Through the following example the present invention is explained more specifically, should understand said embodiment and only be for the present invention is described, rather than limit scope of the present invention by any way.

The specific embodiment

Embodiment 1, and the preparation of infectious coryza of chicken, chicken virus mycoplasma bigeminy lipoid inactivated vaccine is carried out according to the following steps:

First class inoculum preparation: get the streak inoculation of A type haemophilus paragallinarum strain on Carnis Gallus domesticus soup agar plate, at CO₂Volume content is 5%, temperature is to cultivate 16h in 37 ℃ the candle cylinder; 5 colonies typicals selecting the diameter that meets strain characteristic and be 1mm are inoculated in the 6 age in days SPF chick embryo yolk sacs after with the dilution of 10ml sterilization Carnis Gallus domesticus soup; Every embryonic breeding kind 0.2ml continues hatching under 37 ℃ of temperature, collect the yolk liquid of dead embryo in the 30h; After pure check confirms not have assorted bacterium, as first class inoculum.

The second class inoculum preparation: getting first class inoculum, by volume for the amount of semisynthetic medium volume 1/200 is inoculated in the semisynthetic medium, is to cultivate 17h under 37 ℃ of conditions in temperature; After pure check confirms not have assorted bacterium; Be second class inoculum, and place 2～8 ℃ of preservations, must not surpass 6～8h.

Seedling is with the preparation of bacterium liquid: second class inoculum by volume is inoculated in the semisynthetic medium for the amount of semisynthetic medium volume 1/40; Static cultivation 20h in 37 ℃ of greenhouses; Every during this time separated 8h rocks once; Confirm no living contaminants through pure check, adding volume is the formalin-inactivated 24h of semisynthetic medium volume 1/2000, obtains seedling and uses bacterium liquid.

Concentrating and deactivation of bacterium liquid: the seedling that will be up to the standards purely concentrates with the ultrafiltration and concentration method with bacterium liquid; Concentrate the back bacteria containing amount and be no less than 5,000,000,000 cfu/ml; Through the turbidimetry for Determination bacteria containing amount; Add volume more respectively and amass 1/10000 thimerosal with volume for concentrating bacteria liquid for the formaldehyde that concentrates bacteria liquid long-pending 1/1000, deactivation 2 days under 4 ℃ of conditions behind the mixing is through the qualified A type haemophilus paragallinarum inactivation antigen bacterium liquid that obtains of steriling test.

First order seed breeding: CM culture medium 1ml is injected the freeze-drying lactobacillus bottle strain is restored; Be that the chicken virus mycoplasma bacterial classification inoculation of CM culture volume 1/10 is in the CM culture medium with volume then; In temperature is to cultivate 48h under 36 ℃ of conditions, and the pH value before the pH value of treating postvaccinal CM culture medium and the inoculation is compared to descend and stopped in 0.5 o'clock cultivating, after pure check confirms not have the bacterium of mixing; As first order seed, be no more than 60 days in preservation below-25 ℃.

Secondary seed breeding: get first order seed,, put under 36 ℃ of temperature and cultivate 48h,,, be no more than 5 days 2-8 ℃ of preservation as secondary seed through purely after the assay was approved by volume for the amount of CM culture volume 1/10 is inoculated in the CM culture medium.

Seedling prepares with bacterium liquid: be the basis, breed three grades of seeds according to the secondary seed propagation method with the secondary seed that is up to the standards purely again; Be the basis, breed the level Four seed with three grades of seeds that are up to the standards purely again according to the secondary seed propagation method; Further do the seed amplification culture according to the secondary seed propagation method, culture propagation goes down to posterity and was no more than for 8 generations, and last generation is up to the standards purely; Measure through count plate, viable count is not less than 10^8.5Ccu/ml obtains seedling and uses bacterium liquid.

Bacterium liquid concentrates and deactivation: after the seedling that makes is concentrated with 8 times in ultrafiltration and concentration method work with bacterium liquid; Adding volume is the formaldehyde of concentrate volume 1/500; In temperature is deactivation 20h under 37 ℃ of conditions; Every during this time separated 6h shakes concentrate 1 time, obtains the chicken virus mycoplasma antigen liquid through aseptic being up to the standards with deactivation, puts 2～8 ℃ of preservations and is no more than 20 days.

(4) preparation of vaccine: the ratio mixing that with the above-mentioned A type haemophilus paragallinarum inactivation antigen bacterium liquid that makes and C type haemophilus paragallinarum inactivation antigen bacterium liquid is 1: 1 by volume; Obtain haemophilus paragallinarum deactivation hybrid antigen bacterium liquid; Be to pour in the emulsion tank behind 1: 1 the ratio mixing by volume with haemophilus paragallinarum deactivation hybrid antigen bacterium liquid and chicken virus mycoplasma antigen liquid; Stir with the rotating speed of mixer with 750r/min; Simultaneously slowly add poplar bark lipoid, making the final volume percent concentration of poplar bark lipoid in mixture is 2.0%, then the mixer rotating speed is brought up to 2500r/min and continues emulsifying 60 minutes; Promptly obtain vaccine, to be the final content of 1,000,000,000 cfu/ml, chicken virus mycoplasma be 10 to the final bacteria containing amount of A type haemophilus paragallinarum and C type haemophilus paragallinarum in the vaccine^8.0Ccu/ml.

Above-mentioned Carnis Gallus domesticus soup agar plate is preparation like this: get chicken juice 100ml, casein peptone 1g, sodium chloride 0.5g, agar powder 1.5g mixing; Regulate the PH to 7.4 of mixed solution with the NaOH solution of 4mol/L; Sterilized 30 minutes for 116 ℃; The healthy chicken serum mixing that adds nadide (NADH) and filtration sterilization before a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices; Make that the mass concentration of nadide (NADH) in the mixed solution is 0.05%, the concentration of volume percent of the healthy chicken serum of filtration sterilization is 10%, then mixed solution is poured in the plate that diameter is 90mm, set level cooling and promptly obtain Carnis Gallus domesticus soup agar plate; Said Carnis Gallus domesticus method is to get 4 parts of 1 part of adding distil waters of chicken-breasted mud, and 4 ℃ of soaked overnight were boiled 30 minutes, removes the fleshing slag, and clarification filtration obtains.

Above-mentioned semisynthetic medium is like this preparation: get polypepton 5g, casein peptone 30g, sodium glutamate 15g, yeast and soak powder 5g, glucose 3g, sodium chloride 15g; Putting into the 1000ml purified water stirs well; Regulate PH to 7.2～7.4 of gained solution; 116 ℃ of sterilizations 40 minutes, the mass concentration of facing the deactivation healthy chicken serum that adds 60ml before using and 10ml is 0.5% nadide (NADH) aqueous solution.

Embodiment 2, and the preparation of infectious coryza of chicken, chicken virus mycoplasma bigeminy lipoid inactivated vaccine is carried out according to the following steps:

First class inoculum preparation: get the streak inoculation of A type haemophilus paragallinarum strain on Carnis Gallus domesticus soup agar plate, at CO₂Volume content is 5%, temperature is to cultivate 18h in 37 ℃ the environment; 3 colonies typicals selecting the diameter that meets strain characteristic and be 1mm are inoculated in the 7 age in days SPF chick embryo yolk sacs after with the dilution of 10ml sterilization Carnis Gallus domesticus soup; Every embryonic breeding kind 0.2ml continues hatching under 37 ℃ of temperature, collect the yolk liquid of dead embryo in the 30h; After pure check confirms not have assorted bacterium, as first class inoculum.

The second class inoculum preparation: getting first class inoculum, by volume for the amount of semisynthetic medium volume 1/200 is inoculated in the semisynthetic medium, is to cultivate 18h under 37 ℃ of conditions in temperature; After pure check confirms not have assorted bacterium; Be second class inoculum, and place 2～8 ℃ of preservations, must not surpass 6～8h.

Seedling is with the preparation of bacterium liquid: second class inoculum by volume is inoculated in the semisynthetic medium for the amount of semisynthetic medium volume 1/40; Static cultivation 18h in 38 ℃ of greenhouses; Every during this time separated 6h rocks once; Confirm no living contaminants through pure check, adding volume is the formalin-inactivated 24h of semisynthetic medium volume 1/2000, obtains seedling and uses bacterium liquid.

Concentrating and deactivation of bacterium liquid: the seedling that will be up to the standards purely concentrates with the doughnut filter method with bacterium liquid; Concentrate the back bacteria containing amount and be no less than 5,000,000,000 cfu/ml; Through the turbidimetry for Determination bacteria containing amount; Add volume more respectively and amass 1/1000 thimerosal with volume for concentrating bacteria liquid for the formaldehyde that concentrates bacteria liquid long-pending 1/1000, deactivation 2 days under 4 ℃ of conditions behind the mixing is through the qualified A type haemophilus paragallinarum inactivation antigen bacterium liquid that obtains of steriling test.

First order seed breeding: CM culture medium 1ml is injected the freeze-drying lactobacillus bottle strain is restored; Be that the chicken virus mycoplasma bacterial classification inoculation of CM culture volume 1/10 is in the CM culture medium with volume then; In temperature is to cultivate 48h under 37 ℃ of conditions, and the pH value before the pH value of treating postvaccinal CM culture medium and the inoculation is compared to descend and stopped in 0.8 o'clock cultivating, after pure check confirms not have the bacterium of mixing; As first order seed, be no more than 60 days in preservation below-25 ℃.

Secondary seed breeding: get first order seed,, put under 37 ℃ of temperature and cultivate 24h,,, be no more than 5 days 2-8 ℃ of preservation as secondary seed through purely after the assay was approved by volume for the amount of CM culture volume 1/10 is inoculated in the CM culture medium.

Bacterium liquid concentrates and deactivation: after the seedling that makes is concentrated with 8 times in doughnut filter method work with bacterium liquid; Adding volume is the formaldehyde of concentrate volume 1/500; In temperature is deactivation 20h under 37 ℃ of conditions; Every during this time separated 6h shakes concentrate 1 time, obtains the chicken virus mycoplasma antigen liquid through aseptic being up to the standards with deactivation, puts 2～8 ℃ of preservations and is no more than 20 days.

(4) preparation of vaccine: the ratio mixing that with the above-mentioned A type haemophilus paragallinarum inactivation antigen bacterium liquid that makes and C type haemophilus paragallinarum inactivation antigen bacterium liquid is 1: 1 by volume; Obtain haemophilus paragallinarum deactivation hybrid antigen bacterium liquid; Be to pour in the emulsion tank behind 1: 1.5 the ratio mixing by volume with haemophilus paragallinarum deactivation hybrid antigen bacterium liquid and chicken virus mycoplasma antigen liquid; Stir with the rotating speed of mixer with 850r/min; Simultaneously slowly add poplar bark lipoid, making the final volume percent concentration of poplar bark lipoid in mixture is 3.8%, then the mixer rotating speed is brought up to 2500r/min and continues emulsifying 60 minutes; Promptly obtain vaccine, to be the final content of 1,000,000,000 cfu/ml, chicken virus mycoplasma be 10 to the final bacteria containing amount of A type haemophilus paragallinarum and C type haemophilus paragallinarum in the vaccine^8.0Ccu/ml.

The method for preparing of said Carnis Gallus domesticus soup agar plate, semisynthetic medium, CM culture medium is identical with embodiment 1.

Contrast test

Be that adjuvant adds infectious coryza of chicken, the chicken virus mycoplasma bigeminy vaccine that infectious coryza of chicken and chicken virus mycoplasma antigen prepare oil-emulsion vaccine (infectious coryza of chicken, chicken virus mycoplasma bigeminy oil emulsion inactivated vaccine), prepare according to the method for ZL03142556.9 by conventional method with mineral oil; Compare test with the infectious coryza of chicken of the present invention's preparation and the lipoid inactivated vaccine of chicken virus mycoplasma antigen preparation, contrast project and result are as shown in the table:

The method of inspection of related vaccine is following among the embodiment:

(1) physical behavior, outward appearance should be evenly Emulsion of yellow fraction white, anhydrous oil layering.Viscosity is drawn 25 ℃ of left and right sides vaccine 1.0ml with 1ml suction pipe (the end opening internal diameter is 1.2mm, and internal diameter suitable for reading is 2.7mm), makes its vertical outflow naturally, and record flows out the needed time of 0.4ml, should be in 5 seconds.(2) steriling test is undertaken by " People's Republic of China's veterinary drug allusion quotation " method, answers asepsis growth.(3) safety verification, with 8 of the SPF chickens of 40～60 ages in days, every subcutaneous injection vaccine 1ml observed 14, should no abnormal reaction.(4) efficacy test, infectious coryza of chicken part: with 8 of the SPF chickens at 2～3 monthly ages, every subcutaneous injection vaccine 0.5ml.After 1 month, together with 4 of the identical contrast chickens of condition, injection bacterium liquid 0.2ml (50～1,000,000 viable bacteria) in the hole observed 14 under each socket of the eye, and the contrast chicken all falls ill, and immune chicken is protected 6 at least, or 3 morbidities of contrast chicken, and immune chicken protects 7 to be qualified at least.The chicken virus mycoplasma part: get 8 of 40～60 age in days SPF chickens, nape portion or leg muscle vaccinate, every 0.5ml is after 30 days, together with 6 of the identical contrast chickens of condition, at 2～3m³Secret room (room temperature should be 15～30 ℃, and relative humidity is 50～70%) internal spraying attack strong malicious culture 500～600ml, spraying should be no less than 5 minutes at the persistent period, droplet is about 2 μ m.Observed 14, and cutd open inspection and observe the air bag pathological changes, to its scoring, at least 4 chickens of matched group, pathological changes more than 2 parts appears in every air bag, and the average air bag injury protection of immune group chicken rate should be more than 60%, and vaccine is judged to qualified.

Protective rate computational methods and formula calculate protective rate with the air bag lesion degree, and the every side air bag of test chicken is divided into five grades by lesion degree.0 minute-normal air bag, clean transparent and approaching; 1 minute-air bag thickens with slight muddy slightly, and there are minority Lycoperdon polymorphum Vitt or yellow exudate speckle in the part; 2 minutes-part air bag zone is thickened with the air bag moderate by visible Lycoperdon polymorphum Vitt and yellow exudate simultaneously; 3 minutes-large stretch of air bag is discontented with yellow cheesy exudate; 4 minutes-whole air bag is discontented with yellow cheesy exudate, and air bag follows the string.

Air bag protective rate computing formula:

Claims

1. the method for preparing of an infectious coryza of chicken, chicken virus mycoplasma bigeminy lipoid inactivated vaccine is characterized in that, may further comprise the steps:

First class inoculum preparation: get the streak inoculation of A type haemophilus paragallinarum strain on Carnis Gallus domesticus soup agar plate, at CO₂Volume content is 5%, temperature is to cultivate 16～18h in 37 ℃ the environment; 3-5 colonies typical selecting the diameter that meets strain characteristic and be 1mm is inoculated in 6～7 age in days SPF chick embryo yolk sacs after with the dilution of 10ml sterilization Carnis Gallus domesticus soup; Every embryonic breeding kind 0.2ml continues hatching under 37 ℃ of temperature, collect the yolk liquid of dead embryo in the 30h; After pure check confirms not have assorted bacterium, as first class inoculum;

The second class inoculum preparation: getting first class inoculum, by volume for the amount of semisynthetic medium volume 1/200 is inoculated in the semisynthetic medium, is to cultivate 17～18h under 37 ℃ of conditions in temperature; After pure check confirms not have assorted bacterium; Be second class inoculum, and place 2～8 ℃ of preservations, must not surpass 6～8h;

Seedling is with the preparation of bacterium liquid: second class inoculum by volume is inoculated in the semisynthetic medium for the amount of semisynthetic medium volume 1/40; Static cultivation 18～20h in 37～38 ℃ of greenhouses; Whenever rock once during this time at a distance from 6～8h; Confirm no living contaminants through pure check, adding volume is the formalin-inactivated 24h of semisynthetic medium volume 1/2000, obtains seedling and uses bacterium liquid;

Concentrating and deactivation of bacterium liquid: the seedling that will be up to the standards purely concentrates with bacterium liquid; Concentrate the back bacteria containing amount and be no less than 5,000,000,000 cfu/ml; Through the turbidimetry for Determination bacteria containing amount; Add volume more respectively and amass 1/10000 thimerosal with volume for concentrating bacteria liquid for the formaldehyde that concentrates bacteria liquid long-pending 1/1000, deactivation 2 days under 4 ℃ of conditions behind the mixing is through the qualified A type haemophilus paragallinarum inactivation antigen bacterium liquid that obtains of steriling test;

(2) preparation of C type haemophilus paragallinarum inactivation antigen bacterium liquid: its preparation method is identical with the method for preparing of A type haemophilus paragallinarum inactivation antigen bacterium liquid;

First order seed breeding: CM culture medium 1ml is injected the freeze-drying lactobacillus bottle strain is restored; Be that the chicken virus mycoplasma bacterial classification inoculation of CM culture volume 1/10 is in the CM culture medium with volume again; In temperature is to cultivate 24～48h under 36～37 ℃ of conditions, and the pH value before the pH value of treating postvaccinal CM culture medium and the inoculation is compared to descend and stopped in 0.5～0.8 o'clock cultivating, after pure check confirms not have the bacterium of mixing; As first order seed, be no more than 60 days in preservation below-25 ℃;

Secondary seed breeding: get first order seed,, put under 36～37 ℃ of temperature and cultivate 24～48h,,, be no more than 5 days 2-8 ℃ of preservation as secondary seed through purely after the assay was approved by volume for the amount of CM culture volume 1/10 is inoculated in the CM culture medium;

Seedling prepares with bacterium liquid: be the basis, breed three grades of seeds according to the secondary seed propagation method with the secondary seed that is up to the standards purely again; Be the basis, breed the level Four seed with three grades of seeds that are up to the standards purely again according to the secondary seed propagation method; Do the seed amplification culture according to the secondary seed propagation method, culture propagation goes down to posterity and was no more than for 8 generations, and last generation is up to the standards purely; Measure through count plate, viable count is not less than 10^8.5Ccu/ml obtains seedling and uses bacterium liquid;

Bacterium liquid concentrates and deactivation: after the seedling that makes is concentrated with 8 times in bacterium liquid work; Adding volume is the formaldehyde of concentrate volume 1/500; In temperature is deactivation 20h under 37 ℃ of conditions; Every during this time separated 6h shakes concentrate 1 time, obtains the chicken virus mycoplasma antigen liquid through aseptic being up to the standards with deactivation, puts 2-8 ℃ of preservation and is no more than 20 days;

(4) preparation of vaccine: the ratio mixing that with the above-mentioned A type haemophilus paragallinarum inactivation antigen bacterium liquid that makes and C type haemophilus paragallinarum inactivation antigen bacterium liquid is 1: 1 by volume; Obtain haemophilus paragallinarum deactivation hybrid antigen bacterium liquid; Be to pour in the emulsion tank behind 1: 1～1: 1.5 the ratio mixing by volume with haemophilus paragallinarum deactivation hybrid antigen bacterium liquid and chicken virus mycoplasma antigen liquid; Stir with the rotating speed of mixer with 750～850r/min; Slowly add simultaneously poplar bark lipoid; Making the final volume percent concentration of poplar bark lipoid in mixture is 2.0～3.8%; Then the mixer rotating speed is brought up to 2500～3000r/min and continued emulsifying 30～60 minutes, promptly obtain vaccine, to be the final content of 1,000,000,000 cfu/ml, chicken virus mycoplasma be 10 to the final bacteria containing amount of A type haemophilus paragallinarum and C type haemophilus paragallinarum in the vaccine^8.0Ccu/ml;

Said Carnis Gallus domesticus soup agar plate is preparation like this: get chicken juice 100ml, casein peptone 1g, sodium chloride 0.5g, agar powder 1.5g mixing; Regulate the PH to 7.4 of mixed solution with the NaOH solution of 4mol/L; Sterilized 30 minutes for 116 ℃; The healthy chicken serum mixing that adds nadide (NADH) and filtration sterilization before a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices; Make that the mass concentration of nadide (NADH) in the mixed solution is 0.05%, the concentration of volume percent of the healthy chicken serum of filtration sterilization is 10%, then mixed solution is poured in the plate that diameter is 90mm, set level cooling and promptly obtain Carnis Gallus domesticus soup agar plate; Said chicken juice is to get 4 parts of 1 part of adding distil waters of chicken-breasted mud, and 4 ℃ of soaked overnight were boiled 30 minutes, removes the fleshing slag, and clarification filtration obtains;

Said semisynthetic medium is like this preparation: get polypepton 5g, casein peptone 30g, sodium glutamate 15g, yeast and soak powder 5g, glucose 3g, sodium chloride 15g; Putting into the 1000ml purified water stirs well; Regulate PH to 7.2～7.4 of gained solution; 116 ℃ of sterilizations 40 minutes, the mass percentage concentration of facing the deactivation healthy chicken serum that adds 60ml before using and 10ml is 0.5% nadide (NADH) aqueous solution;

Said CM culture medium is like this preparation: be behind 1% the phenol red 1ml mixed dissolution with PPLO meat soup 21.0g, glucose 5.0g, deionized water 1000ml, concentration; In temperature is that 115 ℃ of pressure are to sterilize 20 minutes under the 0.106MPa condition; Put 2～8 ℃ of preservations; Add porcine blood serum 130ml, 25% yeast leachate 100ml and 800,000 units/ml penicillin solution 1.2ml before using, the pH value that uses sodium hydroxide solution to regulate culture medium is 7.8～8.0;

Said concentration is 1% phenol red manufacturing process: the preparation of (1) 1mol/L sodium hydroxide, get the clarifying sodium hydroxide saturated solution 56.0ml under the room temperature, and add the room temperature water for injection 1000ml that boiled and promptly make the 1mol/L sodium hydroxide; (2) take by weighing the phenol red powder of 10.0g and join and left standstill 5～10 minutes after stirring in the 1mol/L sodium hydroxide solution of 20.0ml, will dissolve partly and pour in the 1000ml graduated vessels; (3) left standstill 5～10 minutes after the 1mol/L sodium hydroxide solution that in undissolved phenol red powder, adds 20ml again stirs, will dissolve partly and pour in the 1000ml graduated vessels; (4) if the not dissolving fully of phenol red powder then can add a small amount of 1mol/L sodium hydroxide solution again, but must not surpass 20.0ml, solution is poured in the 1000ml graduated vessels; (5) add the room temperature water for injection that boiled and in graduated vessels, supply solution, shake up back packing bottle,, put 2～8 ℃ of preservations through 116 ℃ of sterilizations 15 minutes to 1000ml;

The manufacturing process of said 25% yeast leachate: get fresh yeast 250g and join fully stirring in the 1000ml distilled water; Make Saccharomyces mycetolysis complete; Boiled 30 minutes, boiled cooling after, with the 2800r/min rotating speed centrifugal 25 minutes; Extracting the supernatant packing, is that 115 ℃, pressure are sterilization 20 minutes under the 0.106MPa condition in temperature.

2. the method for preparing of a kind of infectious coryza of chicken according to claim 1, chicken virus mycoplasma bigeminy lipoid inactivated vaccine is characterized in that, any in the concentrated employing high speed centrifugation of said bacterium liquid, doughnut filtration, the ultrafiltration and concentration mode.

3. the method for preparing of a kind of infectious coryza of chicken according to claim 2, chicken virus mycoplasma bigeminy lipoid inactivated vaccine is characterized in that, the concentrated employing ultrafiltration and concentration mode of said bacterium liquid.