CN102698259B - Preparation method for necrotic enteritis (Type A) inactivated vaccine - Google Patents
Preparation method for necrotic enteritis (Type A) inactivated vaccine Download PDFInfo
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Abstract
The invention relates to a preparation method for a necrotic enteritis (Type A) inactivated vaccine for producing a synthetic culture medium. In China, an inactivated vaccine specific to necrotic enteritis is unavailable; and a sarco-liver-stomach enzyme digesting soup culture medium is generally adopted specific to clostridium disease inactivated vaccines of other animals, mainly comprises beef, liver and the like, and is influenced greatly by the varieties, ages, freshness and digesting degrees of animals; and under the influences of the varieties, ages, freshness and digesting degrees of animals, instable quality, high dispersing degree among batches and complexity in a manufacturing process are caused, the quality of a vaccine is influenced, and particularly the homogeneity of the vaccine is influenced. According to a synthesized culture medium provided by the invention, the defects are overcome, and production of a high-quality necrotic enteritis (Type A) inactivated vaccine is ensured powerfully; and the inactivated vaccine is the developing trend of bacteria high-cell-density culture, and has the advantages of easiness and convenience for operating and stable quality.
Description
Technical field
The present invention relates to a kind of chicken necrotizing enterocolitis（A type）Inactivated vaccine preparation method.Belong to veterinary biologicses manufacture
Field.
Background technology
Chicken necrotizing enterocolitis are also called clostridium enteritis（Clostridial enteritis）, flora is unbalance disease
（dysbacteriosis）Or the excessive disease of small gut bacterial growth（SIBO）.It is by bacillus perfringens（Clostridium
perfringens）A kind of disease that A type or c-type cause, A type bacillus perfringens produce alpha toxin, and c-type produces α and β toxin,
Both toxin are the primary toxins causing necrotic enteritis.The fowl that dies of illness has wheat bran sample with hepatomegaly, intestinal bleeding and intestinal mucosa
Necrosis region is characterized, and the sickness rate of normal chicken group is 1.3%～37.3%, and the sickness rate of specific pathogen free flock may be up to 62%（King
Pool China, Li Canheng. the diagnosis and treatment of chicken necrotizing enterocolitis, Chinese Poultry industry guide, 2002,19（14）：35-36）.
The domestic at present vaccine not still being directed to chicken necrotizing enterocolitis, treats this disease and mainly adopts antibiotic.Domestic A type shuttle
Mushroom production of vaccine uses lonely liver bouillon and meat liver stomach enzymic digestion soup culture medium, its main component beef of this culture medium and
Liver etc., is affected larger by animal varietiess, age, fresh and digestible degree, causes that culture medium quality is unstable, discrete between batch
Degree is big and manufacturing process is loaded down with trivial details, and then affects the quality of vaccine, the particularly homogeneity of vaccine.Large scale fermentation synthesis culture
Base designs according to bacillus perfringens Nutrition and Metabolism feature, with the addition of the life promoting growing microorganism, improving protective antigen content
The long factor, has the advantages that to produce poison amount height, steady quality, easy and simple to handle.Synthetic medium is the pass of bacterial vaccine quality
Key factor, in view of this, it is contemplated that a kind of suitable A type bacillus perfringens CVCC37 strain of design grows, product poison amount is high, one-tenth
Split-phase is to the synthetic medium determining, being easy to on-line checking, monitoring and timely adjustment, preparation A type bacillus perfringens inactivation epidemic disease
Seedling.
Content of the invention
A type bacillus perfringens CVCC37 strain seed liquor is inoculated in by the 1%～2% of culture medium total amount with pancreas casein peptone
With yeast extract powder in the liquid synthetic medium of main raw material(s), 37 DEG C of Anaerobic culturel/or quiescent culture 6h, culture terminates
It is centrifuged 30min by 3000r/min, removes thalline, system is concentrated by ultrafiltration with the millipore of 8Ku molecular cut off respectively and enters
Row be concentrated by ultrafiltration, through 0.6% formalin inactivation detoxification complete after, add final concentration 20% aluminium hydroxide gel be configured to epidemic disease
Seedling.
The present invention describes in detail
1. production of vaccine strain
（1）Source bacillus perfringens CVCC37 strain strain, former C57-1, F21A, virulent strain, supervised by Chinese veterinary medicament
Examine and identified, take care of and supply.
（2）Strain properties
Virulence：Meat liver stomach enzymic digestion soup culture supernatant intravenous injection, the lethal white mice of 0.05ml；0.1～1.0ml is lethal
Rabbit.
Immunogenicity：2.0ml concentrates aluminium glue Seedling immunizing rabbit and can produce 75%～100% protection.
2 culture medium adopt the synthetic medium of present invention design
（1）Formula (W/V) is as follows：
Peptone 1.5g, yeast powder 0.5g, sodium chloride 0.25g, dipotassium hydrogen phosphate 0.45g, glucose 0.5g, iron chloride
0.0001g, cysteine hydrochloride 0.05g, Vc0.001g, deionized water adds to 100ml.
（2）Synthetic medium compound method
Weigh each composition by formula to be dissolved in deionized water, adjust pH value to 8.0～8.5 with the sodium hydroxide of 2mol/L.
116 DEG C of autoclaving 30min.
（3）Synthetic medium is checked
1）Character solution clear, be in brown.
2）Steriling test presses Republic of China Veterinary Pharmacopoeia（Chinese veterinary pharmacopoeia committee. People's Republic of China's veterinary drug
Allusion quotation 2005 editions three. Chinese agriculture publishing house, 2006, hereinafter referred to as《Chinese veterinary pharmacopoeia》）The method of regulation is carried out, should be aseptic
Growth.
3）PH value should be 8.0～8.5.
4）Growth test
(1) seed liquor preparation
By A type bacillus perfringens bacterium CVCC37 strain（China Veterinery Drug Inspection Office provides）Freeze-drying lactobacillus are common with 1ml
Meat soup（China Veterinery Drug Inspection Office provides）After dilution, be inoculated in 10ml meat liver stomach enzymic digestion soup, put 37 DEG C culture 18～
24 hours, inoculation ordinary broth, plain agar inclined-plane, lonely liver bouillon and litmus milk each 2 were managed respectively simultaneously, often pipe 0.2ml,
Culture 48 hours, purely after the assay was approved as first order seed.Preserve at 2～8 DEG C, validity period must not exceed 15.Take one-level
Seed 0.1ml inoculates 10ml meat liver stomach enzymic digestion soup, cultivates 6 hours for 37 DEG C, purely as secondary seed after passed examination.（In
The Ministry of Agriculture of magnificent people's republic. in 2000 version of People's Republic of China's regulations. Chemical Industry Press,
2001, hereinafter referred to as《Code》）.
(2) bacterium solution culture and judgement
Seed liquor is pressed the 500ml triangular flask of 1% inoculation 200ml fluid medium, 37 DEG C of quiescent culture 6 hours, set simultaneously
Do not inoculate negative control for 1.Separately sampled 3000r/min is centrifuged 30min, takes supernatant by existing《Code》Carry out toxicity test.
Virulence should reach 50MLD/ml, and negative control answers asepsis growth.
5）Storage should be now with the current with effect duration fluid medium, places room temperature and at once use after autoclaving.
3 vaccine manufactures
（1）Prepared by seed liquor
By A type bacillus perfringens CVCC37 strain（China Veterinery Drug Inspection Office provides）Freeze-drying lactobacillus 1ml meat liver stomach
Enzymic digestion soup（China Veterinery Drug Inspection Office provides）After dilution, it is inoculated in 10ml meat liver stomach enzymic digestion soup, puts 37 DEG C of cultures
18～24 hours, inoculation ordinary broth, plain agar inclined-plane, lonely liver bouillon and litmus milk each 2 were managed respectively simultaneously, often manage
0.2ml, cultivates 48 hours, purely after the assay was approved as first order seed.Preserve at 2～8 DEG C, validity period must not exceed 15.
Take first order seed 0.1ml inoculation 10ml meat liver stomach enzymic digestion soup, cultivate 6 hours for 37 DEG C, purely as two grades of kinds after passed examination
Son（The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's regulations. chemical industry goes out
Version society, 2001, hereinafter referred to as《Code》）.
（2）Vaccine manufactures
Seed liquor is inoculated in pancreas casein peptone and yeast extract powder as main raw material(s) by the 1% of culture medium total amount
In liquid synthetic medium, 37 DEG C of Anaerobic culturel/or quiescent culture 6 hours.Culture terminates rear bacterium solution 3000r/min centrifugation
30min, after taking the ultrafiltration concentration system through 8Ku molecular cut off for the supernatant to concentrate, adds 0.6% formalin inactivation to take off immediately
Poison 4～6 days, inactivation detoxification adds the aluminium hydroxide gel of final concentration 20% and 0.006% thimerosal to make inactivated vaccine completely afterwards.
（3）Product inspection
After physical behavior this product standing, upper strata is yellowish-brown clear liquid, and lower floor is pale precipitation, after shaking uniformly
Suspension.
Steriling test is pressed existing《Chinese veterinary pharmacopoeia》Note is carried out, and no answers bacteria growing.
The broiler of safety verification subcutaneous vaccination 14 age in days 10, every 2ml, observes 10, should not occur being caused by vaccine
Any locally or systemically react.
The broiler of efficacy test subcutaneous injection 14 age in days 5, every 0.5ml, after 14 days, compare meat together with condition identical
Chicken 5, each intravenous injection 0.2ml（Containing 1MLD）A type clostridium perfringens toxoid, observes 5, and immune group at least 4/5 is protected, empty
White matched group 5/5 is dead.
Formaldehyde, thiomersal measure by existing《Chinese veterinary pharmacopoeia》Annex is carried out, and in product, residues of formaldehyde content should not
More than 0.5%, thimerosal residual content should be less than 0.01%.
The present invention relates to the information of microorganism A according to the present invention type aerogenesis pod clostridium bacterial strain, bacterium number respectively CVCC37,
CVCC52, CVCC2027, CVCC2030, CVCC2014, are all from the veterinary microorganism preservation of China of China Veterinery Drug Inspection Office
Administrative center（China of China Veterinery Drug Inspection Office veterinary microorganism preservation administrative center is write. Chinese veterinary's strain catalogue the
Two editions. Scientia Agricultura Sinica technology publishing house, 2002, p41, p43, p44, p45, p44）.
The positive effect of the present invention is domestic at present to there is no chicken necrotizing enterocolitis inactivated vaccine, and other A type perfringens shuttles
Mushroom vaccine generally uses meat liver stomach enzymic digestion soup or lonely liver bouillon culture medium, its main component beef of this culture medium and
Liver etc., is affected larger by animal varietiess, age, fresh and digestible degree, causes that culture medium quality is unstable, discrete between batch
Degree is big and manufacturing process is loaded down with trivial details, and then affects the quality of vaccine, the particularly homogeneity of vaccine.And synthesis provided by the present invention
Culture medium, overcomes above defect, is by the development trend of antibacterial high-cell-density cultivation, has easy and simple to handle, quality
Stable advantage, is that the chicken necrotizing enterocolitis inactivated vaccine production of high-quality provides sound assurance.
Specific embodiment
Embodiment 1
A type bacillus perfringens CVCC37 strain（Former C57-1 strain）The research of synthetic medium
This synthetic medium formula is filtered out by quiescent culture A type bacillus perfringens CVCC37 strain, this synthesis is trained
The preparation condition of foster base and condition of culture are optimized, and can be compared with the toxigenicity of meat liver stomach membrane digestion soup, and result should
Formula 6 hours toxigenicity of 37 DEG C of quiescent culture can reach 50～100MLD/ml, and Toxin producing C meets or exceeds meat liver stomach membrane digestion
Soup.The adaptability of this synthetic medium is studied, this synthetic medium of result is only applicable to the culture of CVCC37 strain.
1 material
1.1 culture medium raw materials
Pancreas casein peptone（Lot number VM732731-644）, purchased from MERCK company；Yeast extract（Lot number 911948）It is purchased from
OXOID company；Dipotassium hydrogen phosphate, glucose, iron chloride, cysteine hydrochloride, leucine, arginine, histidine, phenylpropyl alcohol ammonia
Acid, VB1、VK1, Vc, methionine be analysis pure chemistry reagent, purchased from Beijing chemical reagents corporation.
1.2 meat liver stomach membrane digestion soup meat soups（Lot number is 0130,0227,0307,0115,0119,0205）, A type test tube makes
Use specification 500ml with specification 25ml, triangular flask, basis set offer is cultivated by China Veterinery Drug Inspection Office.
1.3 strain
Bacillus perfringens CVCC37 strain, CVCC52 strain, CVCC2027 strain, CVCC2030 strain, CVCC2014 strain, 0.3ml/
, identified by China Veterinery Drug Inspection Office, take care of and supply.
1.4 animal
Mice：ICR system, 30～35 ages in days, body weight 16～20g, SPF level, by China Veterinery Drug Inspection Office's laboratory animal
Group is purchased and is provided.
2 methods and result
2.1 basic components screenings
From different combination formulas, by cultivating bacillus perfringens CVCC37 strain, carry out producing malicious effectiveness comparison, screen base
Plinth formula.
Weigh sodium chloride 2.5g, dipotassium hydrogen phosphate 4.5g, be dissolved in 1000ml deionized water, with the sodium hydroxide of 2mol/L
PH value is adjusted to be 8.0～8.5.Peptone and yeast powder are pressed different proportion（W/V）It is designed to 9 formula, be labeled as 1～9（Specifically
Ratio and numbering the results are shown in Table 1）, it is settled to 100ml respectively with above-mentioned buffer, 116 DEG C sterilize 30 minutes, set up meat liver simultaneously
Stomach enzymic digestion soup compares.By 1% addition seed liquor, 37 DEG C of quiescent culture 6 hours, take cultured bacterium solution 3000r/min centrifugation
30min, takes supernatant to survey poison.Result see table 1.
19 groups of basic components toxigenicity of table can survey malicious result
* toxin gelatin buffer is suitably diluted, 2 mices of diluent 0.2ml intravenous inoculation, in 24 hours, make
The whole dead highest extension rate of mice 2/2 is multiplied by 5, the minimum lethal dose of as every ml toxin（MLD）Number.As follows.
From table 1, formula 6 and formula 8 toxigenicity can be higher, are closer to meat liver stomach enzymic digestion soup.
The screening of 2.2 somatomedin
Choose toxigenicity and can make fluid medium close to 2 groups of basic components of meat liver stomach enzymic digestion soup, 116 DEG C of sterilizings
30 minutes, treat that it is down to room temperature, be dispensed in the A type test tube of sterilizing.A type test tube is separately added into through 0.22 μm of filtration sterilization
Glucose, iron chloride, cysteine hydrochloride, leucine, arginine, histidine, Phenylalanine, VB1、VK1、VC, first sulfur ammonia
The somatomedin such as acid（W/V ratio is shown in Table 2）, set up meat liver stomach enzymic digestion soup to compare simultaneously.By 1% addition seed liquor, 37 DEG C of standings
Culture 6 hours, takes cultured bacterium solution 3000r/min centrifugation 30min, takes supernatant to survey poison.Result see table 2.
Table 2 culture medium somatomedin screening test surveys malicious result（MLD/ml）
Can be seen that from table 2 result, glucose, iron chloride, cysteine hydrochloride and Vc culture medium produce poison certain promotion
Effect, basic components 8 are better than formula 6.
2.3 synthetic medium combined sortings
Each production factor is designed to several formula by various combination be respectively added in basal medium, adds by 1% and plant
Sub- liquid, 37 DEG C of quiescent culture 6 hours, its toxigenicity energy is measured by sampling.The results are shown in Table 3.
Table 3 basic components add combinations of. growth factors (W/V) screening comparative test and survey malicious result
* represent 10×Dilution toxin, intravenous inoculation mice 0.2ml, mice 2/2 is dead；And 20×Dilution toxin, mice 1/
2 is dead, therefore virulence is set to 50～100 (MLD/ml).Below "～" in concept with herewith state.
Find out from table 3 result：Suitable culture medium prescription (W/V) is 1.5% peptone+0.5% yeast powder+0.25% chlorination
Sodium+0.45% dipotassium hydrogen phosphate+0.5% glucose+0.0001% iron chloride+0.05% cysteine hydrochloride+0.001%Vc.
The optimization of 2.4 synthetic medium preparation conditions
Synthetic medium condition of high voltage, pH value and cultivation temperature are optimized, determine the most suitable sterilising conditions be 116 DEG C,
30min, optimum pH scope is 8.0～8.5, and adaptation system water is deionized water.The results are shown in Table 4, table 5, table 6.
The product poison result of the different condition of high voltage A type synthetic medium of table 4（MLD/ml）
The product poison result of table 5 different pH condition A type synthetic medium（MLD/ml）
Table 6 is different to prepare the malicious result of A type culture medium product under watering condition（MLD/ml）
The determination of 2.5 synthetic medium condition of culture
Prepare the culture medium of 6 300ml by synthetic medium formula respectively, the amount by 1% inoculates bacillus perfringens respectively
CVCC37 strain seed liquor, wherein 1 puts 37 DEG C of culture 20h, and period takes respectively at 4,6,8,10,12,14,16,18 and 20h priority
Sample 5ml；In addition prepare 5 culture medium, sample 5ml after putting 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C and 39 DEG C of culture 6h respectively, take thereon
Carry out clearly toxicity test, to determine optimal product poison time and cultivation temperature, result shows, synthetic medium cultivates CVCC37 strain
After 6h, virulence reaches 0.01～0.02ml/MLD, and with the prolongation of incubation time, virulence no longer strengthens, when cultivation temperature is 36
DEG C～37 DEG C when, culture medium product poison best results.The results are shown in Table 7, table 8.
The different incubation time toxigenicity of table 7 can survey malicious result
Under the different cultivation temperature of table 8, toxigenicity can survey malicious result
2.6 synthetic mediums the results are shown in Table 9 with the comparative test of meat liver stomach membrane digestion soup.
Table 9 synthetic medium and meat liver stomach enzymic digestion soup virulence comparative result（MLD/ml）
Find out from table 9 result, the alternative conventional medium of A type synthetic medium.
2.7 synthetic medium amplification culture tests
On the basis of a small amount of culture success, further amplification culture volume, make volume of culture respectively reach 1000,
2000th, 3000,4000,5000,6000,7000,8000,9000 and 10000ml, sampled at 6,12,18 and 24 hours respectively, right
The toxigenicity of culture medium can be measured, and result shows when volume of culture expands, and the toxigenicity of culture medium can be constant, is produced poison
The virulence of element all reaches 50MLD/ml.The results are shown in Table 10.
The different volume of culture of table 10 can survey malicious result in the toxigenicity of different incubation times（MLD/ml）
The culture experiment to different A type bacterial strains for 2.8 synthetic mediums
For determining the culture situation to different A type bacillus perfringens bacterial strains for the synthetic medium, take 5 plants of A type aerogenesis pod shuttles
Bacteria strain, bacterium number is respectively CVCC37, CVCC52, CVCC2027, CVCC2030, CVCC2014, is trained at 35 DEG C and 37 DEG C
Support, sampled at 5 hours and 7 hours respectively, its toxigenicity can be measured, result only CVCC37 strain grows in this culture medium
Well, aerogenesis is more, and 5～7 hours virulence of culture can reach 50MLD/ml.Remaining 4 plants of bacterium aerogenesis is few or not aerogenesis, cultivates 5～7
Hour, virulence all can not reach 50MLD/ml.The results are shown in Table 11.
Table 11 synthetic medium can survey malicious result (MLD/ml) to different A type strain culturing toxigenicity
Note：-, represent not aerogenesis；"+"～" +++ " writes on one's behalf gas production respectively from little to big.# represents 10×Dilution toxin, quiet
Arteries and veins Mice Inoculated 0.2ml, equal 0/2 death of mice；Low extension rate is surveyed poison and is not carried out.Thus virulence 0～50 (MLD/ml).
As can be seen from Table 11：Synthetic medium is only effective to A type bacillus perfringens CVCC37 strain.
3. conclusion
This synthetic medium formula is peptone 1.5g, yeast powder 0.5g, sodium chloride 0.25g, dipotassium hydrogen phosphate 0.45g,
Glucose 0.5g, iron chloride 0.0001g, cysteine hydrochloride 0.05g, Vc0.001g, deionized water adds to 100ml., produce poison
Performance is basically identical with meat liver stomach membrane digestion soup, can reach 50～100MLD/ml, but this culture medium is only applicable to perfringens shuttle
The culture of bacterium CVCC37 strain.
Embodiment 2
Synthetic medium culture A type toxin concentration technology test
Alpha toxin is A type bacillus perfringens Major Secretory toxin, is also main immunogens, its molecular weight about 43Ku.Will
Cultured A type bacillus perfringens CVCC37 strain bacterium solution, is centrifuged 30min through 3000r/min, removes thalline, uses difference respectively
The millipore of molecular cut off is concentrated by ultrafiltration system and is concentrated by ultrafiltration, and then carries out concentrating forward and backward volume quantitative, and takes
Concentrate forward and backward and peritoneal effluent sample each 10ml toxicity test.The yield rate of 10Ku molecular cut off is 40%, and it appears vena axillaris
Inoculation 0.2ml, mice 2/2 is dead；And 8Ku yield rate reaches 75%, its peritoneal effluent peritoneal effluent intravenous inoculation 0.2ml, mice 0/2 is dead
Die；The concentration of toxin suitably cultivated by the film bag of therefore 8Ku molecular cut off to A type bacterium（It is shown in Table 12）.
The film bag of table 12 PSPP concentrates result
Embodiment 3
Vaccine manufactures
（1）Prepared by seed liquor
By A type bacillus perfringens CVCC37 strain（China Veterinery Drug Inspection Office provides）Freeze-drying lactobacillus 1ml meat liver stomach
Enzymic digestion soup（China Veterinery Drug Inspection Office provides）After dilution, it is inoculated in 10ml meat liver stomach enzymic digestion soup, puts 37 DEG C of cultures
18～24 hours, inoculation ordinary broth, plain agar inclined-plane, lonely liver bouillon and litmus milk each 2 were managed respectively simultaneously, often manage
0.2ml, cultivates 48 hours, purely after the assay was approved as first order seed.Preserve at 2～8 DEG C, validity period must not exceed 15.
Take first order seed 0.1ml inoculation 10ml meat liver stomach enzymic digestion soup, cultivate 6 hours for 37 DEG C, purely as two grades of kinds after passed examination
Son（The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's regulations. chemical industry goes out
Version society, 2001, hereinafter referred to as《Code》）.
（2）Vaccine manufactures
Seed liquor is inoculated in pancreas casein peptone and yeast extract powder as main raw material(s) by the 1% of culture medium total amount
In liquid synthetic medium, 37 DEG C of quiescent culture 6 hours（Or Anaerobic culturel）.Culture terminates rear bacterium solution 3000r/min centrifugation
30min, after taking the ultrafiltration concentration system through 8Ku molecular cut off for the supernatant to concentrate, adds 0.6% formalin inactivation to take off immediately
Poison 4～6 days, inactivation detoxification adds the aluminium hydroxide gel of final concentration 20% and 0.006% thimerosal to make inactivated vaccine completely afterwards.
（3）Product inspection
After physical behavior this product standing, upper strata is yellowish-brown clear liquid, and lower floor is pale precipitation, after shaking uniformly
Suspension.
Steriling test is pressed existing《Chinese veterinary pharmacopoeia》Note is carried out, and no answers bacteria growing.
The broiler of safety verification subcutaneous vaccination 14 age in days 10, every 2ml, observes 10, does not occur being caused by vaccine
Any locally or systemically react.
The broiler of efficacy test subcutaneous injection 14 age in days 5, every 0.5ml, after 14 days, compare meat together with condition identical
Chicken 5, each intravenous injection 0.2ml（Containing 1MLD）A type clostridium perfringens toxoid, observes 5, immune group 4/5 is protected, blank right
Dead according to group 5/5.
Formaldehyde, thiomersal measure by existing《Chinese veterinary pharmacopoeia》Annex is carried out, and in product, residues of formaldehyde content all accords with
Close regulation.
Embodiment 4
Chicken necrotizing enterocolitis（A type）The research of inactivated vaccine
By cultured CVCC37 strain bacterium solution, 3000r/min centrifugation 30min takes supernatant, through being concentrated by ultrafiltration after system concentration,
Formalin with 0.6% inactivates detoxification, and inactivation detoxification adds the aluminium hydroxide gel of final concentration 20% and 0.006% sulfur completely afterwards
Willow hydrargyrum prepares seedling.Subcutaneous vaccination 14 Day-old Broiler Chickens 5, every 0.5ml, together with blank intravenous injection 0.2ml after 14 days
（Containing 1MLD）A type toxin carry out counteracting toxic substances, observe 5, result immune group 4/5 is protected, and matched group is all dead, shows vaccine
Immunogenicity is good.Its result see table 13.
The survey poison result to 28 Day-old Broiler Chickens for the table 13 A type toxin
This A type toxin is that every 1ml contains 10MLD to the minimum lethal dose of 28 Day-old Broiler Chickens as can be seen from Table 13, that is,
0.1ml/MLD.
14 3 batches of vaccine immunity counteracting toxic substances results of table
As can be seen from Table 14, the inactivated vaccine immunity of the A type bacillus perfringens CVCC37 strain of synthetic medium culture
Originality is good.
Embodiment 4
Chicken necrotizing enterocolitis（A type）Inactivated vaccine safety testing
3 batches of chicken necrotizing enterocolitis by preparation（A type）Inactivated vaccine, subcutaneous injection 14 Day-old Broiler Chickens respectively, every group 10,
Every 2ml（Containing 4 using dosages）, separately set 5 and be only used as blank, observe 10, any local that vaccine causes does not occur
And systemic adverse reactions, vaccine group and blank control group average weight gain no significant difference.Result see table 15,16.
15 3 batches of chicken necrotizing enterocolitis of table（A type）Inactivated vaccine safety testing result
16 3 batches of chicken necrotizing enterocolitis of table（A type）Inactivated vaccine safety testing body weight gains result
Note：
The method of toxin toxicity test：Toxin gelatin buffer is suitably diluted, each dilution factor tail vein injection body
Weigh the white mice 2 of 16～20g, every 0.2ml, be multiplied by 5 with the whole dead highest extension rate of white mice in 24 hours 2/2
Minimum lethal dose contained by this every 1ml toxin（MLD）Number.
Claims (1)
1. a kind of preparation method of chicken necrotizing enterocolitis A type inactivated vaccine is it is characterised in that by A type bacillus perfringens CVCC37
Strain seed liquor is inoculated in the liquid with pancreas casein peptone and yeast extract powder as main raw material(s) by the 1%～2% of culture medium total amount
In body synthetic medium, the culture/of 37 DEG C of anaerobic fermentations or quiescent culture 6 hours, culture terminate after through 3000r/min centrifugation
30min, removes thalline, system is concentrated by ultrafiltration with the millipore of 8Ku molecular cut off respectively and is concentrated by ultrafiltration, through 0.6%
Formalin inactivation detoxification complete after, add final concentration 20% aluminium hydroxide gel be configured to vaccine；
Used in it, the formula (W/V) of synthetic medium is：Peptone 1.5g, yeast powder 0.5g, sodium chloride 0.25g, phosphoric acid
Hydrogen dipotassium 0.45g, glucose 0.5g, iron chloride 0.0001g, cysteine hydrochloride 0.05g, Vc0.001g, deionized water adds
To 100ml.
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