CN101099864A - Method for preparing inactivated vaccine both for preventing chicken Newcastle disease and infectious bronchitis - Google Patents

Method for preparing inactivated vaccine both for preventing chicken Newcastle disease and infectious bronchitis Download PDF

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CN101099864A
CN101099864A CNA2006100180692A CN200610018069A CN101099864A CN 101099864 A CN101099864 A CN 101099864A CN A2006100180692 A CNA2006100180692 A CN A2006100180692A CN 200610018069 A CN200610018069 A CN 200610018069A CN 101099864 A CN101099864 A CN 101099864A
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strain
virus
chicken
infectious bronchitis
newcastle disease
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CN101099864B (en
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崔保安
舒德广
王莉
徐端红
高文明
王学斌
张素梅
魏占勇
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Henan Agricultural University
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Henan Agricultural University
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Abstract

The invention is concerned with a kind of bacterin to prevent fowl infectious disease, relating to a kind of bacterin preparation method to prevent Newcastle Disease and infectious bronchitis. The selected genus is Newcastle Disease virus La Sota individual plant, infectious bronchitis virus M41 individual plant and HN99 kidney type of aberrance individual plant. Dilute two kind of virus and inoculate in chick embryo allantoic cavity, collect liquid of embryo to alive and dead embryos as seed. Dilute and inoculate in allantoic cavity of affectable chick embryo to get liquid of virus embryo. Concentrate with equipment and add with formaldehyde solution to prepare bacterin through emulsion process. This invention can prevent Newcastle Disease, chick kidney type and breathing type infectious bronchitis, and it can reduce the times of injection and the cost of epidemic prevention for easy using and practicality.

Description

The preparation method of the bivalent inactivated vaccine of prevention newcastle disease, infectious bronchitis
Technical field
The present invention relates to prevent the vaccine of fowl infection, relate in particular to a kind of preparation method of preventing the bivalent inactivated vaccine of newcastle disease, infectious bronchitis.
Background technology
Newcastle disease, chicken breathing pattern and renal type infectious bronchitis are very serious to poultry husbandry harm, corresponding individual event inactivated vaccine is only arranged at present,, need separately injection respectively as newcastle disease inactivated vaccine, infectious bronchitis (unit price) inactivated vaccine, use very inconvenient, epidemic prevention cost height.
Summary of the invention
The object of the invention is to provide a kind of preparation method of preventing the bivalent inactivated vaccine of newcastle disease, breathing pattern and renal type infectious bronchitis.
The present invention adopts and realizes above-mentioned purpose with following technical proposals: the preparation method of the bivalent inactivated vaccine of prevention newcastle disease, infectious bronchitis, selecting seed culture of viruses for use is Avian pneumo-encephalitis virus LaSota strain, infectious bronchitis virus M 41And HN 99Kidney form variation strain, the step of preparation bigeminy vaccine is as follows:
(1), with Avian pneumo-encephalitis virus La Sota strain, infectious bronchitis virus M 41And HN 99Kidney form variation strain dilution back inoculation SPF chick embryo allantois intracavity is collected blastochyle, as seed culture of viruses;
(2), with La Sota strain, M 41Strain and HN 99Inoculate the susceptible chick embryo allantoic cavity respectively after the dilution of strain seed culture of viruses, gather in the crops La Sota strain, M respectively 41Strain and HN 99Strain Embryo Gallus domesticus virus allantoic fluid;
(3), viral allantoic fluid is concentrated respectively;
(4), concentrating virus liquid adds the inactivator deactivation respectively, mixed in equal amounts is made single-phase oil emulsion inactivated vaccine with emulsion process then.
Described emulsion process for the Si Ben-80 of the white oil for animals of volume parts 92-95 part, 5-8 part and account for that white oil for animals mixes with the aluminium stearate of Si Ben-80 gross weight 1%-3%, heat fused and the postcooling of sterilizing be oil phase; With 3 kinds of deactivation concentrating virus liquid mixed in equal amounts and fully vibration, add the tween 80 that accounts for its percent by volume 4%-5% and be mixed into water; In the ratio of water and oil phase volume ratio 1: 3-4, single-phase oil emulsion inactivated vaccine is made in emulsifying.
Inactivator is the formalin of concentration of volume percent 0.1%-0.2%, and inactivation time is 12-24 hour.
Nephropathogenic infectious bronchitis HN 99Strain virus is the virus of new isolation identification, and it has following feature:
(1) see that under Electronic Speculum the coronavirus particle is arranged, diameter 90～105nm has cyst membrane, has the pears shape that is about 18nm outstanding on every side, is radial arrangement;
(2), morbidity chicken symptom except that respiratory symptom, most dead chicken all has pathological changes such as kidney enlargement, urate deposition, piebaldism kidney;
(3), viral isolates produces interference to the breeding of NDV La Sota strain virus;
(4), viral isolates does not produce coagulation to 1% chicken red blood cell;
(5), return the test of SPF chicken and respiratory symptom and pathological changes such as renal swelling, piebaldism kidney can occur;
(6), the specific serum neutralization results proves viral isolates of sick chicken and HN 99Positive serum is positive, and with NDV La Sota strain, IBDV B 87Strain, AIV H 9N 2The strain reaction that all is negative;
(7), with HN 99Strain positive serum and EID 50M 41Viruses such as strain, Holte strain, Gray strain, T strain and Local Isolates Hubei strain, Harbin strain, Shandong strain, Beijing strain, Shanghai strain, Tianjin strain are done serum neutralization test, and the result shows, HN 99Strain positive serum and T strain, Hubei strain, Tianjin strain and Shandong strain are positive, with the reaction that all is negative of other strain virus.
It is Avian pneumo-encephalitis virus La Sota strain, infectious bronchitis virus M that the present invention selects seed culture of viruses for use 41And HN 99Kidney form variation strain, wherein HN 99Kidney form variation strain is the virus of new isolation identification, and this Strain immunogenicity is good, can effectively prevent the Nephropathogenic infectious bronchitis infectious disease, and protective rate reaches 80～100%; This Strain high specificity, the specialized prevention Nephropathogenic infectious bronchitis, this is that other Strain are irreplaceable.Vaccine production has also been used the ultrafiltration and concentration technology, this vaccine that administers one injection can prevent three kinds of infectious disease such as newcastle disease, respiratory infectious bronchitis, nephropathy modification infectious bronchitis, could prevent these three kinds of infectious disease relatively with the existing three pin individual event vaccines of making a call to, this invention is economical to be used, simplify immune programme for children, reduced the epidemic prevention cost.
Avian infectious bronchitis virus strain of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 26th, 2006, depositary institution is called for short: CGMCC, deposit number: CGMCC No.1729, classification name: Nephropathogenic infectious bronchitis virus, English name is Avain nephropathogenic Infectious bronchitis virus HN99 Strain, the Latin name is called Tarpeiapulli, depositary institution address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, postcode: 100080.
The specific embodiment
The preparation method of the bivalent inactivated vaccine of prevention newcastle disease, infectious bronchitis, selecting seed culture of viruses for use is Avian pneumo-encephalitis virus La Sota strain, infectious bronchitis virus M 41And HN 99Kidney form variation strain, the step of preparation bigeminy vaccine is as follows:
1, the source of seed culture of viruses
(1) newcastle disease adopts La Sota strain, and imitating inspection is F with strong poison 48E 9, effluent south agriculture university provides.The standard of seed culture of viruses and store method are with the regulation of " the low virulence live vaccine of newcastle disease is made and inspection procedure " in " People's Republic of China's veterinary biologics rules " (following all be called for short " rules ") 1.
(2) the strong malicious M of standard is all adopted in infectious bronchitis manufacturing and check 41Strain and Local Isolates HN 99Strain provides by Agricultural University Of He'nan.
The strong malicious M of standard 41Strain and Local Isolates HN 99Strain virulence and seed culture of viruses standard are as follows:
Viral level M 41Strain and HN 99The strain seed culture of viruses to the infective dose of 10 age in days SPF chick embryo allantois intracavity inoculations all 〉=10 6.5EID 50/ 0.1ml.Infecting criterion is by all or part of death in 24～144 hours of inoculation back Embryo Gallus domesticus, and the Embryo Gallus domesticus of survival dehydration occurs, rolls up, grows little or specificity lesions such as renal swelling, urate deposition are standard with fetus.
Immunogenicity immunity counteracting toxic substances method: malicious valency 〉=10 6.0EID 50The M of/0.1ml 41Strain and HN 99Strain susceptible Embryo Gallus domesticus allantotoxicon, after deactivation, (water: ratio oil phase) was made oil emulsion inactivated vaccine, by 8 of every plumage part immunity 15 age in days SPF chickens, after three weeks, together with 4 of the identical contrast chickens of condition, attacks M respectively in 1: 3 respectively 41Strain and HN 99Strain is poison by force, every chicken trachea injection M 41Strain or HN 99Strong each 0.2ml of malicious allantoic fluid of strain observed 10 days, and the contrast chicken all falls ill, and immune chicken protection is more than 6/8, or immune chicken is all protected contrast chicken morbidity 3/4.
Gathered trachea, lungs and kidney swab in 6～7 days behind above-mentioned immune chicken of virus separation test and the contrast chicken counteracting toxic substances, handle back inoculated into chick embryo respectively, carry out virus and separate, the chicken that separates less than virus is judged to protection.Separate less than virus immunity chicken 〉=8/10, and the chicken of contrast chicken 〉=8/10 is separated to virus.
Pure seed culture of viruses is by 301 pages of checks of appendix of " People's Republic of China's veterinary biologics quality standard " (following all be called for short " standard "), and no antibacterial, mycete and mycoplasma and exogenous virus pollute.
Specificity M 41Strain and HN 99The strain seed culture of viruses respectively with M 41Strain and HN 99In the plant type specific serum and after, inoculated into chick embryo does not cause death or infection.
Be kept at below-15 ℃ noxious dampness storage life 6 months, M 41Strain lyophilizing poison is 3 years, HN 99Strain is tentative to be 2 years, and lyophilizing poison below-80 ℃ can be preserved 10 years at least.
2, chicken is used in test
Chicken is used in the safety test of bigeminy Seedling, check, all adopts SPF chicken or susceptible chicken.
3, attack with strong poison and counteracting toxic substances method
NDV F 48E 9Strain is poison by force, passes for 2 generations with preceding with the SPF embryo, malicious valency ELD 50Be 10 -7.0/ 0.1ml, muscle or subcutaneous injection.
IBV M 41Strain is poison by force, passes for 2 generations with the SPF Embryo Gallus domesticus, its malicious valency 〉=10 6.5EID 50/ 0.1ml to 1-7 age in days SPF chickling, splashes into 0.2ml in the strong poison pipe with 10 times of dilutions, observes 10 days, and it is the morbidity standard that symptoms such as typical bronchitis appear in the chickling more than 80%.
IBV HN 99Strain is poison by force, to malicious valency 〉=10 of Embryo Gallus domesticus 6.5EID 50/ 0.1ml.During counteracting toxic substances seed culture of viruses is done 10 times of dilutions, to splashing into 0.2ml in 10 tracheas of 1～7 age in days SPF chicken, to observe 10 days, cough appears in the chickling more than 80%, bronchitis symptom such as ataxia or bronchus, lungs are congested or hemorrhage, or disease symptoms such as renal swelling, urate deposition are arranged.
4, EID 50, HI, HA assay method HI, HA assay method be with reference to 40 pages of " rules " appendix.EID 50Assay method with 47 pages of " rules " appendix.
5, the foundation of seed lot is used in seedling with seed culture of viruses preparation and production
(1) NDV La Sota strain seedling is with " low virulence live vaccine manufacturing of newcastle disease and inspection procedure " in " rules " 2.1 of the foundation of seed culture of viruses preparation and seed lot.
(2) IBV M 41Strain and HN 99The seedling of strain is with the foundation of seed culture of viruses preparation and seed lot.
The seed culture of viruses breeding with 100 times of sterile saline dilutions, is inoculated 10-11 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml with seed culture of viruses in the allantoic cavity.Embryo alive and dead germ blastochyle are collected in inoculation back 30～72 hours, select aseptic through checking, to embryo toxicity valency 〉=10 6.5EID 50, the negative blastochyle of 1% chicken red blood cell agglutination test is made seed culture of viruses usefulness, indicate harvest date, seed culture of viruses generation.
The seed culture of viruses evaluation is undertaken by aforementioned project, should be up to specification.
Seed culture of viruses is kept at-15 ℃ of following conditions, and the operating period is no more than 6 months.
The seed culture of viruses subculture was no more than for 3 generations.
6, seedling is with the preparation and the check of viral liquid
(1) preparation of NDV La Sota strain virus liquid and check are with " the low virulence live vaccine of newcastle disease is made and inspection procedure " in " rules " 2.2,2.3,2.4.
(2) IBV M 41Strain and HN 99The preparation of strain virus liquid and check
Seedling selects well-developed 10～11 age in days susceptible Embryo Gallus domesticus as the seedling material with material.
Egg inoculation is with M 41Strain and HN 99The strain seed culture of viruses is diluted to 10 with sterile saline respectively -2～10 -3, inoculate the susceptible chick embryo allantoic cavity respectively, every embryo 0.1ml, sealing pin hole in inoculation back is put 37 ℃ and is continued to hatch, and stops egg-turning.
After hatching and observing egg inoculation, according to egg 1 time, discarded dead germ before 30 hours, later on every 4～8 hours photograph eggs once, dead Embryo Gallus domesticus is chosen at any time, and though to death in 72 hours whether, all take out, air chamber upwards places 2～8 ℃, cools off 12～24 hours.
Results are gathered in the crops M respectively 41Strain and HN 99Strain Embryo Gallus domesticus liquid.Refrigerative Embryo Gallus domesticus is taken out, with iodine tincture sterilization air chamber position, divest air chamber position chorion with aseptic method then, break allantocherion and amniotic membrane (yolk is broken), draw Embryo Gallus domesticus liquid, whenever the blastochyle of several embryos is mixed into one group, places the sterilization bottle, every milliliter adds penicillin, each 1000 unit of streptomycin, puts 2～8 ℃ of senses and does 4～12 hours.
Should check Embryo Gallus domesticus in results one by one in the blastochyle, all fetus corruption, blastochyle muddy and have the suspicious person of any pollution to discard need not.
The check M after treatment of Embryo Gallus domesticus virus liquid 41Strain and HN 99Strain Embryo Gallus domesticus virus liquid carries out sterility test (can not transplant) according to 50 pages of " rules " appendix respectively, answers asepsis growth; Blastochyle should not have coagulation to 1% chicken red blood cell; To inoculating EID in the 10 age in days susceptible chick embryo allantoic cavities 50Answer≤10 -6.0/ 0.1ml.What meet above regulation can be as the viral liquid of seedling.
7, viral liquid concentrates and deactivation
(1) concentrator
Use the MINI-PELLICON standard ultrafiltration apparatus of U.S. Millipore Corp..This equipment is made up of two parts device.First virus allantoic fluid pre-filtrating equipment, this device uses flat 293 disk filters, and this filter is made with the 316L rustless steel, all steel interface.Operating cost is low, and viral liquid resid vol is very little, but clamping pre-filtering and aseptic filtration film several pieces.Because NDV La Sota strain and IBV M 41Strain, HN 99The viscosity height of strain chick embryo allantoic liquid, adopt conventional degerming filter membrane flow very low, time length can cause viral liquid to lose poison, therefore, use the AP2529325 model specific pre-filtering film of the MILLIPORE in 0.45 μ m aperture, viral allantoic fluid is carried out coarse filtration, but the impurity in the filtering allantoic fluid and a lot of microorganism.Inactivation of virus step in conjunction with producing the later stage can reach aseptic result.It two is viral liquid ultra-filtration enrichment facilities, and this device uses 316L stainless steel membrane double team tool and pipeline, all adopts the health interface, and surfaces externally and internally electropolishing is handled.Power set use rustless steel peristaltic pump and silica gel peristaltic pump tube.Ultrafiltration and concentration uses the ultrafilter membrane bag of molecular weight as 500K-800K, can hold back virion such as ND, IB.This ultrafilter membrane bag adopts low protein adsorption modified fibre materials, and its protein adsorption quantity only is 1/4～1/5 of an international polyether sulfone materials.Inlet of this ultrafiltration apparatus and refluxing opening and see through and mouthful all to be furnished with diaphragm and diaphragm valve, the pressure of may command system guarantees the safety of ultrafiltration and concentration process and efficient.Film double team tool is a upright structures simultaneously, and system's discharging is easy, and resid vol is little, adds at last and washes viral liquid with sterile saline flushing, top, makes viral liquid loss minimum.
(2) ultrafiltration concentration process method
Coarse filtration is used viral allantoic fluid earlier the 100 orders/cm of sterilization 2The rustless steel funnel filter, remove large granular impurity, or, remove sediment, in absorption supernatant to the disinfectant container with the centrifugal 30min of the centrifuge of 3000～5000rpm/min.
Pre-filtering is with (121 ℃ of high pressure vapor sterilization such as dull and stereotyped filter of 293 disks and pipeline, 30min), after the container that fills viral liquid is connected the air pressurized that charges into degerming by pipeline with filter, (generally being controlled at 0.05 μ pa), virus liquid by the filter membrane of dull and stereotyped filter, flows out from outlet under certain pressure.Collect from the effusive viral liquid in dull and stereotyped filter filtration back with the disinfectant container, finish pre-filtering.
Ultrafiltration and concentration is rinsed ultrafiltration apparatus well with distilled water earlier repeatedly, and the sodium hydroxide solution soaked overnight of using 1N concentration is then washed with the disinfectant distilled water earlier during use more than 12 hours repeatedly, and NaOH is rinsed well, makes the near neutrality of discarded liquid after the flushing.Then pre-filtered viral liquid container is connected with ultrafiltration apparatus by decontamination duct, opens peristaltic pump, make its flow reach 4L/ minute, high like this flow velocity guarantees service life and the best ultrafiltration effect that the ultrafilter membrane bag is long.Viral liquid through ultrafiltration and concentration is contained in the airtight container, and cycles through ultrafiltration apparatus repeatedly, until meeting the requirements of striking point.
(3) before 3 kinds of viruses of concentrated effect mensuration concentrate and after concentrating, use and survey blood clotting valency HA or EID 50, TCID 50Check etc. method, and definite striking point.The definite of striking point shows as follows with the antigen scale:
Regulation in definite " the newcastle disease inactivated vaccine is made and inspection procedure " of NDV striking point, the viral level of La Sota strain allantoic fluid is 10 in the single-phase oil seepage 8.0EID 50, hence one can see that, and the viral level of the striking point of La Sota strain answers 〉=3 * 10 in the bigeminy Seedling 8.0EID 50
IBV M 41Strain and HN 99Determining of strain striking point: from " M 41Strain and HN 99Strain immunogenicity, storage life are measured " test as can be known IBV M 41Strain or HN 99Viral level in the single-phase oil seepage of strain is 10 6.0EID 50, hence one can see that, M in the bigeminy Seedling 41Strain or HN 99The viral level of the striking point of strain is for answering 〉=3 * 10 6.0EID 50
3 kinds of viral liquid concentrate by above-mentioned striking point, and the virus after concentrating should reach the requirement of striking point, should sample at any time to the discarded liquid in the concentration process simultaneously and check, checks its EID 50Should be null value.
(4) 3 kinds of viral liquid after the deactivation of concentrating virus liquid concentrates with check add 10% formalin respectively, and making its ultimate density is 0.1%, begin to calculate inactivation time when Seedling temperature rise to 37 ℃, and deactivation is 16 hours altogether.3 kinds of viral liquid after the deactivation, inoculated into chick embryo carries out HA, EID respectively 50Measure, prove thoroughly deactivation of virus.Make steriling test by " rules " P50 page or leaf method simultaneously.Prove no bacterial growth person, can be as seedling antigen.
8, the emulsifying of bigeminy Seedling and packing
(1) oil phase preparation: white oil for animals is Hangzhou Refinery production, and Si Ben-80 produces for Shanghai Dazhong Pharmaceutical Manufacturer, and aluminium stearate is that oceangoing voyage chemical plant, Shanghai produces.More than three kinds of compositions mix, heat fused is to transparent, and is standby through the autoclaving postcooling.
(2) water preparation: through 3 kinds of deactivation concentrating virus liquid after the assay was approved, mixed in equal amounts is also fully vibrated evenly, adds sterilization cooled tween 80 (Shanghai Dazhong Pharmaceutical Manufacturer's production), and mix homogeneously is standby.
(3) emulsifying of vaccine: use " rules " (P84～85) method, water, oil phase are mixed the back with colloid mill or the method that is used with homogenate pump, carry out emulsifying.
Emulsifying props: colloid mill JTM50 is that Shenyang new opto-mechanical factory makes, and homogenate pump is that Shanghai Dong Hua machinery plant makes., rinse well more than 8 hours with 0.5% formalin soaking disinfection earlier before using with preceding hot distilled water with sterilization.Wash repeatedly with hot water with the back, thoroughly eliminate residual oil seepage.
Concrete emulsion process for the Si Ben-80 of the white oil for animals of volume parts 92-95 part, 5-8 part and account for that white oil for animals mixes with the aluminium stearate of Si Ben-80 gross weight 1%-3%, heat fused and the postcooling of sterilizing be oil phase; With 3 kinds of deactivation concentrating virus liquid mixed in equal amounts and fully vibration, add the tween 80 that accounts for its percent by volume 4%-5% and be mixed into water; In the ratio of water and oil phase volume ratio 1: 3-4, single-phase oil emulsion inactivated vaccine is made in emulsifying.
The concrete composition proportion of each embodiment is as follows:
Embodiment 1 Embodiment 2 Embodiment 3
White oil for animals 92 94 95
Si Ben-80 8 6 5
Aluminium stearate 1％ 2％ 3％
Tween 80 4％ 4.5％ 5％
Water: oil phase (volume ratio) 1∶3 1∶3.5 1∶4
9, the laboratory inspection of bigeminy Seedling: from the bigeminy Seedling of laboratory trial-production, select the successive three batches of vaccines 0203,0204,0205 of lot number, do following test respectively.
(1) physical behavior of bigeminy Seedling check
Dosage form: three batches of bigeminy Seedlings are dripped in the cold water surface, be not separated into w/o type, be separated into the 0/W type.
Viscosity: with the 1ml suction pipe of 1.2 millimeters of outlet internal diameters, draw 1ml bigeminy Seedling at ambient temperature, calculate and vertically emit the required time of 0.4ml.
Stability:, observe vaccine layering situation with bigeminy Seedling centrifugal 15 minutes with 3000rpm.
Vaccine was put under 37 ℃ of conditions through 21 days, observed vaccine layering situation.
(2) steriling test carries out steriling test by the method for 50 pages of " rules " appendix to three batches of bigeminy Seedlings.
(3) safety test is with the SPF chicken in 3 ages in week, and intramuscular injection bigeminy Seedling 2ml (4 plumage part) observed for two weeks, and dissects, the response situation of record test chicken and cut open the inspection situation.
(4) efficacy test
The ND efficacy test: the every batch of vaccine is with 15 of 1 monthly age SPF chickens (ND HI＜1: 4), 10 each subcutaneous or intramuscular injection bigeminy Seedling 20 μ l wherein, in addition 5 in contrast not immune, after 3 weeks, every strong malicious F of chicken muscle injection ND 48E 9/ E2, dosage are 10 5ELD 50, observed 14 days, write down immune chicken and survival of contrast chicken and death condition.
The IB potency test: the every batch of vaccine with 3 age in week 16 of SPF chickens, subcutaneous or intramuscular injection bigeminy Seedling one plumage part (0.3ml), other 8 in contrast not immune.After three weeks wherein 8 immune chickens splash into M in the trachea together with 4 of contrast chickens 41Toxic allantoic fluid (EID 50〉=10 6.5/ 0.1ml) 0.2ml observed for 1 week, write down reaction and the death condition of immune chicken and contrast chicken.Splash into HN in other 8 immune chickens and 4 contrast chicken tracheas 99Strain virus liquid EID 50〉=10 6.5/ 0.1ml) 0.2ml observed for 1 week, write down the reaction of immune chicken and contrast chicken and cutd open the inspection situation.
(5) the different immunizing dose tests of bigeminy Seedling: get three batches of above-mentioned bigeminy Seedlings, every batch of Seedling is respectively by 0.1,0.2,0.3,0.4 dosage, immunity non-immune chicken in 3 ages in week respectively, 3 week the back measure immune chicken antibody level according to said method, and attack strong poison, observe and write down antibody horizontal, reaction and the pathological changes situation of immune chicken and contrast chicken.
10, the lab test results of bigeminy Seedling
(1) bigeminy Seedling physical behavior assay
Dosage form: 0203～0,205 three batch of bigeminy Seedling sample survey drips in the cold water surface, does not disperse substantially, is W/O (Water-In-Oil) type.
Viscosity: with 1 milliliter of suction pipe of 1.2 millimeters of outlet internal diameters, draw 3 crowdes of each 1ml of bigeminy Seedling at ambient temperature, vertically emit the required time of 0.4ml all in 8 seconds, meet in " rules " in " the newcastle disease inactivated vaccine is made and inspection procedure " viscosity requirement about oil emulsion.
Stability: the 3 batches of bigeminy Seedlings centrifugal 30 minutes with 3000rpm, vaccine is not stratified, breakdown of emulsion not.
The bigeminy Seedling was preserved 1 year at 2～8 ℃, preserved half a year for 20 ℃～25 ℃, and vaccine is not stratified, 37 ℃ put one month not stratified, there was the oil reservoir about 2mm on the vaccine surface in two months, sway the back Emulsion uniformly.Result such as table 1.
Table 1 bigeminy Seedling physical behavior assay
Lot number Dosage form Viscosity (second/0.4ml) Different temperatures is preserved the not stratified time
2～8 ℃ of room temperatures (20～25℃) 37℃
0203 W/O 8 12 months 6 months 1 month
0204 W/O 7 12 months 6 months 1 month
0205 W/O 7 12 months 6 months 1 month
(2) steriling test: the method by 50 pages of " rules " appendix is carried out steriling test to 3 batches of bigeminy Seedlings, and the equal asepsis growth of result is as table 2.
Table 2 bigeminy Seedling steriling test result
Batch T.G cultivates G.A cultivates G.P cultivates Judge
0203 - - - Asepsis growth
0204 - - - Asepsis growth
0205 - - - Asepsis growth
(3) safety test: with the SPF chicken in 3 ages in week, intramuscular injection bigeminy Seedling 2ml (4 plumage part).The short time lassitude appears in vaccine injection rear section test chicken, promptly recovers normal after 1～2 hour.Observed for 3 weeks altogether, all test chicken spirit are good, and food and drink are normal.Catch and kill after three weeks, all internal organs no abnormality seens change.Injection site has a small amount of unabsorbed vaccine, does not fester but there is swelling.The evidence vaccine safety, result such as table 3.
Table 3 bigeminy Seedling safety verification result
Lot number Injected dose (ml) The injection chicken General reaction Cut open inspection The result judges
Kind Age in days (my god) Quantity (only) Fabricius bursa Muscle Internal organs Injection site
0203 2 SPF 21 10 - - - - ± Qualified
0204 2 SPF 21 10 - - - - ± Qualified
0205 2 SPF 21 10 - - - - ± Qualified
Annotate: "-" expression is reactionless, and " ± " represents to have not absorb vaccine on a small quantity.
(4) potency test
The ND potency test: the every batch of vaccine is with 10 of 1 monthly age SPF chickens, 5 subcutaneous injections wherein, 5 intramuscular injection, dosage is bigeminy Seedling 20 μ l, in addition 5 in contrast not immune, after 3～4 weeks, every strong malicious F of chicken muscle injection ND 48E 9/ E 2, 10 5ELD 50, to observe 14 days, the contrast chicken is all dead, and three batches of vaccine immune chickens are all protected, and reach the efficacy test standard of " the newcastle disease oil emulsion inactivated vaccine is made and inspection procedure " in " rules ".
The IB potency test: the every batch of vaccine with 3 age in week 16 of SPF chickens, each 8 of subcutaneous and intramuscular injection, every dosage 0.3ml, after three weeks wherein 8 immune chickens (4 intramuscular injection, 4 subcutaneous injections) together with 4 of the identical contrast chickens of condition, intratracheal injection M 41Toxic allantoic fluid (EID 50≤ 10 -6.5/ 0.1ml) 0.2ml observed for two weeks, and the protection of result's immunity chicken is more than 7/8.M 41Strain contrast chicken falls ill entirely, and the morbidity chicken has cough, gets rid of first-class typical bronchitis symptom, and 2 chickens are dissected back bronchus, lungs hyperemia, pathological changes such as hemorrhage, 4 of other 8 immune chickens and contrast chickens, intratracheal injection HN 99Toxic allantoic fluid (the EID of strain 50≤ 10 -6.5/ 0.1ml), observed for two weeks, result's immunity chicken full guard, 3/4 morbidity of contrast chicken, the morbidity chicken has cough, breathes fast, gets rid of first-class bronchitis symptom, and wherein 1 chicken has renal swelling, 1 chicken bronchial, lungs hyperemia and hemorrhage dissection symptom.Meet the requirement of " inactivated vaccine for avian infectious bronchitis " efficacy test standard in " state quality standard ".Detailed results sees Table 4.
Table 4 bigeminy Seedling potency test
The vaccine lot number Imitate the inspection project Immunizing dose (ml) Immunity chicken number Antibody HI (LOg before the ND counteracting toxic substances 2) The counteracting toxic substances protection Control case The result
0203 ND 0.02 10 8.5 10/10 0/5 lives Qualified
M 41 0.3 8 7/8 4/4 morbidity
HN 99 0.3 8 8/8 3/4 morbidity
0204 ND 0.02 10 8.25 10/10 0/5 lives Qualified
M 41 0.3 8 8/8 4/4 morbidity
HN 99 0.3 8 8/8 3/4 morbidity
0205 ND 0.02 10 8.5 10/10 0/5 lives Each lattice
M 41 0.3 8 7/8 4/4 morbidity
HN 99 0.3 8 8/8 3/4 morbidity
Annotate: counteracting toxic substances dosage:
NDV:F 48E 9-E 2, 10 5ELD 50, intramuscular injection.
IBV:M 41E 4And HN 99Strain, toxic allantoic fluid (EID 50≤ 10 -6.5/ 0.1ml) splash into 0.2ml in the trachea, and implement cold stimulation.
11, the potency test of the different immunizing doses of bigeminy Seedling
With 0203,0204,0,205 three batch of vaccine of bigeminy Seedling, use 0.1,0.2,0.3,0.4 immunizing dose respectively, according to the method for 2.6.4 item efficacy test, the immunity test chicken.After 21 days, ND HI is measured in blood sampling, and attacks ND, IB M respectively 41And HN 99Strong poison is observed and is majored in the result.As table 5.
The different immunizing dose tests of table 5 bigeminy Seedling
The vaccine lot number Immunizing dose (ml) TPPA before the counteracting toxic substances Result behind the counteracting toxic substances
ND HI Protection number/counteracting toxic substances number (P/C) Dead or the morbidity of contrast
ND IB ND IB
M41 HN 99
0203 0.1 6.15 7/10 7/8 8/8 5/5 death 4/4 morbidity
0.2 7.0 10/10 8/8 8/8
0.3 7.75 10/10 7/8 8/8
0.4 9.10 10/10 7/8 6/8
0204 0.1 6.0 9/10 8/8 6/8
0.2 7.75 10/10 8/8 6/8
0.3 7.35 10/10 8/8 7/8
0.4 9.0 10/10 8/8 6/8
0205 0.1 6.4 7/10 6/8 8/8
0.2 6.75 10/10 8/8 8/8
0.3 8.0 9/9 8/8 6/8
0.4 9.10 10/10 7/8 8/8
From the result of table 5 as can be seen, three crowdes of bigeminy Seedling immunity 0.1～0.4ml, behind the test chicken counteracting toxic substances, behind the immunity test chicken ND counteracting toxic substances, protection 7/10～10/10, IB M 41And HN 99Counteracting toxic substances protection 6/8～8/8, the counteracting toxic substances result all reaches the required standard of " rules " and " standard ".
12, bigeminy Seedling laboratory results is analyzed
(1) IBV M 41Strain and HN 99The strain basis is planted malicious algebraically and is the highest generation E 5, producing with kind of the highest generation of poison was the 3rd generation; The lyophilizing poison is below-18 ℃, and storage life at least 3 years is produced and used seed culture of viruses (chick embryo allantoic liquid) preserving below-15 ℃, is no more than 6 months.
(2) seedling of bigeminy Seedling is with measuring viral level 〉=10 of NDV LaSota strain chick embryo allantoic liquid preparing with the method in " rules " of viral liquid 8.0EID 50, HA 〉=1: 640; IBV M 41Strain, HN 99The viral level of strain chick embryo allantoic liquid 〉=10 6.0EID 50Three kinds of viral liquid meet the requirement of seedling through the equal asepsis growth of steriling test.
(3) the concentration test result of 3 kinds of viral liquid of bigeminy Seedling
3 kinds of viral liquid is feasible with the technology of U.S. Mi Libo ultrafiltration apparatus concentrating virus in the bigeminy Seedling.Select the ultrafilter membrane bag in suitable aperture for use,,, can reach the purpose that concentrates NDV, 3 kinds of viral liquid of IBV according to giving the striking point of design earlier with the method for measuring viral level or HA.Discarded liquid after concentrating adopts and checks EID 50Method, prove in the relief liquor after concentrating not contain virus that concentrated effect is comparatively desirable.
(4) inactivation test of bigeminy Seedling concentrating virus liquid confirms, 3 kinds of concentrating virus liquid adopt 0.1% formalin, deactivation after 16 hours under 37 ℃ of conditions, La Sota strain, M 41Strain, HN 99Strain virus deactivation liquid 0.2ml inoculated into chick embryo, Embryo Gallus domesticus all is good for and is lived at the appointed time as a result, and 3 kinds of viral liquid all lose activity after deactivation, prove that deactivation is thorough.
(5) after the viral liquid mixed in equal amounts after the emulsifying of bigeminy Seedling and three kinds of deactivations of packing bigeminy Seedling, according to water: oil phase is 1: 3 a ratio, after the emulsifying of colloid mill difference, the fully emulsified and mix homogeneously through refiner can be prepared ideal single-phase oil emulsion inactivated vaccine again.
13, the field test of vaccine
The product lot number, the lot number that use in the field test: 0203,0204,0,205 three batch
Test period and place: Yingyang, Zhengzhou City chicken house in June, 2002
Main content of the test and result:
The three batches of bigeminy Seedlings are each 1000 of immune Luo Man 90 ages in days and 210 age in days laying hens respectively, every intramuscular injection 0.5ml.Observed altogether after the injection 14, all injection chickens all do not have General Symptoms and occur, and it is normal to eat food, drink water, and 210 age in days laying hen laying rate are uninfluenced.The minority chicken time grabs chicken because of injection stress, and do not lay eggs the same day, but promptly recover next day normal.The injection site reaction of all immune chickens is normal, does not have red and swollen or the phenomenon of festering.Chicken use no bad General Symptoms in back and local symptom appearance that three batches of bigeminy Seedlings are split antenatal hen and laid eggs are described, laying rate is unaffected.Evidence, three batches of bigeminy Seedlings are safe in utilization.
In the 3rd week after the immunity of three batches of bigeminy Seedlings, antibody is measured in blood sampling, and ND HI (log 2) rises to 9.0～11.5 by original 7.0～8.25, IB neutralizing antibody valency by rose to 1: 16 in original 1: 8～1: 32.Evidence, three batches of bigeminy Seedlings are after three weeks of immunity, and ND HI, IB neutralizing antibody rise to higher level.Pay a return visit the client after 4 months, these two kinds of infectious disease of ND, IB all do not take place in immune chicken.
Each 500 of the non-immunized chickses of three batches of bigeminy Seedlings immunity 7 ages in days, every chicken injection 0.3ml, the injection back was observed 14, any General Symptoms and local symptom all do not appear in all immune chickens, immunized chicks diet, spirit are normally, grow normally, abnormal conditions such as there is not redness the injection site, fester prove that the bigeminy Seedling is good to the chickling safety.
Above-mentioned three batches of bigeminy Seedling immunized chickses by the method for " rules " and " standard ", are used NDV F respectively after two weeks 48E 9Strong poison 10 5ELD 50Dosage and IBV M 41Strain and HN 99The strong malicious allantoic fluid 0.2ml of strain attacks with intramuscular injection and trachea drop-method respectively, and three batches of equal full guard of bigeminy Seedling ND immunized chicks as a result contrast deadly 5/5, pass a M 41And HN 99All protect 8/8, contrast 3/4 morbidity proves that the bigeminy Seedling is better to the chickling immune effect.
14, trial production and assay in the bigeminy Seedling
Requirement according to " Ministry of Agriculture's new biological product management method for animals ", finish on the breadboard basis, carried out the middle test manufacture of bigeminy Seedling in BJ Animal Biological Medical Products Factory, manufacture experimently 6 batches of bigeminy Seedlings altogether, and the method for reference " People's Republic of China's veterinary biologics rules " (hereinafter to be referred as " rules ") and " People's Republic of China's veterinary biologics quality standard " (hereinafter to be referred as " GB ") has been carried out safety test to 6 batches of vaccines, potency test, physical behavior detects, steriling tests etc., every assay have all reached the requirement of " rules " and " GB ".Detailed results sees Table 6,7,8.
Check of table 6 bigeminy Seedling pilot scale physical behavior and steriling test
The vaccine lot number The dosage form check Viscosity check second/0.4ml 37 ℃ 21 days Steriling test
T.G G.A G.P
0601 Water-In-Oil 7.8 Not stratified - - -
0602 Water-In-Oil 7.8 Not stratified - - -
0603 Water-In-Oil 7.9 Not stratified - - -
0604 Water-In-Oil 7.8 Not stratified - - -
0605 Water-In-Oil 7.9 Not stratified - - -
0606 Water-In-Oil 7.9 Not stratified - - -
Table 7 bigeminy Seedling pilot scale safety verification
The vaccine lot number Test chicken kind and quantity Age in days Injected dose The injection site Observe result on the 14th
General reaction Local response
0601 10 of SPF 21 2ml Chest muscle 10/10 is normal 10/10 -
0602 10 of SPF 21 2ml Chest muscle 10/10 is normal 10/10 -
0603 10 of SPF 21 2ml Chest muscle 10/10 is normal 10/10 -
0604 10 of SPF 21 2ml Cervical region is subcutaneous 10/10 is normal 10/10 -
0605 10 of SPF 21 2ml Cervical region is subcutaneous 10/10 is normal 10/10 -
0606 10 of SPF 21 2ml Cervical region is subcutaneous 10/10 is normal 10/10 -
Annotate: (-) represents no abnormal reaction.
Produce efficacy test as a trial in the table 8 bigeminy Seedling
The vaccine lot number The type of inspection Chicken is used in immunity The immunity number of elements Immunizing dose Injecting method Counteracting toxic substances dosage The counteracting toxic substances result The result judges
Immunoprotection Contrast is dead
0601 NDLa Sota 1 monthly age SPF chicken 10 20ul Intramuscular injection 10 5ELD 50 10/10 5/5 Qualified
IB M 41 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
IB HN 99 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 3/4
0602 NDLa Sota 1 monthly age SPF chicken 10 20ul Intramuscular injection 10 5ELD 50 8/10 5/5 Qualified
IB M 41 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
IB HN 99 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 3/4
0603 NDLa Sota 1 monthly age SPF chicken 10 20ul Intramuscular injection 10 5ELD 50 10/10 5/5 Qualified
IB M 41 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
IB HN 99 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 3/4
0604 NDLa Sota 1 monthly age SPF chicken 10 20ul Subcutaneous injection 10 5ELD 50 10/10 5/5 Qualified
IB M 41 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
IB HN 99 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 3/4
0605 NDLa Sota 1 monthly age SPF chicken 10 20ul Subcutaneous injection 10 5ELD 50 10/10 5/5 Qualified
IB M 41 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
IB HN 99 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 3/4
0606 NDLa Sota 1 monthly age SPF chicken 10 20ul Subcutaneous injection 10 5ELD 50 9/10 5/5 Qualified
IB M 41 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
IB HN 99 3 the week age SPF chicken 8 0.3ml 0.2ml 8/8 4/4
15, bigeminy Seedling large area region test
6 batches of bigeminy Seedlings producing as a trial in the utilization, plant 5 kind chicken houses such as chicken house, the everyday red fowl industry of Luohe City company limited in Henan Province Luohe City sunlight fowl industry company limited, Nanyang Pu Shan kind chicken house, Nanyang Feng Lou kind chicken house, Konka, carried out large-area regional test.Inject 46000 of the kind chickens of kinds such as Luo Man, Hai Lan, Hai Sai, AA altogether.2～3 weeks were observed in injection back, the spirit of injection chicken, searched for food, drink water all normal, and injection laying rate on the same day descends 2～3 percentage points in response to swashing reaction, promptly recovers next day normal.Situations such as the injection site does not have swelling substantially, festers, inflammation.Injection back ND HI is preceding 3～4 log 2 that rise of injection, from regional test user's response situation, prove that the safety of bigeminy Seedling is good, render a service reliable.Detailed results sees Table 9.
Table 9 bigeminy Seedling regional test situation
The vaccine lot number The test chicken house Injection kind, age in days Injection quantity (only) Local response General reaction Before the injection After the injection
ND HI (log2) ND HI (log2)
0601 0602 0603 0604 0605 0606 Luohe City sunlight kind chicken house Konka plants chicken house Luohe City sunlight kind chicken house Nanyang Pu Shan kind chicken house Konka and plants everyday Red Indian race chicken house Nanyang Feng Lou kind chicken house Nanyang Feng Lou kind chicken house Luohe Red Indian race chicken house everyday of Pu Shan kind chicken house Luohe, chicken house Nanyang Hai Lan, 110 Hai Sai, 125 Hai Lan, 110 Hai Lan, open antenatal Hai Sai, 125 Hai Lan, open antenatal AA, 18 Luo Mankai Luo Mankai half a year in puerperal AA half a year in puerperal, 18 2000 3000 2000 3000 3000 3000 5000 10000 10000 5000 No abnormal response is as good as paradoxical reaction not to be had abnormal response and is as good as paradoxical reaction and does not have abnormal response and be as good as paradoxical reaction and do not have abnormal response and be as good as paradoxical reaction and do not have abnormal response and be as good as paradoxical reaction Spirit, the diet normal psyche, the diet normal psyche, the diet normal psyche, the diet normal psyche, the diet normal psyche, the diet normal psyche, the diet normal psyche, the diet and the normal psyche of laying eggs, the diet and the normal psyche of laying eggs, diet is normal 6.0 8～9 6.0 6～7 8～9 6～7 6～8 9～11 9～11 6～8 9.0 10～13 9.0 9～11 10～13 9～11 8～10 10～14 10～14 8～10

Claims (3)

1, the preparation method of the bivalent inactivated vaccine of prevention newcastle disease, infectious bronchitis is characterized in that selecting seed culture of viruses for use is Avian pneumo-encephalitis virus La Sota strain, infectious bronchitis virus M 41And HN 99Kidney form variation strain, the step of preparation bigeminy vaccine is as follows:
(1), with Avian pneumo-encephalitis virus La Sota strain, infectious bronchitis virus M 41And HN 99Kidney form variation strain dilution back inoculation SPF chick embryo allantois intracavity is collected blastochyle, as seed culture of viruses;
(2), with La Sota strain, M 41Strain and HN 99Inoculate the susceptible chick embryo allantoic cavity respectively after the dilution of strain seed culture of viruses, gather in the crops La Sota strain, M respectively 41Strain and HN 99Strain Embryo Gallus domesticus virus allantoic fluid;
(3), viral allantoic fluid is concentrated respectively;
(4), concentrating virus liquid adds the inactivator deactivation respectively, mixed in equal amounts is made single-phase oil emulsion inactivated vaccine with emulsion process then.
2, the preparation method of the bivalent inactivated vaccine of prevention newcastle disease as claimed in claim 1, infectious bronchitis, it is characterized in that, described emulsion process for the Si Ben-80 of the white oil for animals of volume parts 92-95 part, 5-8 part and account for that white oil for animals mixes with the aluminium stearate of Si Ben-80 gross weight 1%-3%, heat fused and the postcooling of sterilizing be oil phase; With 3 kinds of deactivation concentrating virus liquid mixed in equal amounts and fully vibration, add the tween 80 that accounts for its percent by volume 4%-5% and be mixed into water; In the ratio of water and oil phase volume ratio 1: 3-4, single-phase oil emulsion inactivated vaccine is made in emulsifying.
3, the preparation method of the bivalent inactivated vaccine of prevention newcastle disease as claimed in claim 1 or 2, infectious bronchitis is characterized in that, inactivator is the formalin of concentration of volume percent 0.1%-0.2%, and inactivation time is 12-24 hour.
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