Poultry source salmonella and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to avian salmonella and application thereof. The avian salmonella is avian salmonella enteritidis, and the preservation number of the avian salmonella enteritidis is CGMCC (China General Microbiological Culture Collection Center) No.25474. The avian salmonella provided by the invention is easy to proliferate and enrich, has good growth performance, biological safety and genetic stability, and can be used as a host bacterium for proliferation of multiple strains of avian salmonella bacteriophages to realize fermentation of multiple strains of bacteriophages by one strain of host bacterium, so that a high-titer preparation of multiple salmonella bacteriophages is prepared, and the application prospect is wide. The cost is effectively reduced while the production process is simplified.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to avian-derived Salmonella and applications thereof. Background Art

[0002] Salmonella is one of the main pathogens that causes disease in chickens in modern large-scale farms. Chicken salmonellosis is common in farms across the country and can be transmitted vertically, seriously endangering the healthy development of the poultry industry and the safety of animal-derived food. The main poultry-derived Salmonella include: Salmonella pullorum, Salmonella fowl typhoid, Salmonella enteritidis and Salmonella typhimurium, which bring great economic losses to the poultry industry. Among them, Salmonella enteritidis and Salmonella typhimurium are pathogens that are common to humans and animals. Ingestion of animal-derived foods such as meat, eggs, and milk contaminated with Salmonella can cause human enteritis and food poisoning, which has important public health significance.

[0003] For a long time, antibiotics have been the main treatment for salmonellosis in poultry. The irrational use of antibiotics has led to the continuous increase of bacterial resistance, resulting in a decrease in the therapeutic effect of antibiotics on bacterial diseases, and antibiotic residues pose a threat to food safety and public health. As the natural enemy of bacteria, lytic bacteriophages are natural antibacterial agents. Phages have the advantages of specifically lysing pathogens and not destroying the normal flora of animals, and have broad application prospects in the fight against bacterial infections.

[0004] The host specificity of bacteriophages is an important factor that limits the widespread application of phage preparations. A single phage can often only lyse some strains of the same bacteria, and its host spectrum has certain limitations. In practice, multiple phages are usually mixed to prepare a "phage cocktail" to broaden the host spectrum. Different phages are often fermented and enriched with different host bacteria, and then mixed together to prepare a "cocktail". The production process is complicated and the cost is high.

[0005] Therefore, it is hoped to provide a host bacterium that can allow multiple Salmonella phages to proliferate, so as to simplify the production process and reduce production costs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an avian salmonella and its application. The avian salmonella provided by the present invention is easy to proliferate and enrich, has good growth performance, biosafety and genetic stability, can be used as a host bacteriophage for the proliferation of multiple avian salmonella phages, and one host bacteriophage can ferment multiple phages, thereby preparing a high-titer and multiple salmonella phage preparation, effectively reducing costs while simplifying the production process.

[0007] In the first aspect, the present invention provides an avian Salmonella, which is avian Salmonella enteritidis, with strain number S46 and preservation number CGMCC No. 25474, and was preserved on August 3, 2022 at the General Microbiology Center of the China Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Optionally, the length of the Enteritidis Salmonella S46 is 1.40-2.09 μm, with an average length of 1.61 μm, and the width is 0.57-0.73 μm, with an average width of 0.66 μm (see Figure 4 ); Salmonella S46 has slender and curved flagella, which are about 4.39 μm long and 20 nm in diameter (see Figure 6 ); some bacteria may produce a microcapsule with a thickness of about 130 nm (see Figure 5 ) There are about 40 pili of varying lengths on the bacterial body, with the length of the pili ranging from 0.32-0.72 μm (see Figure 7 )). The Salmonella Enteritidis S46 provided by the present invention has flagella, indicating that it has motility.

[0009] Optionally, the Enteritidis Salmonella S46 is of ST11 type, with a total genome length of 4718126 bp, 88 Contigs, 368 Subsystems, 4789 ORFs (Open Reading Frames), and its gene annotation is shown in Fig.11 .

[0010] Optionally, the Enteritidis Salmonella S46 belongs to group AF, the O antigen is 9,12, the first phase H antigen is g,m, the second phase H antigen is not detected, and the serotype of the strain is 9,12:g,m:-.

[0011] Optionally, the Enteritidis Salmonella S46 is sensitive to antibiotics such as gentamicin, florfenicol, spectinomycin, neomycin, fosfomycin and polymyxin B, but is resistant to antibiotics such as amoxicillin, penicillin and ampicillin.

[0012] The enteritidis Salmonella S46 provided by the present invention has biological safety, and the Salmonella phages obtained by using the enteritidis Salmonella S46 as a host bacterium for proliferation do not carry virulence genes and drug-resistant genes.

[0013] The Salmonella Enteritidis S46 provided by the present invention has genetic stability, and the morphology and culture characteristics of each generation remain consistent after being cultured in a culture medium for 40 generations, and the biochemical characteristics and titer are also stable.

[0014] In a second aspect, the present invention provides the use of the avian-origin Salmonella described in the first aspect as a host bacterium for the proliferation of avian-origin Salmonella phage.

[0015] As a preferred technical solution of the present invention, the avian-origin Salmonella phage includes one or more avian-origin Salmonella phages.

[0016] The poultry-derived Salmonella phage of the present invention is a phage that can lyse Salmonella enteritidis S46, especially a Salmonella phage isolated from a poultry farm. As a preferred technical solution of the present invention, the poultry-derived Salmonella phage includes vB_SalP_LD1, vB_SalP_LD2, ​​vB_SalP_LD4, vB_SalP_LD5, vB_SalP_LD6, vB_SalP_LD7, vB_SalP_LD8, vB_SalP_LD9, vB_SalP_LD10, vB_SalP_LD11, vB_SalP_LD12, vB_SalP_LD13, vB_SalP_LD14, vB_SalP_LD15, vB_SalP_LD16, vB_SalP_LD17, vB_SalP_LD18, vB any one or more of vB_SalP_LD19, vB_SalP_LD20, vB_SalP_LD21, vB_SalP_LD22, vB_SalP_LD23, vB_SalP_LD24, vB_SalP_LD25, vB_SalP_LD26, vB_SalP_LD27, vB_SalP_LD28, vB_SalP_LD29, vB_SalP_LD30, vB_SalP_LD32, vB_SalP_LD33, vB_SalP_LD34, vB_SalP_LD35 and vB_SalP_LD36, the above bacteriophages are referred to as SalP_LD19, vB_SalP_LD20, vB_SalP_LD21, vB_SalP_LD22, vB_SalP_LD23, vB_SalP_LD24, vB_SalP_LD25, vB_SalP_LD26, vB_SalP_LD27, vB_SalP_LD28, vB_SalP_LD29, vB_SalP_LD30, vB_SalP_LD32, vB_SalP_LD33, vB_SalP_LD34, vB_SalP_LD35 and vB_SalP_LD36, respectively. Sal P1, Sal P2, Sal P4, Sal P5, Sal P6, Sal P7, Sal P8, Sal P9, Sal P10, Sal P11, Sal P12, Sal P13, Sal P14, Sal P15, Sal P16, Sal P17, Sal P18, Sal P19, Sal P20, Sal P21, Sal P22, Sal P23, Sal P24, Sal P25, Sal P26, Sal P27, Sal P28, Sal P29, Sal P30, Sal P32, Sal P33, Sal P34, Sal P35 and Sal P36. The avian-derived Salmonella phage of the present invention is not limited to the above-mentioned phages, but also includes other phages that can lyse S46.

[0017] The avian-derived Salmonella provided by the present invention can be used as a host bacterium for the proliferation of one or more avian-derived Salmonella phages, so that one host bacterium can ferment multiple phages, thereby preparing high-titer and multiple Salmonella phage preparations, simplifying the production process and effectively reducing costs.

[0018] In a third aspect, the present invention provides a method for preparing an avian-origin Salmonella phage combination, the preparation method comprising: using the avian-origin Salmonella described in the first aspect as a host bacterium to ferment and culture multiple strains of avian-origin Salmonella phage.

[0019] As a preferred technical solution of the present invention, the multiple strains of avian Salmonella phages include vB_SalP_LD1, vB_SalP_LD2, ​​vB_SalP_LD4, vB_SalP_LD5, vB_SalP_LD6, vB_SalP_LD7, vB_SalP_LD8, vB_SalP_LD9, vB_SalP_LD10, vB_SalP_LD11, vB_SalP_LD12, vB_SalP_LD13, vB_SalP_LD14, vB_SalP_LD15, vB_SalP_LD16, vB_SalP_LD17, vB_SalP_LD18, vB_SalP_LD35 and vB_SalP_LD36. The avian Salmonella phage of the present invention is not limited to the above phages, but also includes other phages that can lyse S46.

[0020] In a fourth aspect, the present invention provides a use of the avian-origin Salmonella described in the first aspect in the preparation of a drug against avian-origin Salmonella, wherein the drug comprises multiple strains of avian-origin Salmonella bacteriophages.

[0021] As a preferred technical solution of the present invention, the multiple strains of avian Salmonella phages include vB_SalP_LD1, vB_SalP_LD2, ​​vB_SalP_LD4, vB_SalP_LD5, vB_SalP_LD6, vB_SalP_LD7, vB_SalP_LD8, vB_SalP_LD9, vB_SalP_LD10, vB_SalP_LD11, vB_SalP_LD12, vB_SalP_LD13, vB_SalP_LD14, vB_SalP_LD15, vB_SalP_LD16, vB_SalP_LD17, vB_SalP_LD18, vB_SalP_LD35 and vB_SalP_LD36. The avian Salmonella phage of the present invention is not limited to the above phages, but also includes other phages that can lyse S46.

[0022] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0023] The avian-derived Salmonella provided by the present invention is easy to proliferate and enrich, has good growth performance, biosafety and genetic stability, can be used as a host bacteriophage for the proliferation of multiple strains of avian-derived Salmonella phages, and one host bacteriophage can ferment multiple strains of phages, thereby preparing high-titer and multiple Salmonella phage preparations, effectively reducing costs while simplifying the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0026] Figure 1This is the situation in which different bacteriophages described in Example 2 of the present invention form plaques on Salmonella pullorum S19;

[0027] Among them, A-the plaques are large and transparent, and the bacteria are completely lysed, B-the plaques are transparent, but there is a faint hazy background, C-the lysis is incomplete, and the turbidity of the spot area is large, D-there are some individual phage plaques in the sample area, and E-there are no plaques.

[0028] Figure 2 This is the situation of different bacteriophages forming plaques on avian Salmonella S46 as described in Example 2 of the present invention.

[0029] Figure 3 This is the growth curve of Salmonella S46 and S87 described in Example 2 of the present invention.

[0030] Figure 4 This is a transmission electron microscope image 1 of the Salmonella S46 described in Example 4 of the present invention.

[0031] Figure 5 This is the second transmission electron microscope image of Salmonella S46 described in Example 4 of the present invention.

[0032] Figure 6 This is the third transmission electron microscope image of Salmonella S46 described in Example 4 of the present invention.

[0033] Figure 7 This is the transmission electron microscope picture 4 of the Salmonella S46 described in Example 4 of the present invention.

[0034] Figure 8 These are the culture characteristic results of Salmonella S46 described in Example 5 of the present invention.

[0035] Fig. 9 This is the PCR identification result of Salmonella of S46 described in Example 7 of the present invention.

[0036] Fig.10 This is the PCR identification result of Salmonella Enteritidis S46 described in Example 7 of the present invention.

[0037] Fig.11 This is the gene annotation map of Salmonella S46 described in Example 8 of the present invention.

[0038] Fig.12 This is the culture characteristic of the 10th generation Salmonella S46 described in Example 13 of the present invention.

[0039] Fig.13 This is the culture characteristic of the 20th generation Salmonella S46 described in Example 13 of the present invention.

[0040] Fig.14 This is the culture characteristic of the 30th generation Salmonella S46 described in Example 13 of the present invention.

[0041] Fig.15 This is the culture characteristic of the 40th generation Salmonella S46 described in Example 13 of the present invention. DETAILED DESCRIPTION

[0042] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0043] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0044] The main reagents and preparation methods involved in the embodiments of the present invention are as follows:

[0045] 1.1 Main reagents and sources

[0046] LB liquid medium, MacConkey agar, ordinary nutrient agar, 20% iodine solution and 0.1% brilliant green were purchased from Beijing Luqiao Technology Co., Ltd.; SS agar, XLT4 agar, sodium tetrathiosulfate brilliant green enrichment broth base (TTB) and buffered peptone water (BPW) were purchased from Qingdao Haibo Biotechnology Co., Ltd.; NaCl was purchased from Sinopharm Chemical Reagent Co., Ltd.; tryptone and yeast extract powder were purchased from Oxoid, UK; glycerol was purchased from Yantai Yuandong Fine Chemical Co., Ltd.; bacterial microbiochemical reaction tubes were purchased from Hangzhou Binhe Microbiological Reagent Co., Ltd.; Gram staining kit was purchased from Hunan Beekman Biotechnology Co., Ltd.; bacterial DNA extraction kit and 50×TAE Buffer were purchased from Beijing Aidelai Biotechnology Co., Ltd.; 2×Taq Master Mix and DNA Marker / Ladder were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; Water-DEPC Treated Water was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; agarose was purchased from Beijing Qingke Biotechnology Co., Ltd.; and nucleic acid dyes were purchased from Beijing Polymei Biotechnology Co., Ltd.

[0047] 1.2 Preparation of reagents and culture medium

[0048] 50% glycerol: Add 100 mL of glycerol to a clean conical flask, then add distilled water in the same proportion, mix well, heat and boil until the glycerol is completely dissolved, sterilize with high-pressure steam at 121°C for 15 minutes, cool to room temperature, and refrigerate at 4°C for later use.

[0049] Nutrient agar plate: Add 6.6 g of nutrient agar medium to a clean conical flask, add 200 mL of distilled water, heat and stir continuously until dissolved, sterilize at 121°C high pressure steam for 15 min, cool to about 55°C at room temperature, pour into the autoclaved plate, cool and solidify, and refrigerate at 4°C for later use.

[0050] MacConkey agar plate: Add 11 g of MacConkey agar into a clean conical flask, add 200 mL of distilled water, heat and stir continuously until dissolved, sterilize with high-pressure steam at 121°C for 15 min, cool to about 46°C at room temperature, pour into the autoclaved plate, cool and solidify, and refrigerate at 4°C for later use.

[0051] SS agar plate: Add 12.7 g of SS agar medium to a clean conical flask, add 200 mL of distilled water, heat and stir continuously until it dissolves. When the room temperature cools to about 55°C, pour it into an autoclaved plate, cool and solidify, and refrigerate at 4°C for later use.

[0052] XLT4 agar plate: Add 11.04 g of XLT4 agar medium to a clean conical flask, add 200 mL of distilled water, heat and stir continuously until dissolved. When the room temperature cools to about 55°C, pour into an autoclaved plate, cool and solidify, and refrigerate at 4°C for later use.

[0053] TTB: weigh 17.82g, add 200mL of distilled water, heat and stir continuously until dissolved, sterilize with high pressure steam at 121℃ for 15min, add 4mL of 20% iodine solution and 2mL of 0.1% brilliant green before use, prepare and use immediately, do not keep for a long time.

[0054] BPW: Weigh 4 g, add 200 mL of distilled water, heat and stir continuously until dissolved, sterilize at 121°C with high pressure steam for 15 min, and refrigerate at 4°C for later use.

[0055] LB liquid culture medium: Add 2 g of tryptone, 1 g of yeast extract powder and 2 g of NaCl into a clean conical flask, add 200 mL of distilled water, heat and stir continuously until they are dissolved, sterilize with high-pressure steam at 121°C for 15 min, and refrigerate at 4°C for later use.

[0056] LB solid culture medium: Add 2 g of tryptone, 1 g of yeast extract powder, 2 g of NaCl and 1.5 g of agar powder into a clean conical flask, add 200 mL of distilled water, heat and stir continuously until dissolved, sterilize with high-pressure steam at 121°C for 15 min, cool to about 55°C at room temperature, pour into a high-pressure sterilized plate, cool and solidify, and refrigerate at 4°C for later use.

[0057] 1% agarose gel: Add 1 g of agarose and 100 mL of 1×TAE to a clean conical flask, mix well and heat in a microwave oven to boil. Immediately take out the conical flask and shake it gently to observe whether there is undissolved agarose at the bottom. If undissolved agarose is found, heat it again until all the agarose is dissolved. After cooling to room temperature, add 10 μL of nucleic acid dye, shake well and pour into the gel-making mold with a comb inserted, and cool to solidify at room temperature.

[0058] Example 1

[0059] This example is used to illustrate the separation and purification of poultry-derived Salmonella.

[0060] 1.1 Sources of avian Salmonella

[0061] Diseased samples were collected from poultry farms in different provinces or autonomous regions across the country from 2020 to 2024 for the isolation of Salmonella. Chicken or duck livers were collected aseptically from farms in 10 provinces or autonomous regions, including Shandong, Guangdong, Sichuan, Anhui, Heilongjiang, Jiangsu, Hebei, Guangxi, Liaoning, and Jilin.

[0062] 1.2 Isolation and purification of avian Salmonella

[0063] Poultry liver samples were sterilely taken, inoculated into buffered peptone water, and cultured at 37℃ constant temperature shaker 180r / min for 12h for pre-enrichment; the pre-enrichment solution was inoculated into sodium tetrasulfonate brilliant green enrichment solution, and cultured at 37℃ constant temperature shaker 180r / min for 12h for selective enrichment of Salmonella, and then streaked on Salmonella chromogenic medium, placed in a 37℃ constant temperature incubator for 24h, observed the colony morphology and growth, picked a single suspected colony and streaked on LB solid medium, repeated 3 to 5 times, and purified and cultured. After PCR identification, 84 strains of Salmonella were isolated, including 33 strains of Enteritidis Salmonella, 30 strains of Pullorum Salmonella, and 21 strains of Typhimurium Salmonella. The strain code, serotype, etc. are shown in Table 1.

[0064] 1.3 Storage of avian Salmonella

[0065] The avian Enteritidis Salmonella S46 was deposited on August 3, 2022 at the General Microbiology Center of the China Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 25474.

[0066] Example 2

[0067] This example is used to illustrate the selection of avian-derived Salmonella Enteritidis S46 as a host bacterium for Salmonella phage.

[0068] 2.1 Detection of the lytic activity of different Salmonella phages against Salmonella isolates

[0069] The double-layer plate spot method was used to observe the lysis of 84 Salmonella isolates by different phages using 36 Salmonella phages isolated and purified in the laboratory.

[0070] 84 strains of Salmonella were used to make double-layer plates. About 10 mL of LB solid culture medium was poured into a sterile square plate and solidified at room temperature to serve as the bottom culture medium. The pre-sterilized LB semi-solid culture medium was placed in a 50°C constant temperature water bath. 4 mL of the semi-solid culture medium in the 50°C water bath was added to a 10 mL sterile centrifuge tube. It was cooled to about 46°C and 200 μL of Salmonella solution was added. The mixture was quickly mixed and poured onto the solid plate. It was left for at least 15 minutes to solidify. 10 μL of each of the 36 strains of bacteriophage was spotted on each of the above double-layer plates and cultured at a constant temperature of 37°C overnight to observe whether there were plaques, the size of the plaques, and the degree of transparency. The formation of plaques by different bacteriophages on Salmonella pullorum S19 was shown in the following figure. Figure 1 As shown. Classify and record according to the status of plaques:

[0071] 4+, the plaques are large and clear, and the bacteria are completely lysed, such as Figure 1 -A;

[0072] 3+, the plaques are clear, but there is a faint hazy background, such as Figure 1 -B;

[0073] 2+, the lysis is incomplete, and the turbidity in the spot area is large, such as Figure 1 -C;

[0074] 1+, there are some individual phage plaques in the spotted area, e.g. Figure 1 -D;

[0075] -, no plaques, e.g. Figure 1 -E.

[0076] The number of plaques formed by 36 phage strains on 84 strains of avian Salmonella and the number of clear plaques (3+ and above) are shown in Table 1:

[0077] Table 1

[0078]

[0079]

[0080]

[0081] Note: SE stands for Salmonella Enteritidis; ST stands for Salmonella Typhimurium; SP stands for Salmonella Pullorum.

[0082] From Table 1, we can find that among the 84 Salmonella strains, the one that produces the most clear plaques is the Enteritidis Salmonella S46. Of the 36 phage strains, 31 strains form relatively clear plaques on S46, and 2 strains form fuzzy plaques. The plaques formed by different phages on the poultry-derived Salmonella S46 are shown in the figure below. Figure 2 In addition, 33 strains of S87 in Salmonella pullorum can form plaques, and 28 strains have 3+ or above, so S46 and S87 are proposed to be selected as alternative host bacteria.

[0083] 2.2 Determination of growth performance of candidate strains

[0084] As a culture medium for phages, it must itself have good growth performance and provide sufficient nutrition for phages, thereby ensuring the proliferation of phages and preparing high-titer phage preparations.

[0085] Take 100 μL of bacterial solution in the logarithmic phase, inoculate into 5 mL of LB liquid culture medium, shake at 37°C, 180 rpm, culture for 12-14 hours, and set aside; take 100 μL of the above culture medium into a test tube containing 5 mL of LB liquid culture medium, preheat to 37°C, connect to 2 tubes, numbered 1 and 2 respectively; incubate: shake at 37°C, 180 rpm on a shaker, and shake a tube of LB liquid culture medium without bacteria at the same time as a blank control; take samples from tube 1 at 0h, 1.5h, 3h, 4h, and 6h, and take samples from tube 2 at 8h, 10h, 12h, and 14h, 200 μL each time, and take 200 μL of liquid in the blank control tube at the same time, measure the OD value of the bacterial solution at a wavelength of 620nm, subtract the blank control, and get the corresponding OD value. With the culture time as the horizontal axis and the measured corresponding OD value as the vertical axis, draw the growth curve of Salmonella S46 and S87, as shown below: Figure 3 shown.

[0086] Depend on Figure 3 It can be found that Salmonella S46 has better growth performance than Salmonella S87. Combined with the formation of transparent plaques by phage and the growth characteristics of bacterial colonies, Salmonella S46 is more conducive to the proliferation of phages. Therefore, Salmonella S46 was selected as the host bacteria of Salmonella phage.

[0087] Example 3

[0088] This example measures the titers of different Salmonella phages after proliferation using Salmonella S46.

[0089] The proliferation ability of 36 Salmonella phages stored in the laboratory on Salmonella S46 was determined.

[0090] Inoculate 100 μL of S46 bacterial solution and 100 μL of phage solution in LB liquid medium, culture at 37°C shaker at 180 rpm for 2 hours, centrifuge and determine phage titer using double-layer plate method. Take phage filtrate and dilute it 10 times with PBS, mix 200 μL of phage dilution with 200 μL of host bacterial solution, incubate at 37°C for 5 minutes, pour 4-5 mL of LB semi-solid medium at about 55°C into the mixture of phage dilution and host bacterial solution, and then immediately pour it into a plate with LB solid medium at the bottom, prepare double-layer plates, culture at 37°C for 6 hours, and count plaques. Phage titer (PFU / mL) = number of plaques × 5 × dilution multiple, the results are shown in Table 2.

[0091] Table 2

[0092]

[0093]

[0094] From the results in Table 2, we can find that 34 of the 36 phage strains were titered and could proliferate, and 29 of them had a titer of 10 8 The proliferation titers of 26 strains were above 10 9 The titers of SalP19 and SalP32, which had no spots in the spot test, were also 10 2 The above results indicate that Salmonella S46 is suitable for the proliferation of multiple (or most) avian-derived Salmonella phages and is suitable as a "culture medium" for Salmonella phages.

[0095] Example 4

[0096] This example is used to illustrate the microscopic morphology of Salmonella S46.

[0097] The S46 strain was isolated from Liaocheng, Shandong Province. The white-feathered broiler liver sent for inspection by Wei Zhuang Chicken Farm of Fengxiang Group on April 9, 2021 was identified as Enteritidis Salmonella.

[0098] 4.1 Microscope oil immersion observation:

[0099] Aseptically pick pure culture of Salmonella S46 on nutrient agar or SS agar to prepare smears, perform Gram staining, and observe under an oil immersion microscope. It can be found that Salmonella S46 is mostly single, scattered, rod-shaped bacteria with blunt ends, and Gram staining is negative.

[0100] 4.2 Transmission electron microscopy observation:

[0101] The logarithmic phase S46 bacteria were inoculated into LB liquid culture medium, incubated at 37°C and 180 rpm for 10 h, and two sterile 10 mL centrifuge tubes were taken, 5 mL of bacterial solution was dispensed into each tube, and the tubes were centrifuged at 5000 rpm for 6 min. The tubes were washed with sterile PBS, resuspended by pipetting, and washed 3 times. The two tubes were combined into one tube, and 5 mL of glutaraldehyde, a fixative, was added, and the tubes were adsorbed on a carbon support film and observed by negative staining under a transmission electron microscope. The results are as follows: Figure 4-7 As shown. Using CAD software to measure multiple bacteria, the strain's bacteria length is 1.40-2.09μm, with an average of 1.61μm, and the width is 0.57-0.73μm, with an average of 0.66μm, as shown. Figure 4 ; There are long and thin flagella, which are about 4.39μm long and 20nm in diameter. Figure 6 ; A layer of microcapsule about 130nm thick can be produced around some bacteria. Figure 5 There are about 40 pili of varying lengths of 0.32-0.72 μm on the bacterial body. Figure 7 .

[0102] Example 5

[0103] This example is used to illustrate the culture characteristics of Salmonella S46.

[0104] The purified Salmonella S46 was streaked onto ordinary nutrient agar, MacConkey agar, SS agar, and Salmonella chromogenic medium XLT4 agar, and the colony morphology was observed after constant temperature culture at 37°C for 18-24 hours; the motility of the bacteria was tested by puncture inoculation into semi-solid agar medium; the culture characteristics of Salmonella S46 were shown in the following table. Figure 8 shown.

[0105] Depend on Figure 8 It can be found that Salmonella S46 appears as colorless, translucent, smooth and neatly edged dew-drop-shaped small colonies on nutrient agar and MacConkey medium, red, round, smooth and neatly edged colonies on XLT4 agar, and black colonies with metallic luster on SS plates. When punctured and inoculated in semisolid culture medium, Salmonella S46 spreads and grows in all directions, and the culture medium around the puncture line is turbid, indicating that the bacteria have flagella and are motile.

[0106] Example 6

[0107] This example is used to illustrate the biochemical characteristics of Salmonella S46.

[0108] The isolated and purified Salmonella S46 was aseptically inoculated into bacterial microbiochemical reaction tubes, and biochemical tests such as glucose, lysine, ornithine, sorbitol, lactose, potassium cyanide and urea were performed respectively. Semi-solid puncture was used to detect motility. Among them, liquid paraffin was added to the lysine and ornithine tubes during inoculation, and the results were observed after constant temperature culture for 24-48 hours. During observation, indigo matrix test tubes needed to be added with indigo matrix reagent, and 10% FeCl3 was added to the phenylalanine test. The biochemical test results are shown in Table 3:

[0109] Table 3

[0110]

[0111]

[0112] Note: +, positive; -, negative; Produces acid and gas.

[0113] The results showed that the biochemical characteristics of the bacteria were consistent with those of Salmonella Enteritidis.

[0114] Example 7

[0115] This example is used to illustrate the PCR identification of S46.

[0116] Take 1mL of S46 bacterial solution, centrifuge at 5000r for 5min, discard the supernatant; add 100μL of DEPC water, insert a float, boil for 10min; centrifuge, centrifuge at 12000r for 10min, take the supernatant for later use. Use this as a template for PCR identification, and the primer sequences are shown in Table 4:

[0117] Table 4

[0118]

[0119] PCR reaction system 25 μL: 2×Taq Master Mix 12.5 μL, upstream and downstream primers 1 μL each, DNA template 1 μL, DEPC water to 25 μL.

[0120] PCR reaction conditions: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s, annealing for 30s, 72℃ extension for 40s, 30 cycles in total; 72℃ extension for 10min. 1% agarose gel electrophoresis, electrophoresis results are as follows Fig. 9 and Fig.10 As shown, Fig. 9 The PCR identification results of Salmonella spp. Fig.10 PCR identification results of Salmonella Enteritidis, and M-DL2000 DNA Marker; 1-positive control; 2-S46; 3-negative control.

[0121] Depend on Fig. 9 and 10 It can be shown that the S46 strain is Salmonella Enteritidis.

[0122] Example 8

[0123] This example is used to illustrate the whole genome sequencing of S46.

[0124] The S46 bacterial suspension was sent to Beijing Novogene Technology Co., Ltd. for whole genome sequencing, and the fasta data of Contigs were submitted to the RAST official website. The genome was quickly annotated using the Subsystem Technology Rapid Annotation tool. The genome of the strain is 4718126 bp in length, with 88 Contigs, 368 Subsystems, and 4789 ORFs (Open Reading Frames). The gene annotation of Salmonella S46 is as follows: Fig.11 shown.

[0125] According to the whole genome sequencing results, multilocus sequence typing (MLST) was performed on strain S46. Seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, suc A, thrA) were analyzed. The typing of the seven housekeeping genes is shown in Table 5:

[0126] Table 5

[0127]

[0128] It can be found from Table 5 that each housekeeping gene combination obtains an ST type, and the results show that the strain S46 is of ST11 type.

[0129] Example 9

[0130] This example is used to identify the surface antigen of S46.

[0131] The serotype identification was performed using the Salmonella diagnostic serum kit produced by Ningbo Tianrun Biopharmaceutical Co., Ltd. The kit includes AF, O2, O4, O5, O7, O8, O9, O10, O11, O14, O15, O19, O20, O27, O34, O46, O4,12, 03,19, O9,12, Vi, H multivalent 1, Ha, Hb, Hc, Hd, Hi, H multivalent 2, He,h, Hh, He.nx, Hn, Hx, Hz15, Hg,p, Hg, Hp, Hf, Hm, Hs, Ht, Hu, H multivalent 3, H1,v, H v, Hw, Hz13, Hz28, Hk, Hr, Hy, Hz, Hz10, H multivalent 4, H1,2,3,5, H2, H5, H6, H7, Hz6, Hz29, a total of 60 sera, among which AF includes O2, O4, O7, O8, O9, O10, O11 multivalent sera; H multivalent 1 includes Ha, Hb, Hc, Hd, Hi and other factors; H multivalent 2 includes He.nx, He,h, Hg,p, Hf, Hm, Hs; H multivalent 3 includes Hk, Hr, Hy, Hz, Hz10; H multivalent 4 includes H2, H5, H6, H7, Hz6 and other factors.

[0132] S46 was subjected to glass plate agglutination test with the above 60 diagnostic sera respectively. 20 μL of S46 bacterial solution to be tested was dropped on a clean glass slide, and then 1-2 drops of serum were added to mix with the bacterial solution. The glass slide was gently shaken to observe whether agglutination occurred.

[0133] Results The strain agglutinated with AF, O9, O4,12, O9,12, H multivalent 2, Hg, and Hm, and multiple granular agglutinates appeared, while it was uniformly turbid with other sera without agglutinated particles.

[0134] The results showed that the S46 strain belonged to group AF, the O antigen was 9,12, the first phase H antigen was g,m, the second phase H antigen was not detected, and the serotype of the strain was 9,12:g,m:-.

[0135] Example 10

[0136] This example is used to determine the sensitivity of S46 to antibiotics.

[0137] The antibiotic sensitivity of S46 was determined by disk diffusion method using drug sensitivity disks of gentamicin, lincomycin, cephalothin, cefotaxime, amoxicillin, florfenicol, spectinomycin, cefuroxime, neomycin, ciprofloxacin, penicillin, fosfomycin, polymyxin B, and ampicillin produced by Hangzhou Binhe Microbiological Reagent Co., Ltd.

[0138] Pick the pure culture of S46 into a centrifuge tube containing PBS and prepare a bacterial suspension of 0.5 McFarland units. Dip a sterile cotton swab into the bacterial suspension, and evenly spread it on the sterilized nutrient agar plate with the soaked cotton swab, and let it stand for 5 minutes. Use sterile tweezers to take the drug-sensitive paper, place it on the surface of the plate, and gently press the paper to make it flat. The distance between the paper pieces must not be less than 24mm, and the distance between the center of each paper piece and the edge of the plate must not be less than 15mm. After pasting, place the plate upside down in a 37℃ constant temperature incubator, incubate for 24 hours, and use a vernier caliper to measure the diameter of the inhibition zone. Determine the results according to the standards of the American Clinical Laboratory Standards Institute (CLSI2008).

[0139] The results showed that S46 was sensitive to gentamicin, florfenicol, spectinomycin, neomycin, fosfomycin and polymyxin B, but was resistant to other antibiotics such as amoxicillin, penicillin and ampicillin.

[0140] Embodiment 11

[0141] This embodiment is used to test the purity of S46.

[0142] S46 was inoculated into 2 tubes, TG tube, GA slant and SS agar, 0.2 mL each, 1 tube was cultured at 35-37°C, 1 tube was cultured at 23-25°C, and no foreign bacteria grew after 3-5 days of observation, indicating that the Salmonella S46 isolated and purified by the present invention was pure and free of foreign bacteria.

[0143] Example 12

[0144] This example is used to test the biosafety of S46.

[0145] Salmonella S46 was used as the host bacteria to enrich Salmonella phage Sal P16 and Sal P34, respectively. 100 μL of phage lysate and host bacteria culture fluid were added to 5 mL LB liquid culture medium, and cultured at 37°C, 180 r / min and shaking for 2 h. The culture was centrifuged at 12000 r / min for 2 min and filtered through a 0.22 μm filter membrane to obtain phage lysate. The enriched phages Sal P16 and Sal P34 were sequenced, and the two phages did not carry virulence genes or drug-resistant genes.

[0146] Embodiment 13

[0147] This example is used to test the genetic stability of S46.

[0148] 13.1 Subculturing S46

[0149] Take the first generation of Salmonella S46, streak it on SS medium, place it in a 37°C constant temperature incubator for 24 hours, observe the colony morphology and growth, pick a single black colony in LB liquid medium, place it in a 37°C constant temperature shaker at 180r / min for 12 hours, and obtain the second generation of S46 Salmonella. Pipette 200μL of the second generation of bacterial liquid into LB liquid medium, enrich the bacteria and culture, and obtain the third generation of bacterial liquid. Follow this step to subculture up to the 40th generation.

[0150] 13.2 Stability of morphology and culture characteristics

[0151] A single suspected colony was picked from the SS solid culture medium, and the 1st, 5th, 10th, 15th, 20th, 25th, 30th, 35th, and 40th generations of bacteria were subjected to Gram staining respectively. Observation under an oil microscope revealed scattered, single, red rod-shaped bacteria with blunt ends that were Gram-negative, and the morphological staining results of each generation were consistent.

[0152] The 10th, 20th, 30th and 40th generations of Salmonella S46 were streaked on ordinary nutrient agar, MacConkey agar, SS agar and XLT4 agar, respectively, and the colony morphology was observed after constant temperature culture at 37℃ for 18-24 hours. The semi-solid agar was punctured and inoculated, and its motility was observed after culture at 37℃ for 18-24 hours. The culture characteristics of S46 at the 10th, 20th, 30th and 40th generations are shown in Figure 2. Figure 12-15 As shown, Fig.12 The culture characteristics of the 10th generation S46. Fig.13 The culture characteristics of the 20th generation S46. Fig.14 The culture characteristics of the 30th generation S46. Fig.15 These are the culture characteristics of the 30th generation S46.

[0153] The results showed that the culture characteristics of each generation were consistent. On nutrient agar and MacConkey agar plates, the colonies appeared as colorless, translucent, smooth, and neatly edged dew-drop-shaped small colonies. On SS plates and XLT4 agar, the periphery of the streaked bacterial lawn and the individual colonies turned black.

[0154] 13.3 Stability of biochemical characteristics

[0155] The 10th, 20th, 30th and 40th generations of Salmonella S46 were inoculated into various bacterial microbiochemical tubes, and biochemical tests such as glucose, lysine, ornithine, dulcitol and lactose were performed, and motility was detected by semi-solid puncture. After constant temperature culture for 24 to 48 hours, the biochemical results were observed to be the same as those of the first generation, indicating that Salmonella S46 has biochemical stability.

[0156] 13.4 Stability of titer

[0157] The 1st, 5th, 10th, 15th, 20th, 25th, 30th, 35th, and 40th generation Salmonella S46 bacterial suspensions close to the stable period were diluted in multiples by pouring plate method. 100 μL of bacterial suspension was added to 900 μL PBS and diluted to 10 -6 cfu / mL. Take 100 μL of the diluted liquid and mix it evenly with the LB medium, wait for it to dry and place it in an incubator, count the colonies for 24 hours, and calculate the titer of each generation of bacterial solution. The results are shown in Table 6:

[0158] Table 6

[0159]

[0160]

[0161] From Table 6, we can find that the titer of Salmonella S46 at each generation is 10 8 -10 10 CFU / mL, with relative stability.

[0162] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0163] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A poultry-derived Salmonella, characterized in that: The poultry-derived Salmonella is poultry-derived Salmonella enteritidis, and its preservation number is CGMCC No.25474.

2. Use of the avian-origin Salmonella according to claim 1 as a host bacterium for the proliferation of avian-origin Salmonella phage.

3. The use according to claim 2, characterized in that: The avian-origin Salmonella phage comprises one or more avian-origin Salmonella phages.

4. The use according to claim 2 or 3, characterized in that: The poultry-derived Salmonella phages include vB_SalP_LD1, vB_SalP_LD2, ​​vB_SalP_LD4, vB_SalP_LD5, vB_SalP_LD6, vB_SalP_LD7, vB_SalP_LD8, vB_SalP_LD9, vB_SalP_LD10, vB_SalP_LD11, vB_SalP_LD12, vB_SalP_LD13, vB_SalP_LD14, vB_SalP_LD15, vB_SalP_LD16, vB_SalP_LD17, vB_SalP_LD18, vB_SalP_LD29, vB_SalP_LD30, vB_SalP_LD31 any one or more of vB_SalP_LD19, vB_SalP_LD20, vB_SalP_LD21, vB_SalP_LD22, vB_SalP_LD23, vB_SalP_LD24, vB_SalP_LD25, vB_SalP_LD26, vB_SalP_LD27, vB_SalP_LD28, vB_SalP_LD29, vB_SalP_LD30, vB_SalP_LD32, vB_SalP_LD33, vB_SalP_LD34, vB_SalP_LD35 and vB_SalP_LD36.

5. A method for preparing a combination of avian Salmonella phages, characterized in that: The preparation method comprises: using the avian-origin Salmonella described in claim 1 as a host bacterium to ferment and culture multiple strains of avian-origin Salmonella phages.

6. The preparation method according to claim 5, characterized in that: The multiple strains of avian Salmonella phages include vB_SalP_LD1, vB_SalP_LD2, ​​vB_SalP_LD4, vB_SalP_LD5, vB_SalP_LD6, vB_SalP_LD7, vB_SalP_LD8, vB_SalP_LD9, vB_SalP_LD10, vB_SalP_LD11, vB_SalP_LD12, vB_SalP_LD13, vB_SalP_LD14, vB_SalP_LD15, vB_SalP_LD16, vB_SalP_LD17, vB_SalP_LD18, Two or more of vB_SalP_LD19, vB_SalP_LD20, vB_SalP_LD21, vB_SalP_LD22, vB_SalP_LD23, vB_SalP_LD24, vB_SalP_LD25, vB_SalP_LD26, vB_SalP_LD27, vB_SalP_LD28, vB_SalP_LD29, vB_SalP_LD30, vB_SalP_LD32, vB_SalP_LD33, vB_SalP_LD34, vB_SalP_LD35 and vB_SalP_LD36.

7. Use of the avian Salmonella according to claim 1 in the preparation of a drug against avian Salmonella, wherein the drug comprises multiple strains of avian Salmonella phages.

8. The use according to claim 7, characterized in that: The multiple strains of avian Salmonella phages include vB_SalP_LD1, vB_SalP_LD2, ​​vB_SalP_LD4, vB_SalP_LD5, vB_SalP_LD6, vB_SalP_LD7, vB_SalP_LD8, vB_SalP_LD9, vB_SalP_LD10, vB_SalP_LD11, vB_SalP_LD12, vB_SalP_LD13, vB_SalP_LD14, vB_SalP_LD15, vB_SalP_LD16, vB_SalP_LD17, vB_SalP_LD18, Two or more of vB_SalP_LD19, vB_SalP_LD20, vB_SalP_LD21, vB_SalP_LD22, vB_SalP_LD23, vB_SalP_LD24, vB_SalP_LD25, vB_SalP_LD26, vB_SalP_LD27, vB_SalP_LD28, vB_SalP_LD29, vB_SalP_LD30, vB_SalP_LD32, vB_SalP_LD33, vB_SalP_LD34, vB_SalP_LD35 and vB_SalP_LD36.

Images