CN114940977A

CN114940977A - Salmonella anatipestifer bacteriophage, bacteriophage composition thereof and application of salmonella anatipestifer bacteriophage in prevention and treatment of salmonella anatipestifer infection diseases

Info

Publication number: CN114940977A
Application number: CN202210687863.5A
Authority: CN
Inventors: 潘强; 任慧英; 孙虎芝; 闫艳新; 吴杨
Original assignee: Qingdao Phagepharm Bio Tech Co ltd
Current assignee: Qingdao Phagepharm Bio Tech Co ltd
Priority date: 2021-07-09
Filing date: 2022-06-17
Publication date: 2022-08-26

Abstract

The invention discloses a duck salmonella bacteriophage, a bacteriophage composition and application thereof in treatment and prevention of duck salmonella infection diseases, wherein the bacteriophage is named as PYC03, the preservation number is CGMCC No.22370, and the bacteriophage is preserved in the common microbiology center of China Committee for culture Collection of microorganisms at 12 months 4 and 2021, shows strong lytic property and wide lytic spectrum to duck salmonella, has the functions of reducing duck group death rate, keeping freshness of meat quality and well clearing salmonella in duck intestinal tracts, can be used for preparing medicaments, environmental disinfectants, feed and water additives, food preservatives, detection kits and the like for treating or preventing duck salmonella infection diseases, and avoids the problems of antibiotic residue and drug resistance caused by using antibiotics while solving duck salmonella infection.

Description

Salmonella anatipestifer bacteriophage, bacteriophage composition thereof and application of salmonella anatipestifer bacteriophage in prevention and treatment of salmonella anatipestifer infection diseases

Technical Field

The invention relates to the technical field of microorganisms, in particular to a duck salmonella bacteriophage and application thereof in preventing and treating duck salmonella infection diseases.

Background

Salmonella (Salmonella) is a common zoonotic pathogen, WHO lists Salmonella as a food-borne pathogen with serious harm. According to data statistics, food safety accidents caused by salmonella account for 70-80% of bacterial food poisoning in China. Therefore, the microbial contamination of salmonella and the like has formed a major threat to the safety of our food.

Duck salmonellosis refers to the general term for acute or chronic disease of ducks caused by any one or more members of the genus salmonella. The pathogenic bacteria causing salmonellosis are of various types, belong to each serogroup in salmonella, and are commonly salmonella duck typhosa, salmonella duck, salmonella enteritidis and the like. The disease can occur all the year round, has high sensitivity and pathogenicity to ducklings, generally, ducklings within 3 weeks are most prone to morbidity and mortality, the mortality rate is 20% -80%, the survivors are delayed in development, slow in weight gain, reduced in feed reward, and most of adult ducks become carriers after infection, and are one of the most serious bacterial infectious diseases of ducks. In nature, poultry is the most important storage of salmonella, and the polluted poultry meat and eggs are closely related to the occurrence of salmonellosis of human beings, so that the prevention and control of the salmonellosis of ducks have important significance for promoting the development of duck industry, ensuring food safety and maintaining human health.

Bacteriophage is one of the most abundant organisms in nature, and approximately 10 is deposited on the earth ³¹ I.e. containing up to 10 per 1g of soil or water on average ⁸ The number of bacteriophages has long been recognized as useful as therapeutic drugs and bactericides. The bacteriophage has high specificity to certain bacterial strains, and can not influence the normal operation of organisms or endogenous tissues, so the bacteriophage has no infection to human or animals and plants, can not pollute the environment, and has high safety. After the phage is contacted with the corresponding host bacteria, the adsorption and intracellular proliferation processes of the phage are triggered, and more phage are released through the lysis of the host bacteria. The phage can be amplified in a large amount by killing specific host bacteria in the environment through the method, has an exponential multiplication characteristic, has a far lower drug resistance generation rate than antibiotics, and the bacteriolysis effect of the phage is not limited by the drug resistance of bacteria. In addition, the phage has short screening period, simple preparation process and low cost, and can be used as a killerBiological disinfectants and antibiotic substitutes for bacteria.

At present, phages have been developed gradually and applied to some fields as bactericides or medicines, such as the prevention and treatment of pullorum disease. A patent with a publication number of CN102296051A applied in 2011 by agricultural academy of sciences of Jiangsu province discloses a strong lytic bacteriophage PSPu-4-116, which can be applied to high-efficiency inhibition of salmonella pullorum. The patent with publication number CN110305851A of the university of agriculture in Huazhong discloses a Salmonella pullorum bacteriophage Pu20 with a wide lysis spectrum and application thereof in liquid eggs, and the patent with publication number CN110241091A discloses another Salmonella dublin bacteriophage D1-2 and application thereof in liquid egg samples. The patent with the publication number of CN108359644B applied by the applicant in 2018 discloses a salmonella phage SP4, the cracking rate of the phage on salmonella pullorum is as high as 95.83%, and the control effect on chicken-origin salmonellosis is very remarkable. The applicant also discloses another salmonella phage SPP11 with excellent high temperature resistance and its use in preventing and treating salmonellosis in chicken flocks in the patent of publication No. CN111254121A filed in 2020, and it is seen that phages have been widely developed for preventing and treating salmonellosis in chicken at present.

However, no salmonella duck bacteriophage with high lytic performance specially aiming at salmonella duck exists at present, and the existing bacteriophage products are difficult to be efficiently used for preventing and treating diseases caused by salmonella duck, so that the prior art needs to be further improved.

Disclosure of Invention

Aiming at the problems, the invention provides a new salmonella phage PYC03 and application thereof, wherein the phage can be used for preparing medicines for treating or preventing duck salmonella infection diseases, environmental disinfectants, feed and water additives, food preservatives, detection kits and the like, and the problems of antibiotic residue and pathogenic bacteria drug resistance caused by using antibiotics are avoided while the duck salmonella infection is solved.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a duck salmonella bacteriophage with broad-spectrum strong lytic property, which is separated from duck manure in a certain Shandong place, is named as PYC03 and is preserved in the common microbiology center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu 1 of Beijing Korean-Yang district, the preservation date is 2021 year, 4 months and 12 days, and the preservation number is CGMCC No. 22370.

The phage PYC03 has polyhedral head structure and non-contractive tail, head width of 60nm, length of 68nm, tail length of about 132nm, and its form conforms to the characteristics of long-tail phage family according to the classification method of International Committee for Virus Classification (ICTV), and belongs to long-tail phage.

In a second aspect, the present application also provides a phage composition comprising the salmonella duck phage PYC 03. In practical application, in order to further broaden the cracking spectrum of a phage preparation, fully exert the difference of the cracking spectrum of different phages and perform advantage complementation, the duck salmonella phage PYC03 and other phages can be combined for use, such as being combined with one or two of SP4 and PYC03 which are salmonella phages, so that the bactericidal spectrum is expanded, all salmonella in the environment can be killed as far as possible, and the duck salmonella phage PYC03 can be used for preventing and treating salmonellosis. In addition, the duck salmonella phage PYC03 can be matched with other phages of different types (inhibiting different pathogenic bacteria causing the same type of diseases) to be used for preventing and treating the same type of diseases.

In a third aspect, the application also provides the application of the salmonella phage PYC03 or the phage composition in preparation of medicines for preventing and treating diseases caused by duck-origin salmonella phage infection. The prevention and treatment include prevention and treatment. The term "prevention" is meant herein to include all actions that inhibit or delay the disease by administering the composition. The term "treatment" is meant herein to include all actions that result in an improvement or amelioration of the disease by administration of the composition.

Preferably, the duck-origin salmonella is selected from the group consisting of duck salmonella, duck typhoid salmonella and enteritis salmonella.

In a fourth aspect, the present application also provides a bacteriophage pharmaceutical preparation, the effective ingredient of which comprises the aforementioned salmonella duck bacteriophage or bacteriophage composition; preferably, the bacteriophage pharmaceutical preparation further comprises other bacteriostatic or bactericidal active ingredients; the pharmaceutical preparation is in the form of an oral administration dosage form, an external administration dosage form or an intestinal administration dosage form.

Optionally, the phage drug formulation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active ingredient being administered. In order to formulate the pharmaceutical composition as a liquid formulation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, glucose solution, maltodextrin solution, glycerol, ethanol, various types of culture media, and the like. They may be used alone or in any combination thereof. Other conventional additives, for example, antioxidants, buffers, bacteriostats, and the like, may be added if desired. When also combined with diluents, dispersants, surfactants, binders and/or lubricants, the compositions of the present invention can also be prepared in injectable and oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilizates.

In a fifth aspect, the present application also provides an environmental disinfectant comprising as active ingredients the salmonella bacteriophage PYC03 or the bacteriophage composition as described above; preferably, the concentration of phage is 10 ⁶ PFU/ml or more.

Optionally, the environmental disinfectant also contains other active ingredients for the inhibition or elimination of bacteria in the environment; preferably, the environment in which the environmental disinfectant is applicable includes feed, water, and farming environments including troughs, floors, walls, manure, and bedding.

In a sixth aspect, the present application further provides an application of the above environmental disinfectant in environmental disinfection of a farm, specifically, the environmental disinfectant can disinfect salmonella of a breeding environment and a breeding appliance in a spraying and soaking manner, wherein the breeding environment comprises a trough, a ground, a wall, excrement and padding. The liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, and the like.

In a seventh aspect, the present application also provides a drinking water additive or feed additive, the effective components of which comprise the salmonella phage PYC03 or phage composition; preferably, the concentration of phage is 10 ⁸ PFU/ml or more. Through adding the drinking water additive or the feed additive into water or mixing with feed, the duck group is fed, so that the drinking water and the feed in a duck farm are disinfected and sterilized, the spreading of duck salmonellosis is avoided from the source, and the prevention and the treatment of the salmonellosis are effectively carried out.

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

In an eighth aspect, the application also provides a food preservative, which comprises the salmonella phage PYC03 or the phage composition, and the salmonella phage PYC03 or the phage composition is sprayed on the surface of meat, or a duck meat product is soaked in a solvent added with the food preservative, so that the proliferation of salmonella in the duck product can be inhibited, and the preservation effect is achieved.

In a ninth aspect, the present application also provides a detection kit comprising the aforementioned salmonella bacteriophage. Based on the cracking specificity of the salmonella phage PYC03 to host bacteria, the duck salmonella phage can be applied to the rapid detection of duck salmonella, including but not limited to the detection of duck salmonella in the forms of test paper, test paper boxes and the like, or the screening of target pathogenic bacteria in clinical samples, so that the detection sensitivity is effectively ensured.

The invention has the following beneficial effects:

1. the invention provides a virulent salmonella bacteriophage with a wide lysis spectrum, which can effectively prevent duck salmonellosis and has the effects of reducing the death rate of duck groups, keeping the freshness of meat quality and well clearing salmonella in duck intestinal tracts. The bacteriophage can be used as disinfectant active ingredients, feed and water additives, food preservatives, detection kits and the like, solves the salmonella infection of ducks, and simultaneously avoids the problems of antibiotic residues and pathogenic bacteria drug resistance caused by using antibiotics.

2. The bacteriophage is obtained from nature, is easy to carry out industrial production, and the medicament or disinfectant prepared from the bacteriophage not only can reduce the cost, but also has the advantage of environmental protection.

Drawings

FIG. 1 is a plaque picture of Salmonella anatipestifer phage PYC 03;

FIG. 2 is an electron micrograph of bacteriophage PYC 03;

FIG. 3 is the results of the thermostability test for bacteriophage PYC 03;

FIG. 4 shows the results of pH stability tests of the bacteriophage PYC 03;

FIG. 5 is a one-step growth curve of bacteriophage PYC 03;

FIG. 6 is a graph of the effect of bacteriophage PYC03 on bacterial content in duck shed;

FIG. 7 is a graph of the effect of bacteriophage PYC03 on bacterial biofilm formation;

FIG. 8 shows the effect of the PYC03 phage on the freshness of meat quality.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE 1 isolation and purification of phage

1. The experimental method comprises the following steps: taking a proper amount of duck manure sample collected from a certain farm in Shandong. Adding a small amount of sample into a broth culture medium, adding 1 strain of fresh bacterial liquid of a duck salmonella clinical isolate strain into the culture medium, placing the feces-bacterial liquid mixture in a constant-temperature shaking table at 37 ℃, culturing for about 12 hours at 170rpm, and filtering the culture solution through a sterile microporous filter membrane of 0.22 mu m to obtain a bacteriophage proliferation solution; diluting the phage stock solution by 10 times, taking a proper gradient phage diluent, respectively and uniformly mixing with 1 strain of salmonella bacteria solution 1:1, incubating for 5min at 37 ℃, sucking 200 mu L of mixed solution, placing the mixed solution into upper agar (the agar concentration is 0.7%), rapidly pouring the mixed solution onto a plate with lower agar (the agar concentration is 1.5%) after uniformly mixing, shaking the mixed solution evenly, placing the mixed solution on the plate until a culture medium is solidified, placing the plate in a 37 ℃ incubator for inverted culture for 4-6 h, and obtaining a plaque-forming double-layer plate in the salmonella plate. The obtained salmonella phage was named PYC 03.

A single plaque was picked up on a plaque-forming double-layer agar medium and incubated in 1mL of NB broth for about 30min at a constant temperature of 37 ℃ in a shaker at 170rpm to obtain a phage extract. Uniformly mixing the phage extract with a salmonella (hereinafter referred to as host bacteria) proliferation fluid 1:1 corresponding to plaque formation (incubating for 5min at 37 ℃), sucking 200 mu L of the mixture, placing the mixture on an upper layer of agar, quickly pouring the mixture on a lower layer of agar plate after uniformly mixing, shaking the mixture, flatly placing the mixture on a culture medium for solidification, placing the culture medium in an incubator at 37 ℃ for inverted culture for 4-6 h, and obtaining a plaque-forming double-layer plate again. A single plaque was picked up on the plaque-forming double-layer medium with a sterilized forceps and placed in 1mL of LB broth, and cultured at 170rpm in a constant temperature shaker at 37 ℃ for about 30min to obtain a phage extract. Repeating the steps for 3 times to obtain purified phage leachate, taking the purified phage leachate and host bacterium proliferation solution with equal amount to the purified phage leachate and the host bacterium proliferation solution, carrying out shake culture at 37 ℃ and 170rpm in 5mL of liquid NB culture medium until the liquid becomes clear, centrifuging the clear liquid at 11000rpm for 10min, taking the supernatant, and filtering by using a sterile microporous filter membrane with the diameter of 0.22 mu m to obtain the phage proliferation solution.

2. The experimental results are as follows: as shown in FIG. 1, Salmonella phage PYC03 formed clear plaques on double agar medium plates, with no halo rings around, clearly visible edges, and a diameter of about 1.75 mm.

Example 2 morphological Observation and identification of the Salmonella phage PYC03

1. The experimental method comprises the following steps: and (3) dripping a 20 mu L bacteriophage sample on a copper net with a carbon coating film, naturally precipitating for 15min, slightly drying by using filter paper, then dyeing for 1-2 min by using 2% (W/V) phosphotungstic acid (PTA), slightly drying by using the filter paper, and observing and photographing by using a transmission electron microscope after drying.

2. The experimental results are as follows: as shown in FIG. 2, the bacteriophage PYC03 has a polyhedral head structure and a non-contractive tail, the head has a width of 60nm, a length of 68nm and a tail length of about 132nm, and the bacteriophage morphology of the present application conforms to the characteristics of the Long-tailed bacteriophage family according to the classification method of the International Committee for Virus Classification (ICTV), and belongs to the Long-tailed bacteriophage.

Example 3 temperature stability of Salmonella phage PYC03

1. The experimental method comprises the following steps:

add 5.2 x 10 of the same volume ¹⁰ putting containers of pfu/mL phage PYC03 proliferation solution at 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, keeping the temperature for 20min, 40min and 60min respectively, sampling after the action is finished, immediately putting each sample in an ice bath for cooling, diluting by 10 times, and taking a proper dilution gradient to determine the titer. And drawing a thermal stability curve of the phage by taking the temperature as an abscissa and taking a logarithmic value of the titer of the phage as an ordinate.

2. Experimental results and analysis:

as shown in FIG. 3, the phage PYC03 basically maintained the original activity after 60min at 40 deg.C, 50 deg.C and 60 deg.C, and the titer remained at 1X 10 after 60min at 70 deg.C ⁷ pfu/mL or more, and the phage count is about 1.25X 10 at 80 ℃ for 20min ⁶ pfu/mL, still maintaining a certain activity. This indicates that the phage PYC03 has high thermostability.

Example 4 pH stability of Salmonella phage PYC03

1. The experimental method comprises the following steps:

adding different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9) into sterile test tube10, 11, 12, 13) of NB broth 4.5mL, three tubes each, then the tubes were placed in a 37 ℃ water bath and after the temperature had stabilized, 500. mu.L of 3.42X 10 tubes each were added ⁹ pfu/mL bacteriophage proliferation solution, and mixing uniformly in 37 ℃ water bath for 1h, 2h and 3 h. Immediately after the reaction, an appropriate amount of 1mol/L HCl or NaOH was added to the mixture to adjust the pH of the mixture to about 7, 10-fold dilution was performed, and the titer was measured using an appropriate dilution gradient, with 2 replicates per pH tube. And drawing a bacteriophage pH value stability curve by taking the pH value as an abscissa and the logarithm value of the bacteriophage titer as an ordinate.

2. Results and analysis of the experiments

As shown in FIG. 4, the phage PYC03 was maintained at a pH of 5.0 to 11.0 and its titer was maintained at 10 ⁹ pfu/mL, the activity is relatively stable.

Example 5 one-step growth curves of Salmonella phage PYC03

1. The experimental method comprises the following steps:

1mL of each of the phage growth solution having the multiplicity of infection of 10 and the fresh host bacterium growth solution was mixed well (timing was started), incubated at 37 ℃ for 5min, centrifuged at 13000rpm for 30s, the supernatant was aspirated as much as possible with a micropipette, washed 1 time with 5mL of NB broth (centrifuged at 13000rpm for 30s), and the supernatant was discarded. Suspending and precipitating with preheated NB broth (the total volume is 5mL), mixing well, quickly placing in a shaking table at 37 ℃ for shaking culture at 170rpm, taking out 150 mu L at 0 moment and every 5min, centrifuging at 10000rpm for 1min, diluting with NB broth by 10 times, measuring the titer of phage by using a double-layer plate method, making 3 parallels, taking the average value of the result, drawing a one-step growth curve by using the infection time as a horizontal coordinate and the titer of phage in an infection system as a vertical coordinate, obtaining the incubation period and the outbreak period of the phage PYC03, and calculating the outbreak amount.

The outbreak amount is the total number of bacteria at the end of phage outbreak/the total number of bacteria at the beginning of outbreak

2. Results and analysis of the experiments

From the results shown in FIG. 5, it was found that the titer was substantially unchanged within 10min and stabilized at 10 after the host bacterium was infected with the phage PYC03 ⁶ pfu/mL, indicating that the incubation period of the phage PYC03 is about 10min, after the phage infects host bacteriaWithin 10-60 min, the number of the phage gradually increases, the valence growth begins to be stable within 70min, and the valence can reach 2.00 multiplied by 10 ¹⁰ pfu/mL, the burst phase of phage PYC03 was approximately 50min, with a burst size of 82.

Example 6 determination of phage lysis Profile

1. Preparing materials:

preparing a fresh phage multiplication solution PYC03 and 284 strains of salmonella ducks, salmonella duck typhosa and salmonella enteritidis which are clinically separated from farms in different regions in China; wherein 31 strains of duck salmonella, 28 strains of duck typhoid salmonella and 35 strains of enteritis salmonella are respectively separated in Shandong area; 30 duck salmonella, 36 duck typhoid salmonella and 32 enteritis salmonella are respectively separated in Shanxi area; 38 strains of duck salmonella, 33 strains of duck typhoid salmonella and 21 strains of enteritis salmonella are respectively separated in Guangxi area.

2. The experimental method comprises the following steps:

the detection method of the cracking rate comprises the following steps:

in a 96-well plate, 5. mu.L of 1X 10 was added to each well of the test group ⁸ pfu/mL～1×10 ⁹ pfu/mL phage solution and 5. mu.L of 1X 10 ⁶ cfu/mL～1×10 ⁷ cfu/mL bacterial liquid, adding 190 μ L sterilized nutrient broth, and mixing; adding 5 μ L bacterial liquid and 195 μ L sterilized nutrient broth into control group, and mixing; measuring the OD600 value in each hole at 0h and 4h respectively; calculating the OD value reduction proportion of the phage to different salmonella; the OD value is reduced by more than 50 percent to indicate that the bacterium is completely lysed, namely the mixed solution is clear, and the lysis rate of the phage PYC03 on 284 salmonella strains is counted.

OD value decrease ratio (control OD value-test OD value)/control OD value

Comparison with the existing phage:

the Salmonella anatipestifer which can be cracked by PYC03 is selected and is subjected to cracking spectrum determination by using SP4 in a patent CN108359644B authorized by the company and phage SPP11 disclosed by another patent CN111254121A of the company, and the cracking differences of three different Salmonella bacteriophages are compared.

3. Results and analysis of the experiments

Firstly, from the cracking results in table 1, it can be known that 253 salmonella in 284 clinically isolated duck pathogenic salmonella can be cracked by the bacteriophage PYC03, and the total cracking rate is 89.10%, which indicates that the bacteriophage has the characteristics of strong cracking property and wide cracking spectrum.

In particular, the bacteriophage PYC03 showed high and low differential lysis rates for three different Salmonella species. The phage can crack 92 strains of 99 strains of duck salmonella, 86 strains of 97 strains of duck salmonella typhosa and 75 strains of 88 strains of salmonella enteritidis, and the cracking rates of the phage to the three salmonella are respectively 92.93%, 88.66% and 85.23%. As can be seen, the bacteriophage PYC03 has the highest lysis rate on Salmonella anatipestifer.

Secondly, as can be seen from the experimental results in table 2, the PYC 03-cleavable 253 salmonella strains, the phage SP4 can cleave 74 of 92 duck salmonella strains, 61 of 86 duck salmonella strains and 57 of 75 duck salmonella strains, namely, the cleavage rates of the three salmonella strains cleavable by the PYC03 are 80.43%, 70.93% and 76.00%, respectively. The phage SPP11 can crack 59 strains of 92 strains of Salmonella anatipestifer, 47 strains of 86 strains of Salmonella anatipestifer and 38 strains of 75 strains of Salmonella enteritidis, and the cracking rates are 64.13%, 54.65% and 50.67% respectively.

Overall, the total lysis rate of phage SP4 on PYC 03-cleavable 253 duck-origin salmonella was 75.8%, while the total lysis rate of phage SPP11 on PYC 03-cleavable 253 duck-origin salmonella was 56.9%.

Therefore, the cracking rate of the phage PYC03 on the duck salmonella is far higher than that of the phage SP4 and the phage SPP11, and the phage PYC03 has more excellent cracking performance and wider cracking spectrum on the duck salmonella.

TABLE 1 lysis Profile of PYC03 bacteriophage against 284 strains of Salmonella anatipestifer

TABLE 2 lysis Profile of SP4, PYC03 phage against 253 strains of Salmonella anatipestifer

Example 7 lysis assay of bacteriophage PYC03 on non-host bacteria

1. Experimental methods

Different non-host bacteria, 10 E.coli, 5 staphylococci, 5 proteus and 5 C.welchii strains, were selected and the PYC03 lysis experiment was performed according to the lysis profile determination method of example 6.

2. Results and analysis of the experiments

After the PYC03 and 20 non-host bacteria are mixed and cultured for 4 hours, the OD value reduction ratio is less than 5 percent, which shows that the phage PYC03 can not identify the 10 non-host bacteria of escherichia coli, 5 staphylococcus, 5 proteus vulgaris and 5 non-host bacteria of clostridium welchii, and the result shows that the tested phage has extremely strong host specificity and has no damage to microbial communities.

Example 8 phage PYC03 environmental Disinfection test

1. Experimental methods

In a white feather meat breeding duck farm in Shandong, 9 duck sheds of 1 day old are randomly selected and divided into 3 test groups, 3 control groups and 3 blank control groups. Test groups sprayed 2 times a day with a phage suspension diluted in farm drinking water (10) ⁶ PFU/mL), the amount sprayed was 10mL/m ² The control group was sprayed with the same amount of 84 disinfectant 2 times a day, and the blank control group was operated only conventionally. Detecting the content change of salmonella in the environment before and after disinfection, taking 5 points of 4 points of a central point and a corner as test points, wherein the distance between the sampling point and the ground is 0.5m, the distance between the four points of the corner and the wall is 1m, and the salmonella is placed at each point9cm diameter SS plates. After sampling, the culture dish is placed in a constant temperature incubator at 37 ℃, and the bacterial colony number in each culture dish is recorded after the culture is carried out for 18-24 h.

Counting the total number of colonies C50000N/AT according to the Austenitic formula, wherein C is the total number of colonies per cubic meter (CFU/m) ³ ) (ii) a N is the number of colonies per dish; a is the area of the culture dish (cm) ² ) (ii) a T is sampling time (min).

2. Results and analysis of the experiments

As shown in FIG. 6, the content of Salmonella in the environment of the test group is lower than that of the control group (disinfected by 84 disinfectant) and the blank control group, which shows that the phage PYC03 can obviously reduce the content of Salmonella in the environment and reduce the infection rate of Salmonella, and the phage can be used as a novel biological environment disinfectant.

Example 9 test of treatment of Salmonella anatipestifer with bacteriophage PYC03 as additive for Drinking Water

40 diseased ducks infected with salmonella are randomly divided into 2 groups, and each group comprises 20 diseased ducks which are respectively a control group and a test group. Test group drink containing phage (10) ⁸ PFU/mL), control drinking water without phage. The medicine is continuously applied for 1 week, observed for 1 week, and then the death rate of the sick ducks is counted.

As a result: as can be seen from the results of Table 3 below, the mortality rate of the sick ducks in the test group was 70% lower than that in the control group. This demonstrates that the above phages can be used as a drinking water bio-additive to control salmonellosis. It can be used as feed additive for preventing and treating duck Salmonella bactericidal disease.

TABLE 3 death results of sick ducks

Example 10 preservation of meat quality by bacteriophage PYC03

1. The experimental method comprises the following steps:

sterile fresh duck meat 300g is equally divided into 6 parts, and the average part is divided into two experimental groups and a control group, wherein each group is 3 parts. Preparing high concentration bacteriophage suspension, and diluting with physiological salineReleased to the proper use concentration (about 10) ⁶ pfu/mL). 250 μ L of diluted phage was sprayed on the surface of the test group of duck meat, and the control group was sprayed with the same amount of sterile water. The treated samples were placed in sterile sealed bags and stored at 4℃, and samples were taken at 3d, 7d, 10d and 14d storage respectively for the determination of the Salmonella concentration and the volatile basic nitrogen (TVB-N) value according to the GB5009.228-2016 determination of volatile basic nitrogen in food products, 3 replicates per test group, and 3 replicates were performed.

2. The experimental results are as follows:

as shown in FIG. 8, the concentration of Salmonella in the test group was significantly lower than that in the control group. As shown in FIG. 7, the volatile basic nitrogen (TVB-N) values in the test group are significantly lower than those in the control group, and these results indicate that the bacteriophage PYC03 has an inhibitory effect on Salmonella in duck meat products, which is very important for the preservation of duck meat quality.

Example 11 Whole genome analysis of phage PYC03

Extracting the genome of the phage PYC03, performing whole genome sequencing, wherein the genome sequence obtained by sequencing is OK288022.1 in GenBank of NCBI, and the sequence analysis result is as follows:

(1) the PYC03 genome is 114770bp in total length, the content of G + C is 40.3%, and the content of basic group C, G, A, T is 19.7%, 20.6%, 30.3% and 29.5% in sequence. Whole genome RAST online annotation results show that the genome contains 181 Open Reading Frames (ORFs). Among the 181 Open Reading Frames (ORFs), 54 structural proteins were found, mainly including structural and packaging proteins of bacteriophage (noggin, tailin, tailfiber protein, cervical protein, capsid protein, and terminal enzyme large subunit, etc.), proteins involved in the cleavage of bacteriophage (lyase), proteins involved in DNA replication and modification (restriction endonuclease, DNA binding protein, intron-containing DNA polymerase precursor, HNH endonuclease, DEAD/DEAD cassette helicase, etc.), and other functional proteins (repeatedly infecting immunity protein). Meanwhile, of 181 ORFs, 147 initiation codons were ATG, 8 initiation codons were GTG, and 4 initiation codons were TTG. The genome was analyzed by software tRNAscan-SE for the absence of tRNA genes. The genome does not contain drug resistance genes and virulence genes through the analysis of an online tool CGE server. The genome was analyzed by PHASTER to be free of lysogenically related genes.

(2) In the genome of phage PYC 03: the gene sequence of tail fiber protein related to the recognition of phage host is shown in sequence 1 in the sequence table; the sequence of the highly conserved terminal enzyme large subunit (terminase large subunit) protein gene is shown as sequence 2 in the sequence table; the sequence of the DNA polymerase (DNA polymerase) gene is shown as sequence 3 in the sequence table; the gene sequence of lyase (lysozyme) related to the cleavage capability is shown in sequence 4 in the sequence table, and the related information of the sequences is specifically shown in the following table 4.

TABLE 4 Gene sequence information Table

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Sequence listing

<110> Qingdao Nonbert Biotechnology Ltd

<120> Salmonella anatipestifer phage, phage composition thereof and application thereof in prevention and treatment of Salmonella anatipestifer infection diseases

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<400> 1

atggcactta aaactaaaat tattgtacag cagattctga acatagatga cactacaact 60

actgctagta agtatcctaa gtatacagta gttttaggta attctatcag ttctattact 120

gctggtgaac taacagcggc tgttgaagcc gctgcagagt ctgctgctgc tgctaaagat 180

tctgaaatag cagctaaaga ctctgaaaat aaagctaaag attcggaaat tcaagcgggt 240

attcatgctg gtgcttctga ggcttcagca acccagtctg ctgcttctgc tgctgaatct 300

gaaagacaag ctaacttatc tcaaggtagt gcggaaaact ctgctgcttc tgctttagaa 360

tctaagaatt ttaaagatgc ttcggaactt gctgctcaaa atgcagagca gagtaagatt 420

ttagcagagc aagctcaaag agcggcagaa gctgcccagt ctggtgctca agcttctgaa 480

aataaagcat cagcatttgc tacacaagct gctgcatctt cagcttccgc aggagatttt 540

gctgcagccg ctaaacaatc tgaattaaat gctaaaactt ctgaaaccaa tgccgcaaca 600

tcagaagtgg aagcggaaac ccaagctgaa actgctacta ctgaggcaaa tcgtgctaag 660

gctgaagccg atcgcgcagc tcagattgta gatagtaagt tagataaaga agatatatct 720

ggctttatca aagtctacaa gactaaagaa gaagcggacg ccgacgttag tagccgcgta 780

ctaggtgaaa agatcctagt gtggaaccaa actgactcaa aatatggatg gtataaagta 840

gctggaactg ctgaggctcc agtattagag ttagtagaga cagagcaaaa gctagtttct 900

attaataacg ttcgtgcaga tgacgcaggt aacgtacaga ttactcttcc tggtggtaat 960

ccttccttat ggttgggtga agttacttgg ttcccttatg acaaagattc aggtgttggc 1020

tatcctggtg ttctccctgc tgatggccgc gaagtccttc gtgtagacta tccagatacg 1080

tgggaggcta tcgaagccgg tctgattcct tctgttactg aagaacaatg gcaagctggt 1140

gcaactctct atttctccac tggtaatggt actactactt tccgtctacc tgatatgatg 1200

caggggcagg cattccgtgc tgctgcaaaa ggcgaggaaa acgctggcaa tattaaagag 1260

caaattccgt acattactat gattaacggt aaagctcctg ctgacgatgg tacaattact 1320

ttaggtaatg ctgcagataa aaacgtatgg aatggtgttg atggtgaggt attactaaga 1380

ggtgcttttg gtcttggtgg tactggttta atccttaatg aacctgatgc cgtttccttc 1440

tttaaagcaa tgcgtgcttt tggttctgga tactatagaa atgactctga aagtaaccca 1500

gtgattccta agtattctgc tggattctac tccaaaactg ccgacactca tacttttatc 1560

tgttctgctt atggtaatgg tgttactttc gtagctacta taaatgatgc attattagat 1620

ggagaaaatc ctactgtaca tacaaatatt ctttatggta cagcaaataa acctgatctg 1680

aataccgata ctcaaggagt tttaggagta gagaagggcg gtactggtgc tactacacag 1740

aaaggtgcta gactaaatct ggatactcct gtaggcagca gagctattgg aatgcctaat 1800

aactctgatg tactagcttt catgaaatct tccgcagaaa gcggatatta ttcctctggt 1860

aatatagtta ctggagttcc agaaactgca ggatggtata tgttcgatct ccatgtacat 1920

ggtaagaatg ctgcgggaga aatggagtat ggtaatgtat actgtacaac aagtgctggt 1980

gctatttggt acaccttaat ggaggttggt gtatggcagc catggagacg tttgaccaca 2040

gaacatggta ttattcctat tacttcaggg ggtactggta caaataatgc aaatgacgca 2100

agaataaatc taggtcttgg tcctataaat gcacctactt ttagtggtat gactcttcag 2160

ggtactaatg aaactacttc aggtatagcg gtttttagta atagaaatac ggaagggact 2220

caactttcct attctagaat gtaccatgaa attcagagtg gtgttggtaa aactactatt 2280

cagactacaa gagagggcgg ggcgactaac tatttccaaa ttgatgagta tggtaatatt 2340

gggaatatta actcaattat tgcatatgga tatatgggat taggtgctgc taatgctatg 2400

ggaaatgcct ctattgcgat tggtgactct gactctgggc taaaatggaa tagtgatggt 2460

aacataagta ccatagcaga tggtgtaaaa atagccacat ggacacctca tggattttat 2520

acacataaaa taataagctc agatgttgct aataccgaaa gagggatgta tgtaaacggg 2580

gttaggacta ccggtgcctc cgctcttgta gctggggttg tagaagctgg atctcatgtt 2640

ggttggagag atagagcttc aggtatgctt gttgaattga atactagagg agctgctgcc 2700

aatatctgga aagcaactag atggggtgat caacatgctg gtgcatctga catcgttatt 2760

tatgatgatg gatctcctta ttatagaact cttgtaggcg gtggtgaatt tgggttcaat 2820

ggccttggac aagctacctg tacttcttgg attagtacat ctgatattag gcttaaggca 2880

cagctaaaag agatagtatc tgctaaagat aaggtaaaat ccctacaggg gtacacttat 2940

tttaaacgta atagtttggt tgaagatgag cattcttttt attgtgaaga ggcaggatta 3000

atcgcacagg atgttcaaac tgtactaccg gaagctgtat ataaaatagc taactcagat 3060

cttctcggtg ttaattactc tggtgttacc gcattattgg ctaacgcagt aaaagagatg 3120

ttggcggatg cggaggctca ggaagctcgt atcagtaatc tagaggaaga actggcagag 3180

ttaaaagctc tagtagccac tctggtaaat aagtaa 3216

<210> 2

<211> 1317

<212> DNA

<213> terminal Large subunit of bacteriophage PYC03 (terminase large subunit of PYC03)

<400> 2

atgaaagtct ctagagacta tattaacacc acagatgtta ttgactttgg tattgacaaa 60

cgattctttc gtctaccagt ctctagcatc ctagcacaag agggaattac acctaatgcc 120

cctcagattg caattattaa tgcattggaa gacccaagac atcgtttcgt gactgcatgt 180

gtatcacgtc gtgtaggtaa gtcattcatc gcatatacac ttgggttctt aaaacttctg 240

gaacccaatg tgaaggtgct ggtagttgca cctaactact cactggctaa cattggttgg 300

tcacagattc gtggtcttat taagaagtat ggattacaga ctgaacgtga gaacgccaaa 360

gataaagaga ttgaactagc gaatggttct ctctttaagc tggcttctgc agcgcaggct 420

gactccgcgg ttggtcgttc gtatgacttc atcatctttg acgaagcagc gatttccgat 480

gttggtggtg cagctttcga tattcagtta cgtcctaccc tagataagcc aaactctaaa 540

gctctattta tctctactcc tcgtgggggt aactggttca aagagttcta tgagaaagga 600

tttaacgaaa cgctacctaa ttgggtatct attcatggta catatcgtga taacccacgc 660

gctgacctga acgatatcga ggaagcgcgt cgtactgtta gtaaaaatta cttccgtcaa 720

gaatacgagg ctgacttctc cgtattcgaa ggtcagatct ttgatacctt caatgctatc 780

gagcatgtta aagaccttaa aggtatgcgt cacttcttta aagatgatga ggcattcgaa 840

acgttacttg gtattgacgt tggttatcgt gaccctacag cagttcttac tattaagtat 900

cactacgatg aagatgttta ttacgtactg gaagaatatc agcaagctga aaagactacc 960

gctcaacacg ctgcatatat ccaacactgc atagatcgtt ataatgttga tcgtatattt 1020

gtagactccg ctgctgctca gttccgtcag gacttagctt atgaacatga aatagcttct 1080

gcacctgcta agaaatctgt cctagatggt ttggcatgcc tgcaagcact gttccagcaa 1140

ggtaagatca tcgtagatgc ttcatgcacc tcattgattc acgccctagc gaactacaag 1200

tgggacttcc aggaaggcga agagaaatta tcacgtgaaa aaccacgtca tgatgcgaac 1260

tctcaccttt gtgacgcact gcgttatgga atttactcaa tttcccgtgg gaaataa 1317

<210> 3

<211> 2568

<212> DNA

<213> DNA polymerase of phage PYC03 (DNA polymerase of PYC03)

<400> 3

ttgaaaattg cagtagttga taaagctcta aataacactc gttatgataa acacttccag 60

ctttatggtg agaaagttga tgtattccat atgtgtaacg agaagttatc tggtcgtttg 120

ctgaaaaagc atattacaat cggaactcca gataacccgt tcgatccgaa cgattatgat 180

tttgttgtac tagttggtgc tgaacctttc ctgtatttcg caggtaagaa aggtatcggt 240

gattatactg gtaaacgtgt agagcatggt ggctatgcca actggattgc aagtattagt 300

ccagctcagc tacactttaa acctgaaatg aaaccagttt tcgatgcaac tgttgaaagt 360

attcatgaca ttattaatgg tcgtgagaag attgcaaaag ctggtgatta tcgtcctatt 420

actgatccag atgaagctga agaatacatc aagatggtgt ataacatggt tatcggacct 480

atcgcattcg actccgaaac ctcagctctg tatgctcgtg atggttatct tcttggtgtt 540

tctatgtctc accaagagta tcagggagtc tatattgact ctgacagcat caccgaagta 600

gctgtacatt atctccagaa aattctggat agcaagaatc atcagatcgt ttttcataac 660

ttgaagttcg atatgcactt ttactcctat catctaggac tttccttcga gaaagcacat 720

aaagagcgta gacttcatga taccatgttg caacactatg ttcttgatga acgtcgtggt 780

actcacggct tgaaatctct ggctatgaag tatactgaca tgggcgatta tgactttgaa 840

ctggataagt tcaaggatga ttattgcaag gcacataaga tcaagaaaga ggatttcagc 900

tatgatctga ttccatttga tattatgtgg ccttatgctg caaaagatac cgatgctact 960

ctgcgtctgc ataacttctt cttaccgaaa attgagaaga atgaaaaact ttgcagtcta 1020

tactatgatg ttctgatgcc tggttgcgta ttcctgcaac gtgttgaaga tcgtggtgta 1080

ccgatttctg ttgaccgctt gaaagaagct cagtatcagt taactcacaa cctgaacaag 1140

gctcgtgaaa agctgtatac ttatccagaa gtcgttcagc tagagaaaga tcagaacgaa 1200

gcgtttaacc cgaactctgt taagcaactt cgtgttcttc tgtttgatta tgttggctta 1260

actccaactg gtaaactgac tgatacaggt gcagattcta caaacgcaga agctctgaac 1320

gaactagcaa ctcagcaccc gattgcgaaa actctgctag agattcgtaa gctgactaag 1380

ctgatctcta catatgttga gaagattctt ctgagcatcg atgcagatgg ttgcattcgt 1440

accggtttcc acgaacatat gactacttct ggtcgtttaa gttcttctgg taagctaaac 1500

ctgcaacagt taccacgtga tgaatctatt attaagggtt gtgtagtagc tcctcctgga 1560

tatcgtgtaa tcgcatggga cttaacaact gcggaagttt actatgctgc tgttctgtct 1620

ggtgacagaa atatgcaaca agtatttatc aacatgaaga atgagcctga taaataccct 1680

gacttccatt caaacatcgc acacatggta tttaaactga cttgtgaacc acgtgatgtt 1740

aagaagctgt tcccagctct acgtcaggct gctaaggcaa ttaccttcgg tattctatat 1800

ggttctggcc cagcgaaagt agctcattct gttaacgaag ctcttctgga acaggctgct 1860

aaaacaggcg aaccgtttgt tgaatgtacc gttgcagatg cgaaggacta tatcgaaact 1920

tacttcggtc agttccctca gcttaagcgt tggattgata agtgccacga tcagatcaag 1980

aactttggat tcatctatag tcactttggt cgtaagcgtc gtctgcacaa catccattcc 2040

gaagatcgtg gtgttcaggg tgaagaaatc cgttctggat ttaacgcaat cattcagtct 2100

gcatcttctg atagcctgtt gttaggtgct attgatgctg atgaagaaat cctgtccctg 2160

ggtctaggaa aagagatgaa gatcgtcatg ttggttcacg actccgtggt tgctattgta 2220

cgtgaagatc tgattgatca gtataacgag attctgattc gtaacatcca gaaggatcgt 2280

ggtattagta tccctggctg cccaattggt attgattctg attccgagaa aggtggttct 2340

cgtgactact cttgtggtaa gatgaagaaa cagcacccat caatcgcttg tattgatgat 2400

gatgagtata ctcgttatgt taagggtgta ttactcgatg cagatttcga gtataagaag 2460

ttagctgcaa tggataaaga gcatccagac cacagcaagt acaaggatga taagtttatt 2520

gctgtatgta aagatctgga taatgtgaaa aggattttag gtgcttga 2568

<210> 4

<211> 369

<212> DNA

<213> lyase of the phage PYC03 (lysozyme of PYC03)

<400> 4

atgaaagcat atcaaagtct taagtctggc gtaattaaca ttgttcataa taatcagatc 60

attcaactat attctaaaga tggtgaactt aaacagaaag ttctagtcga agatctagag 120

ggtatcacac ctcactttga ccctgaggcc tttcgcattg tagatgtgga agttgttcct 180

caagtcgagg gtggtcagca cctaaatgtc aatgtgttaa gccgtgatca gcttttggat 240

gcacagaaac atcctgaaaa atatcctcag ttgaccatcc gtgtctctgg ctatgctgtt 300

cgatttaacg cattgacgcg tgaacaacag aacgacgtta ttagtcgtac atttactcag 360

gcgatataa 369

Claims

1. A duck salmonella bacteriophage is characterized by being named as PYC03, and the preservation number of the duck salmonella bacteriophage is CGMCC No. 22370.

2. A phage composition comprising the salmonella phage of claim 1 and an additional phage.

3. Use of a Salmonella anatipestifer bacteriophage of claim 1 or the bacteriophage composition of claim 2 in the manufacture of a medicament for the prevention and treatment of Salmonella anatipestifer infection.

4. The use of claim 3, wherein the Salmonella anatipestifer is selected from Salmonella anatipestifer, and Salmonella enteritidis.

5. A bacteriophage pharmaceutical preparation comprising as an active ingredient the salmonella duck bacteriophage of claim 1 or the bacteriophage composition of claim 2; preferably, the bacteriophage pharmaceutical preparation further comprises other bacteriostatic or bactericidal active ingredients; the pharmaceutical preparation is in the form of oral administration dosage form, external administration dosage form or intestinal administration dosage form.

6. A feed additive or drinking water additive comprising a salmonella duck bacteriophage according to claim 1 or a bacteriophage composition according to claim 2.

7. An environmental disinfectant, characterized in that the active ingredient comprises the Salmonella anatipestifer bacteriophage of claim 1 or the bacteriophage composition of claim 2; preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in the environment; preferably, the phage is used at a concentration of 1X 10 ⁹ PFU/ml or more.

8. The use of the environmental disinfectant in disinfecting duck farm environments as recited in claim 7, wherein said environmental disinfectant is capable of disinfecting the breeding environment, breeding utensils, with salmonella by spraying, soaking, said breeding environment including troughs, floors, walls, manure and bedding.

9. A food preservative comprising the salmonella phage PYC03 of claim 1 or the phage composition of claim 2.

10. A detection kit comprising the salmonella bacteriophage of claim 1 or the bacteriophage composition of claim 2.