CN102366629A - Freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine and industrialized preparation method thereof - Google Patents
Freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine and industrialized preparation method thereof Download PDFInfo
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- CN102366629A CN102366629A CN2011100258490A CN201110025849A CN102366629A CN 102366629 A CN102366629 A CN 102366629A CN 2011100258490 A CN2011100258490 A CN 2011100258490A CN 201110025849 A CN201110025849 A CN 201110025849A CN 102366629 A CN102366629 A CN 102366629A
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Abstract
The invention relates to a bivalent inactivated vaccine, and specifically, relates to a freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine and an industrialized preparation method thereof. The freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine comprises inactivated antigen compositions of aeromonas hydrophila culture inactivated liquid and aeromonas sobria culture inactivated liquid. The invention also provides the industrialized preparation method of the freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine. The industrialized preparation method is characterized by comprising the following steps of mixing uniformly the aeromonas hydrophila culture inactivated liquid and the aeromonas sobria culture inactivated liquid according to a ratio of 1: 1, and carrying out aseptic loading to obtain freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine finished products. The freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine overcomes the defect that a monovalent vaccine has an effect only on a pathogen. The freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine has the effect of resisting two pathogens simultaneously by being applied once, and can be conveniently applied through immersion, injection or feeding after mixing with forage.
Description
Technical field
The present invention relates to bivalent inactivated vaccine, be specifically related to a kind of fresh-water fishes Aeromonas hydrophila, Aeromonas sobria bivalent inactivated vaccine and method for preparing.
Background technology
Freshwater fish bacterial haemorrhage disease is claimed bacterial haemorrhage property septicemia, fulminant hemorrhagic disease, hemorrhagic ascites disease etc. again; Be one of the most serious infectious disease of harm cultured freshwater fish, the Fish of harm mainly contain Fish such as silver carp, flathead, crucian carp, Cyprinus carpio, triangular bream, dace, eel, mandarin fish.This disease morbidity is anxious, and the fresh-water fishes kind of harm is many, and the specification limit of harm fish is big, and popular area is wide, and epidemic season is long, causes comparatively serious loss to freshwater aquaculture industry.This disease pathogen mainly be Aeromonas (
Aeromonas) Aeromonas hydrophila (
A. hydrophila) and Aeromonas sobria (
A. sobria), it extensively is present in fresh water, sewage and soil.Having pathogenicly widely, infect the multiple animal comprise cold blooded animal, cause local infections such as septicemia or skin ulcer, is the especially modal pathogenic bacterium of Fish of aquatic animal.Vaccine is prophylactic effective means; Can stimulate animal body to produce antibody; Can make the special attack of resisting cause of disease of Fish; Ability that also can the anti-distress of enhancing body when improving Fish specific immunity level, the disease prevention and control measure as meeting the environmental friendliness and the strategy of sustainable development has become one of standard production normative content of International Modern culture fishery.China's fish vaccine obtains have only 3 of new veterinary drug certificate at present; The antibacterial class vaccine patent of invention great majority of aquatic animal relate to single Seedling and prepared in laboratory method thereof, and maximum Aeromonas combined vaccine and the batch production method for preparing thereof of freshwater fish harm also do not appeared in the newspapers.
Summary of the invention
The objective of the invention is in order to overcome the shortcoming that single inactivated vaccine only is directed against a kind of pathogen; Provide a kind of bigeminy vaccine can resist the effect of two kinds of pathogen simultaneously to reach applied once, vaccine is through soaking, injecting or mix and stir feedstuff feeding and all can conveniently use.
Fresh-water fishes Aeromonas bivalent inactivated vaccine of the present invention comprises following inactivation antigen compositions: Aeromonas hydrophila culture deactivation liquid and Aeromonas sobria culture deactivation liquid.
According to the further characteristic of fresh-water fishes Aeromonas bivalent inactivated vaccine of the present invention, said Aeromonas hydrophila culture deactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 2,500,000,000 cfu/ml; Said Aeromonas sobria culture deactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 2,500,000,000 cfu/ml.
The present invention also provides the batch production method for preparing of described fresh-water fishes Aeromonas bivalent inactivated vaccine; It is characterized in that: the mixed of Aeromonas hydrophila culture deactivation liquid and Aeromonas sobria culture deactivation liquid being pressed 1:1 is even; Sterile filling promptly obtains fresh-water fishes Aeromonas bivalent inactivated vaccine finished product.
Batch production method for preparing according to fresh-water fishes Aeromonas bivalent inactivated vaccine of the present invention may further comprise the steps:
A. first order seed preparation: Aeromonas hydrophila strain or Aeromonas sobria bacterial classification inoculation nutrient agar panel, 28 ℃ of cultivations, 24 hours, choose lawn inoculation nutrient broth medium, 28 ℃, shake-flask culture 18 hours, preparation primary seed solution;
B. secondary seed preparation: primary seed solution is inoculated the 50L fermentation tank, and culture medium is the toxin producing culture medium, and 28 ℃, the ventilation of 30-60L/min stirred aerobic culture 10-16 hour, the preparation secondary seed solution;
C. seedling bacterium liquid is cultivated: secondary seed solution is inoculated the 500L fermentation tank, and culture medium is the toxin producing culture medium, 28 ℃; The ventilation of 150-200L/min; Stirred aerobic culture 10-14 hour, and obtained preparing the bacterium liquid of vaccine, viable count 5,000,000,000 cfu/ml are above to be qualified;
D. deactivation: bacterium liquid adds 0.35% formalin, and 37 ℃, deactivation 24 hours carries out that steriling test and formaldehyde are residual to be up to the standards, and obtains Aeromonas hydrophila culture deactivation liquid or Aeromonas sobria culture deactivation liquid;
E. join Seedling: respectively two kinds of bacterial cultures deactivation liquid are joined the Seedling jar in the ratio input of 1:1, in-tank mixing liquid stirs;
F. packing: carry out packing under the aseptic condition, roll lid, obtain fresh-water fishes Aeromonas bivalent inactivated vaccine finished product.
Fresh-water fishes Aeromonas bivalent inactivated vaccine batch production mature preparation process among the present invention, cost is not high.Vaccine can be used through soaking, inject, mix and stir modes such as feedstuff feeding; Cultured freshwater fishes such as immunity silver carp, flathead, crucian carp, Cyprinus carpio, triangular bream, dace, eel, mandarin fish; Immune effect is good; The freshwater fish hueppe's disease that can effectively prevent Aeromonas hydrophila and Aeromonas sobria to cause reduces the use of PA class fisheries drug.
The specific embodiment
Following embodiment further specifies of the present invention, but the invention is not restricted to following embodiment.
Embodiment one: the batch production preparation of fresh-water fishes Aeromonas bivalent inactivated vaccine
1, first order seed preparation
Take out the strain that is kept at refrigerator (Aeromonas hydrophila strain, Aeromonas sobria strain), inoculation Nutrient agar (culture medium prescription is seen appendix) was cultivated 24 hours for 28 ℃; Choose lawn with loop carrier and connect Nutrient agar, cultivated 24 hours for 28 ℃, inoculation nutrient broth (culture medium prescription is seen appendix) 300ml/ bottle; 10 bottles, put shaking table 200rmp/min, 28 ℃; Cultivated 18 hours, and obtained the 3L first order seed.
The strain that present embodiment is selected is: Aeromonas hydrophila bacterial strain YYK090901, separate from the Aristichthys nobilis kidney; Aeromonas sobria bacterial strain LYK090702 separates from the Hypophthalmichthys molitrix kidney; Strain all is kept at China's Pearl River Fishery Research Institute of Aquatic Science Research Institute fish diseases chamber at present.
2, secondary seed preparation
20L toxin producing culture medium (culture medium prescription is seen this description appendix) by formulated joins the 50L fermentation tank, 121 ℃; Sterilization in 20 minutes is after the culture medium cooling, with 3L primary seed solution inoculation fermentation jar; 28 ℃, ventilation 40L/min, 250rmp/min; Stir culture 12 hours obtains the secondary liquid seeds.
3, the bacterium liquid of preparation vaccine is cultivated
180L toxin producing culture medium (culture medium prescription is seen this description appendix) by formulated joins the 500L fermentation tank, 121 ℃; Sterilization in 20 minutes after the culture medium cooling, inserts fermentation tank with reaching qualified secondary seed purely; Culture-liquid temp is 28 ℃ in the fermentation tank, filtrated air flow 10.5M
3/ hour (175L/min); 200 rmp/min culture bacteria liquid 12 hours; Cultivate the end aseptic sampling in back, carry out pure and viable count detection (with reference to the 3rd one of " People's Republic of China's veterinary drug allusion quotation " version 2005), viable count need reach and be qualified bacterium liquid more than 5,000,000,000 CFU/mL.
4, deactivation
Qualified bacterium liquid is arrived the deactivation jar through line transportation; The sterile working adds formalin, and the formaldehyde ultimate density is 0.35%, with 37 ℃ of deactivations; Deactivation 24 hours; Whenever stirred once at a distance from 4 hours during this time, the bacterium liquid that deactivation is good is cooled between 1～5 ℃, carries out the residual check of steriling test and formaldehyde (with reference to the 3rd one of " People's Republic of China's veterinary drug allusion quotation " version 2005).
5, join Seedling
Respectively the qualified deactivation liquid of two kinds of antibacterials is joined the Seedling jar in the ratio input of 1:1, in-tank mixing liquid stirs.
6, packing
Carry out packing under the aseptic condition, roll lid, obtain fresh-water fishes Aeromonas bivalent inactivated vaccine finished product.
Through measuring, in the fresh-water fishes Aeromonas bivalent inactivated vaccine of gained, Aeromonas hydrophila culture deactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 2,500,000,000 cfu/ml; Aeromonas sobria culture deactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 2,500,000,000 cfu/ml.
Embodiment two. the immune effect of fresh-water fishes Aeromonas bivalent inactivated vaccine
Healthy Carassius auratus (30-50g/ tail) is divided into three groups at random, every group 100 tail.First group is the injecting immune group, the fresh-water fishes Aeromonas bivalent inactivated vaccine 0.1ml that every tail lumbar injection embodiment one is prepared; Second group is the immersion immunity group, will test fish earlier and in 1%NaCL solution, soak 3-5min, and 10-15min is soaked in inflation in the bigeminy vaccine liquid of 10 times of dilutions then; The 3rd group is matched group.Water temperature is 22-26 ℃.Behind immune 28d; (Aeromonas hydrophila bacterial strain YYK090901 separates from the Aristichthys nobilis kidney with Aeromonas sobria bacterial strain LYK090702 to use the Aeromonas hydrophila bacterial strain YYK090901 of 2 times of LD50 dosage respectively; Aeromonas sobria bacterial strain LYK090702 separates from the Hypophthalmichthys molitrix kidney, and strain is kept at China's Pearl River Fishery Research Institute of Aquatic Science Research Institute fish diseases chamber at present), lumbar injection infection experiment fish; And breeding observing 7 days, morbidity and the death condition of each group test fish of record.
Experimental result shows that the injecting immune group is respectively 84%, 89% to the relative immunity protective rate of bacterial strain YYK090901, LYK090702, and the immersion group is respectively 57%, 59% (seeing table 1).
The effect of table 1 bigeminy Seedling immunity
Claims (3)
1. a fresh-water fishes Aeromonas bivalent inactivated vaccine is characterized in that, said vaccine comprises following inactivation antigen compositions: Aeromonas hydrophila culture deactivation liquid and Aeromonas sobria culture deactivation liquid.
2. fresh-water fishes Aeromonas bivalent inactivated vaccine according to claim 1 is characterized in that: said Aeromonas hydrophila culture deactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 2,500,000,000 cfu/ml; Said Aeromonas sobria culture deactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 2,500,000,000 cfu/ml.
3. the batch production method for preparing of fresh-water fishes Aeromonas bivalent inactivated vaccine as claimed in claim 1 is characterized in that, may further comprise the steps:
A. first order seed preparation: Aeromonas hydrophila strain or Aeromonas sobria bacterial classification inoculation nutrient agar panel, 28 ℃ of cultivations, 24 hours, choose lawn inoculation nutrient broth medium, 28 ℃, shake-flask culture 18 hours, preparation primary seed solution;
B. secondary seed preparation: primary seed solution is inoculated the 50L fermentation tank, and culture medium is the toxin producing culture medium, and 28 ℃, the ventilation of 30-60L/min stirred aerobic culture 10-16 hour, the preparation secondary seed solution;
C. seedling bacterium liquid is cultivated: secondary seed solution is inoculated the 500L fermentation tank, and culture medium is the toxin producing culture medium, 28 ℃; The ventilation of 150-200L/min; Stirred aerobic culture 10-14 hour, and obtained preparing the bacterium liquid of vaccine, viable count 5,000,000,000 cfu/ml are above to be qualified;
D. deactivation: bacterium liquid adds 0.35% formalin, and 37 ℃, deactivation 24 hours carries out that steriling test and formaldehyde are residual to be up to the standards, and obtains Aeromonas hydrophila culture deactivation liquid or Aeromonas sobria culture deactivation liquid;
E. join Seedling: respectively two kinds of bacterial cultures deactivation liquid are joined the Seedling jar in the ratio input of 1:1, in-tank mixing liquid stirs;
F. packing: carry out packing under the aseptic condition, roll lid, obtain fresh-water fishes Aeromonas bivalent inactivated vaccine finished product.
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| CN2011100258490A CN102366629A (en) | 2011-01-25 | 2011-01-25 | Freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine and industrialized preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106913867A (en) * | 2017-03-16 | 2017-07-04 | 中国水产科学研究院珠江水产研究所 | A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine and prepare with scale technology |
| CN106938049A (en) * | 2017-03-16 | 2017-07-11 | 中国水产科学研究院珠江水产研究所 | A kind of Kazakhstan and vibrio alginolyticus bivalent inactivated vaccine and batch production technology of preparing |
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| CN1371748A (en) * | 2002-03-18 | 2002-10-02 | 浙江大学 | Zhonghua soft-shelled turtle aeromonad oral slow-releasing microsphere vaccinum and preparation process thereof |
| CN1762387A (en) * | 2005-08-15 | 2006-04-26 | 大连大学 | Aquaculture creature disease controlling compound vaccine and its preparation method |
| CN101181635A (en) * | 2007-12-10 | 2008-05-21 | 河北师范大学 | Method for preparing pathogenicity hydrosphere unit cell bacterium killed vaccine |
| CN101642567A (en) * | 2009-01-14 | 2010-02-10 | 张秀军 | Aeromonas hydrophila inactivated vaccine and preparation thereof |
| CN101732702A (en) * | 2008-11-17 | 2010-06-16 | 上海海洋大学 | Sturgeon bacterial sepsis syndrome holophagae inactivating vaccine and preparation method thereof |
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2011
- 2011-01-25 CN CN2011100258490A patent/CN102366629A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1371748A (en) * | 2002-03-18 | 2002-10-02 | 浙江大学 | Zhonghua soft-shelled turtle aeromonad oral slow-releasing microsphere vaccinum and preparation process thereof |
| CN1762387A (en) * | 2005-08-15 | 2006-04-26 | 大连大学 | Aquaculture creature disease controlling compound vaccine and its preparation method |
| CN101181635A (en) * | 2007-12-10 | 2008-05-21 | 河北师范大学 | Method for preparing pathogenicity hydrosphere unit cell bacterium killed vaccine |
| CN101732702A (en) * | 2008-11-17 | 2010-06-16 | 上海海洋大学 | Sturgeon bacterial sepsis syndrome holophagae inactivating vaccine and preparation method thereof |
| CN101642567A (en) * | 2009-01-14 | 2010-02-10 | 张秀军 | Aeromonas hydrophila inactivated vaccine and preparation thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106913867A (en) * | 2017-03-16 | 2017-07-04 | 中国水产科学研究院珠江水产研究所 | A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine and prepare with scale technology |
| CN106938049A (en) * | 2017-03-16 | 2017-07-11 | 中国水产科学研究院珠江水产研究所 | A kind of Kazakhstan and vibrio alginolyticus bivalent inactivated vaccine and batch production technology of preparing |
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Application publication date: 20120307 |