CN102643768B - Vaccine for preventing chicken diseases and preparation method thereof - Google Patents
Vaccine for preventing chicken diseases and preparation method thereof Download PDFInfo
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- CN102643768B CN102643768B CN201210124689.XA CN201210124689A CN102643768B CN 102643768 B CN102643768 B CN 102643768B CN 201210124689 A CN201210124689 A CN 201210124689A CN 102643768 B CN102643768 B CN 102643768B
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Abstract
The invention relates to a vaccine for preventing chicken diseases and a preparation method thereof, in particular to a chicken pathogenic flavobacterium strain with preservation code of CCTCCM2012100, and a vaccine containing chicken pathogenic flavobacterium. The preparation method of the vaccine includes diluting the chicken pathogenic flavobacterium strain in sterilized normal saline, and inoculating the strain to a 7-day-old susceptible chick by injecting; placing a piece of liver tissue of the diseased chick in an EP (epoxy) tube containing 1% TritonX-100, and grinding the piece of liver tissue with a grinding bar to make a production strain; emulsifying and packaging after inactivation. The vaccine can be used for preventing avian infectious laryngotracheitis, and avian diseases caused by hepatosis pathogen. The prepared inactivated vaccine has reliable quality, meets requirements, is high in safety, has good immune effect on chickens, is long in maintenance stage, has well protection effect on attach of strong bacteria, and has no influence on egg laying of chickens.
Description
Technical field
The present invention relates to a kind of epiornitic seedling, especially relate to vaccine of a kind of chicken disease and preparation method thereof.
Background technology
For a long time, chicken group, due to gregarious, easily breaks out disease, particularly communicable disease, such as chicken infectious bronchitis, the microbial disease of hepatitis.The common communicable disease of chicken group is very large on the impact of cultivation, if do not prevented, brings huge financial loss by raiser.So the preventative vaccine of communicable disease has just shown out importance especially.Also there is no the vaccine of special infection prevention bronchitis, the microbial disease of hepatitis targetedly at present.
The separation method of bacterial classification is varied in addition, and also different with the feature separation method of itself according to the different sources of bacterium, correct separation and Culture contributes to obtaining suspicious pathogenic bacterium very soon, is vital to the diagnosis of epidemic disease.
Summary of the invention
The technical problem to be solved in the present invention is: the vaccine providing a kind of infection prevention bronchitis, the microbial disease of hepatitis.
For achieving the above object, the invention provides a kind of chicken cause of disease flavobacterium strain, belong to Flavobacterium section, formal name used at school is: Flavobacterium sp, preserving number is CCTCC M 2012100, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center is preserved in, i.e. China typical culture collection center (CCTCC) on April 11st, 2012.
For obtaining chicken cause of disease flavobacterium strain of the present invention, present invention also offers a kind of separation method, the steps include:
The separation of bacterial classification:
Getting the sick chicken gizzard portion that infectious hepatitis occurs organizes a fritter (about 50mg) to be put in the EP pipe that 300ul 1% TritonX-100 is housed, after grinding with grinding rod, respectively get 100ul in containing ammonia benzyl respectively, the LB flat board coating of Ka Na and paraxin, cultivates 24h for 37 DEG C; Result only has colony growth on the flat board containing ammonia benzyl and Ka Na;
Strain expanded culture:
From the flat board of above-mentioned long bacterium, choose single bacterium colony in antibiotic 250ml LB liquid nutrient medium, 24-30 hour cultivated by 37 DEG C of shaking tables, surveys OD600 and is about 2.0; Its microbiotic is simultaneously containing ammonia benzyl and Ka Na;
Strain identification:
Get the bacterium liquid order-checking of above-mentioned enlarged culturing, show that surveyed bacterial classification is Flavobacterium; Be specially the bacterium liquid 30ul getting above-mentioned enlarged culturing and serve the order-checking of extra large handsome Bioisystech Co., Ltd, sequencing primer is 16s primer, and its sequence is 27F:AGAGTTTGATCCTGGCTCA(SEQ ID NO:1); 1492R:GGTTACCTTGTTACGACTT(SEQ ID NO:2); Gained sequence will be checked order at NCBI(http: //blast.ncbi.nlm.nih.gov/BlastAlign.cgi) upper comparison, or at NCBI(http after gained sequence being translated as protein sequence: //blast.ncbi.nlm.nih.gov/BlastAlign.cgi) go up comparison, finally show that surveyed bacterial classification is Flavobacterium;
The preservation of bacterial classification:
Above-mentioned enlarged culturing gained bacterium liquid is carried out 20% glycerine and protect the preservation of bacterium method-80 degree.By the bacterium liquid adding 80ul in the glycerine 20ul after sterilizing, fully after mixing.
Present invention also offers a kind of vaccine of chicken cause of disease flavobacterium strain, can be inactivated vaccine.
The preparation method of vaccine of the present invention is:
Inoculation: injection inoculation 7 age in days susceptible chick after chicken cause of disease flavobacterium strain described in claim 1 is diluted with sterile saline; Get disease chicken gizzard portion to organize a fritter to be put in be equipped with in the EP pipe of 1% TritonX-100, grind with grinding rod, make production bacterial classification;
Deactivation germ liquid: add formaldehyde solution by the above-mentioned production bacterial classification made, limit edged fully shakes up, and puts shaking table deactivation, as antigen for vaccine liquid;
Vaccine is prepared in emulsification: oil phase: injection peanut oil accounts for 10%, aqueous phase: starch accounts for the rear sterilizing of PBS mixing of 2% and 87ml, afterwards the tween-80 of sterilizing is added among aqueous phase, account for 1%, carry out emulsification after above-mentioned adjuvant being added the antigen for vaccine liquid of same volume, shake well makes tween dissolve completely; Sanitas is added before emulsification stops; Sanitas is 1% Thiomersalate, and add-on accounts for 1% of vaccine cumulative volume, ultimate density is ten thousand/;
Packing: vaccine emulsification obtained is sub-packed in sterilizing vaccine bottle, sealing, preserves for 4 DEG C.
Preparation method also comprises the step detecting whether complete inactivation.While guaranteeing complete inactivation germ, reduce formaldehyde usage quantity as far as possible, at different formalin-inactivated final concentration (0.1%, 0.2%, 0.3% and 0.4%) and inactivation time (3h, 6h, 12h, 24h and 48h), repeatedly overlapping experiment has been carried out to viral antigen liquid.Result of study shows: formaldehyde final concentration is 0.4%, and inactivation time is 48h, and inactivating efficacy is best.The method detecting whether complete inactivation can be the bacterium liquid of getting deactivation in the LB flat board coating simultaneously containing ammonia benzyl and Ka Na, cultivates 72 hours for 37 DEG C, whether long bacterium on flat board; Get inactivated bacterial liquid in containing in the LB liquid nutrient medium of ammonia benzyl and Ka Na simultaneously simultaneously, cultivate 72 hours for 37 DEG C, the whether long bacterium of liquid nutrient medium; Above 2 all not long bacterium, then illustrate bacterium liquid complete inactivation.
The present invention also provides the vaccine preparing gained according to above-mentioned preparation method.
Preparation method's tool of the present invention has the following advantages: method is simple, respond well, through multiple batches of experimentation on animals and field test, result shows: vaccine has no adverse reaction to chicken group, chicken group surveys HI antibody horizontal in latter 1 month in immunity, and geometric mean titer ND is 9.8 ~ 10.5log2.Chicken all living creatures is long in order, and open postpartum egg laying performance good, egg productivity is normal with eggshell quality, and within 6 months observation periods, immune group death rate is comparatively with the not immune control group reduction by 0.6% ~ 2.5% of group.Inactivated vaccine quality prepared by above-mentioned preparation method is certain, reliable, meets the requirements.
Vaccine prepared by the present invention can be used for preventing chicken infectious bronchitis, the microbial disease of hepatitis, reaches prophylactic effect.
The result of clinical experiment shows: vaccine of the present invention has no adverse reaction to chicken group, and chicken group surveys HI antibody horizontal in latter 1 month in immunity, and geometric mean titer Flavobacterium is 9.8 ~ 10.5log2.It is good that chicken group opens postpartum egg laying performance, and egg productivity is normal with eggshell quality, and within 6 months observation periods, immune group death rate comparatively reduces by 0.6% ~ 2.5% with group's not immune tetrad seedling control group, on average every voluminous 2 ~ 6 pieces, egg of chicken.After immunity, chicken group can produce higher antibody horizontal, and maintenance phase is longer, attacks have better protecting effect to strong bacterium.
The present invention is by specific works such as lab scale, laboratory work, Process Explorations, the industrialization flow production line scale up test of multiple batches of trial product and 5 batches is studied in Binding experiment room, through multiple batches of experimentation on animals and field test, obtain a large amount of experimental datas, the contrast that meets of experimental result all confirms, " chicken Flavobacterium inactivated vaccine " quality of development is certain, reliable, meets the requirements.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1: strain separating method and standard
1, bacterial classification preparation
(1) separation of bacterial classification:
Getting sick chicken (the Xiamen of Fujian Province Xiang peace Nei Cuo poulty house) liver that infectious hepatitis occurs organizes a fritter (about 50mg) to be put in the EP pipe that 300ul 1% TritonX-100 is housed; after grinding with grinding rod; respectively get 100ul in containing ammonia benzyl respectively, LB flat board coating (the ammonia benzyl: 0.05mg/ml of Ka Na and paraxin; , 37 DEG C cultivate 24h Ka Na: 0.04mg/ml); Result only has colony growth on the flat board containing ammonia benzyl and Ka Na;
(2) strain expanded culture
Single bacterium colony is chosen to simultaneously containing (ammonia benzyl: 0.05mg/ml in the 250ml LB liquid nutrient medium of ammonia benzyl and Ka Na from the flat board of above-mentioned long bacterium; , 37 DEG C shaking tables cultivate 24-30 hour, survey OD Ka Na: 0.04mg/ml)
600be about 2.0;
(3) strain identification
The bacterium liquid 30ul getting above-mentioned enlarged culturing serves the order-checking of extra large handsome Bioisystech Co., Ltd, and sequencing primer is 16s primer, and its sequence is 27F:AGAGTTTGATCCTGGCTCA(SEQ ID NO:1); 1492R:GGTTACCTTGTTACGACTT(SEQ ID NO:1); Gained sequence will be checked order at NCBI(http: //blast.ncbi.nlm.nih.gov/BlastAlign.cgi) upper comparison, or at NCBI(http after gained sequence being translated as protein sequence: //blast.ncbi.nlm.nih.gov/BlastAlign.cgi) go up comparison, finally show that surveyed bacterial classification is Flavobacterium;
(4) preservation of bacterial classification and preservation
20% glycerine is adopted to protect bacterium method, by the bacterium liquid adding 80ul in the glycerine 20ul after sterilizing, fully after mixing ,-80 degree preservations (noticing that glycerine will slowly be inhaled).The bacterial classification preserved delivers to CCTCC, and carry out preservation, preserving number is CCTCC M 2012100, (on April 11st, 2012 is preserved in Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, i.e. China typical culture collection center (CCTCC));
2, bacterial standard (chicken Flavobacterium should meet following standard)
In 2.1 pairs of chicken gizzard, pathogenic index (ICPI) is undertaken by " People's Republic of China's veterinary biologics quality standard " (calendar year 2001 version) (hereinafter referred to as " standard ") annex 316 pages, should be 0.1 ~ 0.4;
The mean time to death (MDT/MLD) of 2.2 pairs of chick minimum lethal doses is undertaken by " standard " annex 316 pages, should be 103 ~ 119 hours;
2.3 red cell agglutination valencys are undertaken by " People's Republic of China's veterinary drug allusion quotation " (version in 2005) (hereinafter referred to as " Chinese veterinary pharmacopoeia ") annex 28 pages, answer >=9log2 (micromethod) containing bacterium chicken blastochyle to red cell agglutination valency;
2.4 securities, 2 ~ 7 Japanese instar chickling 20, is divided into two groups, the 1st group 10, every injection inoculation 5 times dilution containing bacterium liquid 0.05ml, the 2nd group 10, do not inoculate in contrast, two groups of feedings and managements respectively under similarity condition, observe 10 days, should without abnormal response.If any nonspecific death, immune group and control group all should more than 1;
2.5 immunogenicities are to 1 monthly age chicken injection inoculation, and minimum immunity amount answers≤5000EID
50;
2.6 purely should pollute without bacterium, mould (by " Chinese veterinary pharmacopoeia " annex 15 pages), mycoplasma (by " Chinese veterinary pharmacopoeia " annex 19 pages) and external source germ (by " Chinese veterinary pharmacopoeia " annex 20 pages);
2.7 specificity;
2.8 basic bacteria algebraically E2 ~ E5 generations;
2.9 fungi preservation less than-20 DEG C preservations, freeze-drying lactobacillus preservation period is 72 months.
Embodiment 2: preparation production bacterial classification
Injection inoculation 2 ~ 7 age in days susceptible chick after embodiment 1 gained chicken Flavobacterium sp is diluted with sterile saline, get disease chicken gizzard portion to organize a fritter (about 50mg) to be put in be equipped with in the EP pipe of 300ul1% TritonX-100, grind with grinding rod, make production bacterial classification.
Embodiment 3: vaccine manufactures production technique
1, the working concentration of formalin-inactivated agent and inactivation time
While guaranteeing complete inactivation germ, reduce formaldehyde usage quantity as far as possible, at different formalin-inactivated final concentration (0.1%, 0.2%, 0.3% and 0.4%) and inactivation time (3h, 6h, 12h, 24h and 48h), repeatedly overlapping experiment has been carried out to viral antigen liquid.Result of study shows: formaldehyde final concentration is 0.4%, and inactivation time is 48h, and inactivating efficacy is best;
2, the production technique of inactivated vaccine:
For improving production technique, through having carried out different production process repeatedly repeating relatively, establish corresponding specification;
Antigen preparation adopts flavobacterium strain, and inoculation 2 ~ 7 age in days susceptible chicken embryo produces viral antigen liquid, viral antigen liquid germ content>=10 of results
8.0eID
50/ 0.1ml;
Vaccine is prepared in emulsification: oil phase: injection peanut oil accounts for 10%, aqueous phase: starch accounts for the rear sterilizing of PBS mixing of 2% and 87ml, afterwards the tween-80 of sterilizing is added among aqueous phase, account for 1%, carry out emulsification after above-mentioned adjuvant being added the antigen for vaccine liquid of same volume, shake well makes tween dissolve completely; Sanitas is added before emulsification stops; Emulsifying process oil emulsion inactivated vaccine stability is better, modest viscosity;
On repeatedly iterative process sex exploration basis, and the development of Binding experiment room and factorial praluction, through a series of detection validation, confirm that this Technology is stablized, ripening degree is high, and indices all meets " biological products code " and univalent vaccine standard in " import veterinary medical quality standard ".
Embodiment 4: inspection after construction
1, physical behavior
Outward appearance is oyster white emulsion.
Formulation is water-in-oil-type.Get a clean suction pipe, drawing a small amount of vaccine and drip in cold water, except first, should be oil droplet shape, indiffusion;
Stability gets 10ml vaccine in centrifuge tube, through the centrifugal 15min of 3000r/m, should not occur demixing phenomenon; Place 21 under 37 DEG C of conditions, should not breakdown of emulsion;
Viscosity 1ml suction pipe (lower internal diameter is 1.2mm, and upper internal diameter is 2.7mm), draws the vaccine 1ml of about 25 DEG C, makes its vertical natural flow out, and record flows out the time needed for 0.4ml, and it is qualified to be judged within 8 seconds;
2, steriling test is undertaken by " Chinese veterinary pharmacopoeia " annex 15 pages, answers asepsis growth;
3, safety verification gets chick 10 in 3 ~ 7 week age, and every muscle or neck subcutaneous injection vaccine 0.4ml (2 plumage part), observe 14, and any local of occurring because of vaccinate and systemic reaction should not occur;
4, efficacy test
Employing serological method is tested, and when result is against regulation, immunity can be adopted to attack bacterium method and carry out efficacy test;
Serological method is with chick 15 in 3 ~ 7 week age, and the subcutaneous or intramuscular injection vaccine 2ml (10 plumage part) of 10 each necks, another 5 compare.Latter 21 ~ 28 days of inoculation, every chicken is taken a blood sample respectively, separation of serum, carries out HI antibody titer mensuration by " Chinese veterinary pharmacopoeia ".The geometrical mean of immune group HI antibody titer answers >=4log2, and the geometrical mean of not immune control group HI antibody titer answers≤2log2;
Immunity attacks bacterium method with chick 15 in 3 ~ 7 week age, and the subcutaneous or intramuscular injection vaccine 2ml (10 plumage part) of 10 each necks, another 5 compare.Latter 21 ~ 28 days of inoculation, every chicken each intramuscular injection chicken Flavobacterium 1ml, observe 14, control group is all dead, and immune group at least protects 7;
5, formaldehyde content measures and is undertaken by " Chinese veterinary pharmacopoeia " annex 24,25 pages respectively, should conform with the regulations;
6, effect and purposes are for preventing avian infectious hepatitis, and duration of immunity is 6 months;
7, usage and consumption is subcutaneous or intramuscular injection, for seven Japanese instar chicklings inoculations, every only 0.2 ml; For opening the immunization of antenatal (110 ~ 140 age in days) laying hen and kind chicken, every 1ml;
8, precaution
8.1 non-health chickens must guard against injection this product;
The 8.2 poultry diease bacterium hypotypes that will prevent must be consistent with the hypotype that this product indicates, otherwise invalid;
8.3 this product are forbidden to freeze, under should being kept at 2 ~ 8 DEG C of constant temperatures;
8.4 this product as occurred, be sure not to use by the unusual phenomenoies such as breakage, foreign matter or breakdown of emulsion layering;
Vaccine should be returned to room temperature before 8.5 injections;
8.6 suggestions are proper use of under local animal doctor's accurate instruction;
9, preserve 2 ~ 8 DEG C of preservations, validity period is 18 months;
10, specification 100ml/ bottle, 250ml/ bottle, 500ml/ bottle.
Embodiment 5: the simultaneous test of inactivated vaccine
Inactivated vaccine takes injecting immune method and mixed feeding immunization two kinds of modes:
1, injecting immune method is that inactivated vaccine syringe to be directly injected into the neck of chicken subcutaneous.Injection inoculation method: the skin of neck of lightly mentioning chicken with have gentle hands, thrusts from neck stage casing with lower body direction;
Be kept at 4 DEG C inactivated bacterial liquid use before time and equivalent Freund's complete adjuvant fully mix; Use injecting method, the 10 black chickens in age in days Dehua: 0.2mL/ plumage, the black chicken in Dehua becomes chicken (3 monthly age): 1mL/ plumage.Control group uses physiological saline and equivalent Freund's complete adjuvant.Often organize 20, injecting immune carried out viable bacteria infection experiment after 15 days.Be mixed into equably in feed by the Flavobacterium being in logarithmic phase of centrifugation, according to the 50 grams of mixing of the ratio uniform to 1 kg diet of Flavobacterium weight in wet base, the feed of mixing has been fed in 2 hours.All apparatus collect sterilization in time.Feed with chow diet and contrast.Common observation 45 days from injection day.The results are shown in Table 1 and table 2;
Table 1:10 age in days chicken survival rate
| Group | 0 day | 15 days | 20 days | 30 days | 45 days |
| Chow diet is fed; Do not inject | 100% | 95% | 95% | 95% | 95% |
| Chow diet is fed; Injecting normal saline contrasts | 100% | 100% | 95% | 95% | 95% |
| Chow diet is fed; Injection Flavobacterium inactivated vaccine | 100% | 100% | 100% | 100% | 100% |
| Viable bacteria feed is fed; Do not inject | 100% | 100% | 65% | 30% | 25% |
| Viable bacteria feed is fed; Injecting normal saline contrasts | 100% | 100% | 70% | 40% | 30% |
| Viable bacteria feed is fed; Injection Flavobacterium inactivated vaccine | 100% | 100% | 95% | 95% | 95% |
Table 2: become chicken survival rate (3 monthly age)
| Group | 0 day | 15 days | 20 days | 30 days | 45 days |
| Chow diet is fed; Do not inject | 100% | 100% | 100% | 100% | 100% |
| Chow diet is fed; Injecting normal saline contrasts | 100% | 100% | 100% | 100% | 100% |
| Chow diet is fed; Injection Flavobacterium inactivated vaccine | 100% | 100% | 100% | 100% | 100% |
| Viable bacteria feed is fed; Do not inject | 100% | 100% | 75% | 60% | 45% |
| Viable bacteria feed is fed; Injecting normal saline contrasts | 100% | 100% | 85% | 70% | 50% |
| Viable bacteria feed is fed; Injection Flavobacterium inactivated vaccine | 100% | 100% | 100% | 100% | 100% |
2. mixed feeding immunization is by gastral oral immunity method.Often organizing 20 chickens, the deactivation Flavobacterium of centrifugation is mixed in feed equably, allowing the black chicken in Dehua (10 age in days chickens become chicken with 3 monthly ages) be eaten into by inactivated vaccine when eating food simultaneously.According to the 50 grams of mixing of the ratio uniform to 1 kg diet of deactivation Flavobacterium weight in wet base, the feed of mixing has been fed in 2 hours; Within 15 days, carry out viable bacteria infection experiment after this.Be mixed into equably in feed by the Flavobacterium being in logarithmic phase of centrifugation, according to the 50 grams of mixing of the ratio uniform to 1 kg diet of Flavobacterium weight in wet base, the feed of mixing has been fed in 2 hours.All apparatus collect sterilization in time.Feed with chow diet and contrast.From day of feeding, common observation 45 days, the results are shown in Table 3 and table 4;
Table 3:10 age in days chicken survival rate
| Group | 0 day | 15 days | 20 days | 30 days | 45 days |
| Chow diet is fed | 100% | 100% | 95% | 95% | 95% |
| Chow diet is fed; Mixed feeding Flavobacterium inactivated vaccine | 100% | 95% | 95% | 95% | 95% |
| Viable bacteria feed is fed | 100% | 100% | 60% | 30% | 20% |
| Viable bacteria feed is fed; Mixed feeding Flavobacterium inactivated vaccine | 100% | 100% | 90% | 85% | 75% |
Table 4: become chicken survival rate (3 monthly age)
| Group | 0 day | 15 days | 20 days | 30 days | 45 days |
| Chow diet is fed | 100% | 100% | 100% | 100% | 100% |
| Chow diet is fed; Mixed feeding Flavobacterium inactivated vaccine | 100% | 100% | 100% | 100% | 100% |
| Viable bacteria feed is fed | 100% | 100% | 70% | 55% | 50% |
| Viable bacteria feed is fed; Mixed feeding Flavobacterium inactivated vaccine | 100% | 100% | 90% | 85% | 85% |
As can be seen from the result of table 1-4, injecting immune method or mixed feeding immunization compared with the control, all can play reasonable prophylactic effect.In injecting immune method, viable bacteria feed is fed and is injected Flavobacterium inactivated vaccine, and become the survival rate of chicken to reach 100%, 10 age in days chicken survival rate liquid reach 95%(chow diet and feed; The surviving rate of not injecting also is 95%).Can say and provide protection is completely served to chicken.The effect of mixed feeding immunization is slightly more weak than the provide protection of injecting immune method.Comparatively speaking, the better effects if of injecting immune method.
Embodiment 6: clinical trial
1, test materials
1.1 experimental vaccine chicken Flavobacterium inactivated vaccines, Xiamen An Teao biotechnology company limited produces, and product batches is respectively: 201106,201107,201108,201109 and 201110;
1.2 disposable syringes, 50ul the are adjustable amount of moving pipettor, EP manages, mortar, pestle, dull and stereotyped, 250ml flask;
1.3 test chicken house and chicken groups:
1.3.1 Xiamen Xiang peace star plumage opens up into farm 110 age in days Luo Man Parent stock 10200, be divided into 5 groups at random, often organize about 200, use 201106,201107,201108,201109 and 201110 batches of vaccine immunities respectively, every pigeon breast portion intramuscular injection 1 ml, if be control group with batch not immune chicken 120, raise under the same terms;
1.3.2 Tongan Zhu Ba farm 110 age in days Luo Man Parent stock 10200, be divided into 5 groups at random, often organize about 200, use 201106,201107,201108,201109 and 201110 batches of vaccine immunities respectively, every pigeon breast portion intramuscular injection 1ml, if be control group with batch not immune chicken 120, raise under the same terms;
1.4 diagnostic antigen
Antigen is provided by Xiamen An Teao biotechnology company limited;
2, test method
The 2.1 each poulty houses of proof test are got the special cage of corresponding age in days chicken and are raised, and wear pin ring as mark, every batch of vaccination 100 chickens, every chest muscle injection 1ml (every plumage part), and every day observes injection site,
Continuous 14 days, according to following standard determination:
Heavily react: " +++ " injection site serious swelling, spread to whole chest, test chicken is One's spirits are drooping, does not rise sleepingly, even died;
Middle reaction: " ++ " injection site swelling, can not self-healing in 14 days, test chicken lassitude, appetite stimulator person;
Light reaction: the slight swelling in " ± " injection site, between test chicken, spiritedness is depressed, recovers normal person in 7 days;
Reactionless: "-" is without any visible reaction;
Heavily react, middle reaction be dangerous, light reaction, reactionless be safety;
2.2 potency test serological methods different kind chicken house every batch of vaccines respectively immunity open antenatal chicken group, establish non-immunized controls chicken group 100 ~ 120 simultaneously, before immunity, blood sampling once, monthly take a blood sample once after immunity, at every turn all according to 0.3% blood sampling of chicken group quantity, serum antibody titer is measured, until 6 months by HI method;
2.3 vaccinated flock egg laying performances are observed to open and are carried out vaccine immunity in antenatal 3 ~ 4 weeks, namely egg is collected after chicken group lays eggs, continuous 25 weeks (6 months), calculate laying rate, contrast chicken group with the not immune tetrad seedling of same group and the chicken group that raises the same period in former years compares, observe vaccine immunity to the impact of laying eggs;
3, result
After safety verification 5 batches of pilot scale vaccine inoculations, chest muscle injection site does not have swelling, phenomenon of hardening or fester, inoculation chicken drinking-water, search for food and the mental status without any visible reaction;
4, conclusion
The result of clinical experiment shows: vaccine has no adverse reaction to chicken group, and chicken group surveys HI antibody horizontal in latter 1 month in immunity, and geometric mean titer Flavobacterium is 9.8 ~ 10.5log2.It is good that chicken group opens postpartum egg laying performance, and egg productivity is normal with eggshell quality, and within 6 months observation periods, immune group death rate comparatively reduces by 0.6% ~ 2.5% with group's not immune tetrad seedling control group, on average every voluminous 2 ~ 6 pieces, egg of chicken.After immunity, chicken group can produce higher antibody horizontal, and maintenance phase is longer, attacks have better protecting effect to strong bacterium.
The present invention is by specific works such as lab scale, laboratory work, Process Explorations, the industrialization flow production line scale up test of multiple batches of trial product and 5 batches is studied in Binding experiment room, through multiple batches of experimentation on animals and field test, obtain a large amount of experimental datas, the contrast that meets of experimental result all confirms, " chicken Flavobacterium inactivated vaccine " quality of development is certain, reliable, meets the requirements.
SEQUENCE LISTING
<110> Xiamen An Teao biotechnology company limited
Vaccine of a <120> preventing chicken diseases and preparation method thereof
<130> P6581
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> synthetic
<400> 1
agagtttgat cctggctca 19
<210> 2
<211> 19
<212> DNA
<213> synthetic
<400> 2
ggttaccttg ttacgactt 19
Claims (7)
1. a chicken cause of disease flavobacterium strain, preserving number is CCTCC M 2012100.
2. the vaccine containing chicken cause of disease flavobacterium strain described in claim 1, it is characterized in that, described vaccine is inactivated vaccine.
3. utilize the bacterial strain described in claim 1 to prepare a preparation method for inactivated vaccine described in claim 2, it is characterized in that, comprise the steps:
Inoculation: injection inoculation 7 age in days susceptible chick after chicken cause of disease flavobacterium strain described in claim 1 is diluted with sterile saline; Get disease chicken gizzard portion to organize a fritter to be put in be equipped with in the EP pipe of 1% TritonX-100, grind with grinding rod, make production bacterial classification;
Deactivation germ liquid: add formaldehyde solution by the above-mentioned production bacterial classification made, limit edged fully shakes up, and puts shaking table deactivation, as antigen for vaccine liquid;
Vaccine is prepared in emulsification: oil phase: injection peanut oil accounts for 10%, aqueous phase: starch accounts for the rear sterilizing of PBS mixing of 2% and 87ml, be added among aqueous phase by the tween-80 of sterilizing afterwards, carry out emulsification after above-mentioned adjuvant being added the antigen for vaccine liquid of same volume, shake well makes tween dissolve completely; Sanitas is added before emulsification stops;
Packing: vaccine emulsification obtained is sub-packed in sterilizing vaccine bottle, sealing, preserves for 4 DEG C.
4. the preparation method of the inactivated vaccine of claim 3, is characterized in that, also comprises the step detecting whether complete inactivation.
5. the preparation method of the inactivated vaccine of claim 3, is characterized in that, during deactivation germ liquid, the final concentration of formaldehyde is about 0.4%, 37 DEG C of shaking table deactivations 48 hours.
6. the preparation method of the inactivated vaccine of claim 3, is characterized in that, when vaccine is prepared in emulsification, tween-80 add-on accounts for 1% of aqueous phase volume, and sanitas is 1% Thiomersalate, and add-on accounts for 1% of vaccine cumulative volume, ultimate density is ten thousand/.
7. the vaccine prepared according to claim 3-6 either method.
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| CN101787350A (en) * | 2010-01-22 | 2010-07-28 | 西南交通大学 | Functional composite biological agent for high-efficiency degradation of household refuses and application thereof |
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| immunization of broiler chickens against clostridium perfringens-induced necrotic enteritis using purified recombinant immunogenic proteins;yanfen jiang等;《avian diseases》;20091231;第53卷;409-415 * |
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