Broad-spectrum salmonella pullorum phage and application thereof

Abstract

The invention provides a broad-spectrum salmonella pullorum bacteriophage and application thereof, and belongs to the technical field of microorganisms. The salmonella pullorum phage disclosed by the invention is a phage vBSalPLDF9 (Sal P9 for short) and belongs to a salmonella phage, and the preservation number of the salmonella pullorum phage is CGMCC (China General Microbiological Culture Collection Center) No: 45788. The salmonella pullorum bacteriophage provided by the invention is wide in host spectrum, strong in splitting property, stable in heredity, strong in alkali resistance, has certain acid resistance and certain temperature stability, does not contain drug-resistant genes and virulence genes in genome, and has a good application prospect in prevention and treatment of pullorum disease and purification of pullorum disease in breeding chicken farms.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a broad-spectrum Salmonella pullorum bacteriophage and application thereof. Background Art

[0002] Salmonella pullorum can cause acute septicemia in chicks. Young chicks infected through eggs often become ill or die within a week. In the most acute cases, there may be no symptoms and sudden death. Adult chickens are mostly latently infected, with no symptoms and no obvious lesions, but the infection in breeder chickens can be transmitted vertically, causing great economic losses to the poultry industry.

[0003] At present, antibiotics are the main treatment for pullorum, but due to the irrational use of antibiotics, bacterial resistance continues to increase, and the proportion of multi-drug resistant bacteria remains high, resulting in a decrease in the therapeutic effect of antibiotics on pullorum, and antibiotic residues pose a threat to food safety and public health. At the same time, antibiotics can also destroy the balance of intestinal flora and reduce animal immune function. Excessive antibiotic dosage can cause animal poisoning and environmental pollution. In order to ensure food safety, the use of antibiotics is prohibited or restricted in the late stage of broiler breeding and laying period of laying hens, which brings difficulties to the prevention and control of bacterial diseases. Therefore, it is urgent to find an efficient, safe and residue-free bactericidal preparation.

[0004] Bacteriophages are natural enemies of bacteria. As a natural antibacterial agent, bacteriophages have advantages that antibiotics cannot match. In addition, bacteriophages have a long history of clinical treatment and have achieved good results. They have broad application prospects in the fight against bacterial infections. In the field of poultry and livestock farming, bacteriophages can be used to reduce or prevent bacterial infections in animals, and can also be used to control bacterial density in the breeding environment. Therefore, finding relevant bacteriophages that can be effectively used to prevent and treat pullorum and purify pullorum in breeder farms is the key to solving problems related to pullorum. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a Salmonella pullorum phage, which has a wide host spectrum, strong lytic ability and genetic stability, and has good application prospects in the prevention and treatment of pullorum and the purification of pullorum in breeder farms.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A Salmonella pullorum phage, wherein the Salmonella pullorum phage is a phage vB_SalP_ LDF 9, the deposit number is CGMCC No: 45788.

[0007] Preferably, the bacteriophage vB_SalP_LDF The MOI of 9 is 0.00001.

[0008] Preferably, the bacteriophage vB_SalP_LDF The tolerable pH value of 9 is 4~12, and the tolerable temperature is 30~60℃.

[0009] Preferably, the bacteriophage vB_SalP_LDF The incubation period of 9 is 4 minutes and the lysis period is 90 minutes.

[0010] Preferably, the bacteriophage vB_SalP_LDF 9 Stable passage 1~40 generations.

[0011] The invention also provides a preparation containing the Salmonella pullorum phage.

[0012] Preferably, the preparation is a biocide, a cleaner or a disinfectant for the chicken breeding environment.

[0013] The present invention also provides the use of the Salmonella pullorum phage in preparing medicines for treating diseases caused by Salmonella pullorum.

[0014] The present invention also provides the use of the Salmonella pullorum phage in purifying pullorum in a breeder chicken farm.

[0015] The present invention also provides a bactericidal composition for preventing and treating Salmonella pullorum, comprising an effective dose of the Salmonella pullorum phage.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The Salmonella pullorum phage provided by the present invention belongs to the order Caudovirales and the family Laeciophage. The phage has a wide host spectrum, strong lytic ability and genetic stability, strong alkali resistance and certain acid resistance, and certain temperature stability. At the same time, the phage genome does not contain drug resistance genes and virulence genes, and has good application prospects in the prevention and treatment of pullorum and the purification of pullorum in breeder farms.

[0017] Biological Deposit Description Bacteriophage vB_SalP_LDF 9 (Salmonella phage Salmonella phage ), depository: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short), depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No.45788, deposit date is November 29, 2023. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Plaques formed by bacteriophage Sal P9; Figure 2 :Electron microscope photo of bacteriophage Sal P9; Figure 3: Optimal MOI of bacteriophage Sal P9; Figure 4 :Acid-base stability of bacteriophage Sal P9; Figure 5 : Temperature stability of bacteriophage Sal P9; Figure 6 : One-step growth curve of bacteriophage Sal P9; Figure 7 : Circle diagram of the phage Sal P9 genome, where (a) is the genome structure of phage Sal P9; (b) is the gene annotation; Figure 8 :The titer determination results of each generation of bacteriophage Sal P9; Fig. 9 : Electron microscopic images of phage Sal P9 of different generations, where (a) is an electron microscopic image of the 1st generation phage Sal P9; (b) is an electron microscopic image of the 10th generation phage Sal P9; (c) is an electron microscopic image of the 20th generation phage Sal P9; (d) is an electron microscopic image of the 30th generation phage Sal P9; (e) is an electron microscopic image of the 40th generation phage Sal P9; Fig.10 : The results of acid-base stability test of each generation; Fig.11 : Results of temperature stability tests at different generations; Fig.12 : One-step growth curves of different generations; Fig.13 : Survival of chicks in each group; Fig.14 : Dynamic changes of bacterial load in different organs of chicks in each group; (a) is the change of bacterial load in the heart; (b) is the change of bacterial load in the liver; (c) is the change of bacterial load in the spleen; (d) is the change of bacterial load in the lung; (e) is the change of bacterial load in the kidney; Fig.15 : Dynamic changes of phage load in different organs of each group of chicks, including (a) the change of phage load in the heart; (b) the change of phage load in the liver; (c) the change of phage load in the spleen; (d) the change of phage load in the lung; (e) the change of phage load in the kidney. DETAILED DESCRIPTION

[0019] The present invention provides a Salmonella pullorum phage, wherein the Salmonella pullorum phage is a phage vB_SalP_LDF 9 (Sal P9 for short) was isolated from a fecal sample from a chicken farm in Weifang, Shandong Province, and was identified as a Salmonella phage. Salmonella phage, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 29, 2023, with the deposit number CGMCC No: 45788.

[0020] The bacteriophage Sal P9 of the present invention consists of a head and a tail. The total length of the bacteriophage Sal P9 is about 164-180nm. The head is icosahedral symmetrical, with a transverse diameter of about 49-56nm and a long diameter of about 51-59nm. The tail is an elongated cylindrical shape, about 113-125nm long, about 7-11nm in diameter, and has an obvious fibrous structure at the end. According to the 9th report of the International Committee on Taxonomy of Viruses, it can be judged that the bacteriophage belongs to the order Caudovirales and the family Long-tailed Bacteriophage.

[0021] The bacteriophage Sal P9 of the present invention has a wide host spectrum and strong lysis ability against Salmonella pullorum.

[0022] The optimal infection multiplicity (MOI) of the bacteriophage Sal P9 of the present invention is 0.00001. When the MOI of bacteriophage Sal P9 is 0.00001, the titer can reach 10 11 pfu / mL, with strong proliferation ability, a high titer lysate can be obtained with a small amount of seeds.

[0023] The bacteriophage Sal P9 of the present invention has strong alkali resistance and certain acid resistance, and its tolerance pH value is 4-12. When the pH value is 4-12, the titer does not change significantly. When the pH value is 3 and the treatment is carried out for 1 hour, the titer can reach 1.62×10 5 pfu / mL, indicating that bacteriophage SalP9 has strong resistance to gastric acid and is not easily destroyed in the stomach. It can enter the intestine in large quantities to exert its bacteriophagy effect, and oral administration has advantages.

[0024] The bacteriophage Sal P9 of the present invention has certain temperature stability and can tolerate a temperature of 30-60° C. It can maintain high activity at 30-60° C.

[0025] The bacteriophage Sal P9 of the present invention has a short incubation period of only 4 minutes, a lysis period of 90 minutes, and a lysis amount of about 706 pfu / cell, which has the potential of high production efficiency.

[0026] The bacteriophage Sal P9 of the present invention has genetic stability and can be stably propagated for 1 to 40 generations.

[0027] The present invention also provides a preparation containing the above-mentioned Salmonella pullorum bacteriophage, which is a biological fungicide, a cleaner or disinfectant for chicken breeding environment. Those skilled in the art can prepare the preparation containing the above-mentioned bacteriophage according to the disclosure of the present invention and the common sense in the field, and use it as a daily fungicide to specifically kill Salmonella pullorum in the environment and improve the distribution of microorganisms in the environment; it can also be used as a biological fungicide in the breeding, transportation and storage of chicken products, and used to prevent and control Salmonella pullorum contamination during the breeding, transportation and storage of chickens.

[0028] The present invention also provides the use of the above-mentioned Salmonella pullorum phage in the preparation of a drug for treating diseases caused by Salmonella pullorum, which can provide a potential therapeutic drug for bacterial infection caused by Salmonella pullorum, can be used to prevent or treat lesions caused by Salmonella pullorum, and can also be added to feed as a feed additive to specifically and continuously prevent the survival and reproduction of Salmonella pullorum in feed.

[0029] The present invention also provides a bactericidal composition for preventing and treating Salmonella pullorum, comprising an effective dose of the above-mentioned Salmonella pullorum bacteriophage. The Salmonella pullorum bacteriophage of the present invention can also be further prepared into a composition with other bacteriophages for a wider spectrum of antibacterial or antibacterial applications.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] In a specific embodiment of the present invention, the host bacteria involved in the experiment: avian Salmonella HZ210301 (abbreviated as S44), was isolated and identified by the Phage Research Center of Liaocheng University, and was isolated from a duck head in a duck farm in Heze, Shandong in March 2021. It is Enteritidis Salmonella ( salmonella enteritidis ). Infection strain: Salmonella pullorum LC190702 (abbreviated as S18, salmonella pullorum ), which was isolated and identified by the Bacteriophage Research Center of Liaocheng University.

[0034] Samples and sources: The feces samples came from a chicken farm in Weifang, Shandong, and the sampling time was May 2020.

[0035] Reagents: LB solid medium and LB liquid medium were purchased from Beijing Luqiao Technology Co., Ltd.; SM buffer was purchased from Wuhan Kanos Technology Co., Ltd.

[0036] The main experimental instruments are: transmission electron microscope, model JEM-1200EXII, purchased from JEOL, Japan Electronics Co., Ltd.; desktop shaker model SLK-O3000-S, purchased from SCILOGEX, USA.

[0037] Example 1 Salmonella pullorum bacteriophage vB_SalP_LDF9 Isolation, purification and storage of: 1. Bacteriophage vB_SalP_LDF9 Isolation: Take 50 mg of fecal sample and put it into 5 mL LB liquid culture medium, culture it in a constant temperature shaker at 37℃ for 4 hours, centrifuge it at 10000r / min for 5 minutes, filter the supernatant with a 0.22µm filter membrane to sterilize it, and set aside the filtrate. Use the double-layer plate drop method to isolate bacteriophages. Take 200µL of avian Salmonella S44 preserved in the laboratory and mix it with 4-6mL of LB semi-solid culture medium cooled to about 50℃ in a centrifuge tube, pour it on the LB solid culture medium, let it stand for solidification, add 5µL of the filtrate on the double-layer plate, culture it at 37℃ for 6 hours, and observe the formation of plaques.

[0038] 2. Purification of phage: Use a sterile pipette tip to remove a single plaque, place it in SM buffer and vortex to mix, filter it with a 0.22µm filter membrane, take the filtrate and prepare a double-layer plate with the host bacteria S44 again, repeat the single plaque removal and purification operation 4 to 6 times, and form plaques of uniform shape and size on the double-layer plate. The plaques are round and transparent, such as Figure 1 As shown, the purified phage was obtained. It was named according to the international naming rules. vB_SalP_LDF9 , LD stands for Liaocheng University, and F stands for feces. For the convenience of recording, this bacteriophage strain is referred to as Sal P9.

[0039] 3. Preservation of bacteriophages: Mix the bacteriophage proliferation solution with 50% glycerol in a ratio of 6:4, dispense into 2 mL cryovials, and store in a -80°C refrigerator, or make into lyophilized powder and store in a 4°C refrigerator.

[0040] Bacteriophage vB_SalP_LDF9 (Sal P9) was deposited on November 29, 2023 at the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.45788.

[0041] Example 2 Electron microscopic observation of bacteriophage Sal P9: Prepare high-titer phage lysis solution by plate amplification method: take 400µL of host bacterial culture solution grown to the logarithmic phase, mix it with phage lysis solution at the optimal infection multiplicity ratio, prepare double-layer plates, and culture at 37°C for 12 hours. Add 5-7mL of SM buffer to the culture dish, shake on a shaker at 100rpm for 4 hours, collect the eluate, centrifuge at 12000rpm / min for 5 minutes, and filter with a 0.22µm filter membrane to obtain high-titer phage lysis solution.

[0042] The titer of the phage lysate was determined by the double-layer plate method: about 10 mL of LB solid culture medium was poured into a round plate with a diameter of 90 mm, and solidified at room temperature as the bottom culture medium. The phage lysate was diluted 10 times in a sterile 1.5 mL centrifuge tube with sterile LB liquid culture medium, and the pre-sterilized LB semi-solid culture medium was placed in a 50°C constant temperature water bath, 4-6 mL of 50°C semi-solid culture medium was added to a 10 mL sterile centrifuge tube, and 100 µL of host bacteria S44 bacterial solution was added, with a dilution of 10 -12 ~10 -16 100 µL of the lysate was quickly mixed and poured onto the solid plate. Mixed evenly, left for about 15 minutes, and inverted in a 37°C constant temperature incubator for 8-14 hours after solidification. Each dilution was repeated 3 times. Plates with 20 to 300 plaques were selected for counting and the titer of the phage was calculated. The titer of the lysate was determined to be 8.1×10 16 pfu / mL.

[0043] Drop 20µL of the above phage lysis solution on the paraffin slide, place the copper mesh on it for 10 minutes and then remove it to absorb the phage. Dry it at room temperature for 2-3 minutes, use 2% phosphotungstic acid (w / v, pH7.0) for negative staining, absorb the water after 2 minutes, dry it naturally for 5 minutes, observe the phage morphology with an electron microscope, and take pictures at an accelerating voltage of 100kV. Figure 2 Phage Sal P9 consists of two parts: a head and a tail. Measurements of multiple phages showed that the total length of phage Sal P9 was about 164-180 nm. The head was icosahedral symmetrical, with a transverse diameter of about 49-56 nm and a long diameter of about 51-59 nm. The tail was an elongated cylindrical shape, about 113-125 nm long and 7-11 nm in diameter, with an obvious fibrous structure at the end. Figure 2 The total length of the middle phage is 164.1nm, the transverse diameter of the head is 49.2nm, the long diameter is about 51.5nm, the tail length is 112.6nm, and the tail diameter is 7nm. According to the 9th report of the International Committee on Taxonomy of Viruses, it can be judged that the phage belongs to the order Caudovirales and the family Longicaudophage.

[0044] Example 3 Determination of host spectrum of bacteriophage Sal P9: 120 strains of Salmonella pullorum isolated, identified and preserved by the Phage Research Center of Liaocheng University were selected, and the host spectrum of phage Sal P9 was determined by double-layer plate spot method. The strain number information is shown in Table 1. The 120 strains of Salmonella pullorum were isolated from chicken farms in 8 different provinces, including 93 strains in Shandong, 10 strains in Guangdong, 10 strains in Sichuan, 2 strains in Jiangsu, 2 strains in Hebei, 1 strain in Anhui, 1 strain in Heilongjiang, and 1 strain in Guangxi. Among them, 41 strains were isolated from Liaocheng, 16 strains in Heze, 14 strains in Zibo, 5 strains in Weifang, 4 strains in Yantai, 3 strains in Binzhou, 3 strains in Qingdao, 3 strains in Jining, 2 strains in Tai'an, 1 strain in Laiwu, and 1 strain in Weihai.

[0045] Table 1 Information on Salmonella pullorum strains used to determine host spectrum

[0046] Use 120 strains of Salmonella pullorum to make double-layer plates. Pour 10mL of LB solid culture medium into a sterile plate and solidify at room temperature as the bottom culture medium. Place the pre-sterilized LB semi-solid culture medium in a 50℃ constant temperature water bath, take 4~6mL of the semi-solid culture medium in the 50℃ water bath and add it to a 10mL sterile centrifuge tube, add 200µL of Salmonella liquid, mix quickly and pour it on the solid plate, leave it for about 15 minutes, and wait for it to solidify. Take 5µL of Sal P9 phage and drop it on each of the above double-layer plates, culture it at a constant temperature of 37℃ overnight, and observe whether there are plaques and the size and transparency of the plaques. Classify and record according to the state of the drop plaques: +4, large and bright plaques, complete bacterial lysis; +3, plaques are clear but with a faint hazy background; +2, incomplete lysis, with high turbidity in the spot area; +1, presence of some individual phage plaques in the spotted area; -, no plaques.

[0047] The 120 preserved strains of Salmonella pullorum were inoculated, and the results are shown in Table 2.

[0048] Table 2 Results of host spectrum determination of bacteriophage Sal P9

[0049] As shown in Table 2, phage Sal P9 can form large and transparent plaques on 116 strains of Salmonella pullorum, with a coverage rate of 96.7%, indicating that the phage has a wide host spectrum and strong lysis ability against Salmonella pullorum.

[0050] Example 4 Determination of bacteriophage Sal P9 titer: The phage titer was determined by the double-layer plate method: about 10 mL of LB solid medium was poured into a sterile plate and solidified at room temperature as the bottom medium. The pre-sterilized LB semi-solid medium was placed in a 50°C constant temperature water bath. Nine 1.5 mL sterile centrifuge tubes were taken and numbered according to the dilution multiple. 900 µL of sterile LB liquid medium was added to each tube, 100 µL of purified phage filtrate was added to the first centrifuge tube, and vortexed to mix; the pipette tip was changed to take 100 µL to the second tube, and the phage filtrate was diluted 10 times in sequence.

[0051] Take 100µL of phage dilutions of different dilutions and 100µL of avian Salmonella S44 in a 10mL sterile centrifuge tube and mix them. Incubate at 37℃ for 5min. Add 4~6mL of 50℃ LB semi-solid medium to the mixture, and then immediately pour it into a plate with LB solid medium at the bottom. Leave it for about 15min until it solidifies. Prepare double-layer plates, 3 double-layer plates for each dilution, incubate at 37℃ for 8~14h, and count plaques. Select plates with 20~300 plaques for counting. The titer (pfu / mL) = average number of plaques × 10 × dilution multiple. The titer of phage Sal P9 was measured to be 8.4×10 9 pfu / mL.

[0052] Example 5 Determination of the optimal infection multiplicity of bacteriophage Sal P9: The multiplicity of infection (MOI) refers to the ratio of the number of phages to host bacteria that can be adsorbed within a specific period of time. In practical applications, the optimal MOI is usually the ratio of the number of phages to host bacteria that achieves the highest phage titer under certain culture conditions.

[0053] Adjust the host bacterial concentration to 10 8 CFU / mL, dilute the phage Sal P9 lysate in 10-fold gradients using sterile LB liquid culture medium at MOIs of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, etc. Take 100µL of the phage dilution and host bacterial culture solution, add them to the pre-warmed 5mL LB liquid culture medium, and culture at 37°C, 180r / min and shake for 3h. Centrifuge the mixed culture at 12000r / min for 2min, filter with a 0.22µm filter membrane, and obtain the lysate. The double-layer plate method was used to determine the titer of the phage lysate, repeated three times, and the infection multiplicity with the highest titer of the lysate was the optimal infection multiplicity of the phage. The results are shown in Figure 3 .

[0054] Depend on Figure 3It can be seen that when the MOI of phage Sal P9 is 0.00001, the titer is the highest, which is 1.043×10 11 pfu / mL, and the optimal MOI of the phage was 0.00001. This indicated that the phage had a strong proliferation ability and a high titer lysate could be obtained with a small amount of seeds.

[0055] Example 6 Determination of acid-base stability of bacteriophage Sal P9: The pH value of LB liquid medium was adjusted to 1-13 using 1 mol / L HCl and NaOH. 900 µL of LB liquid medium with different pH values ​​was added with 100 µL of phage, mixed evenly, and incubated in a 37°C water bath for 1 h. The phage titer under different pH treatments was determined by the double-layer plate method. The results are shown in Figure 4 .

[0056] Depend on Figure 4 It can be seen that the titer of phage Sal P9 was significantly reduced under pH < 4 conditions, and the titer could reach 1.62×10 5 pfu / mL, the phage is inactivated at pH 1, and there is no significant change in titer at pH 4-12, indicating that phage SalP9 has strong alkali resistance and certain acid resistance. Compared with other Salmonella phages, this phage has stronger acid resistance. Phage SalP9 has strong resistance to gastric acid and is not easily destroyed in the stomach, so it can enter the intestine in large quantities and exert its phagocytic efficacy, which has advantages in oral administration.

[0057] Example 7 Determination of temperature stability of bacteriophage Sal P9: Take 15 sterilized 1.5mL centrifuge tubes, add 500µL of Sal P9 phage lysis solution to each tube, and place 3 tubes in a group, respectively, in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃. Take out one tube from each water bath at different temperatures at 20min, 40min, and 60min, and use the double-layer plate method to determine the phage titer. Repeat three times. The determination results are shown in Figure 5 .

[0058] Depend on Figure 5 It can be seen that phage Sal P9 has a certain temperature stability and can still maintain a high activity after being exposed to 60°C water bath for 1 hour, with a titer of up to 6.40×10 9 pfu / mL, the titer began to decrease after 70℃, and the titer was 1.26×10 4 pfu / mL, it was still active after 40min and 60min, but the activity decreased successively, and the titer was 0 after 20min under 80℃ condition.

[0059] Example 8 Determination of one-step growth curve of bacteriophage Sal P9: The phage Sal P9 lysate was mixed with the host bacteria S44 liquid at the optimal infection multiplicity ratio, placed in a 37°C water bath for 5 minutes, centrifuged at 12000r / min for 5 minutes, the supernatant was discarded, and the precipitate was washed twice with LB liquid medium preheated at 37°C, 1 mL of LB liquid medium preheated at 37°C was added, the precipitate was resuspended, and it was added to 100 mL of LB liquid medium preheated at 37°C, and cultured in a 37°C constant temperature shaker. Samples were taken once at 0, 1, 2, 3, 4, and 10 minutes, and then once every 10 minutes. The phage titer was determined by the double-layer plate method until 120 minutes, and repeated three times. The one-step growth curve of the phage was drawn with time as the horizontal axis and the phage titer as the vertical axis. The one-step growth curve of the phage Sal P9 is shown in Figure 6 .

[0060] The lysis amount was calculated according to the following formula: lysis amount = final phage titer / initial host bacteria number.

[0061] Depend on Figure 6 It can be seen that the incubation period of bacteriophage Sal P9 is 4 minutes, the lysis period is 90 minutes, and then it is a stable period, and the lysis amount is about 706 pfu / cell. This shows that the incubation period of bacteriophage Sal P9 is short, only 4 minutes, and the lysis amount is high, and it has the potential for high production efficiency.

[0062] Example 9 Whole genome sequencing of bacteriophage Sal P9: The purified phage Sal P9 particles were sent to a sequencing company for sequencing. The full length of the phage Sal P9 genome was 43591 bp. The genome circle map is shown in Figure 7 , (a) shows the genome structure of bacteriophage Sal P9; (b) shows the gene annotation. The bacteriophage sequence was compared with the NCBI database, and the sequence submitted for comparison was functionally annotated. The annotation results showed that the bacteriophage genome had 63 CDS (Coding Sequences) encoding 63 proteins.

[0063] The gene sequence of bacteriophage vB_SalP_LDF9 was submitted to the database CARD (https: / / card.mcmaster.ca / ) and the database VFDB (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi) for virulence gene and drug resistance gene detection, respectively. The results showed that the phage genome did not contain drug resistance genes and virulence genes.

[0064] Example 10 Sterility test of bacteriophage Sal P9: Fluid Thioglycollate Medium (TG) was used for the examination of anaerobic and aerobic bacteria. Peptone from casein Agar Medium (GA) was used for the examination of aerobic bacteria. TG and GA were purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., with batch numbers 20220305 and 20220117, respectively.

[0065] Preparation of thioglycollate fluid medium: Weigh 5.85 g of thioglycollate fluid medium granules, dissolve in 200 mL of pure water with heating and stirring, sterilize at 121°C for 15 min, and cool rapidly. Before inoculation, the height of the oxidation layer of the medium must not exceed 1 / 3 of the depth of the medium, otherwise, it must be heated in a 100°C water bath until the pink color disappears, and then cooled rapidly. It is limited to heating once and prevent contamination.

[0066] Preparation of casein agar medium: Weigh 8.0 g of casein agar medium powder, heat and boil to completely dissolve in 200 mL of pure water, divide into portions, sterilize under high pressure at 116°C for 30 min, and set aside.

[0067] The bacteriophage Sal P9 was inoculated into 2 tubes of thioglycollate fluid culture medium (TG tube) and 2 tubes of casein agar (GA) slant, each with 0.2 mL. One tube was placed at 37°C and the other at 25°C for 3 to 5 days to observe whether there was bacterial growth. The results showed that no bacteria grew.

[0068] Embodiment 11 Genetic stability of bacteriophage Sal P9: 1. Subculture of bacteriophage Sal P9 100 µL of phage Sal P9 and host bacterial culture fluid were added to 5 mL of pre-warmed LB liquid medium and cultured at 37°C with shaking at 180 r / min for 2 h for subculture. The mixed culture was centrifuged at 12000 r / min for 2 min and filtered with a 0.22 µm filter membrane to obtain a lysate. The phages of each generation were titered and frozen. The phages of the 10th, 20th, 30th, and 40th generations were observed under an electron microscope and tested for acid-base stability, temperature stability, etc. to determine their genetic stability. The titer determination results of phage Sal P9 at each generation are shown in Figure 8 The titer of each generation was stable, indicating that the phage had genetic stability.

[0069] 2. Electron microscopic observation of phage Sal P9 at different generations The high titer phage lysis solution was prepared by plate amplification method. 20 μL of phage lysis solution of different generations was dropped on paraffin slides, negatively stained with phosphotungstic acid, and observed under transmission electron microscope. The electron microscope photos of the 1st, 10th, 20th, 30th and 40th generations are shown in Fig. 9 In the figure, (a) is an electron micrograph of the 1st generation phage Sal P9; (b) is an electron micrograph of the 10th generation phage Sal P9; (c) is an electron micrograph of the 20th generation phage Sal P9; (d) is an electron micrograph of the 30th generation phage Sal P9; (e) is an electron micrograph of the 40th generation phage Sal P9. This shows that the morphology of the phage is stable and there is no obvious change compared with the 1st to 40th generations.

[0070] 3. Determination of the optimal MOI of each generation of bacteriophage Sal P9 The optimal MOI of the 10th, 20th, 30th and 40th generations of phage Sal P9 was determined in the same manner as in Example 5. The results are shown in Table 3.

[0071] Table 3 Results of determination of optimal MOI of phage Sal P9 at each generation

[0072] As shown in Table 3, the optimal MOI of phage Sal P9 is relatively stable, basically 0.00001. Although the optimal MOI is different at the 20th and 40th generations, the titer is also high when the MOI of the 20th generation is 0.00001, reaching 5.27×10 10 Therefore, the optimal MOI of each generation is basically stable, which is or close to 0.00001. This shows that the phage Sal P9 can obtain high-titer lysis liquid with a small amount of seeds, and the phage has high production efficiency and is conducive to large-scale production.

[0073] 4. Determination of acid-base stability of each generation of bacteriophage Sal P9 The method is the same as in Example 6, and the results are shown in Fig.10 .

[0074] The results showed that the titer of phage Sal P9 did not change significantly at each generation when the pH value was 4-12. When the pH value was 3 for 1 hour, the titers of the 10th, 20th, 30th and 40th generations were 6.2×10 7 pfu / mL, 2.7×10 7 pfu / mL, 9.8×10 7 pfu / mL and 6.7×10 7 pfu / mL, treated at pH 2 for 1 h, the titer at passage 40 was 1.12×10 4pfu / mL, indicating that the acid resistance of phage Sal P9 is enhanced after subculture, which is beneficial for oral phage to resist the destructive effect of gastric acid, so that it can enter the intestine in large quantities to play a phagocytic role and lyse Salmonella pullorum in the intestine.

[0075] 5. Determination of temperature stability of each generation of bacteriophage Sal P9 The method is the same as in Example 7, and the results are shown in Fig.11 .

[0076] The results showed that the titer of phage Sal P9 remained stable when exposed to 30℃~60℃ for 1h. The titer decreased significantly at 70℃, and the temperature tolerance trends of different generations were basically the same. With the passage of generations, the heat resistance of the phage at 70℃ increased. After the 10th, 20th, and 40th generations of phage were exposed to 70℃ for 1h, the titer increased to 10 4 The above indicates that the heat resistance of the phage is enhanced after subculture, which is conducive to its preservation.

[0077] 6. Determination of the one-step growth curve of each generation of bacteriophage Sal P9 The method is the same as in Example 8, and the results are shown in Fig.12 .

[0078] The results showed that the first-step growth curves of each generation were basically the same, and the incubation period was about 4 minutes, indicating that the phage could be lysed in a relatively short period of time. The burst period lasted about 90 minutes, and then entered the stable period. The titer in the stable period was basically at the same level.

[0079] Example 12 Animal protection test of bacteriophage Sal P9 1. Sterility test of phage lysate Take 5 µL of the phage vB_SalP_LDF9 lysate filtered through a 0.22 µm filter membrane and drop it on the LB solid plate. After it is absorbed, invert it and culture it in a 37°C constant temperature incubator for 24 hours. As a result, no colony growth was found on the plate, indicating that the phage lysate is sterile and can be used for therapeutic experiments.

[0080] 2. Experiment on the treatment of SPF chickens infected with Salmonella pullorum using bacteriophage Sal P9 The infection strain selected was Salmonella pullorum S18. 135 7-day-old SPF chicks were randomly divided into a non-infected group, a high-dose phage treatment group, a medium-dose phage treatment group, a low-dose phage treatment group (the three treatment groups are referred to as the high-dose group, the medium-dose group and the low-dose group) and an infected untreated group after anal swabs were taken for bacterial culture verification and sterility verification. Each group had 27 chickens, and the weight of each SPF chicken was weighed. Fifteen chickens were randomly selected from each group and marked to observe the morbidity and mortality of the chickens. The remaining 12 chickens were used to take samples. Except for the non-infected group (negative control group), each group was intraperitoneally injected with 200μL of 1×10 9 CFU / mL of Salmonella pullorum S18 liquid, and the uninfected group was injected with the same dose of sterile saline. Three chickens were taken from each group at 3h, 1d, 4d, 7d, and 10d after the injection of bacteria. After euthanasia, 0.1g of heart, liver, spleen, kidney, lung, and cecum were taken aseptically, diluted 10 times with 900µL sterile saline, and evenly spread on SS culture medium to calculate the bacterial load. At the same time, the double-layer plate method was used to detect the phage load of each organ.

[0081] After each sampling, three different concentrations of phage were used for treatment, the concentrations were: 1×10 10 pfu / mL, 1×10 9 pfu / mL and 1×10 8 pfu / mL, and 200 µL / mouse were injected intraperitoneally, that is, the injection doses of the high, medium, and low dose groups were 2×10 9 pfu / piece, 2×10 8 pfu / each and 2×10 7 pfu / piece. Observe and record the survival status and weight changes of chickens. In addition, cloacal swabs were taken from each group at 3h, 6h, 12h, 1d, 4d, 7d, and 10d for bacterial culture, and PCR was used to verify whether the cloaca was excreting toxins. The test time was 14d.

[0082] 3. Survival of chicks in each group The survival of each group was observed for 14 days. Fig.13 The survival rate of the infected group was 0%, the survival rate of the non-infected group was 100%, and the survival rates of the high-, medium-, and low-dose groups were 86.7%, 26.7%, and 40%, respectively.

[0083] 4. Content of Salmonella in different organs of chicks in each group Dynamic changes of bacterial load in different organs of chicks in each group Fig.14, (a) shows the change of bacterial load in the heart; (b) shows the change of bacterial load in the liver; (c) shows the change of bacterial load in the spleen; (d) shows the change of bacterial load in the lung; (e) shows the change of bacterial load in the kidney. In the heart, liver, and lung, the Salmonella content in each treatment group was significantly lower than that in the infection group (infection without treatment group). In the heart, the bacterial content in the high-dose group was 4 to 6 orders of magnitude lower than that in the infection group, especially on the 10th day after infection, no bacteria were detected. In the lungs and kidneys, the bacterial content of the medium-dose group, high-dose group, and low-dose group were significantly lower than that in the infection group. The bacterial content in the kidneys of the medium-dose group at 7 dpi and the lungs at 10 dpi was higher than that in the infection group, and the bacterial content at 1 dpi and 4 dpi was significantly lower than that in the infection group. No bacteria were detected in the high-dose group at 7 dpi and 10 dpi, and no bacteria were detected in the low-dose group at 10 dpi. This indicates that bacteriophage Sal P9 can effectively inhibit the proliferation of Salmonella pullorum S18 in various organs, reduce the bacterial load in the organs, and even eliminate bacteria in the heart and kidneys. 9 pfu / piece has the best antibacterial effect.

[0084] 4. Phage loads in different organs of chicks in each group The dynamic changes of phage load in different organs of chicks in each group are shown in Fig.15 , (a) shows the changes in phage load in the heart; (b) shows the changes in phage load in the liver; (c) shows the changes in phage load in the spleen; (d) shows the changes in phage load in the lungs; (e) shows the changes in phage load in the kidneys. Fig.14 In the heart and liver, as the bacterial content decreased, the overall phage load also decreased significantly. At 10 dpi, no phage was detected in the heart of the high-dose group. At 4 dpi, the phage load in each organ of the high-dose group was significantly higher than that of the medium and low-dose groups. At 7 dpi and 10 dpi, it was mostly lower than that of the medium and low-dose groups. At 10 dpi, no phage was detected in the heart, spleen and kidney of the high-dose group and in the kidney of the low-dose group. This shows that the phage load is closely related to the bacterial content.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Salmonella pullorum phage, characterized in that: The Salmonella pullorum phage is a phage vB_ SalP_LDF 9, the deposit number is CGMCC No: 45788.

2. The Salmonella pullorum phage according to claim 1, characterized in that The bacteriophage vB_SalP_ LDF The MOI of 9 is 0.00001.

3. The Salmonella pullorum phage according to claim 1, characterized in that The bacteriophage vB_SalP_ LDF The tolerable pH value of 9 is 4~12, and the tolerable temperature is 30~60℃.

4. The Salmonella pullorum phage according to claim 1, characterized in that The bacteriophage vB_SalP_ LDF The incubation period of 9 is 4 minutes and the lysis period is 90 minutes.

5. The Salmonella pullorum phage according to claim 1, characterized in that The bacteriophage vB_SalP_ LDF 9 Stable passage 1~40 generations.

6. A preparation containing the Salmonella pullorum phage according to any one of claims 1 to 5.

7. The preparation according to claim 6, characterized in that The preparation is a biocide, a cleaner or a disinfectant for chicken breeding environment.

8. Use of the Salmonella pullorum phage according to any one of claims 1 to 5 in the preparation of a medicament for treating a disease caused by Salmonella pullorum.

9. Use of the Salmonella pullorum phage according to any one of claims 1 to 5 in the purification of pullorum in breeder chicken farms.

10. A bactericidal composition for preventing and treating Salmonella pullorum, characterized in that: The invention comprises an effective dose of the Salmonella pullorum phage according to any one of claims 1 to 5.

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