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Wide-host-spectrum Salmonella pullorum bacteriophage and application thereof

A Salmonella and phage technology, applied in the field of microbial engineering, can solve problems such as the emergence of drug-resistant strains, antibiotic residues, serious problems, etc., and achieve the effect of high safety and low toxic and side effects

Active Publication Date: 2014-09-17
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Farms often use purification methods and feeding antibiotics to control Salmonella infection, but because the disease spreads vertically and horizontally, these control measures are not ideal, and the extensive use of antibiotics not only leads to the continuous emergence of drug-resistant strains, but also The problem of antibiotic residues is also becoming more and more serious

Method used

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  • Wide-host-spectrum Salmonella pullorum bacteriophage and application thereof
  • Wide-host-spectrum Salmonella pullorum bacteriophage and application thereof
  • Wide-host-spectrum Salmonella pullorum bacteriophage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, the separation and preparation of bacteriophage PSPu-4-116

[0019] The fecal liquid sewage sample among the present invention is collected from Nanjing Lukou laying hen farm cesspool; Phage host bacterium is Salmonella pullorum spu-116 (isolated from the intestinal tract of broiler chickens, separated by the laboratory according to the national standard GB / T 4789.4-2008, identified and saved).

[0020] Fecal sewage samples were filtered through double-layer filter paper, centrifuged at 8000 rpm for 20 min, and then the supernatant was filtered with 0.45 μm and 0.22 μm filter membranes respectively. Take 50mL supernatant, add 500μl overnight culture of phage host bacteria, and then add CaCl 2 After mixing the mother solution to a final concentration of 2mM, add 20mL of TSB-YE liquid medium and place it at 37°C for 12-16h. On the next day, the above-mentioned culture was centrifuged at 12000 rpm for 10 min, and the supernatant was taken and 0.3% chlorofor...

Embodiment 2

[0023] Embodiment 2, amplification culture and purification of phage

[0024] On the double-layer plate forming phage plaques, use the tip of a pipette to pick up a single phage plaque with a larger diameter, inoculate it in 3-5mL TSB-YE liquid medium, add 0.1mL of phage host bacterial solution, Mix well, act at room temperature for 15 minutes, incubate at 37°C for 10-14 hours, centrifuge at 12000 rpm, 4°C for 10 minutes, take the supernatant, and add 0.3% chloroform.

[0025] Take 1 mL of freshly cultured host bacteria and add 0.1 mL of phage lysate (the ratio of a single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Incubate at 37°C for 20 minutes to make the phage particles adsorb to the host bacteria; add 100mL TSB-YE liquid medium, then add CaCl 2 The mother liquor was prepared to a final concentration of 2 mM, cultured with shaking at 37°C for 12-16 hours, centrifuged at 12,000 rpm, and 4°C for 10 minutes, and the supernatant was collected to obt...

Embodiment 3

[0028] Example 3, detection of broad host spectrum of bacteriophage

[0029]In the experiment, 27 strains of bacteria (including Salmonella, Escherichia coli, and Shigella) were selected to analyze the host spectrum of phage PSPu-4-116. The specific operation is as follows: take 100 μl of the overnight culture of 27 strains of bacteria, drop them on the center of 1.5% TSB-YE plate (for Salmonella) or 1.5% LB plate (for E. Apply to form a uniform lawn. Then divide each plate into two areas equally, in one area, take 10 μl of phage PSPu-4-116 and drop it on the surface of the lawn, and in the other area, add 10 μl of physiological saline as a control, and place it upside down at 37°C after the droplet is dry. Cultivate for 12-16 hours and observe the results.

[0030] The results showed (Table 1): the bacteria liquids selected in the test all grew well on the plates. The area where the phage PSPu-4-116 was added dropwise showed a transparent area where no bacteria grew on the...

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Abstract

The invention relates to a Salmonella bacteriophage, particularly a wide-host-spectrum strongly-lytic Salmonella pullorum bacteriophage. The invention also relates to Salmonella pullorum for controlling the bacteriophage in a culture environment. The Salmonella pullorum bacteriophage is named PSPu-4-116, and collected in China Center for Type Culture Collection, and the collection number is CCTCC M2011042.

Description

technical field [0001] The invention relates to a strong lytic phage with broad host spectrum Salmonella pullorum and its application in breeding environment, especially the application capable of specifically killing Salmonella pullorum in the breeding environment. It belongs to the field of microbiological engineering. Background technique [0002] In poultry, Salmonella can cause pullorum, typhoid and paratyphoid fever. Among them, Salmonella pullorum pullorum causes a common multiple infectious disease in chickens, which seriously affects the survival rate of chicks. Most adult chickens are chronic or recessively infected, and generally do not show obvious clinical symptoms. Salmonella pullorum infection is often carried for life, and the eggs produced by infected hens can be as high as 1 / 3. The hatching rate of infected eggs is low, and the survival rate of hatched chicks is also low. Therefore, the economic loss caused by pullorum is huge. Pullorum is distributed w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76
CPCY02A50/30
Inventor 包红朵王冉张辉
Owner JIANGSU ACAD OF AGRI SCI
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