CN111481574B

CN111481574B - Combined phage preparation for treating piglet diarrhea

Info

Publication number: CN111481574B
Application number: CN202010306966.3A
Authority: CN
Inventors: 魏炳栋; 徐永平; 于维; 郑琳; 陈龙; 李淑英; 李纪彬; 杨欢
Original assignee: DALIAN SEM BIOLOGICAL ENGINEERING TECHNOLOGY CO LTD; Jilin Jijia Feed Additive Co ltd; Jilin Academy of Agricultural Sciences
Current assignee: DALIAN SEM BIOLOGICAL ENGINEERING TECHNOLOGY CO LTD; Jilin Jijia Feed Additive Co ltd; Jilin Academy of Agricultural Sciences
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2023-03-14
Anticipated expiration: 2040-04-17
Also published as: CN111481574A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a combined phage preparation for treating piglet diarrhea. Comprises coliphage vB _ EcoP _ E21, coliphage vB _ EcoM _ F2 and Shigella phage vB _ SsoM _ Z31; the preservation number of the Escherichia coli phage vB _ EcoP _ E21 is as follows: CGMCC 18869, and the preservation number of the Escherichia coli phage vB _ EcoM _ F2 is as follows: CGMCC 18871, and the preservation number of Shigella phage vB _ SsoM _ Z31 is as follows: CGMCC 18870. The combined phage preparation is applied in a direct drenching mode, can reduce the abundance of escherichia coli and shigella, effectively prevents and controls the occurrence of diarrhea diseases of piglets in the breeding process, improves the survival rate of the piglets, and reduces the economic loss. The combined phage preparation provided by the invention can replace the traditional feed antibiotics, has the characteristics of safety, high efficiency, no residue and the like, and is an ideal antibiotic substitute.

Description

Combined phage preparation for treating piglet diarrhea

Technical Field

The invention belongs to the technical field of biology, and relates to three phage isolates capable of specifically cracking enterotoxigenic escherichia coli, multiple drug-resistant escherichia coli and shigella dysenteriae strains of piglets and application of the phage isolates in treatment of piglet diarrhea.

Background

The newborn piglet has low self resistance because a stable micro-ecosystem is not established in the intestinal tract, is sensitive to external stimulation and is easily affected by infection of pathogenic microorganisms and various stress factors. The infectious factors causing piglet diarrhea are two main types, namely viral and bacterial, wherein the bacterial diarrhea is mainly caused by escherichia coli and shigella. The escherichia coli infection of the piglets has the characteristics of wide prevalence range, high morbidity and mortality and the like, and the survival rate of the piglets is reduced, the growth of the piglets is blocked and the piglets die. Clinically, the basic method for treating escherichia coli and shigella infections is to use antibacterial drugs. In recent years, with the use of a large amount of antibacterial drugs, especially when the antibacterial drugs are added into feed as feed additives for a long time, the escherichia coli can generate drug resistance, the drug resistance spectrum is continuously expanded, the propagation speed is faster and faster, the number of the multiple drug-resistant escherichia coli is also continuously increased, and the health and public health safety of people are seriously threatened. At present, global breeding has raised the hot tide of 'banning' completely, 7 months and 10 days in 2019, 194 bulletin number issued by rural departments in agriculture, and since 1 month and 1 day in 2020, all varieties of growth-promoting drug feed additives except traditional Chinese medicines are withdrawn, so that the step of China entering the 'nonreactive age' is accelerated.

The bacteriophage is a generic name of viruses which can infect bacteria, fungi and actinomycetes, has a simple structure and wide distribution, and is known as the most abundant biological entity in a biosphere. Compared with the traditional antibiotics, the phage therapy has the advantages of strong self-replication capacity, high host specificity, safety, low research and development cost and the like, and is an ideal antibiotic substitute. However, the phage therapy also has the problems of too narrow lysis spectrum, unstable action effect, weak lysis activity and the like.

The selected phage specially aims at enterotoxigenic escherichia coli, multiple drug-resistant escherichia coli and shigella dysenteriae causing piglet diarrhea, and has high potency, good stability and stronger cracking activity.

Disclosure of Invention

In order to prevent and treat piglet diarrhea caused by escherichia coli, shigella and the like, the invention provides a combined phage preparation for enterotoxigenic escherichia coli, multi-drug-resistant escherichia coli and shigella dysenteriae, and simultaneously provides application of the phage preparation in preventing and treating piglet diarrhea.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the invention provides a combined phage preparation, which comprises three phages, namely a coliphage vB _ EcoP _ E21, a coliphage vB _ EcoM _ F2 and a shigella phage vB _ SsoM _ Z31, wherein the coliphage vB _ EcoP _ E21 is preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 06 days in 2019 with the preservation number of CGMCC NO.18869 and is named as a coliphage in classification; the coliphage vB _ EcoM _ F2 is preserved in China general microbiological culture Collection center (CGMCC) in 2019 at 11 and 06 months, has the preservation number of CGMCC NO.18871 and is named as coliphage in classification; the shigella phage vB _ SsoM _ Z31 is preserved in China general microbiological culture Collection center (CGMCC) in 11 and 06 months 2019, the preservation number is CGMCC NO.18870, and the classification name is shigella phage. China general microbiological culture Collection center address: west road No.1, north chen, chaoyang district, beijing, zip code: 10010.

the observation of a transmission electron microscope of the coliphage vB _ EcoP _ E21 shows that the phage belongs to the order of tailed phage and the family of brachyphagidae, the diameter of the head capsid is 52.1 +/-1.2 nm, the length of the tail is 10.4 +/-1.1 nm, a one-step growth curve shows that the latency of the phage for cracking bacteria is 20min, and the cracking amount is 370PFU/mL; the observation of a transmission electron microscope of a coliphage vB _ EcoM _ F2 shows that the phage belongs to the order of tailed phage and the family of myophagae, the diameter of the head capsid is 160 +/-1.1 nm, the length of the tail is 19 +/-1.4 nm, a one-step growth curve shows that the latency of the phage is 30min, and the cracking amount is 110PFU/mL; transmission electron microscope observation of Shigella phage vB _ SsoM _ Z31 shows that the phage belongs to the order of tailed phage and the family of myophagidae, the diameter of the head is 63.8 +/-1.3 nm, the length of the tail is 102 +/-2.3 nm, and a one-step growth curve shows that the latency of the phage is 10min and the cracking amount is 115PFU/mL.

In the above technical solution, further, the volume ratio of the coliphage vB _ EcoP _ E21, the coliphage vB _ EcoM _ F2 and the shigella phage vB _ SsoM _ Z31 is 1.

The invention also provides aThe combined phage preparation is primarily applied to preventing and treating bacterial diseases of piglet diarrhea, the phage body fluid is used for drenching piglets in the onset period, and the combined phage titer is more than or equal to 10 ¹⁰ PFU/mL is administrated for 1 time a day for 3-7 days continuously, and can effectively treat or prevent piglet diarrhea.

Further, when the preparation is taken, 100mL of 1.2-1.4% sodium bicarbonate solution is taken first, and after 10-15min, 100mL of combined phage preparation lysate is taken.

The invention has the beneficial effects that: the combined phage preparation is applied in a direct drenching mode, can reduce the abundance of escherichia coli and shigella, effectively prevents and controls the occurrence of diarrhea diseases of piglets in the breeding process, improves the survival rate of the piglets, and reduces the economic loss.

The combined phage preparation provided by the invention can replace the traditional feed antibiotics, has the characteristics of safety, high efficiency, no residue and the like, and is an ideal antibiotic substitute.

Drawings

Fig. 1 projection electron microscopy observation of phage morphology, a. Phage vB _ EcoP _ E21, b. VB _ EcoM _ F2, c. VB _ SsoM _ Z31;

figure 2 phage temperature stability, a. Phage vB _ EcoP _ E21, b.vb _ EcoM _ F2, c.vb _ SsoM _ Z31;

figure 3 phage pH stability, a. Phage vB _ EcoP _ E21, b. VB _ EcoM _ F2, c. VB _ SsoM _ Z31;

FIG. 4 bacteriophage one step growth curve, a. Bacteriophage vB _ EcoP _ E21, b. VB _ EcoM _ F2, c. VB _ SsoM _ Z31;

FIG. 5 example 6 rectal temperature profile of test pigs;

FIG. 6 example 6 test swine fecal scoring profile.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1

Isolation and screening of phages

(I) preparation of host bacteria

The invention relates to three host bacteria, wherein Enterotoxigenic Escherichia coli (Enterotoxichia coli k88, CVCC 83902) is purchased from China Veterinary microorganism Culture Collection management Center (CVCC), shigella dysenteriae (CGMCC 21535) is purchased from China General Microbiological Culture Collection management Center (CGMCC); the multi-drug resistant Escherichia coli F2 (JIsnky-E.coli F2) is separated from piglet diarrhea feces of a pig farm of Jilin province Agricultural Academy of Sciences (JAAS), is automatically preserved in an antibiotic substitute laboratory of Jilin province Agricultural Academy of Sciences, and is identified to have drug resistance to 9 antibiotics such as ampicillin, sulbactam, piperacillin/tazobactam, cefuroxime axetil, cefotetan, imipenem, compound neonomamine and the like through drug sensitive tests.

The specific separation process of the multiple drug-resistant escherichia coli F2 is as follows:

collecting a piglet diarrhea fecal sample, putting the piglet diarrhea fecal sample into a foam box with an ice bag, and transporting the piglet diarrhea fecal sample back to a laboratory for strain separation and identification. Picking a small amount of excrement samples by using a cotton swab and streaking the excrement samples on a MacConkey flat plate, placing the MacConkey flat plate at the constant temperature of 37 ℃ for culturing for 12-16h, picking red bacterial colonies which are round, smooth, neat and clear in boundary and consistent in size by using an inoculating loop, continuously streaking the red bacterial colonies on the MacConkey flat plate, and performing purification culture; after 2-3 times of purification, round and smooth red colonies are picked by an inoculating loop and streaked on an eosin methylene blue culture medium, and further purification is carried out after constant temperature culture at 37 ℃ for 12-16 h. And (3) picking a single colony with black metallic luster on an eosin methylene blue culture medium by using an inoculating loop, uniformly coating the single colony on a glass slide, and drying at room temperature. Dropwise adding an ammonium oxalate crystal violet solution, acting for 90s, cleaning with pure water, and sucking residual water drops on the glass slide; adding a gram iodine solution, fully acting for 90s, cleaning with pure water, and sucking residual water drops on the slide; slightly inclining the glass slide at a certain angle, dripping 95% ethanol by a dropper for decoloring for 30s, and cleaning by pure water; the bacterial strain is counterstained with safranine liquid for 30s, washed and dried by pure water, and examined by an oil microscope, and gram stain is negative and is in the shape of short rod-shaped bacteria without spores.

The strain is comprehensively evaluated to be escherichia coli through biochemical reaction tests and 16s rDNA sequence comparison results. And then, monitoring the drug resistance and sensitivity of the E.coli F2 to clinically common antibiotics by using a drug sensitive paper sheet method, wherein the specific operation is carried out according to standard operation established by the national Committee for standardization of clinical laboratories (NCCLS) in 2010, and the E.coli F2 has drug resistance (9/20) to 9 antibiotics such as ampicillin, sulbactam, piperacillin and the like.

Respectively streaking and inoculating the three host bacteria on an LB solid culture medium, carrying out overnight culture at 37 ℃, selecting a single colony on a flat plate, inoculating the single colony in a fresh LB liquid culture medium, carrying out shake culture at 37 ℃ for 5-8h, and selecting logarithmic phase bacterial liquid for separating, amplifying and purifying phage.

(II) treatment of sewage sample

Taking 500mL of wastewater samples from a certain pig farm in Jilin and a certain hospital in Dalian city, mixing, and then pretreating the water samples: adding CaCl ₂ And MgCl ₂ The final concentration was adjusted to 1mmol/L.

(III) amplification of Sewage-like phages

Taking a sewage sample 50mL, centrifuging at 12000rpm for 6min, and filtering with a 0.22-micron filter membrane to obtain a supernatant; adding 30mL of supernatant into an equal volume of LB liquid culture medium and 1-2mL of logarithmic phase host bacterial liquid, uniformly mixing, and carrying out shake culture at a constant temperature of 37 ℃ and 160r/min overnight; taking 50mL of culture solution, centrifuging at 12000rpm for 10min, filtering with a 0.22 μm filter membrane to obtain supernatant, and storing at 4 ℃ to obtain the amplified phage amplification solution to be detected.

(IV) identifying whether the phage is contained

And (3) identifying whether the sewage amplification solution contains the phage or not by adopting a single-layer flat plate dropping method. Uniformly coating 200mL of logarithmic phase bacteria liquid on a monolayer LB solid culture medium, completely airing, dripping 10 mu l of phage amplification liquid to be detected on a plate, and placing the plate right side into an incubator at 37 ℃ for overnight culture. And taking out the sample on the second day to observe whether obvious plaques appear at the drip part or not, and if so, determining that the sewage amplification solution contains the phage to be detected.

(V) Primary isolation of phages

The separation of phage was performed by the double-layer plate method, and two kinds of LB solid media were prepared: a lower layer LB solid culture medium containing 2% agar; the upper layer of LB semi-solid medium containing 1% agar. Heating and dissolving the upper layer culture medium, placing in a 60 ℃ oven for standby, taking 8 10mL centrifuge tubes, respectively adding 200 mu L host bacteria liquid, sucking phage suspension, and performing gradient dilution to 10 ^-7 Respectively taking out 200 mu L of 8 phage diluents with different concentration gradients, adding the phage diluents into a 10mL centrifuge tube for acting for 3-5min, adding 8mL LB semisolid culture medium containing 1% agar, fully mixing uniformly, pouring the mixture on a solidified 2% agar LB solid culture medium flat plate, after solidification, inversely culturing in an incubator at 37 ℃, and observing whether transparent plaques are formed after 8-12 h. If a countable single plaque is formed on the plate, this indicates that this dilution gradient can be used for phage isolation and titer calculation.

(VI) purification of phages

The plaque of primary separation is not uniform in size and shape, and the phage needs to be further purified, so that the phage forms the plaque with uniform size and shape on the plate. Picking up plaques with obvious difference in shape and size by using a sterile 200 mu L gun head, respectively adding the plaques into a centrifuge tube of 1mL sterile SM Buffer solution, standing for 4h at 4 ℃ to fully release the phage into the SM Buffer, centrifuging for 5min at 12000r/min, collecting supernatant, filtering by using a 0.22 mu m filter membrane, performing gradient dilution on phage filtrate, and separating and purifying by adopting a double-layer plate method. Repeating the above operations for 5-6 times until the plaque shape and size are completely consistent, and obtaining the purified phage. The purified three phages are deposited in the general microbiological center of China Committee for culture Collection of microorganisms in 11 th and 06 th of 2019, and are classified and named as coliphage and Shigella phage, wherein the preservation numbers of the coliphage vB _ EcoP _ E21 are as follows: CGMCC 18869, and the preservation number of the Escherichia coli phage vB _ EcoM _ F2 is as follows: CGMCC 18871, shigella phage vB _ SsoM _ Z31 preservation number is: CGMCC 18870. The purified three phage whole genome sequences were submitted to the Genbank library of NCBI website, and the phage genome names and sequence accession numbers are vB _ EcoP _ E21 (MN 604053), vB _ EcoM _ F2 (MN 864145) and vB _ SsoM _ Z31 (MN 655999), respectively.

Enrichment and amplification of (VII) phages

Proliferation is carried out by liquid proliferation method. The specific method comprises the following steps: adding each phage purification solution into host bacteria solution cultured to logarithmic phase according to the proportion of 1. And finally, mixing the three phage enrichment liquids in equal proportion to obtain the combined phage preparation.

Example 2

And (4) measuring the temperature, the pH stability and the lysis spectrum of the phage.

Adding 1mL of phage lysate into 1.5mL of sterile centrifuge tubes, placing the centrifuge tubes in water baths at 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, selecting different time points of 20, 40, 60, 80, 100 and 120min, taking out 100 mu L of phage lysate from the centrifuge tubes, cooling to room temperature, measuring the titer by a double-layer plate method, and repeating three times for each group. The result shows that the phages vB _ EcoP _ E21, vB _ EcoM _ F2 and vB _ SsoM _ Z31 have stronger tolerance to temperature, the titer is basically kept stable at 40 ℃, 50 ℃ and 60 ℃ after 120min of action, except that the titer begins to sharply decrease after the phage vB _ EcoM _ F2 is acted at 70 ℃ for 40min, and the titer is decreased to 0 after 80min of action, the phages vB _ EcoP _ E21 and vB _ SsoM _ Z31 can still maintain certain activity at 80 ℃. The results are shown in FIG. 2.

The pH value of LB culture medium is adjusted to 2-12 by using 1M HCL and 1M NaOH solution, 900 mu L of different pH culture medium is added into a 1.5mL centrifuge tube, then 100 mu L of fresh phage lysate is added, the constant temperature culture is carried out for 1h at 37 ℃, the phage titer is measured by using a double-layer plate method, and each group is divided into three parallels. The results show that the phages vB _ EcoP _ E21, vB _ EcoM _ F2 and vB _ SsoM _ Z31 have certain tolerance to pH, the phages are inactivated at pH values of 2.0 or 12.0, the titer is 0, and the phage activity remains substantially stable at pH values of 4, 6, 8 and 10, indicating that the three phages have strong tolerance to pH values. The results are shown in FIG. 3.

Determination of the fragmentation spectrum: respectively coating different log-phase bacterial liquids to be detected on an LB solid culture medium, after the bacterial liquids are dried in the air, sucking 5 mu l of phage lysis liquid to be dropped on the culture medium, carrying out inverted culture at 37 ℃ for 10-12h, observing whether plaque appears, if plaque appears, indicating that the phage has a lysis effect on the bacterial strain, and the result of a phage lysis spectrum is shown in Table 1. As can be seen from tables 1-1 and 1-2, the bacteriophages vB _ EcoP _ E21, vB _ EcoM _ F2 and vB _ SsoM _ Z31 have strong complementarity on the lysis spectrum, and the combination of the three bacteriophages can mutually compensate the defect of narrow lysis spectrum.

TABLE 1-1 E.coli phages vB _ EcoP _ E21 and vB _ EcoM _ F2 fragmentation spectra

Host bacterium	vB_EcoP_E21	vB_EcoM_F2
			Escherichia coli 6	—	+
Escherichia coli 8	+	+
			Escherichia coli 11	—	+
Escherichia coli F1	—	+
			Escherichia coli F2	—	+
Escherichia coli E13	+	+
			Escherichia coli O157	+	—
Enterotoxigenic Escherichia coli K88	+	—
			Escherichia coli12588	+	—
Escherichia coli236	+	+
			Escherichia coli233	+	+
Escherichia coli238	—	—
			Escherichia coliF18	+	—

Table 1-2 Shigella phage vB _ SsoM _ Z31 fragmentation profiles

Host bacterium	vB_SsoM_Z31
		Shiga toxin-producing Escherichia coli 10668	+
Shigella firigella	+
		Shigella firigella 10865	+
Shigella Sonnei 21535	+
		Shigella dysentery 10983	+

Example 3

And (4) determining the optimal complex infection number of the phage.

The multiplicity of infection (MOI) of a bacteriophage refers to the ratio of the number of phage to host bacteria at the time of infection, i.e., the number of phage infected per bacterium. The determination method comprises the following steps: (1) Selecting a single colony of the host bacteria by using an inoculating loop, inoculating the single colony in an LB liquid culture medium, and performing shaking culture at 37 ℃ and 160r/min until the logarithmic phase of the host bacteria; (2) Mixing phage with known titer with the above bacterial liquid at different ratio of 0.0001, 0.001, 0.01, 0.1, 1 and 10; (3) Shaking-culturing at 37 deg.C for 5h at 160r/min, centrifuging for 2min, collecting supernatant, and filtering with 0.22 μm filter membrane for sterilization; (4) Three groups of parallel filtrates are taken for gradient dilution, the phage titer is measured by a double-layer plate method, and the infection complex group with the highest measured phage titer is the optimal infection complex number. The multiplicity of infection of the phage is shown in Table 2. As can be seen from Table 2, the optimal multiplicity of infection for these three phages was 0.1, 1 and 0.01, respectively.

TABLE 2 optimal multiplicity of infection (MOI) of bacteriophage

Bacteriophage	Optimal multiplicity of infection (MOI)
		vB_EcoP_E21	0.1
vB_EcoM_F2	1
		vB_SsoM_Z31	0.01

Example 4

Determination of one-step growth Curve of bacteriophage

The one-step growth curve of the phage can describe the growth rule of the phage and understand the incubation period and the cracking amount of the phage. The method comprises the following steps: (1) According to the best MOI result, 1mL of host bacteria cultured to logarithmic phase is mixed with the phage, incubated at 37 ℃ for 15min, centrifuged at 12000r/min for 10min, and the supernatant is discarded; (2) Washing twice with 2mL of LB liquid culture medium preheated to 37 ℃ to ensure that free phage not adsorbed with bacteria are completely removed; (3) Resuspending the phage suspension by using 2mL of a 37 ℃ preheated LB liquid culture medium, adding the suspension into 10mL of 37 ℃ preheated LB liquid culture medium, fully and uniformly mixing, quickly placing the suspension at 37 ℃ for shaking culture, starting timing T0=0, continuously sampling for 120min every 10min, removing bacteria by using a 0.22 mu m filter membrane, measuring titer by using a double-layer plate method, and drawing a phage one-step growth curve by using time as an abscissa and the titer as an ordinate. The one-step growth curve of the phage is shown in FIG. 4.

The result shows that the incubation period of the phage vB _ EcoP _ E21 is 20min, and the lysis amount is 370PFU/mL; the incubation period of the phage vB _ EcoM _ F2 is 30min, and the lysis amount is 110PFU/mL; phage vB _ SsoM _ Z31 latency 10min, lysis volume is 115PFU/mL.

Example 5

The safety test selects 30 male Kunming mice (weight 35g +/-2 g) with the age of 8 weeks, randomly divides the mice into two groups, each group comprises 15 mice, wherein the experimental group is irrigated with the combined phage preparation of the invention, each experimental group is irrigated with 10mL of the combined phage preparation every day, and the titer is 10 ¹⁰ And injecting equivalent normal saline into a control group of the phage preparation PFU/mL, carrying out continuous experiment for 7 days, then carrying out cervical dislocation and death on the mice, carrying out autopsy, observing whether pathological changes appear in the lung, the liver, the kidney and the abdominal cavity, preliminarily verifying that the measured phage liquid has no influence on the physiological condition of the mice, and proving that the combined phage preparation has feeding safety.

Example 6

Effect of preventing and controlling colibacillus and shigella diarrhea of piglets

1. Test animals and management

24 weaned piglets (weaned at 28 days old) were selected for the test and randomly divided into three groups, namely a control group, a challenge group and a phage treatment group, 8 pigs in each group were fed with a basal diet free of antibiotics. Before the experiment, 100mL of 1.2-1.4% sodium bicarbonate solution, 10mAfter in, 5mL of escherichia coli and shigella compound bacterial liquid (mixed according to the volume ratio of 1:1 and the concentration of 10) which is re-suspended by using physiological saline is poured into the challenge group and the phage group ⁸ CFU/mL), 5mL of physiological saline sterile solution was administered to the control group, and after 10min, 100mL of phage preparation of the present invention (titer of 10) was administered to the phage treatment group ¹⁰ PFU/mL), challenge group orally administered 100mL of phage-free saline, and experiment was continuously observed for 14 days.

2. Index and method of measurement

During the test period, the weight change, the diarrhea occurrence frequency and degree of each group of animals and the excrement score (0 point, normal excrement, 1 point, soft excrement but molding, 2 points, brown excrement, 3 points, severe water sample excrement, continuous determination for 7 days) are recorded in detail, and the rectal temperature (continuous determination for 7 days) is determined simultaneously; 10mL of blood was collected from the anterior vena cava of all test pigs on

test days

1, 7 and 14, respectively, and plasma was prepared and stored frozen at-20 ℃ for determination of IL-8 and TNF- α levels. After the test was completed, 3 pigs were selected for each group and slaughtered, and digestive tract samples were isolated for determination of the number and pH of E.coli bound to the intestinal mucosa in the duodenum, jejunum, ileum, colon and cecum segments.

3. Test results

3.1 growth Performance

The results of the experimental pig growth performance are shown in Table 3. As can be seen from Table 3, the initial weight difference between the groups was not significant (p > 0.05), the final weight and average daily gain of the challenge group and the treated group were significantly lower than those of the control group (p < 0.05), but the final weight and average daily gain of the treated group were significantly higher than those of the challenge group (p < 0.05), indicating that the phage was able to suppress the loss of weight caused by E.coli diarrhea to some extent.

TABLE 3 test of growth Performance in pigs

3.2 rectal temperature and stool Scoring

The rectal temperature and stool score results for the test pigs are shown in fig. 5 and 6. As can be seen from FIG. 5, after the Escherichia coli liquid is orally taken by the toxicity attacking group and the treating group, the rectal temperature increases, which indicates that the organism generates an inflammatory reaction after Escherichia coli toxicity attacking, the body temperature increases, the rectal temperature reaches the highest value on day 2, and then gradually decreases, and is basically normal on day 7, but the rectal temperature of the toxicity attacking group is lower than that of the toxicity attacking group, which indicates that the bacteriophage can inhibit the growth of Escherichia coli, and the inflammatory reaction of Escherichia coli to the organism is reduced.

As can be seen from figure 6, diarrhea of different degrees can occur in the toxin attacking group and the treating group after the escherichia coli is orally taken, the diarrhea score of the toxin attacking group from the first day to the seventh day is basically more than 1.5, the diarrhea symptom begins to be relieved after the treating group orally takes the traditional Chinese medicine composition for the 1 st day, and the feces score for basically curing the diarrhea after the 5 th day is basically less than 1, so that the bacteriophage product provided by the invention can effectively prevent and treat diarrhea of piglets caused by the escherichia coli.

3.3 blood cytokine content

As can be seen from Table 4, on the 7 th day of the oral administration of Escherichia coli challenge in the challenge group and the treatment group, the cytokines IL-8 and TNF-alpha are obviously increased (p < 0.05), but the difference between the challenge group and the treatment group is not obvious (p > 0.05), and by 14 days, the contents of the cytokines in the challenge group and the treatment group are reduced, compared with the control group, the difference is not obvious (p > 0.05), and the contents of the cytokines in the phage treatment group are lower than that in the challenge group but do not reach the obvious difference level (p > 0.05), which indicates that the phage product provided by the invention can reduce the inflammatory response caused by Escherichia coli infection to a certain extent.

TABLE 4 blood cytokine content

3.4 quantity and pH value of colibacillus combined with mucous membrane of each intestinal segment of digestive tract

As shown in Table 5, when Escherichia coli is taken orally for counteracting, the pH values of the caecum and the colon can be increased remarkably (p is less than 0.05) compared with the control group, but the pH value difference between the jejunum and the ileum is not significant (p is less than 0.05), which indicates that Escherichia coli infection mainly occurs at the rear end of the digestive tract, and the bacteriophage preparation provided by the invention can be taken orally for remarkably reducing the pH value of the colon (p is less than 0.05) but also is increased remarkably (p is less than 0.05) compared with the control group; from the number of escherichia coli combined with intestinal mucosa, each section of mucosa of jejunum, ileum, colon and cecum is combined with escherichia coli in different degrees, the number of mucosa combined with the mucosa of the attacking group is obviously higher than that of the treating group or the control group (p is less than 0.05), and the number of the escherichia coli combined with each section of mucosa of the intestine is obviously reduced (p is less than 0.05) by orally taking the phage preparation provided by the invention, which indicates that the phage product has a strong lysis effect, can achieve the lysis effect at the rear end of the digestive tract, and can effectively prevent and treat diarrhea caused by the escherichia coli.

TABLE 5 pH of each intestinal segment of the digestive tract and the number of mucosally bound Escherichia coli

It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Claims

1. A combined phage preparation comprising bacteriophage vB _ EcoP _ E21, bacteriophage vB _ EcoM _ F2 and shigella vB _ SsoM _ Z31; the preservation number of the Escherichia coli phage vB _ EcoP _ E21 is as follows: CGMCC NO.18869, the preservation number of the coliphage vB _ EcoM _ F2 is as follows: CGMCC NO.18871, wherein the shigella phage vB _ SsoM _ Z31 has a preservation number of: CGMCC NO.18870.

2. The combinatorial phage preparation of claim 1, characterized in that the volume ratio of the coliphage vB _ EcoP _ E21, coliphage vB _ EcoM _ F2 and shigella phage vB _ SsoM _ Z31 is 1.

3. Use of the combined phage preparation according to claim 1 for the preparation of a bacterial disease preparation for the prevention and treatment of diarrhea in piglets.

4. Use according to claim 3, characterized in that the combined phage titer is ≥ 10 ¹⁰ And PFU/mL, the combined phage bacterium liquid is drenched for 1 time every day for 3-7 days continuously.

5. The use according to claim 4, wherein 100mL of 1.2-1.4% sodium bicarbonate solution is administered first, and after 10-15min, 100mL of the combined phage lysate is administered.