CN101948781A - Culture medium for production of riemerella anatipestifer vaccine and preparation method thereof - Google Patents
Culture medium for production of riemerella anatipestifer vaccine and preparation method thereof Download PDFInfo
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- CN101948781A CN101948781A CN 201010268808 CN201010268808A CN101948781A CN 101948781 A CN101948781 A CN 101948781A CN 201010268808 CN201010268808 CN 201010268808 CN 201010268808 A CN201010268808 A CN 201010268808A CN 101948781 A CN101948781 A CN 101948781A
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Abstract
The invention discloses a culture medium for production of a riemerella anatipestifer vaccine, which comprises a basic culture medium and an additive. The basic culture medium comprises the following components in percentage: 1.5 to 1.9 percent of tryptone, 0.2 to 0.7 percent of yeast extract, 0.2 to 0.7 percent of beef extract, 0.2 to 0.7 percent of hydrolytic albumin, 0.1 to 0.4 percent of glucose, 0.5 percent of NaCl, 0.1 to 0.7 percent of monopotassium phosphate, 0.5 to 0.9 percent of Na2HPO4.12H2O, 0.1 to 0.2 percent of (NH4)2SO4 and 0.01 to 0.03 percent of NH4Cl, wherein the percentage of each component is weight/volume percentage. The additive is sterilely extracted infected-area strong cattle blood through a jugular vein blood taking or carotid artery blood bleeding method, is subjected to sterile fiber removal, is subjected to freeze thawing at the temperature of 20 DEG C below zero and at room temperature for three times, is preserved for later use at the temperature of 20 DEG C below zero, or is directly preserved at the temperature of 20 DEG C below zero after the blood is extracted and subjected to sterile fiber removal, and is subjected to freeze thawing for three times before use. The culture medium has the advantages of capacities of reducing cost, increasing nutrition, overcoming the defects of complex operation and high labor cost during culture by using live animals or embryos, and use for large-scale fermentation production of the riemerella anatipestifer vaccines.
Description
Technical field
The present invention relates to a kind of riemerella anatipestifer production of vaccine substratum and preparation method thereof.
Background technology
Infectious serositis of duck is a kind of contagious disease that betides tame duck, goose, turkey and multiple bird, claim riemerella anatipestifer disease, pest of duck Bacillus pasteurii disease, duck septicemia, pest of duck syndromes, pest of duck septicemia and new duck disease again, its cause of disease be riemerella anatipestifer (Riemerella anatipestifer, RA).This disease is acute sepsis or chronic infection, mainly shows as cough clinically, breathes, diarrhea, an eye nasal discharge increase, ataxia and neck tremble, and symptoms such as neck is crooked appear in the chronic case of minority; Typical cytopathic is fibrinous pericarditis, serohepatitis, airsacculitis, meningitis and caseous salpingitis.This disease all can take place throughout the year, and is serious with the morbidity in winter, mainly propagation such as the feed through polluting, drinking-water, the spittle; The duck in age in 1-8 week is extremely sensitive, and is higher with the duckling sickness rate in 2-3 age in week especially, can reach 90%; Mortality ratio is subjected to influence of various factors, and difference is bigger, often is 10-20%, but also have up to 60-75%.Stressors such as stocking density be excessive, it is smooth to ventilate, sanitary condition is poor, abrupt change of climate etc., can bring out the generation of this disease, and mortality ratio improves greatly; Anti-duck excessively can show nervous symptoms, grows slowly, becomes thin, and the price of deed descends, and has a strong impact on the production achievement, causes serious economy loss.
Infectious serositis of duck is at present duck one of the most serious transmissible disease that already works the mischief to be supported in countries in the world, worldwide distributes, and almost adopting intensification to support countries that duck produces at all has discovery, support duck to the world and already caused enormous economic loss.China is a foster duck big country, support the duck industry and in the rural economy of China, be seized of consequence, also be people one of the important sources of required meat egg of living, infectious serositis of duck is geographic popular more serious each foster duck of China, be that China duckery (particularly commodity duck field) is difficult to tackle, cause supports duck one of the main transmissible disease of loss of helping already most, the resistance of RA strengthens gradually in addition, supports duck to China and has already caused the tremendous economic loss.
Because infectious serositis of duck provisions duck already brings serious economy loss, relevant its immunoprophylaxis research has caused increasing concern.Although RA serotype is more; substantially there is not cross-protection between various; brought bigger difficulty for the development of vaccine; but because of vaccine inoculation is the effective measure of silent Salmonella in the prevention pest of duck; therefore the research of RA vaccine has caused veterinary biologics field scientific research personnel's extensive concern, at present the RA vaccine of research report mainly contain the RA inactivated vaccine (have only a kind of serotype the unit price inactivated vaccine, contain the multivalent inactivated vaccine of multiple serotype), RA and E.coli bivalent inactivated vaccine, RA attenuated live vaccines, extract the subunit vaccine that obtains RA subunit composition.But also do not have government permission production to use silent Salmonella vaccine in the pest of duck at present both at home and abroad, be mostly oneself vaccine (be own limited production, oneself uses).
No matter produce the vaccine of which kind of type of silent Salmonella vaccine in the pest of duck, its basis all relates to the cultivation of silent Salmonella in the pest of duck, but the culture condition of silent Salmonella requires high in the pest of duck, on common microbial culture prescription or matrix, be difficult to growth, related culture medium mainly contains following a few class in the Salmonella culturing process of writing from memory in the pest of duck at present: the first kind is the commercialization special culture media, as tryptic soy broth, brain-heart broth etc., this class substratum price is higher; Second class is to add the animal blood of higher proportion or serum (as 10% calf serum or take off the nutrient broth of fine sheep blood) in ordinary culture medium; The 3rd class is to cultivate with the animal or the embryo of living.In the above-mentioned three class culture mediumes, animal or the embryo culture of living is more loaded down with trivial details, workload is big, and can not be used for the large scale fermentation production of batch production; The first kind commercialization special culture media and second class add the high and present effect report that is not useful on the production of batch production large scale fermentation of method cost of higher proportion animal blood or serum in ordinary culture medium.
Summary of the invention
Substratum (the called after: the N-J synthetic medium) that the object of the present invention is to provide a kind of riemerella anatipestifer production of vaccine to use, can be used for the riemerella anatipestifer large scale fermentation, and can reduce in the pest of duck in the production of silent Salmonella vaccine the cost of this production link of large scale fermentation of silent Salmonella in the pest of duck.
Riemerella anatipestifer production of vaccine special culture media of the present invention (N-J synthetic medium) extremely is fit to the riemerella anatipestifer large scale fermentation, comprises the preparation procedure of basic medium composition and proportioning, additive and pretreatment process thereof and ratio, substratum.
A kind of riemerella anatipestifer production of vaccine substratum comprises basic medium and additive, and the component of described basic medium and ratio are: Tryptones, 1.5-1.9%; Yeast extract, 0.2-0.7%; Beef extract, 0.2-0.7%; Lactoalbumin hydrolysate, 0.2-0.7%; Glucose, 0.1-0.4%; NaCl, 0.5%; Potassium primary phosphate, 0.1-0.7%; Dipotassium hydrogen phosphate, 0.1-0.7%; Na
2HPO
412H
2O, 0.5-0.9%; (NH
4)
2SO
4, 0.1-0.2%; NH
4Cl, 0.01-0.03%; Wherein said each percentages of ingredients is a weight/volume percent; Described additive is taked healthy and strong ox blood of Pest-or disease-free area and the aseptic fibre that takes off for the method that adopts jugular vein blood sampling or carotid artery bloodletting is aseptic, in-20 ℃ and room temperature freeze thawing, behind the multigelation 3 times, be stored in-20 ℃ of standby or blood samplings and asepticly directly be stored in-20 ℃ after taking off fibre, take out multigelation 3 times before using.
Described riemerella anatipestifer production of vaccine substratum, the consumption of described additive is: cracking blood cell whole blood 0.8-1.5% by volume adds in the basic medium.
A kind of riemerella anatipestifer production of vaccine preparation method of substratum, may further comprise the steps, at first take by weighing after the required basic medium component 121 ℃ of sterilizations 15 minutes according to the described proportioning of claim 1, composition subject to sterilization is cooled to when being lower than 40-50 ℃ described additive is added in the described basic medium, and substratum gets product after shaking up.
The described riemerella anatipestifer production of vaccine preparation method of substratum, the consumption of described additive is: 0.8-1.5% adds in the basic medium by volume.
For example: the concrete prescription of configuration 100ml basic medium is: Tryptones, 1.8g; Yeast extract, 0.4g; Beef extract, 0.4g; Lactoalbumin hydrolysate, 0.4g; Glucose, 0.2g; NaCl, 0.5g; Potassium primary phosphate, 0.2g; Dipotassium hydrogen phosphate, 0.2g; Na
2HPO
412H
2O, 0.5g; (NH
4)
2SO
4, 0.1g; NH
4Cl, 0.01g; Water adds to 100mL; With 121 ℃ of sterilizations of above-mentioned each component 15 minutes, composition subject to sterilization was cooled to when being lower than 50 ℃, added additive cracking blood cell whole blood 1ml then in above-mentioned basic medium; The N-J synthetic medium gets product after shaking up.
Because riemerella anatipestifer substratum of the present invention adopts the basic medium of multiple composition and the pretreated whole blood of cracking blood cell as additive, can be used for the riemerella anatipestifer large scale fermentation and produce the riemerella anatipestifer vaccine, can reduce culture medium cost simultaneously, overcome, in cultivation, large scale fermentation and the production riemerella anatipestifer vaccine of riemerella anatipestifer, have great advantage or irreplaceable effect with problems such as living animal or embryo culture complicated operation and cost of labor height.
One, reduces cost.Because the composition of basic medium is the low conventional reagent of price, and additive cracking blood cell whole blood is through pre-treatment, the nutrition that blood cell comprised discharges fully, the ratio that adds blood reduces greatly, so cost reduces greatly than commercialization special culture media (tryptic soy broth, brain-heart broth etc.) and adding 10% calf serum or the cost that takes off the nutrient broth medium of fine sheep blood.
Two, increase nutrition, shortened incubation time.Because the blood cell that adds is carried out repeatedly cracked pre-treatment, and after the blood cell cracking, nutritive ingredient is discharged in the substratum fully, increased nutrition greatly on the one hand, bacterium is contacted as early as possible and use required necessary nutrition, so shortened incubation time.
Three, overcome with living animal or embryo culture complicated operation and the high deficiency of cost of labor.Need body animal or embryo one by one be inoculated with living animal or embryo culture, results also are that one by one body animal or embryo collected during thalline, so complicated operation, cost great amount of manpower, cost of labor height.Produce the riemerella anatipestifer thalline and adopt basal culture medium to can be used for the riemerella anatipestifer large scale fermentation, easy and simple to handle, time saving and energy saving, cost of labor is low.
Four, can be used for large scale fermentation and produce the riemerella anatipestifer vaccine.Present various culture mediumes all are under laboratory study or small-scale production condition, do not have and adopt fermentor tank to be used for the example that large scale fermentation is produced the riemerella anatipestifer vaccine, and the present invention can be used for large scale fermentation production riemerella anatipestifer vaccine.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The preparation of 0.8% cracking blood cell whole blood N-J synthetic medium:
The 0.8% cracking blood cell whole blood N-J synthetic medium of configuration 100ml: Tryptones: 1.5g; Yeast extract: 0.4g; Beef extract: 0.5g; Lactoalbumin hydrolysate: 0.6g; Glucose: 0.2g; NaCl:0.5g; Potassium primary phosphate: 0.18g; Dipotassium hydrogen phosphate: 0.18g; Na
2HPO
412H
2O:0.7g; (NH
4)
2SO
4: 0.12g; NH
4Cl:0.02g; Water adds to 100mL; With 121 ℃ of sterilizations of above-mentioned each component 15 minutes, composition subject to sterilization was cooled to when being lower than 50 ℃, added additive cracking blood cell whole blood 0.8ml then in above-mentioned basic medium; The N-J synthetic medium gets product after shaking up.
Described additive cracking blood cell whole blood is taked healthy and strong ox blood of Pest-or disease-free area and the aseptic fibre that takes off for the method that adopts jugular vein blood sampling or carotid artery bloodletting is aseptic, in-20 ℃ and room temperature freeze thawing, behind the multigelation 3 times, be stored in-20 ℃ of standby or blood samplings and asepticly directly be stored in-20 ℃ after taking off fibre, take out multigelation before using and make for 3 times.
Embodiment 2
The preparation of 1% cracking blood cell whole blood N-J synthetic medium:
The 1% cracking blood cell whole blood N-J synthetic medium of configuration 100ml: Tryptones: 1.9g; Yeast extract: 0.7g; Beef extract: 0.7g; Lactoalbumin hydrolysate: 0.7g; Glucose: 0.4g; NaCl:0.5g; Potassium primary phosphate: 0.3g; Dipotassium hydrogen phosphate: 0.3g; Na
2HPO
412H
2O:0.9g; (NH
4)
2SO
4: 0.2g; NH
4Cl:0.03g; Water adds to 100mL; With 121 ℃ of sterilizations of above-mentioned each component 15 minutes, composition subject to sterilization was cooled to when being lower than 40 ℃, added additive cracking blood cell whole blood 1ml then in above-mentioned basic medium; The N-J synthetic medium gets product after shaking up.
Described additive cracking blood cell whole blood is taked healthy and strong ox blood of Pest-or disease-free area and the aseptic fibre that takes off for the method that adopts jugular vein blood sampling or carotid artery bloodletting is aseptic, in-20 ℃ and room temperature freeze thawing, behind the multigelation 3 times, be stored in-20 ℃ of standby or blood samplings and asepticly directly be stored in-20 ℃ after taking off fibre, take out multigelation before using and make for 3 times.
Embodiment 3
Proof test:
1 test objective
Filter out the best medium of infectious serositis of duck inactivated vaccine fermentation usefulness.
2 test design
Behind different substratum fermentation culture serum I type riemerella anatipestifers, measure the bacterium number in its culture and compare its cost.
3 materials
3.1 bacterial classification serum I type riemerella anatipestifer RA-CH-I strain.
3.2 the chicken embryo is available from the chicken embryo of chicken house hatching to 10 ages in days.
3.3 reagent ordinary broth, extractum carnis are available from Hangzhou microorganism reagent company; Tryptic soy broth, peptone, Tryptones, lactoalbumin hydrolysate, yeast extract are available from BD company; NaCl, MgSO
4.7H
2O, glucose, potassium primary phosphate, dipotassium hydrogen phosphate, Na
2HPO
412H
2O, (NH
4)
2SO
4, NH
4Cl, VITMAIN B1 are homemade analytical pure, and new-born calf serum is homemade.
3.4 the tryptic soy broth of the ordinary broth of substratum 10% serum, tryptic soy broth, 10% serum, 1% cracking blood cell whole blood N-J synthetic medium and check are produced with standby after the assay was approved by " Chinese veterinary drug allusion quotation " appendix with substratum is all strict; 1% cracking blood cell whole blood N-J synthetic medium prescription adopts embodiment 2 listed prescriptions.
4 methods
RA is after the blood agar activation culture, be inoculated in the ordinary broth that contains 10% calf serum, putting 37 ℃ cultivated 24 hours, sampling is done purely after the assay was approved, inoculum size by 2% is inoculated in the ordinary broth of 10% serum respectively, tryptic soy broth, the tryptic soy broth of 10% serum, P-L meat soup, in 10% blood-serum P-L meat soup and the 1% cracking blood cell whole blood N-J synthetic medium, cultivate in the culture tank, blastochyle/idiosome is inoculated and collected to chicken embryo fine hair allantoic cavity according to a conventional method, the price of bacterial count result and substratum in mensuration and more several culture and the blastochyle/idiosome mixture, the output height, the conduct typing production technique that cost performance is good.
5 outcome evaluation
5.1 the result judges the production bacterium amount of judging various substratum according to the bacterial count result, selects which kind of substratum for use according to the production bacterium amount and the judgement of substratum price of various substratum.
5.2 the theory and technology maturation that this experiment of validity of test is used, the result judges that index is objective, and each link of whole experiment is strict operates, correctly assesses experimental result, and the result is if any doubtful point repetitive operation 2 times at least.Experimental scale is stable each time, thinks that thus this experimental result is effective.
6 results
See the following form 1:
Bacterial count in the different culture medium culturing things of table 1, substratum price and cost performance
This serum I type riemerella anatipestifer RA-CH-I strain is the highest in the tryptic soy broth and the output in the 1% cracking blood cell whole blood N-J synthetic medium of 10% serum, and output is minimum in P-L meat soup; The price of P-L meat soup and 1% cracking blood cell whole blood N-J synthetic medium is minimum; Best with 1% cracking blood cell whole blood N-J synthetic medium cultivation cost performance.
7 conclusions
This test-results shows, 1% cracking blood cell whole blood N-J synthetic medium is fit to do the substratum of riemerella anatipestifer large scale fermentation, and the production of infectious serositis of duck inactivated vaccine can be with 1% cracking blood cell whole blood N-J synthetic medium as the typing production technique with substratum.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (4)
1. a riemerella anatipestifer production of vaccine substratum is characterized in that, comprise basic medium and additive, the component of described basic medium and ratio are: Tryptones, 1.5-1.9%; Yeast extract, 0.2-0.7%; Beef extract, 0.2-0.7%; Lactoalbumin hydrolysate, 0.2-0.7%; Glucose, 0.1-0.4%; NaCl, 0.5%; Potassium primary phosphate, 0.1-0.7%; Dipotassium hydrogen phosphate, 0.1-0.7%; Na
2HPO
412H
2O, 0.5-0.9%; (NH
4)
2SO
4, 0.1-0.2%; NH
4Cl, 0.01-0.03%; Wherein said each percentages of ingredients is a weight/volume percent; Described additive is taked healthy and strong ox blood of Pest-or disease-free area and the aseptic fibre that takes off for the method that adopts jugular vein blood sampling or carotid artery bloodletting is aseptic, in-20 ℃ and room temperature freeze thawing, behind the multigelation 3 times, be stored in-20 ℃ of standby or blood samplings and asepticly directly be stored in-20 ℃ after taking off fibre, take out multigelation 3 times before using.
2. riemerella anatipestifer production of vaccine substratum according to claim 1 is characterized in that the consumption of described additive is: cracking blood cell whole blood 0.8-1.5% by volume adds in the basic medium.
3. the described riemerella anatipestifer production of vaccine of a claim 1 is with the preparation method of substratum, it is characterized in that, may further comprise the steps, at first take by weighing required basic medium component and mix back 121 ℃ of sterilizations 15 minutes according to the described proportioning of claim 1, composition subject to sterilization is cooled to when being lower than 40-50 ℃ described additive is added in the described basic medium, and substratum gets product after shaking up.
4. riemerella anatipestifer production of vaccine according to claim 3 is characterized in that the consumption of described additive is: in the basic medium of 0.8-1.5% adding by volume with the preparation method of substratum.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250792A (en) * | 2011-06-17 | 2011-11-23 | 四川农业大学 | Culture medium for riemerella anatipestifer |
| CN103656636A (en) * | 2013-12-13 | 2014-03-26 | 福建省农业科学院畜牧兽医研究所 | Method for preparing riemerlla antipestifer capsular antigen and propolis vaccine |
| CN105695375A (en) * | 2016-04-25 | 2016-06-22 | 四川农业大学 | Novel riemerella anatipestifer culture medium and preparation method thereof |
| CN107384830A (en) * | 2017-08-20 | 2017-11-24 | 云南省畜牧兽医科学院 | The cultural method and its culture medium of a kind of riemerella anatipestifer |
| CN108949619A (en) * | 2018-07-02 | 2018-12-07 | 广东省农业科学院动物卫生研究所 | A kind of zymotechnique of riemerella anatipestifer |
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| CN1724647A (en) * | 2005-06-30 | 2006-01-25 | 上海交通大学 | Preparation method of parachicken blood phili bacillus liquid culture medium |
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2010
- 2010-09-01 CN CN201010268808XA patent/CN101948781B/en not_active Expired - Fee Related
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| CN1724647A (en) * | 2005-06-30 | 2006-01-25 | 上海交通大学 | Preparation method of parachicken blood phili bacillus liquid culture medium |
Non-Patent Citations (2)
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| 《中国家禽》 20041231 蔡家利等 鸭疫里默氏杆菌培养特性的研究 第82到85页 1-4 第8卷, 第1期 2 * |
| 《江西农业大学学报》 20021031 邓舜洲等 鸭疫里氏杆菌（2型）灭活疫苗的研制 第569-573页 1-4 第24卷, 第5期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250792A (en) * | 2011-06-17 | 2011-11-23 | 四川农业大学 | Culture medium for riemerella anatipestifer |
| CN102250792B (en) * | 2011-06-17 | 2013-03-06 | 四川农业大学 | Culture medium for riemerella anatipestifer |
| CN103656636A (en) * | 2013-12-13 | 2014-03-26 | 福建省农业科学院畜牧兽医研究所 | Method for preparing riemerlla antipestifer capsular antigen and propolis vaccine |
| CN103656636B (en) * | 2013-12-13 | 2015-08-12 | 福建省农业科学院畜牧兽医研究所 | A kind of preparation method of Riemerellosis Anatipestifer k antigen Proplis_adjuvant vaccine |
| CN105695375A (en) * | 2016-04-25 | 2016-06-22 | 四川农业大学 | Novel riemerella anatipestifer culture medium and preparation method thereof |
| CN107384830A (en) * | 2017-08-20 | 2017-11-24 | 云南省畜牧兽医科学院 | The cultural method and its culture medium of a kind of riemerella anatipestifer |
| CN108949619A (en) * | 2018-07-02 | 2018-12-07 | 广东省农业科学院动物卫生研究所 | A kind of zymotechnique of riemerella anatipestifer |
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| CN101948781B (en) | 2012-07-25 |
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