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A Pseudomonas aeruginosa phage and its application in the prevention of mink hemorrhagic pneumonia

A technology of Pseudomonas aeruginosa and bacteriophage, which can be applied to viruses/phages, medical raw materials derived from viruses/phages, antibacterial drugs, etc., can solve problems such as economic losses in the mink breeding industry, and reduce feeding costs and usage. , the effect of improving the economic benefits of aquaculture

Active Publication Date: 2016-07-06
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium, which causes hemorrhagic pneumonia in mink during the alternating seasons of summer and autumn, with a mortality rate as high as 70%, which has brought huge economic losses to the mink breeding industry

Method used

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  • A Pseudomonas aeruginosa phage and its application in the prevention of mink hemorrhagic pneumonia
  • A Pseudomonas aeruginosa phage and its application in the prevention of mink hemorrhagic pneumonia
  • A Pseudomonas aeruginosa phage and its application in the prevention of mink hemorrhagic pneumonia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Phage Screening and Purification

[0017] 1. Preparation of Pseudomonas aeruginosa

[0018] The disease materials of mink suffering from hemorrhagic pneumonia were collected from a mink farm, and Pseudomonas aeruginosa was isolated using a selective medium. Using molecular biology methods, it was determined that the pathogen was Pseudomonas aeruginosa. Use LB medium to amplify and save phages for isolation.

[0019] 2. Treatment of water samples

[0020] Take hospital wastewater, add CaCl 2 The final concentration is 1mol / L, and centrifuged at 5000rpm for 10min to remove the precipitated particles in the sewage. The supernatant was sterilized by filtration with a 0.22 μm filter membrane. Take 20 ml of the filtrate, mix it with 20 ml of 2×LB medium, inoculate 400 μl of Pseudomonas aeruginosa at an inoculum size of 1%, and enrich and culture at 37° C. for 12 hours. Take 5ml of the above bacterial solution, centrifuge at 5000rpm at 4°C for 10min, take the supernatant...

Embodiment 2

[0028] Biological properties of phages

[0029] 1. Electron microscope observation of phage

[0030] Take the phage suspension purified by ultracentrifugation and drop a little on the copper grid covered with polyvinyl formaldehyde film, stain with 2% phosphotungstic acid (pH7.0) for 5-10min, put the copper grid on dry filter paper, and let it dry naturally. Then observe with a Hitachi JEM2100C electron microscope.

[0031] 2. One-step growth curve of phage

[0032] Add phage and early logarithmic host bacteria to make MOI = 0.1, incubate at 37°C for 15 minutes, centrifuge at 10,000×g for 10 minutes, discard the supernatant, suspend the bacteria in LB medium, centrifuge again, suspend the sediment, wash twice, and use 5ml Suspend the preheated LB medium and mix well, quickly place it in a 37°C shaker (140rpm / min) for culture, start timing, sample 100μl at time 0 and every 10min, centrifuge at 10000×g for 5min, and pipette Clear, 10-fold serial dilution to determine the phag...

Embodiment 3

[0036] Phage safety experiment

[0037] Select 12 male Kunming mice with a body weight of 25-30 g, and randomly divide them into the experimental group and the blank control group, and inoculate the purified phage lysate (10 10 PFU / ml) and normal saline 20ul, after continuous inoculation for 3 days, the behavioral performance of the mice was observed, and the lungs, liver, and kidneys were removed to make pathological sections to determine whether the purified phage lysate had toxic side effects on the mice.

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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 [0001] The invention belongs to the field of biotechnology, and relates to a phage isolate capable of specifically lysing Pseudomonas aeruginosa strains and the application of the phage in the prevention of mink hemorrhagic pneumonia by means of ultrasonic spraying. Background technique [0002] Mink is the world's main fur economic animal breeding species, with high economic value. In recent years, my country's demand for fur has been increasing day by day, and it has transformed from the original fur export country to a major fur import and consumption country. The huge development potential of the domestic fur market has also enabled the rapid development of the fur animal breeding industry that supports the development of the fur industry. Pseudomonas aeruginosa is an opportunistic pathogenic bacterium, which causes hemorrhagic pneumonia in mink in the alternating seasons of summer and autumn, with a mortality rate as high as 70%, which has brought huge ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A61P11/00C12R1/92
Inventor 徐永平曹振辉张建城李振李晓宇王丽丽李纪彬李淑英徐乐
Owner DALIAN UNIV OF TECH
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