A duck rimerella bacteriophage with temperature change resistance and its application in preventing duck serositis and water drug delivery
By screening out the thermoresistant Riemerella anatipestifer phage PRA006, the problem of phage inactivation in temperature-changing environments has been solved, achieving continuous and efficient inhibition and prevention of Riemerella anatipestifer, reducing antibiotic dependence, and providing a safe and effective solution for the prevention and treatment of duck serositis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN KEQIAN BIOLOGY CO LTD
- Filing Date
- 2025-10-20
- Publication Date
- 2026-05-29
AI Technical Summary
Existing bacteriophages are easily inactivated in natural water bodies due to drastic temperature fluctuations, leading to unstable control of Riemerella anatipestifer infection. Furthermore, antibiotic resistance and drug residues are serious problems, and existing vaccines have incomplete coverage and short periods of immune protection.
A thermosensitive Riemerella anatipestifer phage, PRA006, was screened out. It can maintain high activity in the range of 4-50℃ and can be applied to microbial preparations, feed additives and drinking water additives for the prevention and treatment of duck serositis.
It achieves continuous and efficient inhibition of Riemerella anatipestifer in temperature-changing environments, solves the problem of phage activity interruption, reduces antibiotic dependence, and provides a safe and effective prevention and treatment solution.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a thermosensitive strain of Riemerella anatipestifer and its application as a drug for the prevention of duck serositis and for water administration. Background Technology
[0002] Riemerella anatipestifer is the main pathogen causing infectious serositis in ducks, geese and other waterfowl. It mainly enters the body of ducks and other animals through wounds or other routes such as air, contaminated feed and drinking water, and multiplies. It then travels through the bloodstream to reach all organs of the body, causing inflammation in various organs, including the brain, resulting in typical symptoms of air sacculitis, pericarditis, perihepatitis and meningitis. The morbidity and mortality rates are high, seriously endangering the aquaculture industry. Currently, the main control method relies on antibiotics (such as florfenicol and enrofloxacin), but the following problems exist: (1) The resistance rate of Riemerella anatipestifer to multiple antibiotics is increasing year by year, leading to treatment failure. (2) Drug residues: The abuse of antibiotics leads to drug residues in poultry products, threatening food safety and public health. (3) At least 21 serotypes of Riemerella anatipestifer have been found to date. Existing vaccines, such as inactivated vaccines or subunit vaccines, do not cover all serotypes and have a short period of immune protection.
[0003] In recent years, bacteriophages, as naturally occurring bacterial viruses, have gradually become a strong candidate to replace traditional antibiotics due to their high specificity, environmental friendliness, and low likelihood of developing drug resistance. Bacteriophages can specifically infect and lyse their host bacteria, thereby effectively controlling bacterial infections. Furthermore, bacteriophages are widely distributed in the environment, possessing the ability to self-replicate and self-spread, and can form high concentrations of phage particles at the site of infection, further enhancing their bactericidal effect. Given the increasingly serious problem of drug resistance in *Rimerella anatipestifer*, bacteriophages capable of lysing drug-resistant pathogens offer a new therapeutic approach. However, compared to bacteriophages targeting other avian pathogens, there are currently relatively few publicly available effective *Rimerella anatipestifer* bacteriophages.
[0004] On the other hand, when using bacteriophages for biocontrol, especially against pathogens in natural water bodies (such as aquaculture ponds and lakes), we have long faced a core bottleneck: the temperature dependence of bacteriophage activity. Natural water bodies are not constant-temperature environments, especially in shallow lake areas, where diurnal temperature variations can cause drastic fluctuations in water temperature exceeding 10°C within hours (e.g., from above 30°C at noon to below 15°C at night). Most laboratory-screened bacteriophages are easily inactivated in environments with drastic temperature fluctuations within a short period, failing to sustainably and effectively inhibit pathogens. This "time window" of interruption provides pathogens with an opportunity to regroup and multiply, significantly limiting the reliability and stability of phage therapy in open environments. Current phage screening strategies mostly focus on finding strains with high lytic efficacy against specific pathogens, often conducted under constant-temperature conditions (usually the optimal growth temperature for the pathogen). While phages screened using this method show significant in vitro experimental results, their ecological adaptability and sustained effectiveness are often unsatisfactory when introduced into real, dynamically temperature-changing natural environments. Therefore, we lack bacteriophages for Riemerella anatipestifer that can inhibit Riemerella anatipestifer, are safe to use, and are resistant to temperature changes. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a thermosensitive Riemerella anatipestifer phage PRA006, the preservation number of which is: CCTCC NO: M20251329.
[0006] Another object of the present invention is to provide the use of Riemerella anatipestifer phage PRA006 in the preparation of a drug for preventing Riemerella anatipestifer type II infection.
[0007] Another objective of this invention is to provide the application of Riemerella anatipestifer phage PRA006 in the preparation of Riemerella anatipestifer type II antibacterial agent.
[0008] To achieve the above objectives, the present invention adopts the following technical measures:
[0009] The applicant screened a thermosensitive strain of *Riemerella anatipestifer* phage PRA006 from the brains of ducks with duck serositis collected from various disease-affected farms. This strain maintains high activity during temperature cycles of 45℃ and 15℃, and also maintains high activity at 4-50℃ and pH values of 5-10. It exhibits biocompatibility and immunogenicity, making it suitable for use as a vaccine against duck serositis. This phage was deposited at the China Center for Type Culture Collection (CCTCC) on June 10, 2025, with the classification name: *Riemerella anatipestifer* phage (PRA006), accession number: CCTCC NO: M20251329, address: Wuhan University, Wuhan, China.
[0010] The scope of protection of this invention includes:
[0011] A microbial preparation containing Riemerella anatipestifer phage PRA006.
[0012] Preferably, the phage drug formulation described above further comprises a pharmaceutically acceptable carrier.
[0013] Preferably, the dosage form of the microbial preparations described above is a solution, powder, gel, granule, or lyophilized agent.
[0014] Application of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the preparation of Riemerella anatipestifer type II inhibitors.
[0015] Application of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the in vitro inhibition of Riemerella anatipestifer type II.
[0016] Application of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the preparation of feed additives.
[0017] Application of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the preparation of livestock and poultry drinking water additives.
[0018] Application of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the preparation of microbial preparations for release into natural waters.
[0019] The use of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the preparation of drugs for the prevention of Riemerella anatipestifer type II infection.
[0020] Application of Riemerella anatipestifer phage PRA006 or the above-mentioned microbial preparations in the preparation of duck serositis vaccine.
[0021] A drug for the prevention and treatment of bacterial serositis in ducks, said drug containing Riemerella anatipestifer bacteriophage PRA006.
[0022] A feed additive comprising Riemerella anatipestifer phage PRA006.
[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0024] The Riemerella anatipestifer bacteriophage PRA006 of this invention can lyse two different Riemerella anatipestifer strains, and can inhibit bacteria for a long time, with high stability and good safety.
[0025] The bacteriophage provided by this invention exhibits excellent temperature cycling tolerance, solving the problem of easy inactivation of bacteriophage preparations due to diurnal cycles. It has a good killing and control effect on Riemerella anatipestifer in the environment and in ducks. It can be directly applied to the natural environment or sprayed as a microbial preparation, such as in environmental water bodies or soil, to more continuously and efficiently inhibit and kill Riemerella anatipestifer. It can also be used as a feed additive or drinking water additive in the daily diet of livestock and poultry for disease prevention, or as a preventive vaccine in the early stages of potential disease outbreaks. It shows good promise in the clinical application of preventing and treating bacterial serositis in ducks. Attached Figure Description
[0026] Figure 1 This is a graph showing the titer changes of six strains of Riemerella anatipestifer bacteriophage under temperature cycling conditions.
[0027] Figure 2 This is an image of plaques from Riemerella anatipestifer phage PRA006.
[0028] Figure 3 This is the optimal infection multiplicity diagram of Riemerella anatipestifer phage PRA006.
[0029] Figure 4 This is a one-step growth curve of Riemerella anatipestifer phage PRA006.
[0030] Figure 5 This is a temperature tolerance diagram of Riemerella anatipestifer phage PRA006.
[0031] Figure 6 This is a pH tolerance diagram of Riemerella anatipestifer phage PRA006.
[0032] Figure 7 This is a safety test diagram of Riemerella anatipestifer phage PRA006. Detailed Implementation
[0033] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all derived from commercial sources.
[0034] Example 1:
[0035] Screening of Riemerella anatipestifer phage PRA006
[0036] 1. Phage isolation and purification
[0037] Brains were collected from diseased ducks at various farms. 1g of brain tissue was added to 1mL of sterile physiological saline, and the brain was thoroughly ground. The mixture was then centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected and filtered through a 0.22µm filter for sterilization. The supernatant was stored at 4℃ for later use. 0.1mL of *Riemerella anatipestifer* RA08006 and 1mL of the filtrate were added to 1mL of 2×TSB broth containing 5% fetal bovine serum. The mixture was thoroughly mixed and incubated at 37℃ for 12–14 hours. The next day, the culture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was filtered through a 0.22µm filter for sterilization to obtain a phage enrichment solution.
[0038] Phage purification was performed using the double-layer plate method: 0.1 mL of the above phage enrichment solution was serially diluted 10-fold, and 10 samples were taken from each solution. -2 10 -4 10 -6 Add 0.1 mL of the diluent to 0.1 mL of the logarithmic-phase host bacterium RA08006, and then add 3 mL of 0.6% semi-solid LB medium at approximately 45°C. Spread the mixture evenly onto pre-prepared semi-solid medium containing 5% fetal bovine serum (TFB). Incubate at 37°C for 12–14 h, and then observe plaque formation. Repeat the experiment three times. Store the purified phage fluid at 4°C for later use.
[0039] A total of 6 strains of Riemerella anatipestifer bacteriophage were isolated, and 5 of the bacteriophages were named PRA001, PRA002, PRA003, PRA004, PRA005 and PRA006, respectively.
[0040] 2. Screening of thermostable Riemerella anatipestifer phage PRA006
[0041] The water bath temperature was cycled between 45℃ and 15℃, i.e., 45℃-15℃-45℃, with the temperature changed every 6 hours. The six phage strains isolated in step 1 were diluted with SM buffer to a titer of 1×10⁶. 4 After obtaining PFU / mL, place the sample in the water bath and measure the potency once a day for a total of 7 days.
[0042] The results are as follows Figure 1 As shown, bacteriophages PRA003 and PRA005 decreased in titer by approximately 2 titers on day 7, while bacteriophages PRA001, PRA002, and PRA004 decreased by approximately 1.5 titers. Bacteriophage PRA006, however, only decreased by 0.4 titers, exhibiting strong temperature resistance. Therefore, the bacteriophages of this invention can be directly applied to or sprayed in the natural environment, such as in water bodies or soil, to more continuously and efficiently inhibit and kill *Riemerella anatipestifer*. They can also be used as feed additives or drinking water additives in the daily diet of livestock and poultry for disease prevention, or as a preventative vaccine in the early stages of a potential disease outbreak.
[0043] The morphology of the purified phage PRA006 was observed, such as... Figure 2 As shown, the phage plaques are approximately 0.2 cm in diameter and are clear and transparent.
[0044] Therefore, phage PRA006 was selected for preservation and subsequent experiments. This phage was deposited at the China Center for Type Culture Collection (CCTCC) on June 10, 2025, and is classified and named as *Riemerella anatipestifer* phage PRA006, with accession number CCTCC NO: M20251329, located at Wuhan University, Wuhan, China. In this invention, it is referred to as *Riemerella anatipestifer* phage PRA006.
[0045] Example 2:
[0046] Assay for the optimal multiple of infection of Riemerella anatipestifer phage PRA006
[0047] The concentration of the host bacterium *Riemerella anatipestifer* RA2018016 in the logarithmic phase was adjusted to 1×10⁻⁶. 6 CFU / mL was added to the phage stored at 4℃ in Example 1 at ratios of 100, 10, 1, 0.1, and 0.01, respectively. The phage was then added to 10 mL of TSB broth containing 5% fetal bovine serum to ensure a uniform total volume of the culture system. After incubation at 37℃ for 5 h, the culture was centrifuged at 10000 rpm for 10 min, and the supernatant was collected and diluted to an appropriate concentration. The titer was determined using a two-layer method.
[0048] The results are as follows Figure 3 As shown, the titer of Riemerella anatipestifer phage PRA006 is highest at an MOI of 0.1. Therefore, the optimal multiplicity of infection (MOI) for Riemerella anatipestifer phage PRA006 is 0.1.
[0049] Example 3:
[0050] One-step growth curve of Riemerella anatipestifer phage PRA006
[0051] The concentration of the host bacterium *Riemerella anatipestifer* RA2018016 in the logarithmic phase was adjusted to 1×10⁻⁶. 6 CFU / mL, according to the multiplicity of infection ratio of 0.1, was added to the phage stored at 4℃ in Example 1, and added to 10 mL of TSB broth containing 5% fetal bovine serum. The mixture was incubated at 37℃ for 5 h, and the titer was determined by double-layer method every half hour.
[0052] The results are as follows Figure 4 As shown, the titer of the bacteriophage remained at 10 within 0-1.5 hours. 4 The phage titer remained constant at approximately PFU / mL, representing its incubation period. Within 1.5–3 hours, the phage titer increased exponentially, from 10... 4 PFU / mL increased to 10 8 The PFU / mL value represents the burst phase of this bacteriophage; at 5 hours, the final titer was 3.2 × 10⁻⁶. 8 PFU / mL.
[0053] Example 4:
[0054] Host spectrum determination of Riemerella anatipestifer phage PRA006
[0055] 0.1 ml of each of the 17 overnight cultures of *Riemerella anatipestifer* strains was added to 4 ml of TSB semi-solid medium containing 5% fetal bovine serum at approximately 50°C. The mixture was then evenly spread on pre-prepared LB solid medium. Each plate was then divided into two equal regions, with 10 µL of the culture from one region adjusted to a titer of 1 × 10⁻⁶. 8 PFU / ml, the phage stored at 4℃ in Example 1 was dropped onto the surface, and another area was dropped with TSB broth that was not inoculated with bacteria as a control. After the droplets dried, they were inverted and incubated at 37℃ for 12 hours. The results were observed. If phage plaques were produced, it was recorded as "+" and otherwise as "-".
[0056] The results are shown in Table 1: Riemerella anatipestifer phage PRA006 can strongly lyse two Riemerella anatipestifer strains RA202408006 and RA202408017.
[0057] Table 1. Spotting results of Riemerella anatipestifer phage PRA006
[0058]
[0059] Note: "+" indicates the degree of lysis of bacteria by the bacteriophage; the more "+" signs, the higher the degree of lysis. "-" indicates that the bacteriophage has no ability to lyse bacteria.
[0060] Example 5:
[0061] Temperature tolerance test of Riemerella anatipestifer phage PRA006
[0062] Take 10 sterile EP tubes and add 0.5 ml of phage (1×10⁻⁶) stored at 4℃ for later use in Example 1 to each tube. 9 The phages (PFU / mL) were incubated at 4℃, 25℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 60 min, respectively. After the incubation period, the phages were immediately cooled in a water bath, and then the titer of the phages was determined.
[0063] Temperature tolerance test, such as Figure 5 As shown: The titer of *Rimerella anatipestifer* phage PRA006 can be maintained at 10 within a temperature range of 4-50℃. 9 The activity was stable at approximately PFU / mL; the potency was 5×10⁻⁶ at temperatures of 50 and 60°C, respectively. 8 PFU / mL, 1.9×10 5 PFU / mL, the potency drops sharply to 10 at 70℃. 2 PFU / mL; completely inactivated at 80℃. Therefore, phage PRA006 exhibits strong stability over a wide temperature range and can adapt to certain high-temperature environments.
[0064] Example 6:
[0065] pH tolerance assay of Riemerella anatipestifer phage PRA006
[0066] Take 6 0.1 ml portions of phage from Example 1 and store them at 4°C for later use (1×10⁻⁶). 9 The phages (PFU / mL) were placed in 0.9 mL of SM buffer at pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and incubated at 37 °C for 2 h. The titer of the phages after the reaction was then determined using the bilayer plate method.
[0067] Test results as follows Figure 6 As shown: The titer of Riemerella anatipestifer phage PRA006 was 1.8 × 10⁻⁶ within the pH range of 5-10. 8 PFU / mL, 1.1×10 9 PFU / mL, 1.1×10 9 PFU / mL, 1.3×10 9 PFU / mL, 1.0×10 9 PFU / mL, 1.5×10 7 The phages maintained stable activity at PFU / mL and remained active at pH 3 and pH 11, with titers of 10 and 10, respectively. PFU / mL, 1.5×103 PFU / mL. Therefore, phage PRA006 exhibits strong stability over a wide pH range and can adapt to certain strong acid and alkaline environments.
[0068] Example 7:
[0069] Biosafety assessment of Riemerella anatipestifer phage PRA006
[0070] Ten 25-day-old ducks were divided into a control group and a phage group, with five ducks in each group. The control group was injected with 0.2 mL of TSB broth containing 5% fetal bovine serum, while the phage group was subcutaneously injected with 0.2 mL of phage PRA006 (2×10⁻⁶). 9 PFU / mL). Clinical symptoms of each group of ducks were observed daily. After 7 days, the ducks were dissected, and their hearts, lungs, and spleens were collected to observe whether there were any lesions in these organs in both groups.
[0071] The results showed no significant difference in clinical symptoms between the two groups of ducks within one week of phage treatment. No ducks in the phage group died, and no abnormalities such as ruffled feathers, lethargy, emaciation, or loss of appetite were observed. The necropsy results are as follows: Figure 7 As shown, no serositis was observed in either group of ducks, and no lesions were found in other internal organs. These results indicate that the duck phage preparation of *Rimerella anatipestifer* is safe and has no toxic side effects.
[0072] Example 8:
[0073] Application of Riemerella anatipestifer phage PRA006 in the preparation of a vaccine for the prevention of duck serositis
[0074] Ten 37-day-old ducklings were divided into three groups: a control group (n=3), a positive control group (n=3), and a prevention group (n=4). From day 1 to day 3, the prevention group received bacteriophage PRA006 in their drinking water, ensuring a concentration of 10 mg / mL. 6 PFU phage was administered via oral ingestion for 1 hour. On day 4, except for the control group, the other groups received an intraperitoneal injection of 1 mL (1×10⁻⁶). 6 (CFU / mL) Riemereum anatipestifer type II RA08006. After challenge, continue observation for 3 days, recording clinical symptoms and mortality of ducks daily.
[0075] On day 1 post-challenge (day 0 is considered challenge day), one duck in the positive group died. Autopsy revealed pericarditis and perihepatitis in the dead duck. No ducks died in the prevention and negative groups. Within three days post-challenge, ducks in the positive group had loose, yellow-green feces, while ducks in the prevention and negative groups had formed feces. These results indicate that the Riemerella anatipestifer phage preparation PRA006 has a good preventive effect against duck serositis.
[0076] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. An isolated strain of Riemerella anatipestifer bacteriophage ( Riemerella anatipestifer The phage is listed as PRA006, and its preservation number is CCTCC NO: M20251329.
2. A microbial preparation containing the Riemerella anatipestifer phage PRA006 as described in claim 1.
3. The microbial preparation according to claim 2, wherein the microbial preparation comprises a pharmaceutically acceptable carrier.
4. The microbial preparation according to claim 2, wherein the dosage form of the microbial preparation is a solution, powder, gel, granule or lyophilized agent.
5. Any of the following applications of the *Rimerella anatipestifer* bacteriophage PRA006 of claim 1 or the microbial preparation of claim 2: 1) Used to prepare inhibitors of type II Riemerella anatipestifer; 2) Used for in vitro inhibition of Riemerella anatipestifer type II; 3) Used in the preparation of feed additives; 4) Used in the preparation of drinking water additives for livestock and poultry; 5) Used to prepare microbial agents for release into natural water bodies; 6) Used in the preparation of drugs for the prevention of Riemerella anatipestifer type II infection; 7) Used to prepare duck serositis vaccine.
6. A drug for preventing and treating bacterial serositis in ducks, said drug comprising the duck plague Riemerella phage PRA006 as described in claim 1.
7. A feed additive comprising the Riemerella anatipestifer phage PRA006 as described in claim 1.