A duck rimer's bacillus bacteriophage and its isolation method and application

Abstract

This invention discloses a Riemerella anticipestifer phage SD-RA1, its isolation method, and its applications. This phage can simultaneously lyse serotypes 1 and 7 of Riemerella anticipestifer. It was deposited at the China Center for Type Culture Collection (CCTCC) on August 23, 2021, with accession number CCTCC M 20211061. This phage can be used not only to prepare drugs for the prevention and treatment of various livestock and poultry diseases caused by Riemerella anticipestifer infection, but also to prepare livestock and poultry feed additives and environmental disinfectants. The phage and its compositions are safe to use and have no side effects, effectively avoiding antibiotic residues and the problem of inducing drug-resistant Riemerella anticipestifer caused by traditional antibiotic use.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a bacteriophage of *Riemerella anatipestifer*, its isolation method, and its application. Background Technology

[0002] Riemereella anatipestifer (RA) is a contagious disease affecting domestic ducks, geese, turkeys, and other poultry and wild birds. It is also known as new duck disease, duck septicemia, duck plague septicemia, duck disease syndrome, and infectious serositis. The disease progresses through acute or chronic septicemia, with characteristic lesions including fibrinous pericarditis, perihepatitis, air sacculitis, caseous salpingitis, and meningitis. Asymptomatic respiratory infections may also occur. Infected ducks suffer from high mortality, emaciation, ketosis, and decreased quality, resulting in significant economic losses for farmers. There are multiple serotypes of Riemereella anatipestifer (RA), and there is no cross-reactivity or cross-protection between different serotypes. After years of identification and analysis of RA serotypes, domestic research experts have identified serotypes 1, 2, 6, 7, and 10 as the dominant serotypes in most parts of my country. Understanding the prevalent dominant serotypes can provide a reference for the development of RA drugs and comprehensive prevention and control.

[0003] Riemereella anatipestifer is a significant pathogen threatening duck farming, with few effective drugs and a high rate of drug resistance. Therefore, the overuse or exceeding of treatment durations for multiple drugs is common. This results in two consequences: firstly, drug residues in duck carcasses, which can affect human health if ingested; secondly, duck farm waste is discharged into the environment, turning drugs into environmental pollutants and negatively impacting the ecosystem. Therefore, the development of pollution-free antibiotic alternatives, either as substitutes or partial substitutes, is urgently needed.

[0004] Bacteriophages are bacterial viruses widely found in natural environments such as soil, sewage, and feces. They possess advantages such as being natural, safe, highly effective, and residue-free, making them a promising candidate for development as an antibacterial agent. While promising results were achieved in treating bacterial infections with bacteriophages as early as the beginning of the 20th century, research on bacteriophages almost stagnated due to the rapid development of antibiotics. In recent years, however, there has been increasing interest in bacteriophage therapy. Compared to antibiotics, bacteriophages offer several advantages: firstly, they target specific bacteria without disrupting the normal microecological balance and have no side effects; secondly, bacteriophages die upon the loss of their host, leaving no residue in the animal's body or products. Therefore, bacteriophages can serve as a highly effective biological disinfectant and drug with bactericidal properties.

[0005] However, there is currently no effective phage for Riemerella anatipestifer that can be used to prevent and treat Riemerella anatipestifer disease. Therefore, the existing technology needs further improvement. Summary of the Invention

[0006] To address the shortcomings of existing technologies and solve the aforementioned problems, a method for isolating and applying *Riemerella anatipestifer* bacteriophage is proposed. The specific technical solution is as follows:

[0007] This invention provides a bacteriophage of *Rimerella anatipestifer*, named SD-RA1. This bacteriophage can simultaneously lyse serotypes 1 and 7 of *Rimerella anatipestifer*. It was deposited in August 2021 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20211061.

[0008] Furthermore, transmission electron microscopy revealed that the head of the duck plague Riemerella phage SD-RA1 was a regular polyhedral structure with a diameter of approximately 61 nm, and it had a tail with a length of approximately 244 nm, belonging to the family Longtail Phages.

[0009] Furthermore, the SD-RA1 bacteriophage of Riemerella anatipestifer is a virulent bacteriophage that exhibits strong lytic activity against Riemerella anatipestifer types 1 and 7, with a lytic rate exceeding 90%. This provides a source of bacteriophages for the industrial, large-scale, and programmed production of bacteriophages and for the treatment of animal bacterial diseases such as duck serositis caused by Riemerella anatipestifer.

[0010] Furthermore, the titer of the duck plague Riemerella phage SD-RA1 remained above 10⁹ PFU / ml after treatment at 50°C for 60 minutes.

[0011] Furthermore, the *Rimerella anatipestifer* phage SD-RA1 maintained a titer of 10 when cultured at 37°C for 60 minutes within a pH range of 6.0–9.0. 10 At concentrations above PFU / ml, the ability to lyse the host is completely lost at pH 2.0; at pH 12, the titer remains at 10. 3 PFU / ml, phage SD-RA1 is acid-sensitive and alkali-loving.

[0012] The present invention also provides a method for isolating Riemerella anatipestifer bacteriophage as described above, comprising the following steps:

[0013] Wastewater and manure treatment: Collect duck farm manure and wastewater, add an appropriate amount of SM buffer solution and let it precipitate overnight. Take the supernatant after overnight precipitation, centrifuge and filter, and store the centrifuged liquid for later use;

[0014] Phage enrichment: Add an appropriate amount of centrifuged liquid to an appropriate amount of tryptone soybean broth medium, add an appropriate amount of isolated and identified serum type 1 or serum type 7 Riemerella anatipestifer solution, co-culture in a 37℃ constant temperature incubator, and obtain phages after centrifugation and filtration of the culture medium.

[0015] Verification of phage isolation: Phages were isolated using the double-layer plate drop method. The presence of phage plaques proved that phages were isolated from farm manure and sewage, and that they had a lytic effect on serotypes 1 and 7 of Riemerella anatipestifer.

[0016] Furthermore, SM buffer solution was added to the collected duck manure and sewage, wherein the volume ratio of SM buffer solution to manure and sewage was 1:1 to 1:2.

[0017] Furthermore, the specific steps for treating the feces and wastewater are as follows: collect duck farm feces and wastewater, add 1×SM buffer at a ratio of 1:1 to 1:2, and let it precipitate overnight at 2 to 8°C. Then, take the upper liquid and filter it through 8 or 16 layers of gauze. Take the liquid after gauze filtration, centrifuge it at 8000 to 12000 rpm for 3 to 5 minutes, and finally filter it through a 0.22 μm filter membrane for sterilization. Store it at 2 to 8°C for later use.

[0018] Furthermore, the phage enrichment step specifically involves adding the centrifuged liquid obtained from the fecal wastewater treatment step to 2×TSB medium containing 3%–5% fetal bovine serum at a ratio of 1:1 to 1:2. Then, add serotype 1 or serotype 7 *Riemerella anatipestifer* bacterial suspension, adding 3–4 strains to each enrichment tube, with each strain occupying a proportion of 1:20 to 1:50 in the medium. Incubate at 37°C using a constant temperature shaker for 4–6 hours. Centrifuge the culture medium at 8000–12000 rpm for 3–5 minutes, and finally filter through a 0.22 μm filter membrane for sterilization. Store at 2–8°C for later use.

[0019] Furthermore, the filtrate obtained from the phage enrichment step was validated using the double-layer plate drop method, wherein the lower layer of the double-layer plate was TSA solid medium, and the upper layer of the double-layer plate was TSB semi-solid medium containing 3%–5% fetal bovine serum.

[0020] Furthermore, the upper layer of the double-layer plate, TSB semi-solid culture medium, is prepared as follows: 5g of TSB culture medium and 1.5g-2.0g of agar powder are dissolved in 200ml of purified water, the pH is adjusted to 7.2-7.4, and then autoclaved for later use. The lower layer of the double-layer plate, TSA solid culture medium, is prepared as follows: 10-15ml of autoclaved TSA culture medium is poured into each sterile petri dish after cooling to 50℃-55℃ at room temperature and then cooled to room temperature for later use.

[0021] Furthermore, the upper layer of the bilayer plate is a semi-solid culture medium containing 3%–5% fetal bovine serum (FBS). Take a 10 ml centrifuge tube, add 300–500 μl of bacterial suspension, autoclave the semi-solid culture medium, cool it to 50°C–55°C at room temperature, add a final concentration of 3%–5% FBS, pour 5–8 ml into a 10 ml sterile centrifuge tube, and pour the liquid from the centrifuge tube into a Petri dish, which is the upper layer of the bilayer plate.

[0022] Furthermore, the double-layer plate drop method is as follows: the filtrate after the phage enrichment step is dropped onto a double-layer plate, 4-6 μl on each plate. After the liquid on the plate surface dries, the double-layer plate is inverted and placed in a 37℃ CO2 incubator for 10-12 hours. The appearance of phage plaques is observed. The above-mentioned duck plague Riemer's bacteriophage plaques are 1-2 mm in diameter, round and transparent, without a halo.

[0023] The present invention also provides an application of the *Rimerella anatipestifer* bacteriophage as described above, specifically its application in the preparation of a medicament for the prevention or treatment of animal diseases caused by *Rimerella anatipestifer*. Based on this application, the present invention can provide a bactericidal composition for the prevention and treatment of *Rimerella anatipestifer*, the active ingredient of which includes the *Rimerella anatipestifer* bacteriophage SD-RA1 described in this invention.

[0024] The present invention provides an application of the duck plague Riemerella phage as described above, wherein the application is in the preparation of feed for the prevention or treatment of animal diseases caused by duck plague Riemerella phage.

[0025] The present invention provides an application of the above-described Riemerella anatipestifer bacteriophage, characterized in that it is used in the preparation of cleaning agents or disinfectants for the prevention or treatment of animal diseases caused by Riemerella anatipestifer.

[0026] Beneficial effects:

[0027] 1. This invention involves isolating and screening two strains of Riemerella anatipestifer from the brains of ducks exhibiting serositis symptoms at an intensive duck farm in Heze, Shandong Province. These strains were identified as serotypes 1 and 7. Using these two strains as hosts, bacteriophages SD-RA1 for serotypes 1 and 7 of Riemerella anatipestifer were isolated from the farm's manure and wastewater. This bacteriophage SD-RA1 exhibits strong lytic activity against pathogenic serotypes 1 and 7 of Riemerella anatipestifer in poultry farming environments, providing a phage source for the industrial production of bacteriophages for the prevention and treatment of serositis caused by pathogenic serotypes 1 and 7 of Riemerella anatipestifer in poultry farming environments.

[0028] 2. The phage SD-RA1 of serotypes 1 and 7 of Riemerella anatipestifer provided by this invention maintains good activity within a pH range of 6.0–9.0, with no significant change in phage titer. The highest phage titer, reaching 10, is observed at pH 8.0. 10 The PFU / ml concentration exhibits good stability under alkaline conditions, maintaining a potency of 10 at pH 12. 3PFU / ml. Within the pH range of 2.0–5.0, the phage titer decreases significantly with decreasing pH, and at pH 2.0, it completely loses its ability to lyse the host. Phage SD-RA1 exhibits acid-sensitive and basophilic properties.

[0029] 3. The bacteriophage of the present invention is obtained from the natural environment and is easy to produce industrially. The drugs or disinfectants prepared from the above bacteriophages can not only reduce costs, but also the treatment with the bacteriophage or bacteriophage composition does not need to consider the problem of drug residues, and can be widely used as a safe and efficient biological disinfectant and drug.

[0030] 4. The Riemerella anatipestifer bacteriophage and its bacteriophage composition of the present invention have a wide range of applications. They can not only be used to prepare drug preparations for the prevention and treatment of Riemerella anatipestifer disease, but also, by mixing with feed and drinking water, as well as by using as a disinfectant, they can be widely used in various stages of poultry farming that are prone to losses due to Riemerella anatipestifer infection, for daily disinfection of the farming environment, and for antibacterial purposes on fresh products, which is beneficial to the healthy development of poultry farming, especially duck farming. Attached Figure Description

[0031] Figure 1 This is a photograph of the SD-RA1 phage plaque from the present invention;

[0032] Figure 2 This is an electron microscope image of the bacteriophage SD-RA1 of this invention;

[0033] Figure 3 The results of thermal stability testing of the bacteriophage SD-RA1 of this invention;

[0034] Figure 4 This is the pH stability test result of the bacteriophage SD-RA1 of this invention;

[0035] Figure 5 This is the one-step growth curve of the phage SD-RA1 of this invention;

[0036] Figure 6 These are autopsy images of normally dead ducks in the experimental and control groups before slaughter in Example 7 of this invention.

[0037] Figure 7 This is a comparison of feces before and after medication in the meat duck experimental group and control group in Example 7 of the present invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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. 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. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0039] Example 1

[0040] Isolation and identification of serotypes 1 and 7 of Riemerella anatipestifer

[0041] Duck heads exhibiting serositis symptoms were collected from a duck farm. The brains of these ducks were aseptically extracted and streaked onto TSA (containing 5% fetal bovine serum) medium. The culture was incubated at 37°C with CO2 for 16–20 hours. Single colonies were picked from the plates for Wright's staining and microscopic observation. Colonies with intense Wright's staining at both extremes were purified by streaking again. This process was repeated three times until uniform, round, smooth, translucent, dew-like single colonies of uniform size and shape grew. These colonies were then picked and cultured in TSB (containing 5% fetal bovine serum) broth at 37°C with shaking at 180 rpm for 10–12 hours, yielding a homogeneous, turbid bacterial suspension. Biochemical tests showed positive results for oxidase and catalase tests, while negative results for methyl red, indole, and nitrate reduction tests. The bacteria did not ferment sugars or liquefy gelatin, consistent with the physicochemical characteristics of *Riemerella anatipestifer*. Further 16S RNA molecular biological identification confirmed the bacteria as *Riemerella anatipestifer*. Furthermore, the isolated Riemerella anatipestifer was identified as serotype 1 and serotype 7 through a serum plate agglutination test.

[0042] Example 2

[0043] Isolation and identification of SD-RA1 bacteriophage of serotypes 1 and 7 of Riemerella anatipestifer

[0044] 1. Wastewater and manure treatment: Collect wastewater and manure from the duck farm, add SM buffer at a 1:1 ratio, let stand overnight at 2-8℃, take the supernatant and filter it through 8 layers of gauze, centrifuge the filtered wastewater at 8000 rpm for 5 minutes, filter the supernatant through a 0.22μm filter, and store it at 2-8℃ for later use.

[0045] 2. Phage enrichment: Add 1 ml of filtered wastewater to 1 ml of TSB liquid medium and add an appropriate amount of bacterial solution (about 4 kinds of bacteria can be added per tube, 20-40 μl of each kind). Incubate at 37°C in a shaker for 2-4 hours. Finally, filter through a 0.22 μm filter and store at 2-8°C.

[0046] 3. Prepare the lower layer of the double-layer plate: Take the autoclaved TSA medium, cool it to 50℃~55℃ at room temperature, pour 10~15ml into each sterile plate, cool it to room temperature and store it at 2~8℃ for later use.

[0047] 4. Spreading double-layer plates: First, dispense 500 μl of the enriched Riemerella anatipestifer suspension into a 10 ml sterile centrifuge tube. Then, after cooling the autoclaved semi-solid culture medium (with 5% fetal bovine serum) to 50°C at room temperature, pour 5 ml into the above 10 ml centrifuge tube and pour the upper layer plate.

[0048] 5. Phage Isolation and Purification: After the upper agar plate solidifies, aspirate the filtered wastewater liquid and drop it onto the plate. Once the surface liquid dries, invert the plate and incubate it in a 37°C CO2 incubator for 12–16 hours. If phages are present, they will form transparent, regular, circular plaques on the culture medium. Discard a single plaque into 1×SM buffer, shake at 37°C for 4–6 hours, centrifuge at 8000 rpm for 5 minutes, filter the supernatant through a 0.22 μm filter, and then drop it onto a double-layer agar plate to obtain individual plaques. Repeat this process 3–5 times until circular, transparent plaques of uniform size and approximately 1–2 nm in diameter are formed. Figure 1 As shown.

[0049] 6. Phage propagation: Phage propagation uses a "scraping" method to ensure that the phage titer is consistently above 10. 10 PFU / ml. Add 100 μl of purified *Riemerella anatipestifer* phage to a 10 ml sterile centrifuge tube, add 1 ml of *Riemerella anatipestifer*, and then add 5 ml of semi-solid culture medium (containing 5% fetal bovine serum). Pour the mixture onto a plate and allow it to solidify. Invert the plate and incubate at 37°C in a CO2 incubator for 12–16 hours. Once plaques appear on the plate, scrape the upper agar layer into a centrifuge tube, add an appropriate amount of 1×SM buffer, shake at 37°C for 4–6 hours, centrifuge at 8000 rpm for 5 minutes, filter through a 0.22 μm filter, and store at 2–8°C. For long-term storage of the phage, add 30% glycerol and store at -80°C.

[0050] 7. Phage titer assay: Using a sterile pipette tip, take 1 ml of the above phage suspension and add it to a test tube containing 9 ml of SM buffer. Mix thoroughly, then take 1 ml of the diluted phage suspension and add it to another centrifuge tube containing 9 ml of SM buffer. Repeat the above steps to dilute the sample concentration to 10. -2 ~10 -8 Eight dilution gradients were used. Two to three suitable sample dilution gradients were selected. Using a sterile pipette tip, 100 μl of each gradient was added to 100 μl of *Riemerella anatipestifer* proliferation medium, mixed well, and incubated at 37°C for 5 minutes. 5 ml of the upper TSA semi-solid medium (containing 5% fetal bovine serum) was added, mixed well, and poured onto the lower NA solid medium. After solidification, the plates were inverted and incubated at 37°C for 10–15 hours. The morphology of individual and uniform plaques was observed, and the number of plaques was counted. Plates with 30–300 plaques were selected for counting, and the phage titer was calculated using the formula, retaining one significant figure. The experiment was repeated three times, and the average result was taken. Phage titer (PFU / ml) = number of plaques on a plate with appropriate plaque density × dilution factor × 10. The result showed that the SD-RA1 phage titer was 2.8 × 10⁻⁶. 10 PFU / ml.

[0051] Example 3

[0052] Electron microscopic observation of bacteriophages

[0053] 1. Phage concentration:

[0054] With a valence of 2.8 × 10 10 PFU / ml phage SD-RA1 suspension was collected in a 1L blue-necked bottle. DNase and RNase were added to a final concentration of 1μg / ml for 30 minutes at room temperature. Solid sodium chloride was then added to the suspension to a final concentration of 1mol / L, and the mixture was shaken to accelerate dissolution. The mixture was incubated overnight at 4°C to allow for complete separation of the phage from the host bacterial fragments. The supernatant was collected after centrifugation at 12000rpm for 10 minutes at 4°C.

[0055] Add solid PEG8000 to the collected supernatant to a final concentration of 10% (w / v), shake to accelerate dissolution, incubate overnight at 4°C, and the next day dispense into 50ml centrifuge tubes. Centrifuge at 12000rpm for 10 minutes, discard the supernatant, and invert the centrifuge tubes to drain any remaining liquid.

[0056] Add 1 ml of SM buffer to each centrifuge tube, let stand for 1 hour to allow the precipitate to dissolve completely, collect the liquid and store it at 4°C for later use.

[0057] The phage titer was determined according to the phage titer detection method in Example 2, and the result was 1.6 × 10⁻⁶. 11 PFU / ml.

[0058] Phages were treated using the phosphotungstic acid negative staining method: 100 μl of concentrated phage was dropped onto a paraffin slide, and the film-coated side of a copper mesh was placed on the phage drop. After 10 minutes, the copper mesh was removed from the drop and placed on clean filter paper. After drying for 3 minutes, a drop of 2% phosphotungstic acid (PTA) was added to the copper mesh to stain the phages for 10 minutes. Then, the staining solution was absorbed from the side with filter paper. After the sample dried, the morphology of the phages was observed using an electron microscope.

[0059] Depend on Figure 2 It is known that the bacteriophage has a regular polyhedral head structure with a diameter of about 61 nm and a tail with a length of about 244 nm. According to the classification criteria of the Ninth Report of the International Committee on Taxonomy of Viruses, this bacteriophage can be identified as belonging to the family Longtailed Phagesidae, and it is named SD-RA1.

[0060] Example 4

[0061] Biological characteristics of bacteriophages

[0062] 1. Temperature stability determination of bacteriophage SD-RA1

[0063] Centrifuge tubes containing 500 μl of phage were placed in constant temperature water baths at 40℃, 50℃, 60℃, 70℃, and 80℃, respectively. Samples were taken after 30 minutes and 60 minutes, and the changes in phage titer were determined using the double-layer plate method.

[0064] The results are as follows ( Figure 3 After being exposed to 40℃ and 50℃ for 1 hour, phage SD-RA1 essentially maintained its original activity; after 30 minutes at 60℃, its titer decreased by two orders of magnitude, and after 1 hour, it decreased by four orders of magnitude; under high temperatures of 70℃ and 80℃, the phage was essentially inactivated after 30 minutes. The experimental results indicate that phage SD-RA1 can withstand temperatures below 50℃.

[0065] 2. Determination of pH stability of bacteriophages

[0066] The pH of TSB liquid culture medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 using concentrated hydrochloric acid and NaOH solution. For each pH value, 200 μl of the medium was taken and mixed with 200 μl of 10¹⁰ PFU / ml phage solution. The mixture was then incubated at 37°C for 1 hour. From each tube, 200 μl of phage was taken to determine the change in phage titer.

[0067] The results are as follows ( Figure 4 Within the pH range of 6–9, the phage titer remained almost unchanged or decreased slightly, all within 10. 9 At concentrations above PFU / ml, the phage titer is highest, reaching 10 at pH 8.0. 10The PFU / ml concentration exhibits good stability under alkaline conditions, maintaining a potency of 10 at pH 12. 3 PFU / ml. Within the pH range of 2.0–5.0, the phage titer decreases significantly with decreasing pH, and at pH 2.0, it completely loses its ability to lyse the host. Phage SD-RA1 exhibits acid-sensitive and alkali-loving properties. This phage grows most readily in neutral environments.

[0068] 3. Determination of the optimal multiple of infection (MOI) for bacteriophages

[0069] The concentration of the host bacteria was adjusted to 1.0 × 10⁻⁶. 8 With CFU / ml and MOIs set at 0.0001, 0.001, 0.01, 0.1, and 1, equal volumes of phage fluid and bacterial culture (500 μl each) were mixed thoroughly and incubated at 37°C with shaking at 180 rpm for 12–16 hours. The mixed culture was then centrifuged at 12000 rpm for 5 minutes to obtain lysis buffer. Each MOI value was performed in triplicate, and the phage titer was measured. The MOI of the phage was the multiplication factor that produced the highest titer.

[0070] The results showed that the MOI of phage SD-RA1 was 0.01. Under these conditions, the titer of progeny phages produced by phage infection of the host bacteria was 3.6 × 10¹⁰ PFU / ml, which was the highest among the five multiples of infection. Detailed results are shown in Table 1.

[0071] Table 1. Results of SD-RA1 Optimal Multiple of Infection (MOI) assay.

[0072]

[0073] 4. One-step growth curve of bacteriophage

[0074] The host bacteria were cultured for 7–8 hours to achieve a concentration of 1 × 10⁻⁶. 8 At approximately CFU / ml, the phage solution and host bacterial solution were incubated in a 37°C water bath for 5 minutes at the optimal multiplicity of infection ratio to allow the phage to fully adsorb the host bacteria. After centrifugation at 12,000 rpm for 1 minute, the supernatant was discarded, and the unadsorbed phage was thoroughly washed away with sterile TSB liquid medium three times. The washed solution was then added to 100 ml of TSB liquid medium (containing 5% fetal bovine serum), mixed thoroughly, and incubated in a 37°C constant temperature shaker. Sampling was started at 0 minutes and repeated every 10 minutes for 3 replicates to determine the phage titer.

[0075] From such Figure 5 The results show that the incubation period for bacteriophage infection of the host bacteria is 10 minutes, the lysis period is 70 minutes, and the lysis volume is approximately 433.

[0076] Example 5

[0077] Bacteriophage lysis spectrum assay

[0078] 1. Determination of phage lysis profile

[0079] Thirty strains of *Riemerella anatipestifer* and ten strains of *Pasteurella multocida*, already identified in the laboratory, were selected, and the phage lysis profile was determined using the double-layer plate method. The steps were as follows: Host bacterial suspensions were prepared according to the method described in Example 1. 200 μl of the bacterial suspension and 200 μl of phage were incubated at 37°C for 5 minutes. The mixture was then added to the upper semi-solid culture medium (containing 5% fetal bovine serum) to prepare double-layer plates. After the upper layer solidified, the plates were incubated upside down overnight at 37°C. The lysis results were observed, and the statistical results are shown in Table 2.

[0080] 2. Antimicrobial resistance testing of host bacteria

[0081] Antimicrobial susceptibility tests were performed on 30 strains of Riemerella anatipestifer and 10 strains of Pasteurella multocida. The detailed results are shown in Table 2.

[0082] Table 2. Lysis patterns of different serotypes of Riemerella anatipestifer SD-RA1 phage against Riemerella anatipestifer and Pasteurella multocida.

[0083]

[0084]

[0085] As shown in Table 2, phage SD-RA1 could completely lyse all 11 strains of serotype 1 and 8 strains of serotype 7 of *R. anatidae*, with a lysis rate of 100%. It showed no lysis against serotypes 2, 5, 6, and 10 of *R. anatidae*, and no lysis against *Pasteurella multocida*. Analysis of host bacterial resistance revealed that 30 strains of *R. anatidae* and 10 strains of *Pasteurella multocida* exhibited different levels of resistance, with over 90% of *R. anatidae* strains displaying multidrug resistance.

[0086] 3. In vitro lysis assay of bacteriophages (OD value method)

[0087] The experiment consisted of four experimental groups and one control group. In each experimental group, 500 μl of serum-containing Riemerella anatipestifer type 1 bacterial suspension was added to a 10 ml sterile centrifuge tube, with a final bacterial concentration of 1 × 10⁻⁶. 8 CFU / ml, then add 200 μl of phage, the final concentration of phage in the four experimental groups is 1×10⁻⁶. 8 PFU / ml, 1×10 7 PFU / ml, 1×10 6 PFU / ml, 1×10 5PFU / ml. The control group was added with the same amount of sterile TSB broth (with 5% fetal bovine serum) as the experimental group, and cultured at 37°C and 180 rpm in a shaker. The OD value of the mixture was measured at regular intervals until the mixture became clear, and the residual bacterial load in each group was simultaneously determined using the spread plating method at different time points.

[0088] The experimental results are shown in Tables 3 and 4. The results show that phage SD-RA1 has a good lytic effect on serological type 1 Riemerella anatipestifer. The lysis rates of the four different concentrations of phage fluids against serological type 1 Riemerella anatipestifer can all reach over 98%, only the lysis time differs. However, within 8 hours, the phage at all four concentrations can achieve a good killing effect on the host bacteria.

[0089] Table 3 Results of in vitro lysis assay of phage SD-RA1 strain

[0090]

[0091] Table 4. Determination of residual bacterial count in each group after 8 hours of incubation.

[0092]

[0093] Example 5

[0094] Safety testing of bacteriophages in target animals

[0095] Forty ducklings aged 7–14 days were randomly divided into an experimental group and a control group, with 20 ducklings in each group. Mice in the experimental group were intraperitoneally injected with 500 μl (10 μg / mL) of phage SD-RA1 proliferation solution. 9 The control group mice were injected intraperitoneally with the same dose of physiological saline for 7 consecutive days. The mice were observed for changes in mental state, food intake, water intake, and mortality. The mice were observed for 7 consecutive days, and at the end of the observation period, the mice were dissected to observe changes in their organs.

[0096] The results are shown in Table 5. The mice in both the experimental and control groups had normal mental status, food and water intake, and no deaths. Autopsy revealed that the liver, lungs, heart, spleen, and kidneys of the experimental group mice were all normal, with no significant differences compared to the control group. Detailed results are shown in Table 5.

[0097] Table 5. Safety results of bacteriophage SD-RA1 on 7-14 day old ducklings.

[0098]

[0099] Example 6

[0100] Protection against Riemerella anatipestifer in ducklings after challenge with bacteriophage

[0101] 1. The protective effect of oral administration of bacteriophages against challenge with Riemerella anatipestifer in ducklings.

[0102] Eighty 2-4 day old ducklings were randomly divided into four groups: oral administration test group 1, oral administration test group 2, control group 1, and control group 2, with 20 ducklings in each group. Ducklings in test group 1 and control group 1 were injected intraperitoneally with serum type 1 of *Rimerella anatipestifer*, while ducklings in test group 2 and control group 2 were injected intraperitoneally with serum type 7 of *Rimerella anatipestifer*, 0.5 ml (1×10⁻⁶) per duckling. 6 CFU / ml), 6 hours after challenge, groups 1 and 2 were given oral phage SD-RA1 daily, 0.5 ml (1×10⁻⁶ CFU / ml) per animal. 9 PFU / ml), administered for 3 consecutive days; Groups 1 and 2 received intraperitoneal injection of phage SD-RA1 daily, 0.5 ml (1×10⁻⁶ PFU / ml) per animal. 9 The ducklings were given the same dose of physiological saline for 3 consecutive days (PFU / ml) and observed for 7 consecutive days after administration. The mortality rate of the ducklings was recorded.

[0103] The results showed that 90% of the ducklings in control groups 1 and 2 died, while in the oral administration test, 90% of the ducklings in groups 1 and 2 survived after oral administration of phage SD-RA1. This indicates that oral administration of phage SD-RA1 can effectively protect against attacks by serotypes 1 and 7 of Riemerella anatipestifer, and has a significant effect in preventing and treating serositis in ducklings caused by serotypes 1 and 7 of Riemerella anatipestifer. Specific results are shown in Table 6.

[0104] 2. The protective effect of intraperitoneal injection of bacteriophages against challenge with Riemerella anatipestifer in ducklings.

[0105] Eighty 2-4 day old ducklings were randomly divided into four groups: intraperitoneal injection test group 1, intraperitoneal injection test group 2, control group 1, and control group 2, with 20 ducklings in each group. Ducklings in test group 1 and control group 1 were injected intraperitoneally with serum of Riemerella anatipestifer type 1, while ducklings in test group 2 and control group 2 were injected intraperitoneally with serum of Riemerella anatipestifer type 7, 0.5 ml (1×10⁻⁶) per duckling. 6 CFU / ml), 6 hours after challenge, groups 1 and 2 were given intraperitoneal injections of phage SD-RA1 daily, 0.5 ml (1×10⁻⁶ CFU / ml) per animal. 9 The ducklings were given the same dose of physiological saline for 3 consecutive days (PFU / ml) and observed for 7 consecutive days after administration. The mortality rate of the ducklings was recorded.

[0106] The results showed that 90% of the ducklings in control groups 1 and 2 died, while in the intraperitoneal injection experiment, 100% of the ducklings in groups 1 and 2 survived after injection of bacteriophage SD-RA1. This indicates that bacteriophage SD-RA1, after injection, can completely protect against infection by serotypes 1 and 7 of Riemerella anatipestifer, and is significantly effective in preventing and treating serositis in ducklings caused by serotypes 1 and 7 of Riemerella anatipestifer. Specific results are shown in Table 6.

[0107] In summary, these results further demonstrate that phage SD-RA1 administered by injection provides better protection against Riemerella anatipestifer than oral administration.

[0108] Table 6. Results of the protection against Riemerella anatipestifer challenge in ducklings by different routes of administration of phage SD-RA1.

[0109]

[0110] Example 7

[0111] Purification effect of Riemerella anatipestifer bacteriophage on Riemerella anatipestifer in broiler duck farms

[0112] 1. Application objective: To evaluate the purification effect of Riemerella anatipestifer bacteriophage on Riemerella anatipestifer in meat ducks and its impact on production performance.

[0113] 2. Experimental location: A large-scale meat duck farm in Jiangsu Province

[0114] 3. Information on the experimental duck flock: There are 16 houses in the whole farm. Houses 1-9 each have 26,000 ducks, houses 10-12 each have 24,000 ducks, and houses 13-16 each have 11,000 ducks.

[0115] (1) Four houses were selected as the experimental group. Riemerella anatipestifer bacteriophage was administered at a rate of 10,000 birds / bottle, via drinking water, once a day, to be consumed within 2-3 hours, for 2 consecutive days. The birds were used at ages 23-24 and 33-34.

[0116] (2) Four buildings were selected as the control group.

[0117] (3) The experimental group and the control group had the same source of ducklings, the same feed, the same immunization program, and the same feeding and management conditions.

[0118] (4) Grouping situation

[0119]

[0120] 4. Test evaluation indicators:

[0121] Observe the entire duck rearing period, including mortality, changes in manure in the sheds, feed conversion ratio at slaughter, European index, and survival rate.

[0122] 5. Experimental Results

[0123] Table 7 Feed conversion ratio

[0124]

[0125] Summary: According to the statistical results of feed conversion ratio in Table 7, the average feed conversion ratio of the phage group was 1.72, while that of the control group was 1.76. The feed conversion ratio of the phage group was 0.04 lower than that of the control group.

[0126] Table 8 European Index

[0127]

[0128] Summary: According to the results of the European index of meat ducks in Table 8, the average European index of the phage group was 467.7, while the average European index of the control group was 459.6, which was 8.1 higher than that of the control group.

[0129] Table 9 Survival Rate

[0130]

[0131] Summary: According to the average survival rate results in Table 9, the average survival rate of the phage group was 98.31%, while the average survival rate of the control group was 98.55%, which was 0.24% lower than that of the control group.

[0132] 5.4 Autopsy of normally dead ducks in the experimental and control groups before slaughter

[0133] Three to four days before slaughter, select an equal number of dead ducks from each shed, dissect them, observe the symptoms (and take photos for record-keeping, such as...). Figure 6 (As shown), recording ratio

[0134] Summary: Autopsy of dead ducks in the phage group showed that the proventriculus and gizzard were basically normal, with only a few exhibiting minor gizzard ulcers. The control group showed pericarditis and hepatitis, proventricular edema, gizzard ulcers, and impaired bile excretion. This indicates that feeding ducks with bacteriophages can reduce the infection of conditionally pathogenic bacteria such as Escherichia coli.

[0135] 5.5 Photographs of feces

[0136] Photos were taken at fixed points before, during, and after the use of the experimental and control groups.

[0137] Summary: Figure 7 As shown, the stool of the bacteriophage group was normal at all stages, while the control group developed bloody stool at 35 days of age. Figure 7 The oval frame indicated by the letter A in the diagram represents the bloody stool area, demonstrating that bacteriophages can improve the stool of meat ducks and protect intestinal health.

[0138] 6. Conclusion

[0139] 6.1 Production indicators: The feed conversion ratio of the phage group was 0.04 lower than that of the control group, and the European index was 8.1 higher;

[0140] 6.2 Symptoms: Effectively prevents the occurrence of pericarditis and perihepatitis symptoms;

[0141] 6.3 Feces: Phage groups can improve fecal condition.

[0142] In summary, the experimental results show that Riemerella anatipestifer bacteriophage can reduce the infection of conditionally pathogenic bacteria such as Escherichia coli in ducks, improve duck feces, protect intestinal health, and improve breeding indicators.

[0143] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention.

[0144] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A Riemerella anatipestifer bacteriophage, characterized in that, The bacteriophage can simultaneously lyse Riemerella anatipestifer of serotype 1 and serotype 7, and was preserved in China Center for Type Culture Collection in August 2021, with a preservation number of CCTCC MNO.20211061.

2. Use of a Riemerella anatipestifer bacteriophage according to claim 1, characterized in that, Application in preparation of a medicine for preventing or treating animal diseases caused by Riemerella anatipestifer of serotype 1 and serotype 7.

3. Use of a bacteriophage of Riemerella anatipestifer according to claim 1, characterized in that, Application in preparation of a feed for preventing or treating animal diseases caused by Riemerella anatipestifer of serotype 1 and serotype 7.

4. Use of a bacteriophage of Riemerella anatipestifer according to claim 1, characterized in that, Application in preparation of a cleaning agent or a disinfectant for preventing or treating animal diseases caused by Riemerella anatipestifer of serotype 1 and serotype 7.

Images