Escherichia coli bacteriophage capable of enhancing intestinal barrier performance of broiler chickens and having high blood entering efficiency and application of escherichia coli bacteriophage

Abstract

The invention belongs to the field of biotechnology poultry breeding bacterial disease prevention and control, and discloses an Escherichia coli bacteriophage capable of enhancing the intestinal barrier performance of broiler chickens and having high blood entering efficiency and application. The preservation number of the bacteriophage is CCTCC (China Center For Type Culture Collection) NO: M 20242424. When the bacteriophage DCP80 is independently used, the bacteriophage DCP80 has the advantages of increasing the down-implicit ratio and enhancing the intestinal barrier performance of the broiler chickens, also has very high blood entering efficiency, is very strong in acid and alkali resistance, and has a relatively high clinical application value. The DCP80 has a killing effect on escherichia coli of various serotypes; the DCP80 can be used for preventing and treating various poultry bacterial diseases caused by escherichia coli and the like, the fermentation efficiency of the DCP80 is extremely high, the production cost is greatly reduced, and a new prevention and treatment method is provided for poultry breeding under the food safety requirement.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology poultry breeding bacterial disease prevention and control, and specifically relates to an Escherichia coli phage that enhances the intestinal barrier performance of broiler chickens and has high blood entry efficiency and its application. Background Art

[0002] Escherichia coli is ubiquitous in nature and is one of the normal intestinal flora of livestock and poultry. It can synthesize B vitamins and vitamin K, is beneficial to the host, and has an inhibitory effect on many pathogens. However, some specific serotypes of Escherichia coli strains are pathogenic, especially when the host's health declines or under stress conditions. These strains will show pathogenicity. Avian colibacillosis refers to a disease caused by focal and systemic infections caused by some specific serotypes such as O1, O2, O35, O78, etc. Avian pathogenic Escherichia coli (APEC) infection. Avian colibacillosis occurs worldwide. Chickens of different ages and breeds may be infected with this disease throughout the year. Air, water, feed and related equipment can all become the medium of transmission of the disease. Avian colibacillosis is roughly estimated to cause economic losses of about hundreds of millions of dollars to the global poultry industry each year. Therefore, comprehensive and effective prevention and control of avian colibacillosis is a major challenge facing the poultry industry.

[0003] The integrity of the intestinal mucosal tissue structure is the basic guarantee for the digestion and absorption of nutrients and the healthy growth of poultry. It is also the basis for the normal functioning of all physiological functions of the intestine. Indicators such as villus height, crypt depth, and the ratio of villus height to crypt depth (V / C) are often used to evaluate the growth and development of the intestine. The increase in intestinal villus height will increase the absorption area of ​​the small intestine, allowing nutrients to be fully absorbed and utilized; the crypt depth reflects the proliferation rate and maturity of intestinal mucosal cells, and the shallower crypt depth indicates that the proliferation rate of intestinal epithelial cells is accelerated and the absorption function is enhanced; the ratio of villus height to crypt depth can comprehensively reflect the function of the intestine, and a decrease in the ratio indicates a decrease in digestion and absorption function.

[0004] Phages are ubiquitous in all environments, including soil, water, food, and even in the intestinal microbiota of animals and humans, and are easy to be isolated and screened. Bacteria lysed by phages cannot restore their viability, which is beneficial to solving the problem of bacterial resistance compared to the antibacterial effect of most antibiotics. Phages are highly host-specific. Except for a very small number of phages, phages will only lyse the target host bacteria without the risk of interfering with other microorganisms. They are safer than common antibiotic inhibitors. Overall, phages are a kind of biological antibacterial substance with a wide range of sources, high specificity, high safety, and can replace antibiotic inhibitors. Moreover, when bacteria are killed, the concentration of phages also decreases until they completely disappear from the body, so phages are not infectious to humans or animals and plants, and will not pollute the environment, so they are relatively safe. At present, although phage therapy has not replaced antibiotics, phage therapy for bacterial diseases is still a very promising treatment method with advantages that antibiotics cannot match.

[0005] At present, there are reports that phages are related to intestinal health. For example, Wu Liting et al. found that compared with the blank control group, the combination of HN02 phage + lactobacillus can significantly increase the average daily weight gain and villus height / crypt depth of chicks (P<0.05), and significantly reduce the feed-to-weight ratio of chicks and the crypt depth of the jejunum and ileum (P<0.05), but the use of lactobacillus or HN02 phage alone does not have the effect of increasing the villus height / crypt depth. Tang Luyao (2022) found in his study that the phage cocktail he developed (with 5 strains of Salmonella phage and 7 strains of Escherichia coli phage) can significantly increase the villus height by 9.1% (P<0.01) and the VH / CD value by 6.5% (P<0.01) compared with the antibiotic group, and has no significant effect on the crypt depth (P>0.05).

[0006] Phages have a wide range of application scenarios, but phages may have low blood entry efficiency and the possibility of damaging the intestinal mucosa, which will reduce the effectiveness of phage use on the one hand, and affect the homeostasis of intestinal microorganisms and thus affect the growth rate of poultry on the other hand. Therefore, screening out phages that can both promote intestinal barrier performance and have a high blood entry efficiency has a high clinical application value. The DCP80 phage screened by the present invention has the advantage of enhancing the intestinal barrier performance of broiler chickens when used alone, and also has a high blood entry efficiency, and has a high clinical application value. Summary of the invention

[0007] The object of the present invention is to provide an Escherichia coli phage which can enhance the intestinal barrier performance of broiler chickens and has high blood-entry efficiency. The deposit number of the phage is CCTCC NO: M20242424.

[0008] Another object of the present invention is to provide the use of Escherichia coli phage DCP80 in the preparation of drugs for treating or preventing diseases caused by Escherichia coli infection.

[0009] In order to achieve the above object, the present invention adopts the following technical measures:

[0010] The applicant isolated an Escherichia coli phage DCP80 from sewage. The phage can enhance the intestinal barrier performance of broiler chickens, has a high blood entry efficiency, and has a broad-spectrum antibacterial effect on multiple serotypes of Escherichia coli. The phage was sent to the China Center for Type Culture Collection for preservation on November 4, 2024, address: Wuhan University, Wuhan, China; the preservation number is CCTCC NO: M 20242424, and the classification name is: Escherichia phage (Escherichia phage) DCP 80.

[0011] Observation under transmission electron microscope showed that the head of the selected phage showed a polyhedral structure. According to the morphological classification of Bradley and Ackermann, the isolated phage belongs to the order Myovirales.

[0012] The protection scope of the present invention includes:

[0013] A composition comprising Escherichia coli phage DCP80.

[0014] Use of Escherichia coli phage DCP80 or the above composition in the preparation of Escherichia coli inhibitors.

[0015] Use of Escherichia coli phage DCP80 or the above composition in preparing medicine for treating or preventing diseases caused by Escherichia coli infection.

[0016] Application of Escherichia coli phage DCP80 or the above composition in preparing chicken intestinal probiotic drugs.

[0017] In the above application, the Escherichia coli includes serum types O1, O2, O35 and O78.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] 1) The DCP80 bacteriophage provided by the present invention has the ability to increase the villus-cryptoproliferative ratio when used alone, which can enhance the intestinal barrier performance of broiler chickens;

[0020] 2) DCP80 phage has a high blood-entry efficiency;

[0021] 3) DCP80 phage has a broad bactericidal spectrum and can lyse various serotypes of E. coli in poultry farming, including O1, O2, O35, and O78. DCP80 phage can be used to prevent and treat various bacterial diseases in poultry caused by E. coli, such as enteritis, perihepatitis, pericarditis, omphalitis, and air sac inflammation.

[0022] 4) DCP80 bacteriophage has strong acid and alkali resistance and can maintain high activity at pH 2-12, which has high clinical application value;

[0023] 5) DCP80 has a very high fermentation efficiency. When the optimal infection multiplicity M=0.1, the titer can reach 1.0×10 after 6 hours of fermentation. 9 pfu / ml, which greatly reduces production costs and provides a new prevention and control method for poultry farming under food safety requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Transmission electron micrograph of Escherichia coli phage DCP80.

[0025] Figure 2 : Schematic diagram of the temperature stability of Escherichia coli phage DCP80.

[0026] Figure 3 : Schematic diagram of blood entry performance detection of Escherichia coli phage DCP80. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to implementation cases, but the scope of the present invention is not limited to the scope of the embodiments. The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies; the reagents or materials, unless otherwise specified, are all from commercial channels.

[0028] Embodiment 1:

[0029] Phage isolation, purification and sequencing:

[0030] After the collected samples were left to stand overnight at room temperature, 10 mL of water sample was taken from each portion and centrifuged at 6000 rpm for 10 min. The supernatant was filtered and sterilized. 5 mL of the filtrate was mixed with the same volume of 2×TSB liquid culture medium and 1 mL of bacterial solution in the logarithmic phase (10 8 cfu / mL) were evenly mixed and cultured overnight in a shaker at 30°C and 200rpm to enrich the phage. The enriched solution was centrifuged at 6000rpm for 10 minutes, and the supernatant was sterilized with a 0.22μm microporous filter to obtain a filtrate containing phages. The double-layer plate method was used to observe the morphology of phage plaques. When a single plaque grows, the double-layer plate experiment operation is repeated 3-5 times to obtain the purified phage, and its morphology is as follows: Figure 1The phage was sent to the China Center for Type Culture Collection on November 4, 2024, with the address: Wuhan University, Wuhan, China; the deposit number is CCTCC NO: M 20242424, and the classification name is Escherichiaphage DCP80.

[0031] Embodiment 2:

[0032] Determination of the titer of bacteriophage DCP80 at different infection multiplicity:

[0033] Pick a single colony of the host bacteria, inoculate it into a test tube containing 3ml TSB culture medium, and culture it in a shaker at 180rpm at 37℃ overnight to obtain a host bacterial suspension. Take 100μL of the bacterial suspension and inoculate it into 10ml TSB culture medium, and culture it in a shaker at 37℃ and 180rpm until the logarithmic growth phase. Dilute the phage and its corresponding host bacteria to a certain concentration and count them, mix and culture them according to different infection multiplicity ratios (MOI = number of phages / number of bacteria), and add TSB liquid culture medium to make the total volume of each tube the same. Culture it in a shaker at 37℃ and 180rpm for 6h, aseptically take out 5mL of culture medium, centrifuge it at 5000rpm for 10min, collect the supernatant, and determine the phage titer.

[0034] Each group was cultured in duplicate tubes and the average value was taken. The MOI that produced the highest phage titer was the optimal infection multiplicity. The experiment was repeated 3 times. The results showed that the titer of phage DCP80 could reach 1.0×10 after 6 hours of fermentation at the optimal infection multiplicity M=0.1. 9 pfu / ml, the results are shown in Table 1.

[0035] Table 1 MOI determination of Escherichia coli phage DCP80

[0036]

[0037] Embodiment 3:

[0038] Stability test of bacteriophage DCP80:

[0039] Acid-base stability: Take 9 mL of TSB liquid culture medium and adjust it to different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), place it in a constant temperature water bath at 25°C, and after the temperature is balanced, add 1 mL of 4×10 9 PFU / mL pure culture of Escherichia coli phage DCP80 was placed in an environment of 25℃ for 4h.

[0040] After diluting part of the sample, the double-layer plate method was used to determine the phage titer. Each point was cultured in duplicate tubes to obtain the average value. The experiment was repeated 3 times. The results are shown in Table 2, indicating that phage DCP80 can maintain high activity at a pH of 2-12. Temperature stability: Temperature stability test of Escherichia coli phage

[0041] Take several sterile 50mL centrifuge tubes, add 45mL TSB to each tube, and add 2×10 10 PFU / mLDCP80 pure culture solution was placed in a constant temperature water bath at the corresponding temperature and acted at 4℃, 25℃, 37℃, 50℃, 60℃, and 70℃ for 24 hours. After the action time, the sample tube was taken out and immediately placed in an ice bath for cooling. After appropriate dilution, the phage titer was determined by the double-layer plate method. Duplicate culture was performed at each point to obtain the average value. The experiment was repeated 3 times. The test results are shown in Figure 2 shown.

[0042] Table 1 pH stability of Escherichia coli phage DCP80

[0043]

[0044]

[0045] Embodiment 4:

[0046] Bacteriophage DCP80 lysis range test:

[0047] The stock solution of DCP80 was taken and the lysis spectrum of the phage was determined by the drop method.

[0048] 78 strains of Escherichia coli containing various serotypes isolated from different regions of the country were selected and inoculated into centrifuge tubes containing 3mL TSB, and cultured at 37℃180rpm for 8h to obtain bacterial suspensions of each strain. Take 600μL of bacterial suspension and mix it with TSB semi-solid culture medium and spread it on the prepared NA plate, and take 5μLDCP80 culture solution and drop it on the plate. After natural air drying, culture at 37℃ for 8-12h and observe the results. The experiment was repeated three times, and the results are shown in Table 3, indicating that the Escherichia coli that can be killed by DCP80 include O1 type Escherichia coli, O2 type Escherichia coli, O35 type Escherichia coli, and O78 type Escherichia coli, and the lysis rate of Escherichia coli can be as high as 96.15%.

[0049] Table 3 The lysis spectrum of Escherichia coli phage DCP80

[0050]

[0051] Embodiment 5:

[0052] Determination of the ability of bacteriophage DCP80 to enter blood:

[0053] Thirty one-day-old white-feathered broilers with negative blood phages were selected and orally administered with 0.2 mL of a 2×10 9 PFU / mL of DCP80 phage was tested in the blood and intestines of chicks to determine the blood entry performance and residence time of the prepared phage. The average value was statistically analyzed. The results are shown in Figure 3 .

[0054] It was found that after oral administration of phages, phages could be detected in the blood or intestines, and the phage titer decreased with the extension of administration time. After 12 hours, the titer of phage DCP80 in the cecal contents was about 10 7 PFU / mL, but rapidly decreased to ≤10 at 84h 4 PFU / mL, it was almost undetectable after 156 hours. However, in the blood, although its highest titer in the blood was ≤10 5 P FU / mL, but the residence time in the blood is relatively long, and the presence of phages can still be detected in the blood after 156 hours.

[0055] Embodiment 6:

[0056] Verification of bacteriophage DCP80's promotion of intestinal barrier performance and clinical bactericidal effect:

[0057] 40 laying hens aged 2 weeks were randomly divided into 4 groups, 10 in each group. They were DCP80 treatment group, DCP514 treatment group, challenge group and blank group. 9 cfu / ml of pathogenic Escherichia coli CVCC3026 (which can be lysed by Escherichia coli phage DCP80 and Escherichia coli phage DCP514 (CN118178510A)), the blank group was given 0.2 ml PBS and isolated.

[0058] 6 h after the challenge, the DCP80 treatment group was given a final concentration of 1.0×10 9 PFU / mL DCP80 phage 0.2 ml / mouse; DCP514 treatment group: final concentration of 1.0×10 9 PFU / mL DCP514 phage 0.2 ml / animal, treatment for 4 consecutive days. The challenge group and the blank group were fed normally. The average bacterial load in the liver of each group was counted on the second day after the treatment, and the results are shown in Table 4.

[0059] The integrity of the intestinal mucosal tissue structure is the basic guarantee for the digestion and absorption of nutrients and the healthy growth of poultry. It is also the basis for the normal functioning of all physiological functions of the intestine. Indicators such as villus height, crypt depth, and the ratio of villus height to crypt depth (V / C) are often used to evaluate the growth and development of the intestine. The increase in intestinal villus height will increase the absorption area of ​​the small intestine, allowing nutrients to be fully absorbed and utilized; the crypt depth reflects the proliferation rate and maturity of intestinal mucosal cells, and the shallower crypt depth indicates that the proliferation rate of intestinal epithelial cells is accelerated and the absorption function is enhanced; the ratio of villus height to crypt depth can comprehensively reflect the function of the intestine, and a decrease in the ratio indicates that the digestion and absorption function is reduced. The villus height and crypt depth of each group were counted on the second day after the end of treatment. The results are shown in Tables 5 and 6.

[0060] Table 4: Mean bacterial load in the liver

[0061]

[0062] Table 5: Villus length values ​​of each group

[0063]

[0064]

[0065] Table 6: Crypt depth values ​​of each group

[0066]

Claims

1. An isolated Escherichia phage DCP80, the deposit number of which is CCTCC NO: M 20242424.

2. A composition comprising the Escherichia coli phage DCP80 according to claim 1.

3. Use of the Escherichia coli phage DCP80 according to claim 1 or the composition according to claim 2 in the preparation of an Escherichia coli inhibitor.

4. Use of the Escherichia coli phage DCP80 according to claim 1 or the composition according to claim 2 in the preparation of a medicament for treating or preventing a disease caused by Escherichia coli infection.

5. Use of the Escherichia coli phage DCP80 according to claim 1 or the composition according to claim 2 in the preparation of a chicken intestinal probiotic drug.

6. The use according to claim 3 or 4, wherein the Escherichia coli includes serum types O1, O2, O35, and O78.

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