Salmonella phage JN06 and application thereof

Abstract

The invention discloses a salmonella bacteriophage JN06 and an application thereof. The preservation number of the salmonella phage JN06 is GDMCC (China General Microbiological Culture Collection Center) No: 65479-B1, and the salmonella phage JN06 is preserved in Guangdong Province Microbial Culture Collection Center on November 13, 2024. The Salmonella lyase has the advantages that the Salmonella lyase is simple in preparation method and wide in host spectrum, not only can be used for lysing various serotypes of salmonella, particularly can be used for lysing Salmonella Schwarzenge Salmonella vaporariorum, but also can be used for lysing Escherichia coli at the same time. The method has the advantages of short incubation period, high outbreak amount, strong tolerance to temperature and pH value, wider application range, and no virulence gene and drug-resistant gene.

Description

Technical Field

[0001] The present invention relates to the field of bacteriophages, and in particular to a Salmonella bacteriophage JN06 and a use thereof. Background Art

[0002] Salmonella is an important zoonotic and foodborne pathogen that poses a serious threat to public health and food safety. Diseases such as pullorum, fowl typhoid and paratyphoid caused by Salmonella infection have caused huge losses to the breeding industry. The abuse of antibiotics has led to the prevalence of a large number of drug-resistant bacteria, which has brought greater challenges to the prevention and control of Salmonella.

[0003] Bacteriophage (phage) is a natural bacterial killer that is widely distributed in nature and is an important weapon to solve the problem of drug-resistant bacteria pollution. It has been widely used in disease prevention and control and food safety. Liu Baotao et al. (application publication number CN118581050A) disclosed a Salmonella phage vB-SenS-JZ291 and its application. The phage can lyse some Salmonella, such as Typhimurium, Typhimurium 1, 4, [5], 12:i:-unidirectional variants, Enteritis, Pullorum, Thompson and Mbandaka, etc., but does not include Salmonella Schwarzeneggeron, Salmonella delbrueckii, etc.; Wang Shuo et al. (application Publication No. CN118620848A) discloses a Salmonella phage SPYS-2 and its application. SPYS-2 can specifically lyse Salmonella (Enteritidis Salmonella, Typhimurium and colistin-resistant mcr-1-positive Typhimurium 1, 4, [5], 12:i:-variants). It is described that it can be used to prevent and treat Salmonella infections in animal husbandry, excluding Salmonella Schwarzeneggeron, Salmonella Derby, etc.

[0004] CN118048315B discloses a strain of Salmonella phage that has a strong lysis effect on Salmonella, has a cross-lysis effect on Salmonella from different animal sources, but cannot lyse Escherichia coli. CN113416712A discloses a strain of Salmonella phage with a wide lysis spectrum that can lyse Salmonella schwarzeneggeron, but also does not lyse Escherichia coli. CN108103031A discloses a wide-spectrum phage Escherichia coli phage NTHP01 for aquaculture, which, in addition to having a good lysis effect on Aeromonas, can also lyse other bacteria (common bacteria such as Edwardsiella tarda, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Kvassium, Citrobacter freundii, Enterobacter, etc.), excluding Salmonella schwarzeneggeron, Salmonella delbrueckii, etc. CN112143709A discloses a strain of Aeromonas hydrophila phage, which, in addition to having a good lysis effect on Escherichia coli, can also be used as a potent phage for treating Aeromonas hydrophila infection in freshwater fish in my country, and has broad application prospects in aquaculture. It can lyse some Aeromonas hydrophila, but cannot lyse Aeromonas vermifuge and Vibrio alginolyticus. CN110317792A discloses a strain of Vibrio parahaemolyticus phage, which, in addition to having a good lysis effect on Escherichia coli, can also prevent and treat pathogenic Vibrio parahaemolyticus infection in white shrimp, and has broad application prospects in aquaculture, but only points out that it has a good lysis effect on pathogenic Vibrio parahaemolyticus causing acute hepatopancreatic necrosis.

[0005] Phages have the characteristic of high specificity, but this characteristic also limits the development of phages to a certain extent. In actual production and life, the occurrence of diseases is often accompanied by infection with multiple pathogens. Therefore, finding a cross-species broad-spectrum virulent phage is conducive to further promoting the application of phages in disease prevention and control. Summary of the invention

[0006] The object of the present invention is to provide a bacteriophage which can lyse multiple serotypes of Salmonella, in particular, can lyse Salmonella Schwarzengrund, and can also lyse Escherichia coli at the same time.

[0007] To achieve the above-mentioned object, the present invention provides a Salmonella enterica phage JN06, characterized in that the Salmonella enterica phage JN06 has a preservation number of GDMCC No: 65479-B1, and is preserved in Guangdong Provincial Microbiological Culture Collection Center on November 13, 2024.

[0008] The present invention also protects the use of the Salmonella enterica phage JN06 for simultaneously lysing Salmonella and Escherichia coli.

[0009] Furthermore, the Salmonella refers to at least one of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Derby, Salmonella Schwarzeneggeron, Salmonella Infantis, Salmonella Agona, and Salmonella Anuta; and the Escherichia coli is enteropathogenic Escherichia coli.

[0010] The present invention also protects the use of the Salmonella enterica phage JN06 in preparing an antibacterial agent.

[0011] Furthermore, the use refers to use in preparing an antibacterial agent.

[0012] Furthermore, the antibacterial agent refers to an agent that inhibits Salmonella and / or Escherichia coli.

[0013] Furthermore, the Salmonella refers to at least one of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Derby, Salmonella Schwarzeneggeron, Salmonella Infantis, Salmonella Agona, and Salmonella Anuta; and the Escherichia coli is enteropathogenic Escherichia coli.

[0014] The present invention also provides an antibacterial agent, characterized in that it contains the Salmonella enterica phage JN06.

[0015] The Salmonella phage JN06 was isolated from sewage collected from the broiler breeding base of Jiangxi Agricultural University.

[0016] The Salmonella phage JN06 belongs to the family of long-tailed phages, has a polyhedral head, the head is about 50×55nm, and the tail length is about 110nm. The burst volume is 125PFU / infected cell, and the incubation period is 5min. The phage proliferation efficiency is highest when the MOI is 0.001; JN06 has strong adaptability to the environment and can maintain an active state within the pH range of 3-12, while in an environment with a pH of 2, the phage titer becomes 1 / 2 of the original; stable phage activity can be maintained under conditions of 30-60°C, and the titer is reduced from the original 9.8PFU / mL to 9.0PFU / mL, 8.2PFU / mL and 7.3PFU / mL respectively after being treated at 70°C for 15min, 30min and 60min.

[0017] Whole genome sequencing revealed that the genome of Salmonella phage JN06 is 41,745 bp long, with 68 open reading frames encoding lysozyme and holin, but does not contain virulence genes or drug resistance genes.

[0018] The Salmonella phage JN06 has good antibacterial properties in vitro. When the MOI value is greater than 0.1, it can completely inhibit the growth of the host bacteria within 8 hours. In addition, phage JN06 can eliminate Salmonella in chicks and can be used to treat Salmonella infections.

[0019] Salmonella Schwarzengrund is the main serotype of Salmonella in live chickens or chicken, which can infect animals and humans, and can be transmitted from chicken to humans. The Salmonella phage JN06 of the present invention has a wide host spectrum, and can not only lyse multiple serotypes of Salmonella, especially Salmonella Schwarzengrund, but also lyse Escherichia coli at the same time. It has a short incubation period, a high burst volume, strong tolerance to temperature and pH, a wider range of applications, and no virulence genes and drug-resistant genes. The present invention also provides a method for using a phage in treating Salmonella infection in chicks, and a high-titer phage can completely eliminate the Salmonella in the chicks. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The morphology of bacteriophage JN06 on agar plate;

[0021] Figure 2 is a transmission electron micrograph of bacteriophage JN06;

[0022] Figure 3 This is the result of the whole genome analysis of bacteriophage JN06;

[0023] Figure 4 This is a graph showing the determination of the optimal MOI of phage JN06;

[0024] Figure 5 is the one-step growth curve of bacteriophage JN06;

[0025] Figure 6 is the pH stability diagram of bacteriophage JN06;

[0026] Figure 7 is the temperature stability diagram of bacteriophage JN06;

[0027] Figure 8 This is the in vitro antibacterial performance diagram of bacteriophage JN06;

[0028] Fig. 9 This is a graph showing the levels of Salmonella and bacteriophages in chicken feces;

[0029] Fig.10 is the graph of serum inflammatory cytokine content;

[0030] Fig.11 This is a pathological section of duodenum tissue. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0032] Example 1 Isolation and Identification of Salmonella Phage

[0033] (1) Isolation and screening of bacteriophage JN06

[0034] S1. The sample used in the present invention is sewage collected from the sewage pool of the broiler breeding base of Jiangxi Agricultural University in 2023. CaCl2 with a final concentration of 1 mmol / L was added to the sewage sample, and after standing for 2 hours at 37°C, centrifuged at 3000×g for 30 minutes, the supernatant was filtered and sterilized through a 0.22 μm filter, and the filtrate was collected and placed at 4°C for use.

[0035] S2. Take 3 mL of the above filtrate and mix it with 0.5 mL of Salmonella SMT in the logarithmic phase, transfer it to 10 mL of 2×TSB liquid culture medium, place it in a constant temperature shaker at 37°C and culture it at 220 rpm for 12-16 hours.

[0036] Salmonella SMT: Take the intestinal contents of diarrheal chickens, resuspend them with 2ml of 0.85% saline, use an inoculation loop to dip the suspension, streak it on an XLD agar plate, place it in a 37℃ constant temperature incubator for 24h, pick a suspected single colony and inoculate it in LB broth medium for overnight culture at 37℃. Identify it by PCR, use a bacterial DNA extraction kit to extract and separate the purified bacterial liquid DNA, use DNA as a template, detect the invA gene, and identify the Salmonella if the invA gene is positive.

[0037] S3. The obtained culture medium was centrifuged at 4°C, 12000×g for 10 min. The supernatant obtained by centrifugation was filtered through a 0.22 μm filter. The filtrate was taken and repeated the above steps. The enrichment was repeated 3 times to obtain the phage enrichment solution.

[0038] Take 300 μl of overnight cultured host bacteria (Salmonella SMT), transfer to 10 mL of fresh TSB liquid culture medium, and culture to the logarithmic phase. Add 100 μl of the logarithmic phase bacterial solution to 7 mL of TSB semi-solid agar medium cooled to about 50°C, mix quickly and pour on the LB agar plate. After the agar solidifies, drop 10 μl of phage enrichment solution (obtained in S3) on the agar surface containing the host bacteria; after loading, place the agar plate in a 37°C constant temperature incubator for overnight culture and observe the phage plaques.

[0039] (2) Purification of bacteriophage JN06

[0040] The phage enrichment solution that can form clear plaques on the agar plate was diluted 10 times with SM buffer to obtain phage dilution solution. 100 μl of Salmonella SMT (OD 600 =0.5-0.6) and 100 μl of the obtained phage dilution were mixed evenly, placed in a 37°C constant temperature incubator for incubation for 15 min, and then 200 μl of the mixed solution was added to the semi-solid agar medium, mixed evenly and quickly poured into the LB agar plate. After the semi-solid solidified, the plate was placed in a 37°C constant temperature incubator for overnight incubation (12-16 hours), and the plaques were observed and counted.

[0041] Pick a single large and clear plaque on the double-layer plate and add it to 1 mL SM buffer, placed in a 37°C constant temperature incubator for 12 hours, then centrifuged at 4°C, 12000×g for 10 minutes, collected the supernatant and filtered it with a 0.22μm filter, and the liquid obtained after filtration was the phage filtrate; 100μl of phage filtrate and 100μl of host bacteria in the logarithmic phase were added to 10mL of TSB medium, placed at 37°C, 220rpm shaking culture for 5 hours, then centrifuged at 4°C, 12000×g for 10 minutes, collected the supernatant and filtered it with a 0.22μm filter, and the liquid obtained after filtration was the phage stock solution; the phage stock solution was diluted 10 times with SM buffer, and 100μl of phage dilution was taken and mixed with 100μl of host bacteria in the logarithmic growth phase, placed in a 37°C constant temperature incubator for 15 minutes, and then 200μl of the mixed solution was added to the semi-solid agar medium, mixed evenly and quickly poured into the LB agar plate. After the semi-solid solidifies, place the plate in a 37°C constant temperature incubator and culture overnight for 12-16 hours.

[0042] Repeat the above steps until the plaques on the agar plate have the same shape and size, and the purified phage is obtained. The phage is named JN06. The plaques on the double-layer plate are counted, and the titer of the phage can reach up to 10 11 PFU / mL. Figure 1 As shown, the phage forms round clear plaques surrounded by a halo on the double-layer plate.

[0043] (3) Identification of bacteriophage JN06

[0044] ① Electron microscopic morphology of bacteriophage JN06

[0045] Take 20 μl of the purified phage stock solution (the phage titer is 1.5×10 11 PFU / mL) was dropped onto the copper grid, dried at room temperature for 10 min, stained with 2% phosphotungstic acid for 5 min, excess droplets were removed with filter paper strips, dried under incandescent light, and observed and photographed under a transmission electron microscope. The results are shown in the figure. Figure 2 As shown, phage JN06 belongs to the family of long-tailed phages, has a polyhedral head of approximately 50×55 nm, and a tail length of approximately 110 nm.

[0046] ② Whole genome sequencing analysis

[0047] The whole genome of bacteriophage JN06 was extracted using a viral genome extraction kit, and the sample was sent to a sequencing company for whole genome sequencing. Figure 3 As shown, its genome size is 43274bp, with 67 open reading frames (ORFs) and a GC content of 50.27%. It does not contain genes encoding virulence, antibiotic resistance and lysogeny, indicating that bacteriophage JN06 has good biosafety.

[0048] The deposit information of bacteriophage JN06 is as follows:

[0049] Strain name: Salmonella enterica phage JN06;

[0050] Deposit date: November 13, 2024;

[0051] Depository: Guangdong Microbiological Culture Collection Center (GDMCC), Laboratory Building, Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China;

[0052] Deposit number: GDMCC No: 65479-B1.

[0053] Example 2 Determination of lysis spectrum of bacteriophage JN06

[0054] The above test strains were cultured to the logarithmic phase, 100 μl of the logarithmic phase bacterial solution was added to 7 mL of semi-solid culture medium, mixed and quickly poured into LB agar plates, and after the semi-solid solidified, 10 μl of the phage stock solution was dropped on the agar plate, and an equal amount of SM buffer was dropped on another area as a negative control. Incubate in a 37°C constant temperature incubator for 2 hours, and invert the plate for 12-16 hours. Observe the plaques on the agar plate. The appearance of clear empty plaques on the plate indicates that JN06 can lyse the bacteria, which is recorded as "++"; the appearance of fuzzy empty plaques indicates that it can be slightly lysed, which is recorded as "+"; the absence of empty plaques indicates that it cannot be lysed, which is recorded as "-". The results are shown in Table 1. The lysis spectrum of phage JN06 is wide, and it can not only lyse multiple serotypes of Salmonella, but also lyse Escherichia coli.

[0055] Table 1 Lysis range of bacteriophage JN06

[0056]

[0057]

[0058] Note: AMP: ampicillin; AMC: amoxicillin; CTX: cefotaxime; TCY: tetracycline; CEN: gentamicin; NEO: neomycin; FFC: florfenicol; CIP: ciprofloxacin; NA: nalidixic acid; SXT: co-trimoxazole; "-" indicates no resistance. "+++": indicates strong lysis ability, forming completely transparent plaques; "++": indicates strong lysis ability, forming relatively transparent plaques; "+": indicates slightly weak lysis ability, forming fuzzy plaques; "-": indicates no lysis ability.

[0059] Antibiotic resistance refers to the resistance background of the selected strain. For example, the Salmonella Enteritidis strain SMT is resistant to five antibiotics: AMP / AMC / GEN / NEO / NA.

[0060] As can be seen from Table 1, the bacteriophage JN06 of the present invention has a strong lysis ability against two different strains of Salmonella Enteritidis, forming completely transparent plaques.

[0061] The bacteriophage JN06 of the present invention has different lysis performances on 8 different strains of Salmonella Typhimurium, among which ATCC BAA-708, 2100, 1094b, 2112 and 1079b all have strong lysis ability and form completely transparent plaques, while 1054b has stronger lysis ability and forms relatively transparent plaques; ATCC14028 has slightly weaker lysis ability and forms fuzzy plaques, and 2088b has no lysis ability at all.

[0062] The bacteriophage JN06 of the present invention has different lysis abilities to 4 different strains of Salmonella Derby, among which 2042a, 2082b and 2054a have stronger lysis abilities and form brighter plaques; 2042a has no lysis ability.

[0063] The bacteriophage JN06 of the present invention has a strong lysis ability against Salmonella Schwarzengrund 2018, forming completely transparent plaques.

[0064] The bacteriophage JN06 of the present invention has a strong lysis ability against Salmonella Infantis 1073a and forms completely transparent plaques.

[0065] The bacteriophage JN06 of the present invention has different lysis abilities against two strains of enteropathogenic Escherichia coli, among which ATCC7045 has a strong lysis ability and forms completely transparent plaques. CMCCB150071 has a relatively strong lysis ability and forms relatively transparent plaques.

[0066] The bacteriophage JN06 of the present invention exhibits different lysis performances on three strains of Salmonella Agona.

[0067] The bacteriophage JN06 of the present invention exhibits different lysis performances on three strains of Salmonella Anatum, wherein 1084a has a slightly weaker lysis ability and forms fuzzy plaques, 1100 has a stronger lysis ability and forms relatively clear plaques, and 2089b has no lysis ability.

[0068] The bacteriophage JN06 of the present invention has no lysis ability against two strains of Salmonella Heidelberg.

[0069] The bacteriophage JN06 of the present invention has no lysis ability against Salmonella Senftenberg 2057a.

[0070] The bacteriophage JN06 of the present invention has no lysis ability against Salmonella Rissen 2043a.

[0071] The bacteriophage JN06 of the present invention has no lysis ability against Salmonella Sinstorf 2086a.

[0072] The bacteriophage JN06 of the present invention has no lysis ability against Pseudomonas Aeruginosa 9027.

[0073] Example 3 Characterization of bacteriophage JN06

[0074] (1) Optimal multiplicity of infection (MOI) of bacteriophage JN06

[0075] Add 100μl (MOIs were 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, respectively) of bacteriophage JN06 and 100μl of host bacteria (Salmonella SMT) in the logarithmic phase to 10Ml TSB liquid culture medium, incubate at 37°C for 15 minutes, then move to a 37°C constant temperature shaker and shake at 220rpm for 5 hours. Centrifuge at 4°C, 12000×g for 10 minutes, and filter the supernatant with a 0.22μm filter membrane for sterilization. The double-layer agar plate method was used to determine the phage titers obtained at different MOIs. The MOI with the highest phage titer is the optimal multiplicity of infection (MOI). The results are as follows Figure 4 As shown, the optimal infection multiplicity of phage JN06 is 0.001.

[0076] (2) Determination of one-step growth curve

[0077] According to the optimal MOI ratio, add 100μl of phage JN06 and 100μl of host bacteria in the logarithmic phase to a 1.5mL centrifuge tube, incubate at 37℃ for 15min, centrifuge at 4℃, 12000×g for 2min, carefully remove the supernatant, resuspend the precipitate in 20mL TSB liquid culture medium, and culture at 37℃, 220rpm shaking. Take 500μl of culture at 0, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 100, and 120min, centrifuge at 4℃, 12000×g for 30s, aspirate the supernatant, and measure the phage titer using the double-sided plate method. The method for calculating the burst volume is the phage titer at the end of the phage burst / the concentration of bacteria at the beginning of the infection. Figure 5 As shown, the incubation period of bacteriophage JN06 is 5 min, the burst period lasts from 5 min to 80 min, and the burst volume is 125 PFU / cell.

[0078] Example 4 Stability of bacteriophage JN06

[0079] (1) Determination of pH stability

[0080] The SM buffer was adjusted to pH 1-13 with 0.1 mol HCl and NaOH, 100 μl of phage JN06 was added to 900 μl of SM buffer with different pH values, and the mixture was placed in a 37°C constant temperature shaker and cultured at 220 rpm for 1 h. The titer of phage JN06 under different pH conditions was then determined using the double-sided plate method. Figure 6 As shown in Figure 2, JN06 remained relatively stable under pH 3-12 conditions, with a titer of 10 9 PFU / mL or more; at pH 2, about 50% of the phages were inactivated. This shows that JN06 has a high tolerance to acid and alkali

[0081] (2) Determination of temperature stability

[0082] Take 1mL of phage stock solution (titer is 10 9 PFU / mL) was added into a sterilized 2mL centrifuge tube and placed in a constant temperature water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 15min, 30min, and 60min, respectively, and then the phage titer was measured by the double-sided plate method. Figure 7 As shown in the figure, the activity of JN06 remained basically unchanged after being exposed to 30-60℃ for 60min, and was still maintained at 10 9 PFU / mL, at 70℃, the phage activity gradually decreased with the extension of the action time, and the phage activity level dropped to 75% of the original level after 60min, while at 80℃, phage JN06 was completely inactivated. This shows that phage JN06 has good tolerance to temperature and has a wide range of applications.

[0083] Example 5 In vitro antibacterial effect of bacteriophage JN06

[0084] 1 mL of bacteriophage JN06 (MOI of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, respectively) and 1 mL of Salmonella SMT in the logarithmic growth phase were added to 100 mL of TSB liquid culture medium, placed in a 42°C constant temperature shaker for 24 hours, and sampled every 4 hours to measure the reading of the culture medium at OD600nm to monitor the growth of Salmonella SMT. In the positive control group, the bacteriophage was replaced with an equal amount of SM buffer. The experimental results are shown in Figure 2. Figure 8 As shown, the growth of Salmonella SMT was inhibited in all groups with the addition of bacteriophage JN06. When the MOI was 100 and 10, the growth of Salmonella SMT was delayed by nearly 8 hours, indicating that bacteriophage JN06 had a good inhibitory effect on Salmonella SMT.

[0085] Example 6 Therapeutic effect of bacteriophage JN06 on Salmonella infection in chickens

[0086] 1. Experimental animals and management

[0087] 75 one-day-old chicks (three-yellow chickens) were randomly divided into a control group, a phage group, a challenge group, and a treatment group (low-dose treatment group and high-dose treatment group), with 15 chicks in each group. Each group was raised independently and fed a basic diet without antibiotics. After 3 days of feeding, each chicken in the challenge group and the treatment group was orally administered 500 μl 10 10 CFU / mL of Enteritidis SMT, each chicken in the control group was orally administered with an equal volume of normal saline, and each chicken in the phage group was orally administered with 500 μl 10 11 PFU / mL JN06. After 6 hours, each chicken in the low-dose and high-dose treatment groups was gavaged with a titer of 10 7 PFU / mL and 10 11 PFU / mL JN06, treatment for 3 consecutive days, and observation for 4 days after the end of treatment.

[0088] 2. Measurement indicators and methods

[0089] On the 1st, 3rd and 7th days of the experiment, anal swab sampling and mixed sampling on the fecal receiving plate were used to detect the levels of Salmonella and bacteriophage in the feces of the experimental chickens, respectively. At the same time, 5 chickens were randomly selected from each group for dissection, and the duodenum was collected to observe the pathological changes in the intestine; serum was collected to determine the levels of IFN-γ, IL-1β, IL-6 and IL-10.

[0090] 3. Test results

[0091] 3.1. Effect of bacteriophage on the elimination of Salmonella in chickens

[0092] Depend on Fig. 9 A shows that the content of Salmonella in feces of the phage treatment group (Salmonella+MOI0.001, Salmonella+MOI10) was significantly lower than that of the challenge group (Salmonella), especially the high-dose treatment group (Salmonella+MOI10). In the early stage of treatment, the detection amount of Salmonella in the low-dose treatment group (Salmonella+MOI0.001) and the high-dose treatment group was 1.96log and 3.26log less than that of the challenge group, respectively; after 3 consecutive days of treatment, the detection amount of Salmonella in the low-dose treatment group was 3.0log CFU / mL, which was 2.24log less than the 5.24log CFU / mL of the challenge group, while no Salmonella was detected in the fecal samples of the high-dose treatment group. After the phage was discontinued, the Salmonella in the chickens no longer decreased further.

[0093] Depend on Fig. 9From B, we can see that 5.0logPFU / mL of phages can be detected in the phage group from the beginning of treatment to 3 days after the end of treatment, indicating that the phages can stably exist in the body of the chicks; at the same time, it was found that the phage content in the two treatment groups during the treatment period was significantly higher than that in the phage group, indicating that the phages can further grow and reproduce in the chicken body.

[0094] 3.2. Content of inflammatory cytokines in serum

[0095] Depend on Fig.10 It can be seen that the levels of cytokines IFN-γ, IL-6 and IL-1β increased after the chicks were infected with Salmonella, among which the high level of cytokine IFN-γ continued until the 7th day after infection, while the IFN-γ in the phage treatment group was significantly lower than that in the challenge group (p<0.05), and there was no significant difference with the control group (p>0.05). This shows that the phage JN06 provided by the present invention can reduce the inflammatory response caused by Salmonella infection to a certain extent.

[0096] 3.3 Histopathological changes of the duodenum

[0097] Chickens infected with Salmonella can cause duodenal lesions. According to the results of histopathological sections, ( Fig.11 ), the villi of the duodenum in the challenge group became shorter and their integrity was destroyed; with the increase of the course of the disease, the degree of lesions worsened, while the phage treatment group alleviated the relevant pathological changes to a certain extent, especially in the high-dose phage treatment group. On the third day, the villi of the duodenum remained intact and arranged in an orderly manner, with no significant difference from the control group. This shows that phage JN06 can protect the duodenum from Salmonella infection to a certain extent.

[0098] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A salmonella enterica phage JN06, characterized in that: The Salmonella enterica phage JN06 has a deposit number of GDMCC No: 65479-B1 and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 13, 2024.

2. The Salmonella enterica phage JN06 according to claim 1 has the purpose of simultaneously lysing Salmonella and Escherichia coli.

3. The use according to claim 2, characterized in that The salmonella refers to at least one of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Derby, Salmonella Schwarzeneggeron, Salmonella Infantis, Salmonella Agona, and Salmonella Anuta; the Escherichia coli is enteropathogenic Escherichia coli.

4. Use of the Salmonella enterica phage JN06 according to claim 1 in the preparation of an antibacterial agent.

5. The use according to claim 4, characterized in that The use is to prepare an antibacterial agent.

6. The use according to claim 4, characterized in that The antibacterial agent refers to the agent that inhibits Salmonella and / or Escherichia coli.

7. The use according to claim 6, characterized in that The salmonella refers to at least one of Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Derby, Salmonella Schwarzeneggeron, Salmonella Infantis, Salmonella Agona, and Salmonella Anuta; the Escherichia coli is enteropathogenic Escherichia coli.

8. An antibacterial agent, characterized in that Containing the Salmonella enterica phage JN06 according to claim 1.

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