CN103981154B - A kind of Pseudomonas aeruginosa phage and the application in mink hemorrhagic pneumonia is prevented thereof - Google Patents
A kind of Pseudomonas aeruginosa phage and the application in mink hemorrhagic pneumonia is prevented thereof Download PDFInfo
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Abstract
The invention belongs to biological technical field, a kind of Pseudomonas aeruginosa phage and the application in mink hemorrhagic pneumonia is prevented thereof.This Pseudomonas aeruginosa phage on April 14th, 2014 is preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number is CGMCC No.9102, and Classification And Nomenclature is Pseudomonas aeruginosa phage.This Pseudomonas aeruginosa phage application in mink hemorrhagic pneumonia is prevented, it is assumed that the living space of every mink is 1m3；Adopt ullrasonic spraying mode: the titer of Pseudomonas aeruginosa phage is 109PFU/ml, every mink consumption is 10ml；Used 1 time every 1 month of outbreak of disease phase, mink hemorrhagic pneumonia can be prevented.Adopting the method for application of ullrasonic spraying, phage can be directly to the pulmonary reaching pathogenic bacterium field planting, improves phage and controls the efficiency of animal lung bacterial infection, improves fanning economics.
Description
Technical field
The invention belongs to biological technical field, relate to a strain can Specific lytic pseudomonas aeruginosa strains phage separator and adopt ullrasonic spraying mode use this phage mink hemorrhagic pneumonia prevent in application.
Background technology
Mink is the fur economic animal breed variety that the whole world is main, has higher economic worth.In recent years, the demand of fur coat is increased by China day by day, has been converted into main fur coat import and consumption big country from original fur coat exported country.The great potential in domestic fur coat market, also makes the Fur Animal Feeding industry supporting fur coat industrial development develop rapidly.Pseudomonas aeruginosa is a kind of conditionality pathogenic bacterium, replaces season at Xia Qiu, and this microbial mink hemorrhagic pneumonia, mortality rate, up to about 70%, brings huge economic loss to mink aquaculture.
Phage be one can bacterial infection, do not infect the virus of animal and plant cell.Lytic phage (hereinafter referred to as phage), after bacterial infection, can cause host cell to break, and utilizes this characteristic can apply phage and controls antibacterial infection.The 70-80 age in last century, the appearance of bacterial resistance sex chromosome mosaicism, particularly superbacteria, it is subject to common concern about phage 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, shows advantage in controlling animal bacteria disease.
Medicine can be fed directly to pulmonary by inhalation, is the treatment relatively simple effective route of administration of pulmonary disease.Pulmonary administration novel form and preparation mainly have metered dose inhaler, spray, Foradil Aerolizer formoterol fumarate, microsphere and liposome.Wherein, the reasons such as ullrasonic spraying adopts high oscillation intensity and low ventilation level that medicine can be sent into lung deep effectively, and equipment needed thereby is simple, preparation expense is relatively low, are the suitableeest administering modes treating antibacterial pulmonary infection in animal productiong process.
Phage separator in the present invention is to separate from nature to obtain lytic phage, and Pseudomonas aeruginosa has stronger splitting action；After adopting spray pattern to use, it is possible to prevention 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 rate that hemorrhagic pneumonia causes, improve the technical 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 caused by Pseudomonas aeruginosa in prevention mink breeding process, reduce the mortality rate caused by this disease, the technical method of mink farming Business Economic Benefit can be improved.
The technical scheme is that and provide a kind of Pseudomonas aeruginosa phage Pseudomonasaeruginosabacteriophage, this phage is preserved on April 14th, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; preserving number is CGMCCNo.9102, Classification And Nomenclature is Pseudomonas aeruginosa phage.China Committee for Culture Collection of Microorganisms's common micro-organisms centre address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, 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 good preventive effect.
With the Pseudomonas aeruginosa in mink source for host in the present invention, separation obtains 1 Pseudomonas aeruginosa strain fine melt phage, and one step growth curve shows, its incubation period, burst times respectively may be about 25min, 35min, burst size is 115PFU/ infection cell, and cracking ability is stronger；14 bacterial strains in 15 Pseudomonas aeruginosa strains there are is splitting action, has wider fragmentation pattern；Through the mode of collunarium, inoculating phage splitting liquid, compared with matched group, there is not exception in mice behavior, and pathological change all do not occur in pulmonary, liver, spleen, it was shown that Pseudomonas aeruginosa lytic phage lysate is safe and free of toxic and side effects；Use ullrasonic spraying to be atomized this phage, the titer of phage is not made significant difference；Adopt ullrasonic spraying mode: the titer of Pseudomonas aeruginosa phage is >=109PFU/ml, the confined space of every mink is 1m3, consumption is 10ml；Used 1 time every 1 month of outbreak of disease phase, mink hemorrhagic pneumonia can be prevented.
Beneficial effects of the present invention:
(1) separate acquisition one Pseudomonas aeruginosa strain fine melt phage, and fragmentation pattern is wider, can be applicable to Internal-external contral charrin disease；
(2) adopting the method for application of ullrasonic spraying, phage can be directly to the pulmonary reaching pathogenic bacterium field planting, improves phage and controls the efficiency of animal lung bacterial infection, improves fanning economics；
(3) apply this phage and control Pseudomonas aeruginosa, preventing and treating animal lung antibacterial infects substitute antibiotics, can reduce antibiotic and make consumption in mink farming process, reduce the occurrence probability of Resistant strain, improve the action effect of antibiotic therapy other diseases, reduce feeding cost.
Detailed description of the invention
Embodiment 1
The screening of phage and purification
1. the preparation of Pseudomonas aeruginosa
Gather the mink pathological material of disease that hemorrhagic pneumonia is suffered from mink farming field, use selective medium separation Pseudomonas aeruginosa.Adopt molecular biology method, it is determined that these pathogenic bacterium are Pseudomonas aeruginosa.Use the amplification of LB culture medium, stay separation phage to use.
2. the process of water sample
Take hospital wastewater, add CaCl2Final concentration of 1mol/L, 5000rpm are centrifuged 10min, remove the deposit seed in sewage.Take 0.22 μm of filter membrane Entkeimung of supernatant.Take filtrate 20ml, mix with 20ml2 × LB culture medium, by the inoculum concentration of 1%, inoculate the Pseudomonas aeruginosa of 400 μ l, 37 DEG C of enrichment culture 12h.Taking the above-mentioned bacterium solution of 5ml, 4 DEG C, 5000rpm is centrifuged 10min, takes supernatant by 0.22 μm of filter membrane Entkeimung, obtains phage stock solution.
3. the separation of phage
Adopt double-deck agar plate method, separate phage, concrete grammar is as follows: by phage stock solution, 10 times of gradient dilutions, taking the logarithmic (log) phase Pseudomonas aeruginosa mixing corresponding with 300ul of 300ul diluent respectively, hatch 15min for 37 DEG C, what be incubated with 5ml55 DEG C is completely melt that top agar (agar concentration is 0.5%) mixes, uniform spreading, on bottom agar (agar concentration 1.5%) plate, is inverted 37 DEG C of incubated overnight after cooling 15min.Next day, observe the appearance situation of plaque.The single plaque of picking, repeats 3 double-deck agar plate experiments, obtains single phage.Dissolve in the normal saline of 1ml at the single plaque of picking.
4. the amplification of phage
Choose the single bacterium colony of Pseudomonas aeruginosa, be inoculated in 50ml LB liquid medium, 37 DEG C, 140rpm shaken cultivation to exponential phase early stage；Add 1ml and contain the normal saline of plaque, 37 DEG C, 160rpm shaken cultivation overnight；4 DEG C, the centrifugal 10min of 5000rpm, remove bacterial sediment, take supernatant.Join in the Pseudomonas aeruginosa logarithmic (log) phase bacterium solution of 500ml, after bacterium solution is clarified, 4 DEG C, the centrifugal 10min of 5000rpm, take supernatant.Repeated amplification, uses 0.22 μm of membrane filtration, obtains the phage splitting liquid of wanted volume.
5. the purification of phage
NaCl, final concentration of 1mol/L is added in phage splitting liquid, after ice bath 1h, 4 DEG C, the centrifugal 10min of 8000rpm, precipitate bacterial chip, collect supernatant；Volume according to supernatant, adds PEG, final concentration of 10% (m/v), 4 DEG C, the centrifugal 10min of 10000g, removes supernatant, by the phage of equal-volume physiological saline solution precipitation, namely obtains the phage splitting liquid of preliminary purification.
Embodiment 2
The biological characteristics of phage
1. the electron microscopic observation of phage
Take the phage suspensions that ultracentrifugation purifies and drip on the copper mesh being covered with polyvinyl formal film a little, dye 5-10min with 2% phosphotungstic acid (pH7.0), copper mesh is put on dry filter paper, natural drying.Then with Hitachi JEM2100C type electron microscopic observation.
2. the one step growth curve of phage
Add phage and logarithm early stage Host Strains make MOI=0.1,37 DEG C hatch 15min after, 10000 × g is centrifuged 10min, abandon supernatant, LB culture medium suspension thalline, recentrifuge, suspension precipitation, after washing 2 times, precipitate with the LB culture medium suspendible of 5ml preheating and fully mix, being immediately placed in 37 DEG C of shaking tables (140rpm/min) and cultivate, starting timing, 100 μ l are sampled in 0 moment and each 10min, 10000 × g is centrifuged 5min, draws supernatant, and 10 times of gradient dilutions measure phage titre.Each time point is all made the multiple pipe of double and is averaged, and simultaneously with the Host Strains being not added with phage be not added with the phage of Host Strains for comparison, experiment repeats 3 times.Last with infection time for abscissa, the titre infecting system pnagus medius is that vertical coordinate draws one step growth curve, draws the incubation period of phage, burst times and burst size.Wherein burst size computing formula: burst size=outburst phage titre in latter stage/initial infection Host Strains concentration.
3. the fragmentation pattern of phage
Take the phage solution of 10 times of gradient dilutions after the purification of 300 μ l, the Pseudomonas aeruginosa mixing that 15 strains obtained are to be measured is newly separated respectively with 300ul, 37 DEG C hatch 15min after, add the top agar being the completely melt mixing of 5ml, 55 DEG C of insulations, pour into rapidly on the LB culture dish of bottom-layer agar.After cooling 20min, after being inverted 37 DEG C of incubators cultivation 10-12h, observe the appearance situation of plaque.Whether appearance according to plaque, judges the fragmentation pattern of phage respectively.
Embodiment 3
The safety experiment of phage
Selecting 12 body weight is the male mouse of kunming of 25～30g, is randomly divided into experimental group and blank group, respectively the phage splitting liquid (10 of intranasal inoculation purification10PFU/ml) and normal saline 20ul, after vaccinization 3 days, observe the behavior expression of mice, and go lung, liver, kidney to do pathological section, it is determined that whether the phage splitting liquid of purification is to mice toxic side effect.
Embodiment 4
Phage titer is not made significant difference by ullrasonic spraying
Illustrate that according to ultrasound atomizer the phage splitting liquid of atomization purification is collected the phage drop after atomization, measured its titer, compares the change of phage titer before and after atomization.
Embodiment 5
Ullrasonic spraying mode is adopted to use Pseudomonas aeruginosa phage prevention mink hemorrhagic pneumonia experiment 1
Break out peak period (August-November) in hemorrhagic pneumonia, namely 8,9,10, monthly beginning of the month in November, use ultrasound atomizer with ultrasonic atomizatio mode, application of bacteriophage lysate prevention mink hemorrhagic pneumonia.
Test period: on August 1st, 2012；JIUYUE 1 day；October 1；November 1；
Materials and methods: large-scale mink farming field, Liaoning Province, chooses U.S. sable 54, is randomly divided into two groups, often group 27, respectively inserts in 2 confined spaces and (is 27m3).With PPA-ABTNL, (titer is for 109PFU/ml) phage splitting liquid 270ml is as sucking liquid, uses nebulizer to be all atomized by suction liquid in 50min, and atomization relief mink sucks 1h.It is the pure water of 270ml that tester sucks liquid, and atomization condition is ibid.Continuously perform 4 experiments, itemized record mink death condition the beginning of each month, the dead sick ermine of doubtful hemorrhagic pneumonia is cutd open inspection, calculates the mortality rate caused by hemorrhagic pneumonia.
The impact on mink hemorrhagic pneumonia mortality rate of the table 1 ultrasonic atomizatio phage mix preparation
Ullrasonic spraying mode is adopted to use Pseudomonas aeruginosa phage prevention mink hemorrhagic pneumonia experiment 2
Break out peak period (August-November) in hemorrhagic pneumonia, namely 8,9,10, monthly beginning of the month in November, use with ultrasonic atomizatio mode, application of bacteriophage lysate prevention mink hemorrhagic pneumonia.
Test period: on August 1st, 2013；JIUYUE 1 day；October 1；November 1；
Materials and methods: large-scale mink farming field, Liaoning Province, chooses the white ermine of Denmark 54, is randomly divided into two groups, often group 27, respectively inserts in 2 confined spaces and (is 27m3).With PPA-ABTNL, (titer is for 109PFU/ml) phage application liquid 270ml is as sucking liquid, uses ultrasound atomizer to be all atomized by suction liquid in 50min, and atomization relief mink sucks 1h.It is the pure water of 270ml that tester sucks liquid, and atomization condition is ibid.Continuously perform 4 experiments, itemized record mink death condition the beginning of each month, the dead sick ermine of doubtful hemorrhagic pneumonia is cutd open inspection, calculates the mortality rate caused by hemorrhagic pneumonia.
The impact on mink hemorrhagic pneumonia mortality rate of the table 2 ultrasonic atomizatio phage mix preparation
Claims (2)
1. a Pseudomonas aeruginosa phage, it is characterized in that, this Pseudomonas aeruginosa phage is preserved on April 14th, 2014 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number is CGMCCNo.9102, and Classification And Nomenclature is Pseudomonas aeruginosa phage.
2. a Pseudomonas aeruginosa phage as claimed in claim 1 is being prepared for preventing the application in mink hemorrhagic pneumonia medicine.
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| 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 |
| US11497216B2 (en) * | 2017-02-17 | 2022-11-15 | Intron Biotechnology, Inc. | Pseudomonas aeruginosa bacteriophage pse-AEP-4 and use thereof for inhibiting proliferation of Pseudomonas aeruginosa |
| CN110612349B (en) * | 2017-02-17 | 2023-05-05 | 尹特荣生物科技株式会社 | Novel pseudomonas aeruginosa phage Pse-AEP-3 and use thereof for inhibiting pseudomonas aeruginosa proliferation |
| 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 |
| 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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| EP2248890A1 (en) * | 2009-04-30 | 2010-11-10 | Institut Pasteur | Bacteriophages specific to PAK and CHA strains of Pseudomonas aeruginosa and their applications |
| CN103732235A (en) * | 2010-09-17 | 2014-04-16 | 药物技术业制药技术股份有限公司 | Antibacterial phage, phage peptides and methods of use thereof |
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| EP2248890A1 (en) * | 2009-04-30 | 2010-11-10 | Institut Pasteur | Bacteriophages specific to PAK and CHA strains of Pseudomonas aeruginosa and their applications |
| CN103732235A (en) * | 2010-09-17 | 2014-04-16 | 药物技术业制药技术股份有限公司 | Antibacterial phage, phage peptides and methods of use thereof |
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| Title |
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| Bacteriophages Can Treat and Prevent Pseudomonas aeruginosa Lung Infections;Laurent Debarbieux, et al.;《The Journal of Infection Diseases》;20101231;摘要，图1 * |
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