Riemerella anatipestifer phage as well as composition and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a riemerella anatipestifer bacteriophage as well as a composition and application thereof. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 29, 2023, and the preservation number is CGMCC No.45785. The invention further discloses a preparation method of the strain. A composition comprises the bacteriophage and a riemerella anatipestifer inactivated vaccine. The invention discloses a method for quickly establishing immune protection on riemerella anatipestifer in a duck group. An effective amount of the composition is applied to the duck group in need. According to the invention, the specific bacteriophage RDP-RA-22010 and the inactivated vaccine are synergistically compounded, so that the conventional cognition of an immune blank period is broken through. The composition can provide the challenge protection rate up to 90% in the third day after inoculation, while the protection rate of a common vaccine group in the same period is only 40%. And the effective protection time is shortened to 3 days from the traditional 7-14 days. The pathogen inhibition effect and the animal protection rate of the vaccine are far better than the sum of the effects of a phage or a vaccine which are independently used, and reach 1 + 1gt; and 2, synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Riemerella anatipestifer bacteriophage, its composition, and its applications. Background Technology

[0002] Avian serositis is a common poultry disease, primarily caused by Riemerella anatipestifer, which seriously affects poultry health and the economic benefits of poultry farming. Currently, the main treatments for Riemerella anatipestifer infection include: (1) Antibiotic treatment: Commonly used drugs include florfenicol and enrofloxacin, but long-term use can easily lead to drug resistance; (2) Vaccination: Inactivated or subunit vaccines can be used to improve poultry immunity, but they cannot directly eliminate infected pathogens. Furthermore, there is a 10-14 day gap in immunity after vaccination; if infection with Riemerella anatipestifer occurs during this period, it will result in immunization failure. Currently, the usual solution for this gap is to inject antibodies one week after inactivated vaccine vaccination to shorten the gap, but in practice, a relatively long gap has been observed. (3) Phage therapy: This therapy utilizes the specific lysis properties of bacteriophages to treat bacteria. Phages are highly specific, can quickly enter the bloodstream, and have a fast bactericidal speed and obvious effect. However, they are prone to inducing bacterial resistance mutations. In addition, their effect is unstable when used alone, and they are poorly adapted to complex infection environments.

[0003] Therefore, how to safely and effectively shorten or even eliminate the immune gap period of vaccines and achieve a seamless connection between immediate and long-term immunization is a long-standing but unsolved technical problem in this field.

[0004] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain of Riemerella anatipestifer bacteriophage, its composition, and its applications.

[0006] The technical solution of this invention is as follows: On the one hand, the present invention provides a strain of Riemerella anatipestiferbacteriophage, named RDP-RA-22010, which was deposited at the China General Microbiological Culture Collection Center on November 29, 2023, with accession number CGMCC No. 45785.

[0007] On the other hand, the present invention provides a composition comprising the above-mentioned Riemerella anatipestifer phage and Riemerella anatipestifer inactivated vaccine.

[0008] On the one hand, on the 3rd day after intramuscular inoculation of 14-day-old SPF ducks with this composition, at a dose of 1×10 9 When challenged with a highly virulent strain of Riemerella anatipestifer at a dose of CFU / mL, the survival rate of ducks was no less than 90%.

[0009] On the one hand, its valence is not less than 1×10 9 The inactivated vaccine was prepared by mixing phage proliferation fluid with PFU / mL and inactivated bacterial solution at a volume ratio of 1 to 10:1.

[0010] On the one hand, the mixing volume ratio of the phage proliferation solution to the inactivated bacterial solution is 4:1.

[0011] On the other hand, the composition is a water-in-oil emulsion, and the titer of phage RDP-RA-22010 in the final composition is ≥1×10⁻⁶. 8 pfu / mL.

[0012] On the other hand, after intramuscular injection, the composition showed detectable bacteriophages in the blood after 2 hours, reaching peak efficacy at 8-12 hours, with a titer of 10. 6 pfu / mL.

[0013] On the one hand, the present invention provides the use of the above-mentioned Riemerella anatipestifer phage RDP-RA-22010 in at least one of the following (1) to (4): (1) Prepare products that kill Riemerella anatipestifer; (2) Prepare products that inhibit Riemerella anatipestifer; (3) Prepare products for the prevention and / or treatment of poultry diseases caused by Riemerella anatipestifer; (4) In the preparation of drugs for shortening the immunization gap period of inactivated duck disease Riemerella anatipestifer vaccine.

[0014] On the other hand, the present invention provides a method for rapidly establishing immune protection against Riemerella anatipestifer in a flock of ducks, comprising the following steps: Administer an effective amount of the composition as described in any one of claims 1 to 7 to a flock of ducks in need.

[0015] The application of this composition can provide duck flocks with at least 90% protection against virulent Riemerella anatipestifer within 3 days.

[0016] On the other hand, the method of administration is intramuscular injection in the leg, and the dosage is 0.25 mL per duck.

[0017] The beneficial effects achieved by this invention are as follows: This invention breaks with conventional understanding of the immune gap period through the synergistic combination of a specific bacteriophage, RDP-RA-22010, and an inactivated vaccine. This composition provides up to 90% protection against viral challenge as early as day 3 post-vaccination, compared to only 40% protection in the conventional vaccine group at the same time. This effectively shortens the effective protection period from the traditional 7-14 days to 3 days. The pathogen inhibition effect and animal protection rate of this invention far exceed the sum of the effects of using the bacteriophage or vaccine alone, achieving a synergistic effect greater than the sum of its parts (1+1>2). Attached Figure Description

[0018] Figure 1 This is a streak culture result of Riemerella anatipestifer on a culture medium.

[0019] Figure 2 This is a photograph of the plaque of the phage RDP-RA-22010 of this invention.

[0020] Figure 3 This is an electron microscope image of the bacteriophage RDP-RA-22010 of this invention.

[0021] Figure 4 This is a growth curve diagram of the bacteriophage RDP-RA-22010 of the present invention.

[0022] Figure 5 This is a graph showing the effect of the bacteriophage vaccine complex of the present invention on Riemerella anatipestifer.

[0023] Figure 6 This is a graph showing the survival rate results of the virus challenge protection experiment of this invention.

[0024] Figure 7 This is a graph showing the determination of phage content in the blood of SPF ducklings using the phage vaccine complex of this invention.

[0025] Figure 8 This is an experimental survival rate graph showing the shortened immune gap period of the phage vaccine complex of this invention. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, Riemerella anatipestifer phage RDP-RA-22010 is referred to simply as "phage RDP-RA-22010" or "RDP-RA-22010".

[0028] Example 1: Isolation and Identification of Riemerella anatipestifer 1. Isolation and screening of Riemerella anatipestifer Ducks suspected of being infected with Riemerella anatipestifer were collected from Shandong Province. They were then dissected using aseptic techniques. A sample of duck brain was collected using a sterile inoculation loop and streaked onto TSA agar containing 5% sheep blood. The samples were then incubated at 37°C in a 5% CO2 incubator for 24 hours. The incubation results (e.g., ...) were recorded. Figure 1 (As shown in the image), then smooth, milky white, opaque colonies with neat edges and bead-like or semi-circular protrusions were selected for purification. Gram-negative short bacilli were identified after staining. Simultaneously, single colonies were streaked onto MacConkey agar plates, but no bacterial growth was observed. Based on this, it was initially identified as a suspected strain of *Riemerella anatipestifer*. After 16S rRNA amplification, the PCR product was sent to BGI Genomics for sequencing. The sequencing results, after BLAST alignment, confirmed *Riemerella anatipestifer*, and named BRA-22010.

[0029] 2. Serotyping of Riemerella anatipestifer The purified *Riemerella anatipestifer* was inoculated onto a TSA plate containing 5% serum and incubated at 37°C with 5% CO2 for 24 hours. A suitable amount of bacterial growth was scraped off with a sterile inoculation loop and mixed in 1 mL of physiological saline. Then, 10 μL of bacterial suspension was added to a clean glass slide, along with 10 μL of positive serum. The mixture was then agitated using a pipette tip and the agglutination results were read within one minute under bright sunlight.

[0030] The serotype of Riemerella anatipestifer in this patent has been identified as type 1.

[0031] Example 2: Isolation, identification and biological characteristics of bacteriophage RDP-RA-22010 1. Isolation of Riemerella anatipestifer bacteriophage 5g of fresh feces collected from different farms in Shandong Province were soaked in 15mL of sterile water, shaken thoroughly, and soaked for 30min. The soaking solution was then centrifuged, and the supernatant was filtered through a 0.22μm filter to obtain bacteriophage filtrate for later use. 3mL of filtrate and 600μL of fresh bacterial suspension were inoculated into an Erlenmeyer flask containing 30mL of TSB medium and incubated at 37℃ for 24h with shaking at 160rpm to obtain bacteriophage enrichment solution. The enrichment solution was then centrifuged, and the supernatant was filtered through a 0.22μm filter to obtain bacteriophage enrichment solution. 0.2mL of filtrate was then mixed with 0.2mL of bacterial suspension and immediately added to TSA semi-solid agar. The mixture was then plated in double layers and incubated in a 37℃ incubator containing 5% carbon dioxide for 20h. After the appearance of phage plaques, a single phage plaque was picked, soaked in 1mL of physiological saline for 15min, diluted to an appropriate dilution factor, and then plated in double layers again for purification of the phage. A total of 5 purification processes were performed. The phage was isolated by the double plate method, and one phage strain was obtained and named RDP-RA-22010.

[0032] Then, phage plaques were picked from the purified plates and added to 5 mL of TSB medium, along with 0.1 mL of host bacteria. After inoculation, the plates were incubated at 37°C in a CO2 incubator for 10 hours. The prepared phage proliferation solution was then sterilized using a 0.22 μm pore size filter and inoculated into 100 mL of TSB medium, along with 5 mL of host bacteria. The plates were incubated at 37°C in a CO2 incubator for 10 hours. After incubation, a 10-fold serial dilution was performed, and the plates were plated on double plates using an appropriate dilution gradient. The plates were then incubated at 37°C in a CO2 incubator for 20 hours. The titer of the phage proliferation solution was determined to be 7 × 10⁻⁶. 9 PFU / mL. Its plaque photographs are as follows: Figure 2 As shown.

[0033] 2. Morphological observation of Riemerella anatipestifer. Using phosphotungstic acid negative staining: drop bacteriophage fluid (titer 10) onto a paraffin slide. 10 For the phage droplet, place 100 μL of copper mesh (pfu / mL) with the membrane side on it. After 10 min, remove the mesh and allow it to air dry for 3 min. Then, place a drop of 2% phosphotungstic acid (PTA) aqueous solution on the copper mesh for staining. After 10 min, remove the mesh and allow it to air dry for 15 min. Observe the mesh with an electron microscope and take a picture of the clear phage image.

[0034] Depend on Figure 3 Electron micrographs show that the bacteriophage RDP-RA-22010 is a long-tailed bacteriophage with a regular hexagonal head, a head diameter of about 45-55 nm, and a tail length of 150-200 nm.

[0035] 3. Whole genome sequencing of Riemerella anatipestifer phage After enriching and culturing a single bacteriophage, RDP-RA-22010, the cells were centrifuged at 8000g for 15 min at 4°C. 10% PEG8000 and 0.5M NaCl were added and the mixture was allowed to stand overnight. An equal volume of chloroform was then added and mixed thoroughly. After separation, the cells were centrifuged at 5000g for 10 min. The chloroform and PEG layers were removed, and the cells were digested with restriction endonucleases. The cells were then suspended in cesium chloride at a gradient density. The cells were dialyzed three times with TM (Temperature Melting) buffer for 30 min each time. Finally, a portion of the dialyzed cells was reserved for electron microscopy, and the remainder was sent to BGI Genomics for whole-genome sequencing.

[0036] The phage sequence is shown in SEQ ID NO.1. The complete genome sequence of the phage is 31237 bp, with 50 open reading frames. No genes encoding lysogenicity, antibacterial activity, virulence, or toxin-antitoxin related genes were detected.

[0037] 4. Growth curve of RDP-RA-22010 bacteriophage of Riemerella anatipestifer Take 1×10 9 pfu / mL of Rimora anatipestifer phage RDP-RA-22010 and 1×10 8 The host bacteria (cfu / mL) were mixed at a 1:1 volume ratio and incubated at 37 °C for 10 min to allow the phages to adsorb onto the host bacteria. The mixture was then centrifuged at 10000 r / min for 5 min, the supernatant was discarded, and the phage was resuspended in an equal volume of LB broth. This process was repeated twice to remove any unadsorbed phages. 10 mL of this liquid was added to 90 mL of LB broth. Samples (1 mL) were taken at regular intervals (i.e., samples at 0 min, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 420 min) and centrifuged at 10000 r / min for 5 min. The supernatant was then used to determine the phage titer using the double-layer plate method.

[0038] Growth curves as follows Figure 5 As shown, the incubation period for bacteriophages is from 0 to 180 minutes. From 180 to 360 minutes, the phage titer shows a rapid upward trend. During this stage, progeny phages within the host cell mature, the host cell begins to lyse, and a large number of progeny phages are released into the culture medium, leading to a sharp increase in titer. This reflects the phage lysis and release process, and is known as the lysis phase. After 360 minutes, the phage titer tends to stabilize, maintaining at approximately 210 × 10⁻⁶. 7pfu / mL. This is because most host cells have lysed, the release of progeny phages has reached its peak, and there are no new host cells available for phage proliferation. Therefore, the titer no longer changes, and this is the stationary phase of the phage. The entire process of phage proliferation and lysis within the host cell has clear stages, and ultimately releases a large number of progeny phages, demonstrating strong lytic ability.

[0039] Example 3: Lysis spectrum experiment of RDP-RA-22010 bacteriophage of Riemerella anatipestifer The lysis profile of 159 strains of *Riemerella anatipestifer* was tested using RDP-RA-22010. The specific steps were as follows: TSB medium containing 0.7% agar was heated and melted. When the medium cooled to about 55°C, 5 mL was added to a sterile test tube, followed by 200 μL of *Riemerella anatipestifer* suspension. The mixture was quickly shaken and then poured into a petri dish containing the bottom layer of medium. After solidification, 5 μL of RDP-RA-22010 phage solution was added. The dish was then incubated at 37°C for 24 hours. If plaques were observed, the phages could lyse the pathogen; otherwise, they could not.

[0040] Table 1: Detailed Results of Fracturing Spectra

[0041] Note: "-" indicates non-lytic; "+" indicates lytic; "--" indicates unknown serotype. The experimental results are shown in Table 1. The lysis rate of bacteriophage RDP-RA-22010 against 159 strains of bacteria in the laboratory was 63%. This bacteriophage has a high lysis spectrum and has great application potential.

[0042] Example 4: Preparation and in vitro efficacy verification of phage-vaccine complex 1. Preparation of a complex of Riemerella anatipestifer bacteriophage and vaccine S1 Preparation of bacterial suspension: Take one vial of cryopreserved solution from the -80℃ freezer and thaw it rapidly within 30 seconds in a 37℃ water bath. Take 0.5 mL of the cryopreserved solution and inoculate it into 5 mL of TSB medium containing 5% serum. Incubate at 37℃ with shaking for 24 h. Then, perform the first scale-up culture by inoculating the bacterial suspension in the test tube into 200 mL of TSB medium containing 5% serum. Incubate at 37℃ with shaking for 24 h. Then, perform the second scale-up culture by taking 40 mL of the bacterial suspension from the first scale-up culture and inoculating it into 1 L of TSB medium containing 5% serum. Incubate at 37℃ with shaking for 24 h.

[0043] S2 Phage Propagation: Take one vial of phage cryopreservation solution from a -80℃ freezer and thaw it rapidly within 30 seconds in a 37℃ water bath. Take 0.5 mL of the cryopreservation solution and 0.2 mL of the bacterial solution from the first expansion culture in step S1 and inoculate them simultaneously into 5 mL of TSB medium containing 5% serum. Place the tube in a shaker at 37℃ for 24 h. Then, perform the first expansion culture of the phage. Take 5 mL of activated phage solution and 10 mL of the bacterial solution from the first expansion culture in step S1 and inoculate them simultaneously into 200 mL of TSB medium containing 5% serum. Place the tube in a shaker at 37℃ for 24 h. Immediately afterward, perform the second expansion culture. Take 40 mL of the bacterial solution from the first expansion culture in step S1 and 40 mL of the phage solution produced in the first expansion culture in step S2 and inoculate them simultaneously into 1000 mL of TSB medium containing 5% serum. Place the tube in a shaker at 37℃ for 24 h.

[0044] S3 Inactivation: Take the bacterial solution from step S1 and centrifuge at 10,000 rpm for 20 min. Discard the supernatant, then add 200 mL of sterile PBS solution to resuspend the bacterial solution. Next, add formaldehyde at 0.1% of the bacterial solution volume and place in a shaker at 37°C for 24 h to inactivate the bacterial strain.

[0045] S4 Immunogen Preparation: First, centrifuge the phage proliferation solution from step S2, and sterilize the supernatant by passing it through a 0.22-micron filter. Then, take 800 mL of the sterilized phage solution and mix it with 200 mL of the inactivated bacterial solution from step S3. Add sterile Tween-80 to the mixture to achieve a final concentration (by volume) of 2.0%.

[0046] Preparation of S5 oil phase: Add 10g of aluminum stearate to 94 parts of white oil and 4 parts of Span-80 (by volume), heat to dissolve into a transparent liquid, and then bottle and autoclave at 121℃ for 20min.

[0047] S6 vaccine preparation: Mix the oil phase of S5 and the immunogen phase of S4 in a 2:1 ratio (volume ratio). First, add 2000 mL of the oil phase to the premixing tank, adjust the speed to 6000 rpm, and add 1000 mL of the immunogen phase while stirring. Then increase the speed to 20000 rpm and emulsify for 30 min.

[0048] After compounding, the titer of the above-mentioned bacteriophages is ≥1×10⁻⁶. 8 pfu / mL.

[0049] 2. Effect curve of phage vaccine complex on Riemerella anatipestifer BRA-22010 This experiment was designed with two groups: one group containing Riemerella anatipestifer BRA-22010 and the other containing a bacteriophage vaccine complex, to verify the bactericidal ability of the bacteriophage in the complex.

[0050] First, take two conical flasks containing 100 mL of sterile TSB broth. Inoculate one flask with 2 mL of Riemerella anatipestifer and 2 mL of sterile saline, and inoculate the other flask with 2 mL of Riemerella anatipestifer and 2 mL of phage vaccine complex. Incubate at 37°C and 160 rpm in a shaker. After every 1 hour, take 1 mL from each flask and perform a 10-fold serial dilution. Use the pour method to determine the viable count.

[0051] Depend on Figure 5 It can be seen that Riemerella anatipestifer enters the logarithmic phase after 5 hours of culture and enters the stationary phase after 7 hours. After inoculation with Riemerella anatipestifer vaccine complex, Riemerella anatipestifer failed to grow normally and was continuously inhibited for more than 20 hours.

[0052] Example 5: Immunogenicity of phage vaccine complex and animal challenge protection experiment 1. Immunogenicity assay of Riemerella anatipestifer bacteriophage and vaccine complex Ten 150-day-old laying hens were selected, with five in the immunization group and five in the control group. Each hen was subcutaneously inoculated with 0.5 ml of the solution, followed by a booster immunization of 1.0 ml per hen 15 days later. Eggs were collected two weeks later, and the yolks were diluted with physiological saline at a ratio of 1:3 (v / v). An equal volume of chloroform was added, and the mixture was thoroughly shaken, extracted, and centrifuged to separate the supernatant, which was then used as the test sample. The yolk antibody titer was then determined using the agar diffusion assay. The immunization group showed an effective yolk antibody titer of 1:16, while the control group showed a negative yolk antibody titer.

[0053] 2. Virus challenge protection experiment Prepare 40 14-day-old SPF ducklings and randomly divide them into 4 groups: treatment group, prevention group, challenge group, and blank group, with 10 ducklings in each group.

[0054] The treatment group was first challenged with Riemerella anatipestifer BRA11 via intramuscular injection in the leg, and then treated with 0.5 mL of phage and vaccine complex via intramuscular injection in the leg.

[0055] The prevention group first received an intramuscular injection of a phage vaccine complex in the leg, followed by an intramuscular injection of 0.5 mL of live bacteria (1×10⁶) in the leg 14 days later. 9 The bacteria were challenged with CFU / mL of Riemerella anatipestifer BRA11.

[0056] The control group received only a saline injection in the leg without undergoing viral challenge.

[0057] The challenge group received only an injection of Riemerella anatipestifer BRA11 into the leg.

[0058] Depend on Figure 6It can be seen that the phage vaccine complex of this embodiment has a control effect on Riemerella anatipestifer. The challenge group died within 24 hours, the survival rate of the infected group was only 50%, the survival rate of the treated ducklings reached 90%, and the survival rate of the prevented ducklings reached 100%. This shows that the phage and vaccine complex of this embodiment has both therapeutic and preventive effects.

[0059] Example 6: Determination of phage content in the blood of SPF ducklings using phage vaccine complexes First, prepare 20 14-day-old SPF ducklings and randomly divide them into two groups. One group is the experimental group, which receives the drug via intramuscular injection in the leg, with each duckling receiving 0.25 mL of the drug at a potency of 1×10⁻⁶. 9 A phage vaccine complex of pfu / mL was administered, with one group serving as the control group. Each duck was given 0.25 mL of sterile saline once, in the same manner. Blood was collected from the wing veins of ducks at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h post-administration. The blood was then incubated at 4℃ for 1h, followed by centrifugation at 3500 r / min for 10 min at 4℃. The serum was filtered for sterilization, serially diluted 10-fold with sterile saline, and the phage titer in the blood was determined using the double-layer plate method.

[0060] Depend on Figure 7 It can be seen that bacteriophages can be detected in the blood 2 hours after intramuscular injection of the phage vaccine complex, reaching peak titer at 8-12 hours, with a titer of 10. 6 The concentration of phages decreased over time, reaching pfu / mL. Phages were still detectable at 196 hours, but the concentration was low at 10 pfu / mL. 2 pfu / mL. No bacteriophages were detected in the control group. Therefore, the bacteriophages in the bacteriophage vaccine complex of this patent can be administered by injection and reach the target organ through blood circulation to exert a bactericidal effect.

[0061] Example 7: Experiment on the shortening of the immune gap period by phage vaccine complex First, 70 14-day-old SPF ducklings were prepared. Ten ducklings were designated as the challenge group, and the remaining 60 were randomly divided into two groups of 30 each. Group One received the phage vaccine of this patent via intramuscular injection in the leg (0.25 mL of the patented phage and vaccine complex). Then, they were randomly divided into three groups of 10 each. Group 1-1 was challenged on day 3, Group 1-2 on day 14, and Group 1-3 served as a blank control. Group Two received a conventional vaccine without phage via intramuscular injection in the leg (0.25 mL). Then, they were randomly divided into three groups of 10 each. Group 2-1 was challenged on day 3, Group 2-2 on day 14, and Group 2-3 served as a blank control.

[0062] Depend on Figure 8 It can be seen that the survival rate of ducklings challenged with the virus but not immunized was only 50%, while the survival rate of ducklings challenged with the ordinary vaccine group on the 3rd day after vaccination was 40%, indicating that the vaccine failed to take effect at this time, and immunization failed. The survival rate of ducklings challenged with the virus on the 14th day after vaccination was 90%, indicating that the vaccine produced antibodies with good preventive effect. The survival rate of ducklings challenged with the bacteriophage and vaccine groups on the 3rd day after vaccination was 90%, far higher than that of the ordinary vaccine group, thus indicating that the vaccine immunization gap period was shortened and the immunization effect was improved.

[0063] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of *Riemerella anatipestifer* bacteriophage, characterized in that, The name is RDP-RA-22010, and it was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 29, 2023, with the accession number CGMCC No. 45785.

2. A composition, characterized in that: Includes the Riméae davidiana bacteriophage and the Riméae davidiana inactivated vaccine as described in claim 1.

3. The composition according to claim 2, characterized in that: On day 3 following intramuscular inoculation of 14-day-old SPF ducks with this composition, at a dose of 1×10 9 When challenged with a highly virulent strain of Riemerella anatipestifer at a dose of CFU / mL, the survival rate of ducks was no less than 90%.

4. The composition according to claim 2, characterized in that: Its potency is not less than 1×10 9 The inactivated vaccine was prepared by mixing phage proliferation fluid with PFU / mL and inactivated bacterial solution at a volume ratio of 1 to 10:

1.

5. The composition according to claim 4, characterized in that: The volume ratio of the phage proliferation solution to the inactivated bacterial solution is 4:

1.

6. The composition according to claim 2, characterized in that: The composition is a water-in-oil emulsion, and the titer of phage RDP-RA-22010 in the final composition is ≥1×10⁻⁶. 8 pfu / mL.

7. The composition according to claim 2, characterized in that: After intramuscular injection, the composition showed detectable bacteriophages in the blood 2 hours later, reaching peak efficacy at 8-12 hours with a titer of 10. 6 pfu / mL.

8. The use of the RDP-RA-22010 phage of *Rimerella anatipestifer* according to claim 1 in at least one of the following (1) to (4): (1) Prepare products that kill Riemerella anatipestifer; (2) Preparation of bacterial products that inhibit Rimolecular bile duct disease in ducks; (3) Prepare products for the prevention and / or treatment of poultry diseases caused by Riemerella anatipestifer; (4) In the preparation of drugs for shortening the immunization gap period of inactivated duck disease Riemerella anatipestifer vaccine.

9. A method for rapidly establishing immune protection against Riemerella anatipestifer in a duck flock, characterized in that, Includes the following steps: Administer an effective amount of the composition as described in any one of claims 1 to 7 to a flock of ducks in need; The application of this composition can provide duck flocks with at least 90% protection against virulent Riemerella anatipestifer within 3 days.

10. The method according to claim 9, characterized in that: The administration method is intramuscular injection in the leg, and the dosage is 0.25 mL per duck.

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