CN106995803A - One plant is used to prevent and treat the sick bacteriophage of prawn vibrio parahaemolytious and its propagation method - Google Patents
One plant is used to prevent and treat the sick bacteriophage of prawn vibrio parahaemolytious and its propagation method Download PDFInfo
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- 241001515965 unidentified phage Species 0.000 title claims abstract description 80
- 241000607598 Vibrio Species 0.000 title claims abstract description 56
- 241000238557 Decapoda Species 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 32
- 230000004151 fermentation Effects 0.000 claims abstract description 32
- 201000010099 disease Diseases 0.000 claims abstract description 29
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 29
- 241000927735 Penaeus Species 0.000 claims abstract description 13
- 239000013535 sea water Substances 0.000 claims abstract description 8
- 239000006041 probiotic Substances 0.000 claims abstract description 3
- 235000018291 probiotics Nutrition 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 27
- 238000011081 inoculation Methods 0.000 claims description 24
- 239000012530 fluid Substances 0.000 claims description 15
- 239000011521 glass Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 244000052616 bacterial pathogen Species 0.000 claims description 5
- 239000013642 negative control Substances 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 230000003612 virological effect Effects 0.000 claims description 3
- 241000210651 Enterobacteria phage 1 Species 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000034994 death Effects 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims description 2
- 208000015181 infectious disease Diseases 0.000 claims description 2
- 230000007480 spreading Effects 0.000 claims description 2
- 238000003892 spreading Methods 0.000 claims description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 abstract description 8
- 101001124039 Banna virus (strain Indonesia/JKT-6423/1980) Non-structural protein 4 Proteins 0.000 abstract description 7
- 238000004321 preservation Methods 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 3
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 238000000926 separation method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 206010018910 Haemolysis Diseases 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000008588 hemolysis Effects 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00051—Methods of production or purification of viral material
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Abstract
The invention discloses one plant of bacteriophage and its propagation method for being used to prevent and treat prawn vibrio parahaemolytious disease, the vibrio parahaemolytious that the present invention is infected using Penaeus Vannmei successfully filters out one plant of fine melt bacteriophage in the seawater, and the phage titer is up to 1010‑1012Pfu/mL, fermentation period is 8 12h, and therapeutic effect substantially, is significant for disease caused by treatment vibrio parahaemolytious and exploitation bacteriophage probiotics.Depositary institution's code of the bacteriophage is CCTCC China typical culture collection centers, and address is：Wuchang, wuhan Luo Jia Shan Wuhan University China typical culture collection center in the school, preservation date is on December 9th, 2016, and deposit number is CCTCC NO：M 2016740, preservation testing result is survival, and entitled vibrio parahaemolyticus phage VP11, Classification And Nomenclature is Vibrio parahaemolyticus bacteriophage VP11.
Description
Technical field
The invention belongs to biological technical field, it is related to one plant of bacteriophage and its expansion for being used to prevent and treat prawn vibrio parahaemolytious disease
Culture method.
Background technology
Control at present, the Main Means for eliminating pathogenic bacteria are still various methods physically or chemically, including various antibiotic
Use.The drawbacks of method physically or chemically all has obvious.First, elimination of the method for physics to various pathogenic bacteria is made
With a certain link that can be only applied to association area, it is impossible to realize the application of all links in whole field, secondly, in chemical method
Though soda acid processing can obtain certain prevention effect, after harmful bacteria formation biomembrane, the effects of these methods with regard to pole not
It is preferable；Finally, although antibiotic begins to use effect significantly, but is used for a long time, and can not only strengthen the resistance to the action of a drug of pathogen, and
And suppress profitable strain, it can not finally efficiently control, eliminate pathogenic bacteria.
In recent years, the research and development of microbial ecological preparation are paid close attention to by each side, and particularly the research of bacteriophage is by people
Favor.Since bacteriophage was found from 1896, because it efficiently cracks its host, and it is harmless to humans and animals, and enjoy
Concern.Bacteriophage is a kind of bacterial virus, and it can produce specific splitting action to host bacteria, and in Bacteria Culture plate
Upper formation plaque, thus it is named as bacteriophage.Bacteriophage is divided into temperate bacteriophage and virulent phage, wherein strong phagocytosis
Body has very strong lysis efficiency to Host Strains, and its duplicating efficiency and fertility are significantly larger than Host Strains；Virulent phage exists
After the stable propagation of Host Strains interior energy, a phage particle can kill several hundred million Host Strains.Phage titer (titre) is single
The bacteriophage number having in the volume of position in (mL).Due to efficient cracking ability of the bacteriophage to Host Strains, for treating bacterium sense
Dye, the research for particularly treating the phage preparation of drug-resistant bacteria infection has become one of focus for studying both at home and abroad, by
The attention of increasing biotech company and research institution.
A kind of vibrio parahaemolyticus phage of the entitled separation of Patent No. CN200910038392.X and its sterilization and
The national inventing patent of application in fungi-proofing discloses a kind of vibrio parahaemolyticus phage (Vibrio of separation
Parahaemolyticus) and its sterilization and it is fungi-proofing in application.The phage monomer isolated is named as BPH-VP-1, right
Vibrio parahaemolytious has the sterilizing ability of wide spectrum.
Number of patent application is entitled a kind of vibrio parahaemolyticus phages of 201510872234.X and preparation method thereof and should
Be related to a kind of vibrio parahaemolyticus phage and its as sterilization of the biological bactericide in food and aquaculture environment should
With.There is the vibrio parahaemolyticus phage of separation, preserving number CCTCC M 2015577 efficient cracking to live to vibrio parahaemolytious
Property, its isolated culture and complex preparation can be used as vibrio parahaemolytious in food additives or environment disinfectant prevention and control food
Pollution.
The content of the invention
The technical problems to be solved by the invention be to provide one plant be used for prevent and treat prawn vibrio parahaemolytious disease bacteriophage and
Its propagation method, with phage splitting it is strong the characteristics of, the bacteriophage has the characteristics of sterilization speed is fast, yield is high, simultaneously
In fermentation ends, separation of solid and liquid is carried out to zymotic fluid, it is to avoid harm of the vibrio parahaemolytious to environment.
Depositary institution's code of the bacteriophage is CCTCC- China typical culture collection centers, in China typical culture collection
Heart location is：Wuchang, wuhan Luo Jia Shan Wuhan University China typical culture collection center in the school, preservation date is 2016
December 9, deposit number is CCTCC NO：M 2016740, preservation testing result is survival, entitled vibrio parahaemolytious phagocytosis
Body VP11, Classification And Nomenclature is Vibrio parahaemolyticus bacteriophage VP11.
In order to solve the above technical problems, the technical scheme is that：Phagocytosis for preventing and treating prawn vibrio parahaemolytious disease
Body, it is characterised in that comprise the following steps：
Step A：Host Strains are isolated and purified, and vibrio parahaemolytious of the invention is from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis
Separation is obtained；
Step B：The expansion culture of Host Strains, adds 1-5% NaCl, 121 DEG C of high pressures in NB or LB fluid nutrient mediums
Sterilize 20min, is cooled to room temperature, using the following two kinds inoculation method, and the first inoculation method is single bacterium colony inoculation, in sterile bar
Single bacterium colony inoculation under part on picking conservation flat board, fermentation temperature is 28-35 DEG C, and rotating speed is 50-200rpm, fermentation time 14-
18h；Second of inoculation method is liquid inoculation, and the CFU for adding culture volume 10-20% is 106-108Pfu/mL secondary haemolysis
Vibrios bacterium solution is fermented into NB fluid nutrient mediums, and fermentation temperature is 28-35 DEG C, and rotating speed is 50-200rpm, fermentation time
10-16h；
Step C：Bacteriophage separates, and separates and obtains from a large amount of seawater；
Step D：Bacteriophage is spread cultivation, and the bacteriophage separated is spread cultivation；
Step E：By the zymotic fluid of gained, 1000-8000rpm centrifuges 5-20min at 2-10 DEG C so that Host Strains are settled
In bottom, take upper strata to clarify part, produce bacteriophage probiotics, its potency is 1010-1012pfu/mL；
Step F：The cracking growth curve that bacteriophage is used to prevent and treat prawn vibrio parahaemolytious disease is determined；
Step G：Penaeus Vannmei challenge viral dosage, takes the healthy Penaeus Vannmei that size specification is 50 tails/jin, per cylinder 60
Tail, is cultivated in 15 glass jars respectively, wherein 9 are experiment cylinder, is divided into A, B, C group；Remaining 6 are control cylinder, are divided into the moon
Property control D and blank control E groups, every group of 3 repetitions, in each glass jar plus seawater that 120L salinity is 25；What prawn caught an illness
Method is infected using immersion, i.e., under the conditions of 28 DEG C, using the good vibrio parahaemolytious bacterium solution of step B fermentation, according to bacterial concentration
Every group of volume for needing to add bacterium solution is calculated with glass jar volume, A, B, C and negative control D groups is separately added into, finally makes glass
Water body bacteria containing amount is up to 1 × 10 in glass cylinder8Cfu/mL, control E groups it is without any processing, in 3 groups of experimental boxs respectively add bacteriophage 1 ×
105pfu/mL、1×104pfu/mL、1×103Pfu/mL, A group, B groups, C groups, record Penaeus Vannmei death condition.
It is preferred that, vibrio parahaemolytious is the strong pathogenic bacteria of prawn, single bacterium colony inoculation fermentation time 14-18h in the step B；
The inoculation bacterium solution CFU of second of inoculation method is 106-108Between, inoculative proportion 10-20%, fermentation time 10-16h.
It is preferred that, the PFU for being used to be inoculated with bacteriophage in the step D is 104-108Pfu/mL, inoculation volume is zymotic fluid
Between the 1-20% of volume.
It is preferred that, the condition of spreading cultivation is that temperature is 28~37 DEG C in the step D, and rotating speed is 50~200rpm；
It is preferred that, expand incubation time control between 8-12h.
It is preferred that, in the step E, 1000-8000rpm centrifuges 5~20min at a temperature of 2~10 DEG C so that Host Strains
Bottom is fallen to, takes upper strata to clarify part, produces bacteriophage ecological agent, its potency is 1010~1012pfu/mL。
The propagation method of one plant of bacteriophage for preventing and treating prawn vibrio parahaemolytious disease, is used to prevent and treat prawn pair using one plant
Hemolysis vibrion disease bacteriophage, bacteriophage spread cultivation be by 1-20% PFU be 104-108Pfu/mL bacteriophages are added to above-mentioned hair
In the good vibrio parahaemolytious zymotic fluid of ferment, 28-37 DEG C of fermentation temperature, rotating speed 50-200rpm, fermentation time 8-12h.
The advantage of the invention is that：The vibrio parahaemolytious that the present invention is infected using Penaeus Vannmei is isolated in the seawater
One plant of fine melt bacteriophage, the bacteriophage yield is high, and (its potency is up to 1010-1012Pfu/mL), the short (8- of fermentation period
12h), it is significant for disease caused by treatment vibrio parahaemolytious.The bacteriophage is named as VP11, is preserved in Chinese allusion quotation
Type culture collection CCTCC, address：Wuchang, wuhan Luo Jia Shan Wuhan University is in the school in China typical culture collection
The heart, preservation date is on December 9th, 2016, and deposit number is CCTCCNO：M 2016740.Have the following advantages that：1. separation
Bacteriophage can high-titer cracking vibrio parahaemolytious, with the spy such as site-specific sterilization, long-term continuous action, environmentally safe
Point；2. growth cycle is short, culture medium is simple, and cost is low, and yield is high；3. breeding water is handled using bacteriophage, relative to antibiosis
Element, with not polluted water, avoids antibody-resistant bacterium from producing and advantage with low cost；4. it can be used for treating prawn culturing process
Disease caused by middle vibrio parahaemolytious, promotes high yield and the good harvest of prawn culturing；5. in fermentation ends, zymotic fluid is consolidated
Liquid is separated, it is to avoid harm of the vibrio parahaemolytious to environment.
Brief description of the drawings
Further detailed description is done to the present invention with reference to the accompanying drawings and detailed description.
Fig. 1 is bacteriophage and its life of the step of propagation method pnagus medius one that the present invention is used to prevent and treat prawn vibrio parahaemolytious disease
Long curve map.
Embodiment
The bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease of the present invention comprises the following steps：Step A：Point of Host Strains
From purifying, vibrio parahaemolytious of the invention separates from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis to be obtained.Step B：Host Strains
Expand culture, 1-5% NaCl, 121 DEG C of autoclaving 20min are added in NB or LB fluid nutrient mediums, room temperature is cooled to.Have
Two kinds of inoculation methods：One is single bacterium colony inoculation, aseptically the single bacterium colony inoculation on picking conservation flat board, and fermentation temperature is
28-35 DEG C, rotating speed is 50-200rpm, fermentation time 14-18h；Two be liquid inoculation, adds culture volume 10-20%'s
CFU is 106-108Pfu/mL vibrio parahaemolytious bacterium solution is fermented into NB fluid nutrient mediums, and fermentation temperature is 28-35 DEG C,
Rotating speed is 50-200rpm, fermentation time 10-16h；Step C：Bacteriophage separates, and separates and obtains from a large amount of seawater.Step D：Bite
Thalline is spread cultivation, and the bacteriophage separated is spread cultivation.Step E：By the zymotic fluid of gained, the 1000- at 2-10 DEG C
8000rpm centrifuges 5-20min so that Host Strains fall to bottom, takes upper strata to clarify part, produces bacteriophage probiotics,
Its potency is 1010-1012pfu/mL.Step F：The cracking growth curve that bacteriophage is used to prevent and treat prawn vibrio parahaemolytious disease is surveyed
It is fixed, it is shown in Table 1.Step G：Penaeus Vannmei challenge viral dosage, takes the healthy Penaeus Vannmei that size specification is 50 tails/jin, per cylinder 60
Tail, is cultivated in 15 glass jars respectively, wherein 9 are experiment cylinder, is divided into A, B, C group；Remaining 6 are control cylinder, are divided into the moon
Property control D and blank control E groups, every group of 3 repetitions, in each glass jar plus seawater that 120L salinity is 25.What prawn caught an illness
Method is infected using immersion, i.e., under the conditions of 28 DEG C, using the good vibrio parahaemolytious bacterium solution of step B fermentation, according to bacterial concentration
Every group of volume for needing to add bacterium solution is calculated with glass jar volume, A, B, C and negative control D groups is separately added into, finally makes glass
Water body bacteria containing amount is up to 1 × 10 in glass cylinder8Cfu/mL, control E groups it is without any processing, in 3 groups of experimental boxs respectively add bacteriophage 1 ×
105Pfu/mL (A groups), 1 × 104Pfu/mL (B groups), 1 × 103Pfu/mL (C groups), records Penaeus Vannmei death condition.Knot
Really, 1, discovery are shown in Table：Do not apply all dead in the negative control group D of bacteriophage, prawn 72h, and experimental group morbidity prawn disease
Shape is lighter；Bacteriophage concentration is higher, and the occurring degree of prawn is lighter, and survival rate is higher, and experimental group survival rate reaches
More than 85%.
The challenge viral dosage result (72h) of the bacteriophage of table 1 preventing and treating Penaeus Vannmei vibrio parahaemolytious disease
The vibrio parahaemolytious that the present invention is infected using Penaeus Vannmei isolates one plant of fine melt bacteriophage in the seawater,
Potency is up to 10 after bacteriophage fermentation10-1012Pfu/mL, fermentation period is 8-12h.Have the following advantages that：The bacteriophage of separation
Can high-titer lytic phage, with site-specific sterilization, long-term continuous action, it is environmentally safe the features such as；Fermentation week
Phase is short, and culture medium is simple, and cost is low, and yield is high；Breeding water is handled using bacteriophage, relative to antibiotic, with not polluting
Water quality, avoid antibody-resistant bacterium produce and advantage with low cost；Vibrio parahaemolytious is drawn during can be used for treatment prawn culturing
The disease risen, promotes high yield and the good harvest of prawn culturing；In fermentation ends, separation of solid and liquid is carried out to zymotic fluid, it is to avoid pair
Harm of the hemolysis vibrion to environment.
For the detection for the bacteriophage for preventing and treating prawn vibrio parahaemolytious disease, using above-mentioned for preventing and treating the secondary haemolysis arc of prawn
The bacteriophage of bacterium disease, is spread evenly across LB flat boards by the vibrio parahaemolytious of incubated overnight using drop method, treats that flat board is parched, and is added dropwise
The 20-50ul liquid that spreads cultivation, keeps 28-37 DEG C of environment temperature simultaneously quiescent culture 8-18 hours, and observation whether there is plaque appearance；Using
Double-layer agar technique, complete thawing is heated to by NB the or LB semisolid culturemediums of solidification, when temperature is down to 45 DEG C, adds culture
To the vibrio parahaemolytious 0.2mL and pregnant solution 0.1mL of logarithmic phase, pour into LB flat boards upper strata, it is to be solidified after, in 28-37 DEG C of culture
8-18 hours, observation whether there is plaque appearance；It is equal according to above-mentioned drop method and double-layer agar technique two methods testing result
It is positive, then proves there is bacteriophage.
For the potency identification for the bacteriophage for preventing and treating prawn vibrio parahaemolytious disease, based on for preventing and treating prawn vibrio parahaemolytious
The detection of the bacteriophage of disease, continuous ten times of doubling dilutions of bacteriophage filtrate, using double-layer agar technique overnight incubation, are then counted
Plaque number, plaque number be accurate with every NA or LB flat boards 30-300, and experiment needs in triplicate, take three times and put down
Average is the potency of final bacteriophage, wherein, potency reduction formula is：Phage titer=plaque number3* extension rate/
The bacteriophage filtrate volume of addition.If if the plaque number in potency qualification process per NA or LB flat boards be more than at double or into
Times it is less than per NA or LB flat boards 30-300, then needs to be multiplied or reduce bacteriophage filtrate extension rate at double.
The propagation method of one plant of bacteriophage for preventing and treating prawn vibrio parahaemolytious disease, is used to prevent and treat prawn pair using one plant
Hemolysis vibrion disease bacteriophage, bacteriophage spread cultivation be by 1-20% PFU be 104-108Pfu/mL bacteriophages are added to above-mentioned hair
In the good vibrio parahaemolytious zymotic fluid of ferment, 28-37 DEG C of fermentation temperature, rotating speed 50-200rpm, fermentation time 8-12h.
Bacteriophage one step growth curve as shown in Figure 1, bacteriophage is inoculated in Host Strains bacteria suspension, takes the time 0,
25,50,75,100,125,150,175,200,720,1440min, using doubling plate method, bacteriophage quantity detection is carried out, is obtained
Bacteriophage one step growth curve.As a result show, bacteriophage VP11 is strong for vibrio parahaemolytious VP11 cracking performances, with extremely strong
Vibrios ability is killed, up to 1010More than pfu/mL.
It is last it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention and non-limiting technical side
Case, it will be understood by those within the art that, those modify or equivalent substitution to technical scheme, and
The objective and scope of the technical program are not departed from, all should be covered among scope of the presently claimed invention.
Claims (7)
1. one plant of bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease, it is characterised in that comprise the following steps：
Step A：Host Strains are isolated and purified, and vibrio parahaemolytious of the invention separates from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis
Obtain；
Step B：The expansion culture of Host Strains, adds 1-5% NaCl, 121 DEG C of autoclavings in NB or LB fluid nutrient mediums
20min, is cooled to room temperature, using the following two kinds inoculation method, and the first inoculation method is single bacterium colony inoculation, aseptically
Single bacterium colony inoculation on picking conservation flat board, fermentation temperature is 28-35 DEG C, and rotating speed is 50-200rpm, fermentation time 14-18h；
Second of inoculation method is liquid inoculation, and the CFU for adding culture volume 10-20% is 106-108Pfu/mL vibrio parahaemolytious
Bacterium solution is fermented into NB fluid nutrient mediums, and fermentation temperature is 28-35 DEG C, and rotating speed is 50-200rpm, fermentation time 10-
16h；
Step C：Bacteriophage separates, and separates and obtains from a large amount of seawater；
Step D：Bacteriophage is spread cultivation, and the bacteriophage separated is spread cultivation；
Step E：By the zymotic fluid of gained, 1000-8000rpm centrifuges 5-20min at 2-10 DEG C so that Host Strains fall to bottom
Portion, takes upper strata to clarify part, produces bacteriophage probiotics, and its potency is 1010-1012pfu/mL；
Step F：The cracking growth curve that bacteriophage is used to prevent and treat prawn vibrio parahaemolytious disease is determined；
Step G：Penaeus Vannmei challenge viral dosage, takes the healthy Penaeus Vannmei that size specification is 50 tails/jin, per the tail of cylinder 60, point
Do not cultivate in 15 glass jars, wherein 9 are experiment cylinder, be divided into A, B, C group；Remaining 6 are control cylinder, are divided into negative control
Add the seawater that 120L salinity is 25 in D and blank control E groups, every group of 3 repetitions, each glass jar；The method that prawn catches an illness is adopted
With immersion infection, i.e., under the conditions of 28 DEG C, using the good vibrio parahaemolytious bacterium solution of step B fermentation, according to bacterial concentration and glass
Cylinder volume calculates every group of volume for needing to add bacterium solution, is separately added into A, B, C and negative control D groups, finally makes in glass jar
Water body bacteria containing amount is up to 1 × 108Cfu/mL, control E groups are without any processing, add bacteriophage 1 × 10 in 3 groups of experimental boxs respectively5pfu/
mL、1×104pfu/mL、1×103Pfu/mL, A group, B groups, C groups, record Penaeus Vannmei death condition.
2. the one plant as claimed in claim 1 bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease, it is characterised in that：The step
Vibrio parahaemolytious is the strong pathogenic bacteria of prawn, single bacterium colony inoculation fermentation time 14-18h in rapid B；The inoculation bacterium of second of inoculation method
Liquid CFU is 106-108Between, inoculative proportion 10-20%, fermentation time 10-16h.
3. the one plant as claimed in claim 1 bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease, it is characterised in that：The step
The PFU for being used to be inoculated with bacteriophage in rapid D is 104-108Pfu/mL, inoculation volume is between the 1-20% of fermentating liquid volume.
4. the one plant as claimed in claim 1 bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease, it is characterised in that：The step
The condition of spreading cultivation is that temperature is 28~37 DEG C in rapid D, and rotating speed is 50~200rpm.
5. the one plant as claimed in claim 1 bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease, it is characterised in that：Expand training
Time control is supported between 8-12h.
6. the one plant as claimed in claim 1 bacteriophage for being used to prevent and treat prawn vibrio parahaemolytious disease, it is characterised in that：The step
In rapid E, 1000-8000rpm centrifuges 5~20min at a temperature of 2~10 DEG C so that Host Strains fall to bottom, take upper strata to clarify
Part, produces bacteriophage ecological agent, and its potency is 1010~1012pfu/mL。
7. one plant of propagation method for being used to prevent and treat the bacteriophage of prawn vibrio parahaemolytious disease, wherein appoints using such as claim 1 to 6
One plant described in what one is used to prevent and treat the sick bacteriophage of prawn vibrio parahaemolytious, it is characterised in that：Bacteriophage spread cultivation be by
1-20% PFU is 104-108Pfu/mL bacteriophages are added in the above-mentioned vibrio parahaemolytious zymotic fluid fermented, fermentation temperature 28-
37 DEG C, rotating speed 50-200rpm, fermentation time 8-12h.
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