CN103820397A - Muscovy duck parvovirus and application thereof - Google Patents
Muscovy duck parvovirus and application thereof Download PDFInfo
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- CN103820397A CN103820397A CN201410082767.3A CN201410082767A CN103820397A CN 103820397 A CN103820397 A CN 103820397A CN 201410082767 A CN201410082767 A CN 201410082767A CN 103820397 A CN103820397 A CN 103820397A
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Abstract
The invention aims to provide a Muscovy duck parvovirus. The Muscovy duck parvovirus is a YBMDV strain, and is preserved in the China General Microbiological Culture Collection Center of the China Committee Of Culture Collection For Microorganisms in the Institute of Microbiology Chinese Academy of Science at the address of No. 3, No. 1 yard, Beichen west road, Chaoyang District, Beijing, on November 11th, 2013; the preservation number is CGMCC No. 8504. The Muscovy duck parvovirus screened by the invention has the characteristics of low toxicity and high immunogenicity; dead embryo and embryo liquid can be obtained by vaccinating the Muscovy duck parvovirus to a Muscovy duck embryo; virus liquid can be collected after grinding and freeze thawing; a vaccine is prepared by ultrafiltration concentration, formalin inactivation, the addition of an adjuvant, and mixing and emulsification; the Muscovy duck parvovirus can improve antibody of the Muscovy duck, ensure parent source antibody level of an offspring, and prevent green duck parvovirus infection caused by Muscovy duck parvovirus; the vaccine has the advantages of high efficiency and good safety.
Description
Technical field
The invention belongs to poultry sex pheromone triage techniques field, be specifically related to a kind of kind of Duck parvovirus and in the application of preparing in vaccine, antibody.
Background technology
Kind Duck parvovirus disease is the acute or subacute infectious disease of the one of the young kind duck of infringement, be breathe, diarrhoea and pancreas downright bad and hemorrhage be the transmissible disease of principal character; There is hyperinfection, and M & M is all very high, often causes serious financial loss.This disease mainly betides the young bird kind duck in 1～3 week age, the especially young bird kind duck of 10 age in days left and right, and just substantially do not fallen ill above when reaching 3 week age.This disease comes across Fujian China and French western Brittany area early than the middle and later periods eighties 20th century, and early 1990s just recognizes that it is the independent disease that is different from gosling plague.If kind duck to death is dealt with improperly, often cause spreading of virus, produce larger harm.Therefore, just need to filter out the height of tiring, a kind Duck parvovirus that immunogenicity is good is prepared vaccine.And, because causing the vaccine valence using, virus variation reduces, and also need the virus strain that filters out variation to prepare the vaccine that curative effect is higher.
Summary of the invention
The object of this invention is to provide a kind of kind Duck parvovirus filtering out, and in the application of preparing in vaccine; With vaccine prepared by this virus have advantages of efficiently, security is good, protection ratio is high, thereby make up the deficiencies in the prior art.
First the present invention provides a kind of kind Duck parvovirus, for YBMDV strain, the China Committee for Culture Collection of Microorganisms's common micro-organisms center that has been preserved in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013, deposit number is CGMCCNo.8504.
Of the present invention kind of Duck parvovirus is for the preparation of inactivated vaccine;
Of the present invention kind of Duck parvovirus is also for the preparation of immune antibody.
It is little that a kind Duck parvovirus for the present invention's screening has toxicity, and the feature that immunogenicity is high, with a kind Duck parvovirus YBMDP strain inoculation kind duck embryo, gather in the crops respectively dead embryo idiosome and blastochyle, after grinding, freeze thawing, collect virus liquid, after ultrafiltration and concentration, formaldehyde solution deactivation, the adjuvant mixing and emulsifying that refuels is made vaccine.Can improve the antibody of kind of kind duck, guarantee its filial generation maternal antibody level, the duckling parvovirus infections that prevention kind Duck parvovirus causes, this vaccine has advantages of efficiently, security is good.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
One, the screening of YBMDV strain
Acute kind Duck parvovirus disease has been broken out duck field in Zhejiang Province in 2011, get pancreas and the enteron aisle of the duck that dies of illness, pancreas and intestinal tissue are used without physiological saline homogenate and made 20% suspension, the centrifugal 15min of 3000r/min, inoculates respectively 13 ages in days kind duck embryo through allantoic cavity after getting supernatant degerming, hatches 120 hours, collect allantoic fluid and idiosome tissue, after homogenate, multigelation 3 times, gets supernatant liquor frozen.The virus liquid of results has carried out the analyzing and testing of the virus characteristic of the aspects such as viral level, immunogenicity, specificity and pure property after purified, and result shows that this strain viral level is 10
5.7eLD
50/ 0.2ml, minimum immune dosage is 10
2.0eLD
50/ 0.2ml, this virus only with a kind Duck parvovirus generation specific reaction, pollute without bacterium, mycoplasma and exogenous virus, be suitable as seedling strain.The China Committee for Culture Collection of Microorganisms's common micro-organisms center that screening YBMDV strain is preserved in to No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013, deposit number is CGMCC No.8504.
Proterties for the strain of the present invention screening detects, and result shows, this strain virus belongs to parvovirus, circular without cyst membrane, diameter in 20 μ m left and right, insensitive to ether, trypsinase, acid, to red corpuscle without agglutination phenomenon.This virus can cause that the acute septic transmissible disease take hemorrhagic enteritis as feature occurs young kind duck.Vaccine immunity prepared by this strain is grown up after kind duck, can protect kind filial generation that duck produces in immune 4 months to avoid the attack of epidemic isolates.This strain is done after 100 times of dilutions, with the neutralization of equivalent Muscovy Duckling Parvovirus disease antiserum(antisera), and inoculation 13 ages in days kind duck embryo, result shows, neutralization group kind duck embryo is strong living all, and virus control group kind duck embryo is all dead.
The structure gene VP of the YBMDV strain to screening checks order, and result VP total length is 2199bp, 732 amino acid of encoding; The blast sequential analysis of NCBI shows, the VP of the structure gene VP of YBMDV strain and published kind of Duck parvovirus there are differences site, and homology is that 97.7%～98.3%(exists 9-16 amino acid whose difference).
In order to detect above-mentioned amino acid whose difference to the antigenic impact of YBMDV strain, prepare antigen with YBMDV strain and detect its immunogenicity.
Two, the preparation of antigen for vaccine
1. seedling will be produced the strain with seed culture of viruses YBMDV with the preparation of virus liquid, allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo, and every embryo 0.2ml, hatches for 36～37 ℃, 2 photograph embryos inspection every day.Select after inoculation deadly in 48～120 hours, and idiosome has extensively hemorrhagely, and hair follicle is hemorrhage, liver sex change and necrosis, fine hair allantois has the embryo of Mild edema, gets its allantoic fluid and idiosome and puts into tissue mashing machine and pulverize, and the tissue of getting after pulverizing adds physiological saline to mix by 1:2; Put 2～8 ℃ 4～12 hours, collect allantoic fluid, amniotic fluid and idiosome (remove head and four limbs).With blastochyle homogenate, freeze thawing 3 times, the centrifugal 30min of 4000r/min, gets supernatant and is mixed in sterile chamber, puts 2～8 ℃ of preservations.
2. deactivation is placed in YBMDV strain virus liquid in deactivation bottle, is metered into 10% formaldehyde solution, with adding with shaking, it is fully mixed, and the ultimate density of formaldehyde solution is 0.2%.After adding formaldehyde solution, pour in another deactivation bottle, fail to contact inactivator with near the virus of avoiding adhering to bottleneck.37 ℃ of deactivations were taken out after 16 hours, put 2～8 ℃ of preservations.
3. the inspection of semifinished product
(1) steriling test is got the blastochyle of deactivation, carries out steriling test by existing " Chinese veterinary pharmacopoeia " appendix.
(2) viral level is measured virus liquid sterile saline is made to 10 times of serial dilutions, gets 10
-2, 10
-3, 10
-4, 10
-54 extent of dilution, allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo respectively, every embryo 0.2ml establishes 5 pieces of inoculation physiological saline contrasts, every embryo 0.2ml simultaneously.Put 36～37 ℃ and continue to hatch, per sunshine, embryo 2 times, observed 168 hours.With chicken embryo death and occur that chorioallantoic membrane oedema thickens, the systemic bleeding venereal diseases such as head, neck, back become and are judged to infection, calculate ELD
50.
(3) deactivation check is by 10 pieces of virus liquid allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryos after YBMDV strain deactivation, and every embryo 0.2ml, puts 36～37 ℃ and continue to hatch, Continuous Observation 168 hours.
Three, the preparation of inactivated vaccine: carry out vaccine preparation (in following preparation, each liquid component is counted by volume) through work in-process antigen after the assay was approved.
(1) 95 parts of white oils for animals are got in oil phase preparation, and 1 part of aluminum stearate, is placed in oil phase preparation tank and is heated to after 80 ℃, then Jia Siben-805 part, while reaching 115 ℃, maintains 30min to temperature, cooling rear for subsequent use.
(2) water is prepared the YBMDV strain virus liquid suitable proportion mixing that deactivation is up to the standards, and makes to be not less than 10 containing YBMDV strain virus content in every 0.2ml water
4.0eLD
50.Get 5 parts of tween-80s after sterilizing, add in Agitation Tank, add 95 parts of hybrid antigen liquid simultaneously, start agitator motor and stir 20～30min, tween-80 is dissolved completely.
(3) emulsification is got 2 parts of oil phases and is put in high-speed shearing machine, starts motor slow rotation and stirs, and slowly adds 1 part of water simultaneously, with 10000r/min, and emulsification 5 minutes.After emulsification, get 10ml, with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should be no more than 0.5ml.
Four, vaccine inspection after construction
(1) proterties
Outward appearance vaccine should be oyster white emulsion, and inclusion-free and outer packaging should be qualified.
Formulation is water-in-oil-type.Get a clean suction pipe, draw a small amount of vaccine and splash in cold water, except the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, and with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should be no more than 0.5ml.
Viscosity is undertaken by existing " Chinese veterinary pharmacopoeia " appendix, should conform with the regulations.
(2) loading quantity inspection is undertaken by existing " Chinese veterinary pharmacopoeia " appendix, should conform with the regulations.
(3) steriling test is undertaken by existing " Chinese veterinary pharmacopoeia " appendix, should conform with the regulations.
(4) safety verification is with 10 of 21 ages in days kind ducks, and every neck subcutaneous injection vaccine 2.0ml establishes 5 of contrasts simultaneously, under identical condition, raises, and Continuous Observation 14 days, records that test chicken is searched for food, drinking-water and clinical setting.Should not there is not any part and the systemic adverse reactions that are caused by vaccine.
(5) efficacy test
1. serological method is with 10 of 21～35 age in days SPF chickens, every neck subcutaneous injection vaccine, and 0.5ml/ only, separately gets 5 with not immune comparing of age in days chicken, exempts from latter 21 days, and blood sampling separation of serum, measures respectively kind Duck parvovirus fine jade and expands an antibody.Immune group AGP antibody titer, should be not less than 1:4, and control group should be all negative.
2. filial generation maternal antibody with attack poison protection be related to 20 of 90 age in days kind kind ducks, every neck subcutaneous injection vaccine, 2ml/ only, separately gets 10 with not immune comparing of age in days kind kind duck.In latter 15 days identical approach same doses of immunity again immunity once, collect the hatching of breeding eggs hatching of producing while opening April in postpartum, in the time of young bird kind duck 7 age in days, immune group filial generation is got 10, control group filial generation is got 10, attacks malicious YBMDP strain, every intramuscular injection 0.5ml.Immune group filial generation protection ratio should be not less than 8, and control group sickness rate should be not less than 9.
3. the poison protection (screening of strain) of attacking of local epidemic isolates is prepared respectively to inactivated vaccine by each place epidemic isolates, get 20 of 90 age in days kind kind ducks, every neck subcutaneous injection vaccine, 2ml/ only, separately gets 10 with not immune comparing of age in days kind kind duck.In latter 15 days identical approach same doses of immunity again immunity once, collect to open and produce the hatching of breeding eggs hatching of producing in 1 month, in the time of young bird kind duck 7 age in days, immune group filial generation is got 10, control group filial generation is got 10, attacks malicious 6 strain each place strain isolateds, every intramuscular injection 0.5ml.Malicious protection situation is attacked in observation.
Embodiment 1
One, the preparation of antigen for vaccine
1. seedling will be produced the strain with seed culture of viruses YBMDV with the preparation of virus liquid, allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo, and every embryo 0.2ml, hatches for 36～37 ℃, 2 photograph embryos inspection every day.Select after inoculation deadly in 48～120 hours, and idiosome has extensively hemorrhagely, and hair follicle is hemorrhage, liver sex change and necrosis, chorioallantoic membrane has kind duck embryo of Mild edema, gets its allantoic fluid and a kind duck idiosome and puts into tissue mashing machine and pulverize, and the tissue of getting after pulverizing adds physiological saline to mix by 1:2; Put 2～8 ℃ 4～12 hours, collect allantoic fluid, amniotic fluid and idiosome (remove head and four limbs).With blastochyle homogenate, freeze thawing 3 times, the centrifugal 30min of 4000r/min, gets supernatant and is mixed in sterile chamber, puts 2～8 ℃ of preservations.(referring to table 1).
The preparation of table 1 venom
| Strain title | Seedling material | Inoculation embryo number (piece) | Embryo age in days | Results venom (ml) |
| YBMDP strain | Kind duck embryo | 100 | 13 | 1200 |
2. deactivation is poured virus liquid in deactivation bottle into, is metered into 10% formaldehyde solution, and it is fully mixed, and the ultimate density of formaldehyde solution is 0.2%.After adding formaldehyde solution, pour in another deactivation tank, fail to contact inactivator with near the virus of avoiding adhering to tank mouth.37 ℃ of deactivations were taken out after 16 hours, put 2～8 ℃ of preservations.
3. the vaccine inspection of semifinished product
(1) steriling test is got the blastochyle of deactivation, carries out asepsis growth by existing " Chinese veterinary pharmacopoeia " appendix.
(2) viral level is measured virus liquid sterile saline is made to 10 times of serial dilutions, gets 10
-2, 10
-3, 10
-4, 10
-54 extent of dilution, allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo respectively, every embryo 0.2ml establishes 5 pieces of inoculation physiological saline contrasts, every embryo 0.2ml simultaneously.Put 36～37 ℃ and continue to hatch, per sunshine, embryo 2 times, observed 168 hours.With chicken embryo death and occur that chorioallantoic membrane oedema thickens, the systemic bleeding venereal diseases such as head, neck, back become and are judged to infection, calculate ELD
50be 10
5.7eLD
50/ 0.2ml.
(3) deactivation check is by 10 pieces of virus liquid allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryos, and every embryo 0.2ml, puts 36～37 ℃ and continue to hatch, Continuous Observation 168 hours, and duck embryo is all without dead.
Two, the preparation of inactivated vaccine: carry out vaccine preparation (in following preparation, each liquid component is counted by volume, referring to table 2) through work in-process antigen after the assay was approved.
(1) 95 parts of white oils for animals are got in oil phase preparation, and 1 part of aluminum stearate, is placed in oil phase preparation tank and is heated to after 80 ℃, then Jia Siben-805 part, while reaching 115 ℃, maintains 30min to temperature, cooling rear for subsequent use.
(2) water is prepared the virus liquid mixing that deactivation is up to the standards, and detects in every 0.2ml water and is not less than 10 containing YBMDP strain virus content
5.7eLD
50.Get 5 parts of tween-80s after sterilizing, add in Agitation Tank, add 95 parts of hybrid antigen liquid simultaneously, start agitator motor and stir 20～30min, tween-80 is dissolved completely.
(3) emulsification is got 2 parts of oil phases and is put in high-speed shearing machine, starts motor slow rotation and stirs, and slowly adds 1 part of water simultaneously, with 10000r/min, and emulsification 5 minutes.After emulsification, get 10ml, with 3000r/min centrifugal 15 minutes, separated out without water at the pipe end.
(4) packing quantitative separating, seals.
The emulsification of table 2 vaccine and packing
Three, vaccine inspection after construction
(1) proterties
Appearance milky white emulsion.
Formulation water-in-oil-type, gets a clean suction pipe, draws a small amount of vaccine and drips in cold water, is the indiffusion of oil droplet shape.
Stability is drawn vaccine 10ml and is added in centrifuge tube, and with 3000r/min centrifugal 15 minutes, separated out without water at the pipe end.
Viscosity is undertaken by current edition " Chinese veterinary pharmacopoeia " appendix, is 58.6cp.
(2) loading quantity inspection is undertaken by current edition " Chinese veterinary pharmacopoeia " appendix, conformance with standard.
(3) steriling test is undertaken by current edition " Chinese veterinary pharmacopoeia " appendix, asepsis growth.
(4) safety verification is with 10 of 21 ages in days kind ducks, and every neck subcutaneous injection vaccine 2.0ml establishes 5 of contrasts simultaneously, under identical condition, raise, Continuous Observation 14 days, test kind duck is strong living all, and all do not occur any part and systemic reaction causing because of vaccine.
(5) efficacy test
1. use 10 of 28 age in days SPF chickens, every neck subcutaneous injection vaccine 0.5ml, separately gets 5 with not immune comparing of age in days chicken, exempts from latter 21 days, and blood sampling separation of serum, measures kind Duck parvovirus AGP antibody titer.10 chicken antibodies of immune group are tired and are all not less than as 1:4, and 5 chickens of control group are all negative.(referring to table 3).
Table 3 vaccine potency assay
2. filial generation maternal antibody with attack poison protection be related to 20 of 90 age in days kind kind ducks, every neck subcutaneous injection vaccine, 2ml/ only, separately gets 10 with not immune comparing of age in days kind duck.In latter 15 days identical approach same doses of immunity again immunity once, collect the hatching of breeding eggs hatching of producing while opening April in postpartum, in the time of young bird kind duck 7 age in days, immune group filial generation is got 10, control group filial generation is got 10, attacks malicious YBMDP strain, every intramuscular injection 0.5ml.9 of immune group filial generation protections, control group is all fallen ill.
3. the poison of attacking of local epidemic isolates is protected to 20 of (seed selection of strain) 90 age in days kind kind ducks, every neck subcutaneous injection vaccine, 2ml/ only, separately gets 10 with not immune comparing of age in days kind kind duck.In latter 15 days identical approach same doses of immunity again immunity once, collect to open and produce the hatching of breeding eggs hatching of producing in 1 month, in the time of young bird kind duck 7 age in days, immune group filial generation is got 10, control group filial generation is got 10, attacks malicious 6 strain each place strain isolateds, every intramuscular injection 0.5ml.Result shows, kind Duck parvovirus inactivated vaccine of preparing with the YBMDP strain inactivated vaccine prepared by other strain of comparing, can resist each place and separate malicious attack.Its seed culture of viruses immunogenicity is good, is the comparatively ideal seedling strain of a strain.And kind Duck parvovirus inactivated vaccine of preparing with YBMDP strain shows for the poison experiment of attacking of YBMDP strain, the immune effect of vaccine prepared by YBMDP strain is best, and supposition causes (referring to table 4) because YBMDP pnca gene morphs.
Table 4 is to the malicious protection of attacking of local epidemic isolates
Therefore the YBMD strain that, the present invention screens can be used as seedling strain and prepares inactivated vaccine and yolk antibody.
Claims (5)
1. a kind Duck parvovirus, is characterized in that, described kind Duck parvovirus is YBMDV strain.
2. as claimed in claim a kind of Duck parvovirus, it is characterized in that, described kind Duck parvovirus is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013, deposit number is CGMCC No.8504.
3. as claimed in claim a kind of Duck parvovirus, is characterized in that, described kind Duck parvovirus is circular without cyst membrane, and diameter is 20 μ m left and right, insensitive to ether, trypsinase, acid, to red corpuscle without agglutination phenomenon.
4. claimed in claim a kind of Duck parvovirus is for the preparation of inactivated vaccine.
5. claimed in claim a kind of Duck parvovirus is for the preparation of immune antibody.
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| CN104611389A (en) * | 2014-12-10 | 2015-05-13 | 沈阳药科大学 | Fermentation optimizing technology for producing secalonic acid D (SAD) by using P.oxalicum |
| CN105154410A (en) * | 2015-09-21 | 2015-12-16 | 山东农业大学 | Duck parvovirus strain and live vaccine thereof |
| CN105153303A (en) * | 2015-09-21 | 2015-12-16 | 山东农业大学 | Egg yolk antibody for preventing and controlling duck parvovirus and preparation method thereof |
| CN105219735A (en) * | 2015-09-21 | 2016-01-06 | 山东农业大学 | A kind of Duck parvovirus strain and inactivated vaccine thereof and preparation method |
| CN105585632A (en) * | 2016-03-22 | 2016-05-18 | 重庆三杰众鑫生物工程有限公司 | Meat duck parvovirus refined yolk antibody |
| CN105949307A (en) * | 2016-06-18 | 2016-09-21 | 青岛易邦生物工程有限公司 | Egg yolk antibody used for preventing and treating Muscovy duck source gosling plague |
| CN106139140A (en) * | 2016-06-30 | 2016-11-23 | 类延乐 | A kind of Muscovy duck parvovirus subunit vaccine |
| CN108707588A (en) * | 2018-06-05 | 2018-10-26 | 山东信得科技股份有限公司 | A kind of Muscovy duck parvovirus strain and its application |
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| CN104611389A (en) * | 2014-12-10 | 2015-05-13 | 沈阳药科大学 | Fermentation optimizing technology for producing secalonic acid D (SAD) by using P.oxalicum |
| CN105153303B (en) * | 2015-09-21 | 2018-09-25 | 山东农业大学 | A kind of Yolk antibody and preparation method thereof of prevention Duck parvovirus |
| CN105153303A (en) * | 2015-09-21 | 2015-12-16 | 山东农业大学 | Egg yolk antibody for preventing and controlling duck parvovirus and preparation method thereof |
| CN105219735A (en) * | 2015-09-21 | 2016-01-06 | 山东农业大学 | A kind of Duck parvovirus strain and inactivated vaccine thereof and preparation method |
| CN105219735B (en) * | 2015-09-21 | 2018-04-03 | 山东农业大学 | A kind of Duck parvovirus strain and its inactivated vaccine and preparation method |
| CN105154410B (en) * | 2015-09-21 | 2018-04-03 | 山东农业大学 | A kind of Duck parvovirus strain and its live vaccine |
| CN105154410A (en) * | 2015-09-21 | 2015-12-16 | 山东农业大学 | Duck parvovirus strain and live vaccine thereof |
| CN105585632A (en) * | 2016-03-22 | 2016-05-18 | 重庆三杰众鑫生物工程有限公司 | Meat duck parvovirus refined yolk antibody |
| CN105949307A (en) * | 2016-06-18 | 2016-09-21 | 青岛易邦生物工程有限公司 | Egg yolk antibody used for preventing and treating Muscovy duck source gosling plague |
| CN105949307B (en) * | 2016-06-18 | 2019-11-22 | 青岛易邦生物工程有限公司 | It is a kind of for preventing and treating a kind Yolk antibody for duck source gosling plague |
| CN106139140A (en) * | 2016-06-30 | 2016-11-23 | 类延乐 | A kind of Muscovy duck parvovirus subunit vaccine |
| CN106139140B (en) * | 2016-06-30 | 2019-05-31 | 青岛真源生物技术有限公司 | A kind of Muscovy duck parvovirus subunit vaccine |
| CN108707588A (en) * | 2018-06-05 | 2018-10-26 | 山东信得科技股份有限公司 | A kind of Muscovy duck parvovirus strain and its application |
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