Salmonella bacteriophage with cross-species lysis capability, bacteriophage composition and application of salmonella bacteriophage

Abstract

The invention belongs to the technical field of bacteriophages, and discloses a salmonella bacteriophage with cross-species lysis capability, a bacteriophage composition and application of the salmonella bacteriophage, the salmonella bacteriophage is named as salmonella bacteriophage PC553, the preservation number of the salmonella bacteriophage PC553 is CGMCC No.46166, and the preservation The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on August 16, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacteriophage is wide in splitting spectrum and high in titer, shows cross-species strong splitting energy to escherichia coli, can be prepared into drugs, environment disinfectants, feeds, water additives and the like for preventing and treating pigeon salmonellosis and / or colibacillosis for application, effectively prevents and treats spreading of salmonellosis and avoids the problem of secondary infection of escherichia coli, and has a good application prospect in preventing and treating pigeon salmonellosis and / or colibacillosis. The feed additive has the effects of effectively reducing the death and culling rate of pigeons, reducing the positive rate of salmonellosis antibodies, improving the production performance index of pigeons and the like.

Description

Technical Field

[0001] The present invention relates to the field of bacteriophage technology, and in particular to a Salmonella bacteriophage with cross-species lysis ability, a bacteriophage composition and applications thereof. Background Art

[0002] The pigeon industry is the fourth largest poultry industry after chickens, ducks and geese. It is becoming a new growth point for China's poultry industry and has a significant upward trend. At present, my country's pigeon breeding and sales volume rank first in the world, of which the breeding volume accounts for about 80% of the world's total. The pigeon industry has entered a stage of large-scale, intensive and semi-automated production, but there is still a lot of room for improvement in breeding level. Pigeon meat, as a low-fat, high-protein meat, is favored by more and more consumers. With the development of my country's economy and the continuous improvement of people's living standards, the market demand for pigeons in my country is also increasing year by year, which has led to a steady increase in the number of pigeons raised across the country, and has also led to the rapid development of my country's pigeon industry.

[0003] The meat pigeon breeding process requires little investment, has a short cycle and high returns, and is a breeding industry with great development prospects. However, there is still a large gap in the breeding of meat pigeons in terms of variety breeding, breeding technology, precision feeding, disease prevention and control, processing and circulation, which restricts the transformation and upgrading and high-quality development of my country's pigeon industry. Diseases caused by Salmonella infection in the process of meat pigeon breeding have brought huge losses to the pigeon industry. The feces discharged by sick pigeons infected with Salmonella contain a large number of pathogens, which can contaminate feed, bedding, drinking water and utensils, causing young pigeons to be infected after contact. The disease may also be spread through mating, transfer and other channels. After the young pigeons recover from the infection, they can carry bacteria in their bodies for a long time. One-third of the fertilized eggs produced by pigeons with bacteria will be contaminated by pathogens, which plays an important role in the spread of the disease. The yolk contains a large number of pathogens, which can not only be passed on to the offspring pigeons, causing them to become sick and become a source of infection in the same group, but also may contaminate the incubator and pass it to the same or next batch of pigeons through eggshells, feathers, etc., thereby spreading the disease widely. The key to preventing the occurrence of the disease is to prevent the introduction of pathogens and eliminate carriers and chronic patients in the group. At the same time, strict hygiene, disinfection and isolation systems need to be implemented.

[0004] Salmonella phages have the characteristics of high lysis rate, high titer and high stability. They can target and kill Salmonella in the prevention and treatment of Salmonella in pigeons, as well as the purification of Salmonella. After Salmonella phages target and kill Salmonella in the intestines, they can promote the occupation of probiotics, thereby improving the intestinal microbial environment to a certain extent, and can promote the intestinal health and production performance of pigeons. At the same time, when phages remain in the environment, they can still kill Salmonella in the environment. Therefore, phages have great potential in the prevention and control of Salmonella in pigeons.

[0005] However, in clinical breeding sites, meat pigeons are often infected with E. coli after being infected with Salmonella, causing intestinal flora disorder, diarrhea, pericarditis, hepatitis, etc. Therefore, it is of great significance to strengthen the control of E. coli infection while preventing and controlling Salmonella infection in meat pigeons. However, there are very few phage resources that can simultaneously lyse Salmonella and E. coli across species, which cannot meet the needs of application.

[0006] Therefore, the prior art needs to be further improved. Summary of the invention

[0007] In view of the above problems, the present invention provides a self-screened Salmonella phage with cross-species lysis ability, which can lyse Escherichia coli and can be used for the lysis of Salmonella and Escherichia coli and the prevention and treatment of diseases caused by such infections. The phage can be prepared into drugs, environmental disinfectants, feed and water additives, etc. for preventing and treating Salmonella and Escherichia coli infections for application. While solving the problem of pigeon-source Salmonella and Escherichia coli infections, the phage can be added to water and feed for a long time to achieve the effect of purifying salmonellosis in the farm, and can also improve the quality and production performance of meat pigeons.

[0008] To solve the above problems, the technical solutions of this application are as follows: In a first aspect, the present application further provides a Salmonella phage having cross-species lysis capability, wherein the Salmonella phage is named as Salmonella bacteriophage PC553, and its deposit number is CGMCC No.46166.

[0009] The phage was deposited at the General Microbiology Center of the China Culture Collection Administration on August 16, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Electron microscopy showed that the phage PC553 had a polyhedral head structure and a non-contractile tail. The head was 60-63 nm wide and 65-68 nm long, and the tail was about 123-125 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PC553 of the present application conforms to the characteristics of the long-tailed bacteriophage family and belongs to the long-tailed phage.

[0011] In the present application, the bacteriophage PC553 includes mutants with a homology higher than 98% or 99% and substantially the same bactericidal activity after point mutation, deletion mutation or addition mutation. Since phages are very susceptible to mutation during replication, mutants of the above phages are also within the scope of the present application. The sequence of phage PC553 can be obtained by sequencing the biological material deposited according to the present invention by a known method. For those skilled in the art, it does not require creative work to screen out mutants with extremely similar properties according to the phage provided by the present invention.

[0012] In a second aspect, the present application further provides a phage composition, which includes the aforementioned Salmonella phage PC553 and other phages. In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in the lysis spectrum of different phages, and complement each other's advantages, the above-mentioned pigeon-derived Salmonella phage PC553 and other phages can be used in combination.

[0013] Optionally, the other bacteriophages are other existing Salmonella phages, such as one or more of phage SP4, phage SPP11, and phage SP8; wherein the deposit number of phage SP4 is CGMCC No. 14332; the deposit number of phage SPP11 is CGMCC No. 18868; and the deposit number of phage SP8 is CGMCC No. 45256. In addition, other bacteriophages also include other existing Escherichia coli phages.

[0014] By using the above-mentioned phages in combination, the lysis spectrum is broadened, and different Salmonella and Escherichia coli in the pigeon farm environment are killed, so as to better prevent and treat salmonellosis and / or Escherichia coli disease in pigeons.

[0015] Among them, the specific information of bacteriophage SP4 can be found in the patent publication number CN 108359644 B, the specific information of bacteriophage SPP11 can be found in the patent publication number CN 111254121 B, and the specific information of bacteriophage SP8 can be found in the patent publication number CN 116286671A.

[0016] Specifically, bacteriophage SPP11 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, on November 4, 2019, with a deposit number of CGMCC No. 18868. For more information, see the patent announcement number CN 111254121 B.

[0017] Bacteriophage SP4, the deposit number is CGMCC No. 14332, the depositor is the General Microbiology Center of China National Microbiological Culture Collection Administration, the deposit date is July 27, 2017, for specific information, see the patent announcement number CN 108359644 B.

[0018] Bacteriophage SP8, the deposit number is CGMCC No.45256, the depositor is the General Microbiology Center of China National Microbiological Culture Collection Administration, the deposit date is August 17, 2022, for specific information, see the patent announcement number CN 116286671A.

[0019] In addition, the above-mentioned bacteriophage PC553 can also be combined with other different types of bacteriophages (inhibiting different pathogens that cause the same disease) for the prevention and treatment of the same type of disease, such as acute sepsis caused by mixed infection of different pathogens.

[0020] In a third aspect, the present application also provides the use of the aforementioned Salmonella phage or the aforementioned phage composition in the preparation of drugs, feed additives, environmental disinfectants, and food preservatives for preventing and treating pigeon-derived Salmonella and / or pigeon-derived Escherichia coli infections.

[0021] The control includes prevention and treatment. The term "prevention" herein refers to all actions including suppressing or delaying the disease by administering the composition. The term "treatment" herein refers to all actions including improving or ameliorating the disease by administering the composition.

[0022] Preferably, the disease caused by the pigeon-derived Salmonella infection is: pigeon salmonellosis, also known as pigeon paratyphoid, pullorum salmonellosis. According to its symptoms and pathological changes, pigeon salmonellosis is divided into four types: intestinal type, articular type, visceral type, and neurological type.

[0023] The diseases caused by the pigeon-derived E. coli infection are: E. coli sepsis, E. coli peritonitis, E. coli omphalitis, E. coli granuloma, salpingitis, etc.

[0024] In a fourth aspect, a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Salmonella phage or the aforementioned phage composition. Preferably, the phage pharmaceutical preparation also includes other antibacterial or bactericidal active ingredients; the pharmaceutical preparation is in the form of an oral dosage form, an external dosage form, or a parenteral dosage form.

[0025] The application method of the bacteriophage drug preparation is: adding the bacteriophage or its composition as a therapeutic drug to drinking water or feed, or administering it orally, subcutaneously, or intramuscularly to pigeons. The above methods can prevent and treat salmonellosis, improve survival rate, etc.

[0026] Optionally, the phage pharmaceutical preparation also includes a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" used herein refers to a carrier or diluent that does not cause significant stimulation to an organism and does not eliminate the biological activity and characteristics of the active component administered. In order to prepare the pharmaceutical composition as a liquid preparation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, various types of culture media, etc. They can be used alone or in any combination thereof. As needed, other conventional additives, such as antioxidants, buffers and antibacterial agents, etc., can be added. When it is combined with a diluent, a dispersant, a surfactant, an adhesive and / or a lubricant, the composition of the present invention can also be prepared into injection and oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized agents.

[0027] In a fifth aspect, the present application further provides a feed additive or drinking water additive, the active ingredient of which includes the aforementioned Salmonella phage PC533 or the aforementioned phage composition. Preferably, the concentration of the phage is 10 8 PFU / mL or above.

[0028] The drinking water additive or feed additive is added into water or mixed with feed and then fed to pigeons, thereby disinfecting and sterilizing the drinking water and feed in the pigeon farm, avoiding the spread of salmonellosis from the source, and effectively preventing and controlling salmonellosis.

[0029] Optionally, the drinking water additive or feed additive also contains other active ingredients for inhibiting or eliminating bacteria in water; the drinking water additive and feed additive are in the form of liquid dosage form, powder dosage form or solid dosage form, but are not limited to the above three dosage forms.

[0030] In a sixth aspect, the present application further provides an environmental disinfectant, the active ingredient of which includes the aforementioned Salmonella phage or the aforementioned phage composition. Preferably, the concentration of the phage is 10 8 PFU / mL or above.

[0031] Optionally, the environmental disinfectant also contains other active ingredients for inhibiting or eliminating bacteria in the pigeon farm environment; preferably, the environment to which the environmental disinfectant can be applied includes feed, water and breeding environment, and the breeding environment includes pigeon houses, pigeon cages, waterers, troughs and other feeding tools, feces and bedding.

[0032] In the seventh aspect, the present application also provides the use of the above-mentioned environmental disinfectant in the disinfection of pigeon farm environment, wherein the application method is: applying the disinfectant in pigeon slaughterhouses, pigeon product processing workshops and utensils, and pigeon breeding environments to prevent pigeon-derived Salmonella contamination in the environment. The breeding environment includes pigeon houses, pigeon cages, drinking fountains, feeding troughs and other feeding tools, feces and bedding, etc.

[0033] The application method includes but is not limited to disinfecting and decontaminating the water distribution system, breeding facilities, feeding equipment or other environmental surfaces of the pigeon farm in the form of liquid immersion, spraying, and combined use with an aqueous carrier, and disinfecting and preserving feed. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it will not cause harm to humans and poultry.

[0034] In an eighth aspect, the present application further provides a food preservative, which includes the aforementioned Salmonella phage or the aforementioned phage composition. The food preservative can be sprayed on the surface of pigeon meat, or the pigeon meat product can be soaked in a solvent containing the food preservative, so as to inhibit the reproduction of Salmonella and / or Salmonella in the pigeon meat product and achieve a preservation effect.

[0035] The present invention has the following beneficial effects: 1. Aiming at the problems of salmonella infection and Escherichia coli infection in pigeons, the present invention provides a salmonella phage PC553 with cross-species lysis ability, which has the advantages of wide lysis spectrum and high titer. More importantly, the phage also shows the characteristics of strong cross-species lysis ability and wide lysis spectrum for Escherichia coli. Based on the above-mentioned antibacterial characteristics, the phage PC553 can be prepared into drugs, environmental disinfectants, feed and water additives for preventing and treating salmonellosis and / or colibacillosis in pigeons for application, which not only effectively prevents the spread of salmonellosis and avoids the problem of secondary infection of Escherichia coli, but also has the effects of effectively reducing the mortality rate of pigeons, reducing the positive rate of salmonellosis antibodies, and improving the production performance index of pigeons. Long-term use of the phage product can achieve the effect of purifying salmonella in pigeon farms.

[0036] 2. The bacteriophage PC553 of the present invention is obtained from nature and is easy to be industrially produced. The medicine or disinfectant prepared from the bacteriophage can not only reduce costs but also has the advantage of being green and environmentally friendly.

[0037] 3. When the bacteriophage PC553 of the present application is used as an environmental disinfectant, it can significantly reduce the content of Salmonella and E. coli in the pigeon farm environment, thereby reducing the infection rate of Salmonella and E. coli in the pigeon flock, and can be promoted and applied as a new type of biological environmental disinfectant. The bacteriophage PC553 can also be used as a feed additive or drinking water additive to disinfect and sterilize the drinking water and feed in the pigeon farm, avoid the spread of salmonellosis from the source, and effectively prevent and treat salmonellosis. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The results of the temperature stability test of Salmonella phage PC553; Figure 2 The results of the acid-base stability test of Salmonella phage PC553; Figure 3 This is the one-step growth curve test result of Salmonella phage PC553; Figure 4 This is an electron micrograph of Salmonella phage PC533; Figure 5 This is a graph showing changes in body weight during the preventive effect of Salmonella phage PC553 on Salmonella infection (administered by gavage with mixed feed) in Example 7; Figure 6 This is a graph showing the change in the number of pigeon chicks hatched during the preventive effect of Salmonella phage PC553 on pigeon Salmonella infection in Example 8 (drinking water use). DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0039] Example 1 Isolation and purification of a Salmonella phage with cross-species lysis ability 1. Experimental methods (1) Resuscitation culture of host bacteria and preparation of proliferation medium Select the pigeon-derived Salmonella and pigeon-derived Escherichia coli preserved in this laboratory, use a sterilized inoculum stick to dip the freezing solution and streak on the SS culture medium for recovery, and culture it in a constant temperature incubator at 37℃ for 18-24h to obtain a single colony; pick a single colony and inoculate it in 5mL of NB broth, and culture it at 37℃ with shaking at 170rpm / min for 16h to obtain fresh Salmonella and Escherichia coli liquids.

[0040] (2) Phage isolation and purification Take an appropriate amount of pigeon droppings and other samples from Shandong area into a conical bottle, add an appropriate amount of broth culture medium, add pigeon-derived Salmonella liquid, put the mixed liquid into 37℃ 170rpm / min shaking culture for 12h, centrifuge at 11000rpm for 5min, and then filter with a 0.22μm sterile microporous filter membrane to obtain phage proliferation liquid; The phage stock solution was diluted 10-fold, and appropriate gradient phage dilutions were taken and mixed with pigeon-derived Salmonella in a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was placed on the upper agar (agar concentration was 0.7%), and after mixing, it was quickly poured onto the lower agar (agar concentration was 1.5%) plate, shaken and placed flat until the culture medium solidified, and placed in a 37°C incubator for inverted culture for 4 to 6 hours to obtain a double-layer plate with plaque formation.

[0041] A single phage plaque was removed from the above plate and co-cultured with multiple strains of pigeon Escherichia coli in 5 mL of NB broth medium at 37°C and 170 rpm / min for 12 hours to obtain a co-culture proliferation liquid. The co-culture proliferation liquid was used to perform double-layer plate plaque verification on pigeon Escherichia coli to obtain a phage that can lyse Salmonella and Escherichia coli across species.

[0042] Pick a single plaque from the double-layer agar medium that formed plaques in the previous step, and place it in 1mL of NB broth and culture it in a constant temperature shaker at 37℃, 170 rpm for about 30min to obtain a phage extract. Take the phage extract and the corresponding plaque-forming Salmonella (hereinafter referred to as the host bacteria) proliferation solution and mix them evenly in a ratio of 1:1 (incubate at 37℃ for 5min), draw 200μL and place it on the upper agar, mix it and quickly pour it on the lower agar plate, shake it and place it flat until the culture medium solidifies, place it in a 37℃ incubator and culture it upside down for 4~6h, and then obtain a double-layer plate with plaques again. Use sterilized tweezers to pick a single plaque from the double-layer culture medium where plaques are formed and place it in 1mL of LB broth, and culture it in a constant temperature shaker at 37℃, 170 rpm for about 30min to obtain a phage extract. Repeat the above steps 3 times to obtain the purified cross-species lysed phage extract.

[0043] (3) Phage titer determination Take an equal amount of purified phage extract and host bacteria proliferation liquid in 5 mL liquid NB medium, and culture at 37°C, 170 rpm with shaking until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 min, take the supernatant, and filter it with a 0.22 μm sterile microporous filter membrane to obtain phage proliferation liquid, and use the double-layer plate method to determine the titer of the newly isolated phage.

[0044] (4) Phage lysis spectrum determination Twenty pigeon-derived Escherichia coli strains (G. E. coli 2401-G. E. coli 2420) and 20 strains of pigeon-derived Salmonella (G. Salmonella 2401 - G. Salmonella 2420), the lysis rate of the above-isolated and purified phages was determined by the double-layer plate method, and the phages with higher lysis rate were screened.

[0045] 2. Experimental results: As shown in the results in Table 1, according to the above experimental method, five strains of Salmonella phage with cross-species lysis ability were screened using the isolates, and they were numbered PC551-PC555 respectively. All five strains of Salmonella phage formed clear plaques on the double-layer agar medium plate, with no halo around them, and the edges were clearly visible, with a diameter of about 0.5mm~1mm. The titers of these five phages were between 1.00×10 9 PFU / mL-3.26×10 10 PFU / mL, the lysis rate of Escherichia coli was 20%~70%, and the lysis rate of pigeon Salmonella was 80%~90%. According to the titer and the lysis rate of the two host bacteria, the phage PC553 with higher titer and lysis rate was screened out, and the phage was used as the subsequent experimental object.

[0046] Table 1 The titers of five Salmonella phages with cross-species lysis ability and their lysis rates against Escherichia coli (PFU / mL)

[0047] Example 2 Determination of biological characteristics of Salmonella phage PC553 (1) Experimental methods 5.0×10 9 PFU / mL Salmonella phage PC553 proliferation solution was incubated at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃, with three parallel samples at each temperature, and kept warm for 20 min, 40 min, and 60 min respectively. After the reaction was over, the samples were immediately cooled in an ice bath, and then the phage titer at different temperatures was detected by the double-layer plate method. The thermal stability curve of PC553 phage was drawn with temperature as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0048] (2) Experimental results and analysis The results are as follows Figure 1 It can be seen that phage PC553 still maintained its original activity after being exposed to 60°C for 60 min; after being exposed to 80°C for 20 min, the phage still contained 3.42×10 3PFU / mL active phage. It can be seen that the temperature stability of Salmonella phage PC553 is good.

[0049] 2. Acid and alkali stability (1) Experimental methods Take three test tubes containing 4.5 mL NB broth medium with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) respectively, put the test tubes containing broth with different pH values ​​in a 37℃ water bath for 30 min, and after the broth temperature in the test tube reaches a stable state, add 0.5 mL 5.0×10 9 PFU / mL of phage, shake and mix thoroughly, and act in a 37℃ constant temperature water bath for 1h, 2h, and 3h. After the specified time, add an appropriate amount of NaOH or HCl to the mixed solution containing phage to adjust the pH to about 7.0 to stop the effect of acid and alkali on phage. Take each stop solution and use the double-layer plate method to determine the change of phage titer at different pH values ​​and different action times. Draw the phage acid-base stability curve with the lg value of phage titer as the vertical axis and the pH value as the horizontal axis.

[0050] (2) Experimental results and analysis The results are as follows Figure 2 It can be seen that the titer of bacteriophage PC553 can still be maintained at 10 in the pH range of 5.0-10.0. 9 PFU / mL, the activity is relatively stable; at pH 4.0 for 3h, the potency is 10 3 PFU / mL; at pH 11.0 for 1h, the titer is 10 5 PFU / mL; It can be seen that bacteriophage PC553 has strong stability in a wide pH range and can resist a certain range of strong acid and strong alkali environments.

[0051] 3. One-step growth curve (1) Experimental methods Adjust the phage titer and bacterial concentration to an infection multiplicity of 10, take 1 mL of the adjusted phage proliferation solution and bacterial solution, mix thoroughly (start timing at this time), incubate at 37°C for 5 minutes, centrifuge at 13000 rpm for 30 seconds, discard the supernatant, wash once with 5 mL NB broth, centrifuge and discard the supernatant. Add 5 mL of 37°C preheated NB broth medium to fully suspend the precipitate, and quickly place it at 37°C 170 rpm for shaking culture. Starting from time 0, take out 200 μL each time, centrifuge at 11000 rpm for 5 minutes, take the supernatant, and use the double-layer plate method to determine the phage titer at different time points. Do 3 replicates for each gradient and calculate the average value. With the infection time as the horizontal axis and the titer of the phage in the infection system as the vertical axis, draw a one-step growth curve to obtain the incubation period and outbreak period of phage PC553, and calculate the outbreak volume.

[0052] Outbreak volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak (2) Experimental results and analysis The results are as follows Figure 3 It can be seen that after the phage PC553 infects the host bacteria, the incubation period is about 0-30 minutes. Within 40-100 minutes after the phage infects the host bacteria, the number of phages increases sharply, and the titer growth tends to be stable after 100 minutes, at which time the titer can reach 10 9 PFU / mL, it can be seen that the burst period of bacteriophage PC553 is about 60 min, and the burst volume is 106 PFU / cell.

[0053] 4. Phage electron microscopy observation (1) Experimental methods Take 20 μL of phage sample and drop it on a copper mesh with a carbon-coated film. Allow it to precipitate naturally for 15 min, dry it with filter paper, and then stain it with 2% (W / V) phosphotungstic acid (PTA) for 1-2 min. Drain it with filter paper, and observe and photograph it under a transmission electron microscope after it is dried.

[0054] (2) Experimental results and analysis like Figure 4 As shown, bacteriophage PC553 has a polyhedral head structure and a non-contractile tail. The head is 60-63 nm wide and 65-68 nm long, and the tail is about 123-125 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the bacteriophage PC553 of the present application conforms to the characteristics of the long-tailed bacteriophage family and belongs to the long-tailed phage.

[0055] Example 3 Determination of phage lysis spectrum 1. Experimental methods (1) The 40 strains of Salmonella used in patent CN116286671A (as shown in Table 2 below) were selected for lysis spectrum determination, and the differences in lysis rates of SP8, SP4, SPP11 and PC553 were compared.

[0056] (2) Select 40 recently isolated pigeon-derived Salmonella strains (as shown in Table 3) and 20 pigeon-derived Escherichia coli strains (as shown in Table 4) and streak them on SS plates for recovery. Then, pick a single colony for proliferation and culture to obtain fresh bacterial liquid. Prepare fresh phage proliferation liquid PC533 and 40 pigeon-derived Salmonella strains and 20 pigeon-derived Escherichia coli liquids and use the double-layer plate method to detect the phage lysis spectrum.

[0057] At the same time, the phage SP4 in the patent with publication number CN108359644B, the phage SPP11 in the patent with publication number CN111254121B and the phage SP8 in the patent with publication number CN116286671A were used to measure the lysis spectra of the above 40 strains of pigeon-derived Salmonella and 20 strains of pigeon-derived Escherichia coli, and the lysis performance of these three different Salmonella phages was compared with that of phage PC533.

[0058] 2. Experimental results and analysis (1) Comparative analysis of the lysis effects of four bacteriophages on 40 strains of Salmonella in the patent The results are shown in Table 2. For the 40 strains of Salmonella used in patent CN116286671A, PC553 and SP8 can lyse 35 strains, with a lysis rate of 87.5%; while SPP11 and SP4 can lyse 30 strains, with a lysis rate of 75%.

[0059] (2) Comparative analysis of the lysis effects of four phages on 40 strains of pigeon-derived Salmonella and 20 strains of Escherichia coli Salmonella phages SP8, SP4, SPP11 and PC553 can lyse 34, 30, 31 and 32 of the 40 strains of pigeon-derived Salmonella in Table 3, respectively, and their lysis rates are 85%, 75%, 77.5% and 80%, respectively.

[0060] However, bacteriophages SP8, SP4, and SPP11 did not lyse the 20 strains of pigeon-derived E. coli in Table 4, while PC553 lysed 15 of the 20 strains of pigeon-derived E. coli, with a lysis rate of 75%.

[0061] In summary, the lysis rates of Salmonella phage PC553 for the 40 strains of Salmonella in Table 2 and the 40 strains of pigeon-derived Salmonella in Table 3 were all 80% or above, and it had a higher lysis rate (75%) for the 20 strains of pigeon-derived Escherichia coli. The results showed that phage PC553 has a wide lysis spectrum and excellent cross-species lysis ability of Escherichia coli, and has great potential in the prevention and control of Salmonella and Escherichia coli infections in clinical sites.

[0062] Table 2 Lysis spectra of 4 phages against 40 strains of Salmonella in the patent

[0063] Table 3 Lysis spectrum of 40 strains of pigeon-derived Salmonella by 4 phages

[0064] Table 4 Lysis spectra of 4 phages against 20 strains of pigeon Escherichia coli

[0065] Example 4 Whole genome analysis of Salmonella phage PC553 The genome of bacteriophage PC553 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows: (1) The PC553 genome is 43834 kb in length, with a G+C content of 49.50%, and the base contents of C, G, A, and T being 24.71%, 24.79%, 25.20%, and 25.30%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 67 open reading frames (ORFs) with an average OFR length of 654 bp, of which 26 ORFs had known functions and 41 ORFs were hypothetical protein expression sequences. Among these 67 open reading frames (ORFs), 16 structural proteins were found, mainly including the structure and packaging proteins of the phage (head protein, tail protein, tape measure protein, tail fiber protein, neck protein, capsid protein, terminase large subunit and terminase small subunit, etc.), phage lysis-related proteins (lyase, perforin), and proteins related to DNA replication and modification (restriction endonucleases, DNA binding proteins, DNA polymerases, HNH endonucleases, and recombination proteins, etc.). At the same time, among the 67 ORFs, 62 start codons are ATG, 3 start codons are GTG, and 2 start codons are TTG. The software tRNAscan-SE analysis showed that the genome does not contain tRNA genes. The online tool CGE server analysis showed that the genome does not contain drug resistance genes and virulence genes. The PHASTEST analysis showed that the genome does not contain lysogeny-related genes.

[0066] (2) In the genome of bacteriophage PC553: the DNA polymerase protein gene sequence is shown in sequence 1 in the sequence list, and its protein sequence is shown in sequence 2; the tail fiber protein gene sequence related to bacteriophage host recognition is shown in sequence 3 in the sequence list, and its protein sequence is shown in sequence 4; the highly conserved terminase large subunit protein gene sequence is shown in sequence 5 in the sequence list, and its protein sequence is shown in sequence 6; the endolysin protein gene sequence related to cleavage ability is shown in sequence 7 in the sequence list, and its protein sequence is shown in sequence 8. For relevant information, see Table 1 below.

[0067] Table 5 Gene sequence information

[0068] Example 5: Safety of Salmonella phage PC553 for use in pigeons 1. Experimental methods 90 healthy pigeons were randomly divided into three groups, 30 in each group, namely, gavage group, drinking water group, injection group and control group. Each pigeon was given 5×10 PC553 9 PFU were placed in isolators for breeding; the squabs were raised until 28 days old, and their body weight, mental state, and autopsy were observed to observe lesions in the heart, liver, spleen, lung, kidney, brain, and intestine.

[0069] 2. Experimental results and analysis There was no significant difference in weight gain among the gavage group, drinking water group, injection group and control group. The pigeons were in a normal mental state and feeding during the feeding process, and no disease or toxicity symptoms were observed. The tissues, organs and intestines were normal after autopsy. This shows that the use of Salmonella phage according to this method is highly safe.

[0070] Example 6 Therapeutic effect of Salmonella phage PC553 on pigeons infected with Salmonella 1. Experimental methods 120 healthy squabs were selected and randomly divided into 4 groups, 30 in each group, namely, gavage group, drinking water group, positive control group and negative control group; they were raised in isolators respectively.

[0071] Among them, the gavage group, drinking water group, and positive control group were given 5×10 6 CFU Salmonella liquid, the negative control group was orally administered with an equal amount of sterile saline. The gavage group was evenly mixed with Salmonella phage in the feed at a dosage of 20 mL / ton of feed (5×10 8 PFU / pigeon); for the drinking water group, Salmonella phage was evenly mixed into the drinking water at 10 mL / ton (5×108 PFU / pigeon), the phages in the gavage group and the drinking water group were used for 3 consecutive days, and 5 days was discontinued as one course of treatment, and 3 courses of treatment were used; the positive control group and the negative control group did not use phages. The pigeons were raised to 28 days, and the fecal Salmonella positive rate and mortality rate of the pigeons in each group were collected. After the experiment, all chickens were autopsied to observe the Salmonella infection in the liver, and the positive rate of Salmonella infection in the liver was counted.

[0072] 2. Experimental results and analysis The results are shown in Table 6. Compared with the positive control group, the oral phage group and the drinking water phage group can significantly reduce the Salmonella infection. The positive rates of Salmonella in the feces of the two groups were reduced by 66.67% and 70%, respectively. The mortality rates of the oral phage group and the drinking water phage group were reduced by 53.33% and 50%, respectively. Compared with the positive control group, the positive rates of Salmonella in the liver of the oral phage group and the drinking water phage group were reduced by 76.66% and 80%. The results show that bacteriophage PC553 can effectively treat salmonellosis in pigeons, and both oral and drinking water methods can be used.

[0073] Table 6 Statistical table of phage treatment of pigeon Salmonella infection

[0074] Example 7 Preventive effect of Salmonella phage PC553 on mixed infection of Salmonella and Escherichia coli 1. Experimental methods A pigeon farm in Shandong was selected, and the whole process was fed by gavage. It is understood that the pigeons in the farm were seriously infected with Salmonella and E. coli. In this embodiment, Salmonella phage PC553 was used to prevent and control Salmonella and E. coli in the pigeon flock by gavage.

[0075] The specific experimental method is as follows: 2300 pigeons were used in the experimental group and the control group respectively. The Salmonella phage was evenly mixed into the feed at 20 mL / ton of feed (5×10 8 PFU / pigeon), phage was used for 3 consecutive days, and 5 days was discontinued as a course of treatment, and 3 courses of treatment were used. The control group did not use phage, and the pigeons were raised until they were ready for slaughter. During this period, the drugs were used normally according to other drug use procedures in the farm. During the collection period, 200 feces were randomly collected to test the positive rate of Salmonella and body weight, and the average daily number of deaths and the detection rate of Salmonella and Escherichia coli in the livers of dead pigeons were counted.

[0076] 2. Experimental results and analysis Depend on Figure 5It can be seen that the average weight of pigeons in the experimental group (531.49g / pigeon) is significantly higher than the average weight of the control group (495.48g / pigeon), and from the box plot, it can be seen that the dispersion of the weight of pigeons in the experimental group is more concentrated than that in the control group, indicating that the uniformity of the weight of the experimental group is better than that of the control group, and there is a significant difference.

[0077] From the results in Table 7, we can see that the positive rate of Salmonella in feces of the experimental group was 9.5% lower than that of the control group, the average daily number of deaths in the experimental group was 10 less than that of the control group, which was half of that in the control group, the detection rate of Salmonella in dead pigeons in the experimental group was 22.33% lower than that in the control group, and the detection rate of Escherichia coli in dead pigeons in the experimental group was 34.66% lower than that in the control group. The above results show that the use of bacteriophage PC553 can effectively prevent and control the infection of Salmonella and Escherichia coli in meat pigeons, and effectively improve the weight gain and uniformity of pigeons.

[0078] Table 7 Statistics of bacteriophage treatment of pigeon Salmonella infection

[0079] Example 8 Preventive Effect of Salmonella Phage PC553 on Pigeon Salmonella and Escherichia coli Infection 1. Experimental methods A pigeon farm in Jiangsu was selected, which adopted a parent pigeon feeding mode. It is understood that the pigeons released from the farm were seriously infected with Salmonella and E. coli. In this embodiment, Salmonella phage PC553 was used to prevent and control Salmonella and E. coli in pigeons through drinking water medication.

[0080] The specific experimental method is as follows: 4000 pairs of pigeons were used in the experimental group and the control group respectively, and the drinking water group was uniformly mixed with Salmonella phage at 10mL / ton (5×10 8 PFU / pigeon), phage was used for 3 consecutive days, and 5 days was discontinued as one course of treatment, and 3 courses of treatment were used. The control group did not use phage, and the squabs were raised until they were ready for slaughter. During this period, other medication procedures in the farm were used normally. During the collection period, 200 feces were randomly collected to test the positive rate of Salmonella and body weight, and the mortality rate, as well as the detection rate of Salmonella and Escherichia coli in dead pigeons were counted. The changes in the total number of chicks in the first course of treatment (1-8 days old), the second course of treatment (9-16 days old), and the third course of treatment (17-28 days old) were counted.

[0081] 2. Experimental results and analysis The results are shown in Table 8. The positive rate of Salmonella in feces of the experimental group was 12.5% ​​lower than that of the control group, the mortality rate of the experimental group was about 7% lower than that of the control group, the detection rate of Salmonella in dead pigeons was about 43.28% lower than that of the control group, and the detection rate of Escherichia coli was 55% lower than that of the control group; the average weight of the experimental group was 21g higher than that of the control group, and all of them were significantly different.

[0082] Depend on Figure 6 The results show that the number of chicks hatched in the three courses of treatment in the experimental group were 770, 822 and 1090 respectively, and the number of chicks hatched in the three courses of treatment in the control group were 665, 686 and 825 respectively. The number of chicks hatched in each course of treatment in the experimental group increased significantly, which was 105, 136 and 265 more than that of the control group respectively.

[0083] The above experimental results show that using bacteriophage PC553 through drinking water can effectively prevent and control Salmonella and Escherichia coli infections in meat pigeons, and can also effectively increase the weight gain and number of chicks born in pigeons.

[0084] Table 8 Statistics of phage treatment of pigeon Salmonella infection

[0085] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A Salmonella phage with cross-species lysis ability, characterized in that: The Salmonella phage is named as Salmonella bacteriophage PC553, and its deposit number is CGMCC No.46166.

2. A bacteriophage composition, characterized in that: The method comprises the Salmonella phage PC553 as claimed in claim 1 and other phages.

3. The bacteriophage composition according to claim 2, characterized in that The other phages include: one or more of phage SP4, phage SPP11, and phage SP8; wherein the deposit number of phage SP4 is CGMCC No.14332; the deposit number of phage SPP11 is CGMCC No.18868; and the deposit number of phage SP8 is CGMCC No.45256.

4. Use of the Salmonella phage according to claim 1 or the phage composition according to claim 2 in the preparation of a drug, feed additive, environmental disinfectant or food preservative for preventing and treating pigeon-derived Salmonella and / or pigeon-derived Escherichia coli infection.

5. A phage pharmaceutical preparation, the active ingredient of which includes the Salmonella phage according to claim 1 or the phage composition according to claim 2; preferably, the phage pharmaceutical preparation also includes other antibacterial or bactericidal active ingredients; the pharmaceutical preparation is in the form of an oral dosage form, an external dosage form or a parenteral dosage form.

6. A feed additive or drinking water additive, characterized in that: The active ingredient comprises the Salmonella phage according to claim 1 or the phage composition according to claim 2.

7. An environmental disinfectant, characterized in that: The active ingredient comprises the Salmonella phage according to claim 1 or the phage composition according to claim 2.

8. The application of the environmental disinfectant as claimed in claim 7 in the environmental disinfection of a pigeon farm, characterized in that: The environmental disinfectant can disinfect the aquaculture environment and feeding equipment against salmonella and / or E. coli by spraying or soaking, and the aquaculture environment includes feed troughs, floors, walls, feces and bedding.

9. A food preservative, characterized in that: The method comprises the Salmonella phage according to claim 1 or the phage composition according to claim 2.

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