CN103495160B - The preparation method of chicken Mycoplasma synoviae inactivated vaccine - Google Patents
The preparation method of chicken Mycoplasma synoviae inactivated vaccine Download PDFInfo
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Abstract
The present invention relates to a kind of production method of chicken Mycoplasma synoviae inactivated vaccine.Vaccine involved in the present invention adopts the good chicken Mycoplasma synoviae HN01 strain of immunogenicity to be inoculated in appropriate media, and results culture, after formalin deactivation, adds oily adjuvant mixing and emulsifying and make.For preventing chicken Mycoplasma synoviae sick.This vaccine has that safety is good, immune efficacy advantages of higher.
Description
Technical field
The invention belongs to veterinary biologics technical field, more specifically relate to a kind of preparation method of chicken Mycoplasma synoviae inactivated vaccine.
Background technology
Chicken Mycoplasma synoviae is one of most important pathogenic bacterium in avian mycoplasmas, can infected chicken and turkey, mainly cause that joint exudative synovial fluid cyst membrane is scorching and Epitenon synovium is scorching, there is very strong infectiousness, and normal and antibacterial or virus mixed infection cause a disease, and cause huge economic loss to poultry husbandry.Control MS mainly adopts Drug therapy and immunity inoculation.In vitro tests shows, MS is to some antibiotic sensitive, comprise oxytetracycline, enrofloxacin, lincomycin, sharp proceomycin, tetracycline, tylosin, tilmicosin etc., but MS very easily produces drug resistance, therefore medicine controls first to should be noted interval medication, rotation medication and screening sensitive medicaments, avoid the generation of drug resistance, secondly antibiotherapy can not remove the MS infection of chicken group; Therefore immunity inoculation controls this sick more efficiently measure.Vaccine immunity has proved can effectively control this disease in correlation test abroad, and in Australia's extensive use, but there is no the listing of successful vaccine at home at present.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method of chicken Mycoplasma synoviae inactivated vaccine, for the immunoprophylaxis of chicken Mycoplasma synoviae disease.
The object of the invention is to be realized by following technical proposals.
A kind of preparation method of chicken Mycoplasma synoviae inactivated vaccine is prepared according to following step and method:
1, seedling culture medium and preparation method thereof:
(1) basic media components: PPLO meat soup 20.0 ~ 25.5g, lactoalbumin hydrolysate 5 ~ 8g, yeast extract powder 3 ~ 7g, nicotiamide 0.3g, mass concentration are phenol red solution 2ml, the deionized water 700 ~ 800ml of 0.4%.
(2) finished product medium preparation method: by above-mentioned (1) medium component 121 DEG C of autoclavings 15 minutes, 150-200ml porcine blood serum and the 1000 units/ml penicillin of Entkeimung is added after cooling, 20%NaOH adjusts PH7.6 ~ 7.8, aseptic subpackaged, and 2 ~ 8 DEG C save backup.
2, the cultivation of chicken Mycoplasma synoviae bacterium liquid and the preparation of inactivated vaccine:
(1) chicken Mycoplasma synoviae is inoculated in chicken Mycoplasma synoviae finished product culture medium in the ratio of 1:10, cultivates 24 ~ 48h results as primary seed solution at 36 ~ 37 DEG C; In kind primary seed solution being inoculated to go down to posterity in the ratio of 1:10 gathers in the crops as secondary seed solution 1 time;
(2) mechanical agitating fermentation tank continuous Zengjing Granule chicken Mycoplasma synoviae bacterium liquid is adopted: first chicken Mycoplasma synoviae basic media components in fermentation tank is carried out sterilizing, when temperature is down to 37 ~ 38 DEG C, in the ratio inoculation chicken Mycoplasma synoviae secondary seed solution of 1:5 ~ 1:10.Regulate PH, feed supplement, adopt continuous Zengjing Granule mode, PH is 7.6 ~ 7.8,37 DEG C of constant temperature culture, tank pressure is 0.03 ~ 0.05Pa, and dissolved oxygen amount is 40%, treat that PH drops to about 6.5, adding basal medium is 30% of former volume of culture, adjusts PH7.6 ~ 7.8 with 20%NaOH, so carry out twice Zengjing Granule, when second time Zengjing Granule PH drops to 6.4, add formalin deactivation, continuous Zengjing Granule terminates, keep sample inspection, puts 2 ~ 8 DEG C of preservations, be no more than 2.
(3) according to count plate result, chicken Mycoplasma synoviae bacterial concentration reaches 10
8~ 10
9cCU/mL;
(4) preparation of chicken Mycoplasma synoviae inactivated vaccine and vaccine emulsifying: get 96 parts of inactivated bacterial liquid, add 4 parts of tween 80s, for subsequent use as aqueous phase solution after mixing; Get 96 parts of injection white oils, add 4 parts of Si Ben-80, for subsequent use as oil-phase solution after mixing; Oil-phase solution and aqueous phase solution are mixed evenly according to volume ratio 2.8 ~ 3.5:1, namely obtain chicken Mycoplasma synoviae inactivated vaccine.
3, the inspection of vaccine
(1) physical behavior inspection: this vaccine is mineral oil water-in-oil type, the even Emulsion that outward appearance should be creamy white.Draw vaccine 10ml and add centrifuge tube, with 3000r/min centrifugal 15 minutes, the aqueous phase of separating out at the bottom of pipe should≤0.3ml.Get a clean suction pipe, draw a small amount of vaccine and drip in cold water, except first is except cloud spreads, later each equal indiffusion.With lml suction pipe (upper internal diameter 2.7mm, lower internal diameter 1.2mm), draw the vaccine lml of about 25 DEG C, make its vertical natural flow out, record flows out 0.4ml required time, should within 8 seconds.
(2) steriling test: undertaken by existing " Chinese veterinary pharmacopoeia " annex, should without bacterial growth.
(3) safety verification: get 7 age in days SPF chicken 10, each cervical region subcutaneous injection vaccine 1ml, observes 14, should not occur any local of being caused by vaccine and general reaction.
(4) efficacy test: select one to carry out
Immunization method: with 1 monthly age SPF chicken 20, wherein 10 cervical region subcutaneous injection vaccine 0.3ml, do not inoculate for another 10, in contrast.After 30 days, immune chicken and contrast chicken each footpad inoculation chicken Mycoplasma synoviae culture 0.1ml(10 simultaneously
8cCU/ml), observe 14, observe palmula swelling situation, contrast chicken all falls ill, and immune chicken at least protects 8.
Serum Antibody Detection method: immunity as stated above, after 30 days, the contrast chicken 10 identical together with condition is only taken a blood sample, separation of serum, measures chicken virus mycoplasma ELISA antibody titer S/P value.Immunity average S/P value >=0.6 of chicken ELISA antibody, the average S/P value of contrast chicken answers < 0.2.
The invention has the advantages that: (1) employs the continuous Zengjing Granule technique of fermentation tank, not only increases output, but also greatly reduces production cost, simplify production technology, chicken Mycoplasma synoviae can reach 1 × 10 by this technique growth titre
9cCU/ml; (2) this vaccine is all safe to the chicken of all ages and classes, is applicable to the commercial meat bird at various age, laying hen and kind chicken, can reach more than 8/10 to the immune protective rate of the drumsticks paralysis disease that chicken Mycoplasma synoviae causes.Effective duration of immunity that vaccine is once inoculated is 8 months, and at 2 ~ 8 DEG C, storage life is 12 months.
Detailed description of the invention
Embodiment 1
1 preparation culture medium
JIUYUE 21 ~ 22 in 2008, preparation chicken Mycoplasma synoviae basal medium 3000ml.Weigh PPLO meat soup 84g, lactoalbumin hydrolysate 28g, yeast extract powder 12g, nicotiamide 1.2g, mass concentration be 0.4% phenol red solution 8ml, deionized water 3000ml, through 121 DEG C of autoclavings 15 minutes after mixing.
Namely finished product culture medium adds porcine blood serum and the 1000 units/ml penicillin of about 20% on basal medium basis, 20%NaOH adjusts PH7.7, and 4 DEG C save backup.
The cultivation of 2 chicken Mycoplasma synoviae bacterium liquid and the preparation of inactivated vaccine:
(1) breeding of chicken Mycoplasma synoviae secondary seed solution: chicken Mycoplasma synoviae HN01 strain is inoculated in chicken Mycoplasma synoviae finished product culture medium in the ratio of 1:10,48h is cultivated at 37 DEG C, culture pH value drops to 6.6, gathers in the crops as primary seed solution; In kind primary seed solution being inoculated to go down to posterity in the ratio of 1:10 gathers in the crops as secondary seed solution 1 time, and measuring its growth titre is 10
8.5cCU/mL;
(2) mechanical agitating fermentation tank continuous Zengjing Granule chicken Mycoplasma synoviae bacterium liquid is adopted: in the fermentation tank preparation chicken Mycoplasma synoviae finished product culture medium that sterilizing is good, in the ratio inoculation chicken Mycoplasma synoviae secondary seed solution of 1:10.PH7.7 is regulated with 20%NaOH, 37 DEG C of constant temperature culture, tank pressure is 0.05Pa, dissolved oxygen amount is 40%, observe fermentation tank computer display screen, treat that PH drops to about 6.5, in fermentation tank, aseptic interpolation basal medium is 30% of former volume of culture, again regulate PH to 7.7, so carry out twice Zengjing Granule, when second time Zengjing Granule PH drops to 6.4, sampling gives over to sample detection before deactivation, add formalin 0.2% deactivation, period, gap was stirred, and at 37 DEG C, deactivation is after 24 hours, and keep sample inspection, put 4 DEG C of preservations, be no more than 2.Before mensuration deactivation, bacterial concentration is 10
9cCU/mL.
(3) chicken Mycoplasma synoviae deactivation inspection: inactivation time arrives, by the chicken Mycoplasma synoviae bacterium liquid of deactivation sampling 5ml, gets 0.5ml and inoculates chicken Mycoplasma synoviae finished product culture medium 4.5ml, carry out 10 times of serial dilutions to 10
-5, put 37 DEG C and cultivate 10 days, all grow without chicken Mycoplasma synoviae.
3 vaccine formulation
Adopt a step emulsion process, make Water-In-Oil oil emulsion vaccine.
Water-phase component: chicken Mycoplasma synoviae inactivation antigen 96 parts i.e. 4000ml adds 4 parts of tween 80s and 2680ml.
Oil-phase component: get 96 parts of injection white oils and 116250ml, adding 4 parts of Si Ben-80 is 4840ml.
Emulsification method: existing be fed in homogenizer by oil-phase component, by water-phase component charging, circulation stirring, to fully mixing, is emulsified into milky even emulsion inactivated vaccine, lot number 081101, batch 12.5 ten thousand ml.
4 vaccine tests
(1) physical behavior inspection: this vaccine is mineral oil water-in-oil type, the even Emulsion that outward appearance should be creamy white.Centrifugal 15 minutes of 3000r/min is not stratified.With lml suction pipe (upper internal diameter 2.7mm, lower internal diameter 1.2mm), draw the vaccine lml of about 25 DEG C, make its vertical natural flow out, flowing out 0.4ml required time is 4 seconds.
(2) steriling test: undertaken, all without bacterial growth by existing " Chinese veterinary pharmacopoeia " annex.
(3) safety verification: get 7 age in days SPF chicken 10, each cervical region subcutaneous injection vaccine 1ml, observes 14, without subtracting the untoward reaction such as food, depressed, the body temperature reaction of spirit, injection site inflammation, and is all good for alive.
(4) efficacy test: with 1 monthly age SPF chicken 20, wherein 10 cervical region subcutaneous injection vaccine 0.3ml, do not inoculate for another 10, in contrast.After 30 days, the contrast chicken 10 identical together with condition is only taken a blood sample, separation of serum, measures chicken virus mycoplasma ELISA antibody titer S/P value.Result immunity average S/P value >=0.6 of chicken ELISA antibody, the average S/P value < 0.2 of contrast chicken.
Embodiment 2
1 preparation culture medium
12 ~ 13 April in 2009, preparation chicken Mycoplasma synoviae basal medium 5250ml.Weigh PPLO meat soup 147g, lactoalbumin hydrolysate 42g, yeast extract powder 28g, nicotiamide 2.1g, mass concentration be 0.4% phenol red solution 14ml, deionized water 5250ml, through 121 DEG C of autoclavings 15 minutes after mixing.
Namely finished product culture medium adds porcine blood serum and the 1000 units/ml penicillin of about 20% on basal medium basis, 20%NaOH adjusts PH7.8, and 4 DEG C save backup.
The cultivation of 2 chicken Mycoplasma synoviae bacterium liquid and the preparation of inactivated vaccine:
(1) breeding of chicken Mycoplasma synoviae secondary seed solution: chicken Mycoplasma synoviae HN01 strain is inoculated in chicken Mycoplasma synoviae finished product culture medium in the ratio of 1:10,48h is cultivated at 37 DEG C, culture pH value drops to 6.5, gathers in the crops as primary seed solution; In kind primary seed solution being inoculated to go down to posterity in the ratio of 1:10 gathers in the crops as secondary seed solution 1 time, and measuring its growth titre is 10
8.5cCU/mL;
(2) mechanical agitating fermentation tank continuous Zengjing Granule chicken Mycoplasma synoviae bacterium liquid is adopted: in the fermentation tank preparation chicken Mycoplasma synoviae finished product culture medium that sterilizing is good, in the ratio inoculation chicken Mycoplasma synoviae secondary seed solution of 1:10.PH7.7 is regulated with 20%NaOH, 37 DEG C of constant temperature culture, tank pressure is 0.04Pa, dissolved oxygen amount is 40%, observe fermentation tank computer display screen, treat that PH drops to about 6.5, in fermentation tank, aseptic interpolation basal medium is 30% of former volume of culture, again regulate PH to 7.7, so carry out twice Zengjing Granule, when second time Zengjing Granule PH drops to 6.4, sampling gives over to sample detection before deactivation, ratio in 0.2% is added formalin and is carried out deactivation in cultured chicken Mycoplasma synoviae bacterium liquid, period gap stirring, at 37 DEG C, deactivation is after 24 hours, keep sample inspection, put 4 DEG C of preservations, be no more than 2.Before mensuration deactivation, bacterial concentration is 10
9cCU/mL.
(3) chicken Mycoplasma synoviae deactivation inspection: the chicken Mycoplasma synoviae bacterium liquid getting deactivation, in 1:10 ratio inoculation chicken Mycoplasma synoviae finished product culture medium, carries out 10 times of serial dilutions to 10
-5, put 37 DEG C and cultivate 10 days, all grow without chicken Mycoplasma synoviae.
3 vaccine formulation
Adopt a step emulsion process, make Water-In-Oil oil emulsion vaccine.
Water-phase component: chicken Mycoplasma synoviae inactivation antigen 96 parts i.e. 6000ml adds 4 parts of tween 80s and 250ml
Oil-phase component: get 96 parts of injection white oils and 173520ml, adding 4 parts of Si Ben-80 is 7230ml
Emulsification method: existing be fed in homogenizer by oil-phase component, by water-phase component charging, circulation stirring, to fully mixing, is emulsified into milky even emulsion inactivated vaccine, lot number 090602, batch 18.7 ten thousand ml.
4 vaccine tests
(1) physical behavior inspection: this vaccine is mineral oil water-in-oil type, the even Emulsion that outward appearance should be creamy white.Centrifugal 15 minutes of 3000r/min is not stratified.With lml suction pipe (upper internal diameter 2.7mm, lower internal diameter 1.2mm), draw the vaccine lml of about 25 DEG C, make its vertical natural flow out, flowing out 0.4ml required time is 3 seconds.
(2) steriling test: undertaken, all without bacterial growth by existing " Chinese veterinary pharmacopoeia " annex.
(3) safety verification: get 7 age in days SPF chicken 10, each cervical region subcutaneous injection vaccine 1ml, observes 14, without subtracting the untoward reaction such as food, depressed, the body temperature reaction of spirit, injection site inflammation, and is all good for alive.
(4) efficacy test: with 1 monthly age SPF chicken 20, wherein 10 cervical region subcutaneous injection vaccine 0.3ml, do not inoculate for another 10, in contrast.After 30 days, immune chicken and contrast chicken each footpad inoculation chicken Mycoplasma synoviae culture 0.1ml(10 simultaneously
8cCU/ml), observe 14, observe palmula swelling situation.All (10/10) morbidities of result contrast chicken, immune chicken 8/10 is protected.
Claims (1)
1. a production method for chicken Mycoplasma synoviae inactivated vaccine, is characterized in that, comprises the following steps:
(1) by chicken Mycoplasma synoviae in 1: 10 ratio be inoculated in chicken Mycoplasma synoviae finished product culture medium, 36 ~ 37 DEG C cultivate 24 ~ 48h results as primary seed solution; In kind using primary seed solution in 1: 10 ratio inoculate to go down to posterity and gather in the crops as secondary seed solution 1 time; Wherein chicken Mycoplasma synoviae finished product culture medium is: the chicken Mycoplasma synoviae basal medium of following preparation adds 150 ~ 200ml porcine blood serum and the 1000 units/ml penicillin of Entkeimung, and 20%NaOH adjusts pH7.6 ~ 7.8;
(2) mechanical agitating fermentation tank continuous Zengjing Granule chicken Mycoplasma synoviae bacterium liquid is adopted: first chicken Mycoplasma synoviae basic media components in fermentation tank is carried out sterilizing, when temperature is down to 37 ~ 38 DEG C, in the ratio inoculation chicken Mycoplasma synoviae secondary seed solution of 1: 5 ~ 1: 10, regulate pH, feed supplement, adopt continuous Zengjing Granule mode, pH is 7.6 ~ 7.8, 37 DEG C of constant temperature culture, tank pressure is 0.03 ~ 0.05Pa, dissolved oxygen amount is 40%, when pH drops to 6.5, adding basal medium is 30% of former volume of culture, pH7.6 ~ 7.8 are adjusted with 20%NaOH, so carry out twice Zengjing Granule, when second time Zengjing Granule pH drops to 6.4, add formalin deactivation, continuous Zengjing Granule terminates, keep sample inspection, put 2 ~ 8 DEG C of preservations, be no more than 2, wherein chicken Mycoplasma synoviae basic media components is: PPLO meat soup 20.0 ~ 25.5g, lactoalbumin hydrolysate 5 ~ 8g, yeast extract powder 3 ~ 7g, nicotiamide 0.3g, mass concentration are that phenol red solution 2ml, the deionized water 700 ~ 800ml of 0.4% were through 121 DEG C of autoclavings 15 minutes,
(3) chicken Mycoplasma synoviae bacterium liquid viable bacteria concentration reaches 10
8~ 10
9cCU/mL;
(4) preparation of described chicken Mycoplasma synoviae inactivated vaccine and vaccine emulsifying: get 96 parts of inactivated bacterial liquid, add 4 parts of tween 80s, for subsequent use as aqueous phase solution after mixing; Get 96 parts of injection white oils, add 4 parts of Si Ben-80, for subsequent use as oil-phase solution after mixing; Oil-phase solution and aqueous phase solution are mixed evenly according to volume ratio 2.8 ~ 3.5:1, namely obtain described chicken Mycoplasma synoviae inactivated vaccine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105733987A (en) * | 2016-03-21 | 2016-07-06 | 青岛易邦生物工程有限公司 | Mycoplasma synoviae |
| CN105695362A (en) * | 2016-03-21 | 2016-06-22 | 青岛易邦生物工程有限公司 | Fluid medium for culturing mycoplasma synoviae (MS) |
| CN105838641A (en) * | 2016-03-21 | 2016-08-10 | 青岛易邦生物工程有限公司 | Mycoplasma synoviae culture method |
| CN105816868B (en) * | 2016-03-21 | 2020-01-03 | 青岛易邦生物工程有限公司 | Inactivated vaccine for mycoplasma synoviae |
| CN105770881A (en) * | 2016-04-15 | 2016-07-20 | 青岛易邦生物工程有限公司 | Mycoplasma gallisepticum (MG) and mycoplasma synoviae (MS) combined inactivate vaccine |
| CN108949605A (en) * | 2017-06-29 | 2018-12-07 | 南京天邦生物科技有限公司 | A kind of culture medium and preparation method thereof for chicken Mycoplasma synoviae High Density Cultivation |
| CN107384837B (en) * | 2017-09-02 | 2020-05-08 | 河南省农业科学院畜牧兽医研究所 | Chicken mycoplasma synoviae and application thereof |
| CN112779193B (en) * | 2021-02-24 | 2022-09-27 | 福建省农业科学院畜牧兽医研究所 | Virulent strain of mycoplasma synoviae and application thereof |
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