A high temperature resistant pigeon-derived Salmonella typhimurium phage, a phage composition thereof and application thereof

Abstract

The invention discloses a high temperature resistant pigeon-source Salmonella typhimurium phage, a phage composition and an application thereof, wherein the high temperature resistant pigeon-source Salmonella typhimurium phage is named PC204, and its deposit number is CGMCC No. 22368. The phage PC204 belongs to a long-tailed phage, has the characteristics of high temperature resistance, exhibits strong lysis and wide lysis spectrum characteristics to pigeon-source Salmonella typhimurium, can effectively prevent and treat pigeon-source Salmonella typhimurium diseases, has a specific lysis advantage to the pigeon-source Salmonella typhimurium, and can effectively reduce the mortality rate of pigeons and the positive rate of pigeon-source typhoid antibodies. It can be prepared into drugs for preventing and treating pigeon paratyphoid disease, environmental disinfectants, pigeon feed and pigeon water additives, and detection kits for application. The phage is safe and has no toxic side effects, will not produce drug-resistant bacteria, and has a good market application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a high-temperature-resistant pigeon-derived Salmonella typhimurium phage, a phage composition thereof and application thereof. Background Art

[0002] my country's pigeon breeding accounts for about 80% of the world's total, and its breeding technology is at the world's leading level. At present, there are more than 50 million pairs of pigeons in the country, and more than 600 million squabs are produced annually, and the level is increasing every year. Pigeon breeding has become a large-scale special poultry breeding industry after chickens, ducks and geese, and is an important part of my country's special poultry breeding industry. Pigeon salmonellosis is one of the main diseases affecting the development of the pigeon industry.

[0003] Pigeon salmonellosis is mainly caused by Salmonella typhimurium, also known as pigeon paratyphoid. The main mode of transmission is horizontal transmission. The mother pigeon feeds the baby pigeons through the mouth. Salmonella can be directly transmitted from the infected mother pigeon to the baby pigeon through the mouth during feeding. At the same time, its excrement can also become a source of transmission. Pigeons of all ages can be infected. The incidence rate of baby pigeons is higher than that of adult pigeons. The clinical manifestations of sick pigeons are depression, ruffled feathers, lethargy, and nasal tumors that lose their original color; diarrhea, yellow-green loose stools, often contaminating the feathers around the cloaca; decreased or absent appetite, increased drinking water; long-term course of the disease can be seen with weight loss, swollen joints, and increased joint fluid; some sick pigeons also have symptoms such as difficulty breathing and subcutaneous swelling; some sick pigeons have neurological symptoms such as head and neck bending and movement disorders. Salmonella is also an important pathogen that causes foodborne diseases in humans. Contaminated poultry products (meat, eggs, milk, etc.) are common sources of human infection.

[0004] Pigeon paratyphoid can occur all year round, with no obvious seasonality, but often occurs in seasons with changeable climates, especially cold and wet seasons. At the same time, due to the facultative intracellular parasitism of Salmonella, once the disease occurs, it is difficult to be purified, thus causing huge economic losses to the pigeon industry.

[0005] Antibiotics are now widely used to treat pigeon paratyphoid, but the excessive or irregular use of antibiotics has led to the emergence of multi-drug resistant strains of Salmonella in pigeons, as well as drug residues and imbalance of normal intestinal flora in pigeons, threatening human and animal health. With the implementation of the National Action Plan for Reducing and Substituting Antibiotics, the National Action Plan for Containing Antimicrobial Resistance of Animal-Source Bacteria (2017-2020), it is of great importance to find a safe and effective antimicrobial drug.

[0006] Bacteriophages are a type of virus that infects bacteria. They are widely found in nature and have the characteristics of host specificity, rapid sterilization, and no residue. They are non-polluting to humans, animals, and plants, and are highly safe. They have inherent advantages in treating bacterial diseases in farmed animals. The killing effect of bacteriophages on bacteria is not limited by bacterial resistance, and can effectively reduce the impact of bacterial infections on animals and animal products. By reducing the distribution and content of zoonotic pathogens carried by livestock and poultry, it can effectively control the spread of pathogens to humans through animal food. In addition, the screening cycle of bacteriophages is short, the preparation process is simple, the cost is low, and it is easy to produce on a commercial scale.

[0007] Based on the above advantages of bacteriophages, they have been gradually developed as fungicides or drugs and have been applied in some fields, such as the prevention and treatment of pullorum. For example, the patent with publication number CN 108359644 B applied by the applicant in 2018 discloses a bacteriophage SP4, which has a good antibacterial effect on chicken-derived Salmonella and has a more prominent preventive and therapeutic effect on pullorum. The patent with publication number CN 111254121 B applied by the applicant in 2020 discloses another strain of Salmonella phage SPP11 and its application in the prevention and treatment of salmonellosis in chickens. It can be seen that bacteriophages have been widely developed for the prevention and treatment of pullorum disease in chicken farms.

[0008] However, there is currently no bacteriophage with high lysis performance specifically targeting the Salmonella typhimurium that causes pigeon salmonellosis. Existing bacteriophage products are difficult to be used efficiently to prevent and treat pigeon salmonellosis. Therefore, the existing technology needs to be further improved. Summary of the invention

[0009] In response to the above problems, this study screened out a high-temperature-resistant, wide-lysis-spectrum Salmonella typhimurium phage based on actual application conditions, and prepared the phage into drugs for the prevention and treatment of pigeon paratyphoid, environmental disinfectants, pigeon feed and pigeon water additives, and detection kits. While solving the problem of pigeon-source Salmonella infection, the long-term addition of the phage to water and feed can achieve the effect of purifying Salmonellosis in the farm, and can also improve the quality of meat pigeons and the egg production rate and egg quality of laying pigeons.

[0010] The technical solution of the present invention is as follows:

[0011] In a first aspect, the present invention provides a Salmonella typhimurium phage having high temperature resistance and a wide lysis spectrum, which is isolated from pigeon feather contaminants in a pigeon breeding farm in Jiangsu Province, and is named PC204.

[0012] The phage was deposited at the General Microbiology Center of China Culture Collection Administration on April 12, 2021. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 22368.

[0013] Electron microscopy showed that the phage PC204 had a polyhedral head structure and a non-contractile tail. The head was 63-66 nm wide and 66-72 nm long, and the tail was about 138-150 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PC204 of the present application conforms to the characteristics of the long-tailed phage family and belongs to the long-tailed phage.

[0014] In the present application, the bacteriophage PC204 includes mutants with point mutations, deletion mutations or addition mutations having a homology higher than 98% or 99% and maintaining substantially the same bactericidal activity. Since bacteriophages are very susceptible to mutations during replication, the mutants of the above bacteriophages are also within the scope of the present application. The sequence of bacteriophage PC204 can be obtained by sequencing the biological material deposited according to the present invention by known methods. For those skilled in the art, it does not require creative labor to screen out mutants with extremely similar properties according to the bacteriophage provided by the present invention.

[0015] In a second aspect, the present application also provides a phage composition, which includes a high temperature resistant and wide lysis spectrum Salmonella typhimurium phage PC204 and other phages. In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in lysis spectrums of different phages, and complement each other's advantages, the above-mentioned pigeon-derived Salmonella phage PC204 and other phages can be used in combination.

[0016] Optionally, the phage composition includes phage PC204 and one or two of phage SP4 (specifically, see announcement number CN108359644 B) and phage SPP11 (specifically, see announcement number CN 111254121 B). By using the combination of phages, the lysis spectrum is broadened, and different types of Salmonella in the pigeon farm environment are killed, which is used to better prevent and treat pigeon salmonellosis.

[0017] Among them, bacteriophage SPP11 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is November 4, 2019, and the deposit number is CGMCC No. 18868. For more information, please refer to the patent announcement number CN 111254121 B.

[0018] 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.

[0019] In addition, the above-mentioned bacteriophage PC204 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 above-mentioned thermostable Salmonella typhimurium phage PC204 or the above-mentioned phage composition in the preparation of drugs, feed additives, environmental disinfectants and detection kits for preventing and treating Salmonella typhimurium infection. The prevention and treatment 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.

[0021] Preferably, the disease caused by the pigeon-derived Salmonella infection is pigeon salmonellosis, also known as pigeon paratyphoid. Pigeon-derived diseases caused by Salmonella are collectively referred to as pigeon salmonellosis, also known as pigeon paratyphoid. According to their symptoms and lesions, they can be divided into four types: intestinal, articular, visceral, and neurological.

[0022] In a fourth aspect, the present application further provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Salmonella typhimurium phage PC204 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.

[0023] 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.

[0024] 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.

[0025] In a fifth aspect, the present application also provides a pigeon drinking water additive or a pigeon feed additive, wherein the active ingredient comprises the above-mentioned bacteriophage PC204 or bacteriophage composition. Preferably, the concentration of the bacteriophage is 10 8 PFU / mL or above.

[0026] The pigeon drinking water additive or pigeon feed additive is added into water or mixed with feed to feed the 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.

[0027] In a sixth aspect, the present application further provides an environmental disinfectant, the active ingredient of which includes the bacteriophage PC204 or the aforementioned bacteriophage composition; preferably, the concentration of the bacteriophage is 10 8 PFU / mL or above.

[0028] 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.

[0029] In the seventh aspect, the present application also provides the application of the above-mentioned environmental disinfectant in the disinfection of pigeon farm environment. Specifically, the application method is: the disinfectant is used in pigeon slaughterhouses, pigeon product processing workshops and utensils, and pigeon breeding environments to prevent the contamination of pigeon-derived Salmonella in the environment. The breeding environment includes pigeon houses, pigeon cages, drinking fountains, feeding troughs and other feeding tools, feces and bedding, etc.

[0030] 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.

[0031] In an eighth aspect, the present application also provides a detection kit, which includes the aforementioned bacteriophage PC204. Experiments have found that the bacteriophage PC204 has lysis specificity for host bacteria, and the bacteriophage PC204 of the present invention can be applied to the rapid detection of Salmonella typhimurium, including but not limited to the detection of Salmonella typhimurium in the form of test strips, test strip boxes, etc., or the screening of target pathogens in clinical samples, effectively ensuring the sensitivity of the detection.

[0032] The present invention has the following beneficial effects:

[0033] 1. The present invention provides a high-temperature resistant pigeon-derived Salmonella typhimurium phage PC204 to solve the problem of Salmonella typhimurium infection. The phage has high titer and exhibits strong high temperature resistance. Under the condition of 80°C, it also maintains a high titer. At the same time, it exhibits strong lysis ability and a wide lysis spectrum for pigeon-derived Salmonella.

[0034] Based on the above characteristics, the bacteriophage PC204 can be prepared into drugs for preventing and treating pigeon paratyphoid, environmental disinfectants, pigeon feed additives and pigeon water additives, and detection kits for use, which can not only effectively prevent and treat the spread of salmonellosis, but also reduce the mortality rate of pigeons, reduce the positive rate of salmonellosis antibodies, and improve the survival rate of pigeon farms. Long-term use of the bacteriophage product can purify the salmonella in pigeon farms.

[0035] 2. The bacteriophages involved in the present invention are obtained from nature and are easy to be industrially produced. The drugs or disinfectants prepared from the bacteriophages can not only reduce costs, but also have the advantages of being green and environmentally friendly.

[0036] 3. When the bacteriophage of the present application is used as an environmental disinfectant, it can significantly reduce the content of Salmonella in the environment, thereby reducing the Salmonella infection rate in pigeon flocks, and can be promoted and applied as a new type of biological environmental disinfectant. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a plaque image of bacteriophage PC204;

[0038] Figure 2 This is an electron microscope image of bacteriophage PC204;

[0039] Figure 3The pH stability test results of bacteriophage PC204;

[0040] Figure 4 This is the one-step growth curve of bacteriophage PC204. DETAILED DESCRIPTION

[0041] 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.

[0042] Example 1 Isolation and purification of thermostable Salmonella typhimurium phage

[0043] 1. Resuscitation culture of host bacteria and preparation of proliferation medium

[0044] Twenty strains of pigeon-derived Salmonella typhimurium preserved in our laboratory were selected, and the freezing solution was dipped into a sterilized inoculum and streaked on SS culture medium for recovery. The culture was cultured in a constant temperature incubator at 37°C for 18-24 hours to obtain a single colony; a single colony was picked and inoculated into 5 mL of NB broth, and cultured at 37°C with shaking at 170 rpm / min for 16 hours to obtain a fresh pigeon-derived Salmonella typhimurium solution.

[0045] 2. Phage isolation and purification

[0046] Take appropriate amount of pigeon feathers, feces and other samples from Guangdong Province in a bubble sample bottle, add appropriate amount of broth culture medium, put it in a 70℃ water bath for 30 minutes, add 20 strains of pigeon-derived Salmonella typhimurium, put the mixed solution in 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;

[0047] The phage stock solution was diluted 10-fold, and appropriate gradient phage dilutions were taken and mixed with 20 strains of Salmonella typhimurium 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.

[0048] Pick a single plaque from the double-layer agar medium where plaques are formed, and place it in 1 mL of NB broth and culture it in a constant temperature shaker at 37°C, 170 rpm for about 30 minutes to obtain a phage extract. Take the phage extract and the corresponding plaque-forming Salmonella typhimurium (hereinafter referred to as the host bacteria) proliferation solution and mix them evenly in a ratio of 1:1 (incubate at 37°C for 5 minutes), draw 200 μL 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°C incubator and culture it upside down for 4~6 hours, and then obtain a double-layer plate with plaques formed again. Use sterilized tweezers to pick a single plaque from the double-layer culture medium where plaques are formed and place it in 1 mL of LB broth, and culture it in a constant temperature shaker at 37°C, 170 rpm for about 30 minutes to obtain a phage extract. Repeat the above steps 3 times to obtain a purified phage extract.

[0049] 3. Phage titer determination

[0050] 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.

[0051] 4. Experimental results and analysis

[0052] According to the above experimental method, 12 strains of Salmonella typhimurium phages were screened using 20 strains of Salmonella typhimurium isolates, numbered PC197-PC208. All 12 strains of Salmonella typhimurium phages formed clear plaques on double-layer agar plates, with no halo around them and clearly visible edges, with a diameter of about 0.5mm~1.2mm; the titer was between 2.80×10 9 PFU / mL-6.50×10 10 PFU / mL, the results are shown in Table 1.

[0053] Table 1 Titers of 12 newly isolated Salmonella typhimurium phages (PFU / mL)

[0054]

[0055] Example 2 Temperature resistance of six strains of Salmonella typhimurium phage

[0056] 1. Experimental methods:

[0057] The six newly isolated high titer Salmonella typhimurium phages (PC201-PC206) were taken in the same volume of 5×10 9PFU / mL of phage proliferation fluid was added to a 1.5mL centrifuge tube and placed at 50℃, 60℃, 70℃, 80℃, and 90℃, respectively, with two parallel samples at each temperature. After keeping warm for 20 min, 40 min, and 60 min respectively, samples were taken after the reaction ended, and each sample was immediately cooled in an ice bath, and then diluted 10 times, and the titer was determined using an appropriate dilution gradient.

[0058] 2. Experimental results and analysis

[0059] The results are shown in Table 2. All six phage strains maintained high activity within 60°C. After 70°C for 60 minutes, PC202 and PC203 were completely inactivated. After 80°C for 20 minutes, only PC201 and PC204 still maintained 10 5 PFU / mL was relatively high titer, and only PC201 still contained 6.00×10 3 PFU / mL, it can be seen that PC204 has strong heat resistance. After titer and temperature resistance determination, the high titer and heat resistance of Salmonella phage PC204 was finally selected.

[0060] Table 2 The titers (PFU / mL) of the six phage strains stored at different temperatures for different periods of time

[0061]

[0062] Example 3 Morphological Observation and Identification of Salmonella Typhimurium Phage PC204

[0063] 1. Experimental methods

[0064] 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.

[0065] 2. Experimental results and analysis

[0066] like Figure 2 As shown, bacteriophage PC204 has a polyhedral head structure and a non-contractile tail. The head is 63~66nm wide and 66~72nm long, and the tail is about 138~150nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the phage morphology of the present application conforms to the characteristics of the long-tailed bacteriophage family and belongs to the long-tailed phage.

[0067] Example 4 Whole genome analysis of Salmonella typhimurium bacteriophage PC204

[0068] The genome of bacteriophage PC204 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows:

[0069] (1) The PC204 genome is 43821 bp in length, with a G+C content of 49.54% and an A+T content of 50.46%. The base contents of C, G, A, and T are 24.82%, 24.72%, 25.32%, and 25.13%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 69 open reading frames (ORFs). Among these 69 open reading frames (ORFs), 15 structural proteins were found, mainly including the structure and packaging proteins of the phage (capsid protein, capsid and scaffold protein, tail spike, structural protein, phage fiber protein, terminase large subunit, etc.), phage lysis-related proteins (phage lysin), DNA replication and modification-related proteins (DNA polymerase, DNA helicase, DNA polymerase, DNA binding protein, endonuclease, etc.), and other functional proteins (phage repressor protein, dual infection immunity protein, etc.). At the same time, among the 69 ORFs, 64 start codons are ATG, 2 start codons are GTG, 1 start codon is TCA, 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 PHASTER analysis showed that the genome does not contain lysogeny-related genes.

[0070] (2) In the genome of bacteriophage PC204: the gene sequence of the tail spike protein related to phage host recognition is shown in sequence 1 in the sequence listing; the sequence of the highly conserved terminase large subunit protein gene is shown in sequence 2 in the sequence listing; the sequence of the DNA polymerase gene is shown in sequence 3 in the sequence listing; the sequence of the lysin gene related to cleavage ability is shown in sequence 4 in the sequence listing. For specific relevant information, see Table 3 below.

[0071] Table 3 Gene sequence information

[0072]

[0073] Example 5 Determination of the Optimal Multiplicity of Infection (MOI) of Salmonella Typhimurium Phage PC204 against Salmonella

[0074] 1. Experimental methods

[0075] Pick a single Salmonella colony and inoculate it into 5 ml NB broth medium. Cultivate at 37°C and 170 rpm for 12-16 hours to obtain bacterial solution. Determine the bacterial concentration by pouring method. Adjust the bacterial solution concentration to 1×109 cfu / mL, 1×10 8 cfu / mL…1×10 5 cfu / mL. The concentration of phage isolated in Example 2 was adjusted to 1×10 5 ~1×10 7 PFU / mL. According to the ratio of phage number to bacterial number in Table 4, add phage and bacterial solution to NB medium, and proliferate in a shaker at 37°C and 170rpm until the liquid becomes clear, and record the proliferation time. Take an appropriate amount of clear liquid and centrifuge at 11000rpm for 10min, take the supernatant, filter it with a 0.22μm sterile microporous filter membrane, and determine the phage titer in the filtrate by double-layer plate method. The MOI (phage number / bacterial number) with the highest phage titer is the optimal infection multiplicity of the phage.

[0076] 2. Experimental results

[0077] The results in Table 5 show that the PC204 titer reached the highest value at MOI 1:1000, 1.14×10 11 PFU / mL, at this time the initial input of phage is small and the highest reproduction yield can be achieved, which is conducive to large-scale industrial production.

[0078] Table 4 The titer of Salmonella phage PC204 at different infection multiplicity

[0079]

[0080] Example 6 pH Stability of Salmonella Typhimurium Phage PC204

[0081] 1. Experimental methods

[0082] Take three sterile test tubes and add 4.5 mL of NB broth with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 µL of 5×10 10 pfu / mL phage proliferation solution, mix well and incubate in a 37℃ water bath for 1 h, 2 h, and 3 h. After the reaction, immediately add an appropriate amount of 1mol / L HCl or NaOH to the mixture to make the pH value of the mixture about 7, dilute it 10 times, take an appropriate dilution gradient to determine the titer, and set 2 replicates for each pH value test tube. Draw the phage pH stability curve with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0083] 2. Experimental results and analysis

[0084] from Figure 3It can be seen that the titer of bacteriophage PC204 is maintained at 10 in the pH range of 5.0-11.0. 9 pfu / mL, stable activity; at pH 3.0 for 2h, the titer is 10 7 pfu / mL, and can resist a certain strong acid environment. It still maintains a certain activity at pH 1.0 for 1 hour. After 3 hours at pH 12, the titer of the phage is 10 7 pfu / mL, at pH 13 for 3h, the titer can still be maintained at 10 3 pfu / mL, therefore, bacteriophage PC204 has strong stability in a wide pH range and can adapt to a certain range of strong acid and strong alkaline environments.

[0085] Example 7 One-step growth curve of Salmonella typhimurium bacteriophage PC204

[0086] 1. Experimental methods

[0087] 1 mL of phage proliferation liquid and fresh host bacteria proliferation liquid with a multiplicity of infection of 10 were mixed thoroughly (the timing started at this time), incubated at 37°C for 5 minutes, centrifuged at 13000 rpm for 30 seconds, and the supernatant was removed as much as possible with a micropipette, and then washed once with 5 mL of NB broth (centrifuged at 13000 rpm for 30 seconds), and the supernatant was discarded. The precipitate (total volume of 5 mL) was suspended with preheated NB broth and mixed thoroughly, and quickly placed in a 37°C shaker with 170 rpm for shaking culture. 150 μL was taken out at time 0 and every 5 minutes, centrifuged at 10000 rpm for 1 minute, and the phage titer was measured by double-layer plate method after 10-fold dilution with NB broth. Three parallels were made, and the results were averaged. The infection time was used as the horizontal axis and the titer of the phage in the infection system was used as the vertical axis to draw a one-step growth curve to obtain the incubation period and outbreak period of phage PC204, and calculate the outbreak volume.

[0088] Outbreak volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak

[0089] 2. Experimental results and analysis

[0090] from Figure 4 The results show that after phage PC204 infects the host bacteria, the phage lysis cycle is about 60 minutes, the incubation period is about 10 minutes, and the phage outbreak period is about 50 minutes; after 60 minutes, the number of phages remains basically unchanged and enters a stable period, at which time the titer can reach 10 10 pfu / mL, and the burst amount of bacteriophage PC204 was 114.

[0091] Example 8 Determination of the lysis spectrum of Salmonella typhimurium bacteriophage PC204

[0092] 1. Experimental Materials

[0093] Host bacteria: 18 clinical isolates of Salmonella typhimurium (ST1 to ST18) in Example 1 and 27 strains of Salmonella of different serotypes from animals stored in the laboratory (including Salmonella Enteritidis, Salmonella Corvallis, Salmonella Fowl Typhoid, Salmonella Agona, and Salmonella Newport) were selected.

[0094] 2. Experimental methods:

[0095] (1) 45 pathogenic strains of Salmonella of different serotypes isolated from different regions and preserved in the laboratory were selected, and the lysis rates of PC204 on these 45 strains of Salmonella were determined by the double-layer plate method. The detailed lysis spectrum information is shown in Table 5 below;

[0096] (2) Comparison of lysis performance with existing phages: The lysis spectra of the above 45 strains of Salmonella were measured using SP4 (chicken source) in the company's authorized patent CN108359644A and bacteriophage SPP11 (chicken source) disclosed in another authorized patent CN111254121A of the company, respectively, to compare the differences in lysis performance of the three different Salmonella phages on the above 45 strains of Salmonella.

[0097] 3. Experimental results and analysis

[0098] From the results of the fragmentation spectrum experiment in Table 5, we can see that:

[0099] (1) The Salmonella typhimurium phage PC204 was able to lyse 39 of 45 strains of different types and serotypes of Salmonella, with a lysis rate of up to 86.67%. In particular, the lysis rate of 18 of the 45 Salmonella typhimurium strains was 100%, indicating that phage PC204 has a specific lysis advantage against Salmonella typhimurium.

[0100] (2) Bacteriophage SP4 was only able to lyse 28 of the 45 Salmonella strains, with a lysis rate of 62.22%, and was able to lyse 12 of the 18 Salmonella typhimurium strains, with a lysis rate of 66.67%. Salmonella SPP11 was able to lyse 32 of the 45 Salmonella strains, with a lysis rate of 71.11%, and was able to lyse 14 of the 18 Salmonella typhimurium strains, with a lysis rate of 77.78%.

[0101] It can be seen that the total lysis rate of the bacteriophage PC204 of the present application on various Salmonella is much higher than that of bacteriophage SP4 and SPP11. In particular, the lysis rate of bacteriophage PC204 on pigeon-derived Salmonella typhimurium is higher, and it has a specific lysis advantage on it. The above experimental results show that bacteriophage PC204 has a good lysis effect and broad spectrum on pigeon-derived Salmonella typhimurium.

[0102] (3) Based on the differences in the lysis of three phages PC204, SP4 and SPP11 against various Salmonella, in order to improve the use effect of phage products, phage PC204, SP4 and SPP11 can be used in combination, which significantly expands the lysis spectrum of phage products and improves the prevention and treatment effect of diseases caused by pigeon-derived Salmonella infection.

[0103] Table 5 Lysis spectrum of 3 strains of Salmonella phage against 45 strains of pigeon Salmonella

[0104]

[0105] Example 9 Lysis test of Salmonella typhimurium bacteriophage PC204 on non-host bacteria

[0106] 1. Experimental methods

[0107] A total of 25 strains of non-host bacteria of different types, including 10 strains of Escherichia coli, 5 strains of Staphylococcus aureus, 5 strains of Proteus and 5 strains of Clostridium perfringens, were selected, and a lysis spectrum determination experiment of Salmonella typhimurium phage PC204 was carried out according to the lysis spectrum determination method in Example 8.

[0108] 2. Experimental results and analysis

[0109] No clear plaques were found in the double-layer plates of Salmonella typhimurium phage PC204 and 25 strains of non-host bacteria, indicating that phage PC204 could not recognize the above-mentioned 10 strains of Escherichia coli, 5 strains of Staphylococcus aureus, 5 strains of Proteus and 5 strains of Clostridium perfringens. This shows that the test phage PC204 has extremely strong host specificity and has no damaging effect on the microbial community. Its specificity can be used to prepare detection kits.

[0110] Example 10 Environmental disinfection test of Salmonella typhimurium bacteriophage PC204

[0111] 1. Experimental methods

[0112] A certain pigeon hatchery detected a large number of Salmonella in the hatchery, and the sterilization effect of chemical disinfectants was not obvious. This experiment was designed to use bacteriophage PC204 to spray disinfect the hatchery workshop.

[0113] The preparation method of the bacteriophage disinfectant is as follows: take the bacteriophage liquid and the host bacteria in NB broth medium and mix them in a ratio of 1:1, culture at 37°C and 170rpm for 4-6 hours until the liquid is clear and bacterial fragments are visible. Take the clear liquid and filter it to remove the bacterial fragments and residues, and determine the bacteriophage titer to be 1×10 10pfu / ml, the phage liquid was diluted 100 times, and the preparation titer was 1×10 8 pfu / mL of bacteriophage disinfectant, seal and store at 2~8℃ for future use.

[0114] (1) Air disinfection method:

[0115] Find two hatchery rooms of the same size and use the natural sedimentation method to sample the air before and after the hatchery rooms. Take the central point and four corner points as the test points. The sampling point is 0.5m from the ground and the four corner points are 1m away from the wall. Place a 9cm diameter XLD agar plate at each point. Use two XLD agar plates at each sampling point before disinfection. Open the lid of the culture dish and sample for 10 minutes. Use the fog line provided by the pigeon farm for disinfection. Use bacteriophage preparations for disinfection (10mL / m 3 , phage titer 1×10 8 pfu / mL) and benzalkonium bromide disinfection (benzalkonium bromide 1:25 dilution). After 30 minutes of disinfection, two XLD agar plates were placed at each of the five sampling points, and the lid of the culture dish was opened for sampling for 10 minutes. The sampling culture dishes before and after disinfection were placed in a 37℃ constant temperature incubator for 12-24 hours, and the cultured bacteria were counted.

[0116] According to the Ostwald formula, the total number of colonies is C = 50000N / AT, where C is the total number of colonies per cubic meter (CFU / m 3 ); N: number of colonies per dish; A: area of ​​culture dish (cm 2 ); T: sampling time (min).

[0117] 2. Experimental results and analysis

[0118] As shown in Table 6, after disinfection with benzalkonium bromide disinfectant, the elimination rate of Salmonella in the hatchery air was 57.79%; after disinfection with bacteriophage PC204, the elimination rate of Salmonella in the hatchery air was 86.81%. It can be seen that the environmental disinfection effect of bacteriophage PC204 is significantly better than the existing chemical disinfectant such as benzalkonium bromide, and it is safer to use.

[0119] Therefore, the above results indicate that bacteriophage PC204 has an excellent disinfecting effect on Salmonella in hatcheries and can be promoted and applied as a new type of biological environmental disinfectant.

[0120] Table 6 Salmonella colony counts before and after disinfection with bacteriophage PC204 and benzalkonium bromide disinfectant

[0121]

[0122] Example 12 Experiment on the preventive effect of bacteriophage PC204 on salmonellosis in pigeon farms

[0123] 1. Experimental methods

[0124] A pigeon farm in the south with a high positive rate of Salmonella antibody was selected, and two pigeon houses with 1200 pigeons were selected as the experimental group and the control group respectively.

[0125] (1) Preparation of phage freeze-dried powder: skim milk powder and lactose were selected as protective agents, bacteriophage PC204 was added and mixed evenly, and then dried using a Swiss BUCHIB-90 nano spray dryer. The process parameters were: inlet temperature (120°C), air flow rate (120L / min), spray rate (100%), nozzle cover size (4.0μm), and spray-dried particles were collected after spray drying for about 1 hour. Finally, bacteriophage PC204 was made into a spray-dried powder with a concentration of 1×10 9 PFU / g.

[0126] (2) Treatment of the experimental group: Add the prepared bacteriophage PC204 powder to the feed at 500g / ton, starting from the squab stage. Treatment of the control group: No bacteriophage was added to the feed. Other immunization and medication procedures were carried out according to the farm procedures.

[0127] (3) The serum samples of laying pigeons at 28 and 49 days of age in the experimental and control groups were collected for serum plate agglutination test with positive serum of Salmonella typhimurium.

[0128] 2. Experimental results and analysis

[0129] (1) The results are shown in Table 7. For 28-day-old laying pigeons, the positive rate of Salmonella Typhimurium antibody in the control group was 16.7%, and that in the phage group was 0.6%, a decrease of 16.1%; for 49-day-old laying pigeons, the positive rate of Salmonella Typhimurium antibody in the control group was 28.5%, and that in the phage group was 1.0%, a decrease of 27.5%. This indicates that by adding Salmonella Typhimurium phage PC204 to the feed for a long time, the positive rate of Salmonella Typhimurium antibody in the pigeon farm can be significantly reduced, and long-term use can even achieve the effect of purifying Salmonella in the farm.

[0130] (2) By statistically analyzing the mortality rates of pigeons in the phage group and the control group, it was found that the mortality rate of pigeons in the phage group was 15% lower than that in the control group. This indicates that long-term addition of Salmonella typhimurium phage PC204 to feed can reduce the mortality rate of pigeons and has a good control effect on pigeon-borne Salmonella.

[0131] Table 7 Statistics of positive detection of Salmonella typhimurium antibodies and mortality results

[0132]

[0133] Example 13 Safety Test of Bacteriophage

[0134] 1. Experimental methods

[0135] Forty one-day-old pigeons were selected and divided into a phage group and a control group. The phage group was orally administered with 1×10 PC204 10 The mice were fed 0.2 mL of PFU phage and observed for 7 days. The control group was given the same dose of sterile saline orally. The heart, liver, spleen, lung, kidney, brain, and intestinal lesions were observed by autopsy. The spirit and feeding status were observed during the feeding process.

[0136] 2. Experimental results and analysis

[0137] During the entire administration period, no disease or toxicity symptoms were observed in the phage group and the control group, and the mental state and feeding were normal. After detailed clinical autopsy observations, the main organs and intestines of the animals were normal in both the phage group and the control group. This shows that bacteriophage PC204 is highly safe and has no adverse effects on the animal body.

[0138] 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 thermostable pigeon-derived Salmonella typhimurium phage, characterized in that: It was named PC204 and its deposit number is CGMCC No.22368.

2. A bacteriophage composition, characterized in that: The invention comprises the high temperature resistant pigeon-derived Salmonella typhimurium phage as claimed in claim 1 and other phages.

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

4. Use of the high temperature resistant pigeon-derived Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2 or 3 in the preparation of drugs, feed additives, and environmental disinfectants for preventing and treating diseases caused by pigeon-derived Salmonella infection.

5. A phage pharmaceutical preparation, the active ingredient of which comprises the heat-resistant pigeon-derived Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2 or 3.

6. A pigeon feed additive or pigeon drinking water additive, characterized in that: It comprises the high temperature resistant pigeon-derived Salmonella typhimurium phage as claimed in claim 1 or the phage composition as claimed in claim 2 or 3.

7. An environmental disinfectant, characterized in that: The active ingredients include the high temperature resistant pigeon-derived Salmonella typhimurium phage as claimed in claim 1 or the phage composition as claimed in claim 2 or 3.

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 from salmonella by spraying or soaking, and the aquaculture environment includes the ground, walls and feces.

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