CN109453370A - Source of fish vibrio parahaemolytious release oral vaccine and preparation and application - Google Patents
Source of fish vibrio parahaemolytious release oral vaccine and preparation and application Download PDFInfo
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- CN109453370A CN109453370A CN201811249582.1A CN201811249582A CN109453370A CN 109453370 A CN109453370 A CN 109453370A CN 201811249582 A CN201811249582 A CN 201811249582A CN 109453370 A CN109453370 A CN 109453370A
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- vibrio parahaemolytious
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- 229940126578 oral vaccine Drugs 0.000 title claims abstract description 54
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- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 229960005486 vaccine Drugs 0.000 claims abstract description 46
- 238000013268 sustained release Methods 0.000 claims abstract description 24
- 239000012730 sustained-release form Substances 0.000 claims abstract description 24
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 241000894006 Bacteria Species 0.000 claims abstract description 11
- 239000000463 material Substances 0.000 claims abstract description 6
- 230000001900 immune effect Effects 0.000 claims abstract description 5
- 230000028993 immune response Effects 0.000 claims abstract description 3
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- 239000001069 triethyl citrate Substances 0.000 claims description 7
- VMYFZRTXGLUXMZ-UHFFFAOYSA-N triethyl citrate Natural products CCOC(=O)C(O)(C(=O)OCC)C(=O)OCC VMYFZRTXGLUXMZ-UHFFFAOYSA-N 0.000 claims description 7
- 235000013769 triethyl citrate Nutrition 0.000 claims description 7
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 claims description 5
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- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 5
- 239000008188 pellet Substances 0.000 claims description 4
- 229940031551 inactivated vaccine Drugs 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 24
- 238000002649 immunization Methods 0.000 abstract description 17
- 230000003053 immunization Effects 0.000 abstract description 8
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
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- 206010018910 Haemolysis Diseases 0.000 description 1
- 241000321426 Hyporthodus nigritus Species 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
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- 235000018291 probiotics Nutrition 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 108010071967 protein K Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 229920005989 resin Polymers 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 238000000935 solvent evaporation Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/107—Vibrio
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5015—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
- A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
- A61K9/5047—Cellulose ethers containing no ester groups, e.g. hydroxypropyl methylcellulose
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
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- Health & Medical Sciences (AREA)
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- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract
The present invention relates to a kind of preparation and application of vibrio parahaemolytious release oral vaccine, the release oral vaccine is by feed, the vibrio parahaemolytious bacterium solution of inactivation, sustained release agent 10:1:3 in proportion；Preparation step includes: the (1) culture and inactivation of vibrio parahaemolytious, the (2) preparation of oral vaccine, the preparation of (3) release oral vaccine；The application method of source of fish vibrio parahaemolytious release oral vaccine involved in the present invention, method and step include: (1) first immunisation and (2) booster immunization.The present invention is prepared for source of fish vibrio parahaemolytious release oral vaccine, and use oral immunization method, immune effect is preferable, and workload is small, is suitble to large-scale inoculation, simultaneously, the present invention is coated with oral vaccine using slow-release material, can effectively extend the release time of vaccine, resists antigen and is digested enzyme destruction in stomach and anterior intestine and decomposes, so that antigen is successfully reached hindgut and generate immune response, obtains preferable immune effect.
Description
Technical field
The invention belongs to the vaccine of cultured fishes and the technical fields used, and in particular to a kind of source of fish vibrio parahaemolytious is slow
Release oral vaccine and preparation and application.
Background technique
The disease of China's marine fish is most commonly seen in increased trend, especially vibriosis year by year, causes huge
Huge economic loss.Domestic treatment fish bacterial disease is mainly by using antibiotics at present, and pre- bacteriological protection
Property disease mainly use the methods of disinfectant, improver of water quality, probiotics improve breeding water body ecological environment, vaccine
Using actually rare, main cause be obtain formal certification aquatic products vaccine it is seldom, and domestic at present obtain formal certification
Vaccine is all injection-type vaccine, although such vaccine effect is preferable, in immune implementation process, heavy workload is cumbersome,
It easily stress to fish body generation.
For a long time, using antibiotic treatment fish diseases, so that pathogenic bacteria drug resistance enhances, and environmental pollution is caused
And the fish qualities problem such as medicament residue.Country increases supervision to breeding wastewater discharge, and discharge beyond standards are supported
Grow the risk that the enterprise of waste water will face significant fine or shut down.Country, which has ordered, is forbidden to use the serious drug of harm, is
Effective protection ecology of water safety and aquatic product quality is improved, the use of drug should be reduced in aquaculture production to the greatest extent.
It has been widely used in livestock and poultry animal cultivation using vaccine prevention disease, but scale is smaller in aquaculture,
This is related with the particularity that fish live in water.Currently, domestic commercially available aquaculture vaccines are all to vaccinate, fishing injection epidemic disease
The problems such as seedling is there are operating difficulties, process is cumbersome, heavy workload, wound are easy infection.Oral immunity has safe easy, operation
It is convenient, usage amount is small, by fish individual size the advantages such as is not limited, be suitable for being promoted and applied in production.But oral vaccine faces
One important problem, i.e., immunogene is easy to be destroyed and be decomposed in anterior intestine by gastric acid and digestive ferment during oral immunity
Absorption causes its immune effect bad.
About the research of vibrio parahaemolytious oral vaccine, the prior art mainly has both sides to report
(1) medical starch is used to be carrier, be prepared for vibrio parahaemolytious, vibrio alginolyticus by coated fertilizer of acrylic resin
With three oral vaccine of Edwardsiella tarda, turbot protective rate is immunized up to 50%.
(2) emulsification, ionic cross-linking is used to be prepared for outside vibrio parahaemolytious using chitosan and sodium alginate as capsulating material
Memebrane protein K microballoon oral vaccine, oral immunity Larimichthys crocea immune protective rate is up to 60%；Use emulsification-solvent evaporation method with propylene
Acid resin, lecithin and atoleine are the fusion protein of outer membrane protein K and flagellin A that emulsifier prepares vibrio parahaemolytious
Microballoon oral vaccine, is immunized warsaw grouper, and protective rate reaches 50%.
Deficiency existing for above-mentioned two method is: it is carrier, using acrylic resin as coating that method (1), which uses medical starch,
Material is prepared for three oral vaccine of vibrio parahaemolytious, vibrio alginolyticus and Edwardsiella tarda, and phagostimulating effect is poor；Method
(2) it needs to extract outer membrane protein, and outer membrane protein content is low, extracts more difficulty, prepares the micro- of outer membrane protein using emulsion process
Ball vaccine art is complicated, takes time and effort, and production cost is higher, it is difficult to large-scale promotion application.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art place, source of fish vibrio parahaemolytious release oral vaccine and system
Standby and application method.
The present invention realizes that the technical solution of purpose is as follows:
A kind of source of fish vibrio parahaemolytious release oral vaccine and preparation, the release oral vaccine is by commercialization feed, concentration
It is 6 × 1010The vibrio parahaemolytious bacterium solution of cfu/mL, sustained release agent 10:1:3 in proportion, unit are as follows: gram: mL:mL is formulated.
Moreover, the vibrio parahaemolytious bacterium solution have passed through 0.5% formalin-inactivated.
Source of fish vibrio parahaemolytious release oral vaccine and preparation, it is characterised in that: the method for the preparation process is as follows:
(1) inactivate: vibrio parahaemolytious bacterium solution being inactivated using 0.5% formaldehyde；
(2) vaccine preparation:
By commercialization feed: concentration is 6 × 1010The vibrio parahaemolytious ratio of cfu/mL are as follows: 10:1, unit are as follows: gram: mL,
Commercialization feed is put into seed-coating machine, turntable is started, seed-coating machine uses 400 revs/min of revolving speed, will inactivate vibrio parahaemolytious
Vaccine is slowly dropped into the center of turntable, and vaccine is sprayed on feed by the centrifugal force by central turntable, is prepared into vaccine feed, vaccine
Effective concentration is about 6 × 109Cfu/g feed is put into 37 DEG C of baking oven and is dried for standby.
(3) slow release layer coating preparation:
1. prepared by sustained release agent: hydroxypropyl methyl cellulose (HPMC): (EC content is 25- to Aquacoat
30%): triethyl citrate (TEC): the ratio of aqua sterilisa are as follows: 1.25:20:1:80, unit are gram: mL:mL:mL.Magnetic force stirs
It mixes overnight, sustained release agent is made.
2. using seed-coating machine by sustained release agent even application in the surface of vaccine feed；Vaccine feed: the ratio of sustained release agent are as follows:
10:1, unit are as follows: gram: mL repeats the above steps 2 times after 37 DEG C of drying.
3. slow release effect measures: being separately added into the vaccine feed of feed total weight: the ratio of sustained release agent are as follows: 10:3 is utilized
Fluorescence quantitative PCR method detects the copy number of gyrB gene of Vibrio parahaemolyticus, judges the release speed of vibrio parahaemolytious in oral vaccine
Degree, evaluates the slow release effect of oral vaccine.
Picking vaccine feed: the ratio of sustained release agent are as follows: the oral vaccine 10g of 10:3 is placed in the triangle of the sterile water containing 50mL
In bottle, while taking vaccine feed: the ratio of sustained release agent are as follows: the oral vaccine of 10:0 is as control.Be placed in shaking table 50r/min into
Row mixes, and is acquired sample in 7 different time points of 2min, 5min, 10min, 15min, 30min, 60min and 120min,
Using the copy number of gyrB gene of Vibrio parahaemolyticus in fluorescence quantitative PCR detection technique measurement institute's water sampling, oral vaccine is judged
The rate of release of middle vibrio parahaemolytious evaluates the slow release effect of oral vaccine；Use concentration for 1 × 10 simultaneously9The pair of CFU/mL
Hemolysis vibrion bacterium solution makees template, carries out quantitative PCR measurement, the vaccine maximum burst size of control group and vaccine group is judged with this.
1 vibrio parahaemolytious oral vaccine slow release effect measurement result (copy/μ L) of table
Coating group is respectively in the burst size of its vaccine of 2min, 5min, 10min, 15min, 30min, 60min and 120min
17.36%, 16.17%, 22.95%, 27.46%, 60.16%, 88.89% and the 91.77% of control group shows the oral epidemic disease
Seedling have preferable slow releasing function, and when 120min coating group vaccine burst size substantially close to control group.Concentration be 1 ×
109When the vibrio parahaemolytious bacterium solution of CFU/mL directly carries out real-time fluorescence quantitative PCR measurement, the copy number of gyrB gene be can reach
2.3×106Copy/μ L, by conversion, control group reaches the 69.62% of total content in 120min time vaccines burst size, coating group
Reach the 63.64% of total content in 120min time vaccines burst size.
Moreover, the effective vaccine content of the step (2) pellet being prepared into is 6 × 109Cfu/g magnitude is horizontal.
The application method of source of fish vibrio parahaemolytious release oral vaccine, it is characterised in that: the step of this method is as follows:
(1) first immunisation: by release oral vaccine feedstuff feeding fish, feeding volume is identical as chow diet, 2 times a day, continuously
7 days；
(2) booster immunization；Start booster immunization identical with first immunisation method within the 14-21 days after first immunisation.
Advantages of the present invention and effect are:
1, for the present invention compared with existing vaccines for fish preparation method, the present invention is prepared for vibrio parahaemolytious release oral for the first time
Vaccine, and using the method that feeds inoculation, it is easy to operate, workload is small, it is suitble to large-scale inoculation.
2, the present invention has delayed the release time of vaccine, can effectively protect using having the material of slow-release function to be coated
Vaccine is protected, there can be time enough to reach hindgut, be contacted with rat gut mucosa, excites immune response, adaptive immune effect overcomes
Direct oral immunity ineffective problem.
3, the features such as present invention fully takes into account the particularity, living environment, predation of fish, on formula rate repeatedly
It is tested, scientific value, specific aim is very strong, and easy to use, effect highly significant has very high market application value.
Specific embodiment
Below with reference to specific embodiment, the present invention is further described, and specific embodiment is construed as only lifting
Example explanation, is not restrictive, and cannot be limited the scope of protection of the present invention with following illustrations.
A kind of source of fish vibrio parahaemolytious release oral vaccine, the release oral vaccine by commercialization feed, concentration be 6 ×
1010The vibrio parahaemolytious bacterium solution of cfu/mL, sustained release agent 10:1:3 in proportion, unit are as follows: gram: mL:mL is formulated.
In specific implementation of the invention, the vibrio parahaemolytious bacterium solution will pass through 0.5% formalin-inactivated before use.
The preparation of source of fish vibrio parahaemolytious release oral vaccine, the method for the preparation process are as follows:
(1) inactivate: vibrio parahaemolytious bacterium solution being inactivated using 0.5% formaldehyde；
(2) vaccine preparation:
By commercialization feed: concentration is 6 × 1010The vibrio parahaemolytious ratio of cfu/mL are as follows: 10:1, unit are as follows: gram: mL,
Commercialization feed is put into seed-coating machine, turntable is started, pellet machine uses 400 revs/min of revolving speed, will inactivate vibrio parahaemolytious
Vaccine is slowly dropped into the center of turntable, and vaccine is sprayed on feed by the centrifugal force by central turntable, is prepared into vaccine feed, vaccine
Effective concentration is about 6 × 109Cfu/g feed is put into 37 DEG C of baking oven and is dried for standby.
(3) slow release layer coating preparation:
1. prepared by sustained release agent: hydroxypropyl methyl cellulose (HPMC): (EC content is 25- to Aquacoat
30%): triethyl citrate (TEC): the ratio of aqua sterilisa are as follows: 1.25:20:1:80, unit are gram: mL:mL:mL.Magnetic force stirs
It mixes overnight, sustained release agent is made.
2. using seed-coating machine by sustained release agent even application in the surface of vaccine feed；Vaccine feed: the ratio of sustained release agent are as follows:
10:1, unit are as follows: gram: mL repeats the above steps 1-2 times after 37 DEG C of drying.
The application method of source of fish vibrio parahaemolytious release oral vaccine, it is characterised in that: the step of this method is as follows:
(1) first immunisation: daily by release oral vaccine feedstuff feeding fish, feeding volume is identical as chow diet, 2 times a day,
Continuous 7 days；
(2) booster immunization；Start booster immunization identical with first immunisation method within the 14-21 days after first immunisation.
Vibrio parahaemolytious release oral vaccine and its preparating example
The preparation of source of fish vibrio parahaemolytious release oral vaccine, the formula of the release oral vaccine preparation are as follows: 1000g quotient
Product feed is put into seed-coating machine, by 100mL by 0.5% formalin-inactivated vibrio parahaemolytious (concentration be 6 × 1010cfu/
ML), slowly it is sprayed on feed, naturally dry or low temperature drying, vaccine effective content is 6 × 109cfu/g.By 1.3g hydroxypropyl
Ylmethyl cellulose, 20mL Aquacoat (EC content be 25%-30%), 1mL triethyl citrate (TEC),
The mixing of 80mL aqua sterilisa, is stirred overnight, prepares 100mL sustained release agent.By 100mL sustained release agent using seed-coating machine even application in epidemic disease
The surface of seedling pellet, naturally dry or low temperature drying, duplicate packages sustained release agent 2 times.
The application method example 1 of vibrio parahaemolytious release oral vaccine
By the vaccine feed immunity turbot of above-mentioned preparation, turbot specification is 102 ± 11.2 grams.Feeding volume and normal bait
Expect the identical normal bait of feeding volume, continuously feeds 7 days, be spaced 14 days booster immunization 7 days.
Turbot experiment is immunized in aquarium small size pond, designs two groups, control group and sustained release oral immunity group, and every group 3
It repeats.Release oral immune group feeds release oral vaccine 2 times of preparation daily, continuously feeds 7 days, then feeds normal bait
Material；Control group feeds normal granules bait.It is spaced blood sampling in 7 days once, every group is adopted 3 tail fishes, using enzyme-linked immunosorbent assay
(Elisa) antibody level in serum is measured.After 28 days, test fish and control fish carry out vibrio parahaemolytious (bacterial concentration be 3 ×
108Cfu/mL artificial challenge) is carried out, every tail fish injects 0.2mL, the death rate of two groups of fishes is counted after 14 days, and Computation immunity is protected
Shield rate.It the results are shown in Table 2.
The equal section of turbot antibody titers from serum after 2 oral immunity of table
The antibody titer of vibrio parahaemolytious in release oral immune group serum reached maximum value, antibody level at 28 days
All it is apparently higher than control group.The Experimental infection of release oral immune group fish, relative immunity protective rate reach 57.12%.Knot
Fruit proves that resistance of the turbot to vibrio parahaemolytious can be significantly improved by feeding vibrio parahaemolytious release oral vaccine of the invention
Power.
The application method example 2 of vibrio parahaemolytious release oral vaccine
Workshop culture experiment:
Aquaculture model: industrial aquaculture；Cultured fishes: lefteye flounder, size are 89 ± 15.2 grams；Totally 6 test tanks, pilot scale
3, pond is tested, 3, pond is compareed.First immunisation: it feeds 2 times, continuously feeds 7 days daily；Booster immunization: after first immunisation 21 days, often
It feeds 2 times, feeds 7 days.Control pond feeds common bait, and other cultivation and management method are identical.Each pond acquisition after 2 months
5 tail fish serums, using agglutination measure antibody titer, and take 50 tail lefteye flounders carry out vibrio parahemolyticus (bacterial concentration be 3 ×
108Cfu/mL) artificial liver support, every tail fish inject 0.2mL, the death rate in statistics 14 days, Computation immunity protective rate.Test knot
Fruit is shown in Table 3.
The serum antibody titer and immune protective rate of 3 lefteye flounder of table
Experiment pool vibrio parahaemolytious potency section is 128-256, much higher than the 8-16 in control pond；Challenge test shows immune
Protective rate reaches 55.56%.The results show that culture efficiency release oral vaccine immunity experiment pool effect is substantially better than control pond.
Industrial aquaculture, which feeds vibrio parahaemolytious release oral vaccine of the invention, can effectively prevent lefteye flounder infection vibrio parahaemolytious disease,
Improve cultivation survival rate.
Claims (6)
1. a kind of source of fish vibrio parahaemolytious release oral vaccine, it is characterised in that: the vibrio parahaemolytious bacterium including feed, inactivation
10g:1ml:3ml is formulated in proportion for liquid, sustained release agent.
2. source of fish vibrio parahaemolytious release oral vaccine according to claim 1, it is characterised in that: the vibrio parahaemolytious
Bacterium solution have passed through formalin-inactivated 48 hours of 0.5%.
3. source of fish vibrio parahaemolytious release oral vaccine according to claim 1, it is characterised in that: the vibrio parahaemolytious
Concentration is 6 × 1010cfu/mL。
4. a kind of preparation method of source of fish vibrio parahaemolytious release oral vaccine, it is characterised in that: steps are as follows:
(1) the preparation of inactivated vaccine: vibrio parahaemolytious is inactivated using final concentration of 0.5% formaldehyde；
(2) the preparation of oral vaccine:
The vibrio parahaemolytious for taking feed, inactivation, feed is put into seed-coating machine, starts turntable, and pellet machine uses 400 revs/min
Revolving speed, vibrio parahaemolytious vaccine will be inactivated and be slowly dropped into the center of turntable, vaccine is sprayed onto feeding by the centrifugal force by central turntable
On material, it is prepared into oral vaccine, vaccine effective concentration is about 6 × 109Cfu/g feed is put into 37 DEG C of baking oven and is dried for standby；
(3) the preparation of release oral vaccine:
1. prepared by sustained release agent: hydroxypropyl methyl cellulose: Aquacoat: triethyl citrate: the ratio of aqua sterilisa
Example are as follows: sustained release agent is made in 1.25g:20mL:1mL:80mL, magnetic stirrer over night, and the EC content of Aquacoat is
25-30%；
2. using seed-coating machine by sustained release agent even application in the surface of oral vaccine；Oral vaccine: the ratio of sustained release agent are as follows: 10g:
1mL repeats the above steps 2 times after 37 DEG C of drying.
5. the preparation method of source of fish vibrio parahaemolytious release oral vaccine according to claim 4, it is characterised in that: described
(2) the vaccine feed effective vaccine content being prepared into is 6 × 10 to step9Cfu/g magnitude is horizontal.
6. the preparation method of source of fish vibrio parahaemolytious release oral vaccine according to claim 4, it is characterised in that: described
Step (3) 1. the step of in sustained release agent, can effectively extend the release time of vaccine, so that antigen is successfully reached hindgut and produce
Raw immune response, obtains preferable immune effect.
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| CN1931364A (en) * | 2006-09-08 | 2007-03-21 | 陈刚 | Prepn process of oral vaccine for antagonizing bacterial diseases of aquatic animal |
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| CN1931364A (en) * | 2006-09-08 | 2007-03-21 | 陈刚 | Prepn process of oral vaccine for antagonizing bacterial diseases of aquatic animal |
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