Salmonella phage and its composition and application

Abstract

The invention discloses a Salmonella phage, wherein the Salmonella phage is Salmonella phage SaPA-1, and the deposit number is GDMCC.NO:63415-B1. The Salmonella phage provided by the invention is a virulent phage, which can lyse 6 serotypes of Salmonella, such as Typhimurium, Enteritis, Dublin, etc.; it can be rapidly and efficiently amplified by a specific host to produce a high titer of phage titer, and the detection shows that the phage can tolerate a high temperature of 60°C and an acid-base tolerance range of pH 5 to 11; its optimal infection multiplicity is 100, the incubation period is short, about 10min, and it can be stably stored for a long time at 4°C, and has a strong killing effect on Salmonella. The phage genome sequencing results show that there are no pathogenic bacteria virulence factors and antibiotic resistance genes, and it is found through animal in vivo experiments that it is safe for animals and does not cause immune response and body damage.

Description

Technical Field

[0001] The present invention relates to the technical field of Salmonella phage, and in particular to a Salmonella phage and a composition and application thereof. Background Art

[0002] Salmonella is a gram-negative bacillus belonging to the family Enterobacteriaceae and is recognized by the European Union as the second largest zoonotic pathogen. It can not only cause salmonellosis in livestock and poultry, seriously threatening the healthy development of the breeding industry, but also contaminate livestock and poultry products in multiple ways, causing food safety problems and endangering human health. Due to the irregular use of a large number of antibiotics and insufficient regulatory control measures, multidrug-resistant Salmonella has gradually increased. In addition, since 2020, my country has completely banned the addition of antibiotics to feed, and the prevention and control of salmonellosis faces huge challenges. Therefore, in order to control Salmonella, it is urgent to find new antimicrobial agents that can replace antibiotics.

[0003] Bacteriophages are viruses that can infect and use bacterial metabolism to reproduce themselves. They are widely present in nature and in the body. Bacteriophages are small in size, diverse in form, and simple in structure. They are antibacterial substances that exist in large quantities in nature. They kill bacteria mainly by causing a lytic effect. They are promising biological control agents and a reasonable alternative to antibiotics.

[0004] At present, the research on Salmonella phages mainly focuses on biological characteristics, and there are relatively few reports on their genomic analysis and in vivo safety. Therefore, in order to ensure confidence in phages as anti-infective drugs, relevant in vitro studies and in vivo infection model testing are still crucial for production applications. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Salmonella phage, which has good biological properties, has a bactericidal effect on Salmonella, and has genomic safety and in vitro and in vivo safety.

[0006] The technical problem to be solved by the present invention is to provide a Salmonella phage composition, which contains the above-mentioned Salmonella phage.

[0007] The technical problem to be solved by the present invention is to provide a reagent or a kit for a composition of Salmonella phages.

[0008] The technical problem to be solved by the present invention is to provide an application of Salmonella phage.

[0009] To solve the above technical problems, the present invention provides a Salmonella phage, the Salmonella phage is Salmonella sp. phage SaPA-1, and the deposit number is GDMCC.NO: 63415-B1. The phage was deposited in Guangdong Microbiological Culture Collection Center on April 27, 2023, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the deposited taxonomic name is Chivirus sp.

[0010] In one embodiment, the Salmonella phage SaPA-1 is safe in vivo and in vitro. One of the safety features is that the genome of the Salmonella phage SaPA-1 does not contain pathogenic bacteria virulence factors and antibiotic resistance genes. The in vivo safety means that after the Salmonella phage SaPA-1 is injected into the animal body, the animal does not produce an immune response, does not die, and does not suffer from poor growth performance.

[0011] In one embodiment, the Salmonella phage SaPA-1 belongs to the order Caudovirales, family Lacroviridae.

[0012] In one embodiment, the Salmonella phage SaPA-1 has a tadpole-like shape, a head that is an equiaxially symmetrical polyhedron, a diameter of 50 to 100 nm, and an inelastic long tail with a length of 200 to 260 nm. Preferably, the Salmonella phage SaPA-1 has a tadpole-like shape, a head that is an equiaxially symmetrical polyhedron, a diameter of 70 to 80 nm, and an inelastic long tail with a length of 230 to 240 nm. More preferably, the Salmonella phage SaPA-1 has a tadpole-like shape, a head that is an equiaxially symmetrical polyhedron, a diameter of 75 nm, and an inelastic long tail with a length of 235 nm.

[0013] In one embodiment, the Salmonella phage SaPA-1 has good survival characteristics at 60°C. Preferably, after the Salmonella phage SaPA-1 is exposed to 40°C, 50°C, and 60°C for 20 to 40 minutes, the titer of the Salmonella phage SaPA-1 changes very little and basically maintains the original activity; after the Salmonella phage SaPA-1 is exposed to 40°C, 50°C, and 60°C for 60 minutes, or after the Salmonella phage is exposed to 40°C for 120 minutes, the titer of the phage decreases slightly, but can still be maintained at 10 10 Therefore, the Salmonella phage SaPA-1 has good thermal stability.

[0014] In one embodiment, the pH tolerance range of the Salmonella phage SaPA-1 is 5 to 11. The Salmonella phage SaPA-1 exists in the pH range of 5 to 11. When the pH is ≤ 4, the phage is completely inactivated when it is too acidic. When the pH is ≥ 12, the titer of the phage SaPA-1 decreases rapidly and the phage is completely inactivated. The Salmonella phage SaPA-1 remains stable between pH 5 and 11 and can adapt to a certain range of acidic and alkaline environments.

[0015] In one embodiment, the Salmonella phage SaPA-1 is a virulent phage; virulent phages are also called toxic phages. The reproduction of phages is generally divided into five stages, namely, adsorption, invasion, proliferation, maturation and lysis. Any phage that can continuously complete the above five stages in a short period of time and achieve its proliferation is called a virulent phage.

[0016] Furthermore, the Salmonella phage SaPA-1 has the ability to lyse one or more of Salmonella typhimurium, Salmonella Enteritidis, Salmonella Dublin, Salmonella Paratyphi A, Salmonella Paratyphi B and Salmonella Choleraesuis subspecies Choleraesuis.

[0017] In one embodiment, the multiplicity of infection of the Salmonella phage SaPA-1 is 0.01 to 10000. Preferably, the multiplicity of infection of the Salmonella phage SaPA-1 is 1 to 1000. More preferably, the multiplicity of infection of the Salmonella phage SaPA-1 is 100. Characterized by a one-step growth curve, when the host is Salmonella typhimurium ATCC14028, the incubation period of the Salmonella phage SaPA-1 is about 10 minutes, the outbreak time is about 80 minutes, and the lysis amount of the infected host bacteria is about 65 PFU / Cell.

[0018] In one embodiment, the Salmonella phage SaPA-1 maintains a titer without reduction within 1 month when stored at 4°C without the addition of glycerol.

[0019] In order to solve the above problems, the present invention provides a Salmonella phage composition, characterized in that the composition contains the Salmonella phage SaPA-1.

[0020] In order to solve the above problems, the present invention provides a reagent or a kit containing the composition of the Salmonella phage.

[0021] In order to solve the above problems, the present invention provides an application of the Salmonella phage, which includes:

[0022] (1) The Salmonella phage SaPA-1 is used as a biocide; it can be used as a daily bactericide to specifically kill Salmonella in the environment and improve the distribution of microorganisms in the environment; it can also be used as a biocide in the breeding, transportation and storage of livestock and poultry products to prevent pathogenic Salmonella contamination during the breeding, transportation and storage of livestock and poultry; it can also be used in combination with other bactericides and sprayed in food production workshops to prevent Salmonella contamination during food processing.

[0023] (2) The Salmonella phage SaPA-1 is used to provide a potential therapeutic drug or health product for bacterial infections caused by Salmonella;

[0024] (3) The Salmonella phage SaPA-1 is used as a medical device for treating and preventing bacterial infections caused by Salmonella;

[0025] (4) The Salmonella phage SaPA-1 is used as a feed additive. It can be added to feed to specifically and continuously prevent the survival and reproduction of Salmonella in feed, and prevent the contamination of Salmonella in feed storage and animal breeding.

[0026] The implementation of the present invention has the following beneficial effects:

[0027] The Salmonella phage SaPA-1 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 27, 2023, and the deposit number is GDMCC.No:63415-B1. It is a virulent phage that can lyse six serotypes of Salmonella, including Salmonella typhimurium, Salmonella Enteritidis, Salmonella Dublin, Salmonella Paratyphi A, Salmonella Paratyphi B, and Salmonella Choleraesuis subspecies. The phage can be amplified by a specific host to produce a high-titer phage product, and can withstand a temperature of 60°C, an acid-base tolerance range of pH 5 to 11, an optimal multiplicity of infection of 100, a short incubation period of about 10 minutes, and can be stored at 4°C for a long time. The Salmonella phage SaPA-1 has a strong effect in preventing and controlling Salmonella typhimurium, and the Salmonella phage SaPA-1 genome does not contain pathogenic bacteria virulence factors and antibiotic resistance genes. When injected into the body of chicks as a preparation, it does not cause an immune response in the body, does not cause damage, and is safe when used. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an electron microscope image of the Salmonella phage SaPA-1 provided by the present invention.

[0029] Figure 2 The present invention provides the genome map of Salmonella phage SaPA-1.

[0030] Figure 3 This is the infection multiplicity test result of the Salmonella phage SaPA-1 provided by the present invention.

[0031] Figure 4 The one-step growth curve of the Salmonella phage SaPA-1 provided by the present invention.

[0032] Figure 5 This is the temperature stability test result of the Salmonella phage SaPA-1 provided by the present invention.

[0033] Figure 6 This is the pH stability test result of the Salmonella phage SaPA-1 provided by the present invention.

[0034] Figure 7 This is the sterility detection test result of the Salmonella phage SaPA-1 provided by the present invention.

[0035] Figure 8 The present invention provides test results of the effect of the Salmonella phage SaPA-1 on the survival performance of chicks.

[0036] Fig. 9 The present invention provides test results of the effect of Salmonella phage SaPA-1 on serum cytokine levels.

[0037] Fig.10 The test results of the effect of the Salmonella phage SaPA-1 on intestinal microbial flora provided by the present invention;

[0038] Fig.11 The present invention provides test results of the effect of the Salmonella phage SaPA-1 on intestinal microbial flora. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in further detail below.

[0040] Example 1 Isolation and culture of bacteriophage

[0041] (I) Host bacteria activation and cultivation

[0042] The Salmonella used in this study was provided by the laboratory. The Salmonella was taken out from the -80°C refrigerator, placed on ice to dissolve, and then placed on a clean bench. It was taken out with a sterile pipette tip and added to LB liquid culture medium, and then placed in a shaker at 37°C and 160rpm for overnight culture.

[0043] (II) Isolation and purification of bacteriophage

[0044] On June 8, 2021, water samples were collected from the Xianxi Reservoir in Foshan. 40 mL of sewage samples were centrifuged at 4 ° C and 3500 rpm for 10 min to remove sediments in the water samples. The supernatant was filtered and sterilized through a 0.22 μm filter to obtain a water sample filtrate, which was stored at 4 ° C for later use. 20 mL of the water sample filtrate was added to a sterilized conical flask containing 10 mL of LB liquid culture medium, inoculated with 1 mL of Salmonella suspension in the logarithmic phase (OD600 = 0.6), mixed evenly, and placed in a 37 ° C shaker at 160 rpm for 12 to 16 hours to enrich bacteriophages. The enriched liquid was centrifuged at 4 ° C and 7000 rpm for 15 min, the supernatant was collected, filtered and sterilized through a 0.22 μm filter, and the filtrate was collected. If the filtrate passes the sterility test, it may be a bacteriophage stock solution. Take 500 μL of phage stock solution and mix it with 500 μL of host Salmonella solution, let it stand at room temperature for 15 minutes, so that the phage can fully bind to the receptors on the surface of the host bacteria; then add 5 mL of LB semi-solid medium cooled to 55°C, immediately blow and mix, pour it on the solid LB medium plate prepared in advance to form a double-layer agar plate, let it stand at room temperature for 10 minutes, and after the semi-solid solidifies, place it in a 37°C bacterial incubator and invert it for 12 hours to observe the growth of plaques. If plaques appear, it means that the phage has been isolated, and the phage is purified. Select plaques that grow well, have complete morphology, are large and clear on the double-layer agar plate, carefully pick a single complete empty plaque with a sterile pipette tip, inoculate it into the logarithmic phase Salmonella solution, and culture it at 37°C overnight. After the bacterial liquid in the test tube is completely lysed, centrifuge at 4°C and 12000rpm for 10 minutes, take the supernatant, filter and sterilize through a 0.22μm filter, and collect the filtrate; then use the double-layer plate method to identify whether the phage-filtered culture medium contains phages, pick the filtered culture medium containing the target host phage, and use the double-layer plate method for multiple purifications. Finally, plaques of consistent size and shape are formed on the culture medium, and the purification is completed. The phage is named SaPA-1, and the preservation unit is Guangdong Microbiological Culture Collection Center. The preservation time is April 27, 2023, and the preservation number is GDMCC.No:63415-B1.

[0045] Example 2 Phage Identification

[0046] (I) Phage morphology identification

[0047] Add 20μL of purified high-titer phage solution to a 200-mesh coated copper mesh, allow to naturally adsorb for 2 minutes at room temperature, carefully remove excess liquid on the copper mesh with absorbent paper, then add 20μL of 2% phosphotungstic acid solution, stain for 10 minutes, and remove the remaining dye with absorbent paper. After complete drying, observe the stained copper mesh with a Hitachi HT7800 transmission electron microscope, select the appropriate magnification and field of view to take pictures according to the actual situation, and record the morphological structure of the phage. For specific pictures, see Figure 1The phage SaPA-1 is tadpole-shaped, with an equiaxially symmetrical polyhedron head with a diameter of about 75nm and an inextensible long tail of about 235nm. It belongs to the order Caudata and the family Caudata.

[0048] (II) Phage genomics analysis

[0049] 1) Extraction of bacteriophage SaPA-1 genome

[0050] The phage was concentrated by ultrafiltration; the amount of phage concentrate required for the experiment was used to determine whether to perform multiple centrifugal concentrations and collect the concentrate. The phage genome was extracted using a viral gene DNA / RNA extraction kit, and the phage genome was packaged into multiple tubes and stored at -80°C for later use.

[0051] 2) Whole genome sequencing and whole genome feature analysis of bacteriophage SaPA-1

[0052] Whole genome sequence analysis Figure 2 As shown in the figure, the genome of bacteriophage SaPA-1 is composed of 60084bp double-stranded DNA, with 73 open reading frames (ORF) and a GC content of 56.37%. Among the 73 ORFs, only 17 ORFs have annotation functions, and the rest are hypothetical proteins. No pathogenic bacteria virulence factors and antibiotic resistance genes were detected in the genome of bacteriophage SaPA-1. After comparative analysis by NCBI, bacteriophage SaPA-1 has the highest similarity with the bacteriophage with sequence number NC 021783.1, with a coverage rate of 97%.

[0053] Example 3 Detection of biological characteristics of bacteriophage SaPA-1

[0054] (I) Phage SaPA-1 titer determination

[0055] Take 10 μL of the purified phage solution and dilute it 10 times with liquid culture medium. -7 , 10 -8 , 10 -9 , 10 -10 90 μL of phage solution of each dilution was added to 180 μL of Salmonella solution in the logarithmic phase and mixed. Three parallel plates were made for each dilution. The double-layer agar plate method was used to observe the number of plaques in each dilution plate. Plates with a plaque number of 30 to 300 were selected, and the average value of the three valid parallel plates under this gradient was taken. The titer of phage SaPA-1 per milliliter was calculated to be 8.76×10 12 PFU / mL.

[0056] (II) Phage SaPA-1 lysis spectrum

[0057] Take 200 μL of the bacterial solution to be tested and add it to the 55℃ upper agar medium, mix well, pour it into the LB solid agar plate, wait for the plate to dry naturally, take 10 μL of the phage filtrate and drop it on the double-layer plate containing the bacterial solution, and culture it at 37℃ for 6-8h. If round empty plaques appear on the plate, it is determined that the phage can lyse the strain. Among them, the 12 strains of bacteria are: Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CMCC50335, Salmonella Delbei LWCC1148, Salmonella Paratyphi A CMCC50093, Salmonella Paratyphi B CMCC50094, Salmonella Dublin CICC21497, Salmonella Bovine CICC21499, Salmonella Meat CICC21511, Salmonella Enterica serotype chicken CICC21510, Salmonella Duck CICC21498, Salmonella Choleraesuis subspecies DSM4224 and Salmonella Enteritidis arizona subspecies CMCC47001. Finally, it was determined that phage SaPA-1 lysed 6 strains of Salmonella (6 / 12), including: Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Dublin, Salmonella Paratyphi A, Salmonella Paratyphi B, and Salmonella Choleraesuis subsp. Choleraesuis, with a fission rate of 50%. The specific lysis results are shown in the following reference table 1.

[0058] Table 1 Host spectrum of bacteriophage SaPA-1

[0059]

[0060] Note: “+” indicates plaque formation, “-” indicates no plaque formation.

[0061] (III) Determination of the optimal multiplicity of infection (MOI) of bacteriophage SaPA-1

[0062] The MOI was 10 -2 , 10 -1 , 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 The phage stock solution and host bacterial suspension were mixed in a ratio of ; cultured on a shaker at 37°C and 180rpm for 6 hours to obtain a lysate. The lysate was centrifuged at 8000rpm for 15 minutes and the supernatant was collected by filtration; the filtrate was gradiently diluted and the phage titer was determined by the double-layer plate method; host bacteria without phage and phage without host bacteria were used as controls. Among them, the MOI with the highest titer after 6 hours of culture is the optimal multiplicity of infection. The results showed that the titer of the progeny phage produced by infecting the host bacteria was as high as 4.73×10 12PFU / mL, which means that the optimal infection multiplicity of bacteriophage SaPA-1 is 100. For details, please refer to Figure 3 .

[0063] (IV) One-step growth curve of bacteriophage SaPA-1

[0064] Mix the logarithmic host bacteria and phage liquid at the optimal MOI ratio, incubate at 37℃ for 15min; centrifuge at 4℃13000rpm for 30s, discard the supernatant, blow and wash with LB liquid culture medium, centrifuge and discard the supernatant (twice) to remove free phages that have not adsorbed host bacteria. Add LB culture medium (the total liquid must be equivalent to the amount of bacterial liquid during incubation), blow and mix, and take 300μL / tube and divide it into 13 EP tubes, quickly place it at 37℃160rpm shaking culture, and start timing; sample 200μL from time 0 and every 10min, centrifuge at 12000rpm for 30s, and take the supernatant to determine the phage titer. Make a tube culture at each time point to get the average value, make gradient dilutions and pour double-layer plates, and need to make three parallel plates to determine the phage titer. With the infection time as the horizontal axis and the phage titer in the infection system as the vertical axis, draw a one-step growth curve to obtain the latent period, outbreak period and lysis period of the phage. The final results showed that the incubation period of bacteriophage SaPA-1 was about 10 minutes, the outbreak time was about 80 minutes, and the lysis amount of infected host bacteria was about 65 PFU / Cell. Figure 4 The bacteriophage SaPA-1 has a short incubation period, can quickly lyse the host bacteria, and produce a certain amount of progeny phage.

[0065] (V) Thermal stability of bacteriophage SaPA-1

[0066] Add 100 μL of phage solution to each of the 9 sterile EP tubes and place them in a 40℃, 50℃, and 60℃ water bath for 30 min, 60 min, and 2 h. After the action time is over, take out the sample tube and immediately cool it in an ice bath. After the sample is appropriately diluted, add 180 μL of logarithmic phase host bacteria. Use the double-layer plate method to determine the phage titer. Make 3 parallel plates for each dilution gradient and calculate the average value. For specific results, refer to Figure 5 After the phage SaPA-1 was exposed to 40℃, 50℃, and 60℃ for 30 minutes, the titer of the phage changed little and basically maintained its original activity; after the phage was exposed to 40℃, 50℃, and 60℃ for 60 minutes and at 40℃ for 120 minutes, the titer of the phage decreased slightly, but could still be maintained at 10 10 PFU / mL or more. Therefore, the thermal stability of bacteriophage SaPA-1 is good.

[0067] (VI) Phage SaPA-1PH stability

[0068] For the determination of phage pH, LB liquid culture medium with different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) was prepared. Take 0.9mL of liquid LB culture medium with the above pH values ​​and add it to 22 1.5mL sterile EP tubes, with 2 tubes of culture medium for each pH value. Place the EP tube in a constant temperature incubator at 37°C and preheat for 30 minutes. After the temperature is constant, add 0.1mL of phage liquid respectively, and the action time is 1 and 3 hours. The phage titer was detected by the double-layer plate method, and three independent experiments were performed. For specific results, please refer to Figure 6 , bacteriophage SaPA-1 exists in the pH range of 5 to 11. When pH = 4, the phage is completely inactivated when it is too acidic. When pH = 12, the titer of bacteriophage SaPA-1 drops rapidly and the phage is completely inactivated. Bacteriophage SaPA-1 remains stable between pH 5 and 11 and can adapt to a certain range of acidic and alkaline environments.

[0069] (VII) Comparison of storage methods of bacteriophage SaPA-1

[0070] Glycerol at a concentration of 50% was added to the phage suspension to a final concentration of 30%, 20%, 10%, and phage suspensions without glycerol. The phage solutions with different glycerol concentrations were divided into multiple 1.5 mL centrifuge tubes and stored at 22°C, 4°C, -20°C and -80°C, respectively, to compare the storage effects of the phage solutions at different glycerol concentrations and different temperatures. As shown in Reference Table 2, after being stored at different temperatures for 1 month, the phage SaPA-1 had the best storage effect when the storage temperature was 4°C and no glycerol was added.

[0071] Table 2 Comparison of preservation effects of bacteriophage SaPA-1

[0072]

[0073] (VIII) Sterility test of bacteriophage SaPA-1

[0074] Pipette 100 μL of phage liquid and inoculate it on the LB solid plate prepared in advance, spread it evenly; leave it at room temperature, wait for the liquid on the surface of the plate to dry, place it in a 37℃ constant temperature incubator, invert and culture for 24 hours, observe whether there is bacterial growth, set up 3 replicates in total. Figure 7 It can be seen that compared with the positive control, no bacterial colonies appeared on the culture plate after the bacteriophage SaPA-1 was cultured on the LB solid plate for 24 hours, indicating that there was no host bacteria contamination in the bacteriophage SaPA-1.

[0075] 9. Effect of bacteriophage SaPA-1 on the survival performance of chicks

[0076] Sixty healthy Qingyuan Ma chickens, half male and half female, were collected and pre-fed for 2 days. At 3 days old, they were randomly divided into two experimental groups, the bacteriophage SaPA-1 group and the saline control group. Before the experiment, all chicks were weighed and their weight data were recorded. The intraperitoneal injection titer of the bacteriophage SaPA-1 group was 1×10 10 PFU / mL of 200μL bacteriophage SaPA-1, and each chick in the saline control group was intraperitoneally injected with 200μL of saline. After one week of feeding under the same conditions, all chicks were weighed and the data were recorded, the disease conditions of the chicks were counted, the clinical changes and gross pathological changes were observed, and the liver and spleen coefficients were calculated. Organ coefficient = (organ mass / live weight before slaughter) × 100%. It was finally determined that all chicks in the bacteriophage SaPA-1 group did not show abnormal clinical symptoms such as lack of energy, loss of appetite, diarrhea, etc. during the one week of feeding, and their growth was good. The survival rate was 100%, as shown in the following reference Table 3. There was no significant difference in weight gain, weight growth rate, liver coefficient and spleen coefficient between the two groups of chicks in the bacteriophage SaPA-1 group and the saline control group. No pathological changes in tissues and organs were found in the autopsy, which shows that bacteriophage SaPA-1 has good safety for chicks. For specific results, refer to Figure 8 .

[0077] Table 3 Chick survival rate

[0078]

[0079] (X) Effect of bacteriophage SaPA-1 on serum cytokine levels

[0080] Seven days after phage infection, 5 chickens were randomly selected from each group and blood samples were collected from the jugular vein. To separate the serum, the blood samples were naturally coagulated at room temperature for 10-20 min and then centrifuged at 2000-3000 rpm for 20 min. The supernatant was carefully collected and if precipitation occurred during storage, it should be centrifuged again. IL-6 and IFN-γ in serum were determined using a commercial ELISA kit for chicken serum according to the manufacturer's instructions. In each assay, all samples were tested in triplicate. Fig. 9 It can be seen that there was no significant difference in the levels of IL-6 and IFN-γ between the phage SaPA-1 group and the normal saline control group, indicating that the phage has good safety.

[0081] 11. Effects of bacteriophage SaPA-1 on intestinal microbial flora

[0082] The contents of the chicken cecum were scraped for 16S sequencing to quickly identify the abundance of Salmonella. The 16S amplicon sequencing analysis of the intestinal microbial community was performed by the company. Figure 10-11It can be seen that the content of Proteus and Escherichia coli and Shigella in the intestines of chicks in the bacteriophage SaPA-1 group was significantly reduced, and no adverse effects were produced, indicating that bacteriophage SaPA-1 is safe for chicks.

[0083] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A Salmonella phage, characterized in that The Salmonella phage is Salmonella phage SaPA-1, and its deposit number is GDMCC.NO:63415-B1.

2. A composition containing the Salmonella phage according to claim 1, characterized in that: The composition contains the Salmonella bacteriophage SaPA-1.

3. A reagent or kit containing the composition of Salmonella phage according to claim 2.

4. A use of the Salmonella phage according to claim 1, characterized in that: The applications include: (1) The Salmonella phage SaPA-1 is used to prepare a biocide; (2) The Salmonella phage SaPA-1 is used to prepare a therapeutic drug for bacterial infection caused by Salmonella; (3) The Salmonella phage SaPA-1 is used to prepare feed additives.

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