Escherichia coli bacteriophage PD211, bacteriophage composition and application thereof

Abstract

The invention discloses an escherichia coli bacteriophage PD211, a bacteriophage composition and application of the escherichia coli bacteriophage PD211, the preservation number of the bacteriophage PT15 is CGMCC (China General Microbiological Culture Collection Center) No.22362, and the bacteriophage PT15 is preserved in the China General Microbiological Culture Collection Center on April 12, 2021; the invention further provides a bacteriophage composition containing the escherichia coli bacteriophage PD211 and the riemerella anatipestifer bacteriophage PT15, and the bacteriophage and the bacteriophage composition can serve as active ingredients to be used for preparing treatment drugs and feed additives and are applied to prevention and treatment of duck bacterial serositis. The bacteriophage composition has the advantages of being safe, efficient and free of toxic and side effects, can be used as an active ingredient of a novel environment disinfectant, effectively prevents and controls the culture environment and feeding facilities from being polluted, and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an Escherichia coli phage PD211, a phage composition thereof and application thereof. Background Art

[0002] Duck Escherichia coli disease is a systemic or local infectious disease of ducks caused by certain pathogenic Escherichia coli of different serotypes. Duck Escherichia coli disease can occur all year round and is the most common bacterial disease of ducks. It can infect ducks of all ages, and the infection is more serious in young ducks and embryos. It often causes duck septicemia, duck pericarditis, duck hepatitis, duck peritonitis and other inflammations. Escherichia coli belongs to environmental pathogens. The infection of the disease is often caused by excessive Escherichia coli in the environment. When the duck's resistance is reduced, the defense ability is insufficient or completely lost, it is easy to cause the disease, presenting a typical secondary local or systemic infection. Sick ducks have high mortality, slow growth, quality decline, low feed conversion rate and increased treatment costs, which has caused huge economic losses to the duck farming industry.

[0003] Riemerella anatipestifer disease, also known as duck infectious serositis, duck pasteurellosis, etc., is an acute, septicemic infectious disease that infects domestic ducks, wild ducks, goslings, turkeys and many other birds. It is also one of the main bacterial infectious diseases that endanger the duck farming industry. The bacteria mainly infect ducklings aged 1 to 7 weeks. The disease is characterized by fibrinous pericarditis, perihepatitis, air sacculitis, meningitis and caseous salpingitis. The incidence and mortality rate of ducklings infected with Riemerella anatipestifer are high, and it is easy to develop drug resistance. At present, there are at least 21 serotypes of Riemerella anatipestifer identified in China. In recent years, Riemerella anatipestifer has often caused mixed infections in ducks with other pathogens (such as Escherichia coli).

[0004] Bacterial serositis caused by Riemerella anatipestifer and Escherichia coli is a common bacterial disease in ducks. Drug prevention and vaccine immunization are currently important measures to control the above diseases, but both measures have problems. On the one hand, since pathogenic bacteria often have more serotypes and different serotypes may be prevalent in the same duck farm, there is no obvious cross-protection between the serotypes, which leads to some practical difficulties in vaccine immunization and it is difficult to achieve a satisfactory preventive effect. On the other hand, antimicrobial drug treatment is a common method for controlling bacterial diseases and an important way to control epidemic outbreaks of bacterial diseases. However, due to the irregular onset time and high frequency of bacterial diseases, farms often have the problem of using high-concentration antimicrobial drugs to prevent and treat bacterial diseases without authorization, resulting in serious drug resistance and antimicrobial drug residues in bacteria. This will cause serious pollution to the environment, seriously endanger public food safety, and bring harm to the breeding industry and human health.

[0005] Currently, there is no safe and effective drug to prevent and control duck bacterial serositis. Although bacteriophages are beginning to be used in various technical fields, no effective bacteriophages have been found to prevent and control duck bacterial serositis. Therefore, the existing technology needs to be further improved. Summary of the invention

[0006] In view of the above problems, the present invention provides a new strain of Escherichia coli phage PD211 with strong lytic activity and a phage composition with a wide lytic spectrum and strong lytic effect, which is established based on a cocktail prevention and control method and includes Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15. The Riemerella anatipestifer phage and its composition can be used to prepare phage preparations to effectively prevent and control duck serositis caused by Riemerella anatipestifer or co-infection with other pathogens, and can also be used to prepare poultry feed additives, environmental disinfectants and detection kits, etc.

[0007] To solve the above problems, this application provides the following technical solutions:

[0008] In the first aspect, the present invention provides a self-isolated Escherichia coli phage PD211, which was deposited in the China General Microbiological Culture Collection Center on April 12, 2021, and the deposit number of the Escherichia coli phage PD211 is CGMCC No. 22362.

[0009] Under an electron microscope, the morphology of phage PD211 is as follows: its head is a polyhedral head structure, with a width of 87-93nm, a length of 100-107nm, and a tail length of 120-127nm, belonging to a muscle-tailed phage. This phage has excellent lysis performance against pathogenic bacteria Escherichia coli.

[0010] In the present application, the Escherichia coli phage PD211 includes mutants with point mutations, deletion mutations or addition mutations and the same bactericidal activity with a homology higher than 98% or 99%. Since phages are very prone to mutations during replication, mutants of the above phages are also within the scope of protection requested in the present application. The sequence of PD211 can be obtained by sequencing the biological materials deposited according to the present invention by known methods. For those skilled in the art, it does not require creative labor to screen out mutants with extremely similar properties according to the phage provided by the present invention.

[0011] In a second aspect, the present invention provides a phage composition, comprising: Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15.

[0012] The present invention also provides a Riemerella anatipestifer phage PT15, whose deposit number is CGMCC No. 22377. The phage was deposited in the China General Microbiological Culture Collection Center on April 12, 2021. The morphology of phage PT15 was observed under an electron microscope. The phage has a polyhedral head structure and a retractable tail. The head is 61-65nm wide and 63-69nm long, and the tail is 171-208nm long. It is a long-tailed phage.

[0013] The Riemerella anatipestifer phage PT15 includes mutants with the same bactericidal activity that are more than 98% or 99% homologous to point mutations, deletion mutations or addition mutations. Since phages are very prone to mutations during replication, mutants of the above phages are also within the scope of protection requested in this application. The sequence of PT15 can be obtained by sequencing the biological materials preserved according to the present invention by known methods. For those skilled in the art, it does not require creative labor to screen out mutants that are extremely similar to the phages provided by the present invention.

[0014] Preferably, in the above phage composition, the ratio of the number of live Escherichia coli phage PD211 to that of Riemerella anatipestifer phage PT15 is 1:1; preferably, the concentration of each phage is not less than 1×10 8 PFU / mL.

[0015] The whole genome analysis of phages showed that the whole genome sizes of phages PD211 and PT15 were 149506bp and 44128bp, respectively. The G+C content of the phage PD211 genome was 37.70%, with 265 open reading frames (ORFs), and the G+C content of the phage PT15 genome was 34.81%, with 81 open reading frames (ORFs). Phages PD211 and PT15 did not contain drug resistance genes, virulence genes, or lysogeny-related genes.

[0016] In a third aspect, the present invention also provides the use of the above-mentioned Escherichia coli phage PD211 or a phage composition composed of Riemerella anatipestifer phage PT15 and Escherichia coli phage PD211 in the preparation of drugs for preventing and treating duck bacterial diseases, environmental disinfectants, feed additives, and detection kits.

[0017] The duck bacterial diseases include various diseases caused by duck Escherichia coli or duck Escherichia coli and Riemerella anatipestifer, such as duck infectious serositis, duck colibacillosis and the like.

[0018] The application method of the bacteriophage and the composition thereof is as follows: the bacteriophage or the composition thereof is added as a therapeutic drug to the drinking water or feed of ducks suffering from bacterial serositis, or is administered orally, subcutaneously, or intramuscularly to the ducks, wherein the preferred method is the feed mixing method. The above method can prevent and treat duck bacterial serositis, and reduce duck serositis, perihepatitis, and pericarditis, thereby achieving the effects of improving the egg production rate of laying ducks, eggshell quality, duck survival rate, feed-to-meat ratio, etc.

[0019] In a fourth aspect, the present invention further provides a phage preparation, the active ingredient of which includes the aforementioned Escherichia coli phage PD211 or the aforementioned phage composition comprising Riemerella anatipestifer phage PT15 and Escherichia coli phage PD211.

[0020] Preferably, the phage preparation is used by subcutaneous injection, intramuscular injection or oral administration to treat duck bacterial serositis. Experimental verification shows that in 7-day-old ducklings infected with Escherichia coli and Riemerella anatipestifer, 5×10 9 PFU / mL of phage composition, used for 3 consecutive days, can effectively reduce the mortality rate of diseased ducklings and the incidence of serositis within 14 days. In an alternative solution, the above phage preparation can be used by intramuscular injection or oral administration.

[0021] In a fifth aspect, the present invention further provides a feed additive, which comprises the aforementioned Escherichia coli phage PD211 or the aforementioned phage composition comprising Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15; preferably, the concentration of each phage in the feed is at least 1×10 8 PFU / g. The application method is: by mixing with duck feed or duck drinking water and feeding, the duck can prevent bacterial serositis in ducks. Preferably, the concentration of each bacteriophage in the feed is at least 1×10 8 PFU / g, the concentration of each phage in drinking water is at least 1×10 8 PFU / g.

[0022] In a sixth aspect, the present invention further provides an environmental disinfectant, the active ingredient of which includes Escherichia coli phage PD211 or a phage composition comprising Riemerella anatipestifer phage PT15 and Escherichia coli phage PD211. Preferably, the concentration of each phage is 1×10 8 PFU / mL or above.

[0023] Preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in the environment to improve its sterilization or bacteriostatic effect; it may also include other adjuvants, such as adjuvants that can prolong the duration of bacteriophage.

[0024] 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 Escherichia coli or Riemerella anatipestifer 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 ducks.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention provides two strains of bacteriophages with high efficiency in lysis performance isolated by the applicant, namely, Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15. These two groups of phages are used alone to prepare drugs for preventing and treating duck bacterial diseases. The above two or more phages can also be used to prepare a phage composition for effectively preventing and controlling duck bacterial serositis based on the cocktail method, and used as a therapeutic drug or feed additive to prevent and control duck bacterial serositis; it can also be used as an active ingredient of an environmental disinfectant to effectively prevent and control the pollution of the breeding environment and feeding facilities.

[0027] 2. The bacteriophage and bacteriophage composition of the present invention have a short preparation cycle, low cost, and are easy to industrialize. Bacteriophages are mainly screened from nature, with a wide range of sources and a large number, and are natural organisms.

[0028] 3. Phage therapy is not limited by bacterial resistance and is not easy to develop resistance. Therefore, it can be widely used in the breeding process. It can efficiently, quickly and specifically kill Escherichia coli, Riemerella anatipestifer and even duck bacterial serositis caused by resistant strains, effectively reducing the morbidity and mortality rate, and providing a green and natural biological agent for the prevention and control of duck bacterial serositis.

[0029] 4. Compared with a single lytic phage, the phage composition provided by the present invention has a wider sterilization range and higher sterilization activity, and can effectively prevent and control serositis caused by duck Escherichia coli and Riemerella anatipestifer, improve egg production rate and feed-to-meat ratio; and has high safety performance, only infects pathogens of specific species, and will not destroy the normal flora in the duck. The duckling safety test proves that it is safe, has no residue, has no toxic side effects, and will not affect the normal growth and development of ducklings. At the same time, the phage will not cause secondary contamination to the body after entering the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a plaque image of Escherichia coli phage PD211;

[0031] Figure 2 This is a plaque image of Riemerella anatipestifer phage PT15;

[0032] Figure 3 This is an electron microscope image of Escherichia coli phage PD211;

[0033] Figure 4 This is an electron microscope image of Riemerella anatipestifer phage PT15;

[0034] Figure 5 is the thermal stability of Escherichia coli phage PD211;

[0035] Figure 6 The thermal stability of Riemerella anatipestifer phage PT15;

[0036] Figure 7 is the pH stability of Escherichia coli phage PD211;

[0037] Figure 8 pH stability of Riemerella anatipestifer phage PT15;

[0038] Fig. 9 is the one-step growth curve of Escherichia coli phage PD211;

[0039] Fig.10 This is the one-step growth curve of Riemerella anatipestifer phage PT15;

[0040] Fig.11 This is a diagram showing the effect of the bacteriophage composition on the treatment of duck bacterial serositis;

[0041] Fig.12 This is a graph showing the effect of the bacteriophage composition in preventing duck mortality;

[0042] Fig.13 An experiment on using bacteriophages to disinfect the duck farm environment. DETAILED DESCRIPTION

[0043] 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.

[0044] Example 1 Isolation and culture of bacteriophage

[0045] 1. Sample collection

[0046] The feces, sewage and other samples in the present invention were collected from multiple duck farms in Guangdong Province.

[0047] 2. Resuscitation culture of host bacteria and preparation of proliferation medium

[0048] The host bacteria were Escherichia coli and Riemerella anatipestifer isolated, identified and preserved in our laboratory, and isolated from the duck brain or liver of sick ducks in a duck farm in Guangdong Province.

[0049] (1) Use a sterilized inoculation loop to dip into the stored E. coli host bacterial liquid, streak it on the SS agar medium, and culture it 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 it at 37°C with shaking at 170 rpm / min for 12 to 16 hours to obtain a fresh E. coli proliferation liquid for later use.

[0050] (2) Use a sterilized inoculation loop to dip the preserved Riemerella anatipestifer host bacterial liquid, streak inoculate on 10 ml / L tryptic soy agar (TSA) containing newborn calf serum, and culture in a turbid jar at 37°C 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, and culture it at 37°C with shaking at 170 rpm / min for 12 to 16 hours to obtain fresh Riemerella anatipestifer proliferation liquid for use.

[0051] 3. Isolation of bacteriophage

[0052] (1) Isolation of Escherichia coli phage

[0053] Take an appropriate amount of fecal water and put it into a sample bottle, add 200 μL of host bacteria proliferation liquid, and then add an appropriate amount of NB broth, culture at 37°C and 170rpm for 16 to 18 hours to proliferate and enrich phages to obtain culture fluid; take 5 ml of the above culture fluid, centrifuge at 11000rpm for 10 minutes, take the supernatant and filter it with a 0.22μm filter membrane for sterilization to obtain the phage stock solution. The double-layer plate method was used to isolate phages: 100 μL of the phage stock solution was diluted 10 times, 120 μL of the appropriate gradient dilution solution was mixed evenly with 120 μL of the host bacteria proliferation solution, incubated at 37°C for 5 minutes, and then the upper layer agar (agar concentration was 0.7%) at about 45°C was added. After mixing, the lower layer agar (agar concentration was 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 plaques was obtained, which was named Escherichia coli phage PD211.

[0054] (2) Isolation of Riemerella anatipestifer phage

[0055] Take an appropriate amount of fresh manure from the duck farm, add an equal volume of sterile PBS, mix thoroughly, centrifuge at 8000rpm for 10 minutes, and filter the supernatant through a 450nm filter membrane. Add 500μL of Riemerella anatipestifer to the filtrate, then add an equal volume of 2×TSB, and culture at 37℃ for 16h~18h to enrich the bacteriophage. The next day, take out 5-8mL of the liquid and centrifuge at 8000rpm for 10min, and filter the supernatant with a 450nm filter membrane. Repeat the above method to multiply the filtrate once, and the filtrate after the second multiplication is the original solution.

[0056] The phage was isolated by the double-layer plate method. The phage stock solution was diluted 10 times, 120 μL of the phage dilution solution was mixed with 120 μL of the pathogenic bacteria proliferation solution, incubated at 37°C for 5 minutes, and then placed on a TSA upper agar (agar concentration of 0.7%) plate at about 45°C. After mixing, the plate was quickly poured onto a TSA lower agar (agar concentration of 1.5%) plate, shaken and placed flat until the culture medium solidified, and placed at 37°C for inversion and cultured for 10 to 16 hours to obtain a double-layer plate with plaques. The obtained Riemerella anatipestifer phage was named PT15.

[0057] 4. Purification and proliferation of bacteriophage

[0058] (1) Purification and proliferation of Escherichia coli phage PD211

[0059] 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, shake it at 37°C and 170 rpm for 30 minutes, and obtain a phage extract; centrifuge it 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 it 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 deducted and 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 with shaking until the mixed liquid became clear, and then centrifuged and filtered to obtain the phage proliferation liquid.

[0060] Escherichia coli phage PD211 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 1 ).

[0061] (2) Purification and proliferation of Riemerella anatipestifer phage PT15

[0062] The phage was purified by the double-layer plate method. A single plaque was picked from the above double-layer culture medium where the plaque was formed, and placed in 1 mL of SM buffer, 50 μL of chloroform was added, and the mixture was shaken at 37°C and 170 rpm for 1 hour, and then allowed to stand for several hours at 4°C, and then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken and placed in a new small tube. 220 μL of the phage extract was mixed evenly with 220 μL of the corresponding Riemerella anatipestifer proliferation solution, and incubated at 37°C for 10 minutes. The mixture after incubation was added to the upper TSA agar (agar concentration of 0.7%) at about 45°C, and after mixing, it was quickly poured onto the lower TSA agar (agar concentration of 1.5%) plate, and the plate was placed at 37°C for 10 to 16 hours to obtain a double-layer plate where plaques were formed again. Pick a single plaque again and repeat the above steps 3 to 5 times until a plaque with uniform size, morphology and distribution is obtained, which is considered to be a purified phage stock. Pick a single plaque from the purified plaque double-layer culture medium and place it in 1mL TSB broth, shake it at 170rpm for 1h to obtain a phage extract, centrifuge it at 11000rpm for 5min, take 200μL of the supernatant and 200μL of the host bacteria proliferation liquid in 5mL liquid TSB culture medium, shake and culture at 37℃ and 170rpm until the liquid becomes clear, centrifuge the clear liquid at 11000rpm for 10min, take the supernatant, and filter it with a 0.45μm sterile microporous filter membrane to obtain a phage proliferation liquid.

[0063] Riemerella anatipestifer phage PT15 can form clear plaques on double-layer agar plates, with no halo around them, clear and regular edges, and a diameter of about 0.5 mm (see Figure 2 ).

[0064] Example 2 Morphological characteristics and genome analysis of bacteriophage

[0065] 1. Morphological characteristics of bacteriophages

[0066] (1) Morphological observation method

[0067] 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.

[0068] (2) Observation results and analysis

[0069] A. Escherichia coli phage PD211

[0070] like Figure 3 As shown, the head of bacteriophage PD211 has a polyhedral structure, with a width of 87-93nm, a length of 100-107nm, and a tail length of 120-127nm. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of bacteriophage PD211 conforms to the characteristics of the Myotailed Bacteriophage family, and it belongs to the Myotailed Bacteriophage family.

[0071] B. Riemerella anatipestifer phage PT15

[0072] like Figure 4 As shown, phage PT15 has a polyhedral head structure and a retractable tail, with a head width of 61-65nm, a length of 63-69nm, and a tail length of 171-208nm. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of phage PT15 conforms to the characteristics of the Long-tailed Bacteriophage family, and it belongs to the Long-tailed Bacteriophage family.

[0073] 2. Phage genome analysis

[0074] The genomes of the two phage strains were extracted and sequenced. The GenBank number of PD211 in NCBI is OM401812, and the GenBank number of PT15 is ON933637. The following results were obtained by analyzing the genome sequences:

[0075] The PD211 genome is 149506bp long, with a G+C content of 37.6%, and the base C, G, A, and T contents of 18.4%, 19.2%, 31.2%, and 31.2%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 265 open reading frames (ORFs). Among these 265 open reading frames (ORFs), 35 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 terminase large subunit, etc.), the phage lysis-related proteins (lytic enzymes), 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 265 ORFs, 219 start codons were ATG, 16 start codons were GTG, and 15 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.

[0076] In the genome of bacteriophage PD211: the tail fiber protein gene sequence related to phage host recognition is shown in sequence 1 in the sequence list; the sequence of the highly conserved terminase large subunit protein gene is shown in sequence 2 in the sequence list; the sequence of the transferase (Nicotinamide phosphoribosyltransferase) gene is shown in sequence 3 in the sequence list; the sequence of the lysozyme gene related to lysis ability is shown in sequence 4 in the sequence list, and the relevant information is specifically shown in Table 1 below.

[0077] The PT15 genome is 44128bp in length, with a G+C content of 34.81%, and the base C, G, A, and T contents of 18.65%, 16.16%, 28.24%, and 36.96%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 81 open reading frames (ORFs). Among these 81 open reading frames (ORFs), 13 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 phage lysis-related proteins (lytic enzymes), 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 81 ORFs, 76 start codons are ATG, 1 start codon is GTG, and 1 start codon is TTG. The software tRNAscan-SE analysis showed that the genome does not contain tRNA genes. The online tool CGEserver 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.

[0078] In the genome of bacteriophage PT15: the protein gene sequence of the structural protein (Helix-turn-helixdomainprotein) related to phage host recognition is shown in Sequence 5 in the sequence list; the sequence of the highly conserved terminase large subunit protein gene is shown in Sequence 6 in the sequence list; the sequence of the DNA-binding protein gene is shown in Sequence 7 in the sequence list; the relevant information is specifically shown in Table 1 below.

[0079] Table 1 Phage gene sequence information

[0080]

[0081] Example 3 Determination of phage titer

[0082] 1. Determination method:

[0083] 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 (candle jar culture for Riemerella anatipestifer 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.

[0084] Phage titer (pfu / mL) = average plaque number × dilution factor × 10.

[0085] 2. Measurement results and analysis

[0086] The results showed that the titer of Escherichia coli phage PD211 was 3.85×10 10 PFU / mL, the titer of Riemerella anatipestifer phage PT15 is 9.80×10 9 PFU / mL.

[0087] Example 4 Thermal stability of bacteriophage

[0088] 1. Experimental methods:

[0089] Take 500 μL of different types of phage proliferation fluid and dispense them into sterile EP tubes. Place them in a water bath at 40°C, 50°C, 60°C, 70°C, and 80°C for 20 min, 40 min, and 60 min, respectively. Set up 2 replicates for each temperature. Take samples after the reaction is over, and immediately cool the samples in an ice bath, dilute them 10 times, and determine the titer using the double-layer plate method. Plot the thermal stability curves of Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15 with temperature as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0090] 2. Experimental results and analysis

[0091] The results are as follows Figure 5 As shown, the titer of Escherichia coli phage PD211 is relatively stable in the temperature range of 40-60°C. After being exposed to 70°C for 60 minutes, its titer decreases by 6 orders of magnitude; after being exposed to 80°C for 20 minutes, the phage is inactivated.

[0092] like Figure 6As shown in the figure, the titer of Riemerella anatipestifer phage PT15 was relatively stable when the temperature range was 40-60℃ for 1 hour, and the phage still maintained a high activity when the temperature was 70℃ for 40 minutes. This result shows that Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15 have strong heat resistance and can maintain a certain activity in a higher temperature environment.

[0093] Example 5 pH stability of bacteriophage

[0094] 1. Experimental methods:

[0095] Take three sterile test tubes and add 4.5mL of NB broth (applied to PD211) or TSB broth (applied to PT15) with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). 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 test tube, mix well and place in a 37℃ water bath for 1h, 2h, and 3h. After the action 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 PD211 and PT15 were drawn with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0096] 2. Experimental results and analysis

[0097] like Figure 7 As shown, after the Escherichia coli phage PD211 was exposed to pH 5-11 for 3 hours, the titer was still close to the initial titer, and the titer decreased by 5 orders of magnitude or was inactivated after 2 hours of exposure to pH 12 or pH 13.

[0098] like Figure 8 As shown in the figure, the titer of Riemerella anatipestifer phage PT15 was relatively stable after 3 hours of action at pH 5-11. After 1 hour of action at pH 4 or pH 12, the titer began to decrease, and the phage was inactivated at pH 3. This result shows that phage PD211 and PT15 have strong acid-base tolerance and a wide range of adaptability to pH.

[0099] Example 6 Determination of one-step growth curve of bacteriophage

[0100] 1. Experimental methods:

[0101] 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 PD211) or TSB broth (applied to PT15) (centrifuge at 12000rpm for 30 seconds), and discard the supernatant. Use preheated NB broth (applied to PD211) or TSB broth (applied to PT15) 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.

[0102] 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 PD211 and PT15 phages, respectively, and the outbreak amount of phages was calculated.

[0103] Burst volume = phage concentration in stable period / initial bacterial concentration

[0104] 2. Experimental results and analysis

[0105] like Fig. 9 As shown in the figure, after the Escherichia coli phage PD211 infected the host bacteria, the titer remained basically unchanged within 30 minutes, and the titer was maintained at 10 6 PFU / mL, indicating that the incubation period of PD211 was 20 min; and within 30 to 70 min, the phages entered the outbreak period, the number of phages increased sharply, and reached a stable level after 70 min, reaching 10 9 PFU / mL, the burst size of phage PD211 was calculated to be 97.

[0106] like Fig.10 As shown in the figure, the titer of Riemerella anatipestifer phage PT15 remained at 10 within 20 minutes after infecting the host bacteria. 6 PFU / mL, indicating that the incubation period of PT15 is 20 minutes. Within 20 to 70 minutes, it enters the outbreak period, the phage titer increases sharply, and reaches a stable level after 70 minutes, reaching 10 9 PFU / mL, the burst period is about 50min, and the calculation shows that the burst amount of bacteriophage PT15 is 85.

[0107] Example 7 Lysis rate detection experiment of phage composition

[0108] 1. Experimental methods:

[0109] Fresh phage proliferation fluids PD211 and PT15 were prepared, and a total of 40 pathogenic strains (20 strains of Escherichia coli and 20 strains of Riemerella anatipestifer) isolated from the liver and brain tissues of diseased ducks in Guangdong, Jiangsu, Guangxi and other regions were selected and stored in the laboratory. The lysis spectrum of the phage combination was detected by the double-layer plate method. The host bacteria information and lysis spectrum detailed results used in the detection are shown in Table 2.

[0110] Preparation method of phage composition: proliferate two phages according to the phage proliferation method in Example 1, and then mix Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15 at a living body quantity ratio of 1:1 to prepare a phage composition.

[0111] 2. Experimental results and analysis

[0112] The results showed that the Escherichia coli phage PD211 could lyse 17 of the 20 strains of Escherichia coli, with a lysis rate of 85% for Escherichia coli, but did not lyse Riemerella anatipestifer, and the total lysis rate for 40 host bacteria was 42.5%; the Riemerella anatipestifer phage PT15 could lyse 17 of the 20 strains of Riemerella anatipestifer, with a lysis rate of 85% for Riemerella anatipestifer, but did not lyse Escherichia coli, and the total lysis rate for 40 host bacteria was 42.5%; the total lysis rate of the phage combination composed of the above two phages was 85.00%; it can be seen that the phage combination has a good lysis effect and broad spectrum for mixed infection of Escherichia coli and Riemerella anatipestifer, and its lysis ability is maximized, which can make up for the limitations of the lysis spectrum of a single phage.

[0113] Table 2 Phage and its combination lysis results

[0114]

[0115]

[0116] Note: +: Lysed, with clear plaques; -: Unlysed, without plaques.

[0117] Example 8 Safety Test of Bacteriophage

[0118] 1. Experimental methods:

[0119] Sixty one-day-old Cherry Valley ducks were isolated and reared for 2 days before the bacteriophage safety test. They were divided into experimental group 1 (15 ducks), experimental group 2 (15 ducks) and control group 1 (15 ducks), control group 2 (15 ducks). Each duckling in experimental group 1 was gavaged with 1×10 10 PFU / mL phage composition 1 ml, test group 2 each duckling leg subcutaneous injection 1 × 10 10PFU / mL phage composition 1ml; control group 1 each duckling was gavaged with 1ml sterile saline, and control group 2 each duckling was injected with 1ml sterile saline. The ducks in each group were raised in the same environment for 2 weeks to observe clinical signs, and the ducks were dissected to observe visceral lesions.

[0120] 2. Experimental results and analysis

[0121] The results showed that within 2 weeks of treatment with the phage combination, there were no significant differences in the clinical symptoms and autopsy of the ducks in the test group and the control group; none of the test ducks died, and no abnormal phenomena such as ruffled feathers, poor spirits, emaciation, loss of appetite, etc. were observed; the autopsy of the ducks in the test group showed no serositis, no lesions in other internal organs, good local absorption of the injection, and no inflammatory nodules or residues. The above results indicate that the phage combination is safe and has no toxic side effects.

[0122] Example 9: Experimental study on the treatment of duckling serositis caused by Escherichia coli and Riemerella anatipestifer using bacteriophage composition

[0123] 1. Experimental method: 100 ducks aged 7 days were selected and divided into 5 groups (20 ducks / group), including blank group, challenge group and treatment group. The treatment group was further divided into 2 single phage treatment groups and phage combination treatment groups. The two bacterial solutions were mixed in a ratio of 1:1 to form a mixed bacterial solution with a bacterial concentration of 5×10 7 CFU / mL, the challenge group, the single phage treatment group and the phage combination treatment group were injected with challenge (0.2 ml / animal), and half an hour after the challenge, 1 ml 1×10 8 The ducks were treated with a phage composition containing PFU / mL of phage. After 3 consecutive days of treatment, the ducks were kept and observed for 14 days, and the number of duck deaths was recorded every day. All surviving ducks were killed after 14 days to observe whether they had perihepatitis, pericarditis and other pathological conditions.

[0124] 2. Experimental results and analysis:

[0125] The results are as follows Fig.11 As shown, there was no death in the blank group, no diarrhea, pericarditis, perihepatitis and other pathological symptoms; the mortality rate of the attack group reached 90%, and the pathological rate was 100%; the mortality rate of the PD211 treatment group was 50%, and the pathological rate was 70%; the mortality rate of the PT15 treatment group was 45%, and the pathological rate was 75%; the mortality rate of the PD211+PT15 treatment group was 15%, and the pathological rate was 10%. The death protection rate and pathological protection rate of the PD211+PT15 treatment group were both greater than 85%, indicating that the phage combination can significantly reduce the mortality rate of ducklings and effectively reduce the degree of pericarditis and perihepatitis in ducklings.

[0126] Example 10 Experiment on the prevention of bacterial serositis in duck farming by bacteriophage composition

[0127] 1. Experimental methods:

[0128] Four duck houses with 5,000 one-day-old broiler ducks were selected from a duck farm in Guangdong and divided into two single phage groups, one phage combination group, and one control group. The mixture was mixed into the duckling feed, with 500 g of phage mixture per ton of feed, and the phage content was 1×10 8 PFU / g. According to the feeding procedures of the farm, the ducks were fed mixed feed containing bacteriophages at 20 days and 35 days of age, respectively, for 3 consecutive days each time, and observed until they were slaughtered. The control group and the experimental group were raised normally according to the procedures in the farm, and the survival rate, growth rate, uniformity and feed conversion rate of the duck flock were measured.

[0129] Autopsy of ducks that died during the breeding process showed fibrous exudate, pericarditis and perihepatitis.

[0130] 2. Experimental results and analysis

[0131] The results are as follows Fig.12 As shown, during the prevention stage when the phage combination was mixed with feed, the survival rate of the phage combination was 3.8% higher than that of the control group, which was higher than the survival rate of the single phage group; the weight of each duck in the phage combination test group was 68.2g higher than that in the control group (P<0.05); at the same time, the feed conversion rate of the phage combination was also higher than that of the control group; this indicated that the use of the phage combination could improve the survival rate of the duck flock, and according to the daily weight gain and feed conversion rate of the ducks, the use of the phage combination had a certain promoting effect on improving the production performance of the duck flock.

[0132] Example 11 Phage disinfection test for duck farm environment

[0133] 1. Experimental methods

[0134] A Cherry Valley duck farm in Shandong Province randomly selected 15 one-day-old duck houses, including 9 experimental groups, 3 control groups, and 3 blank controls. The experimental groups included 2 single phage groups and a phage combination group (3 phages / 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 and Riemerella anatipestifer in the environment before and after disinfection were detected. The central point of the duck 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 Escherichia coli) and a trypsin soy agar (TSA) medium (for Riemerella anatipestifer) were placed at each point. After sampling, the SS culture dish was placed in a 37℃ constant temperature incubator, and the TSA culture dish was cultured anaerobically. The number of bacterial colonies in each culture dish was recorded after 18-24h of culture.

[0135] 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).

[0136] 2. Experimental results and analysis

[0137] like Fig.13 As shown, the total content of Escherichia coli and Riemerella anatipestifer in the phage composition test group environment is lower than that in the control group (84 disinfectant disinfection) and the control group, and is also lower than the total content of Escherichia coli and Riemerella anatipestifer in the single phage group environment. This shows that the phage composition (PD211+PT15) can significantly reduce the total content of Escherichia coli and Riemerella anatipestifer in the environment and reduce the infection rate of the two bacteria; relative to the 84 disinfectant used in the control group, the phage composition (PD211+PT15) has the advantages of safety, high efficiency, non-toxic side effects, and environmental friendliness. Escherichia coli is always present in the environment and can cause disease in animals under certain conditions. Phages can be present in the disinfection site for a long time as disinfectants, and continue to inhibit bacteria. The phages have no irritating odor and are non-irritating to the respiratory mucosa of the animal body. Therefore, the phages and phage compositions of the present application can be used as a new type of biological environmental disinfectant.

[0138] Example 12 Phage Lysis Test on Non-Host Bacteria

[0139] 1. Experimental methods

[0140] A total of 30 strains of non-host bacteria, including 10 strains of Salmonella, 5 strains of Clostridium perfringens, 5 strains of Staphylococcus, and 5 strains of Proteus, were selected, and a phage lysis experiment was performed according to the lysis spectrum determination method in Example 7.

[0141] 2. Experimental results and analysis

[0142] Escherichia coli phage PD211 and Riemerella anatipestifer phage PT15 did not lyse the 25 non-host bacteria selected above, as shown in Table 3 for details, indicating that phage PT15 and PD211 were unable to recognize the above-mentioned 10 strains of Salmonella, 5 strains of Clostridium perfringens, 5 strains of Staphylococcus, and 5 strains of Proteus, indicating that the tested phages have extremely strong host specificity and have no damaging effect on the microbial community.

[0143] Table 3 Lysis results of phage and its combination on non-host bacteria

[0144]

[0145]

[0146] 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. An Escherichia coli phage PD211, characterized in that: Its accession number is CGMCC No.22362.

2. A bacteriophage composition, characterized in that: include: The Escherichia coli phage PD211 and the Riemerella anatipestifer phage PT15 as claimed in claim 1, wherein the Riemerella anatipestifer phage PT15 has a deposit number of CGMCC No. 22377.

3. Use of the Escherichia coli phage PD211 according to claim 1 or the phage composition according to claim 2 in the preparation of drugs for preventing and treating duck bacterial diseases, feed additives, and environmental disinfectants.

4. The use according to claim 3, characterized in that: The duck bacterial diseases include duck serositis caused by duck Escherichia coli or caused by duck Escherichia coli and Riemerella anatipestifer.

5. A bacteriophage preparation, characterized in that: The active ingredient comprises the Escherichia coli phage PD211 as claimed in claim 1 or the phage composition as claimed in claim 2.

6. The bacteriophage preparation according to claim 6, characterized in that It also contains a pharmaceutically acceptable carrier, and the drug dosage form is an oral dosage form, an external dosage form and an injection dosage form.

7. A feed additive, characterized in that: The method comprises the Escherichia coli phage PD211 as claimed in claim 1 or the phage composition as claimed in claim 2.

8. An environmental disinfectant, characterized in that The active ingredient comprises the Escherichia coli phage PD211 as claimed in claim 1 or the phage composition as claimed in claim 2.

9. The environmental disinfectant according to claim 8, characterized in that It also contains other active ingredients that are used to inhibit or eliminate bacteria in the environment.

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