CN102380092A - Inactivated vaccine for infectious coryza of chickens - Google Patents
Inactivated vaccine for infectious coryza of chickens Download PDFInfo
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- CN102380092A CN102380092A CN2011103729414A CN201110372941A CN102380092A CN 102380092 A CN102380092 A CN 102380092A CN 2011103729414 A CN2011103729414 A CN 2011103729414A CN 201110372941 A CN201110372941 A CN 201110372941A CN 102380092 A CN102380092 A CN 102380092A
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- inactivated vaccine
- infectious coryza
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Abstract
The invention relates to an inactivated vaccine for infectious coryza of chickens. The preparation method of the inactivated vaccine for infectious coryza of chickens comprises the following steps: using sterilized PBS (phosphate-buffered saline) to dilute inactivated haemophilus paragallinarum A type C-hpg-8 strain concentrated bacteria liquid prepared by the conventional method by a certain proportion, then mixing with vaccine adjuvants taking carbomer as a main component according to the proportion, stirring and shaking up so as to prepare the inactivated vaccine for infectious coryza of chickens. After a chicken is immunized by the inactivated vaccine for infectious coryza of chickens, the following advantages can be realized: the antibody titer generated by the inoculated chicken is high, the protection period of the antibody is long, and the protection rate against strong virus is high and the like.
Description
Technical field
The present invention relates to a kind of inactivated vaccine of infectious coryza of chicken, belong to field of biology, particularly the veterinary biologics field.
Background technology
The popular rhinitis inactivated vaccine of existing market mainly contains two types, comprises oily adjuvant and aluminium glue Adjuvanted vaccines.The oil emulsion inactivated vaccine antibody persistent period is longer, but has side reaction in various degree.There is not the side reaction phenomenon in the aluminium glue adjuvant inactivated vaccine, but antibody titer is low, and the persistent period is short.
The inactivated vaccine of infectious coryza of chicken of novel adjuvant preparation of the present invention; Have safe, no side reaction, antibody and produce advantages such as fast and protective rate height; Not only be applicable to chicken crowd routine immunization; Effectively prevent the generation of eqpidemic disease, and be applicable to morbidity chicken crowd's urgent inoculation, to controlling primary disease and preventing that recurrence from having better action.
Summary of the invention
The objective of the invention is in order to carbomer is the vaccine adjuvant that main component is formed; Use it in the production process of inactivated vaccine of infectious coryza of chicken; Make the inactivated vaccine of infectious coryza of chicken of production have safe, no side reaction, antibody produces advantages such as fast and protective rate height; Urgent inoculation when both being suitable for normal prevention and also being suitable for falling ill, to the control primary disease with prevent that recurrence from having better action.
The technology path of embodiment of the present invention:
1. inactivated vaccine of infectious coryza of chicken involved in the present invention, the haemophilus paragallinarum A type C-Hpg-8 strain that contains deactivation concentrate bacterium liquid and are the vaccine adjuvant that main component is formed with the carbomer.
The method for preparing of inactivated vaccine of infectious coryza of chicken; Haemophilus paragallinarum A type C-Hpg-8 strain by the deactivation of conventional method preparation concentrates bacterium liquid, uses behind the sterilization PBS dilution certain proportion with mixing and stir to shake up in 4: 1 ratios as the vaccine adjuvant of main component composition with carbomer and is prepared from inactivated vaccine of infectious coryza of chicken.
The specific embodiment
One, strain is used in the production of vaccine check
Manufacturing and check use strain to be haemophilus paragallinarum A type C-Hpg-8 strain, identify, take care of and supply by China Veterinery Drug Inspection Office.
Two, vaccine manufacturing
1. the vaccine manufacturing and the inspection of semifinished product
(1) production prepares with seed
1) the first order seed breeding is got the strain streak inoculation on Carnis Gallus domesticus soup agar plate with evaluation, is containing 5%～10%CO
2Cultivate after 16～18 hours for 37 ℃ in the environment, select the strong typical lawn (bacterium colony) of several fluorescences and be inoculated in the 5 age in days chick embryo yolk sacs, continue to hatch, collect Embryo Gallus domesticus yolk liquid dead in 30 hours at 37 ℃, after pure inspection is qualified, as-the level seed.Preserving below-20 ℃, should be no more than 1 month, otherwise will pass through the Embryo Gallus domesticus rejuvenation again, but should not surpass for 6 generations.
2) the Embryo Gallus domesticus yolk liquid of infection is got in the secondary seed breeding, and streak inoculation Carnis Gallus domesticus soup agar plate is containing 5%～10%CO
2Under the condition, cultivated 16～18 hours for 37 ℃, select the strong typical lawn (bacterium colony) of fluorescence to be inoculated in the chicken soup culture medium, put 37 ℃ and cultivated 16～20 hours, after pure inspection is qualified, be secondary seed.2～8 ℃ of preservations, must not be above 6～8 hours.
(2) seedling uses culture medium to be semisynthetic medium, and manufacturing approach is seen note 4.
(3) preparation of seedling bacterium liquid
1) bacterium liquid is cultivated the 100ml seed liquor is added in the 4000ml semisynthetic medium, cultivates 18～20 hours at 37 ℃, and its culture bottle secondary that shakes is indirectly confirmed no living contaminants through pure picking up, and adds 0.05% formalin, puts 2～8 ℃ of preservations.
2) concentrating with deactivation of seedling bacterium liquid concentrates with the hollow fiber membrane ultrafiltration device bacterium liquid that pure inspection is qualified; And then process suspension with the PBS of pH7.2; (compare with biological product national standard article " Chinese bacterial turbidity standard ", establishing standard pipe, to contain haemophilus paragallinarum be 2,800,000,000/ml), adds the thimerosal of 0.15% formalin and 0.01% more by volume to make every 1ml contain 5,000,000,000 thalline at least; 2～8 ℃ of sterilizations 7 days, through checking asepsis growth as seedling antigen.
(4) preparation of adjuvant
With the carbomer is the preparation of the vaccine adjuvant of main component composition:
NaCl 5～10g (colourless cube of crystallization or white crystals, Shanghai traditional Chinese medicines),
Na
2HPO
41.5～2g (white powder, Shanghai traditional Chinese medicines),
KCl 0.1～0.5g (colourless cubic crystal often is a long column shape, the Shanghai traditional Chinese medicines),
KH
2PO
40.1～0.5g (being colourless crystallization or white particle sprills, the Shanghai traditional Chinese medicines),
Carbomer 3～7g (white loose sprills, Beijing compatriots escape health Science and Technology Ltd.),
Deionized water 1000ml, dissolving is after 121 ℃ of high pressure 30min sterilization backs are subsequent use.Room temperature is preserved, and is no more than 15 days.
Novel adjuvant of the present invention, wherein carbomer is to contain the crosslinked acrylate copolymer of polyalkenyl polyether, (conventional amount used 0.25%～0.5%) just can produce thickening power efficiently under the very low consumption; Conventional amount used can substitute 3%～7% conventional emulsifier 0.2%～0.4%, can infectious coryza of chicken antigen parcel be adsorbed; After the inoculation; In the chicken body, can slowly discharge, can make antibody titer high, the duration is long.Na
2HPO
4And KH
2PO
4Can regulate pH, play the effect of buffer.The main effect of NaCl and KCl is the ion concentration that keeps certain, to keep activity.
(5) join Seedling
To concentrate rhinitis bacterium liquid with sterilization PBS (0.1mol/L; PH 7.0～7.2) mix and stir in 4: 1 ratios with novel adjuvant behind the dilution certain proportion and shake up, guarantee to contain in every 1ml vaccine 1,000,000,000 bacterium numbers (comparing judgement) with biological product national standard article " Chinese bacterial turbidity standard ".
Three, vaccine test:
1. steriling test carries out asepsis growth by " rules ".
2. safety verification is with 8 of the healthy susceptible chickens at 2～3 monthly ages, and every subcutaneous injection vaccine 1ml observed 14, should no abnormal reaction.
3. efficacy test is with 8 of the healthy susceptible chickens at 2～3 monthly ages, every subcutaneous injection vaccine 0.5ml.After 1 month; Together with 4 of the identical contrast chickens of condition, 16 hours culture 0.2ml (50～1,000,000 viable bacteria) of injection C-Hp8-8 bacterial strain Carnis Gallus domesticus soup in the hole observed 14 under each socket of the eye; The contrast chicken all fall ill (facial occur under a side or the two routine sockets of the eye hole and around swelling and rhinorrhea is arranged or has the person of shedding tears concurrently); The immunity chicken is protected 6 at least, or 3 morbidities of contrast chicken, and immune chicken is protected 7 at least for qualified.
4. formaldehyde, thimerosal assay are undertaken by " Chinese veterinary drug allusion quotation " prescriptive procedure respectively, should meet " pertinent regulations of goods check "≤0.4%.
Positive effect of the present invention
A kind of inactivated vaccine of infectious coryza of chicken that the present invention relates to.The method for preparing of this inactivated vaccine of infectious coryza of chicken; Be that haemophilus paragallinarum A type C-Hpg-8 strain by the deactivation of conventional method preparation concentrates bacterium liquid, use behind the sterilization PBS dilution certain proportion with mixing in proportion and stir to shake up as the vaccine adjuvant of main component composition and be prepared from inactivated vaccine of infectious coryza of chicken with carbomer.Behind this inactivated vaccine of infectious coryza of chicken immunity chicken, can make the antibody titer that produces behind the inoculation chicken high, the antibody protection period is long, counteracting toxic substances protective rate advantages of higher.
Embodiment 1
1 strain
Manufacturing and check use strain to be haemophilus paragallinarum A type C-Hpg-8 strain, identify, take care of and supply by China Veterinery Drug Inspection Office.
The 2 vaccine manufacturing and the inspections of semifinished product
(1) production prepares with seed
1) the first order seed breeding is got the strain streak inoculation on Carnis Gallus domesticus soup agar plate with evaluation, is containing 5%～10%CO
2Cultivate after 16～18 hours for 37 ℃ in the environment, select the strong typical lawn (bacterium colony) of several fluorescences and be inoculated in the 5 age in days chick embryo yolk sacs, continue to hatch, collect Embryo Gallus domesticus yolk liquid dead in 30 hours at 37 ℃, after pure inspection is qualified, as-the level seed.Preserving below-20 ℃, should be no more than 1 month, otherwise will pass through the Embryo Gallus domesticus rejuvenation again, but should not surpass for 6 generations.
2) the Embryo Gallus domesticus yolk liquid of infection is got in the secondary seed breeding, and streak inoculation Carnis Gallus domesticus soup agar plate is containing 5%～10%CO
2Under the condition, cultivated 16～18 hours for 37 ℃, select the strong typical lawn (bacterium colony) of fluorescence to be inoculated in the chicken soup culture medium, put 37 ℃ and cultivated 16～20 hours, after pure inspection is qualified, be secondary seed.2～8 ℃ of preservations, must not be above 6～8 hours.
(2) seedling uses culture medium to be semisynthetic medium, and manufacturing approach is seen note 4.
(3) preparation of seedling bacterium liquid
1) bacterium liquid is cultivated the 100ml seed liquor is added in the 4000ml semisynthetic medium, cultivates 18～20 hours at 37 ℃, and its culture bottle secondary that shakes is indirectly confirmed no living contaminants through pure picking up, and adds 0.05% formalin, puts 2～8 ℃ of preservations.
2) concentrating with deactivation of seedling bacterium liquid concentrates with the hollow fiber membrane ultrafiltration device bacterium liquid that pure inspection is qualified; And then process suspension with the PBS of pH7.2; Making every 1ml contain 5,000,000,000 thalline at least (compares with biological product national standard article " Chinese bacterial turbidity standard "; If it is 2,800,000,000/ml) that standard pipe contains haemophilus paragallinarum; The thimerosal that adds 0.15% formalin and 0.01% more by volume is 2～8 ℃ of sterilizations 7 days, through checking asepsis growth as seedling antigen (referring to " inactivated vaccine of infectious coryza of chicken manufacturing and inspection procedure ").
(4) preparation of adjuvant:
NaCl 5～10g (colourless cube of crystallization or white crystals, Shanghai traditional Chinese medicines),
Na
2HPO
41.5～2g (white powder, Shanghai traditional Chinese medicines),
KCl 0.1～0.5g (colourless cubic crystal often is a long column shape, the Shanghai traditional Chinese medicines),
KH
2PO
40.1～0.5g (being colourless crystallization or white particle sprills, the Shanghai traditional Chinese medicines),
Carbomer 3～7g (white loose sprills, Beijing compatriots escape health Science and Technology Ltd.),
Deionized water 1000ml, dissolving is after 121 ℃ of high pressure 30min sterilization backs are subsequent use.
(5) join Seedling
To concentrate rhinitis bacterium liquid with sterilization PBS (0.1mol/L; PH 7.0～7.2) mix and stir in 4: 1 ratios with novel adjuvant behind the dilution certain proportion and shake up, guarantee to contain in every 1ml vaccine 1,000,000,000 bacterium numbers (comparing judgement) with biological product national standard article " Chinese bacterial turbidity standard ".
Embodiment 2
According to " inactivated vaccine of infectious coryza of chicken manufacturing and inspection procedure " (The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's veterinary biologics rules. Chemical Industry Press; 2000, the present invention is hereinafter to be referred as " rules ") on the method for inspection vaccine is tested.
1. steriling test carries out asepsis growth by " rules ".
2. safety verification is with 8 of the healthy susceptible chickens at 2～3 monthly ages, and every subcutaneous injection vaccine 1ml observed 14, should no abnormal reaction.
3. efficacy test is with 8 of the healthy susceptible chickens at 2～3 monthly ages, every subcutaneous injection vaccine 0.5ml.After 1 month; Together with 4 of the identical contrast chickens of condition, 16 hours culture 0.2ml (50～1,000,000 viable bacteria) of injection C-Hp8-8 bacterial strain Carnis Gallus domesticus soup in the hole observed 14 under each socket of the eye; The contrast chicken all fall ill (facial occur under a side or the two routine sockets of the eye hole and around swelling and rhinorrhea is arranged or has the person of shedding tears concurrently); The immunity chicken is protected 6 at least, or 3 morbidities of contrast chicken, and immune chicken is protected 7 at least for qualified.
4. formaldehyde, thimerosal assay are undertaken by " Chinese veterinary drug allusion quotation " prescriptive procedure respectively, should meet " pertinent regulations of goods check "≤0.4%.
Embodiment 3
1. vaccine safety is measured
8 of the subcutaneous healthy susceptible chickens with 2～3 monthly ages of chest muscle approach inoculation through cervical region of this vaccine, every subcutaneous injection vaccine 1ml observed 14, the no abnormal reaction of test chicken diet and health status, injection site dissection after putting to death, side reactions such as no granulation hyperplasia.
2. immune chicken serum detection of antibodies
Vaccine is through wing root muscle immunity 63 age in days SPF chickens; 0.5ml/ only, totally 8, different time sections blood sampling separation of serum after immunity respectively; Detect the Detection of antigen serum antibody with the Hpg-8 strain, the serum maximum dilution multiple that agglutination occurs with antigen-antibody 50% is decided to be the antibody titer of serum.
Immune chicken serum detection of antibodies result behind table 1 vaccine immunity
(data are to represent antibody valence with serum diluting multiple in the form).
Antibody growth and decline rule after the test chicken immunity; Immunity back 11d can produce protection antibody; Antibody titer reaches the peak during 31～40d; Begin progressively to descend from the later antibody of 40d, antibody valence occurs to 90d and be lower than 4 phenomenons, infer that according to " inactivated vaccine of infectious coryza of chicken rules " its protection period can reach 3 months.
3. the vaccine mensuration of immune duration of immunity once
This vaccine is inoculated 40 of the healthy susceptible chickens of 60 ages in days through chest muscle or leg muscle approach; Every vaccinate 0.5ml; Set up 20 of contrasts; 30d, 60d, 90d, 120d and 150d get 8 of immune group and 4 contrasts at random after immunity respectively, utilize the dilution of 350 times of C-Hpg-8 lyophilizing bacterial strains after under the socket of the eye hole inoculation 0.2ml/ only, the counteracting toxic substances result is following:
Table 2 vaccine is the mensuration of the immune duration of immunity once
According to the challenge test result, the interior protective rate of 90d reaches " inactivated vaccine of infectious coryza of chicken rules " standard behind this vaccine immunity, and matched group 4/4 is obviously fallen ill.
Through above result of the test is reached a conclusion: the inactivated vaccine of infectious coryza of chicken vaccine of novel adjuvant of the present invention preparation has safe, no side reaction, antibody produces soon and advantage such as protective rate height
Note:
1 chicken juice
Get 2 parts of 1 part of adding distil waters of chicken-breasted mud,, boiled 30 minutes, remove the fleshing slag, clarification filtration 2～8 ℃ of soaked overnight.
2 chicken soup culture medium
Chicken juice 100ml
The old 1g of casein
Sodium chloride 0.5g
Transfer pH to 7.2, sterilized 30 minutes for 115 ℃, with the fresh yeast leachate 20ml of preceding adding filtration sterilization, the healthy chicken serum 10ml of deactivation makes seed culture.
3 Carnis Gallus domesticus soup agar
In Carnis Gallus domesticus soup, add 1.5% agar powder thermosol.But will fall dull and stereotyped before the healthy chicken serum of (50～60 ℃ time) adding 0.05% nadide (NADH) and 10% filtration sterilization.
4 semisynthetic mediums
Transfer pH to 7.2～7.4,115 ℃ sterilization 40 minutes with sodium hydroxide solution (2mol/L).Face healthy chicken serum (EK) 30～60ml and 0.5% nadide (NADH) aqueous solution, 8～10ml with preceding adding deactivation.
Claims (3)
1. inactivated vaccine of infectious coryza of chicken is characterized in that haemophilus paragallinarum A type C-Hpg-8 strain that this inactivated vaccine contains deactivation concentrates bacterium liquid and is the vaccine adjuvant that main component is formed with the carbomer.
2. a kind of method for preparing of inactivated vaccine of infectious coryza of chicken according to claim 1; It is characterized in that concentrated rhinitis bacterium liquid, uses behind the sterilization PBS dilution certain proportion with mixing and stir to shake up in 4: 1 ratios as the vaccine adjuvant of main component composition and be prepared from inactivated vaccine of infectious coryza of chicken with carbomer by conventional method preparation.
3. like claim 1 and the said a kind of inactivated vaccine of infectious coryza of chicken of claim 2, it is characterized in that wherein be that the vaccine adjuvant that main component is formed is to be prepared into as follows with the carbomer:
Get NaCl 5～10g, Na
2HPO
41.5～2g, KCl 0.1～0.5g, KH
2PO
40.1～0.5g, carbomer 3～7g is dissolved among the deionized water 1000ml, and dissolving is after 121 ℃ of high pressure 30min sterilization backs are subsequent use.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011103729414A CN102380092A (en) | 2011-11-22 | 2011-11-22 | Inactivated vaccine for infectious coryza of chickens |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103729414A CN102380092A (en) | 2011-11-22 | 2011-11-22 | Inactivated vaccine for infectious coryza of chickens |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103110941A (en) * | 2012-10-09 | 2013-05-22 | 江苏康为世纪生物科技有限公司 | Novel immunological adjuvant and quick immunization scheme |
| CN104212736A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Haemophilus paragallinarum, inactivated vaccine and preparation method of inactivated vaccine |
| CN107354114A (en) * | 2017-09-02 | 2017-11-17 | 河南省农业科学院畜牧兽医研究所 | One plant of haemophilus paragallinarum and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103110941A (en) * | 2012-10-09 | 2013-05-22 | 江苏康为世纪生物科技有限公司 | Novel immunological adjuvant and quick immunization scheme |
| CN103110941B (en) * | 2012-10-09 | 2014-09-03 | 江苏康为世纪生物科技有限公司 | Novel immunological adjuvant and quick immunization scheme |
| CN104212736A (en) * | 2013-09-30 | 2014-12-17 | 郑州后羿制药有限公司 | Haemophilus paragallinarum, inactivated vaccine and preparation method of inactivated vaccine |
| CN107354114A (en) * | 2017-09-02 | 2017-11-17 | 河南省农业科学院畜牧兽医研究所 | One plant of haemophilus paragallinarum and its application |
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Application publication date: 20120321 |