Phage vB_SalP_SE29, phage vB_SalP_SE29 preparations, and applications thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a bacteriophage vB_SalP_SE29, a bacteriophage vB_SalP_SE29 preparation and application thereof.The present application provides the bacteriophage vB_SalP_SE29, and the preservation number of the bacteriophage vB_SalP_SE29 is CGMCC No.46499.The present application provides a chicken salmonella pullorum bacteriophage, and through whole genome sequencing analysis, no gene sequence related to lysogeny conversion, antibiotic resistance and virulence factor is detected in the genome of the bacteriophage, so that the biological safety of the bacteriophage for drug development and bactericidal product research is ensured from a molecular level, and potential biological risks are effectively avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a bacteriophage vB_SalP_SE29, a bacteriophage vB_SalP_SE29 preparation and an application thereof. BACKGROUND

[0002] Pullorum disease (PD) is a highly pathogenic zoonosis caused by Salmonella pullorum, which has seriously threatened the sustainable development of poultry farming worldwide for a long time. The infection rate of chicks under 3 weeks of age is relatively high, and the main characteristics are white and sticky diarrhea, acute septicemia, and typical symptoms such as arthritis, with a mortality rate of up to 100%. The pathogen can spread through vertical and horizontal transmission, easily causing large-scale epidemics in intensive farming environments, seriously affecting the production performance and breeding efficiency of poultry. And with the extensive use of antibiotics, the drug resistance of S. pullorum is deteriorating exponentially. The World Organization for Animal Health (WOAH) 2023 monitoring report shows that the drug resistance rate of Salmonella pullorum to commonly used antibiotics worldwide is over 60%, and in some areas, the drug resistance rate is as high as 85%. The failure of antibiotic therapy not only increases the difficulty of disease prevention and control, but also makes it easy for drug-resistant genes to be transmitted to humans through the food chain, forming a zoonotic drug resistance risk, which seriously threatens public health safety and ecological environment health. Therefore, it is urgent to find a new effective antibacterial drug and treatment for Salmonella pullorum.

[0003] Bacteriophages, as a kind of virus that strictly parasitizes bacteria, play a key role in microbial ecological regulation due to their unique bacterial targeting lysis mechanism and self-replication ability. Studies have shown that bacteriophages are the most numerous biological entities on Earth, with an estimated total number of 1031 particles, covering more than 106 different types, widely distributed in various ecological environments such as soil, water, and animal intestines. This natural biodiversity provides a rich material basis for mining specific antibacterial phage resources. In recent years, with the extensive use of antibiotics and the emergence of superbugs, bacteriophage therapy has once again become the focus of international research. Molecular biology research has confirmed that bacteriophages can specifically recognize the receptors on the surface of bacteria, such as lipopolysaccharides and outer membrane proteins, through structural elements such as tail fiber proteins and receptor binding proteins, and then achieve efficient adsorption and invasion of target strains. During the infection cycle, bacteriophages use the host cell metabolic system to complete nucleic acid replication and protein assembly, and finally release progeny bacteriophages by destroying the peptidoglycan structure of the bacterial cell wall with lysozyme, forming a cascading amplification of bactericidal effect.

[0004] In the field of poultry disease prevention and control, a number of studies have confirmed the effectiveness of phage therapy. A cocktail of phages against avian colibacillosis can increase the survival rate of broilers by 30-40%; a phage preparation against duck Riemerella anatipestifer infection can reduce the mortality rate of duck flocks to below 5%. However, the existing research on phages against chicken salmonellosis still has technical bottlenecks such as insufficient coverage of the lytic spectrum and poor environmental stability. The average lytic rate of the chicken salmonellosis phages reported so far is only 55-60% for clinically isolated strains, and the activity decays significantly in complex farming environments such as high temperature and high salt. Therefore, screening new chicken salmonellosis phages with broad-spectrum lytic activity and strong environmental tolerance is a key direction to break through the existing technical bottlenecks and realize the industrial application of phage therapy. SUMMARY

[0005] Therefore, the present application provides a phage vB_SalP_SE29, a phage vB_SalP_SE29 preparation and applications thereof. The present application provides a chicken salmonellosis phage, which is analyzed by whole genome sequencing, and no gene sequence related to lysogenic conversion, antibiotic resistance and virulence factor is detected in the genome thereof, thereby ensuring the biosafety of the phage for drug development and bactericidal product research from a molecular level and effectively avoiding potential biological risks.

[0006] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a phage vB_SalP_SE29, and the preservation number of the phage vB_SalP_SE29 is CGMCC No. 46499.

[0008] In some embodiments of the present application, the above-mentioned phage vB_SalP_SE29 can maintain good activity at 30-50℃, the activity is lower at 60℃, and the activity is completely inactivated at 70-80℃. It is shown that the vB_SalP_SE29 can maintain stable activity at 30-50℃ and pH 5-9; the genome size is 46,095 bp, and the G+C content is 48%.

[0009] The present application further provides a phage vB_SalP_SE29 preparation, comprising the above-mentioned phage vB_SalP_SE29.

[0010] In some embodiments of the present application, the titer of the phage vB_SalP_SE29 in the above-mentioned phage vB_SalP_SE29 preparation is not less than 3.2x10 9 PFU / mL.

[0011] The present application further provides a preparation method of the above-mentioned phage vB_SalP_SE29 preparation, comprising the following steps:

[0012] S1: preparing a host bacteria suspension;

[0013] S2: taking the host bacteria suspension, adding the bacteriophage vB_SalP_SE29 phage, culturing, centrifuging, and preparing a bacteriophage vB_SalP_SE29 preparation.

[0014] The present application also provides the use of the above-mentioned bacteriophage vB_SalP_SE29, the above-mentioned bacteriophage vB_SalP_SE29 preparation, and / or the bacteriophage vB_SalP_SE29 preparation obtained by the above-mentioned preparation method in any of the following:

[0015] (a) preparing a product for treating and / or preventing pullorum disease; and / or

[0016] (b) preparing a product for inhibiting and / or lysing Salmonella and / or Escherichia coli; and / or

[0017] (c) preparing a product for broad-spectrum anti-Salmonella and / or Escherichia coli.

[0018] In some embodiments of the present application, in the above-mentioned uses, the product comprises one or more of the following: a drug, a feed, and an environmental disinfectant.

[0019] In some embodiments of the present application, in the above-mentioned uses, the administration mode of the drug comprises one or more of the following: oral administration, nasal instillation, eye drop, and injection.

[0020] In some embodiments of the present application, in the above-mentioned uses, the dosage form of the drug comprises one or more of the following: oral dosage form, injection dosage form, respiratory tract dosage form, skin dosage form, mucosal dosage form, and implant dosage form.

[0021] In some embodiments of the present application, in the above-mentioned uses, the environment comprises a breeding environment; the breeding environment comprises one or more of the following: inside and / or on the surface of livestock and poultry, livestock and poultry feed, breeding appliances, and breeding space environment.

[0022] The present application also provides a method for preventing and / or treating pullorum disease for non-diagnostic and therapeutic purposes, comprising administering to a subject any of the following:

[0023] (I) the above-mentioned bacteriophage vB_SalP_SE29; and / or

[0024] (II) the above-mentioned bacteriophage vB_SalP_SE29 preparation; and / or

[0025] (III) the bacteriophage vB_SalP_SE29 preparation obtained by the above-mentioned preparation method.

[0026] The application takes a chicken pullorum Salmonella as a host bacterium, and a new bacteriophage with killing activity on chicken pullorum Salmonella is obtained from sewage in multiple places. The bacteriophage has strong lysis activity in vitro, not only provides a safe, efficient and green new antibacterial alternative for chicken pullorum Salmonella infection, but also opens up a new path for industrialized production and application of bacteriophage preparations by establishing a whole-chain technical system from bacteriophage isolation and identification, characteristic research to product development, which has great theoretical value and practical significance for promoting the development of poultry healthy breeding and veterinary public health field. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below.

[0028] Figure 1 A bacteriophage vB_SalP_SE29 plaque photograph is shown.

[0029] Figure 2 A transmission electron microscope photograph of the bacteriophage vB_SalP_SE29 is shown (80.0K times magnification, scale 200nm).

[0030] Figure 3 An optimal MOI graph of the bacteriophage vB_SalP_SE29 is shown.

[0031] Figure 4 A one-step growth curve graph of the bacteriophage vB_SalP_SE29 is shown.

[0032] Figure 5 A temperature stability graph of the bacteriophage vB_SalP_SE29 is shown.

[0033] Figure 6 A pH sensitivity graph of the bacteriophage vB_SalP_SE29 is shown.

[0034] Figure 7 A chloroform sensitivity graph of the bacteriophage vB_SalP_SE29 is shown.

[0035] Figure 8 A result graph of the bacteriophage vB_SalP_SE29 lysis activity on S. pullorum-29 is shown.

[0036] Figure 9 A genome running gel graph of the bacteriophage vB_SalP_SE29 is shown. Wherein: M is Marker; lanes 1 and 2 are the genome of vB_SalP_SE29.

[0037] Figure 10 A whole genome map of the bacteriophage vB_SalP_SE29 is shown.

[0038] Figure 11 Phage vB_SalP_SE29 genome alignment with other phages.

[0039] BIOLOGICAL DEPOSIT DESCRIPTION

[0040] BIOLOGICAL MATERIAL: vB_SalP_SE29; TAXONOMIC NOMENCLATURE: Salmonella pullorum phage; deposited on 2025.07.09 at China General Microbiological Culture Collection Center; address: No.1 Yuanjiadonglu, Xizhimen, Haidian District, Beijing, China; deposit accession number: CGMCC No.46499. DETAILED DESCRIPTION

[0041] Disclosed are phage vB_SalP_SE29, a preparation of phage vB_SalP_SE29 and application thereof.

[0042] It should be understood that the expression "one or more of the following" includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, individually, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0043] The use of the term "include", "have" or "contain", including grammatical variations thereof, should generally be understood to be open and non-limiting, for example, not excluding other non-recited elements or steps, unless otherwise specifically stated or understood from the context.

[0044] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0045] The use of any and all examples, or exemplary language, e.g., "such as" or "including", provided herein, is intended merely to better illustrate the application and does not indicate that any non-recited element is essential to the practice of the application, unless the context suggests otherwise. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0046] Also, the numerical ranges recited herein are approximate, meaning that the ends of the ranges are inherently imprecise. The numerical ranges are intended to encompass each and every value and sub-range within the range. Accordingly, unless otherwise indicated, all ranges, numbers, values and percentages recited herein are approximations. When numerical ranges are used herein, the approximate nature of the range is understood to encompass all ranges that fall within the range, including the end points of the range. In addition, unless otherwise indicated, one or a combination of ranges or list of values should be construed as not only containing the specific values, but also containing the ranges between the values.

[0047] The application provides a Salmonella pullorum bacteriophage, which is analyzed by whole genome sequencing, and no gene sequence related to lysogenic conversion, antibiotic resistance and virulence factor is detected in the genome, so as to ensure the biosafety of the bacteriophage for drug development and bactericidal product research from the molecular level, and effectively avoid potential biological risks.

[0048] The Salmonella pullorum bacteriophage vB_SalP_SE29 provided by the application is a novel bacteriophage which is isolated and identified for the first time at home and abroad. The bacteriophage has good application flexibility, and can be directly used for the prevention and treatment of chicken white diarrhea as a single active ingredient, and can be compounded with other bacteriophages, synergists or adjuvants to construct a bacteriophage cocktail preparation with synergistic bactericidal effect, so as to further broaden the antibacterial spectrum and enhance the bactericidal efficiency.

[0049] Another object of the application is to provide general biological characteristic analysis of the bacteriophage.

[0050] Another object of the application is to provide an efficient prevention and treatment product for preventing and treating Salmonella pullorum, in which the bacteriophage is an effective component, to develop a bacteriophage preparation with strong lysis ability for Salmonella pullorum, which can be used alone or in combination with other bacteriophages to effectively kill Salmonella pullorum in a farm. This provides a safe and non-toxic bacteriophage solution for controlling Salmonella pullorum in the current breeding environment, and provides a stable bacteriophage source for industrial production of bacteriophage bactericides.

[0051] The application provides a broad-host-range strong lytic bacteriophage of chicken pullorum Salmonella, which can not only effectively kill a clinical isolate S. pullorum-SE29 of chicken pullorum Salmonella in a breeding environment, but also has lytic activity on other clinical isolates (S. pullorum-29, S. pullorum-G) of chicken pullorum Salmonella, enteritis Salmonella (SE-E-1, CMCC B50041), salmonella (M, CVCC2229), Salmonella (N, 414, B9, L2, JSST, L5, CMCL, P, S5, S8) and E. coli (29-1).

[0052] The bacteriophage of the application takes a chicken pullorum Salmonella as a host bacterium and is obtained by being separated from sewage in many places in Changchun City, Jilin Province, and is named as vB_SalP_SE29. The bacteriophage has a positive icosahedron head and a short tail. The bacteriophage can form a transparent plaque on a double-layer LB agar medium, and a translucent halo exists around the plaque, the edge is clear and regular, and the diameter is 6 mm±1 mm. The latent period of the bacteriophage vB_SalP_SE29 is less than 10 min, the bacteriophage reaches a platform stage after 60 min, and a lytic cycle is 60 min. The burst size is about 356 PFU / cell. The bacteriophage can maintain good activity at 30-50 DEG C, has lower activity at 60 DEG C, and is completely inactivated at 70-80 DEG C. It is shown that the bacteriophage vB_SalP_SE29 can maintain stable activity at 30-50 DEG C and pH 5-9. The genome size is 46,095 bp, and the G+C content is 48%.

[0053] In combination with culture characteristics and physiological and biochemical identification results, according to the eighth report of the International Committee on Taxonomy of Viruses (ICTV) published in 2005, the bacteriophage vB_SalP_SE29 belongs to Caudovirales and Podoviridae.

[0054] Based on the beneficial effects, the application provides applications of the bacteriophage vB_SalP_SE29 in any one and multiple aspects as follows:

[0055] Preparation of a chicken pullorum Salmonella inhibitor, preparation of a medicine for treating or preventing a disease caused by chicken pullorum Salmonella, and killing of chicken pullorum Salmonella in a space environment.

[0056] As priority, the disease caused by Salmonella pullorum includes acute septicemic chicken white diarrhea in the chick stage, and the symptoms are mainly loose feathers, loss of appetite, and appearance of "pasty anus"; chronic chicken white diarrhea in adult chickens, and the symptoms are mainly arthritis, myocarditis, and perihepatitis; and the space includes water, ground, sludge, feces, bedding, and feed.

[0057] According to the above application, the application provides a product for preventing and treating Salmonella pullorum.

[0058] An application of the above-mentioned bacteriophage, and the purified bacteriophage can be used for inhibiting Salmonella pullorum.

[0059] In the application of the bacteriophage, the use for inhibiting Salmonella pullorum refers to: the purified bacteriophage is prepared into a liquid, a freeze-dried powder or a capsule particle form to prepare a Salmonella pullorum inhibitor; a drug for treating or preventing the disease caused by Salmonella pullorum is prepared, and is used alone or in combination with other bacteriophages to kill Salmonella pullorum in a breeding environment.

[0060] In the application of the bacteriophage, the breeding environment refers to: an environment in or on livestock and poultry, feed for livestock and poultry, breeding appliances or breeding space.

[0061] In the application of the bacteriophage, the use mode is oral administration, nasal instillation, eye instillation or injection.

[0062] As preferred, the bacteriophage vB_SalP_SE29 in the product can be combined with traditional Chinese medicine or active ingredients thereof to form a composition.

[0063] In the embodiments 1 to 14 of the application, the raw materials and reagents used can be purchased from the market.

[0064] The application is further described below in combination with embodiments:

[0065] Embodiment 1: Isolation of bacteriophage

[0066] The fecal sewage sample in the present application is collected from multiple poultry breeding intensive areas and sewage treatment sites in Changchun City, Jilin Province. The collected samples are thoroughly mixed to ensure the diversity of the samples and the comprehensiveness of the phage resources. First, the mixed sewage is coarsely filtered using double-layer sterilized gauze to effectively intercept suspended solids, particulate matter and large-volume impurities; then, precise filtration is performed using a 0.22 μm sterile filter membrane to obtain a filtrate rich in phages. The obtained filtrate is stored in a low-temperature, light-proof environment at 4°C. S. pullorum-SE29 is selected as the specific host bacteria. The S. pullorum-SE29 strain preserved in glycerol is inoculated into LB solid culture medium, and inverted culture is performed in a 37°C constant-temperature incubator for 12-16 h to amplify the bacterial liquid. Then, 1% (v / v) of the bacterial liquid is inoculated into 5 mL of LB liquid culture medium, and continuous shaking culture is performed until the logarithmic growth phase (OD 600 nm=0.5). 1 mL of the S. pullorum-SE29 (OD 600 nm=0.5) bacterial liquid is added to 50 mL of pre-sterilized sewage culture medium, and shaking culture is performed in a 37°C constant-temperature shaker for 12-16 h. Then, the culture is transferred to a sterile centrifuge tube, and centrifugation is performed at 4°C and 10,000 rpm for 10 min to obtain the supernatant. The supernatant is filtered using a 0.22 μm disposable sterile filter to obtain the phage proliferation stock solution and store it at 4°C.

[0067] Example 2: Phage plaque test and purification

[0068] The phage purification process adopts a point-by-point drop method for preliminary screening and a double-layer agar plate method for purification verification to determine whether the phage proliferation stock solution contains phages capable of lysing S. pullorum-SE29. 100 μL of the S. pullorum-SE29 bacterial liquid in the logarithmic growth phase (OD 600 nm=0.5) is spread on the surface of the LB solid culture medium plate to ensure the formation of a single bacterial layer. After the bacterial liquid is completely absorbed by the culture medium, 10 μL of the phage proliferation stock solution is added dropwise to the plate surface, and 3-5 repeated drop sites are set on each plate to improve the reliability of the results. The plate is inverted and placed in a 37°C constant-temperature incubator for overnight culture. If a circular plaque with a clear edge and a transparent center appears in the drop area, it is preliminarily proved that the phage proliferation stock solution contains phages capable of lysing S. pullorum-SE29. Based on the positive results of the drop method, a double-layer agar plate method is used for systematic purification of the phages. First, S. pullorum-SE29 (OD 600 nm=0.5) is incubated with the diluted phage proliferation stock solution diluted by 10 times. Inversion is performed in a 37°C constant-temperature incubator for 8-12 h. If phage plaques appear, they are transferred to 5 mL of S. pullorum-SE29 (OD 600 nm=0.5) bacterial solution, 37°C, 160 rpm for 4-6 h. The culture solution was centrifuged, and the precipitate was discarded. The supernatant was filtered using a 0.22 μm filter membrane to obtain the purified phage stock solution. The purification was repeated 3-5 times until the shape and size of the plaques were consistent. The final purified phage stock solution was mixed with sterile glycerol at a ratio of 8:2 (v / v) and stored at -80°C. The phage was named vB_SalP_SE29.

[0069] The purified phage was named vB_SalP_SE29 and was deposited at the China General Microbiological Culture Collection Center with the accession number: CGMCC No. 46499.

[0070] Example 3 Determination of the titer of phage vB_SalP_SE29

[0071] The titer (plaque forming unit, PFU) of the purified phage vB_SalP_SE29 was determined by double-layer agar plate method. The specific operation process followed the internationally accepted standard for phage quantification detection to ensure the accuracy and repeatability of the results. 200 μL of S. pullorum-SE29 (OD 600 nm=0.5) bacterial solution was mixed with 100 μL of vB_SalP_SE29 diluent and incubated at room temperature for 15 min. The mixture was thoroughly mixed with semi-solid LB agar medium that had been melted and cooled to 45°C, and poured onto the surface of the lower LB solid agar bottom layer plate. After solidification, the plate was incubated at 37°C for 8 h. Plates with 30-300 plaques were selected for counting. The formula for calculating the titer of the phage was: phage titer (PFU / mL) = number of phage plaques x dilution factor x 10. To ensure the reliability of the results, 3 biological replicates were set for each dilution, and the average value was taken as the final titer result. The final titer of vB_SalP_SE29 was determined to be 4.7 x 10 9 PFU / mL. As shown in Figure 1 , the phage formed characteristic plaques on the LB agar medium: transparent round empty plaques with a translucent halo around them, clear edges, and regular morphology, with an average diameter of 6.0 ± 1.0 mm. This morphological characteristic was consistent with the observation during the spotting method and double-layer plate purification process, further verifying the purification effect and biological stability of the phage.

[0072] Example 4 Transmission electron microscopy morphological observation of phage vB_SalP_SE29

[0073] The high-resolution observation of the ultrastructure of the bacteriophage vB_SalP_SE29 was performed by using phosphotungstic acid negative staining transmission electron microscopy. The specific operation was as follows: the concentrated vB_SalP_SE29 suspension was added dropwise to the hydrophilic 400 mesh carbon film copper grid, and then the excess residual liquid was absorbed with filter paper after natural precipitation. After 2% phosphotungstic acid (pH=7.0) solution was added dropwise on the copper grid for 10 min, the staining solution was quickly absorbed with filter paper and dried, and then the transmission electron microscope (HT-7800) was used for observation. The observation results are shown in Figure 2 The bacteriophage has a typical icosahedral head structure with a full length of about 75±2 nm, a diameter of 64±2 nm, a dense and regular head shell structure, a short and non-contracted tail with a length of about 10±2 nm. According to the morphological classification standard of the bacteriophage in the “Virus Classification-Eighth Report of the International Committee on Taxonomy of Viruses” published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, the structural characteristics of the bacteriophage meet the definition of the Podoviridae family, which further determines its taxonomic position.

[0074] Example 5 Determination of the optimal multiplicity of infection (MOI) of the bacteriophage vB_SalP_SE29

[0075] The optimal multiplicity of infection (MOI) is a key parameter for the interaction between bacteriophage and host bacteria, and has a decisive influence on the proliferation efficiency and lysis kinetics of the bacteriophage. First, S.pullorum-SE29 (OD 600 nm=0.5) was mixed with the bacteriophage / host bacteria in a ratio of 100, 10, 1, 0.1, 0.01, 0.001, and then added to the LB liquid medium for 37°C culture for 6 h. The test was repeated 3 times. The double-layer plate method was used to determine the titers of the bacteriophage at different ratios. When the titer of the bacteriophage was the highest, the ratio of the bacteriophage to the host bacteria was the optimal MOI of the bacteriophage. The results are shown in Figure 3 When MOI=0.01, the titer of vB_SalP_SE29 was the highest. Therefore, the optimal MOI of vB_SalP_SE29 was 0.01.

[0076] Example 6 Determination of the one-step growth curve of the bacteriophage vB_SalP_SE29

[0077] One-step growth curve determination: the host bacteria were cultured in fresh LB liquid medium to logarithmic phase, and the bacteriophage vB_SalP_SE29 was accurately calculated and added to the corresponding volume of bacteriophage vB_SalP_SE29 concentrate at the optimal MOI (0.01), mixed thoroughly, and then placed in a 37°C water bath for 15 min. The unabsorbed bacteriophage was removed by centrifugation, the precipitate was resuspended in fresh LB liquid medium, and the culture was incubated at 37°C on a constant temperature shaker. The bacteriophage titer was determined every 10 min, and sampling was performed 12 times in succession. The titer of the bacteriophage at each time point was determined. The one-step growth curve of the bacteriophage vB_SalP_SE29 infecting bacteria was plotted with the sampling time as the abscissa and the logarithm of the titer of the bacteriophage as the ordinate, and the results are shown in Figure 4 The latent period of vB_SalP_SE29 was less than 10 min, and after 60 min of infection, the bacteriophage entered a plateau phase, at which time the host bacteria lysed to release the maximum number of progeny bacteriophage, and the titer of the bacteriophage in the system tended to be stable. The entire lysis cycle was 60 min, and the burst size was calculated to be about 356 PFU / cell, i.e., an average of 356 progeny bacteriophages were ultimately released from each infected host bacterial cell.

[0078] Example 7 Analysis of environmental tolerance and structural stability of the bacteriophage vB_SalP_SE29

[0079] 1. Temperature stability determination

[0080] vB_SalP_SE29 (10 9 PFU / mL) was incubated in a 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C constant temperature water bath for 1 h, and after cooling at room temperature, the titer of the bacteriophage was determined. Three biological replicates were set up for each temperature gradient. The results are shown in Figure 5 At a temperature range of 30-50°C, the bacteriophage vB_SalP_SE29 could maintain good activity; when the temperature was increased to 60°C, its activity was significantly reduced (p < 0.05); and after high-temperature treatment at 70-80°C, it was completely inactivated. This result indicates that the optimal temperature range for vB_SalP_SE29 is 30-50°C.

[0081] 2. pH tolerance evaluation

[0082] A Brrtton-Robinson (BR) buffer system was used to construct different acid-base environments to study the pH stability of vB_SalP_SE29. Different pH liquids were prepared by mixing 0.2 mol / L NaOH solution with pH 1.81 BR buffer stock solution. 100 μL of BR buffer and an equal amount of vB_SalP_SE29 (10 9PFU / mL) suspension was mixed and incubated at 37°C for 1 h. After incubation, the titer of vB_SalP_SE29 at different pH was determined. Each group was repeated three times. The mean and standard deviation were calculated. The results are shown in Figure 6 Figure 2, vB_SalP_SE29 remained stable at pH 5-9; when the pH was lower than 5 or higher than 10, it was completely inactivated. The results showed that vB_SalP_SE29 could maintain stable activity at pH 5-9.

[0083] 3. Chloroform resistance test

[0084] 1 mL vB_SalP_SE29 (109 PFU / mL) was taken in a sterile EP tube, and chloroform solution was added at a proportion of 1%. After mixing evenly, it was placed at room temperature for 30 min, and the titer of the phage was determined. The control group was set up and repeated three times. The results are shown in Figure 7 Figure 3, after treatment of vB_SalP_SE29 with chloroform solution, the titer decreased from 7.9 x 108 PFU / mL to 3.2 x 108 PFU / mL, and the activity decreased insignificantly (p>0.05). It showed that vB_SalP_SE29 had resistance to chloroform. Combined with the transmission electron microscopy observation results, it could be inferred that the phage did not have a capsular structure, and its coat protein could resist the damage of organic solvents, which provided a basis for the stability mechanism of the phage. 9 9 PFU / mL. The results showed that vB_SalP_SE29 had resistance to chloroform. Combined with the transmission electron microscopy observation results, it could be inferred that the phage did not have a capsular structure, and its coat protein could resist the damage of organic solvents, which provided a basis for the stability mechanism of the phage.

[0085] Example 8. Lytic activity test of phage vB_SalP_SE29 on S. pullorum-SE29

[0086] S. pullorum-SE29 (OD 600 nm = 0.5) was precisely added with phage vB_SalP_SE29 concentrate at a proportion of MOI = 0.01, and then mixed evenly and placed in a 37°C, 160 rpm shaking culture. Timing started from 0 min, and 200 μL was taken every 10 min, and the sampling was continued to 140 min. The absorbance value of the sample was determined by a microplate reader at OD 600 nm wavelength. Each time point was determined in triplicate. The results are shown in Figure 8 Figure 4, vB_SalP_SE29 and S. pullorum-SE29 interacted, and the OD 600 nm value gradually increased in the first 30 min, and the OD 600 nm value gradually decreased to about 0.2 in 30-140 min; while the OD 600 ​nm value continued to rise. It is shown that vB_SalP_SE29 has strong lytic activity, which provides direct evidence for its application as a biological antibacterial agent in the prevention and control of chicken white diarrhea.

[0087] Example 9 Bacteriophage vB_SalP_SE29 nucleic acid extraction

[0088] The concentrated vB_SalP_SE29 was extracted according to the instructions of the viral DNA genome extraction kit (Omega Biotek) to extract the genome. The extracted genome was verified by 1% agarose gel electrophoresis. The extracted bacteriophage nucleic acid was stored in a refrigerator at -80 ℃. The results of bacteriophage vB_SalP_SE29 nucleic acid extraction are shown in Figure 9 As shown, the genome of vB_SalP_SE29 is greater than 15,000 bp.

[0089] Example 10 Bacteriophage vB_SalP_SE29 whole genome sequence determination

[0090] The extracted vB_SalP_SE29 genome sample was subjected to whole genome sequencing analysis by Beijing Nuowu Ziyuan Biotechnology Co., Ltd. using the Illumina novaseq sequencing platform. After the DNA sample was detected to be qualified, the Covaris ultrasonic crusher was used for random breaking, and then end repair, A tailing, sequencing adapter addition, and full-length adapter connection were performed for Illumina sequencing. After PCR amplification, the product was purified by AMPure XP system (Beverly, USA). Subsequently, the library quality was evaluated on Agilent 540 system (Agilent, USA), and QPCR quantification was performed. According to the effective library concentration and the required data volume, the qualified library was pooled on the Illumina platform, and sequencing was performed using the PE150 strategy. The clean reads data after quality control was spliced using SPAdes (v3.15.5) to obtain the complete genome sequence. The bacteriophage packaging method and linear end were predicted using PhageTerm (v3.0.1). As shown in Figure 11 As shown, the genome of vB_SalP_SE29 is composed of multiple modules: nucleic acid metabolism and replication module, structural module, and lysis module (see Figure 10). Among them, the genes related to the structure and function are head-tail adaptor (ORF42), head scaffolding protein (ORF43), tail protein (ORF45, 46, 50, 62, 63), internal virion protein (ORF47, 49) and other proteins, etc.; the genes related to the regulation function of bacteriophage genetic material are endonuclease (ORF33, 35, 54), exonuclease (ORF32), RNA polymerase (ORF15), DNA primase / helicase (ORF19, 21, 25), etc.; the genes related to the bacteriophage lysis function are lysozyme (ORF48), Holin (ORF51) and endolysin (ORF58).

[0091] Example 11 Bacteriophage vB_SalP_SE29 Whole Genome Sequence Analysis Alignment

[0092] The open reading frames (ORFs) of vB_SalP_SE29 were predicted using BLASTp. The function of each ORF sequence was predicted and analyzed by comparing with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / ) and the phage annotation tool Phaster (http: / / phaster.ca / ). The whole genome map of vB_SalP_SE29 was drawn using CGView Server (http: / / cgview.ca / k). The whole genome sequence and annotation information of vB_SalP_SE29 have been uploaded to GenBank with the accession number: PP526725. The whole genome of vB_SalP_SE29 is 46,095 bp in size, with a G+C content of 48%. The base composition content is A (27%), T (25%), C (21%), and G (26%). Its genome is a circular double-stranded DNA. Ten most similar phage genomes were selected from the NCBI database for comparison, including Salmonella phage PST_H2 (99.42%), Salmonella phage UAB_Phi78 (86.06%), Salmonella phage vB_SenAt-pSL2 (85.91%), Escherichia phage vB_EcolP_P433.1 (80.02%), Klebsiella phage pKP-M186-2.1 (78.57%), Escherichia phage ULINTec7 (79.47%), Klebsiella phage IME184 (78.52%), Escherichia phage vB-Eco-KMB14 (80.33%), Escherichia phage ULINTec6 (79.6%), and Klebsiella phage K1-ULIP33 (78.33%). Except for the coverage of 96% with phage PST_H2, the coverage of the rest of the phages is less than 70%. As shown in FIG. 1, vB_SalP_SE29 is most similar to Salmonella phage PST_H2 at the gene level, but it still has its own unique gene sequence. Figure 11

[0093] Example 12 Determination of the lytic spectrum of phage vB_SalP_SE29

[0094] ​Twenty-nine Salmonella strains (including 22 Salmonella, 2 Salmonella enteritidis, 2 Salmonella typhimurium, and 3 Salmonella pullorum) and 10 Escherichia coli strains preserved in the laboratory were selected to determine the vB_SalP_SE29 lysis spectrum by the spot method. As shown in Table 1, vB_SalP_SE29 was sensitive to 17 (17 / 28) Salmonella strains other than the host bacteria, including 3 S. pullorum (3 / 17), 2 S. enteritidis (2 / 17), 2 S. typhimurium (2 / 17), 10 Salmonella (10 / 17), and 1 E. coli (1 / 17). The results showed that vB_SalP_SE29 can lyse 18 strains of Salmonella and 1 strain of E. coli, with a lysis rate of 48.72%.

[0095] Table 1

[0096]

[0097] Example 13 Safety experiment of phage vB_SalP_SE29

[0098] Twenty 1-day-old chicks were randomly divided into two groups, each containing 10 chicks. The experimental group of chicks was orally administered with phage vB_SalP_SE29 (dose of 10 9 pfu / 0.25 mL / chick), while the control group was orally administered with the same volume of PBS. After 7 days of continuous oral administration, 5 chicks were randomly selected from each group and killed, and their internal organs, digestive tracts, and mucous membranes were examined for changes. The remaining 5 chicks were continued to be raised, and their fecal samples were collected daily to monitor the changes in the number of phage vB_SalP_SE29.

[0099] The results of the study showed that the use of phage vB_SalP_SE29 at this dose had no adverse effects on the growth of chicks, and no abnormalities were found during the anatomical examination. In addition, three days after the end of the oral phage vB_SalP_SE29 treatment, the presence of phage vB_SalP_SE29 could not be detected in the feces of the chicks.

[0100] Example 14 Chicken diarrhea treatment experiment

[0101] Sixty 1-week-old chicks were used for infection experiments. The host bacteria (10 5 CFU) were orally administered to the chicks, and all the infected chicks showed diarrhea symptoms the next day. Then, the infected chicks were evenly divided into three groups and treated with phage vB_SalP_SE29 by intramuscular injection, oral administration, and spraying, respectively. Each group of chicks received three different doses of phage suspension (10 6、10 7 and 10 8 The animals were observed for diarrhea for 24 hours after infection and the number of diarrhea episodes was recorded.

[0102] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A bacteriophage vB_SalP_SE29, characterized in that, The preservation number of the bacteriophage vB_SalP_SE29 is CGMCC No. 46499.

2. The bacteriophage vB_SalP_SE29 preparation characterized in comprising the bacteriophage vB_SalP_SE29 of claim 1.

3. The bacteriophage vB_SalP_SE29 preparation of claim 2, characterized in that, The titer of the bacteriophage vB_SalP_SE29 in the preparation of the bacteriophage vB_SalP_SE29 is not less than 3.2 x 10 9 PFU / mL.

4. The method of preparing a preparation of the bacteriophage vB_SalP_SE29 according to claim 2 or 3, characterized in that, comprising the following steps: S1: preparing a host bacteria suspension; S2: taking the host bacteria suspension, adding the bacteriophage vB_SalP_SE29 phage, culturing, centrifuging, and obtaining the bacteriophage vB_SalP_SE29 preparation.

5. The bacteriophage vB_SalP_SE29 of claim 1, the bacteriophage vB_SalP_SE29 preparation of claim 2 or 3, and / or the bacteriophage vB_SalP_SE29 preparation obtained by the preparation method of claim 4 for use in any of the following: (a) preparing a medicine for treating chicken white diarrhea; and / or (b) preparing a product for inhibiting and / or lysing Salmonella; and / or (c) preparing a broad-spectrum anti-Salmonella product.

6. The use according to claim 5, wherein the compound is ###0002### The product comprises a medicine and / or an environmental disinfectant.

7. Use according to claim 6, wherein The administration mode of the medicine comprises one or more of oral administration, nose drop, eye drop, and injection.

8. Use according to claim 6 or 7, wherein the compound is ###0002### The dosage form of the medicine comprises one or more of oral dosage form, injection administration dosage form, respiratory administration dosage form, skin administration dosage form, mucous membrane administration dosage form, and implant administration dosage form.

9. Use according to claim 8, wherein the compound is ###0002### The environment comprises a breeding environment; the breeding environment is a breeding appliance and / or a breeding space environment.

Images