CN110607284A - Escherichia coliphage vB_EcoM_swi3 and its application - Google Patents

Abstract

The present invention discloses an Escherichia coli phage vB_EcoM_swi3 with a preservation number of CCTCC M 2019467. The phage belongs to the family Myocaudidae and has a polyhedral head and a retractable tail structure. The diameter of the head is about 80 nm, and the length of the tail is about 120 nm; Transparent plaques can be formed on the solid medium, without halos around, with clear and regular edges, and a diameter of about 1-1.5mm; the phage is effective against E. The pathogenic Escherichia coli has a good lysis effect, and has very broad application prospects in the prevention and treatment of porcine colibacillosis and chicken colibacillosis, pharmaceutical preparations, feed additives and other aspects.

Description

Escherichia coliphage vB_EcoM_swi3 and its application

technical field

The invention belongs to the field of bioengineering, and in particular relates to a coli phage vB_EcoM_swi3 and its application.

Background technique

Escherichia coli is an opportunistic pathogen, and the pathogenicity is related to the serotype of Escherichia coli, and there are differences among different animals. Among them, porcine colibacillosis and chicken colibacillosis are widespread in the breeding environment. Once infected, they will cause diarrhea, significantly affect the weight gain of animals, and cause the mortality rate of animal groups to rise sharply. Diseased animals can also pass through feces, etc. coli from pigs and chickens in the breeding environment will cause continuous infection of animal populations, causing great economic losses to farmers.

At present, the use of antibiotics is the main means of preventing and treating colibacillosis in farms. However, due to the extensive abuse of antibiotics, it has defects such as destroying the structure of animal intestinal microbial flora, antibiotic residues, severe drug resistance and multi-drug resistance of Escherichia coli. Increased the difficulty of disease prevention and control, making the cost of breeding continues to increase, causing huge resistance to the development of the breeding industry.

Phage is a bacteria-dependent virus that can specifically lyse host bacteria, and its mechanism of action is different from that of antibiotics. Bacteria lysed by phage are not affected by bacterial drug resistance and have no residue. It has the advantages of short development time, low cost, With the advantages of strong specificity, less dosage, and no damage to the normal flora, a series of gratifying results have been achieved in the prevention and control of drug-resistant bacteria. Theoretically, any pathogenic bacteria has its corresponding phage, but the type and quantity of phage we have found so far is only the tip of the iceberg, and there are still a large number of phage resources that have not yet been developed. Discovering phages that can simultaneously lyse bacteria from different animal sources has a broader clinical application prospect. Therefore, the development of phage preparations with the above characteristics is of great significance for the healthy and rapid development of the aquaculture industry.

Contents of the invention

In view of the above problems, the present invention proposes a phage capable of simultaneously and efficiently killing pathogenic Escherichia coli from pigs and chickens in a breeding environment.

In order to achieve the above object, the technical scheme adopted in the present invention is:

The invention provides a coli phage vB_EcoM_swi3, the phage preservation number is CCTCC M2019467, which was preserved in the China Center for Type Culture Collection on June 19, 2019, and is classified as porcine coli phage vB_EcoM_swi3.

The above-mentioned coliphage can form translucent plaques on the solid medium, without halos around, with clear and regular edges, and a diameter of 1-1.5mm; it is found by transmission electron microscopy that the coliphage has an obvious regular polyhedral head Structure and retractable tail structure, the diameter of the head is about 80nm, and the length of the tail is about 120nm. It has been identified that the E. coli phage belongs to the family Myocaudaceae.

A series of performance tests were carried out on the above-mentioned coliphage, and the results are as follows:

a. Through titer determination and optimal multiplicity of infection determination experiments, the titer of the coliphage is 6.4×10 ⁸ pfu/mL, and the optimal multiplicity of infection is 1;

b. Through pH, heat and ultraviolet stability experiments, it can be concluded that the coliphage has relatively stable performance under neutral conditions of pH 6-8, is more sensitive to ultraviolet rays, and has relatively stable activity when the temperature is below 60°C;

c. Through one-step growth curve measurement, the incubation period of the coliphage is about 25 minutes, the outbreak period is about 75 minutes, and the outbreak amount is about 25 minutes;

d. Through the mouse safety experiment, it can be concluded that the coliphage does not have a negative impact on the normal growth of mice, and no abnormalities are found in the anatomical examination, indicating that the coliphage shows high safety;

e. According to the treatment experiment of E. coli-infected mice, the concentration of E. coli E.coli-K88 in the blood of mice after intraperitoneal injection of 10 ⁷ pfu/mL coli phage decreased rapidly, and it could be completely cleared in 8.5 hours. The phages in the feces also disappeared, and the phages in the feces were completely cleared within 99 hours, indicating that the coliphages can effectively eliminate pathogenic Escherichia coli in animals in a short period of time, and there is no phage residue;

f. According to the Escherichia coli environmental disinfection experiment, the coli phage proliferation solution with a concentration of 10 ⁵ pfu/mL can effectively kill the polluted host bacteria in the environment.

The invention also provides the application of the coli phage in the preparation of biological products for simultaneously killing pig-derived and chicken-derived Escherichia coli.

The Escherichia coli phage provided by the present invention is a new strain of phage isolated from nature. As a natural enemy of bacteria, it can specifically lyse pathogenic Escherichia coli from pigs and chickens without destroying the normal flora composition. Moreover, it is not affected by bacterial drug resistance, does not have problems such as drug residues, has high safety, and has good application prospects in the prevention and treatment of colibacillosis.

Preferably, the biological product is a feed additive and a disinfectant/cleaner, wherein the feed additive and disinfectant/cleaner are a single preparation prepared from purified coliphage or mainly purified from the purified coliphage Combination formulations prepared from ingredients.

Preferably, the porcine-derived E. coli and chicken-derived E. coli include pathogenic E. coli, and the porcine-derived E. coli includes enterotoxigenic E. coli.

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

The present invention also provides a disinfectant or cleaning agent, which is a single preparation prepared with purified coliphage or a compound preparation prepared with purified coliphage as the main component, which is used to prevent and treat pigs and chickens in the breeding environment. Contamination of source E. coli.

Preferably, the dosage form of the disinfectant or cleaning agent is liquid, freeze-dried powder or tablet; Feed, drinking water and breeding utensils.

The present invention also provides a feed additive, which is a single preparation prepared with purified coliphage or a compound preparation prepared with purified coliphage as the main component, which is added to livestock and poultry feed to prevent and control the Escherichia coli contamination of porcine and chicken sources.

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

Preferably, the porcine-derived E. coli includes pathogenic E. coli and the chicken-derived E. coli includes enterotoxigenic E. coli, and the enterotoxigenic E. coli includes K88 type Escherichia coli, the chicken-derived pathogenic Escherichia coli includes type O78 Escherichia coli.

The colibacillus phage provided by the present invention can be used as an active ingredient of pharmaceutical preparations for preventing and treating colibacillosis, and various product components for inhibiting the proliferation of E. coli in the environment, including feed additives, drinking water additives and meat product cleaners, etc. .

The coliphage provided by the invention is isolated by the following method:

I. Experimental materials:

Fecal sewage samples were collected from the sewage of pig slaughterhouses in Linyi City, Shandong Province. The phage host bacteria were pig-derived pathogenic Escherichia coli E.coli-K88, which were isolated, identified and preserved from the feces of diseased pigs by our laboratory.

II. Experimental method:

Add 5ml of the collected sewage sample to 50ml of LB liquid medium, then add 500μL of Escherichia coli E.coli-K88 bacterial solution, mix well and put it into a 37°C constant temperature incubator for overnight cultivation. The culture was first coarsely filtered with four layers of gauze, the filtrate was centrifuged at 4000rpm for 10min, the supernatant was centrifuged at 12000rpm for 15min, and then the supernatant was filtered with a 0.22μm filter to obtain the phage stock solution. Spread 200 μL of Escherichia coli E.coli-K88 proliferation solution evenly on the surface of LB solid medium with a cotton swab, let it dry for 5 minutes, add 10 μL of phage stock solution dropwise, and at the same time add 10 μL of normal saline as a control, and culture it upside down at 37°C for 10 -12h, it was observed that no bacterial growth and plaques appeared in the spots, which contained bacteriophages capable of cleaving E.coli-K88.

Compared with prior art, advantage and positive effect of the present invention are:

1. The present invention provides a newly discovered Escherichia coli phage, which has the characteristics of better tolerance to physical and chemical factors, short incubation period, high cracking performance, and good safety. Products and means of bacterial diseases;

2. The coli phage provided by the present invention can efficiently and simultaneously kill pig-derived and chicken-derived pathogenic E. coli in the breeding environment and in livestock and poultry, especially pig-derived enterotoxigenic E. coli and chicken-derived pathogenic Escherichia coli;

3. The coliphage provided by the present invention has good hydrophilicity, can be administered through a waterline, and is easy to prepare infusion, spray or rinse solution; the coliphage can also be prepared as a single preparation or combined with other phages It can be compounded and used as a compound preparation. In addition, the coliphage can also be widely used in feed additives, disinfectants, cleaning agents and the like.

Description of drawings

Fig. 1 is the transmission electron microscope picture of coliphage vB_EcoM_swi3 provided by the embodiment of the present invention;

Fig. 2 is a graph showing the pH stability results of coliphage vB_EcoM_swi3 provided by the embodiments of the present invention;

Fig. 3 is the thermostability assay result chart of coliphage vB_EcoM_swi3 provided by the embodiment of the present invention;

Fig. 4 is the result figure of the ultraviolet stability determination of coliphage vB_EcoM_swi3 provided by the embodiment of the present invention;

Fig. 5 is a one-step growth curve determination result diagram of coliphage vB_EcoM_swi3 provided by the embodiment of the present invention;

Fig. 6 is a diagram of the sterilization results of coliphage vB_EcoM_swi3 in animals provided by the embodiment of the present invention;

Fig. 7 is a dynamic change diagram of coliphage vB_EcoM_swi3 in feces provided by the embodiment of the present invention.

Detailed ways

In order to introduce the coliphage and its application provided by the embodiments of the present invention in more detail, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the implementation of the present invention. example, not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1: Isolation and preparation of Escherichia coli phage vB_EcoM_swi3

(1) Experimental materials:

Fecal sewage samples were collected from pig slaughterhouse sewage in Linyi City, Shandong Province. The phage host bacteria was E. coli-K88, which was isolated, identified and preserved from diseased pig feces by our laboratory.

(2) Experimental method:

Add 5ml of the collected sewage sample to 50ml of LB liquid medium, then add 500μL of Escherichia coli E.coli-K88 bacterial solution, mix well and put it into a 37°C constant temperature incubator for overnight cultivation. The culture was first coarsely filtered with four layers of gauze, the filtrate was centrifuged at 4000rpm for 10min, the supernatant was centrifuged at 12000rpm for 15min, and then the supernatant was filtered with a 0.22μm filter to obtain the phage stock solution. Spread 200 μL of Escherichia coli E.coli-K88 proliferation solution evenly on the surface of LB solid medium with a cotton swab, let it dry for 5 minutes, add 10 μL of phage stock solution dropwise, and at the same time add 10 μL of normal saline as a control, and culture it upside down at 37°C for 10 -12h, it was observed that no bacterial growth and plaques appeared in the spots, which contained bacteriophages capable of cleaving E.coli-K88.

Example 2: Purification and Mass Propagation of Escherichia coli Phage vB_EcoM_swi3

Q1. Purification of coliphage vB_EcoM_swi3

Purify the samples identified to contain phages in the above Example 1. The specific operation is as follows: Take 100 μL of the phage stock solution and perform a 10-fold dilution. Take 100 μL of the phage stock solution at each dilution and mix it with an equal amount of overnight cultured E.coli-K88. Add 3ml of 0.7% LB semi-solid medium heated to 50°C and mix well, pour it into the upper layer of the LB agar plate, let it stand for 5 minutes, wait for it to solidify, and culture it upside down at 37°C for 4-5 hours to obtain the formation of phage plaques double-layer flat panel. Pick out a single phage plaque, add 3ml of LB liquid medium, bathe in 40°C water for 30min, centrifuge at 12000rpm for 5min, take the supernatant and filter it with a 0.22μm filter to obtain the phage filtrate. Take 100 μL of phage filtrate, dilute to 10 ^-6 , add an equal volume of host bacteria E.coli-K88, and culture in double-layer plate method. This process was repeated 5 times to obtain purified phage. Phages can form translucent plaques on solid medium without halos around them, with clear and regular edges and a diameter of 1-1.5 mm.

Q2, Propagation of the purification of coliphage vB_EcoM_swi3

Add 1mL of phage stock solution and 1mL of freshly cultured host strain E.coli-K88 into 100ml of LB liquid medium, culture with shaking at 37°C for 9h, add 5% chloroform by volume, continue shaking for 30min, centrifuge at 13000rpm for 15min, and take supernatant to obtain a large amount of phage proliferation fluid;

Add DNase I and RNaseA to the phage proliferation solution to a final concentration of 1 μg/mL, shake and mix well, incubate at 37°C for 30 minutes, add NaCl to a final concentration of 1mol/mL and mix well, let stand at 4°C for 1 hour, and centrifuge at 13,000 rpm for 20 minutes , take the supernatant, add PEG6000 to a final concentration of 10% (W/V), overnight at 4°C, centrifuge at 13000rpm for 25min, discard the supernatant, take the precipitate, and resuspend the obtained precipitate with an appropriate volume of normal saline, and wash and centrifuge thoroughly inner wall of the tube. Add an equal volume of chloroform, shake gently for 30s, and centrifuge at 8000rpm for 15min at 4°C to separate the organic phase and the aqueous phase, take the aqueous phase, and obtain the purified phage proliferation solution, and detect the purified phages by the double-layer plate method;

The purified phage is named vB_EcoM_swi3, and it is preserved in the China Type Culture Collection Center. The address of the preservation unit is: Wuhan University, Wuhan, China. The preservation number is CCTCC M 2019467. The classification name is: Escherichia coli phage, and the preservation date is June 19, 2019.

Example 3: Morphological Observation by Transmission Electron Microscopy of Escherichia coli Phage vB_EcoM_swi3

Take 20 μL of the purified phage proliferation solution and drop it on the copper grid, let it stand for about 15 minutes, and absorb the excess liquid with filter paper. Add 15 μL of 2% phosphotungstic acid (PTA) dropwise on the copper mesh for 5 minutes, absorb excess dye solution with filter paper, and observe with a transmission electron microscope after drying.

The transmission electron microscope results of coliphage vB_EcoM_swi3 are shown in Figure 1. Coliphage vB_EcoM_swi3 has a clear polyhedral head structure and a stretchable tail structure. The diameter of the head is about 80 nm, and the length of the tail is about 120 nm. According to the "Classification of Viruses-The Tenth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2018, the bacteriophage belongs to the family Myoviridae.

Example 4: Cleavage Spectrum Detection of Escherichia coli Phage vB_EcoM_swi3

In the experiment, 10 pathogenic Escherichia coli clinical isolates (including 8 isolates from diseased pigs and 2 isolates from diseased chickens) were selected to determine the lysis profile of coliphage vB_EcoM_swi3. The specific operation is as follows: 200 μL of 10 strains of Escherichia coli proliferation solution were evenly spread on the LB solid medium plate, and after the bacteria liquid was completely absorbed, 5 μL of phage filtrate was added dropwise on the plate, and 5 μL of normal saline was added dropwise as a control, and left to dry naturally. After drying, culture them upside down in a constant temperature incubator at 37°C for 4-6 hours, observe and record the results.

The results showed that the 10 Escherichia coli strains selected in the experiment grew well on the LB solid medium plate and formed a lawn. Among them, there is no bacterial growth in the area where coliphage vB_EcoM_swi3 is added to the plate of 8 strains of Escherichia coli, indicating that the isolated coliphage vB_EcoM_swi3 can lyse 8 strains of 10 strains of Escherichia coli, and the lysis rate is 80%, of which 6 strains are pig The source of Escherichia coli, 2 strains of chicken Escherichia coli.

Table 1 The lysis profile test results of coliphage vB_EcoM_swi3

Example 5: Titer determination of coliphage vB_EcoM_swi3

The Escherichia coli phage vB_EcoM_swi3 proliferation solution was diluted 10 times sequentially, and 100 μL of the proliferation solution of each dilution was mixed with an equal volume of the host bacteria E.coli-K88 bacteria solution, and the plaques were counted by the double-layer plate method. Three parallels were performed for each dilution. The phage titer was calculated according to the number of plaques, and the titer of Escherichia coli phage vB_EcoM_swi3 was measured to be 6.4×10 ⁸ pfu/mL.

Example 6: Determination of optimal multiplicity of infection of coliphage vB_EcoM_swi3

Take coli phage vB_EcoM_swi3 proliferation fluid and the host bacteria E.coli-K88 bacterial fluid cultured to the logarithmic phase, and count them. Mix according to the ratio of multiplicity of infection 0.001, 0.01, 0.1, 1, 10, 100 and 1000 respectively, add 5 mL of LB broth, shake and culture at 37 ° C for 4 h, centrifuge at 12000 rpm for 20 min, and filter with a 0.22 μm filter to obtain phage For the proliferation solution, the phage titer was determined by the double-layer plate method, and three parallels were performed for each group, and the optimal multiplicity of infection was calculated according to the results of the phage titer determination.

The results showed (Table 2) that when the multiplicity of infection was 1, the titer of the coliphage vB_EcoM_swi3 reached a maximum of 8.2×10 ⁸ pfu/mL. Therefore, the optimal MOI of coliphage vB_EcoM_swi3 is 1.

Table 2 The best multiplicity of infection assay results of coliphage vB_EcoM_swi3

Phage count Host bacteria count 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: pH Stability Determination of Escherichia coli Phage vB_EcoM_swi3

Take 4.5 mL of LB liquid medium with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) and add coliphage at a concentration of 10 ⁸ pfu/mL 500 μL of vB_EcoM_swi3 proliferation solution was incubated in a water bath at 37°C, and 1 ml of samples were taken at 1 h, 2 h, and 3 h. The samples taken were sequentially diluted 10 times, and 100 μL of the proliferation solution of each dilution was mixed with an equal volume of E.coli-K88 bacterial solution, and the phage titer was determined by the double-layer plate method, with three parallels for each group, according to Statistical Results The pH stability curve of the phage was plotted.

The results showed (Fig. 2) that coliphage vB_EcoM_swi3 was relatively stable in the pH range of 6-8, and when pH<6 or pH>8, the activity of coliphage vB_EcoM_swi3 decreased rapidly with the increase of pH. When the pH is lowered to 2, the phage acts for 1-3 hours, and the titer is only 10 ³ pfu/mL; when the pH rises to 13, the titer of coliphage vB_EcoM_swi3 is 10 ⁵ pfu/mL after 1 hour of action; After that, the phage titer decreased to 10 ³ pfu/mL. Therefore, coliphage vB_EcoM_swi3 is stable under neutral conditions at pH 6-8.

Example 8: Thermostability assay of coliphage vB_EcoM_swi3

Incubate 100 μL of coliphage vB_EcoM_swi3 proliferation solution with a concentration of 10 ⁸ pfu/mL in water baths at 40°C, 50°C, 60°C, 70°C, and 80°C, and take samples at 20 minutes, 40 minutes, and 60 minutes respectively. Doubling dilution, take 100 μL of phage proliferation solution of each dilution and mix with equal volume of E.coli-K88 bacterial solution, and measure the titer of E. coli phage vB_EcoM_swi3 by double-layer plate method. Statistical results Plot the thermostability curve of phage.

The results showed (Figure 3) that the titer of coliphage vB_EcoM_swi3 remained basically unchanged at 40°C and 50°C; at 60°C, the activity of coliphage vB_EcoM_swi3 was basically unaffected within 20 minutes, but more than 20 minutes Finally, as the action time prolongs, the phage activity decreases rapidly. When the action time is 60 minutes, the phage titer decreases by more than three orders of magnitude; when the temperature is 70°C or 80°C, the phage activity decreases rapidly as the action time prolongs. Therefore, when the temperature is below 60°C, the activity of coliphage vB_EcoM_swi3 is relatively stable.

Example 9: UV Stability Determination of Escherichia coli Phage vB_EcoM_swi3

Take 4 mL of coliphage vB_EcoM_swi3 proliferation solution with a concentration of 10 ⁸ pfu/mL, place it in a clean plate, and place it at a distance of 40 cm from the ultraviolet lamp for 60 min of irradiation. Take 100 μL of phage proliferation solution every 10 minutes, and perform 10-fold dilution. Take 100 μL of each dilution of phage proliferation solution and mix it with an equal volume of E.coli-K88 bacterial solution, and use the double-layer plate method to determine the phage titer. Three parallels were set at each time point, and the UV stability curve of phage was drawn according to the measurement results.

The results showed ( FIG. 4 ), after the coliphage vB_EcoM_swi3 was continuously irradiated with ultraviolet light for 5 minutes, the activity of the phage began to decrease. The titer of phage decreased by about one order of magnitude when exposed to ultraviolet light for 20 minutes. When continuously irradiated by ultraviolet light for 40 minutes, the titer of phage decreased by about two orders of magnitude. When the phage was continuously irradiated for 60 min, the titer of the phage decreased to 10 ⁵ pfu/mL. It shows that coliphage vB_EcoM_swi3 is more sensitive to ultraviolet light.

Example 10: One-step growth curve of coliphage vB_EcoM_swi3

Escherichia coli phage vB_EcoM_swi3 proliferation solution and overnight cultured host bacteria E.coli-K88 200μL each were mixed thoroughly according to the optimal multiplicity of infection ratio, incubated at 37°C for 5min, centrifuged at 13000rpm for 30s, washed twice with LB liquid medium, and discarded. supernatant. Resuspend the pellet with 7 mL of LB liquid medium, and culture with shaking at 37°C. At different time points (starting from time zero, every 5 minutes in the first 1 hour, every 20 minutes in the second hour, and every 30 minutes in the 3rd to 4th hours), 200 μL of the proliferation solution was taken, centrifuged at 16,000 rpm for 1 minute, and the supernatant was taken by double-layer plate method. To measure the phage titer, do three parallels at each time point, draw a one-step growth curve according to the measurement results, and calculate the incubation period, outbreak period and outbreak amount of the phage.

The results showed ( FIG. 5 ), the incubation period of coliphage vB_EcoM_swi3 was about 25 minutes, the outbreak period was about 75 minutes, and the outbreak amount was about 25.

Example 11: Safety test of coliphage vB_EcoM_swi3

(1) Experimental animals: 20 4-week-old Kunming mice were purchased from Qingdao Daren Fucheng Animal Husbandry Co., Ltd.

(2) Experimental method: 4-week-old Kunming mice were randomly divided into 2 groups, with 10 mice in each group. The mice in the test group were injected intraperitoneally with 10 ⁸ pfu of coliphage vB_EcoM_swi3 proliferation fluid, and the mice in the control group were injected with the same volume of sterile LB liquid medium. After injection, the mice were raised under the same environmental conditions, and the mental state of the mice was observed.

(3) Experimental results: 12 hours after injection, 5 mice in each group were killed, and the changes in internal organs and intestines were observed; blood samples and feces samples were collected from the remaining 5 mice regularly, and the titer of phage in the samples was determined by double-layer plate method , to detect the metabolic dynamics of coliphage vB_EcoM_swi3 in mice.

The final results showed that this dose of phage had no effect on the growth of mice, no abnormalities were found in the anatomical examination, and 23 hours after intraperitoneal injection of phage, no phage could be detected in blood and feces.

Example 12: Escherichia coli-infected mice experiment treated with coliphage vB_EcoM_swi3

(1) Experimental animals: 50 4-week-old Kunming mice.

(2) Experimental method: 50 4-week-old Kunming mice were used for the infection test, and were divided into 5 groups (3 treatment groups, 1 control group, and 1 infection group), with 10 mice in each group. Pick a single clone of E.coli-K88, inoculate it in 3ml LB liquid medium, culture overnight at 37°C, centrifuge at 12000rpm for 5min, remove the supernatant, and wash 3 times with PBS. Resuspend the bacteria in PBS and adjust the concentration of the bacteria to 2×10 ⁸ cfu/ml. The infection group and the three treatment groups were given intragastric administration of 10 ⁸ CFU of host bacteria E.coli-K88 to establish a mouse model of Escherichia coli infection, and the control group was intragastrically administered an equal volume of PBS. Two hours later, 10 ⁵ pfu, 10 ⁶ pfu and 10 ⁷ pfu of Escherichia coliphage vB_EcoM_swi3 were injected intraperitoneally into the three treatment groups, respectively, and equal volumes of PBS were injected into the control group and infection group. They were raised under the same environmental conditions, and the mental state and survival of the mice in each group were observed, and the dead mice were autopsied to observe the lesions of internal organs. At the same time, after 0.5h, 1.5h, 2h, 5h, 6.5h, 8.5h, 10h, 23h, 27h, and 51h of inoculation, the blood and feces of the surviving mice in each group were collected to detect the presence of bacteria and phages in the mice. metabolic dynamics.

(3) Experimental results: The results show (Figure 6), E.coli-K88 has entered the blood of mice 30 minutes after intragastric administration, and it increases exponentially with time. After 1.5 hours of intragastric administration, the blood The concentration of bacteria in the medium reached 10 ⁸ CFU, and the mice inoculated with the bacteria were listless, their activities were significantly reduced, and the phenomenon of clustering appeared. After 2 hours of intraperitoneal injection of coli phage vB_EcoM_swi3, the concentration of E.coli-K88 in the blood of mice in the three treatment groups decreased rapidly. The higher the concentration of inoculated phage, the faster the number of bacteria in the blood decreased. It can be cleared within 1 hour, and the bacteria in the blood of mice in the middle dose group and the low dose group can be completely cleared within 51 hours.

The phage in the feces prolongs as the inoculated phage dose increases, and the phage in the high-dose group can be completely eliminated within 99 hours (Fig. 7). All the mice in the infection group died on the 2nd day after inoculation. Among the three treatment groups, one mouse in the low-dose group died, and all the other mice survived. It shows that intraperitoneal injection of 10 ⁶ pfu coli phage vB_EcoM_swi3 can effectively treat the infection caused by E.coli-K88.

Example 13: Escherichia coli phage vB_EcoM_swi3 environmental disinfection experiment

Adjust the concentration of the freshly proliferated host bacteria E.coli-K88 to 10 ⁶ CFU/ml, spray evenly on the floor of the pig house and the surface of the trough, and then divide the spraying area into three areas, and the purified E. coli The bacteriophage vB_EcoM_swi3 proliferation solution was sprayed to disinfect the ground and trough of the piggery in the three districts at the concentration of 10 ⁵ PFU, 10 ⁶ PFU and 10 ⁷ PFU respectively, and the concentration of the host bacteria E.coli-K88 in the environment was detected after 2 hours. content.

The test results showed that in the three areas where the E.coli-K88 bacterial solution was sprayed with 10 ⁶ CFU in the pig house, the number of host bacteria in the trough and the ground dropped below 45 CFU, indicating that the phage proliferation solution of 10 ⁵ PFU can effectively kill Destroy the polluting host bacteria in the environment.

Comparative Example 1: Isolation of porcine pathogenic coliphage JS09 and detection of its lysis profile

(1) Experimental materials:

Host bacteria ETEC EK99-F41, pig farm sewage, 40 different strains of porcine pathogenic Escherichia coli, 1 strain of bovine pathogenic Escherichia coli and 5 strains of engineered Escherichia coli;

(2) Isolation experiment of porcine pathogenic coliphage JS09:

Collect 100 ml of sewage from a pig farm, centrifuge at 12,000 rpm for 15 minutes, take the supernatant, filter and sterilize with a 0.22 μm filter to obtain a filtrate. The host strain ETEC EK99-F41 (10 ⁹ CFU/ml) was added to the filtrate, and cultured with shaking at 37°C overnight. The bacterial solution was centrifuged at 12000 rpm for 10 min, the supernatant was taken, and sterilized by filtering with a 0.22 μm filter to obtain a filtrate. Evenly spread the host bacterium ETEC EK99-F41 on the LB plate, after drying, take 10 μl of the filtrate dropwise on the surface of the plate, incubate overnight at 37°C, observe the appearance of phage plaques, and isolate and obtain phage JS09;

(3) Cleavage spectrum detection of porcine pathogenic coliphage JS09:

46 Escherichia coli clinical isolates from different sources were selected in the experiment, and the lysis profile of the isolated phage JS09 was determined. The specific operation is as follows: take 100 μl of different E. coli culture solutions and spread evenly on the surface of LB agar plate, let it stand to dry, add 10 μl of bacteriophage JS09 (10 ⁹ PFU/ml) dropwise on the surface, culture it upside down at 37°C overnight, and observe the The appearance of bacterial plaque was obtained, and the lysis spectrum of Escherichia coli from different sources was obtained, and the results were recorded. The results show that the lytic phage JS09 can lyse 11 of the 40 strains of pig-derived pathogenic E. coli, and can also lyse 5 strains of E. coli engineered bacteria. It has no lytic effect on bovine E. coli, and its lysis rate is 21.7%. .

Comparative Example 2: Detection of lysis profile of chicken pathogenic coliphage EcP5

(1) Experimental materials:

Chicken pathogenic Escherichia coli and its lytic phage EcP5, 45 different strains of chicken pathogenic Escherichia coli;

(3) Experimental method:

In the experiment, 45 different clinical isolates of chicken pathogenic Escherichia coli were selected to measure the lysis spectrum of the lytic phage EcP5. , let stand for 15min. The formation of phage plaques was observed by the double-layer agar plate method, and the lysis profiles of phages to different chicken pathogenic Escherichia coli were obtained, and the results were recorded;

The results showed that the lytic phage EcP5 could lyse 18 out of 45 strains of chicken pathogenic Escherichia coli, and the lysis rate was 40.0%.

Table 3 Example 1-13 coliphage vB_EcoM_swi3 and comparative example 1-2 phage lysis spectrum comparison

Lysis rate (%) lysed E. coli source Examples 1-13 80% Escherichia coli from chicken, Escherichia coli from pig Comparative example 1 21.7% Lysis of porcine E. coli only Comparative example 2 40% Only lysed chicken E. coli

As can be seen from the results in Table 3, comparative example 1 only cracks pig-derived E. coli and commonly used E. coli engineering bacteria in the laboratory, and has no ability to lyse bovine-derived E. coli; comparative example 2 can only crack chicken-derived E. coli alone, and cannot Escherichia coli from other sources were lysed at the same time, and the lysis rate of Comparative Example 1-2 was lower than that of coliphage vB_EcoM_swi3 in Examples 1-13 of the present invention. It can be seen that the phage provided by the present invention is lysing large intestines from different animal sources. Bacillus has an advantage.

In addition, the bacteriophage vB_EcoM_swi3 provided by the present invention also has the advantages of good tolerance to physical and chemical factors, short incubation period, high cracking performance, and good safety. It can be made into different dosage forms and used as feed additives, disinfectants or cleaning agents, etc. Therefore, it can be seen that the coliphage vB_EcoM_swi3 provided by the present invention has a very broad application prospect in preventing and controlling the pollution of pig and chicken E. coli in the farming environment and in the breeding process of livestock and poultry and other animals.

sequence listing

<110> Qingdao Agricultural University

<120> Coliphage vB_EcoM_swi3 and its application

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 6468

<212>DNA

<213> Coliphage vB_EcoM_swi3

<400> 1

1 TCAGTCCCCT CTAACATACG CAATTACAAC TGTACGAGTT GAGTTTTTCCA 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 TGGTCCACAAA 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 (10)

1. Escherichia coli phage vB_EcoM_swi3, characterized in that the phage preservation number is CCTCC M 2019467, which was deposited in the China Center for Type Culture Collection on June 19, 2019, and was named porcine coli phage vB_EcoM_swi3. 2. the application of the coliphage as claimed in claim 1 in the preparation of simultaneously killing pig-derived and chicken-derived Escherichia coli biological products. 3. application according to claim 2, is characterized in that, described biological product is feed additive and disinfectant/cleaning agent, and wherein said feed additive and disinfectant/cleaning agent are the single preparation with purified coliphage A preparation or a compound preparation prepared with the purified coliphage as the main component. 4. The application according to claim 2, characterized in that the porcine-derived Escherichia coli and chicken-derived Escherichia coli include pathogenic Escherichia coli, and the porcine-derived Escherichia coli includes enterotoxigenic Escherichia coli. 5. The application according to claim 4, wherein the enterotoxigenic Escherichia coli comprises K88 type Escherichia coli, and the chicken-derived pathogenic Escherichia coli comprises O78 type Escherichia coli. 6. Disinfectant or cleaning agent is characterized in that, is the single preparation prepared with purified coliphage as claimed in claim 1 or the composite preparation prepared as main component with purified coliphage as claimed in claim 1 It is used to prevent and control the pollution of E. coli from pigs or chickens in the breeding environment. 7. The disinfectant or cleaning agent according to claim 6, characterized in that, the dosage form of the disinfectant or cleaning agent is liquid, freeze-dried powder or tablet; Table, ground of livestock and poultry breeding sites, air of livestock and poultry breeding sites, livestock and poultry feed, drinking water and breeding utensils. 8. Feed additive, it is characterized in that, for the single preparation prepared with purified coliphage as claimed in claim 1 or the composite preparation prepared as main component with purified coliphage as claimed in claim 1, add in In livestock and poultry feed, it is used to prevent and control the pollution of E. coli from pigs and chickens during the breeding process. 9. The feed additive according to claim 8, characterized in that the feed comprises at least pig feed or chicken feed. 10. The disinfectant or cleaning agent according to claim 6 or the feed additive according to claim 8, characterized in that, the porcine-derived Escherichia coli and chicken-derived Escherichia coli include pathogenic Escherichia coli, and the pig-derived Escherichia coli The source pathogenic E. coli includes enterotoxigenic E. coli, the enterotoxigenic E. coli includes K88 type E. coli, and the chicken source pathogenic E. coli includes O78 type E. coli.