Pig pasteurella phage and application thereof

Abstract

The invention discloses a swine pasteurella phage and application thereof, and relates to the technical field of biology. The swine pasteurella bacteriophage vBP1 is preserved in the China General Microbiological Culture Collection Center on December 10, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46287. The invention discloses a novel swine pasteurella bacteriophage, which has typical lytic bacteriophage characteristics. The bacteriophage has a specific splitting effect on swine pasteurellosis, can be used for preventing and / or treating swine pasteurellosis, and has an important practical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a Pasteurella bacteriophage of pigs and its application. Background Art

[0002] Pasteurella disease of pigs is a common infectious disease caused by Pasteurella multocida. This disease can cause symptoms such as difficulty in breathing, persistent high fever, and loss of appetite in pigs, and even lead to death in severe cases.

[0003] Currently, the main methods for preventing and treating Pasteurella disease of pigs include vaccination and antibiotic treatment. However, these traditional methods have certain limitations. For example, the protective effect of vaccines may be weakened due to strain variation; while long-term use of antibiotics may lead to the generation of bacterial drug resistance, further exacerbating the difficulty of disease prevention and control.

[0004] In recent years, bacteriophages have gradually attracted attention as a new biological control means. Bacteriophages are viruses that specifically infect and kill specific bacteria, with high specificity and self-replicating ability. Compared with traditional antibiotics, phage therapy can effectively avoid the problem of drug resistance and has no toxic and side effects on the animal body. In addition, bacteriophages can continuously act at the infection site until the target bacteria are completely cleared. Although phage therapy has shown many advantages, the research and development of specific bacteriophages for Pasteurella disease of pigs is still in its infancy. Therefore, the present invention aims to provide an efficient and stable Pasteurella bacteriophage of pigs to make up for the deficiencies of the existing technology and provide a new solution for the prevention and treatment of Pasteurella disease of pigs. Summary of the Invention

[0005] The object of the present invention is to provide a Pasteurella bacteriophage of pigs and its application to solve the problems existing in the above-mentioned prior art. This bacteriophage has a specific lysing effect on Pasteurella multocida and can be used for the prevention and / or treatment of Pasteurella disease of pigs.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Pasteurella bacteriophage (Pasteurella virus) vB_P1, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 10, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46287.

[0008] The present invention also provides the application of the above-mentioned Pasteurella bacteriophage vB_P1 in the preparation of drugs for preventing and / or treating Pasteurella infection of pigs.

[0009] The present invention also provides the application of the above-mentioned Pasteurella phage vB_P1 in the preparation of a Pasteurella bactericide for pigs.

[0010] The present invention also provides the application of the above-mentioned Pasteurella phage vB_P1 in the preparation of a pig feed additive.

[0011] The present invention also provides a drug for preventing and / or treating Pasteurella infection in pigs, and the active ingredient includes the above-mentioned Pasteurella phage vB_P1.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the drug is an injection, powder, gel, granule or freeze-dried preparation.

[0014] Furthermore, the drug also includes other active ingredients that have an antibacterial effect on Pasteurella.

[0015] The present invention also provides a Pasteurella bactericide for pigs, and the active ingredient includes the above-mentioned Pasteurella phage vB_P1.

[0016] Furthermore, the dosage form of the Pasteurella bactericide for pigs is a spray, powder, gel, granule or freeze-dried preparation.

[0017] The present invention discloses the following technical effects:

[0018] The present invention has discovered a novel Pasteurella phage for pigs, named vB_P1, which has typical lytic phage characteristics. This phage vB_P1 has a stable titer in an environment with a temperature of 40-60 °C and a pH of 5-12, and can lyse Pasteurella. After identification, phage vB_P1 is a new species of the order Podovirales, the family Podoviridae, and the genus Wuhanvirus.

[0019] The Pasteurella phage provided by the present invention has the characteristics of high efficiency, safety and no side effects, can effectively prevent and treat Pasteurella disease in pigs, is beneficial to reducing the use of antibiotics, and avoiding the generation of drug resistance problems, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is a double-layer plate diagram of phage vB_P1;

[0022] Figure 2 It is the electron micrograph of phage vB_P1;

[0023] Figure 3 It is the schematic diagram of the genome of phage vB_P1;

[0024] Figure 4 It is the phylogenetic tree diagram;

[0025] Figure 5 It is the test result diagram of the temperature stability of phage vB_P1;

[0026] Figure 6 It is the test result diagram of the optimal multiplicity of infection of phage vB_P1;

[0027] Figure 7 It is the test result diagram of the acid-base stability of phage vB_P1;

[0028] Figure 8 It is the one-step growth curve of phage vB_P1;

[0029] Figure 9 It is the in vitro antibacterial curve diagram of phage vB_P1. Detailed implementation manners

[0030] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0035] Example 1 Isolation, Identification and Preservation of Porcine Pasteurella Bacteriophage

[0036] 1. Sample Collection

[0037] Samples were collected from the manure and sewage of the pig farm.

[0038] 2. Bacteriophage Isolation

[0039] The drop method was used for bacteriophage isolation. The specific operation is as follows: appropriate amounts of sewage, bedding, etc. were respectively placed into 5 mL of LB liquid medium, cultured in a constant temperature shaker at 37 °C for 4 h, centrifuged at 10000 r / min for 5 min, and the supernatant was filtered and sterilized using a 0.22 μm filter membrane to obtain the original bacteriophage solution. The double-layer plate drop method was used to isolate the bacteriophage. 200 μL of Porcine Pasteurella preserved in the laboratory was mixed with 5 mL of TSB semi-solid medium cooled to about 55 °C in a centrifuge tube, poured onto the TSA solid medium, and left to stand until it solidified. The back of the prepared double-layer plate was divided into 8 equal parts, and 5 μL of each original bacteriophage solution was added dropwise to each area, and cultured at 37 °C for 6 h to observe whether plaques appeared.

[0040] 3. Specific Lysis Performance Detection and Purification

[0041] The isolated bacteriophage was identified by the double-layer plate method to confirm its specific lysis effect on Porcine Pasteurella. The specific operation is as follows: a single plaque was picked up using a sterile pipette tip, placed in SM buffer and left to stand for 12 h, filtered through a 0.22 μm filter membrane, and the filtrate was used to prepare a double-layer plate with the host bacteria again ( Figure 1 ), and the operation of picking up a single plaque for purification was repeated 6 times to obtain the purified bacteriophage, named vB_P1.

[0042] 4. Bacteriophage Identification

[0043] 4.1 Bacteriophage Morphology Observation

[0044] Preparation of a high-titer phage lysate by the plate amplification method. Take 400 μL of the host bacterium broth grown to the logarithmic phase, mix it with the phage lysate at the optimal multiplicity of infection ratio, prepare a double-layer plate, incubate it at 37 °C for 12 h, add 10 mL of SM buffer to the culture dish, shake it on a shaker at 100 r / min for 4 h, collect the eluate, centrifuge it at 12000 r / min for 5 min, and filter it through a 0.22 μm filter membrane to obtain the high-titer phage lysate for subsequent microscopic observation. The phage lysate is negatively stained with 2% phosphotungstic acid (w / v, pH 7.0), observed using a transmission electron microscope, and micrographs are taken at an accelerating voltage of 80 kV to observe the morphology of the phage, and the head and tail lengths of the phage are measured using ImageJ software.

[0045] Result analysis: As Figure 2 shown, phage vB_P1 has an icosahedral head with a diameter of 64 ± 3 nm and a cylindrical tail with a diameter of 7 ± 2 nm and a length of 12 ± 3 nm, and it can be determined that this phage belongs to the order Podovirales and the family Podoviridae.

[0046] 4.2 Molecular biological identification

[0047] After detection, the genome length of phage vB_P1 is 35964 bp, and the GC content is 41.08% ( Figure 3 ).

[0048] According to the criteria of the International Committee on Taxonomy of Viruses (ICTV), when the genomic sequence similarity between two phages is less than 95%, they should be classified as different species. In the NCBI database, the similarity between phage vB_P1 and other phages was compared and analyzed using BLAST. The results showed that the highest sequence similarity was with MZ995506 under the genus Wuhanvirus. Referring to the classification guidelines of BAVS, viruses with a nucleotide sequence similarity exceeding 50% in the virus population can be classified into the same genus. Therefore, it can be determined that vB_P1 is a new species of the genus Wuhanvirus. A phylogenetic tree was constructed using the maximum likelihood method, and the results are shown in Figure 4 .

[0049] 5. Biological preservation

[0050] Phage vB_P1 was deposited on December 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 46287.

[0051] Example 2 Titration of the titer of Pasteurella multocida phage

[0052] Method for determining phage titer: The double-layer plate method was used to determine the phage titer. The purified phage filtrate was taken for 10-fold serial dilution. 200 μL of the phage dilution was mixed with 200 μL of the host bacteria (Pasteurella multocida) suspension, incubated at 37 °C for 5 min, and then 4 - 5 mL of LB semi-solid medium at about 55 °C was poured into the mixture of the phage dilution and the host bacteria suspension, and immediately poured into a plate with LB solid medium at the bottom to prepare a double-layer plate. Incubated at 37 °C for 6 h, and the number of plaques was counted. Phage titer (PFU / mL) = number of plaques × 5 × dilution factor.

[0053] Result analysis: The titer of phage vB_P1 measured by the double-layer plate method was 9.1×10 5 PFU / mL.

[0054] Example 3 Determination of temperature stability of Pasteurella multocida phage

[0055] Five tubes of phage lysate (500 μL / tube) were placed in water baths at 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C respectively. Samples were taken at 20 min, 40 min, and 60 min respectively, and the phage titer was measured by the double-layer plate method, with three repetitions.

[0056] Result analysis: With the increase of temperature and the prolongation of time, the activity of phage vB_P1 decreased to a certain extent. Among them, within the temperature range of 40 - 60 °C, the phage activity remained basically unchanged. The titer began to decline gradually at 70 °C after 20 min of action, but all had activity. The titer was 0 under the condition of 80 °C ( Figure 5 ).

[0057] Example 4 Optimal multiplicity of infection of Pasteurella multocida phage

[0058] Determination of the optimal multiplicity of infection: The optimal multiplicity of infection of the phage was counted, and the concentration of the host bacteria was adjusted to 10 8 CFU / mL, and the MOIs were set to 100, 10, 1, 0.1, and 0.01 respectively. 500 μL of the phage dilution and 500 μL of the host bacteria suspension were taken, mixed well, and cultured at 37 °C with shaking at 180 r / min for 3 h. The mixed culture was centrifuged at 12000 r / min for 2 min and filtered through a 0.22 μm filter membrane to obtain the lysate. The phage titer of the lysate was measured by the double-layer plate method, with three repetitions. The multiplicity of infection with the highest phage titer of the lysate was the optimal multiplicity of infection of the phage.

[0059] Result analysis: When the multiplicity of infection of phage vB_P1 was 0.1, the phage titer was the highest, which was 2.56×10 8 PFU / mL ( Figure 6 ), so the optimal multiplicity of infection of phage vB_P1 was 0.1.

[0060] Example 5 Bacteriolytic Spectrum of Bacteriophage vB_P1

[0061] The lysis experiment was performed on 23 isolates of Pasteurella multocida by the spot inoculation method to determine the host range of bacteriophage vB_P1. According to the results of host spectrum determination (Table 1), the lysis rate was found to be 56.5%.

[0062] Table 1 Results of Host Spectrum Determination of Bacteriophage vB_P1 against 23 Isolates of Pasteurella multocida

[0063] Strain Lysis result 24012 + 24016 - 24017 + 24018 + 24019 + 24020 + 24021 - 24022 + 24023 + 24024 - 24025 + 24026 - 24027 + 24028 - 24029 + 24030 + 24031 - 24032 - 24033 - 24034 - 24035 + 24036 - 24037 +

[0064] Note: "+" indicates lysable, and "-" indicates non-lysable.

[0065] Example 6 Detection of Acid-Base Stability of Pasteurella multocida Bacteriophage

[0066] The pH value of TSB liquid medium was adjusted to 1 - 13 with HCl (1 mol / L) and NaOH (1 mol / L) respectively. 900 μL of TSB medium with different pH values was taken and 100 μL of bacteriophage vB_P1 was added respectively, and mixed evenly. After reacting at 37 °C for 2 h, the phage titer under different pH actions was determined by the double-layer plate method, and repeated three times.

[0067] The results showed that when the pH was 5 - 12, the phage titer remained basically unchanged. When pH = 1, pH = 2, and pH = 13, bacteriophage vB_P1 was completely inactivated, indicating that both bacteriophages had the characteristics of being alkali-tolerant and acid-intolerant ( Figure 7 ).

[0068] Example 7 One-Step Growth Curve

[0069] Determination of one-step growth curve: The host bacterial liquid (10 7 CFU / mL) was mixed with the bacteriophage vB_P1 lysate (10 6 PFU / mL) at the optimal multiplicity of infection ratio, incubated in a 37 °C water bath for 5 min, then centrifuged at 12,000 rpm for 5 min, the supernatant was discarded, washed twice with pre-warmed (37 °C) TSB liquid medium, 1 mL of pre-warmed (37 °C) TSB liquid medium was added, the precipitate was resuspended, and it was added to 100 mL of pre-warmed (37 °C) TSB liquid medium, cultured in a 37 °C constant temperature shaker, sampled every 10 min, and the phage titer was measured by the double-layer plate method for 120 min, and repeated three times. The results are as Figure 8 shown.

[0070] Example 8 In Vitro Bacteriostatic Experiment of Pasteurella multocida Bacteriophage

[0071] 1. Experimental Method of In Vitro Bacteriostatic Experiment

[0072] Preparation of bacterial solution: Dilute the culture of Pasteurella multocida in the logarithmic growth phase with TSB medium to make the OD 600 value about 0.15.

[0073] Group setting: Take 4 sterile centrifuge tubes, and add 10 mL of the diluted bacterial solution to each tube. Set 4 groups, namely the control group (added with an equal volume of TSB medium), experimental group 1 (MOI = 0.001), experimental group 2 (MOI = 0.01), and experimental group 3 (MOI = 0.1). Mixed culture: Add the corresponding proportion of vB_P1 phage liquid to each group, mix well, and then aliquot into 1.5 mL centrifuge tubes.

[0074] Culture and determination: Place the centrifuge tubes in a shaker at 37 °C and 180 r / min for culture. Take samples every 3 h, measure the absorbance at 595 nm, continuously measure for 24 h, and draw the in vitro antibacterial curve.

[0075] 2. Experimental results

[0076] The in vitro antibacterial curve is as Figure 9 shown. The results show that the phage vB_P1 has good in vitro antibacterial activity against Pasteurella multocida.

[0077] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Pasteurella virus of swine, vB_P1, characterized in that, The porcine Pasteurella bacteriophage vB_P1 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 10, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46287.

2. Use of the porcine Pasteurella bacteriophage vB_P1 as described in claim 1 in the preparation of a medicament for preventing and / or treating porcine Pasteurella infection.

3. Use of the porcine Pasteurella bacteriophage vB_P1 as described in claim 1 in the preparation of a porcine Pasteurella bactericide.

4. Use of the porcine Pasteurella bacteriophage vB_P1 as described in claim 1 in the preparation of a pig feed additive.

5. A drug for preventing and / or treating swine pasteurella infection, characterized in that, The active ingredient comprises the porcine Pasteurella bacteriophage vB_P1 as described in claim 1.

6. The medicament according to claim 5, wherein The medicament further comprises a pharmaceutically acceptable excipient.

7. The medicament according to claim 6, characterized in that, The dosage form of the medicament is an injection, powder, gel, granule or lyophilized product.

8. The drug according to claim 5, characterized in that, The medicament further comprises other active ingredients having an antibacterial effect on porcine Pasteurella.

9. A bactericide for Pasteurella suis, characterized in that, The active ingredient comprises the porcine Pasteurella bacteriophage vB_P1 as described in claim 1.

10. The pasteurella bactericide for pigs according to claim 9, characterized in that, The dosage form of the porcine Pasteurella bactericide is a spray, powder, gel, granule or lyophilized product.

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