Salmonella bacteriophage PC192, and bacteriophage composition and application thereof

Abstract

The invention discloses a salmonella bacteriophage PC192, a bacteriophage composition and application of the salmonella bacteriophage PC192. The salmonella bacteriophage PC192 for preventing and controlling avian bacterial diarrhea has the preservation number of CGMCC No.22367. The salmonella bacteriophage PC192 is preserved in China General Microbiological Culture Collection Center on April 12, 2021, and the preservation number of the salmonella bacteriophage PC192 is CGMCC No.22367. The invention also discloses a bacteriophage composition formed by assembling the salmonella bacteriophage PC192, the escherichia coli bacteriophage PD205 and the clostridium welchii bacteriophage PM11, the salmonella bacteriophage PC192 and the bacteriophage composition can be used as active ingredients for effectively preventing and treating the poultry bacterial diarrhea, and can also be used as an environment disinfectant, so that the pollution of a breeding environment is effectively controlled, and the breeding cost is reduced. Compared with a single lytic bacteriophage, the bacteriophage has the characteristic of wide lysis spectrum, widens the bactericidal range and improves the bactericidal activity.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Salmonella phage PC192, a phage composition thereof and application thereof. Background Art

[0002] With the rapid development of intensive poultry farming, bacterial diseases have increasingly become the primary diseases that endanger the development of the poultry industry. Bacterial diarrhea is an important bacterial disease that endangers the poultry industry. It not only affects the egg production of laying poultry and the weight gain of meat poultry, but can also lead to the death of poultry flocks, causing serious economic losses to the breeding industry. Bacterial diseases that cause diarrhea in poultry include avian Escherichia coli disease, avian salmonellosis, and avian Clostridium perfringens disease.

[0003] Avian salmonellosis is a general term for acute or chronic diseases of poultry caused by a species of Salmonella in the genus Salmonella, which are divided into pullorum, fowl typhoid and fowl paratyphoid. Chickens are the largest host of poultry Salmonella, and the mortality rate of chickens can reach up to 80%; the most common of these is pullorum, which is an acute, septicemic infectious disease in chicks. The morbidity and mortality rates of chicks under 2 weeks of age are very high, and adult chickens infected with Salmonella often show chronic symptoms. However, these non-typhoidal Salmonella that colonize in the intestines of poultry often use eggs and poultry products as the main transmission medium, and are the primary pathogens that cause foodborne diseases in humans. When poultry are infected with Salmonella, not only will their production performance be reduced, but they may even die or be carried into eggs, increasing production costs for the breeding industry, etc.

[0004] Avian colibacillosis is a general term for different types of poultry diseases caused by certain pathogenic or conditionally pathogenic Escherichia coli. It is one of the most serious bacterial diseases that endanger the poultry industry. The disease can infect many types of poultry, such as chickens, ducks, and turkeys of all ages, but chicks are more susceptible than adult chickens, and broilers are more susceptible than laying hens. The respiratory tract, digestive tract, and mating can all be routes of transmission of the disease, but the digestive tract is the main route. The clinical symptoms of affected poultry mainly include loss of appetite, depression, ruffled feathers, swaying gait, lameness, and weight loss. When the digestive tract is affected, symptoms such as diarrhea or green feces will occur.

[0005] Clostridium perfringens disease, also known as Clostridium perfringens disease, is a common acute and fatal diarrheal disease with high morbidity and mortality, causing serious economic losses. According to statistics, the economic losses caused by Clostridium perfringens worldwide are about US$2 billion per year. Clostridium perfringens is widely distributed in nature, growing in soil, sewage, food, and feces. It can form a sandwich in the body of animals and has strong resistance. Poultry infected with Clostridium perfringens type A and C are susceptible to avian necrotic enteritis. Clinical symptoms include depression, closed eyes, decreased feed intake, diarrhea, decreased weight gain rate, and even reddish-brown feces or dark brown tar-like feces, which seriously affect the growth and health of animals and cause great economic losses to farmers.

[0006] The abuse of antibiotics in clinical practice has led to serious bacterial resistance, making it more difficult to prevent and treat poultry bacterial diarrhea with antibiotics. In addition, according to the implementation of my country's policy of prohibiting the addition of antibiotics to feed additives, poultry diarrhea has become increasingly serious. Bacteriophages are a new type of safe and residue-free antibacterial biological agent that has the same killing effect on drug-resistant bacteria and has a good application prospect.

[0007] However, at present, there are few and single Clostridium perfringens phages that can effectively prevent and treat Clostridium perfringens disease, which greatly restricts its scope of application. It is necessary to continue to develop Clostridium perfringens phages with fast reproduction speed and wide lysis spectrum; at the same time, it is also necessary to continue to develop phage compositions with a wider application range, wider lysis spectrum, and the use of the complementary advantages of each phage, so as to better prevent and treat poultry bacterial diarrhea diseases caused by a variety of different bacterial pathogens. Summary of the invention

[0008] In view of the above problems, the present invention provides a Salmonella phage PC192 and a phage composition including Salmonella phage PC192 with a wide lysis spectrum and strong lysis effect, which is established based on a cocktail phage control method. The Salmonella phage PC192 can efficiently lyse Salmonella, and the phage composition can effectively prevent and control diarrhea caused by various pathogenic bacteria such as avian Escherichia coli, Salmonella, and Clostridium perfringens; at the same time, the Salmonella phage PC192 and its phage composition can also be used to prepare pharmaceutical preparations, poultry feed additives, environmental disinfectants, kits, etc.

[0009] In order to solve the above problems, the technical solution of the present invention is as follows:

[0010] In the first aspect, the present invention provides a Salmonella phage PC192 for preventing and controlling poultry bacterial diarrhea, which has a deposit number of CGMCC No. 22367 and was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on April 12, 2021. The morphology of phage PC192 was observed under an electron microscope. The phage has a polyhedral head structure and a retractable tail. The head is 60-67nm wide and 60-67nm long, and the tail is 167-200nm long. It is a long-tailed phage.

[0011] The present invention also provides a Clostridium perfringens phage PM11 for preventing and controlling poultry bacterial diarrhea, with a deposit number of CGMCC No. 22372. The phage was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on April 12, 2021. Under an electron microscope, the morphology of the phage PM11 is observed to be a polyhedral structure with a head diameter of about 44nm and 6 short tail filaments, belonging to a short-tailed phage.

[0012] The present invention also provides an Escherichia coli phage PD205 for preventing and controlling bacterial diarrhea in poultry, with a deposit number of CGMCC No. 22361, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on April 12, 2021. The morphology of phage PD205 observed under an electron microscope is as follows: it has a polyhedral head structure and a retractable tail, the head is 80-87nm wide and 113-120nm long, and the tail is 107-113nm long, belonging to a muscle-tailed phage.

[0013] The whole genome analysis of phages showed that the whole genome sizes of phages PD205, PC192 and PM11 were 168652bp, 39095bp and 109054bp, respectively. The G+C content of phage PD205 genome was 37.60%, with 269 open reading frames (ORFs), the G+C content of phage PC192 genome was 48.29%, with 47 open reading frames (ORFs), and the G+C content of phage PM11 genome was 39.09%, with 183 open reading frames (ORFs). Phages PD205, PC192 and PM11 did not contain drug resistance genes, virulence genes, or lysogeny-related genes.

[0014] In a second aspect, the present invention provides a phage composition, comprising the aforementioned Salmonella phage PC192 and other phages.

[0015] Optionally, the phage composition comprises: the Salmonella phage PC192 and the Clostridium perfringens phage PM11 with a preservation number of CGMCC No.22372.

[0016] In a third aspect, the present invention provides a phage composition for preventing and controlling poultry bacterial diarrhea, comprising the Escherichia coli phage PD205, the Salmonella phage PC192 and the Clostridium perfringens phage PM11 as described above.

[0017] Optionally, the ratio of the number of live Escherichia coli phages to Salmonella phages to Clostridium perfringens phages is 1:1:1; the content of each phage is not less than 1×10 8 PFU / mL.

[0018] In the present application, Clostridium perfringens phage PM11 includes mutants with the same bactericidal activity of more than 98% or 99% homology after point mutation, deletion mutation or addition mutation. Since phages are very prone to mutation during replication, mutants of the above phages are also within the scope of protection requested in the present application. The sequence of PM11 can be obtained by sequencing the biological materials preserved according to the present invention by known methods. For those skilled in the art, it is not necessary to pay creative labor to screen out mutants with extremely similar properties according to the phage provided by the present invention. Escherichia coli phage PD205 and Salmonella phage PC192 also include mutants with the same bactericidal activity of more than 98% or 99% homology after point mutation, deletion mutation or addition mutation, respectively, which are explained in the same way as Clostridium perfringens phage PM11.

[0019] In a fourth aspect, the present invention also provides the use of the Salmonella phage PC192 or the above phage composition in the preparation of drugs for preventing and treating poultry bacterial diarrhea, environmental disinfectants, and feed additives. The term "prevention" herein refers to all actions including inhibiting or delaying the disease by administering the phage. The term "treatment" herein refers to all actions including improving or ameliorating the disease by administering the phage.

[0020] The poultry bacterial diarrhea includes salmonellosis caused by Salmonella or poultry diarrhea caused by co-infection of Salmonella and other pathogens (such as Escherichia coli and Clostridium perfringens). The poultry bacterial diarrhea includes poultry necrotic enteritis, diarrhea caused by Salmonella, enteritis caused by Escherichia coli infection or other types of enteritis caused by mixed infection.

[0021] In addition, the present invention also provides the use of the Escherichia coli phage PD205 or a composition containing the phage in preparing a drug for preventing and treating avian bacterial diarrhea and an environmental disinfectant.

[0022] The present invention also provides the use of the Salmonella phage PC192 or a phage composition containing the phage in preparing a medicine for preventing and treating avian bacterial diarrhea and an environmental disinfectant.

[0023] In a fifth aspect, the present invention provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Salmonella phage PC192 or the aforementioned phage composition; preferably, the phage composition includes Escherichia coli phage PD205, Salmonella phage PC192 and Clostridium perfringens phage PM11.

[0024] The bacteriophage drug preparation further includes excipients; the excipients are one or more of SM buffer, sodium alginate, sucrose, maltodextrin, and glucose. Preferably, the bacteriophage drug preparation further includes bacteriophages of specific pathogens of different types of bacteria.

[0025] In the phage pharmaceutical preparations provided in other embodiments, the active ingredient includes the aforementioned Escherichia coli phage PD205 or a composition containing the phage; or the active ingredient includes the Salmonella phage PC192 or a phage composition containing the phage.

[0026] Specifically, the phage pharmaceutical preparation is in the form of powder, liquid, lyophilized agent, capsule, aerosol, pill or tablet.

[0027] The application method of the drug preparation is: adding the drug preparation to the drinking water of poultry suffering from bacterial diarrhea, or administering it to the poultry through oral administration, intramuscular injection, or adding it to feed, wherein the drinking water method is preferably used to prevent and treat poultry bacterial diarrhea, reduce poultry diarrhea rate, increase poultry survival rate, increase egg production rate and feed-to-meat ratio, etc. The poultry includes chickens, ducks, geese, etc.

[0028] Experimental results show that the use of 5×10 9 The phage composition with a concentration of PFU / mL is mixed with drinking water and fed for 3 consecutive days. The diarrhea rate of sick chicks can be effectively reduced by about 90% within 14 days.

[0029] In a sixth aspect, the present invention further provides an environmental disinfectant, the active ingredient of which includes Salmonella phage PC192 or the aforementioned phage composition (a combination of two or three strains of Clostridium perfringens phage PM11, Escherichia coli phage PD205 and Salmonella phage PC192).

[0030] Preferably, the active ingredients of the environmental disinfectant include three phages: Clostridium perfringens phage PM11, Escherichia coli phage PD205, and Salmonella phage PC192; further preferably, the concentration of each phage is 5×10 8 Preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in the environment to improve its sterilization or antibacterial effect; it may also include other adjuvants, such as adjuvants that can prolong the duration of bacteriophage.

[0031] The application method of the above disinfectant is: use the disinfectant in duck slaughterhouses, duck product processing workshops and equipment, and duck breeding environments to prevent contamination by Salmonella phages or mixed contamination by Salmonella, Clostridium perfringens, and Escherichia coli in the environment. The breeding environment includes sheds, feed troughs, floors, walls, feces, and bedding, etc. Including but not limited to liquid immersion, spraying, and combined use with aqueous carriers to disinfect and decontaminate water distribution systems, breeding facilities, feeding equipment, or other environmental surfaces, and disinfect and preserve feed. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it will not cause harm to humans and poultry.

[0032] In a seventh aspect, the present invention further provides a feed additive for preventing bacterial diarrhea in poultry, wherein the active ingredient of the feed additive comprises: Salmonella phage PC192 or the phage composition.

[0033] Preferably, the effective ingredients of the feed additive include three strains of bacteriophages: Clostridium perfringens phage PM11, Escherichia coli phage PD205 and Salmonella phage PC192.

[0034] The bacteriophage composition and application thereof provided by the present invention have the following beneficial effects:

[0035] 1. The present invention provides three strains of self-isolated Clostridium perfringens phages, Salmonella phages PC192, PM11 and Escherichia coli phages PD205 with excellent lysis performance, and provides a phage composition for preventing and controlling poultry bacterial diarrhea based on the compounding of the three phages. The composition uses Escherichia coli phages PD205, Salmonella phages PC192 and Clostridium perfringens phages PM11 as active ingredients for effectively preventing and treating poultry bacterial diarrhea, and can also be used as an environmental disinfectant to effectively control the pollution of the breeding environment; compared with a single lytic phage, the composition has the characteristics of a wide lysis spectrum, a widened bactericidal range, and improved bactericidal activity, and can effectively prevent and control diarrhea caused by Escherichia coli, Salmonella and Clostridium perfringens in poultry; at the same time, it can improve the egg production rate of laying hens, improve the feed-to-meat ratio of broilers, enhance the immunity of chickens, and reduce the mortality rate caused by bacterial diarrhea.

[0036] 2. The phage compositions involved in the present invention are all obtained from nature, with a wide source, a large number, easy to separate, and belong to natural organisms. The chick safety test proves that it is safe, has no residue, has no toxic side effects, and will not affect the normal flora in the chicks. Therefore, the phage composition with lytic Escherichia coli phage, Salmonella phage and Clostridium perfringens phage as the main components has the advantages of high specificity, small adverse reactions, not easy to produce drug resistance, no residue and low cost, and provides a new green biological preparation for the prevention and control of avian bacterial diarrhea.

[0037] 3. The bacteriophage composition designed by the present invention has a wide range of applications. It can not only be used in the breeding process, but can also specifically, efficiently, quickly and safely kill diarrhea caused by pathogenic Escherichia coli, Salmonella, Clostridium perfringens and even drug-resistant strains, effectively reducing the diarrhea rate of chicks and the mortality rate of diseased chickens; it can also be used as an environmental disinfectant to prevent and control the spread and proliferation of Escherichia coli, Salmonella and Clostridium perfringens; it can also be used as a biological antibacterial agent for the disinfection of poultry products, inhibiting the growth of pathogenic strains and even drug-resistant strains during product processing and preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a plaque image of Escherichia coli phage PD205;

[0039] Figure 2 This is a plaque image of Salmonella phage PC192;

[0040] Figure 3 This is a plaque image of Clostridium perfringens phage PM11;

[0041] Figure 4 This is an electron microscope image of Escherichia coli phage PD205;

[0042] Figure 5 This is an electron microscope image of Salmonella phage PC192;

[0043] Figure 6 This is an electron microscope image of Clostridium perfringens phage PM11;

[0044] Figure 7 is the thermal stability of Escherichia coli phage PD205;

[0045] Figure 8 The thermal stability of Salmonella phage PC192;

[0046] Fig. 9 is the thermal stability of Clostridium perfringens phage PM11;

[0047] Fig.10 is the pH stability of E. coli phage PD205;

[0048] Fig.11 is the pH stability of Salmonella phage PC192;

[0049] Fig.12 is the pH stability of Clostridium perfringens bacteriophage PM11;

[0050] Fig.13 is the one-step growth curve of Escherichia coli phage PD205;

[0051] Fig.14is the one-step growth curve of Salmonella phage PC192;

[0052] Fig.15 is the one-step growth curve of Clostridium perfringens phage PM11;

[0053] Fig.16 This is a diagram showing the effect of the bacteriophage composition on the treatment of poultry diarrhea;

[0054] Fig.17 This is a diagram showing the effect of the bacteriophage composition in preventing poultry diarrhea;

[0055] Fig.18 Effect of bacteriophage compositions on environmental disinfection. DETAILED DESCRIPTION

[0056] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0057] Example 1 Isolation and Preparation of Bacteriophage

[0058] 1. Sample collection

[0059] The samples of liquid feces, sewage, chicken intestines, etc. in the present invention were collected from multiple chicken farms in Shandong Province.

[0060] 2. Resuscitation culture of host bacteria and preparation of proliferation fluid

[0061] The host bacteria were Escherichia coli, Salmonella and Clostridium perfringens isolated, identified and preserved in our laboratory, and isolated from the intestines or livers of sick chickens from a chicken farm in Shandong Province.

[0062] (1) Use a sterilized inoculation loop to dip the stored E. coli and Salmonella liquid, streak inoculate on SS agar medium, and culture in a constant temperature incubator at 37°C for 16 to 24 hours to obtain a single colony. Pick a single colony and inoculate it into a 5 mL NB broth test tube. Culture at 37°C, 170 rpm / min for 12 to 16 hours to obtain fresh E. coli and Salmonella proliferation liquid, which can be temporarily stored at 4°C for later use.

[0063] (2) Use a sterilized inoculation loop to dip into the preserved Clostridium weiheni host bacteria liquid, streak inoculate on 1% glucose-5% sheep blood medium, and culture anaerobically in a 37°C incubator for 16 to 24 hours to obtain a single colony. Pick a single colony and inoculate it into a test tube containing 5 mL of TSB liquid culture medium. Culture anaerobically at 37°C, 170 rpm / min, and culture anaerobically for 12 to 16 hours to obtain a fresh Clostridium weiheni proliferation liquid, which is temporarily stored at 4°C for later use.

[0064] 3. Isolation of bacteriophage

[0065] (1) Separation of Escherichia coli phage and Salmonella phage

[0066] Take an appropriate amount of fecal water and put it into a bubble sample bottle, add 200 μL of Escherichia coli and Salmonella proliferation liquid each, mix them together, then add an appropriate amount of NB broth, culture at 37°C and 170 rpm on a shaker for 16 to 18 hours to proliferate and enrich the phages to obtain a culture solution; take 5 ml of the above culture solution, centrifuge at 11000 rpm for 10 minutes, take the supernatant and filter it with a 0.22 μm filter membrane for sterilization to obtain the phage stock solution. The double-layer plate method was used to separate the phage: 100 μL of the phage stock solution was diluted 10 times, 120 μL of the appropriate gradient dilution solution was mixed with 120 μL of the host bacteria proliferation solution, incubated at 37°C for 5 minutes, and then the upper agar (agar concentration of 0.7%) at about 45°C was added. After mixing, the lower agar (agar concentration of 1.5%) was quickly poured onto the plate, and the plate was shaken and placed flat until the culture medium solidified. After inverted culture in a 37°C incubator for 6 to 8 hours, a double-layer plate with plaque formation was obtained. By using different host bacteria (E. coli or Salmonella), two phages were finally isolated and obtained, which were named E. coli phage PD205 and Salmonella phage PC192, respectively.

[0067] (2) Isolation of Clostridium perfringens phage

[0068] Take an appropriate amount of chicken farm manure sewage or chicken intestinal tissue, add 500μL of Clostridium perfringens liquid, then add TSB medium to 200mL, and culture anaerobically at 37℃ for 16h~18h to enrich bacteriophages. The next day, take out 5-8mL of liquid, centrifuge at 11000rpm for 10min, and filter the supernatant through a 0.22μm sterile microporous filter membrane to obtain the bacteriophage stock solution. The phage was isolated by the double-layer plate method. The phage stock solution was diluted 10 times, and 120 μL of the dilution was taken and mixed with 120 μL of the pathogenic bacteria proliferation solution. After incubation at 37°C for 5 minutes, it was placed on a TSA upper agar plate (agar concentration of 0.7%) at about 45°C. After mixing, it was quickly poured onto a TSA lower agar plate (agar concentration of 1.5%), shaken and placed flat until the culture medium solidified, and placed at 37°C for anaerobic culture for 3 to 6 hours to obtain a double-layer plate with plaques. The obtained Clostridium perfringens phage was named PM11.

[0069] 4. Purification and proliferation of bacteriophage

[0070] (1) Purification and proliferation of Escherichia coli phage PD205 and Salmonella phage PC192

[0071] Using the double-layer plate method, pick a single, smooth-edged and translucent plaque from the double-layer plate where plaques are formed, place it in 1 ml NB broth, and shake it at 37°C and 170 rpm for 30 minutes to obtain a phage extract; centrifuge at 11000 rpm for 5 minutes, take 120 μL of the centrifuged phage extract and mix it thoroughly with 120 μL of the host bacteria proliferation liquid, incubate at 37°C for 5 minutes, add it to the upper agar at about 45°C, mix it well, and quickly pour it onto the plate containing the lower agar culture medium. After the upper agar solidifies, invert it and culture it in a 37°C constant temperature box for 6 to 8 hours. Repeat the above steps 3-5 times to purify the phage repeatedly until plaques of uniform size, morphology and distribution are obtained. From the purified plaque double-layer plate, a single plaque was inoculated into 1 ml NB broth, cultured at 37°C and 170 rpm for 30 min, centrifuged at 11000 rpm for 5 min, 100 μL of supernatant and 100 μL of corresponding host bacteria proliferation liquid were added to 5 ml broth, cultured at 37°C and 170 rpm / min, shaken until the mixed liquid became clear, and then centrifuged to obtain phage proliferation liquid. The proliferation liquids of Escherichia coli phage PD205 and Salmonella phage PC192 were prepared according to the above method.

[0072] Escherichia coli phage PD205 can form clear plaques on double-layer agar plates, with no halo around them, clear and regular edges, and a diameter of about 1 mm (see Figure 1 ).

[0073] Salmonella phage PC192 can form medium and clear plaques on double-layer agar plates, surrounded by a halo, with regular edges and a diameter of about 2 mm (see Figure 2 ).

[0074] (2) Purification and proliferation of Clostridium perfringens phage PM11

[0075] The double-layer plate method is used to purify the phage. A single plaque is picked from the above double-layer culture medium where the plaque is formed, and it is placed in 1 mL of TSB broth, and shaken at 37°C and 170 rpm for 1 hour to obtain a phage extract. 220 μL of the phage extract is mixed evenly with 220 μL of the corresponding above Clostridium perfringens proliferation liquid, and incubated at 37°C for 5 minutes. The incubated mixture is added to a TSA upper agar (agar concentration of 0.7%) at about 45°C, and after mixing, it is quickly poured onto a TSA lower agar (agar concentration of 1.5%) plate, and the plate is placed at 37°C for inverted anaerobic culture for 6 to 12 hours, and a double-layer plate with plaques is obtained again. Repeat the above steps 3-5 times to purify the phage repeatedly until plaques of uniform size, morphology, and distribution are obtained. Use sterile tweezers to pick up a single plaque from the purified plaque double-layer culture medium and place it in 1 mL of TSB broth. Shake at 170 rpm for 1 hour to obtain a phage extract. Centrifuge at 11000 rpm for 5 minutes. Take 200 μL of the supernatant and 200 μL of the host bacteria proliferation liquid in 5 mL of liquid TSB medium. Culture anaerobically at 37°C and 170 rpm until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 minutes, take the supernatant, and filter it using a 0.22 μm sterile microporous filter membrane to obtain the phage proliferation liquid.

[0076] The Clostridium perfringens phage PM11 can form clear plaques on double-layer agar plates, with no halo around them, clear and regular edges, and a diameter of about 2 mm (see Figure 3 ).

[0077] Example 2 Morphological characteristics of bacteriophage

[0078] 1. The morphological observation method is:

[0079] Take 20 μL of phage sample and drop it on a copper mesh with a carbon-coated film. Wait for it to precipitate naturally for 15 minutes. After blotting it with filter paper, stain it with 2% (W / V) phosphotungstic acid (PTA) for 1-2 minutes. After blotting it with filter paper, observe and take pictures with a transmission electron microscope.

[0080] 2. Observation results:

[0081] like Figure 4As shown, the head of Escherichia coli phage PD205 is a regular icosahedron, with a width of 80-87nm, a length of 113-120nm, and a tail length of 107-113nm. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of phage PD205 conforms to the characteristics of the Myotailed Bacteriophage family and belongs to the Myotailed Bacteriophage family.

[0082] like Figure 5 As shown, the head of Salmonella phage PC192 is a polyhedral structure, with a head width of 60-67nm, a length of 60-67nm, and a tail length of 167-200nm. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of phage PC192 conforms to the characteristics of the Long-tailed Bacteriophage family and belongs to the Long-tailed Bacteriophage family.

[0083] like Figure 6 As shown, the head of Clostridium perfringens phage PM11 is a polyhedral structure with a diameter of about 54nm and 6 short tail filaments. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of phage PM11 conforms to the characteristics of Brachyurae and belongs to the Brachyurae family.

[0084] Example 3 Whole genome analysis of three phage strains

[0085] The genomes of the three phages were extracted and sequenced. The GenBank number of PD205 in NCBI is ON922919, the GenBank number of PC192 is ON922920, and the GenBank number of PM11 is OP022426. The following genome sequences were analyzed:

[0086] The PD205 genome is 168652bp long, with a G+C content of 37.6%, and the base C, G, A, and T contents of 18.1%, 19.5%, 31.9%, and 30.5%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 269 open reading frames (ORFs). Among these 269 open reading frames (ORFs), 84 structural proteins were found, mainly including the structure and packaging proteins of the phage (head protein, tail protein, tail fiber protein, neck protein, capsid protein, and large subunit of terminase, etc.), the lysis-related proteins of the phage (lytic enzymes), the proteins related to DNA replication and modification (restriction endonucleases, DNA binding proteins, intron-containing DNA polymerase precursors, HNH endonucleases, DEAD / DEAH box helicases, etc.), and other functional proteins (repeated infection immunity proteins). At the same time, among the 269 ORFs, 259 start codons were ATG, 5 start codons were GTG, and 2 start codons were TTG. The software tRNAscan-SE analysis showed that the genome did not contain tRNA genes. The online tool CGEserver analysis showed that the genome did not contain drug resistance genes and virulence genes. The PHASTER analysis showed that the genome did not contain lysogeny-related genes.

[0087] The PC192 genome is 39095bp in length, with a G+C content of 48.29%, and the base C, G, A, and T contents of 25.68%, 22.61%, 24.90%, and 26.81%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 47 open reading frames (ORFs). Among these 47 open reading frames (ORFs), 32 structural proteins were found, mainly including the structure and packaging proteins of the phage (head protein, tail protein, tail fiber protein, neck protein, capsid protein, and large subunit of terminase, etc.), the lysis-related proteins of the phage (lytic enzymes), the proteins related to DNA replication and modification (restriction endonucleases, DNA binding proteins, intron-containing DNA polymerase precursors, HNH endonucleases, DEAD / DEAH box helicases, etc.), and other functional proteins (repeated infection immunity proteins). At the same time, among the 47 ORFs, 45 start codons are ATG, 1 start codon is GTG, and 0 start codons are TTG. The software tRNAscan-SE analysis showed that the genome does not contain tRNA genes. The online tool CGE server analysis showed that the genome does not contain drug resistance genes and virulence genes. The PHASTER analysis showed that the genome does not contain lysogeny-related genes.

[0088] The PM11 genome is 17894bp in length, with a G+C content of 28.17%, and the base C, G, A, and T contents of 17.40%, 10.76%, 31.46%, and 40.37%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 19 open reading frames (ORFs). Among these 19 open reading frames (ORFs), 9 structural proteins were found, mainly including the structure and packaging proteins of the phage (head protein, tail protein, tail fiber protein, capsid protein, etc.), the lysis-related proteins of the phage (lytic enzymes), the proteins related to DNA replication and modification (intron-containing DNA polymerase precursors, HNH endonucleases, etc.), and other functional proteins. At the same time, among the 19 ORFs, 16 start codons were ATG, 1 start codon was GTG, and 2 start codons were TTG. The software tRNAscan-SE analyzed that the genome did not contain tRNA genes. The online tool CGE server analysis showed that the genome did not contain drug resistance genes and virulence genes. The PHASTER analysis showed that the genome did not contain lysogeny-related genes.

[0089] In the genome of bacteriophage PD205: the gene sequence of the tail fiber protein related to phage host recognition is shown in Sequence 1 in the sequence listing; the sequence of the highly conserved terminase large subunit protein gene is shown in Sequence 2 in the sequence listing; the sequence of the DNA polymerase gene is shown in Sequence 3 in the sequence listing; the sequence of the lysozyme gene related to cleavage ability is shown in Sequence 4 in the sequence listing, and the relevant information is specifically shown in Table 1 below.

[0090] In the genome of bacteriophage PC192: the gene sequence of tail fibers protein related to phage host recognition is shown in sequence 5 in the sequence listing; the sequence of the highly conserved terminase large subunit protein gene is shown in sequence 6 in the sequence listing; the sequence of the DNA polymerase gene is shown in sequence 7 in the sequence listing; the sequence of the lysozyme gene related to cleavage ability is shown in sequence 8 in the sequence listing, and the relevant information is specifically shown in Table 1 below.

[0091] In the genome of bacteriophage PM11: the gene sequence of tail fiber protein related to phage host recognition is shown in sequence 9 in the sequence list; the sequence of DNA polymerase gene is shown in sequence 10 in the sequence list; the sequence of lysozyme gene related to lysis ability is shown in sequence 11 in the sequence list. The relevant information is specifically shown in Table 1 below.

[0092] Table 1 Phage gene sequence information

[0093]

[0094] Example 4 Determination of phage titer

[0095] 1. Experimental methods:

[0096] Dilute the proliferated phage stock solution 10 times, take 240 μL of the above appropriate dilution phage and 240 μL of the host bacterial suspension, mix them into a sterile EP tube, and mix them thoroughly. After incubation at 37°C for 5 minutes, use the double-layer plate method to plate, and after the agar solidifies, invert and culture in a 37°C constant temperature box for 3 to 6 hours (anaerobic culture of Clostridium perfringens phage), and observe the results. Make 2 parallels for each dilution gradient, and count the dilutions between 30 and 300 plaques. Take the average of 2 parallels for each dilution to calculate the phage titer.

[0097] Phage titer (pfu / mL) = average plaque number × dilution factor × 10.

[0098] 2. Experimental results and analysis

[0099] The phage titer of PD205 is 3.80×10 10 PFU / mL, the phage titer of PC192 was 8.60×10 10 PFU / mL, the phage titer of PM11 is 1.20×10 10 PFU / mL.

[0100] Example 5 Thermal stability of bacteriophage

[0101] 1. Experimental methods:

[0102] Take 500 μL of different types of phage proliferation liquid and dispense them into sterile EP tubes, and place them in a water bath at 40°C, 50°C, 60°C, 70°C, and 80°C for 20 min, 40 min, 60 min, and 120 min, respectively. Set up 2 replicates. After the reaction is over, take a sample and immediately cool it in an ice bath, dilute it at a 10-fold ratio, and determine the titer using the double-layer plate method. With temperature as the horizontal axis and the logarithm of the phage titer as the vertical axis, the thermal stability curves of Escherichia coli phage PD205, Salmonella phage PC192, and Clostridium perfringens phage PM11 were plotted.

[0103] 2. Experimental results and analysis

[0104] like Figure 7As shown, the titer of Escherichia coli phage PD205 is relatively stable in the temperature range of 40-60°C. After being exposed to 70°C for 40 minutes, its titer decreases by 4 orders of magnitude; after being exposed to 80°C for 20 minutes, the phage is inactivated.

[0105] like Figure 8 As shown, the titer of Salmonella phage PC192 was stable when placed at 40-60°C for 120 minutes, and its titer decreased by 3 orders of magnitude when exposed to 70°C for 60 minutes; the phage was inactivated when exposed to 80°C for 20 minutes.

[0106] like Fig. 9 As shown, the titer of Clostridium perfringens phage PM11 was relatively stable when it was exposed to the temperature range of 40-70℃ for 2h, and the phage was inactivated when it was exposed to 80℃ for 20min.

[0107] The results show that Escherichia coli phage PD205, Salmonella phage PC192 and Clostridium perfringens phage PM11 have strong heat resistance and can maintain a certain activity in a higher temperature environment, among which Clostridium perfringens phage PM11 has more outstanding heat resistance.

[0108] Example 6 pH stability of bacteriophage

[0109] 1. Experimental methods

[0110] Take sterile test tubes and add 4.5mL of NB broth (applied to phage PD205 and PC192) or TSB broth (applied to phage PM11) with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), three tubes each, and then place the test tubes in a 37℃ water bath. After the temperature stabilizes, add 500μL of different types of phage proliferation liquid to each, mix well and bathe at 37℃ for 1h, 2h, and 3h. After the reaction is over, immediately add an appropriate amount of 1mol / L HCl or NaOH to each mixed solution to make the pH value of the mixed solution about 7. The double-layer plate method was used to determine the phage titer under different pH values, and 2 replicates were set for each pH value test tube. Finally, the pH stability curves of phage PD205, PC192, and PM11 were drawn with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0111] 2. Experimental results and analysis

[0112] like Fig.10 As shown, after 3 hours of exposure to pH 5-11, the titer of Escherichia coli phage PD205 was still close to the initial titer, while after 2 hours of exposure to pH 12 or pH 13, the titer decreased by 5 orders of magnitude or became inactivated.

[0113] like Fig.11As shown, the activity of Salmonella phage PC192 was stable after being exposed to pH 4-11 for 3 hours, and its potency was significantly reduced or inactivated after being exposed to pH 3 and pH 13 for 3 hours.

[0114] like Fig.12 As shown in the figure, after 3 hours of action at pH 4-11, the titer of PM11 of Clostridium perfringens was relatively stable. After 2 hours of action at pH 3, the titer began to decrease, and the titer decreased by 5 orders of magnitude at pH 13. The above results show that phages PD205, PC192 and PM11 have strong acid-base tolerance and can adapt to a wide range of pH environments.

[0115] Example 7 Determination of one-step growth curve of bacteriophage

[0116] 1. Experimental methods

[0117] Take 1 mL of each of the different types of phage proliferation fluid and the corresponding fresh host bacteria proliferation fluid with a multiplicity of infection of 10, mix thoroughly (start timing at this time), incubate at 37°C for 5 minutes, centrifuge at 12000rpm for 30 seconds, use a micropipette to remove the supernatant as much as possible, and then wash once with 5 mL of NB broth (applied to PD205 and PC192) or TSB broth (applied to PM11) (centrifuge at 12000rpm for 30 seconds), and discard the supernatant. Use preheated NB broth (applied to PD205 and PC192) or TSB broth (applied to PM11) to suspend the precipitate (total volume of 5 mL) and mix thoroughly, quickly place it in a 37°C shaker with 170rpm shaking culture, take out 150 μL at time 0 and every 10 minutes, centrifuge at 10000rpm for 1 minute, and use the double-layer plate method to measure the titer, make 2 parallels, and take the average of the results.

[0118] With the infection time as the horizontal axis and the phage titer in the infection system as the vertical axis, a one-step growth curve was drawn to obtain the latent period and outbreak period of PD205, PC192 and PMQ01 phages, respectively, and the outbreak amount of phages was calculated.

[0119] Burst volume = phage concentration in stable period / initial bacterial concentration

[0120] 2. Test results and analysis

[0121] like Fig.13 and 14 As shown in the figure, after the Escherichia coli phage PD205 and the Salmonella phage PC192 infected the host bacteria, the titers remained basically unchanged within 30 minutes, and the titers were maintained at 10 6 PFU / mL and 10 7PFU / mL, indicating that the incubation period of PD205 and PC192 was 30 min; and within 40 to 90 min, both entered the outbreak period, with the number of phages increasing sharply, reaching a stable level after 90 min, reaching 10 9 PFU / mL and 10 10 PFU / mL, and the burst sizes of phages PD205 and PC192 were calculated to be 90 and 102, respectively.

[0122] like Fig.15 As shown in Figure 2, the titer of Clostridium perfringens phage PM11 remained at 10 within 40 minutes of infecting the host bacteria. 6 PFU / mL, indicating that the incubation period of PM11 is 40 minutes. Within 40 to 50 minutes, it enters the outbreak period, the phage titer increases sharply, and reaches a stable level after 60 minutes, reaching 10 9 PFU / mL, the burst period is about 20min, and the calculation shows that the burst volume of phage PM11 is 86. In summary, the burst volume of the three phages is high, especially phage PC192, which indicates that the yield of these phages is high in the same period and is suitable for industrial mass production.

[0123] Example 8 Lysis rate detection experiment of bacteriophage composition

[0124] 1. Detection of lysis rates of three different phage strains

[0125] ① Experimental method: Prepare fresh phage proliferation fluids PD205, PC192 and PM11, select a total of 90 pathogenic strains (30 strains of Escherichia coli, 30 strains of Salmonella and 30 strains of Clostridium perfringens) isolated from the liver and intestinal tissues of diseased chickens in Shandong, Jiangsu, Guangxi and other regions, and use the double-layer plate method to detect the lysis spectrum of the phage combination. The host bacteria information and lysis spectrum detailed results used in the test are shown in Table 2.

[0126] The results showed that the Escherichia coli phage PD205 lysed 24 of the 30 strains of Escherichia coli, with a lysis rate of 80% for Escherichia coli, but the total lysis rate for 90 pathogenic bacteria was 26.67%; the Salmonella phage PC192 lysed 28 of the 30 strains of Salmonella, with a lysis rate of 93.33% for Salmonella, but the total lysis rate for 90 pathogenic bacteria was 31.11%; the Clostridium perfringens phage PM11 could lyse 26 of the 30 strains of Clostridium perfringens, with a lysis rate of 86.67% for Clostridium perfringens, but the total lysis rate for 90 phage strains was 28.89%; and the total lysis rate of the phage combination for 90 pathogenic bacteria was 86.67%. It can be seen that the phage combination has a good lysis effect and broad spectrum for mixed infections of Escherichia coli, Salmonella and Clostridium perfringens, and its lysis ability is maximized, which effectively makes up for the limitations of the lysis spectrum of a single phage.

[0127] 2. Comparison of lysis performance with existing bacteriophages:

[0128] The phage PMQ06 disclosed in the patent application number CN202010691495.2 applied by the applicant was used to lyse the above 30 strains of Clostridium perfringens phage, and the lysis of the phage PMQ06 was compared with the Clostridium perfringens phage PM11 of the present application. The method is described above.

[0129] Experimental results: The Clostridium perfringens phage PMQ06 can only lyse 17 of the 30 strains of Clostridium perfringens mentioned above, with a lysis rate of 56.67%; while the Clostridium perfringens phage PM11 can lyse 26 of the 30 strains of Clostridium perfringens, with a lysis rate of 86.67%; it can be seen that the lysis rate of Clostridium perfringens phage PM11 against the above-mentioned chicken-derived Clostridium perfringens is significantly higher than that of PMQ06.

[0130] Table 2 Phage and its combination lysis results

[0131]

[0132]

[0133]

[0134]

[0135] Note: +: lysis; -: no lysis.

[0136] Example 9 Safety Test of Bacteriophage

[0137] 1. Experimental methods:

[0138] Sixty one-day-old SPF chickens were divided into experimental group 1 (15 chickens), experimental group 2 (15 chickens) and control group 1 (15 chickens) and control group 2 (15 chickens). Each chick in experimental group 1 was gavaged with 1×10 10 Each chick in the experimental group 2 was intramuscularly injected with 1×10 10 1 ml of the phage composition was added; 1 ml of sterile nutrient broth was administered orally to each chick in control groups 1 and 2. The chickens in each group were raised in the same environment for 2 weeks, and the clinical signs were observed, and the chickens were dissected to observe visceral and intestinal lesions.

[0139] 2. Experimental results and analysis

[0140] Two weeks after treatment with the phage composition, there were no significant differences in the clinical symptoms and autopsy results of the chickens in the two test groups compared with the control group. The chickens grew well, no deaths occurred, and no abnormal phenomena such as diarrhea, lethargy, drowsiness, ruffled feathers, and loss of appetite occurred. The autopsy results showed that there were no lesions in the internal organs and intestines of the chickens in the test group. The above results indicate that the phage composition is safe and has no toxic side effects.

[0141] Example 10 Experimental study on the treatment of chicken diarrhea caused by Escherichia coli, Salmonella and Clostridium perfringens using a bacteriophage composition

[0142] 1. Experimental methods:

[0143] A total of 120 7-day-old SPF chickens were divided into 6 groups (20 chickens / group), including a blank group, a challenge group, and a treatment group. The treatment group was further divided into 3 single phage treatment groups and 1 phage combination treatment group. The three bacterial liquids (Escherichia coli liquid, Salmonella liquid, and Clostridium perfringens liquid) were mixed at a ratio of 1:1:1 to 5×10 8 The chickens in the challenge group and the treatment group were challenged with the mixed bacterial solution of 1×10 CFU / mL orally (0.2 ml / chicken). After 50% of the chickens showed symptoms such as diarrhea or anal paste, the chickens in the treatment group were given 1×10 8 PFU / mL phage was used for oral treatment (0.2 ml / bird). After 5 consecutive days of treatment, the chickens were kept and observed for 14 days to observe the mortality, diarrhea and weight gain of the chickens. The live chickens were dissected to observe the pericardium, liver and intestinal lesions.

[0144] 2. Experimental results:

[0145] The results showed that there was no death in the blank group, and no symptoms of diarrhea, pericardial perihepatic and intestinal lesions; Fig.16As shown, the PD205 treatment group, PC192 treatment group, and PM11 treatment group can significantly reduce the diarrhea and mortality of chickens, respectively; and the therapeutic effect of the phage combination of the above three phage strains was greatly improved, the diarrhea rate of chickens in the treatment group decreased by 85%, and the mortality rate of chickens decreased by 76%, and the phage combination also reduced the degree of visceral and intestinal tissue lesions.

[0146] Obviously, the effect of the phage combination of PD205, PC192 and PM11 in reducing diarrhea rate and mortality rate is significantly better than that of a single phage.

[0147] Example 11 Experiment on the use of bacteriophage composition to prevent bacterial diarrhea in poultry farming

[0148] 1. Experimental methods:

[0149] Five duck houses with 10,000 25-day-old meat ducks were selected from a large-scale duck farm in Shandong Province and set up as follows: 4 experimental groups and 1 control group. The 4 experimental groups were divided into 3 different single phage groups (PD205 prevention group, PC192 prevention group, PM11 prevention group) and 1 phage combination group.

[0150] Phage PD205, PC192, PM11 and a combination of phages (the ratio of PD205, PC192, and PM11 was 1:1:1) were added to drinking water at a concentration of 1×10 7 pfu / mL. The chickens were fed with water containing bacteriophages within 3 hours after 25 days of age, and the water was used for 5 consecutive days until slaughter. The control group was fed with normal drinking water. The control group and the experimental group were fed normally according to the farm procedures to observe the diarrhea of ​​the chickens, and the survival rate, daily weight gain, and feed-to-meat ratio of the ducks were calculated.

[0151] 2. Experimental results and analysis

[0152] like Fig.17 As shown in the figure, after using drinking water with a bacteriophage added for prevention, the diarrhea rate of the duck farm was significantly reduced, but the degree of reduction was not as good as that of the experimental group using the bacteriophage combination. After using drinking water with a bacteriophage combination for prevention for 5 times, the diarrhea rate of the bacteriophage combination decreased by 16.00% compared with the blank control group, and the survival rate increased by 5.00%. The difference in diarrhea rate between the bacteriophage combination and the blank control group was extremely significant (P<0.01), indicating that the use of bacteriophage combination has a significant effect on preventing poultry bacterial diarrhea; at the same time, according to the daily weight gain of the chicken flock, daily feed consumption and feed-to-meat ratio, the use of bacteriophage combination can also improve the production performance of the chicken flock, laying the foundation for the promotion of bacteriophage as a green additive in the future.

[0153] Example 12: Experiment on disinfection of duck / chicken farm environment by bacteriophage

[0154] 1. Experimental methods

[0155] A white-feathered broiler farm in Shandong randomly selected 18 one-day-old chick houses, including 12 experimental groups, 3 control groups, and 3 blank control groups. The experimental groups included 3 single phage groups (3 phages / group) and a phage combination group. The experimental groups used drinking water from the farm to dilute the phage suspension (1×10 6 PFU / mL), 2 times / day, spraying volume is 10mL / m 2 The control group was sprayed with the same amount of 84 disinfectant twice a day, and the blank control group was raised according to routine operations. The changes in the content of Escherichia coli, Salmonella and Clostridium perfringens in the environment before and after disinfection were detected. The central point of the chicken house and 4 points in the corners were used as test points. The sampling point was 0.5m from the ground, and the four points in the corners were 1m away from the wall. A 9cm diameter SS plate (for testing Escherichia coli and Salmonella) and a 1% glucose-5% sheep blood culture medium (for testing Clostridium perfringens) were placed at each point. After sampling, the SS culture dish was placed in a 37℃ constant temperature incubator, and the 1% glucose-5% sheep blood culture dish was anaerobically cultured at 37℃. The number of bacterial colonies in each culture dish was recorded after 18-24h of culture.

[0156] According to the Ostwald formula, the total number of colonies was C = 50000N / AT, where C is the total number of colonies of the three bacteria per cubic meter (CFU / m 3 ); N: number of colonies per dish; A: area of ​​culture dish (cm 2 ); T: sampling time (min).

[0157] 2. Experimental results and analysis

[0158] like Fig.18As shown in the figure, the total content of Escherichia coli, Salmonella and Clostridium perfringens in the phage combination test group was lower than that in the control group (disinfection with 84 disinfectant) and the blank control group, and was also lower than that in the single phage group. This shows that the phage combination (PD205+PC192+PM11) can significantly reduce the total content of Escherichia coli, Salmonella and Clostridium perfringens in the environment and reduce the infection rate of the three bacteria. In addition, compared with the 84 disinfectant used in the control group, the phage combination has the advantages of safe use, efficient disinfection, no toxic side effects, and environmental friendliness. The advantages include no irritating odor when disinfecting with the bacteriophage composition, no irritation or damage to the respiratory mucosa of animals, no tolerance to pathogens when used for a long time, and the bacteriophage as a disinfectant can exist in the disinfection place for a long time, continuously inhibiting bacteria, and the disinfectant remaining in the phage disinfection (such as the feed trough, etc.) can be licked by animals, which is safer than 84 disinfectant. It can be seen that the bacteriophage disinfectant has a wider range of disinfection objects and disinfection environments, and has a better application prospect. Therefore, the bacteriophage and the composition (PD205+PC192+PM11) can be used as a new type of biological environmental disinfectant.

[0159] Example 13 Phage Lysis Test on Non-Host Bacteria

[0160] 1. Experimental methods

[0161] A total of 25 non-host bacteria, including 5 strains of Riemerella anatipestifer, 5 strains of Staphylococcus, 5 strains of Pseudomonas aeruginosa, 5 strains of Proteus, and 5 strains of Pasteurella, were selected, and lysis experiments were performed on the three bacteriophages according to the lysis spectrum determination method in Example 8.

[0162] 2. Experimental results and analysis

[0163] The results showed that Escherichia coli phage PD205, Salmonella phage PC192 and Clostridium perfringens phage PM11 did not lyse the 25 non-host bacteria selected above. Detailed information is shown in Table 3. The results showed that Escherichia coli phage PD205, Salmonella phage PC192 and Clostridium perfringens phage PM11 could not recognize the above-mentioned 5 strains of Riemerella anatipestifer, 5 strains of Staphylococcus aeruginosa, 5 strains of Pseudomonas aeruginosa, 5 strains of Proteus, and 5 strains of Pasteurella multocida, which shows that the tested phages have extremely strong host specificity and have no damaging effect on the microbial community.

[0164] Table 3 Lysis results of phage and its combination on non-host bacteria

[0165]

[0166] The above-mentioned embodiments only express several preferred embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several modifications, improvements and equivalent substitutions can be made, which should be included in the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A Salmonella phage PC192, characterized in that: Its accession number is CGMCC No.22367.

2. A bacteriophage composition, characterized in that: Including the aforementioned Salmonella phage PC192 and other phages.

3. The bacteriophage composition according to claim 1, characterized in that include: The Salmonella phage PC192 and the Clostridium perfringens phage PM11 with the deposit number of CGMCC No.22372.

4. The bacteriophage composition according to claim 1, characterized in that include: The Salmonella phage PC192, the Clostridium perfringens phage PM11 with a preservation number of CGMCC No.22372, and the Escherichia coli phage PD205 with a preservation number of CGMCC No.22361.

5. The bacteriophage composition according to claim 2, characterized in that The ratio of the live numbers of Escherichia coli phage, Salmonella phage and Clostridium perfringens phage is 1:1:

1.

6. Use of the Salmonella phage PC192 according to claim 1 or the phage composition according to any one of claims 2 to 5 in the preparation of drugs for preventing and treating avian bacterial diarrhea, environmental disinfectants and feed additives.

7. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient comprises the Salmonella phage PC192 according to claim 1 or the phage composition according to any one of claims 2-5.

8. An environmental disinfectant, characterized in that The active ingredient comprises the Salmonella phage PC192 according to claim 1 or the phage composition according to any one of claims 2 to 5; preferably, the concentration of each phage is 1×10 8 PFU / mL or above; preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in the environment.

9. A feed additive for preventing bacterial diarrhea in poultry, characterized in that: The active ingredient comprises the Salmonella phage PC192 according to claim 1 or the phage composition according to any one of claims 2-5.

10. A detection kit, characterized in that: The invention comprises the Salmonella phage PC192 as claimed in claim 1.

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