that the protection rate of 3 batches of the porcine epinakalin inactivated vaccines on pigs is 100 percent. Therefore, the detection results of the two methods are consistent, the method for detecting the serum neutralizing antibody after the rabbit immunization can be used for evaluating the immune efficacy of the inactivated vaccine against the Seneca virus instead of the pig immunization challenge method, the biological safety is high, and the detection period is short. In addition, the method provided by the invention uses rabbits to replace experimental pigs, and when virus challenge verification is carried out after inactivated vaccine immunization, in clinical symptoms, white or red bulges with millet sizes appear at the junction of noses, lips and noselips of the rabbits infected with SVA and having diseases with few hairs or no hairs, so that the confusion of vesicular symptoms and diseases such as foot-and-mouth disease after the pigs are infected with SVA can be avoided, the diseases such as the pig SVA infection and the pig foot-and-mouth disease are distinguished, and the interference of the foot-and-mouth disease virus in the vaccine efficacy verification process is avoided.
Example 5 pathogenicity of foot-and-mouth disease Virus in rabbits
In this example, foot and mouth disease virus liquids A and O (both having a titer of 10) were used7.5TCID500.1ml, provided by national engineering laboratory of Jinyubaoling biological medicine Co., Ltd.) to carry out virus attack test on the rabbits, and research the pathogenicity of the foot-and-mouth disease virus to the rabbits, specifically comprising the following steps:
5.1, selecting test animals: selecting 12 healthy susceptible rabbits of 1.0-2.0 kg, detecting that the neutralizing antibody of the foot-and-mouth disease virus serum is less than 1:2, and purchasing the neutralizing antibody from Dalatex permanent agriculture and animal husbandry development limited company in Ordos city;
5.2, preparing the toxin for counteracting toxic pathogen: has a poison price of 107.5TCID500.1ml of A-type and O-type foot-and-mouth disease virus liquid for counteracting virus of rabbits;
5.3, counteracting toxic substances: randomly dividing 12 healthy susceptible rabbits into 3 groups, wherein each group comprises 4 rabbits, respectively using A-type and O-type foot-and-mouth disease virus liquids to attack viruses by adopting 2.0 ml/nose dropping approach, using the rest 1 group as blank control, continuously observing for 15 days after attacking viruses, and judging the patient to have disease if the patient has symptoms similar to rabbit infected endo-Baker virus; the challenge results are shown in table 7 below.
Table 7: test results of toxicity counteracting dose
As can be seen from the data in Table 7 above, the use poison value is 107.5TCID500.1ml of A-type and O-type foot-and-mouth disease virus liquid attacks the rabbits, and within an observation period after virus attack, 2 groups of rabbits do not have clinical symptoms similar to those of the rabbits attacked by the Seneca virus, namely, the clinical symptoms of millet-sized white or red bulges at the junction of the nose, the lip and the nose lip of the rabbit without showing little hair or no hair are not shown, so that the rabbit is used as the Seneca virus vaccine efficacy test animal to avoid the interference of the foot-and-mouth disease virus which can cause the pig vesicular lesion.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for testing the efficacy of an inactivated vaccine against porcine epikavirus disease by using a rabbit based on antibody detection comprises the following steps:
1) immunizing a plurality of healthy susceptible rabbits by using the porcine epinakavirus inactivated vaccine;
2) after 28 days of immunization, the neutralizing antibody titer of the seneca virus serum in all rabbit sera is detected;
3) when the neutralizing antibody titer of the seneca virus serum in the serum of at least 4/5 rabbit individuals is more than or equal to 1:64, the protective efficacy of the inactivated vaccine for the porcine seneca virus is judged to be qualified.
2. The method of claim 1, wherein the serum neutralizing antibody titer of seneca virus in the serum of the healthy susceptible rabbit in step 1) is < 1: 2.
3. The method according to claim 1 or 2, wherein the dose of the immunization in step 1) is at least 1ml per mouse, and the route of the immunization is performed by leg intramuscular injection.
4. The method of any one of claims 1 to 3, wherein the method of detecting the titer of serum neutralizing antibodies in step 2) includes, but is not limited to, the fixed virus dilution serum method in the neutralization assay.
5. The method according to any one of claims 1 to 4, wherein the porcine epikavirus inactivated vaccine is judged to be qualified in protective efficacy when the epikavirus seroneutralizing antibody titer in the serum of all rabbit individuals is greater than or equal to 1:64 in step 3).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010410668.9A CN111551747A (en) | 2020-05-15 | 2020-05-15 | Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010410668.9A CN111551747A (en) | 2020-05-15 | 2020-05-15 | Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111551747A true CN111551747A (en) | 2020-08-18 |
Family
ID=71998709
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010410668.9A Pending CN111551747A (en) | 2020-05-15 | 2020-05-15 | Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111551747A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112915215A (en) * | 2021-04-14 | 2021-06-08 | 齐鲁动物保健品有限公司 | Method for testing efficacy of inactivated vaccine against A-type Sendai virus disease by using mice |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104215781A (en) * | 2014-09-04 | 2014-12-17 | 天津瑞普生物技术股份有限公司 | Porcine parvovirus (PPV) inactivated vaccine immune efficacy evaluation method |
| CN105606804A (en) * | 2016-01-20 | 2016-05-25 | 福州大北农生物技术有限公司 | Porcine circovirus inactivated vaccine efficacy testing method |
| CN106645716A (en) * | 2010-01-28 | 2017-05-10 | 普莱柯生物工程股份有限公司 | Method for testing efficacy of porcine circovirus 2-type vaccine |
| CN106771186A (en) * | 2016-12-05 | 2017-05-31 | 青岛易邦生物工程有限公司 | A kind of antibody test method of hog cholera genetic engineering subunit vaccine effect |
| CN106771187A (en) * | 2016-12-05 | 2017-05-31 | 青岛易邦生物工程有限公司 | A kind of hog cholera genetic engineering subunit vaccine efficacy test method |
| CN107589256A (en) * | 2017-09-30 | 2018-01-16 | 天津瑞普生物技术股份有限公司 | The method of inspection of the type of Haemophilus parasuis 4,5 type bivalent inactivated vaccine effect |
| CN107894508A (en) * | 2017-08-31 | 2018-04-10 | 中国农业科学院兰州兽医研究所 | A kind of solid phase competitive ELISA kit and its application for the detection of Senecan antiviral antibody |
| WO2019014144A1 (en) * | 2017-07-12 | 2019-01-17 | Boehringer Ingelheim Vetmedica, Inc. | Senecavirus a immunogenic compositions and methods thereof |
| CN109490529A (en) * | 2018-11-20 | 2019-03-19 | 金宇保灵生物药品有限公司 | The blocking ELISA antibody assay kit of Senecan virus |
-
2020
- 2020-05-15 CN CN202010410668.9A patent/CN111551747A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645716A (en) * | 2010-01-28 | 2017-05-10 | 普莱柯生物工程股份有限公司 | Method for testing efficacy of porcine circovirus 2-type vaccine |
| CN104215781A (en) * | 2014-09-04 | 2014-12-17 | 天津瑞普生物技术股份有限公司 | Porcine parvovirus (PPV) inactivated vaccine immune efficacy evaluation method |
| CN105606804A (en) * | 2016-01-20 | 2016-05-25 | 福州大北农生物技术有限公司 | Porcine circovirus inactivated vaccine efficacy testing method |
| CN106771186A (en) * | 2016-12-05 | 2017-05-31 | 青岛易邦生物工程有限公司 | A kind of antibody test method of hog cholera genetic engineering subunit vaccine effect |
| CN106771187A (en) * | 2016-12-05 | 2017-05-31 | 青岛易邦生物工程有限公司 | A kind of hog cholera genetic engineering subunit vaccine efficacy test method |
| WO2019014144A1 (en) * | 2017-07-12 | 2019-01-17 | Boehringer Ingelheim Vetmedica, Inc. | Senecavirus a immunogenic compositions and methods thereof |
| CN107894508A (en) * | 2017-08-31 | 2018-04-10 | 中国农业科学院兰州兽医研究所 | A kind of solid phase competitive ELISA kit and its application for the detection of Senecan antiviral antibody |
| CN107589256A (en) * | 2017-09-30 | 2018-01-16 | 天津瑞普生物技术股份有限公司 | The method of inspection of the type of Haemophilus parasuis 4,5 type bivalent inactivated vaccine effect |
| CN109490529A (en) * | 2018-11-20 | 2019-03-19 | 金宇保灵生物药品有限公司 | The blocking ELISA antibody assay kit of Senecan virus |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112915215A (en) * | 2021-04-14 | 2021-06-08 | 齐鲁动物保健品有限公司 | Method for testing efficacy of inactivated vaccine against A-type Sendai virus disease by using mice |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988170B (en) | Porcine rotavirus strain, inactivated vaccine prepared from same and application of inactivated vaccine | |
| CN106282128B (en) | One plant causes weak porcine pseudorabies virus gene delection attenuated vaccine strain and its application by the passage of cell low temperature and drug screening | |
| CN102220287B (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN107177001B (en) | Egg yolk antibody for preventing and treating porcine epidemic diarrhea and preparation method thereof | |
| CN104017776B (en) | A kind of sheep infective pustule virus cell weak-toxic vaccine and its preparation method and application | |
| CN108392628A (en) | A kind of porcine mycoplasmal pneumonia inactivated vaccine and preparation method thereof | |
| CN103497934B (en) | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof | |
| CN102068695A (en) | Method for producing quadruple inactivated vaccine for newcastle disease, infectious bronchitis, avian influenza (H9 subtype) and infectious bursal disease | |
| CN103468647B (en) | Swine flu H1N1 and H3N2 subtype bivalent inactivated vaccine | |
| CN111808826B (en) | Porcine type-A seneca virus SVA/CH-Fuj strain and application thereof | |
| CN106729692A (en) | Bovine viral diarrhoea, infectious bovine rhinotrachetis, bovine parainfluenza triple inactivated vaccine and preparation method thereof | |
| CN111729091B (en) | Method for checking efficacy of porcine Seika virus inactivated vaccine by using rabbit | |
| CN110302374B (en) | Canine vaccine and preparation method and application thereof | |
| CN104888213A (en) | Preparation method of classical swine fever spleen-lymph-sourced compound living vaccine | |
| CN111551747A (en) | Method for testing efficacy of porcine epikavirus inactivated vaccine by using rabbit based on antibody detection | |
| CN104130981A (en) | Application of avian infectious bronchitis virus vaccine strain in preparation of inactivated vaccine | |
| CN109207436A (en) | One plant of 4 type aviadenovirus strain of I group and its application | |
| CN103937753A (en) | H9N2 subtype avian influenza virus strain as well as inactivated vaccine and application thereof | |
| CN104388396B (en) | Porcine pseudorabies poison strain and inactivated vaccine prepared therefrom and application | |
| CN102805862A (en) | Preparation method for SFTS bunyavirus purification and inactivation vaccines through VERO cell culture | |
| CN102139104A (en) | Production method for triple inactivated vaccine for newcastle disease, avian influenza (H9 subtype) and infectious bursal disease | |
| CN101380470B (en) | Pig parvovirus live vaccine | |
| CN107338227A (en) | Bovine parainfluenza virus PBIV3 B strains and its application | |
| CN112717128A (en) | Combined vaccine for preventing hand-foot-and-mouth disease and preparation method and application thereof | |
| CN102747045B (en) | Swine influenza virus H1N1 SIVTJ inactivated vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |