CN107007827A - A kind of preparation method of poultry bacillus coli vaccine - Google Patents
A kind of preparation method of poultry bacillus coli vaccine Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of poultry bacillus coli vaccine, it is related to technical field of vaccines.The poultry bacillus coli vaccine that the present invention is provided, by extracting corresponding bacterium respectively from the diseased chicken liver of every plant, by amplification cultivation, inactivation, centrifugation, the process being resuspended, the bacterium amount being prepared into is 5 × 109‑2×1010CFU/mL bacillus coli vaccine, it has with strong points, and vaccine protective rate is high, and the harmfulness of vaccine is low; available for the whole production cycle of poultry, laying poultry particularly can be used for, to peak poultry of laying eggs; very little is influenceed, specific aim is good, and the excellent characteristics that production capacity is not also wasted.
Description
Technical field
The invention belongs to technical field of vaccines, and in particular to a kind of preparation method of poultry bacillus coli vaccine.
Background technology
Family avian escherichia coli be poultry all incidental bacterial disease of whole life cycle, be mainly presented with granuloma,
Air bag inflammation, peritonitis, cellulitis, omphalitis and salpingitis etc., Escherichia coli every year to China's poultry farming cause 10% with
On loss（Annual more than about 10,000,000,000 yuan losses）, and Escherichia coli are also easy to produce drug resistance, in the late two decades, poultry farming
Over range, the overdose problem of industry are very prominent, and certain influence is also resulted in public health security.Wish always in the industry
This sick generation, but the research and application of bacillus coli vaccine in decades can be controlled using bacillus coli vaccine, but always
There is no big breakthrough.Its main cause is Escherichia coli sero-group（More than 162）Many serotypes, blood serum subtype are more（It is thousands of
More than individual）, the vaccine between different sero-groups does not have cross protection or cross protection very poor, is exactly the epidemic disease of same sero-group type
Seedling, the vaccine than O157 groups, because H antigens, K antigens, adhesin, toxin, protection element, iron absorb mechanism and invaded in this group
Malicious difference is attacked, its protective rate to poultry also can very big difference.In China, production of vaccine manufacturer production ability is very big, 100 tons
With regard to that can produce 200,000,000 part vaccines, the yield of one month is exactly tens parts to fermentation tank within several days, and the poultry farms of China are supported
Small scale is grown, main scale is exactly thousands of to tens of thousands of, and often there are the baby chick of poultry farm not of the same race, Escherichia coli in areal
Also vary, e. coli serotype is more and does not have cross protection or cross protection very between different strains vaccine and wild bacterial strain
Difference, the bacterial strain of selection is only effective to some field or several fields of adopted bacterium, then invalid for local or nonlocal other plants,
Vaccine factory liquid production ability is huge but vaccine is poor for applicability, and this will result in the huge waste of vaccine, once produce several hundred million
Part but only has tens of thousands of parts effective.To solve this contradiction, vaccine factory attempts at most to choose 4 in different regions, 5 bacterial strains
Accomplish in a vaccine, it is intended to expand the spreadability of bacterial strain, but because the coli strain type of various regions is too many and fails eventually, institute
So that after the more than ten years are produced as a trial, vaccine factory has abandoned an avian escherichia coli production substantially.But colibacillosis is in itself, due near
Drug Resistance of E. coli is continuously increased over 20 years, is fallen ill but more serious.
The content of the invention
The technical problem to be solved in the present invention is that there is provided a kind of family's avian escherichia coli epidemic disease for above-mentioned the deficiencies in the prior art
The preparation method of seedling, solves many e. coli serotypes and does not have cross protection between different strains vaccine and open country bacterial strain or intersect
Protection is very poor, and vaccine factory liquid production ability is huge but vaccine is poor for applicability and the problem of easily causing the huge waste of vaccine, has
Targetedly strong, vaccine protective rate is high, and epidemic prevention effective time is long, and production capacity is not the characteristics of also waste.
In order to solve the above technical problems, the technical solution used in the present invention is：A kind of system of poultry bacillus coli vaccine
Make method, comprise the following steps：
（1）For there is the plant of demand, corresponding bacterium is extracted respectively from the diseased chicken liver of each plant, mark is simultaneously
It is standby；
（2）By the bacterium of each plant, cultivated, inactivated, centrifuged, be resuspended, obtain coli somatic；
（3）Coli somatic is prepared into for 5 × 10 by bacterium amount according to being actually needed for each plant9-2×1010CFU/mL
Bacillus coli vaccine.
Further, step（1）In, oneself vaccine only makees in each plant, using one-to-one mode, takes specific aim small
Yield solid culture.
Further, step（2）Described in the specific method cultivated be：By the bacterium of each plant, respectively in nutrition
Rule on plate, grow and differential medium is transferred to after bacterium, or directly rule on differential medium, be put into 37 DEG C of insulating boxs
In, after growing and meeting defined bacterium colony, the related multiple bacterium colonies of picking are put into amplification cultivation in nutrient broth, 18-30 small
Shi Hou, the Escherichia coli bacteria liquid of well-grown different breeding is respectively coated and is inoculated on nutrition plate, is supported according to each
The poultry quantity of field this batch is grown by a certain amount of coated Escherichia coli plate, is put into 18-30 hour of 37 DEG C of insulating box cultures.
Further, step（2）Described in the specific method that inactivates be：Well-grown Escherichia coli plate is taken out,
In super-clean bench, the Escherichia coli on plate are soaked with physiological saline, lawn is gently scraped with spreader, rinses and collect, obtain
To the Escherichia coli bacteria liquid of different breeding, 3.5-4.55 ‰ formaldehyde is added according to bacterium solution amount, 37 DEG C of shaking table vibrations is put into and goes out
It is living 3-5 days, carry out inactivation detection.
Further, step（2）In, centrifugal mixer speed is 4000-8000r/min, and centrifugation time is 10min；It is resuspended
Operate and be：Bacterium mud is collected, is resuspended 1-3 times with physiological saline, centrifugation, removes inactivation formaldehyde and coli somatic rupture is produced
A large amount of endotoxins and exotoxin.
Further, the differential medium is one kind in SS agar mediums, Yihong methylene blue culture medium.
Further, further serology or genetics identification are done to bacterium colony as needed（PCR）, to confirm large intestine bar
Bacterium.
Further, it is described inactivation detection concrete operations be：Picking inactivated bacterial liquid carries out the line detection of nutrition plate,
After 24-48 hours, Escherichia coli Growth is seen whether, without Escherichia coli Growth, it was demonstrated that Escherichia coli inactivate successfully.
Further, step（3）In, bacillus coli vaccine is divided into Escherichia coli water seedling or oil-emulsion inactivated vaccine, large intestine bar
Bacterium water seedling is directly diluted coli somatic by physiological saline and is made；Oil emulsion inactivated vaccine is made according to standard practice instructions.
It is using the beneficial effect produced by above-mentioned technical proposal：A kind of making of poultry bacillus coli vaccine of the present invention
Method, with strong points, vaccine protective rate is high, and vaccine harmfulness is low, and production capacity is not the characteristics of also waste, meanwhile, solid is put down
Ware culture is fewer than nutrition class impurity contained by bacterium solution obtained by conventional nutrient rich liquid culture, and vaccine is also small using side effect, can use
It in the whole production cycle of poultry, particularly can be used for laying poultry, to peak poultry of laying eggs, influence very little, do a seedling and prevent
Epidemic disease effective time was at 3 to 4 months.Epidemic prevention effective time can reach 6 to 8 months twice, and some field kind chicken house and father and mother for generations
In generation, plants chicken house, and continuous using twice to three times, the epidemic prevention time is 1 year.
Embodiment
With reference to specific embodiment, the present invention is further detailed explanation.
Embodiment 1：
A kind of preparation method of poultry bacillus coli vaccine, comprises the following steps：
（1）For there is a certain plant of demand, bacterium is extracted from diseased chicken liver；
（2）Rule on nutrition plate, grow and SS agar mediums are transferred to after bacterium, or directly drawn on SS agar mediums
Line, is put into 37 DEG C of insulating boxs, after growing and meeting defined bacterium colony, identification：
Serological Identification：Escherichia coli bacteria liquid is dripped on to a glass plate for pulling each and every one grid, a drop mark is then added
Pseudotype number（Totally 167 kinds）Serum, stirred with sterile toothpick, static 1 to 2 minutes, be exactly the serotype if there is muddiness, do not have
Have muddy reaction, with regard to whether the serotype, the serotype of Escherichia coli is confirmed with this；
PCR is identified：DNA of bacteria is extracted first with standard DNA extracts kit, PCR reaction systems are then set up, by the system
The amplification of Escherichia coli target gene segment needed for being carried out in PCR instrument is put into, if characterizing gene fragment can be amplified, is proved
This bacterium is exactly Escherichia coli；
The related multiple bacterium colonies of picking are put into 18 hours of amplification cultivation in nutrient broth, by well-grown Escherichia coli bacteria liquid
It is respectively coated and is inoculated on nutrition plate, is put down a certain amount of coated Escherichia coli according to this batch of poultry quantity of the plant
Ware, is put into 18 hours of 37 DEG C of insulating box cultures；Well-grown Escherichia coli plate is taken out, in super-clean bench, physiology is used
Escherichia coli on saline sook plate, lawn is gently scraped with spreader, is rinsed and is collected, and obtains the large intestine bar of the plant
Bacterium bacterium solution, according to the formaldehyde of bacterium solution amount addition 3.5 ‰, is put into 37 DEG C of shaking tables vibration inactivations, after 3 days, carries out inactivation detection；It will go out
The Escherichia coli bacteria liquid lived carries out centrifugation 10min with 8000r/min rotating speeds, collects bacterium mud, is resuspended 1 time with physiological saline, from
Gains in depth of comprehension are to coli somatic；
（3）Coli somatic is prepared into for 2 × 10 by bacterium amount according to being actually needed for the plant10CFU/mL large intestine bar
Bacterium water seedling.
Certain plants chicken house for generations, and 10,000 sets of imports breeder for generations, 2015 second half year issue more than 50 client father and mother and plant chick in generation,
There is nail navel, draws white excrement situation in 95% client, occurs mortality after 5 days, the death rate occurs about between 8-20% within first 20 days,
Subsequent chick and young chicken appearance growth are irregular, develop value 15% or so not up to standard.In the end of the year 2015, field censorship is for generations for generations for this
Disease chicken, father and mother's Dai Mao eggs etc., extract through bacterium, cultivate and identify, be Escherichia coli.Bacillus coli vaccine is made according to this method
Afterwards, interval is immunized for 20 days twice, and vaccine during use, has no obvious egg drop reduction.This for generations the end of the year of field 2016 feed back,
This issues more than 100 client at a year and a day in 2016, and 2% death, Fu Mudai are had more than within first 10 days without a kind chicken house reflection
The growth of breeder also complies with the kind and raises requirement.
Embodiment 2：
A kind of preparation method of poultry bacillus coli vaccine, comprises the following steps：
（1）For there is the plant of demand, corresponding bacterium is extracted respectively from the diseased chicken liver of plant, mark and standby；
（2）By the bacterium of each plant, rule respectively on nutrition plate, grow and Yihong methylene blue culture medium is transferred to after bacterium,
Or directly rule on the methylene blue culture medium of Yihong, it is put into 37 DEG C of insulating boxs, after growing and meeting defined bacterium colony, picking is related
Multiple bacterium colonies be put into 30 hours of amplification cultivation in nutrient broth, by the Escherichia coli bacteria liquid of well-grown different breeding
It is respectively coated and is inoculated on nutrition plate, according to each this batch of poultry quantity of plant by a certain amount of coated Escherichia coli
Plate, is put into 30 hours of 37 DEG C of insulating box cultures；Well-grown Escherichia coli plate is taken out, in super-clean bench, with life
The Escherichia coli on saline sook plate are managed, lawn is gently scraped with spreader, rinses and collect, the big of different breeding is obtained
Enterobacteria bacterium solution, according to the formaldehyde of bacterium solution amount addition 4 ‰, is put into 37 DEG C of shaking tables vibrations and inactivates 5 days, carry out inactivation detection；It will go out
The Escherichia coli bacteria liquid lived carries out centrifugation 10min with 4000r/min rotating speeds, collects bacterium mud, is resuspended 3 times, obtained with physiological saline
To coli somatic；
（3）Coli somatic can be prepared into for 5 × 10 according to being actually needed for each plant by bacterium amount9CFU/mL's is big
Enterobacteria water seedling or oil-emulsion inactivated vaccine.
In step（1）In, oneself vaccine only makees in each plant, using one-to-one mode, takes the small yield of specific aim to consolidate
Body culture, with strong points, vaccine protective rate is high, and production capacity is not also wasted.
In step（2）In, in inactivation detection, picking inactivated bacterial liquid carries out the line detection of nutrition plate, cultivates 48 hours
Afterwards, without Escherichia coli Growth, it was demonstrated that Escherichia coli inactivate successfully.
In step（2）In, it is 3 times to overhang number of times, and a large amount of of generation are ruptured in order to ensure removing inactivation formaldehyde and thalline
Endotoxin and exotoxin, substantially reduce the harmfulness of vaccine, available for the whole production cycle of poultry, particularly can be used to produce
Tanka fowl, to peak poultry of laying eggs, influences very little.
Plant to raise in field in a sea blue Huen father and mother's generation and have 9000 sets of 250 age in days, average disease chicken more than 30 dead daily, week
Death and culling rate is up to more than 210, and average egg production is 80%, and sampling separation of bacterial is accredited as Escherichia coli, makes big using this method
Enterobacteria seedling, is immunized once, has no to lay eggs during using vaccine and be decreased obviously, and this is reflected after one month, and all death and culling rates decline
For 5 or so, such case is maintained 5 months always, and laying rate has risen, average out to 83%.
Embodiment 3：
A kind of preparation method of poultry bacillus coli vaccine, comprises the following steps：
（1）For there is the plant of demand, corresponding bacterium is extracted respectively from the diseased chicken liver of each plant, mark is simultaneously
It is standby；
（2）By the bacterium of each plant, rule respectively on nutrition plate, grow and SS agar mediums are transferred to after bacterium, or
Directly rule, be put into 37 DEG C of insulating boxs on SS agar mediums, wait grow meet as defined in after bacterium colony, related many of picking
Individual bacterium colony is put into 25 hours of amplification cultivation in nutrient broth, and the Escherichia coli bacteria liquid of well-grown different breeding is distinguished
Coating is inoculated on nutrition plate, is put down a certain amount of coated Escherichia coli according to each this batch of poultry quantity of plant
Ware, is put into 25 hours of 37 DEG C of insulating box cultures；Well-grown Escherichia coli plate is taken out, in super-clean bench, physiology is used
Escherichia coli on saline sook plate, lawn is gently scraped with spreader, is rinsed and is collected, and obtains the large intestine of different breeding
Bacillus bacterium solution, according to the formaldehyde of bacterium solution amount addition 4.5 ‰, is put into 37 DEG C of shaking tables vibrations and inactivates 4 days, detected；It will inactivate
Escherichia coli bacteria liquid centrifugation 10min is carried out with 5000r/min rotating speeds, collect bacterium mud, be resuspended 2 times with physiological saline, obtain big
Enterobacteria thalline；
（3）Coli somatic can be prepared into for 10 according to being actually needed for each plant by bacterium amount10CFU/mL large intestine
Bacillus water seedling.
In step（2）In, differential medium can also be used such as the training of Yihong methylene blue except that can use SS agar mediums
Support the conventional culture mediums such as base.
In step（3）In, water seedling is directly diluted thalline by physiological saline and is made, and obtained Escherichia coli water seedling can be straight
Connect and use, Escherichia coli water seedling is put thalline after a period of time and can precipitated under gravity, and the used time shakes up again.
The blue grey laying hen in more than 8000 200 age in days sea in certain plant, totally 2 chickens, each 4000, incidence is the same：
Death and culling rate is high, mainly draws white excrement, is stained with hip and water is let out, cut open inspection is mainly core bag liver and yolk peritonitis, uses large intestine bar
During bacterium medicine, death and culling rate is reduced, and the first quarter moon state of an illness just recurs after medication.Afterwards in test, 4000 medications, 4000 separation large intestine bars
Bacterium is cooked this plant bacillus coli vaccine, and obvious egg drop reduction is there are no when vaccine is used.After three months, medication group extremely washes in a pan 352
Only, medication 6 times, 2800 yuan, symptom does not mitigate.Another vaccine group, medication is once, 410 yuan, dead to wash in a pan 43, draws white loose stool to be stained with
Hip, water the phenomenon such as are let out and disappeared, and do not recur, chicken feather integrity degree is generally better than medication group.
Obtained poultry bacillus coli vaccine in above-described embodiment, with strong points, vaccine protective rate height, vaccine
The characteristics of harmfulness is low, available for the whole production cycle of poultry, particularly can be used for laying poultry, to peak poultry of laying eggs,
Very little is influenceed, a seedling is done and prevents epidemic effective time at 3 to 4 months.Epidemic prevention effective time can reach 6 to 8 months twice, and some
Field kind chicken house and Parent stock, are continuously used twice to three times, the epidemic prevention time is 1 year, and production capacity is not wasted for generations
And less economic loss.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, the present invention
Claimed scope is by appended claims, specification and its equivalent thereof.
Claims (9)
1. a kind of preparation method of poultry bacillus coli vaccine, it is characterised in that comprise the following steps：
（1）For there is the plant of demand, corresponding bacterium is extracted respectively from the diseased chicken liver of each plant, mark is simultaneously
It is standby；
（2）By the bacterium of each plant, cultivated, inactivated, centrifuged, be resuspended, obtain coli somatic；
（3）Coli somatic is prepared into for 5 × 10 by bacterium amount according to being actually needed for each plant9-2×1010CFU/mL
Bacillus coli vaccine.
2. the preparation method of poultry bacillus coli vaccine according to claim 1, it is characterised in that step（1）In, each
Oneself vaccine only makees in plant, takes the small yield solid culture of specific aim.
3. the preparation method of poultry bacillus coli vaccine according to claim 1, it is characterised in that step（2）Described in train
Foster specific method is：By the bacterium of each plant, rule respectively on nutrition plate, grow and discriminating culture is transferred to after bacterium
Base, or directly rule on differential medium, it is put into 37 DEG C of insulating boxs, after growing and meeting defined bacterium colony, picking is related
Multiple bacterium colonies be put into amplification cultivation in nutrient broth, after 18-30 hour, by the large intestine bar of well-grown different breeding
Bacterium bacterium solution, which is respectively coated, to be inoculated on nutrition plate, will be a certain amount of coated big according to each this batch of poultry quantity of plant
Enterobacteria plate, is put into 18-30 hour of 37 DEG C of insulating box cultures.
4. the preparation method of poultry bacillus coli vaccine according to claim 1, it is characterised in that step（2）Described in go out
Specific method living is：Well-grown Escherichia coli plate is taken out, in super-clean bench, soaked with physiological saline on plate
Escherichia coli, lawn is gently scraped with spreader, is rinsed and is collected, and the Escherichia coli bacteria liquid of different breeding is obtained, according to bacterium
Liquid measure adds 3.5-4.55 ‰ formaldehyde, is put into 37 DEG C of shaking table vibrations and inactivates 3-5 days, carries out inactivation detection.
5. the preparation method of poultry bacillus coli vaccine according to claim 1, it is characterised in that step（2）In, centrifugation
Stir speed (S.S.) is 4000-8000r/min, and centrifugation time is 10min；Operation, which is resuspended, is：Bacterium mud is collected, 1- is resuspended with physiological saline
3 times.
6. the preparation method of poultry bacillus coli vaccine according to claim 3, it is characterised in that the differential medium
For one kind in SS agar mediums, Yihong methylene blue culture medium.
7. the preparation method of poultry bacillus coli vaccine according to claim 3, it is characterised in that as needed to bacterium colony
Further serology or genetics identification are done, to confirm Escherichia coli.
8. the preparation method of poultry bacillus coli vaccine according to claim 4, it is characterised in that the inactivation detection
Concrete operations are：Picking inactivated bacterial liquid carries out the line detection of nutrition plate, after 24-48 hours, has seen whether Escherichia coli life
Long, inactivating should be without bacterium colony generation on qualified rear nutrition plate.
9. the preparation method of poultry bacillus coli vaccine according to claim 1, it is characterised in that step（3）In, large intestine
Coli vaccine is divided into Escherichia coli water seedling or oil-emulsion inactivated vaccine, and Escherichia coli water seedling directly dilutes large intestine bar by physiological saline
Bacterium thalline is made；Oil emulsion inactivated vaccine is made according to standard practice instructions.
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| CN201710256514.7A CN107007827A (en) | 2017-04-19 | 2017-04-19 | A kind of preparation method of poultry bacillus coli vaccine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108676776A (en) * | 2018-05-30 | 2018-10-19 | 中国水产科学研究院珠江水产研究所 | A kind of enrichment and separation method of neutrophil cell |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4157390A (en) * | 1975-06-12 | 1979-06-05 | Internationale Octrooi Maatschappij "Octropa" B.V. | Process for vaccine preparation |
| CN103007266A (en) * | 2012-11-06 | 2013-04-03 | 湖北省农业科学院畜牧兽医研究所 | Predominant serum type vaccines in duck colibacillosis area, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108676776A (en) * | 2018-05-30 | 2018-10-19 | 中国水产科学研究院珠江水产研究所 | A kind of enrichment and separation method of neutrophil cell |
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