Escherichia coli phage, Salmonella phage, microbial preparations and their applications from white cashmere goats in northern Shaanxi

Abstract

The present invention discloses Escherichia coli phage, Salmonella phage, microbial preparation and application thereof from white cashmere goats in northern Shaanxi, and belongs to the field of microbial technology. Escherichia coli phage PE004 and Salmonella phage PS006 and microbial preparation thereof can not only be used to prepare new biological drugs for preventing and / or treating diarrhea in white cashmere goats in northern Shaanxi, but also can be used for cashmere goat feed additives, bactericides and injection preparations, etc. While solving the diarrhea infection and death of cashmere goats caused by Escherichia coli and Salmonella, it avoids the problems of antibiotic drug residues in animal bodies and livestock products, environmental pollution, and inducing bacterial multidrug resistance caused by the use of antibiotics, and at the same time complies with the national policies of antibiotic replacement and reduction, healthy growth of livestock and poultry, and green production in animal husbandry.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and in particular to Escherichia coli phages, Salmonella phages, microbial preparations and applications thereof, derived from white cashmere goats in northern Shaanxi. Background Art

[0002] The northern Shaanxi white cashmere goat is a new cashmere goat breed approved by the Ministry of Agriculture in 2003. The goat industry, which is mainly based on the northern Shaanxi black goat, has become the leading industry for agricultural development and income increase of farmers and herdsmen in northern Shaanxi (Yulin, Yan'an) and surrounding areas (some counties and districts in Shanxi). It plays an irreplaceable and important role in regional economic development, income increase of farmers and herdsmen, and maintaining social stability. However, in spring and winter or when the temperature changes, Escherichia coli disease is rampant, causing diarrhea, sepsis, and even death in lambs, causing huge economic losses to goat farmers. Pathogenic Escherichia coli induces a variety of fatal diseases in newborn lambs of cashmere goats, and also causes mastitis in ewes, which seriously endangers the health of ewes and lambs, and even causes large-scale death of lambs, with a high mortality rate. Effective prevention and control of cashmere goat Escherichia coli disease is an important problem to ensure animal health and industrial development.

[0003] At present, the treatment for bacterial diseases is the use of antibiotics. Although some antibiotics (oxytetracycline, sulfonamides, etc.) can treat this disease, they cannot solve the fundamental problem. With people's over-reliance on antibiotics, drug-resistant strains continue to emerge, and even some super-resistant bacteria appear. The abuse of antibiotics will also cause antibiotics to remain in animals, thus threatening human health. Therefore, the use of antibiotics has become a restrictive factor affecting the development of healthy livestock and poultry breeding. Finding a feasible biological control method to replace antibiotics or exploring a new way of antibiotic-free breeding is a trend in the future development of animal husbandry. The problem of antibiotic resistance has become an important topic that humans have to face in the 21st century, and the post-antibiotic era is bound to come. In view of the increasingly serious threat caused by the spread of antibiotic resistance, the World Health Organization (WHO) and the European Commission (EC) have recognized the importance of studying antimicrobial resistance agents and emphasized the need to develop appropriate control strategies. Therefore, exploring and researching green, safe and efficient antibiotic substitutes, and providing new ideas and methods for healthy animal breeding and antibiotic-free breeding, is the key to developing healthy breeding in my country's goat industry, enhancing the international competitiveness of my country's goat industry and ensuring a healthier life for humans.

[0004] Bacteriophages are a type of bacterial virus that can infect and lyse bacteria. In particular, phages have the characteristics of strong specificity, rapid proliferation, no residue, and low resistance to drug resistance. They can accurately and quickly lyse host bacteria to exert a bactericidal effect, and are the preferred strategy to replace antibiotics. Bacteriophages can also be used to prevent cross-contamination of pathogens in contact materials and food processing facilities during food production. At present, there are relatively few studies on mature phage biological agents in our country, and there are few reports on phage biological control related to goat diseases. Summary of the invention

[0005] The purpose of the present invention is to provide Escherichia coli phage, Salmonella phage, microbial preparation and application thereof from white cashmere goats in northern Shaanxi, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a strain of Escherichia coli phage PE004 (Myoviridae pahge) from white cashmere goats in northern Shaanxi. The phage was deposited in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Administration on April 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45967.

[0008] The second technical solution of the present invention is a Salmonella phage PS006 (Siphophagesphage) from white cashmere goats in northern Shaanxi. The phage was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on April 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45968.

[0009] The third technical solution of the present invention is a microbial preparation, comprising the Escherichia coli phage PE004 derived from the northern Shaanxi white cashmere goats and / or the Salmonella phage PS006 derived from the northern Shaanxi white cashmere goats.

[0010] The fourth technical solution of the present invention is the use of the Escherichia coli phage PE004 from the northern Shaanxi white cashmere goats, the Salmonella phage PS006 from the northern Shaanxi white cashmere goats or the microbial preparation in the preparation of drugs for treating and / or preventing lamb diarrhea.

[0011] The fifth technical solution of the present invention is a drug for treating and / or preventing lamb diarrhea, comprising the Escherichia coli phage PE004 from the northern Shaanxi white cashmere goats, the Salmonella phage PS006 from the northern Shaanxi white cashmere goats, or the microbial preparation.

[0012] The sixth technical solution of the present invention is the use of the Escherichia coli phage PE004 derived from white cashmere goats in northern Shaanxi, the Salmonella phage PS006 derived from white cashmere goats in northern Shaanxi, or the microbial preparation in the preparation of a fungicide.

[0013] The seventh technical solution of the present invention is a bactericide or bacteriostatic agent, including the Escherichia coli phage PE004 derived from the northern Shaanxi white cashmere goats, the Salmonella phage PS006 derived from the northern Shaanxi white cashmere goats, or the microbial preparation.

[0014] The eighth technical solution of the present invention is a feed additive, comprising the Escherichia coli phage PE004 from the northern Shaanxi white cashmere goats, the Salmonella phage PS006 from the northern Shaanxi white cashmere goats, or the microbial preparation.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] The present invention provides a coliform phage PE004 from white cashmere goats in northern Shaanxi, a salmonella phage PS006 from white cashmere goats in northern Shaanxi, a microbial preparation and its application. Coliform phage PE004 and salmonella phage PS006 and their microbial preparations can not only be used to prepare a new biological drug for preventing and / or treating diarrhea in white cashmere goats in northern Shaanxi, but also can be used for cashmere goat feed additives, bactericides and injection preparations. Coliform phage PE004 and salmonella phage PS006 are mixed to form a phage cocktail mixed preparation with better prevention and treatment effect. The phage cocktail mixed preparation is safe to use and has no side effects. While solving the diarrhea infection and death of lambs caused by Escherichia coli and salmonella, it avoids the problems of antibiotic drug residues in animal bodies and livestock products, environmental pollution, and the problem of inducing bacterial multidrug resistance caused by the use of antibiotics, and at the same time complies with the national policies of antibiotic replacement and reduction, healthy growth of livestock and poultry, and green production in animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The plaques are formed by the purified Escherichia coli phage PE004;

[0019] Figure 2 The plaques are formed by the purified Salmonella phage PS006;

[0020] Figure 3The morphological structure of Escherichia coli phage PE004 under transmission electron microscopy;

[0021] Figure 4 The morphological structure of Salmonella phage PS006 under transmission electron microscopy;

[0022] Figure 5 is the thermal stability of E. coli phage PE004;

[0023] Figure 6 is the thermal stability of Salmonella phage PS006;

[0024] Figure 7 is the pH stability of E. coli phage PE004;

[0025] Figure 8 is the pH stability of Salmonella phage PS006;

[0026] Fig. 9 is the UV sensitivity of Escherichia coli phage PE004;

[0027] Fig.10 The UV sensitivity of Salmonella phage PS006;

[0028] Fig.11 is the one-step growth curve of E. coli phage PE004;

[0029] Fig.12 is the one-step growth curve of Salmonella phage PS006;

[0030] Fig.13 It is a bacteriolysis test of Escherichia coli phage PE004;

[0031] Fig.14 This is a bacteriolysis test for Salmonella phage PS006. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0038] An embodiment of the present invention provides a strain of Escherichia coli phage PE004 (Myoviridae pahge) from white cashmere goats in northern Shaanxi. The phage was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on April 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45967.

[0039] The embodiment of the present invention also provides a Salmonella phage PS006 (Siphophagesphage) from white cashmere goats in northern Shaanxi, which was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on April 24, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 45968.

[0040] The embodiment of the present invention further provides a microbial preparation, including the Northern Shaanxi white cashmere goat-derived Escherichia coli phage PE004 and / or the Northern Shaanxi white cashmere goat-derived Salmonella phage PS006.

[0041] The embodiments of the present invention also provide the use of the Shaanxi white cashmere goat-derived Escherichia coli phage PE004, the Shaanxi white cashmere goat-derived Salmonella phage PS006 or the microbial preparation in the preparation of a drug for treating and / or preventing lamb diarrhea.

[0042] The embodiment of the present invention also provides a medicine for treating and / or preventing lamb diarrhea, comprising the Shaanxi North white cashmere goat-derived Escherichia coli phage PE004, the Shaanxi North white cashmere goat-derived Salmonella phage PS006 or the microbial preparation.

[0043] In some specific embodiments, the administration of the drug includes oral administration and injection; the effective dose of bacteriophage in the oral drug is 1×10 8 PFU / mL; the effective dose of phage in the injection drug is 1×10 6 PFU / mL.

[0044] In some specific embodiments, the drug is in the form of a solution, a powder, a gel, a granule or a lyophilized agent.

[0045] In some specific embodiments, the lamb diarrhea is caused by Escherichia coli and / or Salmonella infection. "Prevention": The bacteriophage or phage cocktail mixed preparation inhibits or eliminates lamb diarrhea. "Treatment": The bacteriophage or phage cocktail mixed preparation improves or improves lamb diarrhea.

[0046] In some specific embodiments, the application method of bacteriophage is as follows: adding bacteriophage or phage cocktail mixed preparation or processed agent thereof to diarrhea lamb feed as a therapeutic drug, or administering it orally, or giving it by intramuscular injection, or adding it to drinking water, so as to prevent and treat diarrhea in Shaanxi white cashmere goat lambs and improve the survival rate of lambs. The bacteriophage is easy to operate and has strong applicability.

[0047] In order to formulate the phage cocktail mixture into a liquid preparation, an acceptable carrier must be sterile and phage compatible. Examples include saline, sterile water, buffered saline and glycerol. They can be used alone or in any combination thereof. If necessary, other conventional additives, such as antioxidants, buffers and antibacterial agents, etc., can be added. When combined with a diluent, a dispersant, a surfactant, a binder and / or a lubricant, the composition of the present invention can also be prepared as an injection (e.g., an aqueous solution, a suspension and an emulsion), or a pill, a capsule, a granule or a tablet.

[0048] The embodiments of the present invention also provide the use of the Escherichia coli phage PE004 derived from white cashmere goats in northern Shaanxi, the Salmonella phage PS006 derived from white cashmere goats in northern Shaanxi, or the microbial preparation in the preparation of a fungicide.

[0049] The embodiment of the present invention also provides a bactericide or bacteriostatic agent, including the Shaanxi white cashmere goat-derived Escherichia coli phage PE004, the Shaanxi white cashmere goat-derived Salmonella phage PS006, or the microbial preparation.

[0050] In some specific embodiments, the effective dose of bacteriophage in the bactericide is 1×10 6 PFU / mL.

[0051] In some specific embodiments, the sheep house environment, feeding utensils, feed, pens, feeding troughs, walls, bedding, etc. can be disinfected by spraying or soaking.

[0052] How to use the antibacterial agent: Soak or spray the surface of fresh mutton for disinfection to inhibit the reproduction of pathogenic Escherichia coli and / or Salmonella during product processing or preservation.

[0053] Method of using the fungicide: The fungicide is used in slaughterhouses, livestock and poultry product processing workshops and equipment, and sheep houses to prevent pathogenic E. coli and Salmonella contamination in the environment. It includes but is not limited to disinfecting and decontaminating the drinking water system, breeding facilities, walls, floors, bedding, feeding utensils or other surfaces of utensils by liquid immersion, spraying, and combined use with aqueous carriers, and disinfecting feed. It can be used to replace antibiotics or traditional disinfection products, and the bacteriophage will not cause harm to humans and sheep. The liquid immersion and spraying forms include but are not limited to detergents, fungicides, detergents, etc.

[0054] The embodiment of the present invention further provides a feed additive, comprising the Shaanxi North White Cashmere Goat-derived Escherichia coli phage PE004, the Shaanxi North White Cashmere Goat-derived Salmonella phage PS006, or the microbial preparation.

[0055] The phage is mixed with cashmere goat feed and then fed to prevent or treat lamb diarrhea. Preferably, the effective dose of phage in the feed is 1×10 8 PFU / g.

[0056] By using two phages that are highly specific to the two main pathogens that cause diarrhea in white cashmere goats in northern Shaanxi to synergistically exert their bactericidal effects, the phage lysis spectrum is expanded, and the prevention and treatment effects of diarrhea in cashmere goats are improved. The phage and phage cocktail mixed biological preparations have a wide lysis spectrum and broad-spectrum bactericidal effects, and can be used as active ingredients in drugs, bactericides, cashmere goat feed additives, and biological antibacterial agents for the prevention and treatment of diarrhea in cashmere goats. They can be widely used in cashmere goat breeding processes and various links in mutton processing and production processes, quickly and efficiently killing pathogenic Escherichia coli and Salmonella, effectively reducing the diarrhea rate and mortality rate of cashmere goats, and improving the survival rate of white cashmere goats. Lamb health and meat product quality safety.

[0057] The above-mentioned bacteriophages and phage cocktail mixed biological preparations have low cost, no irritating odor, and no environmental pollution problems, providing a green, safe, efficient and new biological preparation for the prevention and treatment of diarrhea in white cashmere goat lambs in northern Shaanxi.

[0058] Example 1

[0059] 1.1 Phage isolation and purification

[0060] Add samples of feces and sewage from the Shaanxi white cashmere goat farm to LB culture medium, mix thoroughly and centrifuge to collect the supernatant and culture it with cashmere goat Escherichia coli culture solution at 37°C overnight. After centrifugation of the culture solution, collect 100 μL of the supernatant and 100 μL of freshly cultured Escherichia coli in the logarithmic growth phase and shake it gently to mix it evenly. Let it stand at 37°C for 20 minutes, and separate Escherichia coli phages by double-layer plate method. If plaques grow on the plate, pick up the plaques with a gun tip and put them into 2 mL of freshly cultured Escherichia coli culture in the logarithmic growth phase. Continue to culture at 37°C for 8 hours. After centrifugation of the culture solution, collect 100 μL of the supernatant and 100 μL of freshly cultured Escherichia coli in the logarithmic growth phase and shake it gently to mix it evenly. Let it stand at 37°C for 20 minutes, and purify Escherichia coli phages by double-layer plate method. Repeat this more than 3 times until transparent plaques of uniform shape and size are formed on the double-layer agar plate. Test results: After repeatedly picking up plaques with a gun tip to purify the phage, the plaques formed were uniform in size, neat and transparent, with smooth edges, round, and 2-3mm in diameter.

[0061] Salmonella phage was isolated from diarrhea lambs. The separation and purification method of Salmonella phage was the same as that of Escherichia coli phage. The phages formed by the two phages on the double-layer plate were shown in Figure 2. Figure 1 and Figure 2 .

[0062] 1.2 Phage titer determination

[0063] The double-layer agar plate culture method is used to determine the titer of phages. 0.1 mL of gradient diluted phages is thoroughly mixed with 0.1 mL of host bacteria cultured to the logarithmic growth phase. After standing at 37°C for 20 minutes, it is quickly mixed with LB semi-solid agar medium kept at about 55°C and spread on the bottom agar as the upper layer. After solidification, it is inverted and cultured for 6 hours to count plaques. Each dilution of phage is repeated 3 times, and the plates with 30-300 plaques are selected for counting, and the average value is taken to calculate the titer of phages.

[0064] The calculation formula of phage titer is: phage titer (PFU / mL) = number of plaques × dilution factor × 10.

[0065] Test results: The titer of Escherichia coli phage determined by double-layer agar plate method was 4.32×10 13 PFU / mL; the titer of Salmonella phage is 8.50×10 13 PFU / mL.

[0066] 2. Determination of phage biological characteristics

[0067] 2.1 Phage Concentration

[0068] 1 mL of phage and 200 mL of host bacteria in the logarithmic growth phase were mixed and cultured at 37°C and 220 rpm for 6 h until the mixture became clear. The mixture was centrifuged for 10 min and filtered. NaCl was added to make the final concentration of 1 mol / L, and the NaCl was dissolved by slow stirring. The mixture was placed in an ice bath overnight. The mixture was centrifuged at 4°C and 8000 rpm for 10 min. 10% polyethylene glycol 8000 (PEG-8000) was added to the supernatant, which was slowly inverted several times to fully dissolve and then placed in an ice bath overnight. The mixture was centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the phage precipitate was collected. The phage precipitate was resuspended with SM solution (500 μL of SM solution was used for every 100 mL of phage supernatant). The upper hydrophilic phase was recovered after extraction with an equal volume of chloroform 3 times, filtered, and stored at 4°C for later use.

[0069] 2.2 Phage morphology and structure

[0070] Take 50 μL of phage concentrate and 50 μL of 4% paraformaldehyde-0.5% glutaraldehyde solution, mix thoroughly and fix, take 50 μL and drop it on the copper grid and let it stand for 30 minutes, and absorb the excess liquid with filter paper; add 2% phosphotungstic acid staining solution to the copper grid for staining, let it stand for 10 minutes; dry at room temperature for 10 minutes, observe the phage morphology with a transmission electron microscope and take pictures.

[0071] Test results: The head of Escherichia coli phage PE004 is a regular polyhedron with a diameter of about 79nm; it has a retractable long tail with a length of 114nm and a diameter of 13nm. It belongs to the order Caudovirales and the family Myoviridae. Figure 3 The head of Salmonella phage PS006 is a regular polyhedron with a diameter of about 86nm. It has an inextractable long tail with a length of 99nm and a diameter of 19nm. It belongs to the Caudovirales order and the Siphophage family (Siphophages). Figure 4 ).

[0072] Escherichia coli phage PE004 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on April 24, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45967.

[0073] Salmonella phage PS006 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on April 24, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 45968.

[0074] 2.3 Determination of the optimal multiplicity of infection

[0075] Escherichia coli phage (1×10 11 PFU / mL) and Salmonella phage (1×10 10 PFU / mL) and its host bacteria (1×10 13 CFU / mL) for proliferation culture, determine the phage titer and host bacteria concentration, and perform gradient dilution. Then, according to the phage / bacteria ratio of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100, the phage and host bacteria liquid were mixed and allowed to stand at room temperature for 20 minutes, transferred to LB medium, and cultured at 37°C with shaking for 8 hours; the culture medium was centrifuged at 4°C, 12,000×g for 15 minutes, and then filtered. The titer of the filtrate was determined by the double-layer plate method, and the phage / bacteria ratio with the highest titer was the optimal multiplicity of infection.

[0076] Test results: When the infection multiplicity was 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, the titer of Escherichia coli phage PE004 was 3.0×10 9 , 3.2×10 10 , 1.2×10 8 , 6.7×10 8 , 5.42×10 10 , 3.09×10 10 and 4.88×10 10 PFU / mL, the phage titer was the highest at 1, so the optimal infection multiplicity of E. coli phage PE004 was 1 (Table 1). When the infection multiplicity was 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, the titer of Salmonella phage PS006 was 1.31×10 11 , 1.77×10 12 , 1.63×10 11 , 1.40×10 11 , 1.99×1011 , 1.60×10 11 and 1.92×10 11 The phage titer was highest at 0.001, so the optimal infection multiplicity of Salmonella phage PS006 was 0.001 (Table 2).

[0077] Table 1 Optimal infection multiplicity test of Escherichia coli phage PE004

[0078]

[0079]

[0080] Table 2 Optimal infection multiplicity test of Salmonella phage PS006

[0081]

[0082] 2.4 Phage thermal stability assay

[0083] Escherichia coli phage PE004 culture (titer 6.48×10 13 PFU / mL) and Salmonella phage PS006 culture (titer 1.7×10 12 The phages were diluted with SM buffer for a certain amount of time and then the titer of phages was determined by double-layer agar plate culture method. Each group of experiments was repeated 3 times.

[0084] Test results:

[0085] The activity of E. coli phage PE004 remained basically unchanged at 40℃ for 90 min, but decreased by 2 orders of magnitude at 50℃ for 90 min, 3 orders of magnitude at 60℃ for 90 min, 7 orders of magnitude at 70℃ for 90 min, and 8 orders of magnitude at 80℃ and 90℃ for 90 min, indicating that E. coli phage PE004 is tolerant to high temperature of 90℃ ( Figure 5 ).

[0086] The activity of Salmonella phage PS006 remained basically unchanged after being treated at 60℃ for 30min. The titer decreased by 5 orders of magnitude after being treated at 70℃ for 90min, by 6 orders of magnitude after being treated at 80℃ for 90min, and by 10 orders of magnitude after being treated at 90℃ for 90min. The titer of phage decreased rapidly between 70℃ and 90℃, and was relatively stable between 30℃ and 60℃, indicating that Salmonella phage PS006 can tolerate a certain high temperature ( Figure 6 ).

[0087] 2.5 Phage pH stability

[0088] Escherichia coli phage PE004 culture (titer 1.4×10 12 PFU / mL) and Salmonella phage PS006 culture (titer 1.7×10 12 PFU / mL) were mixed with sterile LB liquid culture medium with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and cultured overnight in a 37°C water bath. After the samples were appropriately diluted with SM buffer, the titer of the phage was determined by double-layer agar plate culture method. Each group of experiments was repeated 3 times.

[0089] Test results:

[0090] The total titer of E. coli phage PE004 in the environment with pH value of 1-10 remained almost unchanged at 1.2×10 12 PFU / mL, indicating that the phage has a wide range of adaptability to pH ( Figure 7 ).

[0091] The titer of Salmonella phage PS006 remained basically unchanged at pH 1 and pH 2, but decreased slightly; the titer decreased by one order of magnitude at pH 3 and pH 4; and the titer decreased by two orders of magnitude at pH 5 to pH 10, indicating that Salmonella phage PS006 has strong pH adaptability and that the phage activity is better in an acidic environment than in an alkaline environment ( Figure 8 ).

[0092] 2.6 Phage sensitivity to UV light

[0093] The phage culture was placed in a sterile culture dish with a diameter of 90 mm on a sterilized clean bench (40W, DL-CJ-2ND), and the phage was 20 cm away from the UV lamp. After turning on the UV lamp, 100 μL of phage was taken at 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, and 8 h, and after SM gradient dilution, it was mixed with the host bacteria cultured to the logarithmic growth phase, and the titer was determined by the double-layer plate method. Each group of experiments was repeated 3 times.

[0094] Test results:

[0095] The titer is 4.68×10 11The titer of E. coli phage PE004 (PFU / mL) remained unchanged or decreased in the first 30 minutes of UV treatment; within 1 to 5 hours of UV treatment, the titer decreased by 1 order of magnitude; after 8 hours of UV treatment, the titer of the phage decreased by 2 orders of magnitude, indicating that E. coli phage PE004 has a strong tolerance to UV and still has a strong ability to break bacteria after 8 hours of UV treatment ( Fig. 9 ).

[0096] The titer is 2.6×10 12 The titer of Salmonella phage PS006 in PFU / mL decreased by one order of magnitude within the first 2 hours of UV treatment; after 8 hours of UV treatment, the titer of the phage decreased by 2 orders of magnitude, indicating that the phage has a strong tolerance to UV ( Fig.10 ).

[0097] 2.7 One-step growth curve drawing of bacteriophage

[0098] The host bacteria cultured to the logarithmic growth phase were mixed with the phage according to the optimal infection multiplicity (MOI) ratio, incubated at 37°C for 5 minutes, and centrifuged at 4°C, 10,000×g for 1 minute to remove the unadsorbed phage particles. The precipitate was suspended with fresh LB liquid culture medium (preheated to 37°C) and cultured at 37°C with shaking. This time was recorded as T0. Samples (100μL) were taken every 5 minutes for the first 15 minutes, and then every 15 minutes. After gradient dilution, the titer of the phage was calculated by the double-layer plate culture method, and the one-step growth curve of the phage infection host bacteria was drawn (with the infection time as the horizontal axis and the phage titer in the infection system as the vertical axis). The latent period (the period from the adsorption of phage to bacteria to the release of progeny phage particles), the outbreak period (bacteria are lysed, and a large number of progeny phage particles are formed and released) and the lysis amount (lysis amount = phage titer in the stable period after the outbreak / initial concentration of host bacteria) during the phage infection of bacteria were calculated based on the one-step growth curve.

[0099] Test results:

[0100] The titer of E. coli phage PE004 did not change within the first 10 minutes of infection, so the incubation period of E. coli phage PE004 was 10 minutes. The titer of the phage increased rapidly within 10-90 minutes after infecting the host, and basically became stable after 90 minutes, reaching 2.12×10 14 PFU / mL, the outbreak period is 80min, and the burst volume of E. coli phage PE004 is 122. This shows that the incubation time of phage PE004 is very short and the burst volume is large ( Fig.11 ).

[0101] The phage titer of Salmonella phage PS006 did not change within the initial 15 minutes of infection, indicating that the incubation period of Salmonella phage PS006 was 15 minutes. The phage titer increased rapidly within 15-80 minutes after infecting the host. After 80 minutes, the phage titer basically became stable and reached 4.95×10 11 PFU / mL, the outbreak period was 60min, and the outbreak volume of Salmonella phage PS006 was 185. This indicates that phage PS006 has a short incubation time and a large outbreak volume ( Fig.12 ).

[0102] Example 2

[0103] Phage lysis rate determination

[0104] 42 strains of Escherichia coli from cashmere goats were selected as the lysis test subjects of Escherichia coli phage PE004. Escherichia coli phage PE004 was mixed with different Escherichia coli in test tubes containing LB liquid culture medium and cultured for 8-24 hours. The turbidity changes of the bacterial solution and the phage mixture in the test tube were observed, and the test tube with only Escherichia coli added but not phage PE004 was used as a blank control. At the same time, the lysis spectrum of Escherichia coli phage PE004 provided in the present application on Escherichia coli was determined in combination with the double-layer plate method.

[0105] Test results: E. coli phage PE004 can lyse 36 of 42 strains of E. coli, with a lysis rate of 85.71%. The mixture of phage and E. coli gradually becomes clear after several hours of culture, or transparent plaques appear after double-layer agar plate culture, while the control group is still a turbid solution ( Fig.13 ), indicating that the phage has a good lysis ability against Escherichia coli from cashmere goats.

[0106] 37 strains of Salmonella from cashmere goats were selected as the lysis test subjects of Salmonella phage PS006. Salmonella phage PS006 was mixed with different Salmonella in test tubes containing LB liquid culture medium and cultured for 8-24 hours, and the turbidity changes of the bacterial solution and phage mixed solution in the test tube were observed. The test tube with only Salmonella phage added but not phage was used as a blank control. At the same time, the lysis spectrum of Salmonella phage PS006 provided in this application on Salmonella was determined in combination with the double-layer plate method.

[0107] Results: Salmonella phages could lyse 29 of 37 strains of Salmonella, with a lysis rate of 78.38%. The mixture of phages and Salmonella gradually became clear after several hours of culture, or transparent plaques appeared after double-layer agar plate culture, while the control group was still a turbid solution ( Fig.14 ), indicating that the phage has a good lysis ability against Salmonella from cashmere goats.

[0108] Table 3 Lysis spectrum of bacteriophage PE004 against 42 strains of Escherichia coli from cashmere goats

[0109] Strain number Whether to crack Strain number Whether to crack Strain number Whether to crack E01 + E15 + E29 + E02 + E16 - E30 + E03 - E17 + E31 + E04 + E18 + E32 + E05 + E19 + E33 - E06 + E20 + E34 + E07 - E21 - E35 + E08 + E22 + E36 + E09 + E23 + E37 + E10 + E24 + E38 + E11 + E25 + E39 - E12 + E26 + E40 + E13 + E27 + E41 + E14 + E28 + E42 +

[0110] Note: +: lysis, clear after in vitro lysis, or transparent plaques after double-layer plate culture; -: unlysis.

[0111] Table 4 Lysis spectrum of bacteriophage PS006 against 37 strains of Salmonella from cashmere goats

[0112]

[0113]

[0114] Note: ++: lysis, the solution becomes clear after in vitro lysis, and transparent plaques are observed after double-layer plate culture; +: lysis, and transparent plaques are observed after double-layer plate culture; -: no lysis.

[0115] Example 3 Safety test of bacteriophage

[0116] Twenty-four female BALB / c mice were randomly divided into three groups, namely, two phage groups and one control group, with 8 mice in each group. The phage group was inoculated with Escherichia coli phage and Salmonella phage by intraperitoneal injection, and the control group was injected with an equal amount of saline by intraperitoneal injection; the phage content in the cecum of the mice was detected from the 1st to the 10th day after treatment, and the changes in the mouse organs were observed by autopsy.

[0117] Test results: There was no effect on the daily activities and health of mice after intraperitoneal inoculation of phages. As the number of days of phage inoculation increased, the phage titer in the cecum of the mice in the test group continued to decrease. After the fourth day of treatment, the phage titer of the cecal contents became 0, that is, no phages could be detected in the mice at this time (Table 5). This shows that as time goes by, mice are able to excrete phages from the body without causing any residue in the body. In addition, the daily performance of mice in the phage group and the control group was observed, and it was found that the mental state, feeding, and drinking water of the mice in the phage group were normal. After dissection, the heart, liver, spleen, kidney, lung, small intestine, and cecum of the mice were examined, and no abnormalities or adverse symptoms such as bleeding spots were found. Based on the above test results, it can be seen that Escherichia coli phage and Salmonella phage are safe and non-toxic to mice.

[0118] Table 5 Changes of phage titer in mouse cecal contents over time

[0119]

[0120]

[0121] Example 4 Experiment on the prevention of diarrhea in lambs by bacteriophage

[0122] A cashmere goat farm was selected for the experiment. The lambs in the farm had diarrhea many times before and after weaning, and there was a phenomenon that the lambs died. 240 lambs were divided into 8 groups, 30 in each group, and divided into 6 experimental groups and 2 control groups. Within the first 7 days of the experiment, 1×10 8 PFU / mL of Escherichia coli phage, 1×10 8 PFU / mL of Salmonella phage, 1×10 8 PFU / mL of cocktail preparations of Escherichia coli phage and Salmonella phage. After that, the same concentration of phage preparations were added to the water tank of the sheep house 1-2 times a week; the control group was not treated. The experiment lasted for 30 days, and the number of lambs with diarrhea and death during the experiment was recorded.

[0123] The preparation method of the cocktail mixture of Escherichia coli phage PE004 and Salmonella phage PS006 is as follows: phage PE004 and phage PS0062 are mixed in a volume ratio of 1:1.

[0124] Experimental results: During the 30-day experimental period, 8 and 9 lambs in the two control groups had diarrhea, and 1 and 1 died; 4 and 5 lambs in experimental group 1 and experimental group 2 had diarrhea, and 0 and 1 died; 5 and 6 lambs in experimental group 3 and experimental group 4 had diarrhea, and 0 and 1 died; 3 and 2 lambs in experimental group 5 and experimental group 6 had diarrhea, and no death. Compared with the control group, the average mortality rate of the experimental group decreased by nearly 1.22% (from 2.33% to 1.11%), and the average diarrhea rate decreased by nearly 14.44% (from 28.33% to 13.89%). The use of Escherichia coli phage and Salmonella phage alone can significantly reduce the number of diarrhea and deaths in cashmere goat lambs, and the mixed preparation of Escherichia coli phage and Salmonella phage cocktail is more effective than using them alone.

[0125] Table 4 The results of bacteriophage prevention on lamb diarrhea

[0126]

[0127]

[0128] Example 5 Experiment on bacteriophage treatment of lamb diarrhea

[0129] In a cashmere goat farm, 30-day-old lambs developed diarrhea, with body temperature of 41-42°C, lethargy, reduced food intake, bubbles in feces and watery stools. The phage cocktail preparation 1×10 8 PFU / mL drinking water for 7 days, combined with intramuscular injection of phage cocktail preparation 1×10 6 PFU / mL, and the number of lambs with diarrhea and death was recorded.

[0130] Test results: Before using the phage cocktail, 11 lambs had diarrhea and 1 died. Compared with not using the phage cocktail, after using the phage cocktail, 3 lambs had diarrhea and 0 died. The water feces in the flock decreased. Through continuous observation, diarrhea did not reappear, the feed intake was normal, the spirit of the flock improved significantly, the feces was normal, the fur was white and shiny, and the lambs were lively and active. The results show that the use of phage cocktail can reduce the mortality rate of lambs.

[0131] Example 6 Phage disinfection test on cashmere goat breeding farm environment

[0132] In a large-scale cashmere goat farm, lambs had diarrhea and lamb deaths. The cashmere goats were divided into two groups, an experimental group and a control group, and the experimental group and the control group were kept in different sheep sheds. For the lambs in the experimental group, 1×10 6 PFU / mL phage cocktail mixed preparations were sprayed for disinfection once a week, and the phage cocktail mixed preparations were used to immerse the feed at the same time; the sheep sheds in the control group were not treated, and the number of lambs with diarrhea within 3 months was observed and recorded. Experimental results: In the sheep sheds of the experimental group treated with phage cocktail mixed preparations for environmental disinfection, 15.13% (18 / 119) of the lambs had diarrhea in the first month and a half, and no sheep died of diarrhea at the end of the 3-month experiment; while the overall proportion of lambs with diarrhea in the sheep sheds in the control group within 3 months was 26.80% (26 / 97), and the mortality rate was 2.06 (2 / 97).

[0133] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Escherichia coli phage (Myoviridae phage) PE004 from white cashmere goats in northern Shaanxi, characterized in that: The phage was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 24, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45967.

2. A Salmonella phage (Siphophagesphage) PS006 from white cashmere goats in northern Shaanxi, characterized by: The phage was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 24, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45968.

3. A microbial preparation, characterized in that: It includes the Escherichia coli phage PE004 derived from the northern Shaanxi white cashmere goats as described in claim 1 and / or the Salmonella phage PS006 derived from the northern Shaanxi white cashmere goats as described in claim 2.

4. Use of the Escherichia coli phage PE004 from white cashmere goats in northern Shaanxi as claimed in claim 1, the Salmonella phage PS006 from white cashmere goats in northern Shaanxi as claimed in claim 2, or the microbial preparation as claimed in claim 3 in the preparation of a drug for treating and / or preventing lamb diarrhea.

5. A medicine for treating and / or preventing lamb diarrhea, characterized in that: It includes the Escherichia coli phage PE004 derived from the northern Shaanxi white-cashmere goats as described in claim 1, the Salmonella phage PS006 derived from the northern Shaanxi white-cashmere goats as described in claim 2, or the microbial preparation as described in claim 3.

6. The drug according to claim 5, characterized in that The administration methods of the drug include oral administration and injection; the effective dose of bacteriophage in the oral drug is 1×10 8 PFU / mL; the effective dose of phage in the injection drug is 1×10 6 PFU / mL.

7. Use of the Escherichia coli phage PE004 from white cashmere goats in northern Shaanxi as claimed in claim 1, the Salmonella phage PS006 from white cashmere goats in northern Shaanxi as claimed in claim 2, or the microbial preparation as claimed in claim 3 in the preparation of a fungicide.

8. A bactericide or bacteriostatic agent, characterized in that: It includes the Escherichia coli phage PE004 derived from the northern Shaanxi white-cashmere goats as described in claim 1, the Salmonella phage PS006 derived from the northern Shaanxi white-cashmere goats as described in claim 2, or the microbial preparation as described in claim 3.

9. The fungicide according to claim 8, characterized in that The effective dose of bacteriophage in the bactericide is 1×10 6 PFU / mL.

10. A feed additive, characterized in that: It includes the Escherichia coli phage PE004 derived from the northern Shaanxi white-cashmere goats as described in claim 1, the Salmonella phage PS006 derived from the northern Shaanxi white-cashmere goats as described in claim 2, or the microbial preparation as described in claim 3.

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