CN109865136A - A kind of silvery pomfret bivalent inactivated vaccine - Google Patents
A kind of silvery pomfret bivalent inactivated vaccine Download PDFInfo
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- CN109865136A CN109865136A CN201910205843.8A CN201910205843A CN109865136A CN 109865136 A CN109865136 A CN 109865136A CN 201910205843 A CN201910205843 A CN 201910205843A CN 109865136 A CN109865136 A CN 109865136A
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Abstract
The present invention provides a kind of silvery pomfret Mermaid luminous bacillus and Vibrio anguillarum disease bivalent inactivated vaccine, includes antigen and vaccine adjuvant, wherein antigen is Mermaid luminous bacillus and the Vibrio anguillarum of inactivation；The deposit number of the Mermaid luminous bacillus is CGMCC NO.16907.Silvery pomfret Mermaid luminous bacillus prepared by the present invention and Vibrio anguillarum disease bivalent inactivated vaccine preparation process be simple, stable yield, at low cost, can largely obtain the inactivated vaccine with higher immune effect.The immune protective rate of the inactivated vaccine prepared using this method is up to 97.8%; the vaccine is pure biological agent; and have the characteristics that safe and environment-friendly; medicament residue will not be generated and polluted the environment; and this method can fundamentally contain the generation and prevalence of hueppe's disease caused by silvery pomfret Mermaid luminous bacillus disease and Vibrio anguillarum, provide technical support for large scale preparation silvery pomfret Mermaid luminous bacillus and Vibrio anguillarum disease bivalent inactivated vaccine.
Description
Technical field
The invention belongs to microorganism vaccine preparation fields, and in particular to a kind of silvery pomfret Mermaid luminous bacillus and Vibrio anguillarum
Sick bivalent inactivated vaccine.
Background technique
Silvery pomfret (Pampus argenteus) be under the jurisdiction of Perciformes (Perciformes), silvery pomfret section (Stromateidae),
Silvery pomfret category (Pampus) is not only one of main economic fish of China coast, is loved by consumers, and in India, section's prestige
Biggish specific gravity is also accounted in the fishery economic of the country such as spy, Iran.In the past 20 years, wild due to overfishing and environmental pollution
The raw continuous atrophy of silvery pomfret resource, fishery harvesting silvery pomfret figure tend to minimize.Therefore, carrying out silvery pomfret artificial breeding and nursery just seems
It is particularly important.Since country cultivation silvery pomfret is started late, though having broken through artificial propagation bottleneck, apart from industrialization, there are also very
Long road.Especially the adult fish culture stage is easy infection Mermaid luminous bacillus, visually observes except reduction of ingesting, mobility
Decline outer non-evident sympton, can fall ill in the short time causes to be annihilated.
Vibriosis (vibriosis) is the most common bacteriosis in aquaculture.The main pathogenic bacteria of vibriosis it
First is that Vibrio anguillarum (V.anguillarum), it can cause silvery pomfret hueppe's disease.Vibriosis has popular extensive, morbidity
The features such as rate and death rate superelevation, brings crushing blow to the artificial breeding and industrialization of silvery pomfret.
The treatment of silvery pomfret bacteriosis depends on antibiotic at present, can not be from although there is certain effect
Fundamentally control disease, and long-term administration will appear many drawbacks: the cause of disease is complicated sometimes for fish disease, and there are blind for a large amount of medications
Mesh, many diseases can treat currently without targetedly drug；Chemoprophylaxis effect is very weak, and sick fish ingests
It is very poor, effective drug dose can not be absorbed；In addition, the drug of fish body intake often remains wherein, it is strong to endanger the mankind
Health, and long-term administration can bring environmental pollution, and lead to the appearance of a large amount of microbial resistances.
Since silvery pomfret figure is flat-sided, travelling is rapid, and stress be strong for environmental stimuli, existing injection-type vaccine effect
Fruit is bad, and existing vaccine is specific since immunogene does not have, and single seedling uses multiple multi-dose, and can bring to silvery pomfret need not
Want stress, cause its food ration reduce.Therefore, the soak type bivalent inactivated vaccine that exploitation is effective, stimulation is small is extremely urgent,
This must be overcome the problems, such as by innovative technology.
Summary of the invention
The object of the present invention is to provide a kind of silvery pomfret Mermaid luminous bacillus and Vibrio anguillarum disease bivalent inactivated vaccine, to replace
For the hueppe's disease that antibiotic treatment silvery pomfret Mermaid luminous bacillus and infection Vibrio anguillarum generate, produced to solve treatment
Raw medicament residue and drug resistance and the problems such as reduce stress stimulation, to make up the deficiencies in the prior art.
Bivalent inactivated vaccine provided by the present invention includes antigen and vaccine adjuvant, and wherein antigen is the beauty of inactivation
Fish luminous bacillus and Vibrio anguillarum；
The Mermaid luminous bacillus is the isolated Mermaid luminous bacillus from silvery pomfret enteron aisle
ZM504 plants of (Photobacterium damselae) is deposited on December 07th, 2018 positioned at the Chaoyang District, Beijing City North Star
The China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 1 institute in West Road, deposit number CGMCC
NO.16907。
In inactivated vaccine of the invention, wherein the concentration of inactivation thallus is respectively 107-1010CFU/ml。
The vaccine adjuvant, preferably rhizoma atractylodis leachate are decocted after taking rhizoma atractylodis distilled water immersion, filtering, filtrate
Rhizoma atractylodis leachate is obtained after concentration.
Inactivated vaccine provided by the present invention, preparation method are as follows:
1) Mermaid luminous bacillus and Vibrio anguillarum bacterial strain are seeded in respectively in LB liquid medium, 28 DEG C are cultivated for 24 hours,
Bacterial concentration is detected up to 10 with blood counting chamber9CFU/ml or spectrophotometer detection OD600 terminate training when reaching 0.6 or more
It supports, obtains the scale-up medium of bacterial strain；
2) 0.5% formalin is added by volume, 24 hours is inactivated in room temperature to the scale-up medium of bacterial strain, during which
Concussion 8-10 times, obtains the inactivated bacterial liquid of bacterial strain；
Silvery pomfret Mermaid luminous bacillus prepared by the present invention and Vibrio anguillarum disease bivalent inactivated vaccine preparation process are simple, produce
Amount is stablized, at low cost, can largely obtain the inactivated vaccine with higher immune effect.The inactivated vaccine prepared using this method
Immune protective rate up to 97.8%, which is pure biological agent, and has the characteristics that safe and environment-friendly, and it is residual will not to generate drug
It stays and pollutes the environment, and this method can fundamentally contain that silvery pomfret Mermaid luminous bacillus disease and Vibrio anguillarum cause
Hueppe's disease generation and prevalence, be that large scale preparation silvery pomfret Mermaid luminous bacillus and Vibrio anguillarum disease bigeminy inactivate
Vaccine provides technical support.
Specific embodiment
The present invention is described in detail below with reference to embodiment.
Embodiment 1: the Mermaid luminous bacillus CGMCC NO.16907 that the present invention screens
1, the acquisition and processing of sample
Morbidity silvery pomfret is derived from Ningbo City, Zhejiang Province Xiangshan County bay aquatic fry growing company, is cut with the dissecting tool after sterilizing
Silvery pomfret abdomen is opened, enteron aisle is taken out, is rinsed well fat, the blood etc. on enteron aisle outer wall with PBS buffer solution.At leisure by stomach
Intestinal content squeezes out, and then longitudinally cuts off gastrointestinal tract with sterilizing eye scissors, several times using PBS buffer solution cleaning, removes residual
Stay intestinal content.Inner wall of intestine mucous membrane is slowly scraped off using rubber scraper later.Isolated Dissolve things inside and mucous membrane are mixed
It is combined, is put into the 10mL centrifuge tube containing PBS buffer solution, be uniformly mixed.It is dipped with oese molten in a small amount of enteron aisle
Object dirty solution is inoculated in LB agar medium, 28 DEG C of constant temperature incubations for 24 hours after, the consistent dominant colony of picking morphological feature carries out
It isolates and purifies, the bacterial strain after purifying 4 times is subjected to customary physiological biochemistry and 16S rDNA gene sequencing, determines dominant bacteria
Strain is Mermaid luminous bacillus (Photobacterium damselae subsp.damselae).
2, the culture of silvery pomfret enteric microorganism
Used culture medium is LB culture medium (pure water 1000mL, peptone 10g, yeast powder 5g, NaCl 10g, agar
15g/L, pH 7.4~7.6).
3, morphological feature
Bacterium colony is circle on nutrient agar, and surface is smooth wet, the smooth of the edge, colony diameter 1-2mm.Leather
Blue Albert'stain Albert is negative, thallus ellipticity, there is the life of 1-2 root flagellum side pole, movable, does not produce gemma.
4, morphological feature
Bacterium colony is circle on nutrient agar, and surface is smooth wet, the smooth of the edge, colony diameter 1-2mm.Leather
Blue Albert'stain Albert is negative, thallus ellipticity, there is the life of 1-2 root flagellum side pole, movable, does not produce gemma.
5, physiological and biochemical property
The physio-biochemical characteristics of bacterial strain are as follows: glucose is positive, sucrose is negative, maltose is negative, mannitol is negative, fine
Tie up disaccharides feminine gender, rhamnose feminine gender, oxidase positive, amylase positive, contact enzyme positive, beta galactosidase feminine gender, the Portugal a-
Polyglycoside enzyme is negative.
6, the pathogenic detection of the bacterial strain screened
From the healthy silvery pomfret of farm's picking age in days 60, long (2.0 ± 1.1) cm of average body, raise in the whole of 10L seawater
It manages in case, every group of 20 tails, continuous charge during experiment, 26-29 DEG C of water temperature, water is changed in daily bait throwing in, temporarily after feeding 7d, will separate pure
For the Mermaid luminous bacillus of change after LB culture medium, 28 DEG C of culture 24-48h, bacteria suspension is resuspended to obtain with antiseptic sea water in centrifugation, if
Determine bacterial concentration gradient, carry out intraperitoneal injection infection, periodically observe and record, takes fall ill after artificial challenge dying silvery pomfret spleen, kidney
Dirty tissue is coated on nutrient agar plate, separates the consistent bacterium of a large amount of colonial morphology height, bacterium colony
Form, size and physiological and biochemical property are identical as Mermaid luminous bacillus.
Table 1: the pathogenic detection of silvery pomfret Mermaid luminous bacillus of artificial screening
ZM504 plants of Mermaid luminous bacillus (Photobacterium damselae) for finally screening acquisition, in 2018
On December 07, the China Committee for Culture Collection of Microorganisms being deposited in positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Common micro-organisms center, deposit number are CGMCC NO.16907.
After Mermaid luminous bacillus is prepared into inactivated vaccine, the healthy silvery pomfret of 30-60 age in days is randomly selected, is transferred to
In inactivated vaccine, after 10-20 minutes, experiment silvery pomfret is taken out, feeds bait, and normally with the health without vaccine immersion treatment
Silvery pomfret is control.By inactivated vaccine immersion and without the silvery pomfret of immersion treatment after 28 days, with 1 × 108The beauty of CFU/ml concentration
Mermaid luminous bacillus viable bacteria carries out challenge test, changes water supplement bacterium solution daily, periodically observes and records the morbidity of each group silvery pomfret and death
Situation, the results showed that appearance in the 3rd day is dead after infection for control group silvery pomfret, and the 8th day when is all dead, and immune protective rate is
0%, and the 8th talent starts to occur dead, the 8th day immune protective efficiency after Immunization to inactivated vaccine immersion group after infection
It is 100%.Exempt from the result shows that the inactivated vaccine of the Mermaid luminous bacillus preparation of screening effectively can carry out antibiotic property to silvery pomfret
Epidemic disease.
Embodiment 2: Mermaid luminous bacillus and Vibrio anguillarum prepare bigeminy vaccine
One, the preparation of inactivated vaccine
The Mermaid luminous bacillus that the present invention is screened first and Vibrio anguillarum are (purchased from Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center CGMCC, bacterial strain deposit number 1.3793) it is inoculated in 500mlLB culture medium (albumen respectively
Peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH 7.4~7.6), 28 DEG C shake culture 24 hours, blood counting chamber detection
Bacterial concentration is up to 109CFU/ml or OD600 value terminates culture when reaching 0.6 or more, obtains Mermaid luminous bacillus and eel arc
The scale-up medium of bacterium；
Then 0.5% 25ml formalin is added by volume, it is small in room temperature inactivation 48 to the scale-up medium of Vibrio anguillarum
When, it during which shakes 5-8 times, obtains the inactivated bacterial liquid of bacterial strain；
It takes rhizoma atractylodis with decocting after distilled water immersion 60min, filters, be concentrated into final concentration of raw medicine 1g/mL, obtain rhizoma atractylodis leaching
Liquid out；
Two kinds of inactivation thallus are mixed with rhizoma atractylodis leachate by 1:1:1, water aqua type rhizoma atractylodis adjuvant silvery pomfret mermaid hair is made
Polished rod bacterium and Vibrio anguillarum bigeminy vaccine；Mermaid luminous bacillus and Vibrio anguillarum immunogene are 10 in vaccine9CFU/ml, rhizoma atractylodis leaching
Liquid is 1g/mL out.
Sterility testing is carried out to the inactivated vaccine: above-mentioned 80 μ l of bivalent inactivated vaccine being taken to be coated on LB solid medium, is applied
After cloth is uniform, 28 DEG C culture 48 hours after have no that bacterium colony is grown, eligible is the bigeminy of Mermaid luminous bacillus and Vibrio anguillarum
Inactivated vaccine；
The safety of the inactivated vaccine is detected: taking inactivated vaccine that silvery pomfret, control group is injected intraperitoneally with 0.2ml/ tail
Inject the same dose of sterile PBS.Then it observes the silvery pomfret vaccinated and all survives, and feed in normal breeding observing l4 days
And activity is normal, internal organ dissect is not shown any abnormalities, and shows that the inactivated vaccine of preparation is safe and reliable.
After carrying out Sterility testing and safety detection to inactivated vaccine, eligible, that is, Mermaid luminous bacillus and Vibrio anguillarum
Bivalent inactivated vaccine, save backup under the conditions of 4 DEG C.
Two, the effect detection of inactivated vaccine
1, the Immunoprotection test of bivalent inactivated vaccine
The immune effect of the inactivated vaccine of preparation is detected using the method for immersion immunity: choosing 30-60 age in days
Healthy 450 tail of silvery pomfret, is randomly divided into 3 groups, carries out immersion immunity test, side using the inactivated vaccine prepared in specific embodiment 2
Method is as follows: test is divided into 3 groups, and the 1st group is control group, and the 2nd group is booster immunization group, and the 3rd group is not reinforce immune group, by the 2nd
Group and the 3rd group of silvery pomfret are soaked in through 1O times of diluted inactivated bacterial liquid (2 × 108CFU/m1 15min is impregnated in).2nd group of silvery pomfret
Booster immunization is primary in the same way again after 10 days, other rearing conditions are consistent with not immune silvery pomfret.30th after immune
It carries out challenge test to 3 groups of silvery pomfrets, and the 1st group of silvery pomfret is all dead, and the 2nd group of immune protective rate is up to 100%, the 3rd group
Immune protective rate is 89%.
2, the Immunoprotection test of vaccine
The immune effect of the inactivated vaccine of preparation is detected using the method for intraperitoneal injection: being randomly selected 30-60 days
150 tail of healthy silvery pomfret in age, point 3 test groups, processing 1 are control group, intraperitoneal injection sterilizing 100 μ l of PBS, processing 2 at random
For booster immunization group, the inactivated vaccine of 100 μ l embodiments 2 preparation is injected intraperitoneally, injects the preparation of 100 μ l embodiments 2 after 10d again
Inactivated vaccine, processing 3 is does not reinforce immune group, the inactivated vaccine of intraperitoneal injection 100 μ l embodiments 2 preparation.It is 30 days immune
Afterwards, challenge test is carried out to 3 groups of silvery pomfrets, the 1st group of silvery pomfret is all dead, and the 2nd group of immune protective rate is up to 99.8%, the 3rd group
Immune protective rate is 88.5%.
It is above-mentioned the experimental results showed that, bivalent inactivated vaccine prepared by the present invention has efficient immune effect, Neng Gouyou
The premunition of the raising silvery pomfret of effect, has good market promotion prospect.
3, influence of the bigeminy vaccine to silvery pomfret stress ability
In view of silvery pomfret stress reaction is strong, can usually cause because of misoperation or environmental stimuli in breeding process
Fish body is injured, causes certain death rate.Therefore, influence of the inactivated vaccine to fish body stress ability, which also becomes, measures vaccine product
An important factor for matter.
Using rhizoma atractylodis leachate as the Mermaid luminous bacillus of adjuvant and Vibrio anguillarum bigeminy vaccine (epidemic disease made of the present invention
Seedling 1) with order 33539 Mermaid luminous bacillus mermaid subspecies freeze-dried powder of ATCC and 1.3793 conduct of Vibrio anguillarum CGMCC
Common inactivated vaccine (vaccine 2) made of immunogene, influence of both detections to silvery pomfret stress stimulation.
When stimulating in view of fish by stressor, Determination of cortisol can be increased significantly, and then blood-sugar content is promoted to increase
Add, the increase of blood glucose provides more energy for muscle and nerve, so that fish adaptation stress environment.Therefore in serum blood glucose and
Cortisol is the important indicator for evaluating fish anti-stress ability.
It is detected to the content of blood glucose and cortisol in rear silvery pomfret serum is vaccinated, selects current year artificial incubation
With a batch silvery pomfret, 6.8 ± 0.78g of weight uses fish as test.After 1 week raise and train, pick out that physically fit, specification is consistent
Silvery pomfret, be divided into 2 groups and tested.Every group sets 3 in parallel, and each parallel plastic barrel for being 500L, each plastic barrel puts 40 in a suitable place to breed
Silvery pomfret is used in tail test, and 2 groups are respectively used to impregnate 5 minutes in 8% NaCl solution, and then 1:5 is diluted with water two by volume
Kind bigeminy vaccine 1,2 obtains vaccine solution, then silvery pomfret is put in inflation in bigeminy vaccine solution and impregnates 1h.After culture experiment
Two groups of experiment fish blood samples are taken to carry out stress tests.
Measure two processing groups, two kinds of vaccines stress in rear silvery pomfret serum cortisol, glucose content, so that reflection is not
Influence with Mermaid luminous bacillus made of immunogene and Vibrio anguillarum bigeminy vaccine to silvery pomfret stress stimulation.The survey of glucose
Surely hexokinase method is selected, the use of instrument is Hitachi Hitachi7600；Radioimmunology is selected in the measurement of cortisol.
Influence of 2: the two kinds of bigeminy vaccines of table to silvery pomfret stress ability
The result shows that using rhizoma atractylodis leachate as the Mermaid luminous bacillus of adjuvant and Vibrio anguillarum bigeminy made of the present invention
Vaccine (vaccine 1) is less than the emergency stimulation of silvery pomfret to be frozen with the 33539 Mermaid luminous bacillus mermaid subspecies of ATCC purchased
Dry powder and Vibrio anguillarum CGMCC 1.3793 are as common inactivated vaccine (vaccine 2) (table 2) made of immunogene；Perhaps be because
Separated Mermaid luminous bacillus is isolated from silvery pomfret intestinal tissue, belongs to the bacterial strain separated self, therefore silvery pomfret will not be made to produce
Raw strong stress reaction.
4, influence of the bigeminy vaccine to silvery pomfret ingestion ability
The Mermaid luminous bacillus that the present invention is filtered out from silvery pomfret enteron aisle can effectively reduce in production process because of vaccine
Using and lead to silvery pomfret loss of appetite, the problems such as reducing of ingesting.Using rhizoma atractylodis leachate as the beauty of adjuvant made of the present invention
Fish luminous bacillus and Vibrio anguillarum bigeminy vaccine (vaccine 1) and the 33539 Mermaid luminous bacillus mermaid subspecies of ATCC ordered
Freeze-dried powder and Vibrio anguillarum CGMCC 1.3793 are as common inactivated vaccine (vaccine 2) made of immunogene, and both detections are to silvery pomfret
The influence of feeding behaviour.
The same a collection of silvery pomfret of current year artificial incubation is selected, 7.12 ± 0.54g of weight uses fish as test.Through 1 week tame and docile
After supporting, physically fit, the consistent silvery pomfret of specification is picked out, is divided into 2 groups and is tested.Every group sets 3 in parallel, is each in parallel
The plastic barrel of 500L, each plastic barrel put the test of 40 tails in a suitable place to breed with silvery pomfret, and 2 groups are respectively used to impregnate 5 in 8% NaCl solution
Minute, then Mermaid luminous bacillus and Vibrio anguillarum bigeminy vaccine vaccine 1,2 is diluted with water in 1:5 by volume.Silvery pomfret is put in
1h is impregnated in inflation in two kinds of bigeminy vaccine solution.Two groups of experiment silvery pomfrets are normally fed after culture experiment, during which
Guarantee that bait is sufficient.It weighs before feeding daily to bait, is denoted as W0, residual erbium is collected after the completion of ingesting, and is weighed after dry,
It is denoted as W1.Culture experiment carries out 7 days altogether, counts two experimental group daily food ration (food rations (g)=W1-W0)；And it calculates flat
Mean value compares and analyzes.
Influence of 3: the two kinds of bigeminy vaccines of table to silvery pomfret food ration
It is above-mentioned the experimental results showed that, bivalent inactivated vaccine prepared by the present invention has efficient immune effect, Neng Gouyou
The premunition of the raising silvery pomfret of effect, reduces the influence to silvery pomfret stress stimulation and ingestion ability, and booster immunization once more helps
In playing effect, there is good market promotion prospect.
Claims (4)
1. a kind of bivalent inactivated vaccine, which is characterized in that the inactivated vaccine includes antigen and vaccine adjuvant, wherein antigen
Mermaid luminous bacillus and Vibrio anguillarum for inactivation；The deposit number of the Mermaid luminous bacillus is CGMCC
NO.16907。
2. bivalent inactivated vaccine as described in claim 1, which is characterized in that in the inactivated vaccine, Mermaid luminous bar
The concentration of bacterium and Vibrio anguillarum is respectively 107-1010CFU/ml。
3. bivalent inactivated vaccine as described in claim 1, which is characterized in that the vaccine adjuvant is rhizoma atractylodis leachate, is
It decocts, filters after taking rhizoma atractylodis distilled water immersion, obtain rhizoma atractylodis leachate after filtrate concentration.
4. the preparation method of bivalent inactivated vaccine described in claim 1, which is characterized in that the method includes following step
It is rapid:
1) Mermaid luminous bacillus and Vibrio anguillarum bacterial strain are seeded in respectively in LB liquid medium, 28 DEG C of cultures for 24 hours, use blood cell
Tally detects bacterial concentration up to 109CFU/ml or spectrophotometer detection OD600 terminate culture when reaching 0.6 or more, obtain
The scale-up medium of bacterial strain；
2) 0.5% formalin is added by volume, the scale-up medium of bacterial strain is inactivated 24 hours in room temperature, during which shakes 8-
10 times, obtain the inactivated bacterial liquid of bacterial strain.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101461940A (en) * | 2009-01-09 | 2009-06-24 | 中国水产科学研究所南海水产研究所 | Photobacterium damsela vaccine as well as preparation method and use thereof |
| WO2009080767A1 (en) * | 2007-12-21 | 2009-07-02 | Intervet International B.V. | Fish vaccine |
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|---|---|---|---|---|
| WO2009080767A1 (en) * | 2007-12-21 | 2009-07-02 | Intervet International B.V. | Fish vaccine |
| CN101461940A (en) * | 2009-01-09 | 2009-06-24 | 中国水产科学研究所南海水产研究所 | Photobacterium damsela vaccine as well as preparation method and use thereof |
| CN102139103A (en) * | 2011-04-07 | 2011-08-03 | 淮海工学院 | Preparation and application methods of photobacterium damsela vaccines of cynoglossus semilaevis |
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