CN103981154A - Pseudomonas aeruginosa bacteriophage and application thereof in prevention of hemorrhagic pneumonia of mink - Google Patents
Pseudomonas aeruginosa bacteriophage and application thereof in prevention of hemorrhagic pneumonia of mink Download PDFInfo
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- CN103981154A CN103981154A CN201410235510.7A CN201410235510A CN103981154A CN 103981154 A CN103981154 A CN 103981154A CN 201410235510 A CN201410235510 A CN 201410235510A CN 103981154 A CN103981154 A CN 103981154A
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Abstract
The invention belongs to the field of biotechnology, and discloses a pseudomonas aeruginosa bacteriophage and an application thereof in prevention of hemorrhagic pneumonia of a mink. The pseudomonas aeruginosa bacteriophage is preserved in China General Microbiological Culture Collection Center on April 14, 2014, has the preservation number of CGMCC No. 9102, and is classified and named pseudomonas aeruginosa bacteriophage. As for the application of the pseudomonas aeruginosa bacteriophage in prevention of hemorrhagic pneumonia of the mink, assume that the living space of each mink is 1m<3>; an ultrasonic spraying mode is adopted; the titer of the pseudomonas aeruginosa bacteriophage is 10<9>PFU/ml, and the dose for each mink is 10ml; hemorrhagic pneumonia of the mink can be prevented by using the pseudomonas aeruginosa bacteriophage once per month in a disease outbreak period. By adopting the ultrasonic spraying application mode, the bacteriophage can directly arrive in the lung where bacteria is colonized, thus improving the efficiency of controlling bacterial infection of the lung of an animal by the bacteriophage, and improving the breeding economic benefits.
Description
Technical field
The invention belongs to biological technical field, relate to the phage isolate that a strain can specificity cracking pseudomonas aeruginosa strains and adopt ullrasonic spraying mode to use the application of this phage in the prevention of mink hemorrhagic pneumonia.
Background technology
Mink is the main fur economic animal breed variety in the whole world, has higher economic worth.In recent years, China increases day by day to the demand of fur coat, from original fur coat export State, is converted into main fur coat import and consumption big country.The great potential in domestic fur coat market, also makes the Fur Animal Feeding industry that supports fur coat industrial development develop rapidly.Pseudomonas aeruginosa is a kind of conditionality pathogenic bacterium, at Xia Qiu, replaces season, this microbial mink hemorrhagic pneumonia, and mortality ratio, up to 70% left and right, has brought huge financial loss to mink aquaculture.
Phage be a kind of can bacterial infection, do not infect the virus of animal and plant cell.Lytic phage (hereinafter to be referred as phage), after bacterial infection, can cause that host cell breaks, and utilizes this characteristic can apply phage and controls bacterium infection.The 70-80 age in last century, the appearance of bacterial drug resistance problem, particularly superbacteria, relevant phage is subject to common concern as the research of antibiotic preparation.At present, in the U.S. and some East European countries, phage has been successfully applied in animal food production process, and has some launch, aspect control animal bacteria disease, is showing advantage.
Inhalation can directly be delivered to lung by medicine, is the comparatively simple and effective route of administration for the treatment of pulmonary disorder.Pulmonary administration novel form and preparation mainly contain metered dose inhaler, sprays, Foradil Aerolizer formoterol fumarate, microballoon and liposome.The reasons such as wherein, ullrasonic spraying adopts high vibration intensity and low ventilation level can effectively medicine be sent into lung deep, and required equipment is simple, preparation expense is lower are in animal production process, to treat the suitableeest administering mode of bacterium pulmonary infection.
Phage isolate in the present invention is to obtain lytic phage from nature separation, and Pseudomonas aeruginosa is had to stronger splitting action; After adopting spray pattern to use, can prevent mink hemorrhagic pneumonia.
Summary of the invention
The invention provides a kind of application lytic phage, adopt the method for application of ullrasonic spraying, reduce the mortality ratio that hemorrhagic pneumonia causes, improve the technological method of mink farming Business Economic Benefit.
Technical scheme
The invention provides a kind of employing ullrasonic spraying mode and use lytic phage, the hemorrhagic pneumonia being caused by Pseudomonas aeruginosa in prevention mink breeding process, reduces the mortality ratio being caused by this disease, can improve the technological method of mink farming Business Economic Benefit.
Technical scheme of the present invention has been to provide a kind of Pseudomonas aeruginosa phage Pseudomonas aeruginosa bacteriophage, this phage is preserved on April 14th, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; preserving number is CGMCC No.9102, Classification And Nomenclature is Pseudomonas aeruginosa phage.China Committee for Culture Collection of Microorganisms's common micro-organisms centre address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101.This Phagus Podoviridae, head diameter is about 40nm, and head length is about 5nm.Adopt ullrasonic spraying mode to use this Pseudomonas aeruginosa phage lysate, mink hemorrhagic pneumonia is had to good preventive effect.
The Pseudomonas aeruginosa in the mink of take in the present invention source is host, separation obtains 1 Pseudomonas aeruginosa strain fine melt phage, and one step growth shows, its latent period, burst times are about respectively 25min, 35min, burst size is 115PFU/ cells infected, and cracking ability is stronger; 14 bacterial strains in 15 Pseudomonas aeruginosa strains are had to splitting action, have wider fragmentation pattern; Through the mode of collunarium, inoculation phage splitting liquid, compares with control group, and mouse behavior does not occur that extremely, pathological change does not all appear in lung, liver, spleen, show Pseudomonas aeruginosa lytic phage lysate safely, have no side effect; Use this phage of ullrasonic spraying atomization, tiring of phage do not made significant difference; Adopt ullrasonic spraying mode: the tiring as>=10 of Pseudomonas aeruginosa phage
9pFU/ml, the enclosed space of every mink is 1m
3, consumption is 10ml; Every 1 month of outbreak of disease phase, used 1 time, can prevent mink hemorrhagic pneumonia.
Beneficial effect of the present invention:
(1) separated acquisition one Pseudomonas aeruginosa strain fine melt phage, and fragmentation pattern is wider, can be applicable to inside and outside and controls charrin disease;
(2) adopt the method for application of ullrasonic spraying, phage can be directly to the lung that reaches pathogenic bacterium field planting, improves the efficiency that phage is controlled animal lung bacterial infection, improves fanning economics;
(3) apply this phage and control Pseudomonas aeruginosa, substitute antibiotics control animal lung bacterium infects, and can reduce the usage quantity of microbiotic in mink farming process, reduces the occurrence probability of Resistant strain, improve the action effect of antibiotic therapy other diseases, reduce feeding cost.
Embodiment
Embodiment 1
The screening of phage and purifying
1. the preparation of Pseudomonas aeruginosa
Gather mink farming field and suffer from the mink pathological material of disease of hemorrhagic pneumonia, use the separated Pseudomonas aeruginosa of selective medium.Adopt molecular biology method, determine that these pathogenic bacterium are Pseudomonas aeruginosa.Use the amplification of LB substratum, stay separated phage to use.
2. the processing of water sample
Get hospital wastewater, add CaCl
2final concentration is 1mol/L, and the centrifugal 10min of 5000rpm removes the deposit seeds in sewage.Get 0.22 μ m filter membrane Entkeimung for supernatant.Get filtrate 20ml, mix with 20ml2 * LB substratum, the inoculum size by 1%, the Pseudomonas aeruginosa of inoculation 400 μ l, 37 ℃ of enrichment culture 12h.Get the above-mentioned bacterium liquid of 5ml, 4 ℃, the centrifugal 10min of 5000rpm, gets 0.22 μ m filter membrane Entkeimung for supernatant, obtains phage stoste.
3. the separation of phage
Adopt double-deck agar plate method, separated phage, concrete grammar is as follows: by phage stoste, 10 times of gradient dilutions, get respectively 300ul diluent and mix with the corresponding logarithmic phase Pseudomonas aeruginosa of 300ul, hatch 15min for 37 ℃, mix with the top-agar of fusing completely (agar concentration is 0.5%) of 5ml55 ℃ of insulation, uniform spreading, on lower floor's agar (agar concentration 1.5%) plate, is inverted 37 ℃ of incubated overnight after cooling 15min.Next day, the appearance situation of observing plaque.The single plaque of picking, repeats 3 double-deck agar plate experiments, obtains single phage.At the single plaque of picking, dissolve in the physiological saline of 1ml.
4. the amplification of phage
Choose the single bacterium colony of Pseudomonas aeruginosa, be inoculated in 50ml liquid LB substratum, 37 ℃, 140rpm shaking culture are early stage to logarithmic phase; The physiological saline that adds 1ml to contain plaque, 37 ℃, 160rpm shaking culture spend the night; 4 ℃, the centrifugal 10min of 5000rpm, remove bacterial sediment, gets supernatant.Join in the Pseudomonas aeruginosa logarithmic phase bacterium liquid of 500ml, after the clarification of bacterium liquid, 4 ℃, the centrifugal 10min of 5000rpm, get supernatant.Repeated amplification, is used 0.22 μ m membrane filtration, obtains the phage splitting liquid of wanted volume.
5. the purifying of phage
In phage splitting liquid, add NaCl, final concentration is 1mol/L, after ice bath 1h, and 4 ℃, the centrifugal 10min of 8000rpm, precipitation bacterial chip, collects supernatant; Volume according to supernatant, adds PEG, and final concentration is 10% (m/v), and 4 ℃, the centrifugal 10min of 10000g, remove supernatant, with the phage of equal-volume physiological saline solution precipitation, obtains the phage splitting liquid of preliminary purification.
Embodiment 2
The biological characteristics of phage
1. the electron microscopic observation of phage
Get phage suspension that ultracentrifugation purifies and drip on the copper mesh that is covered with polyethylene formaldehyde film a little, with 2% phospho-wolframic acid (pH7.0) dyeing 5-10min, copper mesh is put on dry filter paper to seasoning.Then use the JEM2100C of Hitachi type electron microscopic observation.
2. the one step growth of phage
Add the early stage Host Strains of phage and logarithm to make MOI=0.1, hatch after 15min for 37 ℃, the centrifugal 10min of 10000 * g, abandon supernatant, LB substratum suspension thalline, recentrifuge, suspension precipitation, wash after 2 times, by the LB substratum suspendible precipitation of 5ml preheating, also fully mix, be placed in rapidly 37 ℃ of shaking tables (140rpm/min) and cultivate, start timing, in 0, constantly sample 100 μ l with each 10min, the centrifugal 5min of 10000 * g, draws supernatant, and 10 times of gradient dilutions are measured phage titre.Each time point is all made the multiple pipe of double and is averaged, and the phage that does not add the Host Strains of phage and do not add Host Strains of take is contrast simultaneously, experiment repetition 3 times.Finally take infection time as X-coordinate, and the titre that infects system pnagus medius is that ordinate zou is drawn one step growth, draws latent period, burst times and the burst size of phage.Burst size calculation formula wherein: burst size=outburst phage titre/initial infection in latter stage Host Strains concentration.
3. the fragmentation pattern of phage
Get the phage solution of 10 times of gradient dilutions after the purifying of 300 μ l, the Pseudomonas aeruginosa to be measured with new separated 15 strains that obtain of 300ul mixes respectively, hatch after 15min for 37 ℃, add the top-agar of fusing completely of 5ml, 55 ℃ of insulations to mix, on the LB culture dish pouring into rapidly at bottom-layer agar.After cooling 20min, be inverted 37 ℃ of incubators and cultivate after 10-12h, observe the appearance situation of plaque.According to the appearance of plaque whether, judge respectively the fragmentation pattern of phage.
Embodiment 3
The safety experiment of phage
Select 12 male mouse of kunming that body weight is 25～30g, be divided at random experimental group and blank group, respectively the phage splitting liquid (10 of intranasal inoculation purifying
10pFU/ml) and physiological saline 20ul, vaccinization, after 3 days, is observed the behavior performance of mouse, and is gone lung, liver, kidney to do pathological section, determines that whether the phage splitting liquid of purifying is to mouse toxic side effect.
Embodiment 4
Ullrasonic spraying does not make significant difference to phage titer
According to ultrasonic atomizer explanation, the phage splitting liquid of atomization purifying, collects the phage drop after atomization, measures it and tires, the relatively variation of phage titer before and after atomization.
Embodiment 5
Adopt ullrasonic spraying mode to use Pseudomonas aeruginosa phage prevention mink hemorrhagic pneumonia experiment 1
8,9,10, monthly beginning of the month in November in hemorrhagic pneumonia outburst peak period (August-November),, use ultrasonic atomizer in ultrasonic atomizatio mode, application of bacteriophage lysate prevention mink hemorrhagic pneumonia.
Test period: on August 1st, 2012; September 1; October 1; November 1;
Materials and methods: large-scale mink farming field, Liaoning Province, choose 54 of U.S. sables, be divided at random two groups, 27 every group, respectively insert in 2 enclosed spaces and (be 27m
3).The PPA-ABTNL of take (tires as 10
9pFU/ml) phage splitting liquid 270ml is as inhalant liquid, and use spraying gun is by the whole atomizations of inhalant liquid in 50min, and atomization relief mink sucks 1h.Contrast inhalant liquid is the pure water of 270ml, and atomization condition is the same.Carry out continuously 4 experiments the beginning of each month, record in detail mink death condition, the sick ermine of the death of doubtful hemorrhagic pneumonia is cutd open to inspection, calculate the mortality ratio being caused by hemorrhagic pneumonia.
The impact of table 1 ultrasonic atomizatio phage mixed preparation on mink hemorrhagic pneumonia mortality ratio
Adopt ullrasonic spraying mode to use Pseudomonas aeruginosa phage prevention mink hemorrhagic pneumonia experiment 2
8,9,10, monthly beginning of the month in November in hemorrhagic pneumonia outburst peak period (August-November),, use in ultrasonic atomizatio mode application of bacteriophage lysate prevention mink hemorrhagic pneumonia.
Test period: on August 1st, 2013; September 1; October 1; November 1;
Materials and methods: large-scale mink farming field, Liaoning Province, choose 54 of the white ermines of Denmark, be divided at random two groups, 27 every group, respectively insert in 2 enclosed spaces and (be 27m
3).The PPA-ABTNL of take (tires as 10
9pFU/ml) phage application liquid 270ml is as inhalant liquid, and use ultrasonic atomizer is by the whole atomizations of inhalant liquid in 50min, and atomization relief mink sucks 1h.Contrast inhalant liquid is the pure water of 270ml, and atomization condition is the same.Carry out continuously 4 experiments the beginning of each month, record in detail mink death condition, the sick ermine of the death of doubtful hemorrhagic pneumonia is cutd open to inspection, calculate the mortality ratio being caused by hemorrhagic pneumonia.
The impact of table 2 ultrasonic atomizatio phage mixed preparation on mink hemorrhagic pneumonia mortality ratio
Claims (2)
1. a Pseudomonas aeruginosa phage, it is characterized in that, this Pseudomonas aeruginosa phage on April 14th, 2014 be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number is CGMCC No.9102, Classification And Nomenclature is Pseudomonas aeruginosa phage.
2. the application of Pseudomonas aeruginosa phage in mink hemorrhagic pneumonia prevention, is characterized in that, adopts ullrasonic spraying mode: the tiring as>=10 of Pseudomonas aeruginosa phage
9pFU/ml, the enclosed space of every mink is 1m
3, consumption is 10ml; Every 1 month of outbreak of disease phase, used 1 time, can prevent mink hemorrhagic pneumonia.
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Cited By (7)
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|---|---|---|---|---|
| CN105749266A (en) * | 2016-04-26 | 2016-07-13 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof |
| CN106390111A (en) * | 2016-11-25 | 2017-02-15 | 于彦强 | Preparation method of mink hemorrhagic pneumonia inactivated vaccine and application thereof |
| CN108070572A (en) * | 2018-01-25 | 2018-05-25 | 青岛诺安百特生物技术有限公司 | A kind of width fragmentation pattern pyocinophages and its disinfection application |
| CN110144333A (en) * | 2019-05-27 | 2019-08-20 | 青岛诺安百特生物技术有限公司 | One Pseudomonas aeruginosa strain bacteriophage and its application |
| CN110612349A (en) * | 2017-02-17 | 2019-12-24 | 尹特荣生物科技株式会社 | Novel pseudomonas aeruginosa bacteriophage Pse-AEP-3 and use thereof for inhibiting proliferation of pseudomonas aeruginosa |
| CN110621775A (en) * | 2017-02-17 | 2019-12-27 | 尹特荣生物科技株式会社 | Novel pseudomonas aeruginosa bacteriophage Pse-AEP-4 and use thereof for inhibiting proliferation of pseudomonas aeruginosa |
| CN115322973A (en) * | 2022-08-16 | 2022-11-11 | 广西大学 | Pseudomonas aeruginosa bacteriophage HCZ001 and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105749266A (en) * | 2016-04-26 | 2016-07-13 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof |
| CN105749266B (en) * | 2016-04-26 | 2021-02-02 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and clostridium botulinum poisoning bivalent inactivated vaccine and preparation method thereof |
| CN106390111A (en) * | 2016-11-25 | 2017-02-15 | 于彦强 | Preparation method of mink hemorrhagic pneumonia inactivated vaccine and application thereof |
| CN110612349A (en) * | 2017-02-17 | 2019-12-24 | 尹特荣生物科技株式会社 | Novel pseudomonas aeruginosa bacteriophage Pse-AEP-3 and use thereof for inhibiting proliferation of pseudomonas aeruginosa |
| CN110621775A (en) * | 2017-02-17 | 2019-12-27 | 尹特荣生物科技株式会社 | Novel pseudomonas aeruginosa bacteriophage Pse-AEP-4 and use thereof for inhibiting proliferation of pseudomonas aeruginosa |
| CN108070572A (en) * | 2018-01-25 | 2018-05-25 | 青岛诺安百特生物技术有限公司 | A kind of width fragmentation pattern pyocinophages and its disinfection application |
| WO2019144830A1 (en) * | 2018-01-25 | 2019-08-01 | 青岛诺安百特生物技术有限公司 | Broad lysis spectrum pseudomonas aeruginosa phage and disinfecting application thereof |
| CN110144333A (en) * | 2019-05-27 | 2019-08-20 | 青岛诺安百特生物技术有限公司 | One Pseudomonas aeruginosa strain bacteriophage and its application |
| CN110144333B (en) * | 2019-05-27 | 2020-04-17 | 青岛诺安百特生物技术有限公司 | Pseudomonas aeruginosa bacteriophage and application thereof |
| CN115322973A (en) * | 2022-08-16 | 2022-11-11 | 广西大学 | Pseudomonas aeruginosa bacteriophage HCZ001 and application thereof |
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