CN110607284A

CN110607284A - Escherichia coli phage vB _ EcoM _ swi3 and application thereof

Info

Publication number: CN110607284A
Application number: CN201911012910.0A
Authority: CN
Inventors: 张灿; 韩丽丽; 任慧英; 刘文华
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2019-12-24

Abstract

The invention discloses a coliphage vB _ EcoM _ swi3 with the preservation number of CCTCC M2019467, which belongs to the Myocaviridae family, and has a polyhedral head and a telescopic tail structure, wherein the diameter of the head is about 80nm, and the length of the tail is about 120 nm; clear and bright plaques can be formed on a solid culture medium, no halo is formed around the plaques, the edges are clear and regular, and the diameter is about 1-1.5 mm; the bacteriophage has good cracking effect on swine-origin and chicken-origin escherichia coli in a breeding environment, particularly on swine-origin and chicken-origin pathogenic escherichia coli, and has very wide application prospect in multiple aspects of pharmaceutical preparations, feed additives and the like for preventing and treating swine colibacillosis and chicken colibacillosis.

Description

Escherichia coli phage vB _ EcoM _ swi3 and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a Escherichia coli bacteriophage vB _ EcoM _ swi3 and application thereof.

Background

Escherichia coli belongs to conditional pathogenic bacteria, the pathogenicity is related to the serotype of the Escherichia coli, and differences exist among different animals. The colibacillosis of pigs and the colibacillosis of chickens are widely existed in the breeding environment, once infection causes diarrhea and obviously affects the weight gain of animal organisms, so that the death rate of animal groups is increased rapidly, the ill animals can be infected to other healthy breeding animals through ways such as excrement and the like to cause large-area infection, and the colibacillosis of pig origin and chicken origin in the breeding environment can cause the continuous infection of the animal groups and great economic loss to farmers.

At present, antibiotics are used as a main means for preventing and treating colibacillosis in a farm, but due to massive abuse of the antibiotics, the defects of damage to the microbial flora structure of animal intestinal tracts, antibiotic residues, serious drug resistance and multiple drug resistance of escherichia coli and the like exist, the difficulty of disease prevention and control is increased, the breeding cost is continuously increased, and huge resistance is caused to the development of the breeding industry.

The bacteriophage is a bacteria-dependent virus, can specifically crack host bacteria, has a different action mechanism from antibiotic sterilization, is not influenced by the drug resistance of bacteria, has no residue, has the advantages of short development time, low cost, strong specificity, small dosage, no damage to the composition of normal flora and the like, and has a series of favorable achievements in the prevention and treatment work of drug-resistant bacteria. Theoretically, any pathogenic bacteria have corresponding phage, but the types and the number of the found phage are only one in iceberg, and a large number of phage resources are not developed yet. The development of phage capable of simultaneously cracking various bacteria of different animal sources has wider application prospect clinically, so that the development of phage preparations with the characteristics is significant for the healthy and rapid development of the breeding industry.

Disclosure of Invention

Aiming at the problems, the invention provides the phage capable of killing the pig-source and chicken-source pathogenic escherichia coli in the breeding environment at the same time and efficiently.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a colibacillus phage vB _ EcoM _ swi3, which has a phage preservation number of CCTCC M2019467, is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 19 months in 2019, and is classified and named as a porcine colibacillus phage vB _ EcoM _ swi 3.

The coliphage can form transparent plaques on a solid culture medium, the periphery of the coliphage does not have a halo, the edge of the coliphage is clear and regular, and the diameter of the coliphage is 1-1.5 mm; the observation of a transmission electron microscope shows that the coliphage has an obvious regular polyhedron head structure and a telescopic tail structure, the diameter of the head is about 80nm, the length of the tail is about 120nm, and the coliphage is identified to belong to the Myocapnoraceae.

A series of performance assays were performed on the above E.coli phages with the following results:

a. the titer of the coliphage is 6.4 multiplied by 10 as determined by titer determination and optimal multiplicity of infection determination experiments⁸pfu/mL, optimal multiplicity of infection of 1;

b. the coliphage has stable performance under the neutral condition of pH6-8, is sensitive to ultraviolet rays, and has stable activity at the temperature below 60 ℃;

c. the incubation period of the coliphage is about 25min, the outbreak period is about 75min, and the outbreak amount is about 25 by one-step growth curve determination;

d. the mouse safety experiment shows that the colibacillus phage does not have negative influence on the normal growth of the mouse, and no abnormal condition is found in anatomical inspection, which indicates that the colibacillus phage has higher safety;

e. the experiment shows that 10 percent of the total amount of the components is injected into the abdominal cavity through the abdominal cavity⁷The concentration of Escherichia coli E.coli-K88 in the blood of the mice after pfu/mL of Escherichia coli phage is rapidly reduced, the mice can be completely cleared within 8.5 hours, the phage in the blood disappears, and the phage in the excrement is completely cleared within 99 hours, which shows that the Escherichia coli phage can effectively clear pathogenic Escherichia coli in animal bodies in a short time without phage residue;

f. through Escherichia coli environment disinfection experimentTo give a concentration of 10⁵The Escherichia coli phage multiplication liquid with pfu/mL can effectively kill the host bacteria polluted in the environment.

The invention also provides application of the coliphage in preparation of a biological product for killing swine-origin and chicken-origin colibacillosis simultaneously.

The coliphage provided by the invention is a new phage separated from nature, can specifically crack pathogenic colibacillus of pig origin and chicken origin as a natural enemy of bacteria without destroying the normal flora composition, is not influenced by bacterial drug resistance, has no problems of drug residue and the like, has high safety, and has good application prospect in the aspect of preventing and treating colibacillosis.

Preferably, the biological product is a feed additive and a disinfectant/detergent, wherein the feed additive and the disinfectant/detergent are a single preparation prepared from purified coliphage or a compound preparation prepared from the purified coliphage as a main component.

Preferably, Escherichia coli of porcine origin and Escherichia coli of chicken origin include pathogenic Escherichia coli, including enterotoxigenic Escherichia coli.

Preferably, the enterotoxigenic escherichia coli includes K88 type escherichia coli, and the chicken-derived pathogenic escherichia coli includes O78 type escherichia coli.

The invention also provides a disinfectant or a cleaning agent, which is a single preparation prepared by using the purified coliphage or a compound preparation prepared by using the purified coliphage as a main component, and is used for preventing and treating the pollution of colibacillus of pig origin and chicken origin in a breeding environment.

Preferably, the disinfectant or the cleaning agent is in the form of liquid, freeze-dried powder or tablets; the breeding environment comprises livestock bodies, livestock body surfaces, livestock breeding place ground, livestock breeding place air, livestock feed, drinking water and breeding devices.

The invention also provides a feed additive, which is a single preparation prepared by using the purified Escherichia coli phage or a compound preparation prepared by using the purified Escherichia coli phage as a main component, and is added into livestock and poultry feed for preventing and treating the pollution of Escherichia coli from pig sources and chicken sources in the breeding process.

Preferably, the feed comprises at least pig feed or chicken feed.

Preferably, the swine-origin escherichia coli includes and the chicken-origin escherichia coli includes pathogenic escherichia coli including enterotoxigenic escherichia coli including K88 type escherichia coli, and the chicken-origin pathogenic escherichia coli includes O78 type escherichia coli.

The coliphage provided by the invention can be used as an effective component of a pharmaceutical preparation for preventing and treating colibacillosis, and can be used for inhibiting the multiplication of escherichia coli in the environment, and comprises various products such as feed additives, drinking water additives, meat product detergents and the like.

The coliphage provided by the invention is obtained by separation through the following method:

I. experimental materials:

the liquid dung sewage sample is collected from pig slaughter house sewage in linyi city, Shandong province, and the phage host bacterium is swine pathogenic escherichia coli E.coli-K88, and is separated, identified and stored from diseased pig manure by the laboratory.

Experimental method ii:

adding 5ml of collected sewage sample into 50ml of LB liquid culture medium, then adding 500 mu L of Escherichia coli E.coli-K88 bacterial liquid, mixing uniformly, and placing in a constant temperature incubator at 37 ℃ for overnight culture. The culture is roughly filtered by four layers of gauze, the filtrate is centrifuged at 4000rpm for 10min, the supernatant is centrifuged at 12000rpm for 15min, and then the supernatant is filtered by a 0.22 mu m filter to obtain phage stock solution. Uniformly coating 200 mu L of Escherichia coli E.coli-K88 proliferation solution on the surface of LB solid culture medium by using a cotton swab, airing for 5min, then dropwise adding 10 mu L of phage stock solution, simultaneously dropwise adding 10 mu L of normal saline as a control, carrying out inverted culture at 37 ℃ for 10-12h, and observing that a sample without bacteria growing at a spotting position and with a plaque contains a phage capable of cleaving E.coli-K88.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the invention provides a newly discovered coliphage, which has the characteristics of better tolerance of physicochemical factors, short latent period, high cracking performance, good safety and the like, and is a novel product and means for preventing and treating bacterial diseases in livestock and poultry breeding;

2. the coliphage provided by the invention can efficiently and simultaneously kill pig-origin and chicken-origin pathogenic escherichia coli in a breeding environment and in livestock and poultry bodies, particularly pig-origin enterotoxigenic escherichia coli and chicken-origin pathogenic escherichia coli;

3. the escherichia coli bacteriophage provided by the invention has good hydrophilicity, can be administered through an assembly line, and is easy to prepare an infusion solution, a spray solution or an leacheate; the coliphage can also be prepared into a single preparation or be compounded with other phage to prepare a composite preparation for use, and in addition, the coliphage can also be widely applied to feed additives, disinfectants, cleaning agents and the like.

Drawings

FIG. 1 is a TEM image of coliphage vB _ EcoM _ swi3 provided in the present invention;

FIG. 2 is a graph showing the results of pH stability of Escherichia coli phage vB _ EcoM _ swi3 according to the present invention;

FIG. 3 is a graph showing the results of measuring the thermostability of Escherichia coli phage vB _ EcoM _ swi3 according to an example of the present invention;

FIG. 4 is a graph showing the results of UV stability measurement of Escherichia coli phage vB _ EcoM _ swi3 according to the present invention;

FIG. 5 is a graph showing the results of one-step growth curve measurement of Escherichia coli phage vB _ EcoM _ swi3 according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of the sterilization of Escherichia coli phage vB _ EcoM _ swi3 in animals according to the present invention;

FIG. 7 is a diagram showing the dynamic changes of Escherichia coli phage vB _ EcoM _ swi3 in feces.

Detailed Description

In order to more clearly and fully introduce the E.coli bacteriophage and the application thereof provided in the embodiments of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: isolation and preparation of Escherichia coli phage vB _ EcoM _ swi3

(1) Experimental materials:

the liquid dung sewage sample is collected from pig slaughter house sewage in linyi city, Shandong province, and the phage host bacterium is Escherichia coli E.coli-K88, and is separated, identified and stored in the laboratory from the diseased pig manure.

(2) The experimental method comprises the following steps:

Example 2: purification and Mass propagation of E.coli phage vB _ EcoM _ swi3

Purification of Q1, E.coli phage vB _ EcoM _ swi3

The sample identified as containing phage in example 1 above was purified as follows: diluting phage stock solution 100 μ L by 10 times, mixing diluted phage stock solution 100 μ L with equal amount of overnight-cultured E.coli-K88, adding 3ml of 0.7% LB semisolid culture medium heated to 50 deg.C, mixing, and pouring into containerStanding the upper layer of the LB agar plate for 5min, after the solidification, carrying out inverted culture at 37 ℃ for 4-5h to obtain a plaque-forming double-layer plate. Picking single plaque, adding LB liquid culture medium 3ml, water bathing at 40 deg.C for 30min, centrifuging at 12000rpm for 5min, taking supernatant, and vacuum filtering with 0.22 μm filter to obtain phage filtrate. Taking 100 mu L of phage filtrate, diluting to 10^-6Adding equal volume of host bacterium E.coli-K88, and culturing by double-layer plate method. This process was repeated 5 times to obtain purified phage. The phage can form transparent plaques on a solid culture medium, the surrounding of the phage does not have a halo, the edge of the phage is clear and regular, and the diameter of the phage is 1-1.5 mm.

Purified propagation of Q2, E.coli phage vB _ EcoM _ swi3

Adding 1mL of phage stock solution and 1mL of freshly cultured host bacterium E.coli-K88 bacterial solution into 100mL of LB liquid medium, performing shake culture at 37 ℃ for 9h, adding 5% by volume of chloroform, continuing shake culture for 30min, centrifuging at 13000rpm for 15min, and taking supernatant to obtain a large amount of propagation solution of phage;

adding DNase I and RNaseA into a phage proliferation solution to a final concentration of 1 mu g/mL, shaking and uniformly mixing, incubating for 30min at 37 ℃, adding NaCl to a final concentration of 1mol/mL, fully mixing, standing for 1h at 4 ℃, centrifuging for 20min at 13000rpm, taking supernatant, adding PEG6000 to a final concentration of 10% (W/V), centrifuging for 25min at 13000rpm overnight at 4 ℃, discarding supernatant, taking precipitate, re-suspending the obtained precipitate with physiological saline with a proper volume, and fully washing the inner wall of a centrifuge tube. Adding chloroform with the same volume, gently shaking for 30s, centrifuging at 8000rpm at 4 deg.C for 15min to separate organic phase and water phase, collecting water phase to obtain purified phage proliferation solution, and detecting purified phage by double-layer plate method;

the purified phage is named vB _ EcoM _ swi3 and is preserved in China center for type culture Collection, and the address of the preservation unit is as follows: china, Wuhan university, the preservation number: CCTCC M2019467, classification name: porcine Escherichia coli phage (Escherichia coli phase) with a deposit date of 2019, 6 months and 19 days.

Example 3: transmission electron microscope morphology observation of Escherichia coli phage vB _ EcoM _ swi3

20 μ L of the purified phage-proliferated solution was dropped on a copper mesh, left to stand for about 15min, and excess solution was removed by blotting with filter paper. 15 mu L of 2% phosphotungstic acid (PTA) is dripped on a copper net for dyeing for 5min, and the redundant dye solution is sucked by filter paper, dried and observed by a transmission electron microscope.

The transmission electron microscope result of the Escherichia coli phage vB _ EcoM _ swi3 is shown in FIG. 1, and the Escherichia coli phage vB _ EcoM _ swi3 has an obvious regular polyhedron head structure and a telescopic tail structure, wherein the head diameter is about 80nm, and the tail length is about 120 nm. The phage belongs to the Myoviridae family according to the criteria of "viral Classification-International Commission on Virus Classification-tenth report of the International Commission on Virus Classification of viruses (ICTV) 2018.

Example 4: detection of lysis spectrum of Escherichia coli phage vB _ EcoM _ swi3

Test selection 10 pathogenic E.coli clinical isolates (8 of them isolated from diseased pigs and 2 from diseased chickens) were assayed for the lytic spectrum of the E.coli phage vB _ EcoM _ swi 3. The specific operation is as follows: respectively and uniformly coating 200 mu L of 10 escherichia coli proliferation solutions on an LB solid culture medium plate, after the bacterial solutions are completely absorbed, dropwise adding 5 mu L of phage filtrate on the plate, simultaneously dropwise adding 5 mu L of normal saline as a control, naturally drying, inversely culturing for 4-6h in a constant-temperature incubator at 37 ℃, and observing and recording results.

The results showed that the 10 experimentally selected strains of E.coli grew well on LB solid medium plates, forming a lawn. The region of 8 Escherichia coli plates dripped with Escherichia coli phage vB _ EcoM _ swi3 has no bacteria growth, which indicates that the separated Escherichia coli phage vB _ EcoM _ swi3 can crack 8 of 10 Escherichia coli strains with a cracking rate of 80%, wherein 6 Escherichia coli strains are porcine Escherichia coli strains, and 2 Escherichia coli strains are chicken Escherichia coli strains.

TABLE 1 results of lysis Spectrum test of Escherichia coli phage vB _ EcoM _ swi3

Example 5: titer determination of E.coli phage vB _ EcoM _ swi3

Sequentially diluting the multiplication solution of the coliphage vB _ EcoM _ swi3 by 10 times, taking 100 mu L of the multiplication solution of each dilution, respectively mixing the multiplication solution with the equal volume of the host bacterium E.coli-K88 bacterial solution, performing plaque counting by a double-layer plate method, and performing 3 parallels on each dilution. Calculating the titer of the phage according to the number of plaques, and measuring the titer of the Escherichia coli phage vB _ EcoM _ swi3 to be 6.4 multiplied by 10⁸pfu/mL。

Example 6: determination of the optimal multiplicity of infection of the E.coli phage vB _ EcoM _ swi3

And (3) taking a multiplication solution of the coliphage vB _ EcoM _ swi3 and a host bacterium E.coli-K88 bacterium solution cultured to a logarithmic phase, and counting. Respectively mixing according to the ratio of 0.001, 0.01, 0.1, 1, 10, 100 and 1000, adding 5mLLB broth, shaking and culturing for 4h at 37 ℃, centrifuging at 12000rpm for 20min, performing suction filtration and sterilization on a 0.22 mu m filter to obtain phage proliferation solution, measuring phage titer by a double-layer plate method, performing parallel treatment on each group, and calculating the optimal complex infection number according to the measurement result of the measured phage titer.

The results show (Table 2), when the multiplicity of infection is 1, Escherichia coli phage vB _ EcoM _ swi3 titer reached 8.2X 10⁸pfu/mL. Thus the optimal multiplicity of infection for E.coli phage vB _ EcoM _ swi3 is 1.

TABLE 2 determination of the optimal multiplicity of infection of E.coli phage vB _ EcoM _ swi3

Number of phages	Number of host bacteria	Multiplicity of infection	Phage titer after 4h
				6.4×10⁸	3.9×10⁵	1000	9.7×10⁶
6.4×10⁸	3.9×10⁶	100	7.9×10⁷
				6.4×10⁸	3.9×10⁷	10	8.7×10⁷
6.4×10⁸	3.9×10⁷	1	8.2×10⁸
				6.4×10⁸	3.4×10⁹	0.1	1.56×10⁸
6.4×10⁸	3.4×10¹⁰	0.01	9.8×10⁶
				6.4×10⁷	3.4×10¹⁰	0.001	9.4×10⁶

Example 7: determination of the pH stability of the E.coli phage vB _ EcoM _ swi3

4.5mL of LB liquid medium with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) were added to 10 concentrations of each⁸pfu/mL of Escherichia coli phage vB _ EcoM _ swi3 proliferation solution 500. mu.L, 37 ℃ water bath incubation, respectively in 1h, 2h, 3h sample 1 mL. Sequentially diluting the sampled samples by 10 times, uniformly mixing 100 mu L of proliferation liquid with E.coli-K88 bacterial liquid with the same volume, measuring the titer of the phage by a double-layer plate method, and drawing a pH stability curve of the phage according to the statistical result, wherein each group of three groups of proliferation liquid is parallel.

The results show (FIG. 2) that the E.coli phage vB _ EcoM _ swi3 was stable in the pH range 6-8 when pH was adjusted<6 or pH>At 8, the activity of Escherichia coli phage vB _ EcoM _ swi3 decreased rapidly with increasing pH. When the pH is reduced to 2, the phage acts for 1-3h, and the titer is only 10³pfu/mL; when the pH is raised to 13, the Escherichia coli phage vB _ EcoM _ swi3 has a titer of 10 after 1h of action⁵pfu/mL; after 2h of action, the phage titer was reduced to 10³pfu/mL. Thus, the E.coli phage vB _ EcoM _ swi3 was stable under neutral conditions at pH 6-8.

Example 8: determination of the thermostability of E.coli phage vB _ EcoM _ swi3

Respectively adding 100 μ L of the extract to a concentration of 10⁸Incubating pfu/mL Escherichia coli phage vB _ EcoM _ swi3 proliferation liquid in water bath at 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, sampling at 20min, 40min and 60min respectively, diluting the samples by 10 times in sequence, taking 100 mu L of each diluted phage proliferation liquid to be mixed with E.coli-K88 bacterial liquid with the same volume, measuring the titer of the Escherichia coli phage vB _ EcoM _ swi3 by a double-layer plate method, setting three parallels at each temperature, and drawing the thermal stability of the phage according to statistical resultsCurve line.

The results show (FIG. 3), Escherichia coli phage vB _ EcoM _ swi3 at 40 ℃ and 50 ℃ conditions, its titer basically remains unchanged; under the condition of 60 ℃, the activity of the Escherichia coli bacteriophage vB _ EcoM _ swi3 is basically not influenced within 20min, but after the activity exceeds 20min, the activity of the bacteriophage rapidly decreases along with the extension of action time, and when the activity is acted for 60min, the titer of the bacteriophage decreases by more than three orders of magnitude; at temperatures of 70 ℃ or 80 ℃, the activity of the phages decreases rapidly with the extension of the action time. Therefore, the activity of the Escherichia coli phage vB _ EcoM _ swi3 is stable at a temperature of 60 ℃ or lower.

Example 9: ultraviolet stability assay of E.coli phage vB _ EcoM _ swi3

Taking 4mL of 10⁸pfu/mL of the Escherichia coli phage vB _ EcoM _ swi3 proliferation solution was placed in a clean dish and irradiated for 60min at a distance of 40cm from the ultraviolet lamp. And (3) taking 100 mu L of phage multiplication liquid every 10min, diluting by 10 times, taking 100 mu L of phage multiplication liquid of each dilution, respectively mixing the phage multiplication liquid with E.coli-K88 bacterial liquid with the same volume, measuring phage titer by using a double-layer plate method, setting three parallels at each time point, and drawing a phage ultraviolet stability curve according to the measurement result.

As a result, it was revealed (FIG. 4) that the activity of the E.coli phage vB _ EcoM _ swi3 began to decrease after continuous irradiation with ultraviolet light for 5 min. The titer of the phage decreased by about one order of magnitude when irradiated with ultraviolet light for 20 min. When irradiated continuously for 40min in the UV, the titer of the phage decreased by about two orders of magnitude. When the phage was continuously irradiated for 60min, the titer of the phage was reduced to 10⁵pfu/mL. It shows that the Escherichia coli phage vB _ EcoM _ swi3 is sensitive to ultraviolet rays.

Example 10: one-step growth curve of E.coli phage vB _ EcoM _ swi3

200 mul of each of the Escherichia coli phage vB _ EcoM _ swi3 proliferation solution and overnight-cultured host bacterium E.coli-K88 bacterium solution are fully and uniformly mixed according to the optimal infection complex number proportion, incubated at 37 ℃ for 5min, centrifuged at 13000rpm for 30s, washed twice by LB liquid medium weight, and the supernatant is discarded. The pellet was resuspended in 7mL of LB broth and shake-cultured at 37 ℃. 200 mu L of proliferation solution is respectively taken at different time points (from zero time, every 5min for the first 1h, every 20min for the second 2h and every 30min for the third 3-4 h), centrifuged at 16000rpm for 1min, supernatant is taken, the titer of the phage is measured by a double-layer plate method, three parallel growth curves are drawn at each time point, and the incubation period, the outbreak period and the outbreak amount of the phage are calculated according to the measurement result.

The results show (FIG. 5) that the E.coli phage vB _ EcoM _ swi3 has a latency of about 25min, a burst period of about 75min and a burst amount of about 25.

Example 11: safety test of Escherichia coli phage vB _ EcoM _ swi3

(1) Experimental animals: 20 Kunming mice at 4 weeks of age were purchased from Qingdao Daren Fucheng stockbreeding Co Ltd.

(2) The experimental method comprises the following steps: 4-week-old Kunming mice were randomly divided into 2 groups of 10 mice each. Experimental group mice intraperitoneal injection 10⁸pfu coliphage vB _ EcoM _ swi3 proliferation liquid, control group intraperitoneal injection of equal volume of sterile LB liquid medium. After injection, mice were kept under the same environmental conditions and observed for mental status.

(3) The experimental results are as follows: after 12h of injection, 5 mice per group were sacrificed and changes in internal organs and intestinal tracts were observed; blood samples and feces samples are collected from the remaining 5 mice at regular time, the titer of phage in the samples is determined by a double-layer plate method, and the metabolic dynamic change of the coliphage vB _ EcoM _ swi3 in the mice is detected.

The final results showed that the dose of phage had no effect on mouse growth, no abnormalities were seen by dissection, and no phage could be detected in blood and feces 23h after intraperitoneal injection of phage.

Example 12: escherichia coli phage vB _ EcoM _ swi3 experiment for treating Escherichia coli infected mice

(1) Experimental animals: 50 Kunming mice at 4 weeks of age.

(2) The experimental method comprises the following steps: 50 Kunming mice, 4 weeks old, were used for infection testing and were divided into 5 groups (3 treatment groups, 1 control group, 1 infection group) of 10 mice each. coli-K88 monoclonal, inoculated in 3ml LB liquid medium, cultured overnight at 37 deg.C, 1200Centrifuge at 0rpm for 5min, remove supernatant, and wash 3 times with PBS. PBS was used to resuspend the cells and the concentration of the cells was adjusted to 2X 10⁸cfu/ml. The infected group and 3 treatment groups were separately intragastrically administered 10 times⁸The host bacterium E.coli-K88 of CFU, establishing an Escherichia coli infected mouse model, and gavage the same volume of PBS to the control group. After 2h, three treatment groups are respectively injected into the abdominal cavity 10⁵pfu、10⁶pfu and 10⁷pfu coliphage vB _ EcoM _ swi3, control and infected groups were injected i.p. with equal volume of PBS. The mice in each group were kept under the same environmental conditions and observed for mental status and survival, and dead mice were subjected to a necropsy for observation of lesions in each internal organ. Meanwhile, after inoculating bacteria for 0.5h, 1.5h, 2h, 5h, 6.5h, 8.5h, 10h, 23h, 27h and 51h, blood and feces of each group of surviving mice are collected, and the metabolic dynamics of bacteria and phage in the bodies of the mice are detected.

(3) The experimental results are as follows: the results show (fig. 6) that e.coli-K88 had been bled after 30min from the gavage mice and increased exponentially with time, and the concentration of bacteria in the blood reached 10 after 1.5h from gavage⁸CFU, cachexia of vaccinated mice, marked reduction in activity, and the appearance of a bunchy mass. And 2h later, injecting escherichia coli phage vB _ EcoM _ swi3 into abdominal cavity, rapidly reducing the concentration of E.coli-K88 in blood of the mice of the three treatment groups, wherein the higher the concentration of inoculated phage is, the faster the bacterial quantity in blood is reduced, removing bacteria in the blood of the mice of the high-dose treatment group for 8.5h, and completely removing bacteria in the blood of the mice of the medium-dose group and the low-dose group for 51 h.

Phage in feces were present for extended periods of time with increasing phage doses administered, and the high dose groups were completely cleared within 99h (FIG. 7). The infected mice died at the inoculation 2d, and of the three treatment groups, the low dose group died 1 mouse, and all the remaining mice survived. Explanation of intraperitoneal injection 10⁶pfu E.coli phage vB _ EcoM _ swi3 can effectively treat infection caused by E.coli-K88.

Example 13: escherichia coli phage vB _ EcoM _ swi3 environment disinfection experiment

Adjusting the concentration of the freshly proliferated host bacterium E.coli-K88 to 10⁶CFU/ml by sprayingUniformly spraying the mixture on the ground of a pigsty and the surface of a trough in a fog mode, dividing a spraying area into three areas, and respectively spraying 10 parts of purified Escherichia coli phage vB _ EcoM _ swi3 proliferation solution⁵PFU、10⁶PFU and 10⁷And (3) respectively disinfecting the ground and the trough of the pigsty in the three areas by the concentration of PFU in a spraying mode, and detecting the content of host bacteria E.coli-K88 in the environment after 2 hours.

The detection result shows that the number of the pig house is 10⁶The number of the feed trough and the ground host bacteria in three areas of E.coli-K88 bacteria liquid sprayed by CFU is reduced to below 45CFU, which shows 10⁵The phage multiplication liquid of PFU can effectively kill the polluted host bacteria in the environment.

Comparative example 1: separation of porcine pathogenic escherichia coli phage JS09 and detection of lysis spectrum thereof

(1) Experimental materials:

host bacteria ETEC EK99-F41, pig farm sewage, 40 different porcine pathogenic escherichia coli, 1 bovine pathogenic escherichia coli and 5 escherichia coli engineering bacteria;

(2) isolation experiment of porcine pathogenic escherichia coli phage JS 09:

collecting sewage 100ml from pig farm, centrifuging at 12000rpm for 15min, collecting supernatant, and sterilizing with 0.22 μm filter to obtain filtrate. Adding host bacterium ETEC EK99-F41 (10)⁹CFU/ml), shake culture overnight at 37 ℃. Centrifuging the bacterial liquid at 12000rpm for 10min, collecting supernatant, and filtering and sterilizing with 0.22 μm filter to obtain filtrate. Uniformly coating the host bacterium ETEC EK99-F41 bacterial liquid on an LB plate, airing, then taking 10 mu l of filtrate to be dripped on the surface of the plate, carrying out inverted culture at 37 ℃ overnight, observing the occurrence condition of plaque, and separating to obtain a phage JS 09;

(3) detecting a lysis spectrum of the porcine pathogenic escherichia coli phage JS 09:

experimental 46 clinical isolates of e.coli from different sources were selected and the lysate profile of the isolated phage JS09 was determined. The specific operation is as follows: respectively taking 100 mul of different Escherichia coli culture solutions, uniformly coating on the surface of LB agar plate, standing, air drying, and dropwise adding 10 mul of phage JS09(10 mul)⁹PFU/ml), inverted culture at 37 ℃ overnight, and observedAnd (5) observing the occurrence condition of the plaques to obtain the lysis spectrum of the phage on escherichia coli from different sources, and recording the result. The result shows that 11 strains of 40 strains of porcine pathogenic escherichia coli can be lysed in the lytic phage JS09, 5 strains of escherichia coli engineering bacteria can also be lysed, no lysis effect is caused on bovine pathogenic escherichia coli, and the lysis rate is 21.7%.

Comparative example 2: cleavage spectrum detection of chicken pathogenic escherichia coli phage EcP5

(1) Experimental materials:

chicken-origin pathogenic escherichia coli and lytic phage EcP5 thereof, 45 strains of different chicken pathogenic escherichia coli;

(3) the experimental method comprises the following steps:

the experimental selection of 45 different chicken pathogenic escherichia coli clinical isolates to determine the lysis profile of lytic phage EcP5 was performed as follows: mu.l of lytic phage EcP5 was mixed with 100. mu.l of 45 different strains of chicken pathogenic E.coli, respectively, and allowed to stand for 15 min. Observing the formation condition of the plaque by adopting a double-layer agar plate method to obtain a lysis spectrum of the phage on pathogenic escherichia coli of different chickens, and recording the result;

the result shows that the lytic bacteriophage EcP5 can split 18 strains of 45 strains of chicken pathogenic escherichia coli, and the splitting rate is 40.0%.

TABLE 3 comparison of lysis spectra of E.coli phage vB _ EcoM _ swi3 of examples 1-13 with those of comparative examples 1-2

	Cracking Rate (%)	Source of lytic E.coli
			Examples 1 to 13	80％	Escherichia coli derived from chicken and Escherichia coli derived from pig
Comparative example 1	21.7％	Coli of porcine origin only
			Comparative example 2	40％	Coli only from chicken

As can be seen from the results in Table 3, comparative example 1 only cracks Escherichia coli from swine and Escherichia coli engineering bacteria commonly used in laboratories, and has no cracking ability on Escherichia coli from cattle; comparative example 2 can only cleave Escherichia coli of chicken origin alone, can not cleave Escherichia coli of other sources at the same time, and comparative examples 1-2 are all lower in cleavage rate than Escherichia coli bacteriophage vB _ EcoM _ swi3 in examples 1-13 of the present invention, and thus it can be seen that the bacteriophage provided by the present invention is advantageous in cleaving Escherichia coli of different animal origin.

In addition, the bacteriophage vB _ EcoM _ swi3 provided by the invention also has the advantages of good physical and chemical factor tolerance, short latent period, high cracking performance, good safety and the like, and can be prepared into different dosage forms to be used as feed additives, disinfectants or cleaning agents and other products, so that the Escherichia coli bacteriophage vB _ EcoM _ swi3 provided by the invention has very wide application prospects in the aspect of preventing and treating pollution of pig-source and chicken-source Escherichia coli in the breeding process of animals such as livestock and poultry in breeding environments.

Sequence listing

<110> Qingdao agricultural university

<120> Escherichia coli phage vB _ EcoM _ swi3 and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 6468

<212> DNA

<213> Escherichia coli phage vB _ EcoM _ swi3

<400> 1

1 TCAGTCCCCT CTAACATACG CAATTACAAC TGTACGAGTT GAGTTTTCCA TTTTGAGCGC

61 CTGACTACCC AGTCCTATTC TTTCCATGCC TTTAGCCAGT TTACTCATCT GACTAATAAC

121 ACAGTTGAAC GGTTTACTAA ACTTAGTCGA CTGGAACTTA ATGTCGACGG ACATCTTTGG

181 AATAATCTTA TCTGGTAAGT CTGCCTTTGC AGTTGACTCG CCTGTTATCT CCAGAATAGC

241 CTGACCTTCG TCGTACCCAG TAAGCTGGTC CATGACTTCC AGGAACTCAG GAGTGATGGC

301 CTGCATATCC ATTTCCATAT TGAAGAACTT CGATACGTCT GGCCATTTGG CTGCTAGTAC

361 CGGAGCATCC ATCTGTACAC CATCCGCAAA GCCGAAGAAC ATCTGGCCGC GGGCGTATCC

421 TACACTTGTG GGTTCCATCT TCAGCTTAAC CAGAGCCTCA AGCATTGGTT TGGCTATGGC

481 ACATTCAAAT TCAAAATCTA CGTTCAACTT CTCGCGTATC ATATACTTAC CGTTAGTGGC

541 GTAAGCATAA CCATCGCGCA GAAGTAAAGA TGTCGCCCAT ACCTGCGGTG CTTCGTTAGG

601 TACCCACTTG GCTAAGGCAG TTAATACCGG GAGTAATTCG CCATGCGCTG AATACCAGTT

661 GTTAACTGTA GCAAGTGGCG GTATTGGTTC ATTAAGCGCT TGCATTCTTG TTTTAAGTAC

721 ACCTGCTGTG ACTGTGAGGT TTCCAGCGGC TGTAATGTTA AAAGTAATCT GTTCAGTTCT

781 TGCCGCATTA ACGGCCTTAC GAAATGATTC AGCATCCACT TGCAGGTCTG GAAATCCTTT

841 ACATGGCGCT CGTAGTAATA CGTTCTTGTA GTATGAATGG ATTGTTCCGT CTGCAATCCA

901 TATTTTGCCC ATAAGACCAC TATCAGTATA CTTGATGGGA CGAGCTTGTT GAACGGCAGT

961 CTGTATTTGT TTGGTGTCAT AGTTAAACTT GCTCATTTAT ACGGCCATTT GGAGTTTGAT

1021 GTTTGGCATT GGTTTGTACT TCTCAACCAT TGGCGTAACA TCGGCAGGAG TGAAGTCAAA

1081 CACTGACACG TCATAAATTG CATTAATGTC AGGACGACCT TCAAAGTTAG TGTCCAGTGT

1141 AGTATCAGAA TTGCTTACGA TACGTTGCAG GAAAATCTTT GCCGCATCGA TATGATTCTG

1201 ATAGATGTGA TAATCACCCA GCGTAACAGT CATATCACCT ACCCCAGTAC CCAGCTCAGC

1261 AGCAATGGTA ATCTGCAACA TCTGATGCAA CAGAATATCG TGAGGCAGAC CAAGGATAAC

1321 ATCCGCGCTA CGCATGTGGA ACATCAGCTC TAATTCATTA CGGTTGTTAA TGAACAGCTG

1381 AAAACCATGG TAACAAGGCG GAAGGGCCAT GGCGTCCATC TCTTGTGGGT TCCATGCCGT

1441 AATGTATGCC CGGCGGTCAG TAGGCTTAAC CTTCAGCGTT TCAATAACGT CTTTAAGCTG

1501 GTTAGTAAGA ACACCATTCT TATAGAATGG CTTAATCCAT GCACGCCGTA CACAGGACCT

1561 AAGTCACGAT TGTCTGGTGT ACCCCAACGT TTGTTTGCAT CCTGCAGGTT ACCTTCCCAC

1621 CAGTTGCAGC CAAAGCCTTT GAGTGTGTCG ACGTTTGAAA TGCCGTTGAT AAAGCAGTAA

1681 AGCTCAGCAG CAACAGCTTT GATGTTAACA GGTTTAAATA CCGGCACCAT CAGTTTGTTG

1741 TCGTTAAAAG CCAGCTTAAA CGCTGTACCA AAACAACGGC GTGTACCAAC ACCTGTACGG

1801 TCTACAACAT CCTCGCCTTT ACGCAGGATA AAAGAAATGA TGTCGCTGTA AAGAGAATCG

1861 ACAGTGCTGT ATGTTTTCAT TTAGCCTGGG TTGCTACCCG TTCAAATAGT TCTCTGTCAT

1921 CGAGCGCCAT AACCGTTTCC CCAGTCGTAG TTCATGATTT GTGGGTAGGG TCGTTTAATC

1981 CATACCTTGA TGCGCTTAGG AATAGCCAGC TCACCCTTAC GGCGAATAAA CTCATCGCAT

2041 GTTTCAGGCG GTTCTGTTCC TGCAGCATTA CGCCACCAGG CATCTGCCAT TTGCTTAGCA

2101 TTGCCATGGG CCTCTATATT TACCCAGGTT GTATACACTT CCAGGTTTGA GTAATACACA

2161 ACCTTTATAC TGTCAGGTCG TCCCTGCTTC TGGAATAAAG AATAGTTTAC CAGCGTAACA

2221 TCTTTATCCA CAACACGAAG CTGACCGTTA GTCATTACCT CTTCTTCTGA TGAAGTGAAG

2281 GCAATGGCAG GTCCGTTAAC TGTAAACTCA AATTCATGTC CGCACATCGG ATATACACCA

2341 TTCTCGTCTG GCGTACTTCC TTTAGCCGGA TGCTTACATG TACGGGCTGC ATAGCCACTT

2401 ACCTGGTGAC AGATAGGGCA CTCTTTACCG CCAGGTGTTT TCTCTGACCG TTTCTTTTTC

2461 TTCTTACCAA GACGAGGTGG AACAGCCGGG TCGTCAATAG GCCCAAGGTC AATGGTGTTA

2521 GATGTAAAGT CCATAACAAG ACAGTCATGC TTGCCAGGCG CTGTACGTAA TCCTCGACCA

2581 AGTATCTGCA CCCACAAAGC AACAGACGTG GACGGGCGCA AGATGCCGAG CATATCAATC

2641 TCAGGATAGT CAAATCCTGT AGTCAATACG TTTACGTTTA CACAGACCCG TGCTCGTCCG

2701 CTTGTAAACT CTTCCAGTGC GGCTTCACGT TCTTTCTTTG TAAGACCGCC ATGCACAACT

2761 ACAGTCTGCC ACCCTCGCAG ATTAAATTCG TCTGCTATGT TGTGGGCGTG CTCAACACTT

2821 GTGGCAAATA CAAGAACGTG GTTCCTGTCT TTACCGTAGT GCATCATCTC ACGAATAGCC

2881 GCGACTGTTA TACTATGCTG GTTGGCTACC TTCTCAAGCT CTGACGGGAT GTAGTCACCC

2941 ATCCTTTCGC CAACATTACT GGTGTCAATC TTGGTTAACA CTTTCTTGTT GACCAGGCGC

3001 GACAGGAAGC CTTCATTTAC CAGTCGGGTG AATGCGTCAG GTGTTGTAAG GTCATAGCAG

3061 ATGTCGGTAA ATATACCACA GTCCAGCAAG TGACCACCTG CCAGGCGATA CGGAGTTGCT

3121 GTGAGTCCTG TTACCTTTAC CTTAGGATTA AGCTCTTTGA AGTGAGCAAT AACCTTACGG

3181 TATGTTGTTT CAGACTTCTC AGGAACAAGA TGGGCTTCGT CAATGAATAT AAGATTGAAC

3241 TTACCTGCTT CTTCCAGGTT CTTAATAATT GACTGTACAG ATGCTACAAC GATACGGCCT

3301 GAAAAGTCTT TATGTCCTGC ACCTGAAGAG TATATTGATA CTGGTGCTTG TGGCCAGTGA

3361 CGAACAATCG CTTTGGCGTC TTGTTCAACC AGCTCCTTGA CATGTGTCAA TATAAGAATG

3361 CGCTGACGGG GATAGCTGGT AAGAATATCC TTCATCAGCA TACCAAGCAC AGGTGATTTA

3421 CCTGAACCTG TCGGCATGAC AATAAGTGGA TTACCACTAT AAGAATTGAA GTACGACCAC

3481 CAGGCTGTGA CCGCTTCTTG TTGATACCAG CGAGCTTCAA AAGCCATTCA AACGGACTTG

3541 GCATTGTTAA ACTCCTCAAC GCTGAATTCA TCGTATACAA TATTATGTGC CCACAAATGG

3601 GCGACGGCAT TTGCCATACT GTCTTTCCAT TTGGATTGTG AAGCTGTATG AGCCAATGCA

3661 ACTACCCGAA CCAGGTTAAG GTTGGCTATC AGCATATCTG CACAATGTTC GCACGGCGGG

3721 TAGGTTATGT AGATGGTAAC CGGGCCTAAT GAAGGAGTGT CTAACTCTCT CCTTATATTA

3781 GCTAAGGTGT TTATCTCGCT ATGAATAGTT AGAGCGTTCT TAAGGCTATT AGGCACTATT

3841 CTTATAGAGT CTAACTGCGT AGGCAGACCA TTATATCCAG TGGAAATAAT ACGCTTGTTC

3901 TGGTCCACAA GAACGCTACC GACTTGACGT TTCGGGTCCT TCGACCAGCT CGCAACTTCA

3961 CAAGCTATGC GCATAAACCG CCTGTCCCAC TTGTCCATCT TCTTCTTTTG CATATGAAGC

4021 GCTGGGCTAT TTTTGAGCTG ACGTACGAAG ACGAGACCCT CGAAGTTCAG TCCAAACCTA

4081 AGAAACCTGC AAGAGCGCCA TCAGGCCGTT CCTTCAGCAC AACTGTGCCT GAAGAGTGGA

4141 TGACTTTCGA CGACGCCCTG ACACGTTGTA TTCGCAATCG TGAAATGAAG ATTAAAGCTG

4201 TACAGGGTAA TATCACCTTT GTGCCGGCTT TGATTGTTCC TCGCCAGTGG TACTTTGTTG

4261 ACCTGGACAA CCATGATGAT AATCCAGACA TTGAAGCAAC CCACAAAGCG ATAATTGAGG

4321 GGACCCGTGG TGCTTATGCT GAGACGTCAA TCTCTGGCAA AGGGCAACAC ATTGCAATCC

4381 CTTTACCCTG GACTGCAGCA ACATCTAAAG AGAAAGACGA ACAGCTGGAC ATCAAAATCC

4441 AGAAGGCTGA GATGTTCCTG GTAATGACAG GCAATGTATT GAGCGAGTTC AATGCTAATC

4501 CTGTTTCAGC CCGTGAATGG CATGCAGCAA TTGAGAAGTT CTTTCTTGAA GCAAGCGCAC

4561 CTGATATTGA TGTTGAGTTT GAGCAAGACG AAGACCGTGG CGAAGAGTAT GACAAAGAGC

4621 TGTACGAACA GTTAGCCGAC AATACTCGCT GGGCTTTAGA GCACTATCTG AATGAGCATG

4681 CACCAGACGG TGTTGGTGTA AGTAATGACG GTTCTGAACG TTTGAGCCGC ATACTCAAAG

4741 ACTTGCTTCG TGTAACCCGT AACTACGAAG TTACACAACG CATCTTCATG GCCAGCAAAG

4801 CTGCAGCATA TGAAGGTCGC AGACCAAGTC GACACAACCT GTCTGTTGAT AAGTACCATC

4861 AGTGGTTCAG TCGTGTCGGT AAGACTGTGC TGAAAGAAAT GCAACGTGAC GGCTTATTCG

4921 TTAAGACCAA GTTTGCTCTT GACATCAACA AAGAGTTAGC CAAGAATTCA GGTGTCTCCC

4981 TGGCTGAGAA TGTTGAGTTC AAGGTCAACG ATGACATTCT GCCTTCTGAT GTATTCGTCA

5041 GAACGGCCCC GTCTGGGTTT AAACGGCTTA TTGCTGAGAT ACAGGATAAC ATATCTCCGG

5101 CTAACAGGGT TAACGACTAT GCCATCGGCA CAGCTCTTAA CATCCTGAGC AACTGCGCTG

5161 GTCGCAAGTA TGTTTGTCCA GTAGGCGGTC ATGCTAACTT CCTGGTAACC AACATCATTC

5221 TTGTGGGTGG TTCGTCAATC GGTAAATCGC TGTATACCGA GTTGTTCCCA CAAATCCAGG

5281 CAAGTGTGCC AGACACATCG CCTATCAGTC TTAACCGTAT CCCAAGGGAA CAGACTTTTG

5341 CAACCAGGAC GTTTGCTGAA CTGATGTCCA ACCCGTCATA CCATTCAGTC CAGTTGTTCT

5401 ACCCTGAATT TGGTCTGGCT TTAGGTTCTG GCTTACGCAT GAATCCTAAC AACCCGGACA

5461 ACTTTCAGAA AGCTCTGATG GACGCTTCAA CCAAGCGTAA GGTTGGTGGT ATACTGACAG

5521 GTATCAAACG TGCTAATGCT GACAATGATG TCAAGACAGT AAGCGAACCA TGCTACTCTA

5581 TCCTGGGCGA CTCAACTCAA GAGCTTATCC TTGACAACAC CCGGAAGCAA GACTTCTCAA

5641 GTGGCTTTTT GCCCCGCTTC CTGTTCATCC CTAACTATGA ACGAGCGGAG TTTGCCAAAC

5701 CTGAGAAGGT AGGACGTCGC CAGCAGATTC GCCGTACTCA ATTCTCCAAA GAGTTGATTG

5761 AGAAGCTGGA AGCAATCGCT TATGTGAATG CTATCCCGCC TAACGGTAAA CTAAGGCACG

5821 ACCCGATTCC TATTTACGAC GAATCGGACG ATGATGACTT CCTGTACGAG TACCAGTGCA

5881 ATATTAATGA CATGCGCAAG TATTACAGGG ATAACGAAGT AGCATCTGCC TTTGTGGGCC

5941 GAATGGGTGA GTATGTATTC AATATAGCAG CACTGATTGG ATTACTCGAC AACTGGGATA

6001 CTCCTGTAAT GACCAGGGAC AACATCGAAT GGGCTTACAA ATATGTGCTT CGTTGTATAA

6061 CCGCTTGGGT CAATAACACA AGCAGGATAG TCGCACCACC AACTACCAAC GCCGAAGTAG

6121 TGGACGGCTT CCTAAAAGTC TACAGGGACA TTTTGGCCCT TTACGCCAAA AGCGGATGGA

6181 ACGGTATTGT TAAGCATTAT CCTGAGGGTG TTGTGAGAAG TATCCGGGAA GAACACCTCC

6241 AGATGAATGG TTTAAGCATA TATGCTATTC GTCAGTCTTT GAGCTGGTTT AAATCGAATT

6301 ACGGTTTTAA TATCACAGCT AACAACAAAG TCATTGACGG TATGCTGCAG GATATGATTG

6361 ACCAGGACTA TTTAGCAGTT CAAATATATA AGCCCACAAG AGGCCGATCT GCTAACATTT

6421 ACTGCCTGAC AGAGCGAGGA TATGACACTG CTAAGAAGTT AAAATAA

Claims

1. The coliphage vB _ EcoM _ swi3 is characterized in that the preservation number of the phage is CCTCC M2019467, the phage is preserved in the China center for type culture collection in 6 months and 19 days in 2019, and the phage is classified and named as porcine coliphage vB _ EcoM _ swi 3.

2. Use of the coliphage of claim 1 for the preparation of a biological product for killing both porcine-and chicken-derived colibacilli.

3. The use according to claim 2, wherein the biological product is a feed additive and a disinfectant/detergent, wherein the feed additive and disinfectant/detergent are a single preparation prepared from purified coliphage or a combined preparation prepared from purified coliphage as a main ingredient.

4. Use according to claim 2, wherein the E.coli of porcine origin and E.coli of chicken origin comprise pathogenic E.coli, including enterotoxigenic E.coli.

5. The use of claim 4, wherein the enterotoxigenic Escherichia coli comprises Escherichia coli K88, and the pathogenic Escherichia coli of chicken origin comprises Escherichia coli O78.

6. The disinfectant or the cleaning agent is characterized in that the disinfectant or the cleaning agent is a single preparation prepared by using the purified Escherichia coli bacteriophage of claim 1 or a compound preparation prepared by using the purified Escherichia coli bacteriophage of claim 1 as a main component, and is used for preventing and treating the pollution of Escherichia coli of pig origin or chicken origin in a breeding environment.

7. The disinfectant or cleaning agent as claimed in claim 6, wherein the dosage form of the disinfectant or cleaning agent is liquid, lyophilized powder or tablet; the breeding environment comprises livestock bodies, livestock body surfaces, livestock breeding place ground, livestock breeding place air, livestock feed, drinking water and breeding devices.

8. The feed additive is characterized in that the feed additive is a single preparation prepared by using the purified Escherichia coli bacteriophage of claim 1 or a compound preparation prepared by using the purified Escherichia coli bacteriophage of claim 1 as a main component, and is added into livestock and poultry feed for preventing and treating pollution of Escherichia coli of pig origin and chicken origin in the breeding process.

9. The feed additive of claim 8 wherein said feed comprises at least pig feed or chicken feed.

10. The disinfectant or detergent of claim 6 or feed additive of claim 8, wherein said E.coli of porcine and E.coli of chicken origin comprise pathogenic E.coli, said pathogenic E.coli of porcine origin comprise enterotoxigenic E.coli, said enterotoxigenic E.coli comprises E.coli type K88, and said pathogenic E.coli of chicken origin comprise E.coli type O78.