CN1931364A - Prepn process of oral vaccine for antagonizing bacterial diseases of aquatic animal - Google Patents
Prepn process of oral vaccine for antagonizing bacterial diseases of aquatic animal Download PDFInfo
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- CN1931364A CN1931364A CN 200610068922 CN200610068922A CN1931364A CN 1931364 A CN1931364 A CN 1931364A CN 200610068922 CN200610068922 CN 200610068922 CN 200610068922 A CN200610068922 A CN 200610068922A CN 1931364 A CN1931364 A CN 1931364A
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Abstract
The preparation process of oral vaccine for antagonizing bacterial diseases of aquatic animal includes the following steps: adding formaldehyde into pathogenic bacteria fermenting liquid to deactivate; centrifuging and adding sterilized normal saline; and mixing the deactivated bacteria liquid and prepared mixed liquid containing sodium alginate in 0.2-2g %, starch in 0.3-10 %, beta-1-3D glucosan in 0.1-2 % and chitosan in 0.1-2 % in the volume ratio of 1 to 9 to obtain the oral vaccine for antagonizing bacterial diseases of aquatic animal. When used, the vaccine is mixed to the feed for aquatic animal in the ratio of 1 to 9. The vaccine may reach immunity protecting rate of 20 % to 60 %.
Description
Technical field:
The present invention relates to the preparation method of a kind of bacterial diseases of aquatic animal prevention, relate to a kind of preparation method of oral vaccine for antagonizing bacterial diseases of aquatic animal specifically with vaccine.
Background technology:
Bacteria pathogeny has become one of main pathogen of aquatic animal at present, the threat of culture fishery bacterial disease is global, almost encroach on all aquatic animal, endanger greatly especially, limited the sustainable development of aquaculture to a certain extent.The pathogen that causes the bacterial disease of aquatic animal has tens kinds, and how effectively preventing is the difficult problem of the whole aquaculture of puzzlement.
High biologies such as the Fish in the aquatic animal have complete immune system, using for reference the successful experience of vaccine at the prevention of damage by disease of human and poultry poultry, will be the effective way that the control disease takes place by utilizing immunity inoculation to prevent the infecting of bacteria pathogeny of aquatic animal.Usually the immunity inoculation mode of aquatic animal mainly contains injection, immersion/spraying and mode such as oral because oral immunity is easy to operate, do not have injury to cultivated animals, and immunity inoculation is not subjected to the advantages such as restriction of time, place, fish body size, generally is expected.Because antigen is easy to destroyedly in the oral immunity process, the intestinal that can not effectively arrive fish stimulates body to produce stronger immunne response.The antigen dose of oral immunity requirement is far longer than injection and immersion immunity in addition.How to improve the characteristic of oral vaccine, prevent that the antigenicity effectiveness destroyed and the raising vaccine of vaccine from being the oral immunity key of success.
The objective of the invention is to overcome the deficiencies in the prior art; the preparation method of the oral vibrio vaccine of a kind of aquaculture is provided, can reduces oral vaccine and be degraded, add immunopotentiating agent simultaneously again by digestive system; can improve the service efficiency of vaccine greatly, improve the oral vaccine protective rate.Application of the present invention alleviates the harm of Aquatic product bacterial disease, reduces and cultures risk, improves the survival rate of culturing.Promote the sound development of aquaculture.
Preparation method of the present invention adds formalin-inactivated in the pathogen fermentation liquid, the centrifugal supernatant of abandoning or adopting adds physiological saline solution suspension precipitation, and the thalline final concentration is about 40～10,000,000,000/ml.Inactivated bacterial liquid is joined in 0.2～2g% Na-alginate, 0.3～10% starch, 0.1～2% the β-1-3D glucosan and 0.1～2% chitosan mixed liquor of preparation by 1: 9 volume ratio, make the oral vaccine of bacterial diseases of aquatic animal, dosage when this vaccine uses is for using after with 1: 9 ratio mixing with the aquatic animal feedstuff, and can be by pelletize, use dry back.Oral this vaccine can improve specificity and nonspecific immunity simultaneously, the aquatic animal that is used for throwing something and feeding (as Fish, Trionyx sinensis Wiegmann, etc.), immune protective rate can reach 20%～60%.
The present invention can reduce antigen by adding Na-alginate and chitosan and cross in digestive tract destroyed, the stimulation time of enhancement antigen, improve antigenic effectiveness, and with respect to injection and immersion immunity vaccine, oral vaccine have practicality and characteristics easily.
Concrete grammar of the present invention is the deactivation of (1) pathogen: the cultivated animals pathogen after will fermenting is (as the cause of disease Vibrio anguillarum, Vibro harveyi, tarda, vibrio parahaemolytious, sick Vibrio damsela, Aeromonas hydrophilas etc.) add 0.4～0.9% formaldehyde in the bacterium liquid, room temperature is placed 24～120 hours with the complete inactivation pathogen.(2) removal of formaldehyde in the bacterium liquid: with the bacterium liquid behind the formalin-inactivated by centrifuge centrifugal 30～10 minutes with 3000g～10000g, after removing supernatant, add physiological saline solution flushing precipitation, and then under 3000g～10000g condition centrifugal 30～10 minutes, remove supernatant, purpose is the formaldehyde of removing in the solution, and adding aseptic 0.3-0.5% normal saline at last fixed molten is about 40～10,000,000,000/ml to cell concentration.(3) oral vaccine is with the preparation of solution: measure the 90ml distilled water, regulating pH by the NaOH that adds 3mol is 8～11, and be heated to 45 ℃, stir and slowly add 0.2～2g sodium alginate simultaneously, after treating that sodium alginate dissolves fully, with HCl pH value of solution is adjusted back to pH6～7, β-1-3D the glucosan and the stirring of 0.1～2g chitin that add 0.3～10 starch that restrains, 0.1～2g in solution make it fully dissolve mixing, heated and boiled fully dissolved it in 3～5 minutes, was cooled to below 37 ℃ under the gnotobasis.(4) preparation oral vaccine: under the sterile working, ready prepd 40～10,000,000,000/ml deactivation vibrio thalline is joined the oral vaccine that has prepared for 10 milliliters and use in the solution, stir under the 37-45 ℃ of water bath condition and make thalline and the abundant mixing of solution, this is the oral vaccine of the liquid state used of aquaculture bacterial disease.(5) use of vaccine: the feedstuff that adds corresponding aquatic animal before the liquid oral vaccine for preparing uses with 1: 9 ratio.Perhaps the oral vaccine of liquid state is added the prescription feedstuff of corresponding aquatic animal with 1: 9 ratio, by the comminutor pelletize, be dried to the particles with different sizes material aquatic animal of throwing something and feeding behind the mix homogeneously.The oral vaccine of the preparation aquatic animal (as Fish, Trionyx sinensis Wiegmann, etc.) that is used for throwing something and feeding, immune protective rate 20%～60%.
Specific embodiment:
Embodiment 1
To add the formaldehyde of final concentration 0.5% in the bacterium liquid after the 2216E fermentation of Vibrio anguillarum, room temperature is placed 40 hours with the complete inactivation pathogen.With the bacterium liquid behind the formalin-inactivated by centrifuge with 3000g after centrifugal 30 minutes, after removing supernatant, the physiological saline solution flushing precipitation of adding 0.85%, and then under the 3000g condition centrifugal 30 minutes, remove supernatant, add aseptic 0.35%NaCl aqueous solution at last and decide molten to 5,000,000,000/ml Vibrio anguillarum cell concentration.Prepare the preparation of oral vaccine simultaneously: measure the 90ml distilled water with solution, regulating pH by the NaOH that adds 3mol/l is 10, and be heated to 45 ℃, stir and slowly add the 0.7g sodium alginate simultaneously, after treating that sodium alginate dissolves fully, with HCl pH value of solution is adjusted back to pH6, the β-1-3D glucosan and the stirring of 0.5g chitin that add 3 starch that restrain, 0.4g in solution make it fully dissolve mixing, heated and boiled fully dissolved it in 3 minutes, was cooled to below 37 ℃ under the gnotobasis.The preparation oral vaccine will join the oral vaccine that has prepared for 10 milliliters with ready prepd 5,000,000,000/ml deactivation vibrio thalline and use in the solution under the sterile working, stir under 37 ℃ of water bath condition and make thalline and the abundant mixing of solution, this is the oral vaccine of the liquid state used of aquaculture bacterial disease.The oral vaccine of liquid state is added the prescription feedstuff of corresponding aquatic animal with 1: 9 ratio, by the comminutor pelletize, be dried to the particles with different sizes material aquatic animal of throwing something and feeding behind the mix homogeneously.30 grams and 150 of sea-farming restrain left and right sides turbot and Paralichthys olivaceus was thrown something and fed 14 days continuously to utilize the oral vaccine of preparation to be used for throwing something and feeding in 2004, take behind the vaccine one month, utilize 10
8The immune protective rate result that the Vibrio anguillarum of cfu/ml carries out artificial counteracting toxic substances detection immunity back fish is as follows:
| The turbot of 30 grams | Immune protective rate is 52% |
| The Paralichthys olivaceus of 30 grams | Immune protective rate is 47% |
| The turbot of 120 grams | Immune protective rate is 39% |
| The Paralichthys olivaceus of 120 grams | Immune protective rate is 50% |
Embodiment 2
To add the formaldehyde of final concentration 0.5% in the bacterium liquid after the 2216E fermentation of Vibro harveyi, room temperature is placed 40 hours with the complete inactivation pathogen.With the bacterium liquid behind the formalin-inactivated by centrifuge with 3000g after centrifugal 30 minutes, after removing supernatant, the physiological saline solution flushing precipitation of adding 0.85%, and then under the 3000g condition centrifugal 30 minutes, remove supernatant, add aseptic 0.35%NaCl aqueous solution at last and decide molten to 5,000,000,000/ml Vibrio anguillarum cell concentration.Prepare the preparation of oral vaccine simultaneously: measure the 90ml distilled water with solution, regulating pH by the NaOH that adds 3mol/l is 10, and be heated to 45 ℃, stir and slowly add the 0.7g sodium alginate simultaneously, after treating that sodium alginate dissolves fully, with HCl pH value of solution is adjusted back to pH6, the β-1-3D glucosan and the stirring of 1g chitin that add 5 starch that restrain, 0.6g in solution make it fully dissolve mixing, heated and boiled fully dissolved it in 3 minutes, was cooled to below 37 ℃ under the gnotobasis.The preparation oral vaccine will join the oral vaccine that has prepared for 10 milliliters with ready prepd 5,000,000,000/ml deactivation vibrio thalline and use in the solution under the sterile working, stir under 37 ℃ of water bath condition and make thalline and the abundant mixing of solution, this is the oral vaccine of the liquid state used of aquaculture bacterial disease.The oral vaccine of liquid state is added the prescription feedstuff of corresponding aquatic animal with 1: 9 ratio, by the comminutor pelletize, be dried to the particles with different sizes material aquatic animal of throwing something and feeding behind the mix homogeneously.30 grams and 150 of sea-farming restrain left and right sides Paralichthys olivaceuss and Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) was thrown something and fed 14 days continuously to utilize the oral vaccine of preparation to be used for throwing something and feeding, and take behind the vaccine one month, utilize 10
8The immunity guarantor expansion rate result that the Vibro harveyi of cfu/ml carries out artificial counteracting toxic substances detection immunity back fish is as follows:
| The Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) of 30 grams | Immune protective rate is 58% |
| The Paralichthys olivaceus of 30 grams | Immune protective rate is 34% |
| The Lateolabrax japonicus (Cuvier et Va-lenciennes) (Lateolabracis) of 120 grams | Immune protective rate is 49% |
| The Paralichthys olivaceus of 120 grams | Immune protective rate is 40% |
Claims (4)
1, a kind of preparation method of oral vaccine for antagonizing bacterial diseases of aquatic animal is characterized in that its preparation process:
(1) deactivation of pathogen: add formaldehyde in the aquatic animal pathogen bacterium liquid after fermentation, room temperature is placed deactivation in 24～120 hours;
(2) the bacterium liquid behind the formalin-inactivated is removed supernatant by centrifuge is centrifugal after, add physiological saline solution flushing precipitation, and then the centrifugal supernatant of removing, adding physiological saline solution at last, to make cell concentration be about 40～10,000,000,000/ml;
(3) by weight percentage, with the 0.2-2% sodium alginate, 0.3～10% starch, 0.1～2% β-1-3D glucosan and 0.1～2% chitin, the distilled water of 84%-99.2% is mixed into oral vaccine solution, stirring the bacterium liquid that makes 40～10,000,000,000/ml and oral vaccine under 37 ℃ of water bath condition with the ratio abundant mixing of solution with 1: 9, is the oral vaccine of aquaculture bacterial disease liquid state at this moment.
2, as the preparation method of the described a kind of bacterial diseases of aquatic animal prevention of claim 1, it is characterized in that, add in the feedstuff of corresponding aquatic animal with 1: 9 ratio before the liquid oral vaccine for preparing uses and use with vaccine.
3, as the preparation method of claim 1 described a kind of bacterial diseases of aquatic animal prevention with vaccine; it is characterized in that; the prescription feedstuff that the oral vaccine of liquid state is added corresponding aquatic animal with 1: 9 ratio; manufacture particles with different sizes by comminutor behind the mix homogeneously, dried one-tenth oral vaccine bait.
4, as the preparation method of claim 1 described a kind of bacterial diseases of aquatic animal prevention, it is characterized in that above-mentioned pathogen is a Vibrio anguillarum, Vibro harveyi, tarda, vibrio parahaemolytious, sick Vibrio damsela, Aeromonas hydrophila with vaccine.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102058884A (en) * | 2011-01-26 | 2011-05-18 | 广东海大集团股份有限公司 | Grass carp injection vaccine synergist and preparation method thereof |
| CN103463387A (en) * | 2013-08-30 | 2013-12-25 | 兴化市恒威生物技术有限公司 | Medicinal composition for treating turtle cavity disease and preparation method thereof |
| CN104208672A (en) * | 2014-05-13 | 2014-12-17 | 华东理工大学 | Preparation method of inactivated vibrio anguillarum vaccine |
| CN105031636A (en) * | 2014-09-15 | 2015-11-11 | 新乡医学院 | Bi-combined inactivate vaccine of aeromonas hydrophila and aeromonas veronii and preparation method thereof |
| CN104689310B (en) * | 2014-09-15 | 2017-06-09 | 新乡医学院 | A kind of Aeromonas hydrophila and Aeromonas veronii bigeminy oral slow-releasing microsphere vaccine and preparation method |
| CN107568117A (en) * | 2017-09-15 | 2018-01-12 | 广东海大畜牧兽医研究院有限公司 | A kind of fish immunity method |
| CN108837155A (en) * | 2018-06-27 | 2018-11-20 | 灵格兽医技术有限公司 | A kind of animal oral immunity vaccine carrier and preparation method thereof |
| CN109453370A (en) * | 2018-10-25 | 2019-03-12 | 天津市水产研究所 | Source of fish vibrio parahaemolytious release oral vaccine and preparation and application |
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2006
- 2006-09-08 CN CN 200610068922 patent/CN1931364A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102058884A (en) * | 2011-01-26 | 2011-05-18 | 广东海大集团股份有限公司 | Grass carp injection vaccine synergist and preparation method thereof |
| CN102058884B (en) * | 2011-01-26 | 2012-07-11 | 广东海大集团股份有限公司 | Grass carp injection vaccine synergist and preparation method thereof |
| CN103463387A (en) * | 2013-08-30 | 2013-12-25 | 兴化市恒威生物技术有限公司 | Medicinal composition for treating turtle cavity disease and preparation method thereof |
| CN104208672A (en) * | 2014-05-13 | 2014-12-17 | 华东理工大学 | Preparation method of inactivated vibrio anguillarum vaccine |
| CN105031636A (en) * | 2014-09-15 | 2015-11-11 | 新乡医学院 | Bi-combined inactivate vaccine of aeromonas hydrophila and aeromonas veronii and preparation method thereof |
| CN104689310B (en) * | 2014-09-15 | 2017-06-09 | 新乡医学院 | A kind of Aeromonas hydrophila and Aeromonas veronii bigeminy oral slow-releasing microsphere vaccine and preparation method |
| CN105031636B (en) * | 2014-09-15 | 2018-06-19 | 新乡医学院 | A kind of Aeromonas hydrophila and Aeromonas veronii bivalent inactivated vaccine and preparation method |
| CN107568117A (en) * | 2017-09-15 | 2018-01-12 | 广东海大畜牧兽医研究院有限公司 | A kind of fish immunity method |
| CN108837155A (en) * | 2018-06-27 | 2018-11-20 | 灵格兽医技术有限公司 | A kind of animal oral immunity vaccine carrier and preparation method thereof |
| CN109453370A (en) * | 2018-10-25 | 2019-03-12 | 天津市水产研究所 | Source of fish vibrio parahaemolytious release oral vaccine and preparation and application |
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Application publication date: 20070321 |