Bacteriophage of rimerella anatis and use thereof

Abstract

The application discloses two duck Riemerella anatipestifer bacteriophages and application thereof, and particularly relates to the technical field of bacteriophages, and the duck Riemerella anatipestifer bacteriophage RAP7ARASD24 is preserved in the Guangdong Microbial Culture Collection Center on July 2, 2026, and the preservation number is GDMCC NO. 67616-B1; the duck Riemerella anatipestifer bacteriophage RAP16ARASD8 is preserved in the Guangdong Microbial Culture Collection Center on July 2, 2026, and the preservation number is GDMCC NO. 67617-B1. The bacteriophages provided in the application have a wide host range for duck Riemerella anatipestifer, strong lysis ability (can recognize part of Elizabethkingia meningitidis), and a recognition rate of up to 94% or more for duck Riemerella anatipestifer of different serotypes.

Description

Technical Field

[0001] This application relates to the field of phage technology, and in particular to Riemerella anatipestifer phage and its applications. Background Technology

[0002] Riemerella anatipestifer is a common bacterial infectious disease in waterfowl farming, primarily infecting ducks, geese, and other waterfowl, with particularly severe damage to ducklings. It commonly manifests as fibrinous pericarditis, perihepatitis, air sacculitis, and meningitis, characterized by high morbidity and mortality, resulting in significant economic losses to the poultry industry. Riemerella anatipestifer has a complex and diverse serotype profile; at least 21 serotypes have been identified to date, and there is a lack of effective cross-protection between different serotypes, further complicating disease control.

[0003] Currently, the prevention and control of Riemerella anatipestifer infection mainly relies on antibiotics and vaccines. However, with the widespread use and even abuse of antibiotics in the aquaculture industry, the problem of antibiotic resistance in Riemerella anatipestifer is becoming increasingly prominent. Studies have shown that this bacterium exhibits varying degrees of resistance to many commonly used antibiotics such as tetracycline, erythromycin, and neomycin, and resistance genes can spread between strains through mobile elements such as plasmids, further exacerbating the difficulty of prevention and control. In addition, although vaccines can provide immune protection to some extent, due to the diversity and high variability of serotypes, existing vaccines cannot cover all circulating strains, resulting in unstable immunization effects and limiting their widespread application.

[0004] Bacteriophages are viruses capable of specifically lysing bacteria. They possess advantages such as strong host specificity, non-disruption of normal flora, self-replication, and low susceptibility to drug resistance, demonstrating great potential in controlling multidrug-resistant bacterial infections. While research on phages targeting Riemerella anatipestifer has made some progress in recent years, challenges remain, including a narrow lytic spectrum, poor environmental tolerance, and insufficient industrial production capacity, limiting their application in practical production.

[0005] Currently, some phages of Riemerella anatipestifer have been isolated and reported, but most of these phages have limited host range, making it difficult to meet the needs of large-scale application. Summary of the Invention

[0006] To improve the lysis ability against Riemerella anatipestifer and to meet its numerous serotypes, this application provides a phage of Riemerella anatipestifer and its applications.

[0007] Firstly, this application provides a strain of Riemerella anatipestifer phage RAP7ARASD24, employing the following technical solution: A strain of *Rimerella anatipestifer* phage RAP7ARASD24, deposited at the Guangdong Provincial Microbial Culture Collection Center on July 2, 2026, with accession number GDMCCNO. 67616-B1, and classified as follows: Riemerella anatipestifer phage RAP7ARASD24.

[0008] In some embodiments, the RAP7ARASD24 bacteriophage of Riemerella anatipestifer has a head with polyhedral three-dimensional symmetry and a short, contracted tail. The head diameter is 75-90 nm, the tail length is 8-10 nm, and the tail diameter is 2-5 nm.

[0009] Secondly, this application provides a strain of Riemerella anatipestifer phage RAP16ARASD8, using the following technical solution: A strain of *Rimerella anatipestifer* phage RAP16ARASD8, deposited at the Guangdong Provincial Microbial Culture Collection Center on July 2, 2026, with accession number GDMCCNO. 67617-B1, and classified as follows: Riemerella anatipestifer phage RAP16ARASD8.

[0010] In some embodiments, the RAP16ARASD8 bacteriophage of Riemerella anatipestifer has a head with polyhedral three-dimensional symmetry and a short, contracted tail. The head diameter is 45–60 nm, the tail length is 4–7 nm, and the tail diameter is 3–4 nm.

[0011] By adopting the above-mentioned technical solutions, the *R. anatipestifer* bacteriophages RAP7ARASD24 and RAP16ARASD8 of this application are virulent bacteriophages isolated from nature, possessing unique morphological characteristics of the short-tailed virus family. They have a broad host range for *R. anatipestifer*, strong lytic ability, and a recognition rate of over 94% for different serotypes of *R. anatipestifer*, without damaging the normal microbial flora, and exhibit excellent environmental tolerance and stability. More importantly, both can partially recognize *Elizabeth Elizabeth Kinsella meningoseptica*, which has high resistance to various antimicrobial drugs. This is of great significance for expanding the host spectrum of phage therapy and developing alternative or adjuvant treatment options for multidrug-resistant *Elizabeth Kinsella meningoseptica* infections.

[0012] Thirdly, this application provides a composition for inhibiting Riemerella anatipestifer, employing the following technical solution: A composition for inhibiting Riemerella anatipestifer, comprising Riemerella anatipestifer bacteriophage RAP7ARASD24 and / or Riemerella anatipestifer bacteriophage RAP16ARASD8.

[0013] By adopting the above technical solution, this application can broaden the host lysis spectrum of the bacteriophage by combining RAP7ARASD24 and RAP16ARASD8 of Riemerella anatipestifer bacteriophage, and recognize and lyse more strains of Riemerella anatipestifer with more serotypes, thus overcoming the limitation of the lysis spectrum that may exist with a single bacteriophage.

[0014] Optionally, it also includes Enterococcus faecalis phage P35AEFLN26, the preservation number of which is CCTCC NO: M2025336.

[0015] By adopting the above technical solution, the composition of this application further incorporates Enterococcus faecalis phage P35AEFLN26, which can significantly expand the antibacterial spectrum and application range of the composition. Enterococcus faecalis is a common opportunistic pathogen in waterfowl farming environments, often forming mixed infections with Riemerella anatipestifer, and it has strong drug resistance, making it difficult to effectively eliminate with traditional antibiotics. After combining Enterococcus faecalis phage P35AEFLN26 with Riemerella anatipestifer phage, the composition can not only efficiently lyse Riemerella anatipestifer, but also specifically inhibit Enterococcus faecalis, achieving synergistic control of mixed infections.

[0016] Fourthly, this application provides a biological bactericide, which adopts the following technical solution: A biocidal agent comprising RAP7ARASD24 and / or RAP16ARASD8 of Rimolectomyces anatipestifer bacteriophage.

[0017] By adopting the above technical solution, the biological bactericide provided in this application uses RAP7ARASD24 and / or RAP16ARASD8 bacteriophages of Riemerella anatipestifer as the core active ingredients, giving full play to the unique advantages of bacteriophages as natural antibacterial biological agents.

[0018] Optionally, the formulation of the biocidal agent is one of the following: solution, powder, gel, granule, or lyophilized agent.

[0019] Optionally, it may also include a chemical disinfectant, including dodecyl dimethyl benzyl ammonium chloride.

[0020] By adopting the above technical solution, the composition of this application further introduces the chemical bactericide dodecyl dimethyl benzyl ammonium chloride, realizing the synergistic combination of biological bacteriophage and chemical disinfectant, significantly improving the overall bactericidal efficacy and environmental adaptability of the composition. Dodecyl dimethyl benzyl ammonium chloride, as a quaternary ammonium salt cationic surfactant, has the characteristics of rapid bactericidal effect, broad-spectrum bacteriostasis and good penetration. It can rapidly destroy the bacterial cell membrane structure and has a killing effect on a variety of pathogenic microorganisms in the environment. When combined with Riemerella anatipestifer bacteriophage, on the one hand, the chemical bactericide can be used to quickly reduce the bacterial load in the environment and control the infection pressure in the early stage of an outbreak; on the other hand, the specific proliferation ability and continuous bacteriostatic effect of the bacteriophage can be used to achieve targeted elimination and long-term control of Riemerella anatipestifer.

[0021] Fifthly, this application provides a drug, which adopts the following technical solution: A drug comprising RAP7ARASD24 and / or RAP16ARASD8 of Riemerella anatipestifer bacteriophage.

[0022] By adopting the above technical solution, the drug provided in this application uses RAP7ARASD24 and / or RAP16ARASD8 bacteriophages of Riemerella anatipestifer as active ingredients, providing a novel, highly effective, and safe biological agent for the treatment and prevention of Riemerella anatipestifer infection.

[0023] Sixthly, this application provides a feed additive, which adopts the following technical solution: A feed additive comprising RAP7ARASD24 and / or RAP16ARASD8 of Rimolectomyces anatipestifer bacteriophage.

[0024] By adopting the above technical solution, the feed additive provided in this application uses RAP7ARASD24 and / or RAP16ARASD8 phages of Riemerella anatipestifer as its core functional components, providing a green, safe, and sustainable feed solution for the prevention and control of Riemerella anatipestifer disease in waterfowl farming. This feed additive exhibits good pelleting tolerance and storage stability. After being prepared into powder through spray drying or freeze drying processes, the phage activity remains good, and the titer does not decrease significantly, allowing it to adapt to environmental conditions during feed processing and storage.

[0025] Seventhly, this application provides a reagent kit, which adopts the following technical solution: A kit comprising RAP7ARASD24 and / or RAP16ARASD8 phages of Riemerella anatipestifer.

[0026] By adopting the above technical solution, the kit provided in this application uses RAP7ARASD24 and / or RAP16ARASD8 bacteriophages of Riemerella anatipestifer as specific recognition elements, providing an efficient tool for rapid, sensitive, and accurate detection of Riemerella anatipestifer. This kit utilizes the highly specific recognition mechanism between bacteriophages and their host bacteria, enabling accurate detection of target pathogens from complex samples, effectively avoiding false positives or cross-reactions that may occur in traditional detection methods.

[0027] In summary, this application includes at least one of the following beneficial technical effects: 1. The RAP7ARASD24 and RAP16ARASD8 bacteriophages of Riemerella anatipestifer provided in this application are virulent bacteriophages isolated from nature. They have unique morphological characteristics of the short-tailed virus family. They have a wide host range for Riemerella anatipestifer, strong lysis ability, and a recognition rate of more than 94% for different serotypes of Riemerella anatipestifer. They do not damage the normal microbial community and show excellent environmental tolerance and stability. 2. The composition of this application further incorporates Enterococcus faecalis phage P35AEFLN26, which significantly expands the composition's antibacterial spectrum and application range. Enterococcus faecalis is a common opportunistic pathogen in waterfowl farming environments, often forming mixed infections with Riemerella anatipestifer, and it exhibits strong drug resistance, making it difficult to effectively eradicate with traditional antibiotics. By combining Enterococcus faecalis phage P35AEFLN26 with Riemerella anatipestifer phage, the composition not only efficiently lyses Riemerella anatipestifer but also specifically inhibits Enterococcus faecalis, achieving synergistic control of mixed infections. Attached Figure Description

[0028] Figure 1 This is a photograph of the plaque morphology of *Riemerella anatipestifer* bacteriophage in Example 1 of this application; Figure 2 This is a schematic diagram of the morphological structure of *Rimerella anatipestifer* bacteriophage under a transmission electron microscope in Example 1 of this application. Figure 3 This is a distribution map of the unique endonuclease cleavage sites of the phage genome of *Riemerella anatipestifer* in Example 2 of this application; Figure 4 This is a phylogenetic tree diagram of the genome of Riemerella anatipestifer bacteriophage in Example 2 of this application; Figure 5 This is a stability diagram of the duck plague Riemerella phage in Example 6 of this application under different temperature conditions; Figure 6 This is a stability diagram of the *Rimerella anatipestifer* bacteriophage from Example 7 of this application under different pH conditions; Figure 7This is a stability graph of the duck plague Riemerella phage in Example 8 of this application before and after preparation under different powder preparation conditions; Figure 8 This is a diagram showing the lytic activity of the duck plague Riemerella phage against duck plague Riemerella in Example 9 of this application. Figure 9 This is a lysis diagram of non-host pathogenic bacteria by the bacteriophage of *Rimerella anatipestifer* in Example 10 of this application; Figure 10 This is a lysis diagram of non-pathogenic beneficial bacteria by the bacteriophage of *Rimerella anatipestifer* in Example 11 of this application; Figure 11 Example 13 of this application describes the disinfection effect of Riemerella anatipestifer bacteriophage and its composition on the farm environment. Detailed Implementation

[0029] 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] The raw materials used in the embodiments of this application can all be obtained from commercially available or preserved institutions, wherein: Riemerella anatipestifer phage RAP7ARASD24: Preservation name: Riemerella anatipestifer phage RAP7ARASD24 ( Riemerella anatipestifer Phage RAP7ARASD24). Depository: Guangdong Provincial Center for Microbial Culture Collection. Abbreviation: GDMCC. Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Deposit Date: July 2, 2026. Depository Registration Number: GDMCC NO. 67616-B1; Riemerella anatipestifer phage RAP16ARASD8: Preservation name: Riemerella anatipestifer phage RAP16ARASD8 ( Riemerella anatipestifer Phage RAP16ARASD8). Depository: Guangdong Provincial Center for Microbial Culture Collection. Abbreviation: GDMCC. Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Deposit Date: July 2, 2026. Depository Registration Number: GDMCC NO. 67617-B1; Enterococcus faecalis phage P35AEFLN26: Preservation name: Enterococcus faecalis phage P35AEFLN26 ( Enterococcus faecalisPhage P35AEFLN26). Depository Institution: China Center for Type Culture Collection (CCTCC). Abbreviation: CCTCC. Address: School of Life Sciences, Wuhan University. Deposit Date: March 3, 2025. CCTCC Registration Number: CCTCC NO: M2025336.

[0031] Preparation Example 1 Preparation of tryptic soy peptone (TSB) liquid culture medium: Mix 15g tryptic peptone, 5g soy peptone, 5g sodium chloride and 1000mL distilled water, and autoclave at 121℃ for 20min to obtain TSB liquid culture medium.

[0032] Preparation Example 2 Preparation of tryptic soy peptone agar (TSA) solid medium: Mix 15g tryptic peptone, 5g soy peptone, 5g sodium chloride, 15g agar and 1000mL distilled water, and autoclave at 121℃ for 20min to obtain TSA solid medium.

[0033] Preparation Example 3 Preparation of TSB semi-solid agar medium: Mix 15g tryptone, 5g soybean peptone, 5g sodium chloride, 7g agar and 1000mL distilled water, and autoclave at 121℃ for 20min to obtain TSB semi-solid agar medium.

[0034] Preparation Example 4 Preparation of blood agar medium: Mix 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar and 1000mL distilled water, autoclave at 121℃ for 20min, and when it cools to 50℃, add 5% (V / V) sterile defibrinated sheep blood and shake well. Quickly pour into sterile petri dishes to obtain blood agar medium.

[0035] Preparation Example 5 Preparation of SM buffer: Mix 5.8g sodium chloride, 2g magnesium sulfate, 50mL 1mol / L Tris-HCl, 0.25g gelatin and 1000mL distilled water, and autoclave at 121℃ for 20min to obtain SM buffer.

[0036] Example 1: Isolation, Preparation, Purification, and Cultivation Isolation of *R. anatipestifer*: Pathogenic samples collected from farms in Dezhou and Zaozhuang, Shandong Province, were inoculated onto blood agar medium and incubated at 37°C with 5% carbon dioxide for 24 hours. Typical single colonies were picked and inoculated onto blood agar medium, incubated at 37°C for 24 hours, and the above operation was repeated at least 3 times to obtain single colonies. Single colonies were selected for 16S rRNA gene amplification and sequencing. The sequencing results were compared with BLAST to confirm that they were all *R. anatipestifer*, and named *R. anatipestifer* ARASD24 and *R. anatipestifer* ARASD8, respectively. Single colonies were picked and inoculated into 3 mL of TSB liquid medium prepared in Example 1, and incubated at 37°C for 12 hours to obtain logarithmic-phase bacterial suspensions of *R. anatipestifer* ARASD24 and ARASD8.

[0037] Isolation of bacteriophages: Wastewater collected from farms in Dezhou City and Zaozhuang City, Shandong Province, was filtered through double-layer filter paper and centrifuged at low speed at room temperature. After filtration through a 0.22 μm filter membrane, 10 mL of the filtrate was added to 10 mL of the TSB liquid culture medium obtained in Preparation Example 1, along with 1 mL of the host bacterial suspension (Riemerella anatipestifer ARASD24 logarithmic phase suspension or Riemerella anatipestifer ARASD8 logarithmic phase suspension). The culture was incubated at 37 °C for 8 h. The culture was then centrifuged at 8000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain the supernatant, which yielded the bacteriophage host bacterial suspension (Riemerella anatipestifer ARASD24 suspension or Riemerella anatipestifer ARASD8 suspension). Using the double-layer plate method, 0.5 mL of phage host bacterial suspension (Riemerella anatipestifer ARASD24 suspension or Riemerella anatipestifer ARASD8 suspension) was added to 5 mL of TSB semi-solid agar medium obtained in Preparation Example 3 at 48°C and mixed well. The mixture was then poured onto TSA solid medium plates obtained in Preparation Example 2. 10 μL of the filtered supernatant was then dropped onto the solidified double-layer plate, air-dried under aseptic conditions, and incubated at 37°C for 16 h to obtain phage droplets.

[0038] Phage purification: Phage droplets were picked and added to 1 mL of the SM buffer obtained in Preparation Example 5, shaken for 1 min, and serially diluted 10-fold. 1 mL of each appropriate serial dilution was added to 0.5 mL of the logarithmic-phase host bacteria (Riemerella anatipestifer ARASD24 and ARASD8), mixed thoroughly, and allowed to stand for 15 min. Then, 5 mL of 48°C semi-solid TSB medium was added, and the mixture was immediately poured onto TSA plates. After solidification, the plates were incubated at 37°C for 16 h. Double-layer plates containing single phage plaques were obtained. Single phage plaques were picked and added to 1 mL of the SM buffer obtained in Preparation Example 5, and purified 5 times as described above. Finally, single phage plaques of uniform morphology and size (either a single plaque of Riemerella anatipestifer ARASD24 or a single plaque of Riemerella anatipestifer ARASD8) were obtained on the double-layer plates. (See [link to documentation]). Figure 1 A single plaque of *R. anatidae* ARASD24 was named bacteriophage RAP7ARASD24, and a single plaque of *R. anatidae* ARASD8 was named bacteriophage RAP16ARASD8. A single plaque was picked and placed in 50 mL of TSB medium containing 1 mL of the host bacterium (logarithmic-phase *R. anatidae* ARASD24 or logarithmic-phase *R. anatidae* ARASD8), and incubated at 37°C for 12 h. The culture was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the pure bacteriophage cultures (pure cultures of bacteriophage RAP7ARASD24 and bacteriophage RAP16ARASD8).

[0039] Pure cultures of *Riemerella anatipestifer* bacteriophages were irradiated with a transmission electron microscope, and the results were observed. Figure 2 .Depend on Figure 2 It is known that bacteriophage RAP7ARASD24 has a polyhedral symmetrical head and a short, contracted tail. The head diameter is 75-90 nm, the tail length is 8-10 nm, and the tail diameter is 2-5 nm. Bacteriophage RAP16ARASD8 also has a polyhedral symmetrical head and a short, contracted tail. The head diameter is 45-60 nm, the tail length is 4-7 nm, and the tail diameter is 3-4 nm. In the figure, the head diameter of RAP7ARASD24 of *R. anatidae* bacteriophage is 78 nm, the tail length is 9 nm, and the tail diameter is 3 nm; the head diameter of RAP16ARASD8 of *R. anatidae* bacteriophage is 49 nm, the tail length is 6 nm, and the tail diameter is 3 nm.

[0040] Example 2: Genome extraction, sequencing, and whole-genome analysis Genome extraction and sequencing: Phage nucleic acids were extracted and sequenced using a phage DNA extraction kit (Norgen Biotek). Nucleotide sequencing revealed that phage RAP7ARASD24 had the nucleotide sequences shown in SEQ ID No. 1-3, and phage RAP16ARASD8 had the nucleotide sequences shown in SEQ ID No. 4-6.

[0041] Genome sequence analysis: Phage sequencing revealed that the genome of phage RAP7ARASD24 is linear double-stranded DNA, with a total genome length of 43193 bp and a G+C content of 35.7%. The genome of phage RAP16ARASD8 is also linear double-stranded DNA, with a total genome length of 43080 bp and a G+C content of 34.9%.

[0042] Biological characteristics analysis of genomic restriction enzyme sites: The unique endonuclease species and sites of the whole genome of *Riemerella anatipestifer* bacteriophage were analyzed using SnapGene software. The distribution of unique endonuclease cleavage sites in this genome is shown in [Figure 1]. Figure 3 .

[0043] Genetic evolutionary analysis: Phage genomes were analyzed using NCBI phylogenetic tree analysis; results are shown below. Figure 4 .

[0044] Depend on Figure 4 It can be seen that the bacteriophage RAP7ARASD24 is genetically closest to Riemerella anatipestifer. Riemerella Phage vB_RanS_CRP12 is genetically closest to phage RAP16ARASD8 as Riemerella anatipestifer phage. Riemerella anatipestifer Phage vB_RanS_PT03 (MZ995504.1).

[0045] Combining the unique genomic information of Riemerella anatipestifer bacteriophages with their morphological characteristics under transmission electron microscopy, and based on the classification criteria established by the International Committee on Taxonomy of Viruses (ICTV), bacteriophages RAP7ARASD24 and RAP16ARASD8 are Riemerella anatipestifer bacteriophages, belonging to the order Caudovirales and the family Podoviridae.

[0046] Example 3: Determination of Optimal Multiple of Infection (MOI) Potency determination: Using the SM buffer obtained in Preparation Example 5 as a diluent, the pure phage culture media obtained in Example 1 (pure phage RAP7ARASD24 culture media and pure phage RAP16ARASD8 culture media) were serially diluted 10-fold, and then further serially diluted to 10-fold. 8Times. Take l0 respectively. 5 l0 6 l0 7 and l0 8 1000 μL of the diluted phage culture was mixed thoroughly with 300 μL of the host bacteria culture obtained in Example 1 (Riemerella anatipestifer ARASD24 logarithmic phase culture or Riemerella anatipestifer ARASD8 logarithmic phase culture), and allowed to stand for 15 min to allow for sufficient binding to the receptors on the bacterial surface. The mixture was then added to 4 mL of TSB semi-solid agar medium obtained in Preparation Example 3, cooled to 50°C, and immediately spread onto a solidified agar plate. After the agar solidified, the plate was inverted and incubated at 37°C for 8 h. Three replicates were prepared for each dilution, and the average number of plaques from the three replicates at that dilution was used for counting. The phage titer (PFU / mL) was calculated as: average number of plaques × dilution factor.

[0047] MOI determination: A single plaque (either *R. anatipestifer* ARASD24 or ARASD8) obtained in Example 1 was picked and inoculated into a test tube containing 3 mL of TSB liquid medium obtained in Example 1. The culture was incubated at 37°C for 4 h to obtain a host bacterial suspension (either *R. anatipestifer* ARASD24 or ARASD8 suspension). The host bacterial suspension was then transferred at a ratio of 1:100 to 10 mL of TSB liquid medium obtained in Example 1 and incubated at 37°C until the early logarithmic development phase. The pure phage cultures (RAP7ARASD24 and RAP16ARASD8) obtained in Example 1 and host bacteria (MOI = number of phages / number of bacteria) were added in proportions of 1000, 100, 10, 1, 0.1, 0.01, and 0.001, respectively. TSB liquid medium obtained in Preparation Example 1 was added to ensure the total volume of each tube was the same, and the tubes were incubated at 37°C for 12 h. After incubation, the tubes were centrifuged at 10000g for 10 min, and the supernatant was collected. After filtration through a 0.22 μm filter, the phage titer of the filtrate was determined using the double-layer plate method. Double replicates were performed at each point, and the average value was taken. The MOI that produced the highest phage titer was considered the optimal MOI. The experiment was repeated three times.

[0048] Table 1. Titer of Riemerella anatipestifer phage at different multiplicity of infection.

[0049] As shown in Table 1, the titer of RAP7ARASD24 phage of Riemerella anatipestifer reached its highest level (6.3 × 10⁻⁶). 10 At a concentration of PFU / mL, its MOI = 0.01. The titer of RAP16ARASD8 phage of Riemerella anatipestifer reached its highest level (4.7 × 10⁻⁶ PFU / mL). 10When the concentration of PFU / mL is 0.01, the MOI is 0.01. RAP7ARASD24 and RAP16ARASD8 phages of *R. anatidae* have low initial input amounts and rapid proliferation efficiency, making them suitable for industrial production.

[0050] Example 4: Toxicological Experiment The following experiments were conducted on RAP7ARASD24 and RAP16ARASD8 bacteriophages of Riemerella anatipestifer, respectively: ① Twenty healthy, specific pathogen-free (SPF) grade experimental mice of similar age and weight were selected, with half males and half females. After three days of acclimatization, they were randomly divided into two groups (phage group and control group), with 10 mice in each group (5 males and 5 females). The phage dilution obtained in Example 3 (Riemerella anatipestifer phage RAP7ARASD24 dilution or Riemerella anatipestifer phage RAP16ARASD8 dilution) was further diluted with SM buffer (obtained in Preparation Example 5) to obtain a phage titer of 1×10⁻⁶. 10 Phage test solution A (PFU / mL, phage RAP7ARASD24 test solution A or phage RAP16ARASD8 test solution A). Phage groups received a tail vein injection of 100 μL (1×10⁻⁶) of phage test solution A. 10 The mice were administered sterile saline via tail vein injection (PFU / mL), while the control group was injected with an equal volume of sterile saline. Food intake, activity levels, body temperature, weight changes, and survival were observed. Ten days later, the mice were euthanized by neck dislocation, and their internal organs were examined.

[0051] ② Forty healthy SPF-grade experimental mice of similar age and weight were selected, with half males and half females. After three days of acclimatization, they were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group), with 10 mice in each group (5 males and 5 females). The phage dilution obtained in Example 3 (Riemerella anatipestifer phage RAP7ARASD24 dilution or Riemerella anatipestifer phage RAP16ARASD8 dilution) was further diluted with SM buffer (obtained in Preparation Example 5) to obtain a phage titer of 1×10⁻⁶. 9 Phage test solution B (PFU / mL, phage RAP7ARASD24 test solution B or phage RAP16ARASD8 test solution B) was administered orally to phage groups 1, 2, and 3 (0.2 mL / mouse, 0.5 mL / mouse, and 1 mL / mouse, respectively). The control group was administered an equal volume of physiological saline orally. The mice were observed for 15 consecutive days. The mice were then euthanized by cervical dislocation, and their internal organs were examined.

[0052] ③ Forty one-day-old chicks were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group), with ten chicks in each group. Phage group 1, phage group 2, and phage group 3 were given 0.4 mL / chick, 1 mL / chick, and 2 mL / chick of phage test solution B orally, respectively. The control group was given an equal volume of physiological saline orally. The administration was continued for 15 days. The experimental chicks were then euthanized and their internal organs were examined.

[0053] ④ Forty one-day-old Cherry Valley ducks were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group), with 10 ducks in each group. Phage group 1, phage group 2, and phage group 3 were administered 0.4 mL / duck, 1 mL / duck, and 2 mL / duck of phage test solution B orally, respectively. The control group was administered an equal volume of physiological saline orally. The administration was continued for 15 days. The experimental ducks were then euthanized and their internal organs were examined.

[0054] The results above indicate that, in all treatment groups, intravenous injection of large doses of *R. anatidae* phage RAP7ARASD24 or RAP16ARASD8 had no effect on the daily behavior of mice. During the experiment, mice did not exhibit any clinical symptoms such as lethargy, rough coat, decreased appetite, weight loss, or death. Autopsy examination revealed no abnormalities in tissues or internal organs. In other oral treatments, different doses of *R. anatidae* phage RAP7ARASD24 or RAP16ARASD8 had no effect on the daily behavior of the tested animals. No symptoms such as depression, fever, vomiting, or diarrhea were observed. Autopsy examination of chicks and ducks revealed no abnormalities in internal organs. This demonstrates that the *R. anatidae* phage RAP7ARASD24 and RAP16ARASD8 of this application are biosafety indicative of their potential applications in various fields, including but not limited to feed additives.

[0055] Example 5: Temperature Stability Test The pure phage culture media obtained in Example 1 (pure phage RAP7ARASD24 or pure phage RAP16ARASD8 culture media) were aliquoted into sterile EP tubes and incubated in water baths at 4℃ (CK), 55℃, 65℃, and 75℃ for 2h, 24h, and 48h, respectively. After the incubation period, the sample tubes were removed and immediately placed in an ice bath to cool. After appropriate dilution, the phage titer was determined using the double-layer plate method. The experiment was repeated three times, and the results are shown below. Figure 5 .

[0056] Depend on Figure 5It was found that RAP7ARASD24 and RAP16ARASD8, bacteriophages of *R. anatipestifer*, exhibited a certain degree of temperature tolerance. Compared with the control, RAP7ARASD24 showed a survival rate of 42.3% after treatment at 55℃ for 48 hours and a survival rate of 10% after a 2-hour water bath at 65℃. 8 PFU / mL potency, still retains 10 after 2 hours of water bath at 75℃. 2 The titer of Riemerella anatipestifer phage RAP16ARASD8 did not decrease after treatment at 55°C for 24 hours, did not change after a 2-hour water bath at 65°C, and still maintained 10 PFU / mL titer after a 24-hour water bath at 75°C. 5 PFU / mL potency.

[0057] Example 6: pH stability test The RAP7ARASD24 dilution of *Riemerella anatipestifer* phage obtained in Example 3 was diluted with SM buffer (obtained in Preparation Example 5) to obtain a phage titer of 5.19 × 10⁻⁶. 8 Phage assay solution C, PFU / mL. The RAP16ARASD8 dilution of *Riemerella anatipestifer* phage obtained in Example 3 was diluted with SM buffer (obtained in Preparation Example 5) to obtain a phage titer of 7.7 × 10⁻⁶. 8 Phage test solution D (PFU / mL). Phage test solution C or solution D was adjusted to pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0, respectively. After treatment for 1 h, 4 h, 8 h, 24 h, and 96 h, the titer was measured. Results are shown below. Figure 6 .

[0058] Depend on Figure 6 It was found that the titers of *R. anguillarum* bacteriophages RAP7ARASD24 and RAP16ARASD8 did not change significantly between pH 5 and pH 10, indicating good stability under neutral, acidic, and slightly alkaline conditions. Specifically, *R. anguillarum* bacteriophage RAP7ARASD24 remained active after treatment at pH 2 for 24 hours, demonstrating its good tolerance to acidic conditions. *R. anguillarum* bacteriophage RAP16ARASD8 retained its titer at pH 2 for 8 hours. 3 The PFU / mL titer remained active after treatment at pH 12 for 24 hours, indicating that the phage has good tolerance to acidic and alkaline conditions.

[0059] Example 7: Bioactivity Tests of Spray-on Powder and Lyophilized Powder Bioactivity test of the spray powder: 2% starch (w / v) and 4% sorbitol (w / v) were dissolved in phage test solution C or phage test solution obtained in Example 7, and incubated with phage at 37°C and 180 r / min for 2 h for phage coating. The coated phage solution was sprayed onto defatted rice bran carrier at a ratio of 1:4 (w / w), and stirred to ensure uniform mixing. It was then dried at 50°C for 2 h with a relative humidity <10% and passed through a 40-mesh sieve. After preparation, samples were taken and the phage titer was determined using the double-layer plate method. The experiment was repeated three times, and the results are shown in […]. Figure 7 .

[0060] Bioactivity test of the lyophilized powder: 2% starch (w / v) and 4% sorbitol (w / v) were dissolved in phage test solution C or phage test solution obtained in Example 7, and incubated with phage at 37°C and 180 r / min for 2 h for phage coating. The coated phage solution was added to a material tray, freeze-dried under vacuum for 50 h, and then pulverized through a 40-mesh sieve. The moisture content of the lyophilized powder was <10%. After preparation, samples were taken and the phage titer was determined using the double-layer plate method. The experiment was repeated 3 times, and the results are shown in […]. Figure 7 .

[0061] Depend on Figure 7 It can be seen that there was no significant difference in potency between RAP7ARASD24 and RAP16ARASD8 of R. anatipestifer bacteriophages before and after preparation of spray powder and lyophilized powder, respectively, indicating that these two bacteriophages have good tolerance and stability to the powder preparation conditions of spray powder and lyophilized powder.

[0062] Example 8: Pyrolysis Experiment The phage lysis profile was determined using the double-layer plate drop method: A total of 192 single colonies of 12 different serotypes of *Riemerella anatipestifer* were selected and inoculated into test tubes containing 3 mL of TSB liquid medium obtained in Example 1. The cultures were incubated at 37°C for 8 hours to obtain bacterial suspensions for each strain. 300 μL of the bacterial suspension was mixed with TSB semi-solid medium obtained in Example 3 and plated onto ordinary agar plates. The phage dilutions obtained in Example 3 (*Riemerella anatipestifer* phage RAP7ARASD24 dilution or *Riemerella anatipestifer* phage RAP16ARASD8 dilution) were further diluted using SM buffer (obtained in Example 5) to obtain a phage titer of 1 × 10⁻⁶. 6 Phage test solution E (PFU / mL, phage RAP7ARASD24 test solution E or phage RAP16ARASD8 test solution E) was prepared. 5 μL of phage test solution E (phage RAP7ARASD24 test solution E or phage RAP16ARASD8 test solution E) was dropped onto each plate. After air drying, the plates were incubated at 37°C for 8 hours. The results are shown in Tables 2-3 and 3-4. Figure 8 .

[0063] Table 2. Lytic ability of RAP7ARASD24 bacteriophage of Riemerella anatipestifer.

[0064] From Table 2 and Figure 8 It is known that the phage RAP7ARASD24 of Riemerella anatipestifer has a wide host range and can recognize Riemerella anatipestifer of various tested serotypes. Its lysis rate is above 87% and the total lysis rate can reach 94%. It can be well applied in the preparation of biocides for lysing Riemerella anatipestifer.

[0065] Table 3. Lytic ability of RAP16ARASD8 bacteriophage of Riemerella anatipestifer

[0066] From Table 3 and Figure 8 It is known that the RAP16ARASD8 bacteriophage of Riemerella anatipestifer has a wide host range and can recognize various tested serotypes of Riemerella anatipestifer. Its lysis rate is above 86% and the total lysis rate can reach above 94%, which makes it suitable for use in the preparation of biocides for lysing Riemerella anatipestifer.

[0067] Example 9: Lysis test of non-host pathogenic bacteria Determination of phage lysis profiles using the double-layer plate drop method: 160 single colonies of non-host pathogenic bacteria were selected, including 20 strains each of *Elizabeth glomerulonephritis meningoseptica* (A1), *Clostridium perfringens* (A2), *Acinetobacter baumannii* (A3), *Klebsiella pneumoniae* (A4), *Escherichia coli* (A5), *Salmonella* (A6), *Staphylococcus aureus* (A7), and *Pseudomonas aeruginosa* (A8). Each colony was inoculated into a test tube containing 3 mL of TSB and cultured to the logarithmic phase under optimal conditions to obtain bacterial suspensions for each strain. 300 μL of the bacterial suspension was mixed with semi-solid culture medium and plated onto ordinary agar plates. 5 μL of phage test solution E obtained in Example 9 (phage RAP7ARASD24 test solution E or phage RAP16ARASD8 test solution E) was then dropped onto each plate. After air drying, the bacteria were incubated overnight under their optimal culture conditions, and the results were observed. (See attached table for details.) Figure 9 .

[0068] Depend on Figure 9It was found that both RAP7ARASD24 and RAP16ARASD8 of *R. anatipestifer* could recognize some *Elizabeth kinesi* meningoseptica, but could not recognize the other tested bacteria. This indicates that the tested phages have a broad lysis range, which is of great significance for expanding the host spectrum of phage therapy and developing alternative or adjuvant treatment options for multidrug-resistant *Elizabeth kinesi* meningoseptica infections.

[0069] Example 10: Lysis test of non-pathogenic beneficial bacteria Sixty single colonies of non-pathogenic beneficial bacteria were selected, including 10 strains each of non-pathogenic rhizobium (B1), non-pathogenic Bacillus licheniformis (B2), non-pathogenic Bacillus subtilis (B3), non-pathogenic Bacillus megaterium (B4), Bacillus coagulans (B5), and Clostridium butyricum (B6). Each colony was inoculated into test tubes containing 3 mL of TSB and cultured to the logarithmic growth phase under optimal conditions to obtain bacterial suspensions. 300 μL of each bacterial suspension was mixed with semi-solid culture medium and plated onto ordinary agar plates. 5 μL of the phage test solution E obtained in Example 9 (phage RAP7ARASD24 test solution E or phage RAP16ARASD8 test solution E) was dropped onto each plate. The plates were incubated overnight under optimal conditions for each bacterial strain, and the results were observed. The results are shown in the table below. Figure 10 .

[0070] Depend on Figure 10 It was found that neither the RAP7ARASD24 nor the RAP16ARASD8 bacteriophages of *R. anatidae* could recognize any of the 60 tested non-pathogenic beneficial bacteria strains. This indicates that the tested bacteriophages did not damage the beneficial bacterial microbial community.

[0071] Example 11: Preparation of the composition Equal volumes of test solution B for phage RAP7ARASD24 and test solution B for phage RAP16ARASD8 obtained in Example 5 were mixed uniformly to obtain composition 1.

[0072] The phage test solution B obtained in Example 5 (phage RAP7ARASD24 test solution B or phage RAP16ARASD8 test solution B) and the pure culture medium of Enterococcus faecalis phage P35AEFLN26 were mixed in equal volumes to obtain composition 2.

[0073] Equal volumes of test solution B of phage RAP7ARASD24, test solution B of phage RAP16ARASD8, and pure culture medium of Enterococcus faecalis phage P35AEFLN26 obtained in Example 5 were mixed uniformly to obtain composition 3.

[0074] The phage test solution B obtained in Example 5 (phage RAP7ARASD24 test solution B or phage RAP16ARASD8 test solution B) and dodecyl dimethyl benzyl ammonium chloride were mixed in equal volumes and uniformly. The final application mass concentration of dodecyl dimethyl benzyl ammonium chloride in the composition was 0.1% (W / V), thus obtaining composition 4.

[0075] Example 12: The preventive and therapeutic effects of bacteriophages and their compositions on animals infected with Riemerella anatipestifer. (1) The effect of bacteriophages and their combinations on preventing duck infection with Riemerella anatipestifer. Three hundred healthy white-feathered meat ducks aged 10 days and of similar weight were selected and randomly divided into 6 groups after three days of acclimatization: phage group (phage RAP7ARASD24 group or phage RAP16ARASD8 group), composition group 1, composition group 2, composition group 3, positive control group, and negative control group, with 50 meat ducks in each group.

[0076] Before the experiment, the duck house, feed troughs, and waterers were thoroughly cleaned and disinfected, and the entire duck house was fumigated for disinfection. The experimental ducks had free access to feed and water, and the house was cleaned and disinfected regularly.

[0077] Bacteriophage groups (phage RAP7ARASD24 group or phage RAP16ARASD8 group) were orally administered 1 mL / bird / day of phage test solution B (phage RAP7ARASD24 test solution B and phage RAP16ARASD8 test solution B) obtained in Example 5. Composition 1-Composition 3 groups were orally administered 1 mL / bird / day of Composition 1-3 obtained in Example 12. Positive and negative control groups were orally administered an equal volume of sterile saline. After two consecutive days of feeding, each treatment group (except the negative control group) was subcutaneously injected with 1×10⁻⁶ ppm of phage solution. 8 0.5 mL of CFU / mL *Riemerella anatipestifer* (*Riemerella anatipestifer* ARASD24 logarithmic phase or *Riemerella anatipestifer* ARASD8 logarithmic phase) was administered; the negative control group received a subcutaneous injection of an equal volume of sterile saline in the neck of the ducks. All groups were fed a basal diet during the experiment. Seven days after injection, the mortality rate of each group was recorded, and all ducks underwent necropsy to determine the lesion rate of internal organs. Results are shown in Tables 5-6. Mortality rate = (number of dead ducks / total number of ducks) × 100%, lung lesion rate = (number of ducks with visceral lesions / total number of ducks) × 100%, protection rate = 1 - mortality rate. Results are shown in Tables 4-5.

[0078] Table 4. Efficacy of RAP7ARASD24 bacteriophage for preventing R. anatipestifer infection in broiler ducks.

[0079] Table 5. Efficacy of RAP16ARASD8 bacteriophage for preventing R. anatipestifer infection in broiler ducks.

[0080] Table 4 shows that, compared with the positive control group which did not receive prior oral administration of Riemerella anatipestifer phage RAP7ARASD24 for prevention, the mortality rate of ducks in the experimental group that received prior prevention with Riemerella anatipestifer phage RAP7ARASD24 or its combination was no higher than 12%, and the rate of visceral lesions was no higher than 20%. The protection rate of Riemerella anatipestifer phage RAP7ARASD24 and its combination for the tested ducks reached 88%. This indicates that Riemerella anatipestifer phage can effectively prevent infection of ducks with Riemerella anatipestifer.

[0081] Table 5 shows that, compared with the positive control group which did not receive prior oral administration of Riemerella anatipestifer phage RAP16ARASD8 for prevention, the mortality rate of ducks in the experimental group that received prior prevention with Riemerella anatipestifer phage RAP16ARASD8 or its combination was no higher than 8%, and the rate of visceral lesions was no higher than 16%. The protection rate of Riemerella anatipestifer phage RAP16ARASD8 and its combination for the tested ducks reached 92%. This indicates that Riemerella anatipestifer phage RAP16ARASD8 can effectively prevent Riemerella anatipestifer infection in ducks.

[0082] (2) The effect of Riemerella anatipestifer bacteriophage and its combination on the treatment of ducks infected with Riemerella anatipestifer. One hundred and eighty Cherry Valley ducks of similar weight and 10 days old were selected and randomly divided into six groups after three days of acclimatization: bacteriophage group (bacteriophage RAP7ARASD24 group or bacteriophage RAP16ARASD8 group), composition group 1, composition group 2, composition group 3, positive control group, and negative control group, with 30 ducks in each group.

[0083] Before the experiment, the duck house, feed troughs, and waterers were thoroughly cleaned and disinfected, and the entire duck house was fumigated for disinfection. The experimental ducks had free access to feed and water, and the house was cleaned and disinfected regularly.

[0084] Except for the negative control group, the other experimental groups of ducks were injected subcutaneously into the neck with 1×10 80.5 mL of *Riemerella anatipestifer* (log phase ARASD24 or ARASD8) at CFU / mL was injected intramuscularly. Two hours after injection, ducks in the phage group (phage RAP7 / ARASD24 or phage RAP16 / ARASD8) received an intramuscular injection of phage test solution B (phage RAP7 / ARASD24 and Rage RAP16 / ARASD8) obtained in Example 5, 1 mL / day. Ducks in composition groups 1-3 received an intramuscular injection of composition 1-3 obtained in Example 12, 1 mL / day. Ducks in the positive control group received an intramuscular injection of an equal volume of sterile saline, 1 mL / day. Ducks in the negative control group received an intramuscular injection of an equal volume of sterile saline, 1 mL / day. Injections were administered for 3 consecutive days. All groups were fed a basal diet during the experiment. Seven days after the experiment, the mortality rate of each group of experimental ducks was calculated, and all experimental ducks were necropsized to determine their visceral lesion rate. The results are shown in Table 5. Mortality rate = (number of experimental ducks that died / total number of experimental ducks) × 100%. Lung lesion rate = (number of experimental ducks with visceral lesions / total number of experimental ducks) × 100%. Protection rate = 1 - mortality rate, the results are shown in Tables 6-7.

[0085] Table 6. Efficacy of RAP7ARASD24 bacteriophage for treating R. anatipestifer infection in broiler ducks.

[0086] Table 7. Efficacy of RAP16ARASD8 bacteriophage for treating R. anatipestifer infection in broiler ducks.

[0087] As shown in Table 6, compared with the positive control group that was not injected with Riemerella anatipestifer phage RAP7ARASD24, the mortality rate of the ducks in the experimental group injected with Riemerella anatipestifer phage RAP7ARASD24 was significantly lower. The protection rate of Riemerella anatipestifer phage RAP7ARASD24 in the experimental ducks reached 83.3%, and the rate of visceral lesions was no higher than 34%. The protection rate of the combined product in the tested ducks reached 86.7%. This indicates that Riemerella anatipestifer phage RAP7ARASD24 has a good therapeutic effect on ducks infected with Riemerella anatipestifer.

[0088] As shown in Table 7, compared with the positive control group that was not injected with Riemerella anatipestifer phage RAP16ARASD8, the mortality rate of ducks in the experimental group injected with Riemerella anatipestifer phage RAP16ARASD8 was significantly lower. The protection rate of Riemerella anatipestifer phage RAP16ARASD8 in the experimental ducks reached 86.7%, and the rate of visceral lesions was no higher than 34%. The protection rate of the combined phage reached 90% in the tested ducks. This indicates that Riemerella anatipestifer phage RAP16ARASD8 has a good therapeutic effect on ducks infected with Riemerella anatipestifer.

[0089] (3) The effect of Riemerella anatipestifer bacteriophage and its composition on disinfection of duck farm environment Twenty-one duck houses were randomly selected from a white-feathered broiler duck farm and randomly divided into seven groups: a phage group (phage RAP7ARASD24 or phage RAP16ARASD8), composition 1, composition 2, composition 3, composition 4, a control group, and a blank group, with three duck houses in each group. The pure culture medium of *Riemerella anatipestifer* phage RAP7ARASD24 was prepared in Example 3, while the corresponding pure phage cultures of compositions 1, 2, 3, and 4 were prepared in Example 12.

[0090] The pure phage cultures of phage groups (phage RAP7ARASD24 or phage RAP16ARASD8), composition 1, composition 2, composition 3, and composition 4 were diluted with farm drinking water to a final phage concentration of 1x10⁻⁶. 6 Culture medium containing PFU / mL. At 10 mL / mL 2 The control group was sprayed twice daily; the control group was sprayed daily with an equal amount of 0.3% dodecyl dimethyl benzyl ammonium chloride (w / v); the blank group was fed according to routine procedures. Five test points were selected: the center point of the duck house and four corner points. The sampling points were 0.5m above the ground, and the four corner points were 1m away from the wall. A 9cm diameter TSA culture medium was placed at each point to detect changes in bacterial content before and after disinfection. After sampling, the TSA culture dishes were incubated at 37℃ and 5% CO2 for 18-24 hours, and the bacterial colony count in each dish was recorded. The total colony count was recorded using the Ostwald formula: C = 50000N / AT, where C: total colony count per cubic meter (CFU / m³). 3 N: Colony count per dish; A: Area of ​​the petri dish (cm²) 2 T: Sampling time (min), results are shown in Figure 11 .

[0091] Depend on Figure 11It can be seen that the number of colonies treated with RAP7ARASD24 and RAP16ARASD8 bacteriophages of R. anatipestifer and their combination was lower than that of the blank group and the control group, and lower than that of the single bacteriophage group. This indicates that the bacteriophage combination can effectively reduce the number of bacteria in the environment and has a greater antibacterial advantage than using a single bacteriophage.

[0092] Example 13: Preparation and use of kits for bacteriophages and their compositions The kit contains 5-10 mL of phage test solution B obtained in Example 5 (phage RAP7ARASD24 test solution B or phage RAP16ARASD8 test solution B), 1 L of TSB liquid culture medium obtained in Example 1, 1 L of TSB semi-solid culture medium obtained in Example 3, and 1 L of TSA solid culture medium obtained in Example 2.

[0093] The kit is used as follows: Take the phage test solution B obtained in Example 5 (phage RAP7ARASD24 test solution B or phage RAP16ARASD8 test solution B), and use the double-layer plate drop method to determine the lysis spectrum of the tested phage. Pick a single colony to be tested and inoculate it into the TSB liquid medium obtained in Preparation Example 1, and incubate for 8 hours to obtain the bacterial suspension of the test strain. Take 300 μL of the bacterial suspension of the test strain and mix it with 5 mL of TSB semi-solid medium obtained in Preparation Example 3, and spread it on the TSA solid medium plate obtained in Preparation Example 2. Take 10 μL of the host bacterial suspension (Riemerella anatipestifer ARASD24 logarithmic phase bacterial suspension or Riemerella anatipestifer ARASD8 logarithmic phase bacterial suspension) and drop it onto the plate. After air drying, incubate at the target temperature according to the growth characteristics of the test strain, and observe the results to determine whether the target strain is present.

[0094] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A strain of Riemerella anatipestifer phage RAP7ARASD24, characterized in that, The *Rimerella anatipestifer* bacteriophage RAP7ARASD24 was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 2, 2026, with accession number GDMCCNO. 67616-B1, and classified as follows: Riemerella anatipestifer phageRAP7ARASD24.

2. A strain of Riemerella anatipestifer phage RAP16ARASD8, characterized in that, The *Rimerella anatipestifer* phage RAP16ARASD8 was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 2, 2026, with accession number GDMCCNO. 67617-B1, and classified as follows: Riemerella anatipestifer phageRAP16ARASD8.

3. A composition for inhibiting Riemerella anatipestifer, characterized in that, Includes the RAP7ARASD24 bacteriophage of Riemerella anatipestifer as described in claim 1 and / or the RAP16ARASD8 bacteriophage of Riemerella anatipestifer as described in claim 2.

4. The composition for inhibiting Riemerella anatipestifer according to claim 3, characterized in that, It also includes Enterococcus faecalis phage P35AEFLN26, whose accession number is CCTCC NO:M2025336.

5. A biological bactericide, characterized in that, Includes the RAP7ARASD24 bacteriophage of Riemerella anatipestifer as described in claim 1 and / or the RAP16ARASD8 bacteriophage of Riemerella anatipestifer as described in claim 2.

6. The biocidal agent according to claim 5, characterized in that, It also includes chemical disinfectants, including dodecyl dimethyl benzyl ammonium chloride.

7. A drug, characterized in that, Includes the RAP7ARASD24 bacteriophage of Riemerella anatipestifer as described in claim 1 and / or the RAP16ARASD8 bacteriophage of Riemerella anatipestifer as described in claim 2.

8. A feed additive, characterized in that, Includes the RAP7ARASD24 bacteriophage of Riemerella anatipestifer as described in claim 1 and / or the RAP16ARASD8 bacteriophage of Riemerella anatipestifer as described in claim 2.

9. A reagent kit, characterized in that, Includes the RAP7ARASD24 bacteriophage of Riemerella anatipestifer as described in claim 1 and / or the RAP16ARASD8 bacteriophage of Riemerella anatipestifer as described in claim 2.

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