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Clostridium welchii bacteriophage as well as bacteriophage composition and application thereof

A technology for Clostridium welchii and Clostridioides welchii disease, applied in the field of microbiology, can solve problems such as antibiotic residues, and achieve the effects of fast onset of action, wide cracking spectrum, and low cost

Active Publication Date: 2020-09-22
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The phage is safe to use and has no side effects. It is used to solve the infection caused by Clostridium welchii and the water problem caused by the large proliferation of Clostridium welchii in water, avoiding the antibiotic residue caused by the use of antibiotics and the induction of drug resistance. Clostridium welchii problems

Method used

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  • Clostridium welchii bacteriophage as well as bacteriophage composition and application thereof
  • Clostridium welchii bacteriophage as well as bacteriophage composition and application thereof
  • Clostridium welchii bacteriophage as well as bacteriophage composition and application thereof

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Experimental program
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Effect test

Embodiment 1

[0035] The isolation and purification of embodiment 1 phage

[0036] 1. Isolation and identification of pathogenic bacteria

[0037] From a chicken farm in Shandong, sick chickens with necrotic enteritis were collected for dissection, and pathogenic bacteria were isolated from the chicken intestines. The pathogenic bacteria were anaerobically cultured in 1% glucose-5% sheep blood culture medium, and the obtained pathogenic bacteria were identified by microscopic examination and molecular identification by corresponding PCR.

[0038] Results: On the medium of 1% glucose-5% sheep blood, a large off-white colony was isolated, with a double-layer hemolytic ring on the edge of the colony, which was identified as Clostridium welchii type A by PCR with specific primers for Clostridium welchii.

[0039] 2. Isolation of Clostridium welchii phage

[0040] Take 50mL of fecal sewage from chicken farms or 5g of chicken intestines and livers, add 500μL of Clostridium welchii type A isolat...

Embodiment 2

[0044] Morphological characteristics of embodiment 2 Clostridium welchii bacteriophage PMQ06

[0045] The above-mentioned isolated phage PMQ06 suspension was dropped on a copper grid covered with polyvinyl formaldehyde film, stained with 2% phosphotungstic acid for 5-10 minutes, and observed with a Hitachi HT7700 transmission electron microscope after drying.

[0046] Such as figure 1 As shown, it was observed under the electron microscope that the head of phage PMQ06 has a polyhedral structure, the diameter of the head is about 46 nm, and there are 6 short tail filaments. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of bacteriophage PMQ06 conforms to the characteristics of the Brachyphage family and belongs to the Brachyphage family.

Embodiment 3

[0047] The potency determination of embodiment 3 Clostridium welchii bacteriophage PMQ06

[0048] Dilute the proliferated phage stock solution 10 times, and take 10 -6 、10 -7 、10 -8 , 3 dilutions. Take 120 μL of the above-mentioned 3 dilutions of phage and 120 μL of the host bacterial suspension, mix them into sterile EP tubes, and mix well. After incubating at 37°C for 5 minutes, adopt the soft agar plate method and invert the incubator at 37°C for anaerobic culture for 5-6 hours, and observe the results. Two parallels were made for each dilution gradient, and the dilution of phage plaques between 30 and 300 was used as the standard for counting, and the average value of the two parallels for each dilution was taken to calculate the titer of phage.

[0049] Phage titer (pfu / mL) = average number of plaques × dilution factor × 10.

[0050] The results showed that the phage titer of PMQ06 was 3.40×10 9 PFU / mL.

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Abstract

The invention discloses a clostridium welchii bacteriophage as well as a bacteriophage composition and application thereof, the bacteriophage is a clostridium welchii bacteriophage PMQ06 and is preserved in China General Microbiological Culture Collection Center on May 15, 2020, and the preservation number is CGMCC No.19977. The bacteriophage has the advantages of high-efficiency bactericidal activity, wide lysis spectrum and high temperature stability, can be used as an active ingredient for preparing drugs for preventing and treating chicken clostridium welchii disease, environmental disinfectants, feed additives, bacteriostats and the like, can be widely applied to various links in the breeding process and the product processing and production process, and has broad market prospects. The pathogenicity clostridium welchii and even the drug-resistant clostridium welchii are rapidly, efficiently and safely killed, the mortality rate of morbidity chickens is effectively reduced, and thefeed additive has the advantages of being safe, free of residues and free of side effects.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a Clostridium welchii phage, its phage composition and application thereof. Background technique [0002] Clostridium welchii (C. welchii), belonging to the Bacillus family, the genus Clostridium, the species Clostridium welchii. It is widely distributed in nature and can be found in soil, sewage, feed, food, feces, and human and animal intestines. Under certain conditions, it can cause a variety of serious diseases. Forms capsules when grown in human and animal living tissues or in serum-containing media, has no flagella, and cannot move. The bacteria can produce a variety of pathogenic exotoxins, which are more harmful to humans and animals. [0003] Chicken necrotic enteritis is mainly caused by the infection of Clostridium welchii type A, and a small amount is caused by the infection of clostridium welchii type C, which is a common disease caused by Clostridium welchii in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A23K10/18A23K50/75A01N63/40A01P1/00C12R1/92
CPCC12N7/00A61K35/76A61P31/04A23K10/18A23K50/75A01N63/40C12N2795/10221C12N2795/10231C12N2795/10232A61K2300/00
Inventor 潘强任慧英孙虎芝刘广芹葛超
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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