CN102749427A - Method for testing efficacy of duck hemorrhagic ovaritis inactivated vaccine - Google Patents
Method for testing efficacy of duck hemorrhagic ovaritis inactivated vaccine Download PDFInfo
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Abstract
The invention provides a test method for efficacy of duck hemorrhagic ovaritis inactivated vaccine. The method comprises steps of: immunizing ducks using the duck hemorrhagic ovaritis inactivated vaccine for two times, wherein the second immunization is carried out two weeks after the first immunization; challenging the immunized ducks with duck hemorrhagic ovaritis virus three to five weeks after the second immunization; collecting duck serum after the challenge; inoculating the isolated serum into SPF chicken eggs or duck eggs; and calculating the number of eggs which die within 24-168 hours and are tested positive for the duck hemorrhagic ovaritis virus, wherein if the positive incidence of the isolated virus is more than 80% in contrast group ducks while the negative incidence of the isolated virus is more than 70% in the immunized group ducks, the vaccine efficacy is determined to pass the test. The test method for efficacy of duck hemorrhagic ovaritis inactivated vaccine of the invention has the advantages of simple in operation and accurate in test results, and thereby can be wildly used.
Description
Technical field
The present invention relates to a kind of method for testing efficacy of inactivated vaccine for animals, relate in particular to the method for inspection that a kind of duck hemorrhagic oaritis inactivated vaccine is renderd a service, belong to veterinary biologics inspection technology field.
Background technology
Duck hemorrhagic oaritis (Duck Hemorrhagic Ovaritis; DH) be a kind of novel duck infectious disease popular recently, acute, the height contact, its cause of disease be duck hemorrhagic oaritis virus (Duck Hemorrhagic Ovaritis Virus, DHOV); The member who on virus taxis, belongs to flaviviridae; Infect the different days duck, cause that the ovarian hemorrhage of sexal maturity duck, orchiatrophy, egg production, rate of fertilization and incubation rate descend, the duck death rate increases.
In the research of virus taxis status; Through the F4 of isolated viral strain is carried out sequence homology analysis and evolutionary analysis for the go down to posterity genome sequence of virus and known virus of duck embryo, find that the genetic distance of tembusu virus of this virus and flaviviridae Flavivirus is nearest.Aspect diagnosis, people's report can adopt clinical symptoms such as Cao Zhenzhen, Wan Chun and, Su Jingliang, Teng Qiao are vast, reach histopathology variation, pcr amplification and virus substantially and method such as separate and make a definite diagnosis this disease.About these sick prevention and control, the strain that Beijing City Agriculture and Forestry Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd., Qilu Animal Health Products Co., Ltd., Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute and academy of agricultural sciences, Chongqing City animal and veterinary research institute have been separated during having reported and having utilized this disease to break out prepares the method for vaccine.Lin Jian, Liu Yuehuan etc. have reported the artificial challenge's model that utilizes laying ducks successfully to set up duck hemorrhagic oaritis, for experiment basis has been established in the evaluation of vaccine.That Shang Weijian has is objective, feasible, the play-by-play of the vaccine potency evaluation method of strong operability and science.
Summary of the invention
One of the object of the invention provides age in days scope and the sex of a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation duck.
Two of the object of the invention provide duck hemorrhagic oaritis inactivated vaccine effect evaluation with the tissue samples of vaccine immunity number of times, employing time, collection, attack malicious approach and embryo is used in check.
Three of the object of the invention provides a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation method.
The used virus stain called after of the present invention duck hemorrhagic oaritis virus HB strain, preserving number is CCTCC V201122; Said duck hemorrhagic oaritis virus is ssRNA virus, and cyst membrane is arranged, can aggegation dove red blood cell; It is spherical that virion is substantially, diameter 40～60nm; The said virus resistibility of environment to external world is stronger, in 4 ℃, deposits several weeks, in-20 ℃, deposits some months, and its appeal is all unaffected; Said virus is responsive to ether, chloroform; Most of detergents can be with its rapid deactivation; Under 37 ℃ of conditions, stifling 6h just can be its deactivation with 0.1% formalin.This virus can be at 9～13 age in days duck embryo allantoic cavities, embryo CAM, 9～10 age in days chick embryo allantoic cavities, embryo CAM, and 6 age in days chick embryo yolk sacs are cultivated in 7～8 age in days duck embryo yolk bags, also can on DEF and BHK21 cell, cultivate.
The preparation method of the used duck hemorrhagic of the present invention oaritis inactivated vaccine has declared patent of invention in 2011, application number is 201110255115.1.
The present invention provides a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy; This method is used duck hemorrhagic oaritis inactivated vaccine immune duck; Immunity is 2 times altogether, carries out 22 weeks after head exempts from and exempts from, and two exempt from 3～5 weeks of back attacks poison with duck hemorrhagic oaritis virus to immune duck; Attack the poison back and gather duck serum; With the serum inoculation SPF chicken embryo or the duck embryo that separate; Calculate the embryo number of interior death of 24-168h and duck hemorrhagic oaritis virus-positive, it is more than 80% that control group duck virus is separated positive rate and the virus of immune group duck separation negative rate is to be judged to vaccine potency more than 70% to be up to the standards.
Further, the inventive method confirms that according to the neurological susceptibility experimental result it is 120～560 ages in days that duck hemorrhagic oaritis inactivated vaccine efficacy test uses the age in days of duck.
Preferably, the sex of efficacy test use duck is female.
Wherein, the dosage of 2 immune duck hemorrhagic oaritis inactivated vaccines be 0.5ml/ only～2ml/ only.
Wherein, attack poison and uses duck hemorrhagic oaritis virus to be DHOV-HB strain (deposit number is CCTCC V201122, has been to disclose in 201110255115.1 the Chinese patent at application number), attacking the toxic agent amount is 3 * 10
4ELD
50
Wherein, inoculated into chick embryo or duck embryo are through yolk sac inoculation 6～8 age in days SPF chicken embryo or duck embryos.
Wherein, the inoculum concentration of chicken embryo or duck embryo is the 0.2ml/ embryo.
Wherein, attack poison back 4-5 day, gather duck serum.
Wherein, described duck is that meat duck, egg are with duck or SPF duck.
Further; The invention provides a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy; This method is used duck hemorrhagic oaritis inactivated vaccine immunity 120～560 age in days ducks, carries out 22 weeks after head exempts from and exempts from, and twice immunizing dose only is 0.5～2ml/; Two exempt from 3～5 weeks of back attacks poison with the DHOV-HB strain, attacks malicious approach and be oral, intramuscular injection or intravenous injection, and attacking the toxic agent amount is 3 * 10
4ELD
50Attack poison back 4-5 day, gather duck serum, through yolk bag approach inoculation 6～8 age in days SPF chicken embryo or duck embryos; 0.2ml/ embryo; 37 ℃ of hatchings, interior embryo death of calculating 24～168h and detection are judged to the viral separation positive for the embryo number of the duck hemorrhagic oaritis virus infections positive, and control group duck virus separation positive rate is more than 80% and the virus of immune group duck separation negative rate is to be judged to vaccine potency more than 70% to be up to the standards.
Blood sampling time, attack malicious approach and gather sample: 2 weeks carried out secondary immunity after the duck hemorrhagic oaritis inactivated vaccine first immunisation; 3～5 all oral administration, muscle or intravenous route are attacked poison behind the secondary immunity; Attack poison back 4-5 day; Take a blood sample, separation of serum, being used for virus separation control group duck virus separation positive rate is more than 80% and the virus of immune group duck separation negative rate is to be judged to vaccine potency more than 70% to be up to the standards.
The present invention has proposed to be used for age in days and the sex that duck hemorrhagic oaritis inactivated vaccine efficacy test uses duck first; The immune time of vaccine potency when check vaccine, attack malicious approach, the time of drawing materials, the tissue samples of collection, check use embryo, proposed the qualified standard of vaccine potency test method, criterion and vaccine quality of immunoprotection-virus separation.Solved the blank of duck hemorrhagic oaritis inactivated vaccine effect evaluation method and criterion.The method that the present invention confirms is objective, comprehensive, strong operability, can be used for the laboratory and the clinical efficacy evaluation of duck hemorrhagic oaritis inactivated vaccine.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, it is commercially available that used biology, chemical reagent is routine among the embodiment, the conventional means that used technological means is well known to those skilled in the art among the embodiment.Beijing duck used among the embodiment is available from duck hemorrhagic oaritis negative antibody duck field (Nan Kou of Beijing Golden Star Duck Center kind duck field); Sheldrake is available from duck hemorrhagic oaritis negative antibody duck field (dress circle village under four seas town, Yanqing County, Beijing Pearl Spring); The SPF duck is raised to testing required age in days in the SPF chicken isolator by the SPF duck's egg hatching available from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
Embodiment 1 duck hemorrhagic oaritis inactivated vaccine efficacy test is with the age in days of duck
The Beijing duck of 42,50,100,120,200,300,400,560 and 600 ages in days is divided into 9 groups, 10 every group, every group of Beijing duck (available from the Nan Kou of Beijing Golden Star Duck Center kind duck field, duck hemorrhagic oaritis antibody is negative) that is same age in days.Test duck oral route is attacked the DHOV-HB strain virus of malicious same dose, contains 3 * 10 approximately
4ELD
50/ only, attack malicious back 5 days through wing vein collection duck blood, aseptic separation of serum.Serum is inoculated 6 age in days SPF chicken embryos through the yolk bag approach, and 5 pieces/duck, the 0.2ml/ embryo is put 37 ℃ of continuation and hatched to 168h.24h discards with interior dead chicken embryo, and the RT-PCR that calculates interior death of 24～168h and DHOV detects positive chicken embryo number.Having the RT-PCR of 1 piece and above chicken embryo death and DHOV to detect positive person in 5 pieces of chicken embryos of every part of serum is designated as this duck virus and separates positive.According to the neurological susceptibility experimental result, confirm that it is 120～560 ages in days that duck hemorrhagic oaritis inactivated vaccine efficacy test uses the age in days of duck.
The test stone of susceptible duck or duck embryo
(1) 5 of susceptible ducks are from without the immunity of duck hemorrhagic oaritis inactivated vaccine or the popular history duck of duck hemorrhagic oaritis crowd's duck is arranged, and strain is used in the check of 100 times of dilutions of every oral 1.0 milliliters of aseptic 0.5% lactoalbumin hydrolysate Hank ' s liquid of duck.Attacked poison back 5 days, blood sampling, separation of serum carries out virus to be separated.The virus separation rate should at least 4/5 positive.
(2) 10 pieces of susceptible embryos are from without immunity of duck hemorrhagic oaritis inactivated vaccine or the kind egg that has duck hemorrhagic oaritis pestilence history kind duck crowd to be produced, 37 ℃ of hatching to 10 ages in days.Inoculate the kind poison of any generation of 500 times of dilutions of 0.5% aseptic lactoalbumin hydrolysate Hank ' s liquid (LH) through allantoic cavity, every embryo 0.1ml puts and continues hatching under 36～37 ℃ of conditions.The duck embryo should be in inoculation back death in 24～96 hours at least 9/10.It is systemic bleeding that dead duck embryo is observed pathology.
Embodiment 2 duck hemorrhagic oaritis inactivated vaccine effect evaluations are with the sex of duck
The Beijing duck oral route of 80 120 age in days male and female half and half is attacked the DHOV-HB strain virus of malicious same dose, contain 3 * 10 approximately
4ELD
50/ only.Attack malicious back 1～8 day through wing vein collection duck blood, 10/day, aseptic separation of serum.Serum is inoculated 6 age in days SPF chicken embryos through the yolk bag approach, 5 pieces of chicken embryos of serum inoculation of every duck, and the 0.2ml/ embryo is put 37 ℃ of continuation and is hatched to 168h.24h discards with interior dead chicken embryo, calculates death and the positive chicken embryo of DHOV number in 24～168h.Having 1 piece and above chicken embryo death and DHOV positive person to be designated as this duck virus in 5 pieces of chicken embryos of every part of serum separates positive.Female Beijing duck virus is separated positive rate apparently higher than male Beijing duck.The positive rate of the virus infections chicken embryo that the poison of attacking against each other afterwards occurs behind the male and female duck serum inoculated into chick embryo every day carries out the paired t check of two samples, and the P value is less than 0.05, and there were significant differences aspect neurological susceptibility for sex.
Immunoprotection-the isolation of virus (1) of embodiment 3 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 140 age in days Beijing ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012001, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 1ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 4 weeks, and attacking poison strain is the DHOV-HB strain, and oral route is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV and not the Beijing duck of immune duck hemorrhagic oaritis inactivated vaccine susceptible (10, female) attack poison for control group.Attacked the poison back the 5th day, and gathered duck serum, every duck serum is through 5 piece of 6 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 90%, and the incidence of disease of immune group duck is not 80%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (2) of embodiment 4 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 120 age in days Beijing ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012002, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 0.5ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 3 weeks, and attacking poison strain is the DHOV-HB strain, and intramuscular injection path is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV attack poison with immune duck hemorrhagic oaritis inactivated vaccine susceptible Beijing duck (10, female) not for control group.Attacked the poison back the 5th day, and gathered duck serum, every duck serum is through 5 piece of 6 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 70%, and the incidence of disease of immune group duck is not 90%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (3) of embodiment 5 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 560 age in days Beijing ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012003, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 2ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 5 weeks, and attacking poison strain is the DHOV-HB strain, and oral route is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV and not the Beijing duck of immune duck hemorrhagic oaritis inactivated vaccine susceptible (10, female) attack poison for control group.Attacked the poison back the 5th day, and gathered duck serum, every duck serum is through 5 piece of 7 age in days SPF duck embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead duck embryo discards in 24 hours, in 24～168 hours in the duck embryo the dead and RT-PCR positive of 1 piece and above duck embryo be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 80%, and the incidence of disease of immune group duck is not 100%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (4) of embodiment 6 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 300 age in days Beijing ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012004, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 1ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 4 weeks, and attacking poison strain is the DHOV-HB strain, and the intravenous injection approach is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV and not the Beijing duck of immune duck hemorrhagic oaritis inactivated vaccine susceptible (10, female) attack poison for control group.Attacked the poison back the 5th day, and gathered duck serum, every duck serum is through 5 piece of 8 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 70%, and the incidence of disease of immune group duck is not 90%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (5) of embodiment 7 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 400 age in days Beijing ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012005, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 1.5ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 3 weeks, and attacking poison strain is the DHOV-HB strain, and oral route is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV and not the Beijing duck of immune duck hemorrhagic oaritis inactivated vaccine susceptible (10, female) attack poison for control group.Attacked poison back 5 days, and gathered duck serum, every duck serum is through 5 piece of 8 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 90%, and the incidence of disease of immune group duck is not 90%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (6) of embodiment 8 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 200 age in days Beijing ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012006, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 0.5ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 5 weeks, and attacking poison strain is the DHOV-HB strain, and intramuscular injection path is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV and not the Beijing duck of immune duck hemorrhagic oaritis inactivated vaccine susceptible (10, female) attack poison for control group.Attacked poison back 5 days, and gathered duck serum, every duck serum is through 5 piece of 7 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 80%, and the incidence of disease of immune group duck is not 90%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (7) of embodiment 9 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 160 age in days sheldrakes of duck hemorrhagic oaritis inactivated vaccine (lot number 2012001, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 0.5ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 5 weeks, and attacking poison strain is the DHOV-HB strain, and oral route is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish simultaneously do not infect DHOV and not the sheldrake of immune duck hemorrhagic oaritis inactivated vaccine susceptible (10, female) attack poison for control group.Attacked poison back 4 days, and gathered duck serum, every duck serum is through 5 piece of 6 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 70%, and the incidence of disease of immune group duck is not 80%, explains that this batch vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (8) of embodiment 10 duck hemorrhagic oaritis inactivated vaccine effect evaluations
Use 10 of the immune 120 age in days SPF ducks of duck hemorrhagic oaritis inactivated vaccine (lot number 2012001, develop with Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. the Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute), be female, as the vaccine immunity group.The immunizing dose of vaccine immunity group and method are: first immune seedling dosage is 0.5ml/, the chest muscle injection.Head exempts from 2 weeks of back, exempts from identical approach two according to same dose, and two exempt from the back attacked poison in 5 weeks, and attacking poison strain is the DHOV-HB strain, and oral route is attacked poison, and every duck is attacked the toxic agent amount and is about 3 * 10
4ELD
50Establish the SPF duck (10, female) that does not infect DHOV simultaneously and attack poison for control group.Attacked poison back 4 days, and gathered duck serum, every duck serum is through 5 piece of 6 age in days SPF chicken embryo of yolk bag approach inoculation, 0.2ml/ embryo, 37 ℃ of hatchings, the continuous observation to 168 hours.Dead chicken embryo discards in 24 hours, in 24～168 hours in the chicken embryo 1 piece and above chicken embryo death and the RT-PCR positive be calculated as this duck hemorrhagic oaritis virus and separate positive.The malicious protection ratio of attacking of immune group duck is 80%, and the incidence of disease of immune group duck is not 90%, explains that this batch vaccine potency is up to the standards.
Embodiment of the invention 3-10 all uses the method for RT-PCR to detect virus, and the primer of this method is according to the NS5 gene design Flavivirus universal primer (upper reaches: 5 '-TCA AGG AAC TCC ACA CAT GAG ATG TAC T-3 ' of bibliographical information; Downstream: 5 '-GTG TCC CAT CCT GCT GTG TCA TCA GCA TAC A-3 '), theoretical expanding fragment length 988bp.
The RNA that carries out chick embryo allantoic liquid or idiosome according to Trizol kit instructions extracts; Establish the positive and negative quality-control sample simultaneously, carry out RT-PCR according to the kit instructions, the PCR product is got 10 μ l and is carried out 1% agarose gel electrophoresis; Amplified fragments is about 1Kb; And positive control is about 1Kb, and no band occurs about negative control 1Kb, can judge that then testing sample is a duck hemorrhagic oaritis virus-positive.
Claims (10)
1. duck hemorrhagic oaritis inactivated vaccine method for testing efficacy; It is characterized in that use duck hemorrhagic oaritis inactivated vaccine immune duck, immunity is 2 times altogether; Carry out 22 weeks after head exempts from and exempt from, two exempt from 3～5 weeks of back attacks poison with duck hemorrhagic oaritis virus to immune duck; Attack the poison back and gather duck serum; With the serum inoculation SPF chicken embryo or the duck embryo that separate; Calculate the embryo number of interior death of 24-168h and duck hemorrhagic oaritis virus-positive, it is more than 80% that control group duck virus is separated positive rate and the virus of immune group duck separation negative rate is to be judged to vaccine potency more than 70% to be up to the standards.
2. the method for claim 1 is characterized in that, it is 120～560 ages in days that efficacy test uses the age in days of duck.
3. the method for claim 1 is characterized in that, it is female that efficacy test uses the sex of duck.
4. the method for claim 1 is characterized in that, the dosage of said 2 immunity be 0.5ml/ only～2ml/ only.
5. the method for claim 1 is characterized in that, the described poison of attacking uses duck hemorrhagic oaritis virus to be the DHOV-HB strain, and attacking the toxic agent amount is 3 * 10
4ELD
50
6. the method for claim 1 is characterized in that, said inoculated into chick embryo or duck embryo are through yolk sac inoculation 6～8 age in days SPF chicken embryo or duck embryos.
7. the method for claim 1 is characterized in that, the inoculum concentration of said chicken embryo or duck embryo is the 0.2ml/ embryo.
8. the method for claim 1 is characterized in that, attacks poison back 4-5 day, gathers duck serum.
9. the method for claim 1 is characterized in that, described duck is that meat duck, egg are with duck or SPF duck.
10. a duck hemorrhagic oaritis inactivated vaccine method for testing efficacy is characterized in that, uses duck hemorrhagic oaritis inactivated vaccine immunity 120～560 age in days ducks, carries out 22 weeks after head exempts from and exempts from, and twice immunizing dose only is 0.5～2ml/; Two exempt from 3～5 weeks of back attacks poison with the DHOV-HB strain, attacks malicious approach and be oral, intramuscular injection or intravenous injection, and attacking the toxic agent amount is 3 * 10
4ELD
50Attack malicious 4-5 after day, gather duck serum, through yolk bag approach inoculation 6～8 age in days SPF chicken embryo or duck embryos; 0.2ml/ embryo; 37 ℃ of hatchings, interior embryo death of calculating 24～168h and detection are judged to the viral separation positive for the embryo number of the duck hemorrhagic oaritis virus infections positive, and control group duck virus separation positive rate is more than 80% and the virus of immune group duck separation negative rate is to be judged to vaccine potency more than 70% to be up to the standards.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104849463A (en) * | 2014-11-18 | 2015-08-19 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis bivalent vaccine potency test method |
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| CN104849463B (en) * | 2014-11-18 | 2017-02-22 | 天津瑞普生物技术股份有限公司 | Avian infectious bronchitis bivalent vaccine potency test method |
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| CN102749427B (en) | 2015-12-09 |
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