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Novel phage composition of Clostridium perfringens and application of phage composition

A technology of Clostridium perfringens and bacteriophage, which is applied in the field of microorganisms, can solve problems such as affecting the growth performance of animals, economic losses, etc., and achieve the effect of reducing the amount of use and improving economic benefits.

Active Publication Date: 2019-12-31
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the farming industry, necrotizing enteritis and subclinical infections caused by Clostridium perfringens seriously affect the growth performance of animals and cause huge economic losses

Method used

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  • Novel phage composition of Clostridium perfringens and application of phage composition
  • Novel phage composition of Clostridium perfringens and application of phage composition
  • Novel phage composition of Clostridium perfringens and application of phage composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Screening of Clostridium perfringens phage

[0040] (1) Isolation of the perfringing shuttle

[0041] Take the content of broiler necrotic enteritis, dilute it with normal saline, inoculate it on the blood plate by streaking, take the colony with hemolytic ring, inoculate it in the milk medium, and cultivate it at a constant temperature of 45°C to produce a turbulent fermentation strain, which was identified by mass spectrometry to obtain BCP1 , 4 degrees refrigerated storage.

[0042] (2) Isolation and purification of Clostridium perfringens

[0043] Sample pretreatment: if it is a solid sample such as feces, take an appropriate amount and mix it with SM buffer, shake for 2 hours, centrifuge at 4000rpm for 15 minutes, filter the supernatant with a funnel covered with double-layer filter paper, and put it in a new container. Centrifuge the filtrate at 8000rpm for 15min, draw the supernatant with a syringe and connect it to a 0.22μm filter to filter the super...

Embodiment 2

[0052] Example 2 Analysis of biological properties of Clostridium perfringens phage

[0053] Take phages with large and transparent plaques (PA8, 4YP) as an example. The other two phages were analyzed in the same way as PA8 and 4YP.

[0054] 1. Materials and methods

[0055] 1. Clostridium perfringens titer determination

[0056] 1) First, add the fresh host bacterial solution BCP1 and the phage stock solution into the TSC liquid medium in an appropriate proportion for 12 hours, centrifuge at 12,000 r / min for 4 minutes, and filter with a 0.22 μm filter;

[0057] 2) Take 12 sterilized centrifuge tubes and fill them with sterilized water, 900 μL per tube, pipette 100 μL Clostridium perfringens phage into the first centrifuge tube, pipette and mix well, and remove the bacteriophage from the first tube Take 100 μL of liquid and transfer it to the second tube, pipette and mix well, then suck 100 μL of liquid into the third tube, repeat the above steps, and dilute to the 12th tub...

Embodiment 3

[0094] Example 3 Clostridium perfringens infection and phage therapeutic effect

[0095] AA chicks were raised to 10 days old, and 40 healthy AA broilers were selected, raised in cages, divided into 2 groups, 20 in each group, 4 in each cage, free to eat and drink, and the environment was comfortable.

[0096] Cultivate Clostridium perfringens BCP1 bacteria solution 10 8 cfu / mL, use a pipette to take 1, 2, 3, 4 mL into the esophagus of a group of broiler chickens, and the results show that 3 mL can cause half of the group to die.

[0097] In the same way, 3mL of BCP1 was used to challenge group 2, and after 2 hours, 2mL of PA8 phage 10 was taken with a pipette. 9 pfu / mL, esophageal gavage, continuous observation for 7 days, the number of deaths per day were 2, 2, 0, 1, 0, 0, 0, and the cumulative mortality rate was 25%. It shows that phage targeted therapy has definite curative effect.

[0098] Due to the uncertainty of clinically isolated strains, the difficulty and timeli...

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Abstract

The invention discloses a novel phage composition of Clostridium perfringens and application of the phage composition. According to the present invention, four novel phages of Clostridium perfringensare selected and obtained, biological properties and genomics are analyzed, and the phage composition is obtained by utilizing the four novel phages of Clostridium perfringens; and the phage composition is used to prepare products of lytic Clostridium perfringens, is used for the prevention and treatment of Clostridium perfringens infection in breeding companies, can be specifically used to prevent or treat epidemic Clostridium perfringens infection without affecting other normal flora, has fewer side effects, and substitutes for the use of antiboitics effectively.

Description

technical field [0001] The invention relates to a novel Clostridium perfringens phage composition and application thereof, belonging to the field of microorganisms. Background technique [0002] Clostridium perfringens is an anaerobic bacterium that normally exists in the intestines of livestock, poultry and humans, and is widely distributed in nature. The bacterium can produce six main virus-causing toxins (α, β, ε, ι, β2 and enterotoxin), which can cause infection and even sudden death in animals and humans. Not only does it seriously harm the poultry industry, but it is also the third leading cause of human foodborne bacteria. [0003] In the breeding industry, necrotizing enteritis and subclinical infections caused by Clostridium perfringens seriously affect the growth performance of animals and cause huge economic losses. Necrotic enteritis in animals is usually prevented by antibiotics added to the feed. However, the widespread use of antibiotics has led to a gradual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04
CPCC12N7/00A61K35/76A61P31/04C12N2795/00021C12N2795/00032
Inventor 刘玉庆
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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