CN103721254A - Method for preparing swine A-type clostridium perfringens aluminum hydroxide inactivated vaccine - Google Patents
Method for preparing swine A-type clostridium perfringens aluminum hydroxide inactivated vaccine Download PDFInfo
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- CN103721254A CN103721254A CN201310749053.9A CN201310749053A CN103721254A CN 103721254 A CN103721254 A CN 103721254A CN 201310749053 A CN201310749053 A CN 201310749053A CN 103721254 A CN103721254 A CN 103721254A
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Abstract
The invention relates to a method for preparing a swine A-type clostridium perfringens aluminum hydroxide inactivated vaccine. The method comprises the following steps: (1) scraping the intestinal content of a dead swine due to clostridium flatulence; (2) cooling a milk culture medium which is milk sterilized under high pressure to 80 DEG C, pouring a disease material into the culture medium, and keeping on anaerobic culture; (3) centrifugally filtering and precipitating to obtain a clear toxin solution; (4) adding formaldehyde which is 0.4 percent of the total weight of the toxin solution, inactivating and detoxifying; (5) mixing the inactivated and detoxified toxin solution and an aluminum hydroxide adjuvant in a weight ratio of 4:1, and stirring at high speed to synthesize the vaccine. The method has the beneficial effects that the A-type clostridium vaccine is prepared by milk serving as the culture medium and is low in cost, and the culture process is simple, convenient and quick; aluminum hydroxide serving as the adjuvant is capable of enhancing the effect of the inactivated vaccine and reducing adverse response after vaccine injection; the inactivated vaccine is high in immune protection rate, so that the swine A-type clostridium perfringens aluminum hydroxide inactivated vaccine is safe and effective.
Description
Technical field
The invention belongs to biomedicine field, relate in particular to the preparation method of a boar A type bacillus perfringens aluminium hydroxide inactivated vaccine.
Background technology
Bacillus aerogenes capsulatus (Clostridium Perfringens), claims again clostridieum welchii (clostridium welchii), and this bacterium can produce multiple extracellular toxin and enzyme.Main toxin according to clostridieum welchii synthesized secretion, can be divided into A, B, and C, D, E type, wherein A type can infect people, forms blackleg, and mortality rate differs, and B, C, D type is special and intestinal infection animal is in close relations.All energy ramps in the various ordinary culture mediums of this bacterial strain, the most outstanding biochemical characteristic, to litmus milk culture medium " explosion fermentation ", can be used for quick diagnosis.This bacterium can form obvious pod membrane in vivo, and atrichia can not move.At nature, often with spore form, exist, but sporulation is more difficult.The resistance of spore is very strong, and 10% formalin 10 minutes could be lethal by it.This bacterium is distributed widely in nature, spreads all over all over the world, and be also one of flora of being everlasting in intestinal simultaneously,, can cause various serious diseases under certain condition.This bacterium can produce multiple strong extracellular toxin, and that finds at present has 12 kinds (α, beta, gamma, η, δ, ε, θ, ι, κ, λ, μ, ν), wherein, α, β, ε and ι are main lethal toxins.According to main lethal toxin neutralization test antitoxic with it, this bacterium can be divided into A, B, C, D, five types of E.
A type bacillus perfringens mainly causes gastrointestinal pathological changes, is people and animals' gas gangrenes, rabbit clostridium property dysentery, and the Main Pathogenic Bacteria of horse necrotic enteritis and people's alimentary toxicosis, is also the Main Pathogenic Bacteria that causes deer hemorrhagic enteritis.In the multiple extracellular toxin that this type bacterium produces, alpha toxin most importantly.
In order to control bacillus perfringens viral prevalence, many scholars once studied the simply connected of bacillus perfringens and concatenate inactivated vaccine for prevention on cellular level.Old stannum congruence has been carried out the research of " rabbit hemorrhagic disease " and A type bacillus perfringens connection Seedling; Three different local cattle source bacillus perfringens of the employings such as Yao Xiangyan were developed the concentrated aluminium hydroxide inactivated vaccine of Clostridium perfringens from bovines, confirm that this Seedling safety is good, immune latter 30 days with 2 lethal dose strong virus attacks, can obtain 100% protection, immunity is attacked and still can be obtained 90% protection with same dosage in latter 200 days; Yan Xijun Deng Dui Jilin deer hemorrhagic enteritis popularity is investigated, and has developed afterwards deer clostridieum welchii unit price inactivated vaccine, and vaccine is used safety, and immune effect is reliable; Tan Shiwen etc. had once carried out lamb dysentery, colibacillosis, the sudden cellulitis of sheep, the development of Intestinum caprae seu ovis toxemia tetrad oil adjuvant inactivated vaccine; Tu Weiying etc. have developed Clostridium perfringen enterotoxemia vaccine; Xu is the medium rabbit hemorrhagic disease of having developed, pasteurella multocida disease, and the concentrated inactivated vaccine of A type bacillus perfringens sick three, reaches more than 80% the protective rate of bacillus perfringens; Peaceful beautiful face etc. has been developed four kinds of clostridial disease vacines of sheep, and the duration of immunity of this Seedling is limited to 1 year, and enterotoxemia was fixed tentatively as half a year.
The vaccine of having developed in use remains at some needs the problem further solving: 1. domestic animal reaction of inoculation is heavier; 2. injected dose is large, uses and has inconvenience more; 3. preparation process is loaded down with trivial details, and the time is longer.Therefore, preparation process and screening culture medium and adjuvant be need to improve, to prepare fast efficient vaccine, vaccine immunogenicity and collective's immunne response level improved.
The kind of adjuvant is a lot, by its mechanism of action, is divided into absorption adjuvant, particulate adjuvants, immunoregulatory factor etc.; Also can be divided into plant origin by its source, microorganism and biogenetic derivation, artificial synthetic organic materials and inorganic salt etc.Some is formulated by the material of separate sources.
Summary of the invention
The preparation method that the object of this invention is to provide a boar A type bacillus perfringens aluminium hydroxide inactivated vaccine, to overcome prior art above shortcomings.
The object of the invention is to be achieved through the following technical solutions:
The preparation method of one boar A type bacillus perfringens aluminium hydroxide inactivated vaccine, comprises the following steps:
1) will by the lethal sick pig of clostridium, select pathological material of disease, scraping intestinal contents, cold preservation or freezing;
2) with plain chocolate as culture medium, 121 ℃ of autoclavings in pressure cooker, until temperature while dropping to 80 ℃, is poured pathological material of disease in pressure cooker into, anaerobism is cultivated;
3) by step 2) the bacterium liquid cultivated of mesohigh anaerobism filters centrifugally, and obtaining supernatant is toxin solution;
4) in the toxin solution obtaining, add 0.4% formaldehyde, shake well, deactivation detoxification in step 3); And
5) toxin solution of deactivation detoxification is mixed to synthetic vaccine after high-speed stirred according to the weight ratio of 4:1 with aluminum hydroxide adjuvant.
Further, in described step 1), cryogenic temperature is-20 ℃.
Further, described step 2) in, anaerobism condition of culture for cultivating 10-18 h at 37 ℃.
Further, in described step 3), filtering centrifugal condition is centrifugal 20min under 3000 r/min.
Further, in described step 4), deactivation detoxification condition is deactivation detoxification 3-5 days at 37 ℃.
Further, in described step 4), between deactivation During Detoxification every 4 hours intermittent oscillatings.
Beneficial effect of the present invention is: 1, scraping intestinal contents is cause of disease, produces the toxin solution of volume with advantage milk explosion fermentation.
2, take milk as culture medium and take aluminium hydroxide as adjuvant, finished product is cheap, and materials are convenient, produces fast, and boosting vaccine is little, does not affect spirit, appetite, and injection site is without swelling, without downright bad.
3, this vaccine can be preserved transportation under larger temperature range, in 0-30 ℃ of environment, preserves invariant color in 6 months, not stratified, does not affect duration of immunity.
4, the present invention selects plain chocolate as culture medium in vaccine preparation technology, with pressure cooker, carries out autoclaving, and anaerobism is cultivated and explosion fermentation, and the vaccine quality of cultivating is out good, and protective rate is high.
The specific embodiment
The preparation method of the boar A type bacillus perfringens aluminium hydroxide inactivated vaccine described in the embodiment of the present invention, comprises the following steps:
1) will by the lethal sick pig of clostridium, select pathological material of disease, scraping intestinal contents, cold preservation or freezing;
2) with plain chocolate as culture medium, 121 ℃ of autoclavings in pressure cooker, until temperature while dropping to 80 ℃, is poured pathological material of disease in pressure cooker into, anaerobism is cultivated;
3) by step 2) the bacterium liquid cultivated of mesohigh anaerobism filters centrifugally, and obtaining supernatant is toxin solution;
4) in the toxin solution obtaining, add 0.4% formaldehyde, shake well, deactivation detoxification in step 3); And
5) toxin solution of deactivation detoxification is mixed to synthetic vaccine after high-speed stirred according to the weight ratio of 4:1 with aluminum hydroxide adjuvant;
In described step 1), cryogenic temperature is-20 ℃; Described step 2), in, anaerobism condition of culture for cultivating 10-18 h at 37 ℃; In described step 3), filtering centrifugal condition is centrifugal 20min under 3000 r/min; In described step 4), deactivation detoxification condition is deactivation detoxification 3-5 days at 37 ℃; In described step 4), between deactivation During Detoxification every 4 hours intermittent oscillatings.
The method of inspection of pig A type clostridieum welchii vaccine
1) this product standing after, upper strata is colourless liquid, lower floor is milky white precipitate, is even suspension after jolting.
2) steriling test is tested by the method for < < Chinese veterinary pharmacopoeia > > regulation, answers asepsis growth.
3) safety verification is with 2-6 monthly age 5 of healthy susceptible pigs, each subcutaneous injection vaccine 6ml, and Continuous Observation 10 days, should all be good for work.
4) efficacy test is with 2-6 monthly age 5 of healthy susceptible pigs, and each subcutaneous injection vaccine 2ml, establishes simultaneously and do not inoculate 5 of contrast pigs.Immunity blood sampling in latter 14 days, separation of serum, measures respectively anti-clostridium HI antibody, the anti-clostridium HI of immune swine antibody all should >=1:32, contrast pig anti-clostridium HI antibody all should≤1:4.
Residues of formaldehyde quantitative determination
By < < Chinese veterinary pharmacopoeia > > appendix, measure, should meet the regulation of veterinary biologics general rule.
Pig A type clostridieum welchii inactivated vaccine safety testing
(1) the minimum vaccinated safety testing of single dose of age in days pig that uses is got 20 of the healthy susceptible pigs of 30 ages in days, is divided at random 4 groups, 5/group.Front 3 groups of vaccines of 3 batches that neck intramuscular injection is prepared by method provided by the invention respectively (the 1st batch, the 2nd batch and the 3rd batch), 2ml/ is only; Do not inoculate in contrast for the 4th group.Under the same terms, raise, observe 10, whether normally observe dog spirit, diet, feces every day, injection site and whole body have no adverse reaction.
Result: after single dose of intramuscular injection, the pig mental status, diet, feces are all normal, and injection site and whole body have no untoward reaction, all pigs are strong living all.
(2) safety testing of single dose repeated inoculation vaccine is got 20 of the healthy susceptible pigs of 30 ages in days, is divided at random 4 groups, 5/group.Front 3 groups of vaccines of 3 batches that intramuscular inoculation is prepared above respectively, only, after 14 days, once, 2ml/ is only for booster immunization for 2ml/; Do not inoculate in contrast for the 4th group.Under the same terms, raise, observe 10, whether normally observe pig spirit, diet, feces every day, injection site and whole body have no adverse reaction.
Result: after intramuscular routes repeated inoculation vaccine, the pig mental status, diet, feces are all normal, and injection site and whole body have no untoward reaction, all pigs are strong living all.
(3) one times the vaccinated safety testing of overdose is got 20 of the healthy susceptible pigs of 30 ages in days, is divided at random 4 groups, 5/group.3 batches of vaccines of front 3 groups of difference intramuscular inoculations, 6ml/ is only; Do not inoculate in contrast for the 4th group.Under the same terms, raise, observe 10, whether normally observe pig spirit, diet, feces every day, injection site and whole body have no adverse reaction.
(4) vaccine is got 10 of farrowing sows to the safety testing of farrowing sow, is divided at random 2 groups, 5/group.The 1st group in antenatal 20 days intramuscular inoculation vaccines, and 8ml/ only; Do not inoculate in contrast for the 2nd group.Under the same terms, raise, observe 10, whether normally observe pig spirit, diet, feces every day, injection site and whole body have no adverse reaction, and its farrowing situation of tracing observation.
Result: farrowing sow is antenatal 20 ages after the inoculation of muscle overdose, and the sow mental status, diet, feces are all normal, and injection site and whole body have no untoward reaction, and all pigs are strong living all, and farrowing is normal.
(5) safety test that conclusion has been carried out the inoculation of single dose with 3 batches of pig A type clostridieum welchii inactivated vaccines is, the safety test of the safety testing of single dose repeated inoculation, an overdose inoculation, and result is all safe.Show that this vaccine is safe for immunity inoculation pig.
Pig A type clostridieum welchii inactivated vaccine potency test
Get 3 batches of pig A type clostridieum welchii inactivated vaccines prepared by the above inventor, get 20 of healthy susceptible pigs, be divided at random 4 groups, 5/group.1, only, matched group does not carry out immunity to 3 crowdes of vaccine 2ml/ of 2,3 groups of pig difference intramuscular inoculations.3 immunity combination matched groups, at latter 14 days oral clostridium culture fluid 15ml alive counteracting toxic substances of immunity, are observed 10, record incidence.
Result: 3 batches of vaccine test result no significant differences.After vaccine immunity 14 days, the live protective rate of culture fluid counteracting toxic substances of clostridium is to 100%.
The present invention is not limited to above-mentioned preferred forms; anyone can draw other various forms of products under enlightenment of the present invention; no matter but do any variation in its shape or structure; every have identical with a application or akin technical scheme, within all dropping on protection scope of the present invention.
Claims (6)
1. the preparation method of a boar A type bacillus perfringens aluminium hydroxide inactivated vaccine, is characterized in that, comprises the following steps:
1) will by the lethal sick pig of clostridium, select pathological material of disease, scraping intestinal contents, cold preservation or freezing;
2) with plain chocolate as culture medium, 121 ℃ of autoclavings in pressure cooker, until temperature while dropping to 80 ℃, is poured pathological material of disease in pressure cooker into, anaerobism is cultivated;
3) by step 2) the bacterium liquid cultivated of mesohigh anaerobism filters centrifugally, and obtaining supernatant is toxin solution;
4) in the toxin solution obtaining, add 0.4% formaldehyde, shake well, deactivation detoxification in step 3); And
5) toxin solution of deactivation detoxification is mixed to synthetic vaccine after high-speed stirred according to the weight ratio of 4:1 with aluminum hydroxide adjuvant.
2. the preparation method of pig A type bacillus perfringens aluminium hydroxide inactivated vaccine according to claim 1, is characterized in that: in described step 1), cryogenic temperature is-20 ℃.
3. the preparation method of pig A type bacillus perfringens aluminium hydroxide inactivated vaccine according to claim 1, is characterized in that: described step 2), anaerobism condition of culture for cultivating 10-18 h at 37 ℃.
4. the preparation method of pig A type bacillus perfringens aluminium hydroxide inactivated vaccine according to claim 1, is characterized in that: in described step 3), filter centrifugal condition and be under 3000 revs/min centrifugal 20 minutes.
5. the preparation method of pig A type bacillus perfringens aluminium hydroxide inactivated vaccine according to claim 1, is characterized in that: in described step 4), deactivation detoxification condition is deactivation detoxification 3-5 days at 37 ℃.
6. the preparation method of pig A type bacillus perfringens aluminium hydroxide inactivated vaccine according to claim 5, is characterized in that: in described step 4), between deactivation During Detoxification every 4 hours intermittent oscillatings.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108273051A (en) * | 2018-02-02 | 2018-07-13 | 鄂尔多斯市旭和畜牧有限责任公司 | The rapidly and efficiently preparation method of pig A type C.perfringens inactivated vaccines |
| CN111500482A (en) * | 2019-01-31 | 2020-08-07 | 内蒙古金宇保灵生物技术研究院有限公司 | Sheep A-type clostridium perfringens strain, inactivated vaccine thereof and vaccine preparation method |
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- 2013-12-31 CN CN201310749053.9A patent/CN103721254A/en active Pending
Patent Citations (3)
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| CN1269243A (en) * | 1999-04-01 | 2000-10-11 | 农业部兰州生物药厂 | Deactivated clostridium perfringens disease vaccine for ox and its preparation |
| CN101708332A (en) * | 2009-11-30 | 2010-05-19 | 哈药集团生物疫苗有限公司 | Rabbit triple inactivated vaccine, preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108273051A (en) * | 2018-02-02 | 2018-07-13 | 鄂尔多斯市旭和畜牧有限责任公司 | The rapidly and efficiently preparation method of pig A type C.perfringens inactivated vaccines |
| CN111500482A (en) * | 2019-01-31 | 2020-08-07 | 内蒙古金宇保灵生物技术研究院有限公司 | Sheep A-type clostridium perfringens strain, inactivated vaccine thereof and vaccine preparation method |
| CN111500482B (en) * | 2019-01-31 | 2023-09-05 | 内蒙古金宇保灵生物技术研究院有限公司 | Clostridium perfringens type A strain of sheep, inactivated vaccine and vaccine preparation method |
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Application publication date: 20140416 |