CN111705042A

CN111705042A - Pasteurella phage vB _ PmuP _ PS02, phage composition and application thereof

Info

Publication number: CN111705042A
Application number: CN202010622010.4A
Authority: CN
Inventors: 潘强; 任慧英; 孙虎芝; 闫艳新; 崔天丽
Original assignee: Qingdao No Antibiotics Biotechnology Co ltd
Current assignee: Qingdao No Antibiotics Biotechnology Co ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2020-09-25
Anticipated expiration: 2040-07-01
Also published as: CN111705042B

Abstract

The invention discloses a pasteurella phage vB _ PmuP _ PS02, a phage composition thereof and application thereof, wherein the pasteurella phage vB _ PmuP _ PS02 is preserved in China general microbiological culture Collection center at 15/05 in 2020 with the preservation number of CGMCC No. 19972. The bacteriophage and the compounded bacteriophage composition thereof can be used for preparing medicaments for preventing and treating various livestock diseases caused by pasteurella infection, particularly have obvious effect on swine plague and progressive atrophic rhinitis of pigs caused by swine pasteurella disease, and can also be used for preparing pig feed additives, environment and feed disinfectants and the like. The bacteriophage and the bacteriophage composition thereof are safe to use, have no side effect, and effectively avoid the problems of antibiotic residue caused by the traditional use of antibiotics and the induction of drug-resistant pasteurella.

Description

Pasteurella phage vB _ PmuP _ PS02, phage composition and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a pasteurella phage, a composition containing the phage and application of the phage.

Background

Pasteurella multocida is an important zoonotic pathogen and can infect various livestock, poultry and wild animals to cause Pasteurella multocida. Common pasteurellosis includes mainly hemorrhagic septicaemia and various respiratory diseases in animals.

The swine is one of susceptible animals of pasteurella multocida, and the pasteurella multocida serotypes which are prevalent in the swinery are mostly A type and D type, so that swine plague and progressive atrophic rhinitis of the swine are mainly caused. In addition, Pasteurella multocida is often co-infected with pathogens such as Streptococcus suis, porcine reproductive and respiratory syndrome virus, Actinobacillus pleuropneumoniae, and the like. The outbreak and the prevalence of pasteurella multocida cause huge economic losses in China and the pig industry all over the world. Therefore, the development of a novel phage preparation with remarkable curative effect and safe use for preventing and treating swine pasteurellosis is imminent.

The bacteriophage is a virus capable of specifically infecting bacteria, and can be separated from natural environments such as sewage, excrement, soil and the like. Phage therapy has several advantages, firstly, the multiplication efficiency is high, one phage can generate hundreds of filial generations, and the capability is also a remarkable advantage of the treatment mode and plays a crucial role in treatment; the bacteriophage has no side effect in treatment, which is different from antibiotic treatment, and the treatment by using the antibiotic can not only eliminate the bacteria causing infection, but also influence other flora in intestinal tract, cause micro-ecological imbalance and reduce immunity of organism, and the bacteriophage has specificity and can not influence bacteria of other species; the bacteriophage treatment does not need to consider the residue problem, is a strict host-dependent organism and does not remain in the animal body, so the bacteriophage can be used as a high-efficiency biological disinfectant and medicament with a bactericidal effect.

However, no effective pasteurella phages have been available for controlling pasteurellosis, and the prior art is therefore in need of further improvement.

Disclosure of Invention

In order to solve the problems, the invention provides a novel pasteurella phage vB _ PmuP _ PS02 with broad-spectrum strong lytic property, and a phage composition formed by compounding the phage; the bacteriophage can be used for preparing medicines for preventing and treating various livestock diseases caused by Pasteurella multocida infection, especially has remarkable effect on swine plague and progressive atrophic rhinitis of swine caused by swine Pasteurella multocida disease, and can also be used for preparing pig feed additives, environment and feed disinfectants, etc. The bacteriophage and the bacteriophage composition thereof are safe to use, have no side effect, and effectively avoid the problems of antibiotic residue caused by the traditional use of antibiotics and the induction of drug-resistant pasteurella.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a broad-spectrum strong-lytic pasteurella phage vB _ PmuP _ PS02, which is separated from pig manure of a certain farm in Qingdao Shandong, is preserved in China general microbiological culture Collection center (CGMCC) at 15/05 of 2020 with the preservation number of CGMCC No. 19972.

The Pasteurella phage vB _ PmuP _ PS02 can form plaques with the diameter of about 0.5-1.5 mm on a double-layer agar culture medium, and the boundary is clear. Observed under an electron microscope: the length of The head of The phage is 63nm in diameter and 55nm in width, The length of The non-flexible tail is 27nm, and The phage can be determined to be a brachyphagidae and named as vB _ PmuP _ PS02 according to The classification standard reported by The ninth time of The International Committee on Taxomy of Virus, ICTV.

In a second aspect, the present application also provides the use of the pasteurella phage described above in the manufacture of a medicament for the prevention and treatment of disease caused by pasteurella infection. The term "prevention" is meant herein to include all actions that inhibit or delay the disease by administering the bacteriophage. The term "treatment" is meant herein to include all actions that would improve or ameliorate the disease by administration of the bacteriophage.

Preferably, the disease caused by pasteurella infection includes pasteurellosis of poultry and livestock caused by pasteurella infection. Preferably, the pasteurella is selected from pasteurella of poultry and livestock origin. Wherein the pneumonia and septicemia of animals such as pig, cattle, rabbit, and sheep, and fowl cholera are described.

In a third aspect, the invention also provides a phage composition comprising pasteuria phage vB _ PmuP _ PS02 as described above and other phage. The phage composition can be compounded with other pasteurella phage vB _ PmuP _ PS02 to prepare various products for preventing and treating pasteurellosis, such as drugs, disinfectants, preservatives and the like.

Preferably, a phage composition comprises pasteuria phage vB _ PmuP _ PS02 and pasteuria phage vB _ PmuP _ PS 01. By the complementary compounding of the two high-cracking-rate phages, the cracking spectrum of the two phages to various animal source pasteurella is further widened, and the method has better application prospect.

The pasteurella phage vB _ PmuP _ PS01 is separated from pig manure of a certain farm in Qingdao Shandong, and is preserved in China general microbiological culture Collection center (CGMCC) at 05-15 days of 2020 with the preservation number of CGMCC No. 19971. The head of the phage vB _ PmuP _ PS01 is a polyhedron with the diameter of about 55nm, the non-flexible tail is 20nm long, and the phage vB _ PmuP _ PS01 is a brachyphagidae phage and is also a Pasteurella phage with high cracking performance.

Preferably, the above phage composition further comprises one or more of mutants of phage vB _ PmuP _ PS 02; the mutant has homology of not less than 90% with the corresponding phage and keeps basically the same bacteriostatic activity.

Since bacteriophages are very susceptible to mutations during replication, it is preferred that mutants of the aforementioned bacteriophages are also within the scope of the present application. The determination of homology can be suitably carried out by computer programs well known in the art, and the mutants of vB _ PmuP _ PS02 have at least 90% homology with the natural sequence of the phage; more preferably, the mutants are 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the native sequence of the respective phage. Wherein the sequence of vB _ PmuP _ PS02 can be sequenced by known methods according to the biological material deposited according to the invention. The mutants of the phage may be point, deletion or addition mutations, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases may be changed relative to the original phage sequence. It is not necessary for the skilled person to inventively work to select a mutant with a similar trait from the phages provided according to the invention.

In a fourth aspect, the present invention also provides a bacteriophage pharmaceutical preparation, the active ingredient of which is mainly the pasteurella bacteriophage or the bacteriophage composition. Preferably, the phage drug formulation may also include a phage associated with other specific pathogenic bacteria.

Optionally, the phage pharmaceutical preparation is in the form of oral administration or injection, preferably, the injection is intraperitoneal injection. The dosage form of the pharmaceutical preparation is specifically solution, powder, gel, granule, and lyophilized preparation.

Optionally, the phage drug preparation 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 and ethanol. 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 a swine feed additive comprising the pasteurella phage vB PmuP PS02 or phage composition as described above, fed to a herd of swine after being mixed with swine feed to achieve the effect of preventing or treating pasteurellosis in swine⁸PFU/g。

In a sixth aspect, the present application also provides a disinfectant, the effective component of which is mainly the pasteurella phage vB _ PmuP _ PS02 or phage composition, preferably, the titer of the phage is 1 × 10⁸PFU/ml or more. The environmental disinfectant also contains other active ingredients for inhibiting or eliminating viruses and bacteria in the environment. The solvent used for the disinfectant is physiological saline or PBS.

The disinfectant can be used for environmental disinfection and preservation of poultry and livestock breeding places, can replace antibiotics or traditional disinfection products, and the bacteriophage and the metabolite of the environmental disinfectant thereof cannot cause damage to human bodies or other animals. The environment disinfectant can be used for comprehensively disinfecting breeding environments, feeding appliances and the like by virtue of pasteurella through spraying and soaking. The culture environment comprises a trough, the ground, a wall, excrement and padding. The liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, and the like. Preferably, the farm is a piggery.

In a seventh aspect, the invention also provides a test kit comprising the pasteuria phage vB _ PmuP _ PS02 or phage composition as described above. The skilled artisan can use the pasteurella bacteriophages or phage compositions thereof described above to prepare test kits for detecting pasteurella specifically infected therewith, or for controlling diseases caused by pasteurella infection in its host, based on the present disclosure and general knowledge in the art.

In an eighth aspect, the present invention also provides a biological bacteriostatic agent for disinfecting fresh food of poultry and livestock, which comprises the above pasteurella phage vB _ PmuP _ PS02 or phage composition as the active ingredient. The use method of the biological bacteriostatic agent comprises the following steps: the surface of the fresh products of poultry and livestock is soaked or sprayed for disinfection, so as to inhibit the proliferation of pasteurella in the processing or fresh-keeping process of the products.

The invention has the following beneficial effects:

1. the phage vB _ PmuP _ PS02 has a strong cracking effect on pasteurella, can effectively prevent and control pasteurella diseases of various animal farms, has the most obvious effect on swine pasteurella diseases, greatly reduces the incidence rate of swine plague and swine progressive atrophic rhinitis caused by pasteurella multocida, and can sterilize pasteurella for feeding environment, feed, drinking water and the like.

The phage composition formed by compounding the pasteurella phage vB _ PmuP _ PS01 and the pasteurella phage vB _ PmuP _ PS02 further widens the lysis spectrum of the pasteurella of various animal sources by the complementary matching of the two strains of high-quality phage, and has wider application prospect.

2. The bacteriophage is obtained from nature, and is easy to be industrialized, and the medicine or disinfectant prepared by the bacteriophage can reduce the cost; moreover, the treatment of the bacteriophage or the bacteriophage composition does not need to consider the problem of drug residue, and can be widely applied as a safe and efficient biological disinfectant and drug.

3. The pasteurella bacteriophages and the bacteriophages composition thereof have wide application, can be used for preparing pharmaceutical preparations for preventing and treating pasteurella diseases, can be widely used in various links easily causing loss due to pasteurella infection in the breeding process of livestock and poultry, daily disinfection of breeding environment, bacteriostasis of fresh products and the like by mixing with feed and drinking with water and being used as a disinfectant, and are beneficial to the healthy development of poultry and livestock breeding industry, especially pig breeding industry.

Drawings

FIG. 1 is a plaque photograph of bacteriophage vB _ PmuP _ PS 02;

FIG. 2 is an electron micrograph of bacteriophage vB _ PmuP _ PS 02;

FIG. 3 shows the result of the thermostability detection of bacteriophage vB _ PmuP _ PS 02;

FIG. 4 shows the result of pH stability test of the phage vB _ PmuP _ PS 02;

FIG. 5 shows the effect of bacteriophage vB _ PmuP _ PS02 on the OD600 value of Pasteurella.

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 of phage culture and biological Properties

Recovery culture of bacterial strain and preparation of bacterial suspension

And (3) selecting the frozen bacteria liquid of the pasteurella, streaking the frozen bacteria liquid in three zones on a TSA (TSA) plate (added with 5% newborn calf serum), separating a single colony, and culturing for 16-24 h in a 37 ℃ incubator. A single colony was picked and inoculated into 5ml TSB broth (supplemented with 5% newborn calf serum), and cultured with shaking at 180rpm in an air shaker at 37 ℃ for 19h to give a single bacterial suspension.

(II) isolation and purification of bacteriophages

Taking pig manure sample, padding, sewage, fur and other samples, adding a proper amount of TSB broth, oscillating at 37 ℃, 170rpm for 30min, centrifuging at 10000rpm for 5min, and filtering supernatant by a 0.22 mu m bacterial filter for later use.

Each of the flasks was filled with 80% of Pasteurella pasteurella, 20% of the supernatant obtained by centrifugation (5% of newborn calf serum) was added, the mixture was stirred well, incubated overnight at 180rpm in an air shaker at 37 ℃ and centrifuged at 10000rpm for 5min, and sterilized by filtration through a 0.22 μm bacterial filter. Mixing the filtrate with host bacteria, incubating at 37 deg.C for 5min, pouring into double-layer flat plate, standing until it is solidified, and placing into 37 deg.C incubator for inverted culture overnight. If the bacteriophage exists, transparent and regular circular plaques are formed on the culture medium, namely the plaques are formed. And (3) digging single plaques, incubating in 1ml of TSB broth in an air oscillator at 37 ℃ for 30min, centrifuging at 10000rpm for 5min, taking the supernatant, obtaining the single plaques by using a double-layer plate method, and repeating the steps for 3-5 times until circular transparent plaques are formed, the boundaries are clear, and the plaque diameter is about 0.5-1.5 mm.

(III) propagation and titer determination of phages

Adding 100 mu l of host bacteria and phage spot-removing leaching solution into 5ml of TSB broth (added with 5% newborn calf serum), culturing for 5-6 h at 37 ℃ in an air oscillator at 180rpm, and obtaining phage proliferation solution after the mixed solution becomes clear. Diluting phage proliferation solution by 10 times, measuring titer by double-layer plate method, and diluting each dilution3 replicates were made separately. Taking the dilution of 10^-6The results of counting 3 replicates were 243, 247 and 245 plaques, respectively, and the titer was calculated to be 2.45 × 10⁹PFU/ml。

(IV) Transmission Electron microscopy for observing the morphology of the phage

Is taken to be higher than 1 × 10⁹Mu.l of the PFU/ml phage sample was dropped onto a microporous copper mesh, precipitated for 15min, and excess liquid was blotted off with filter paper. 15 μ l of 2% phosphotungstic acid (PTA) was dropped on the copper mesh, dyed for 5min, and excess dye solution was sucked off with filter paper, dried, observed with a transmission electron microscope and photographed.

The electron microscope pictures are polyhedrons with a head length of 63nm and a width of 55nm and a non-flexible tail length of 27nm, and The phage can be determined to be The brachyphagidae according to The classification standard reported by The International Committee on Taxomy of viroses, ICTV for The ninth time, and is named as vB _ PmuP _ PS 02.

(V) detection of thermostability of phage

2.45 × 10⁹The proliferation solution of PFU/ml phage vB _ PmuP _ PS02 is respectively acted in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C for 20min, 40min, 60min, and each temperature is set as two parallel groups. The titer of the phage was determined by a double-layer plate method.

The result shows that the phage vB _ PmuP _ PS02 basically keeps the original activity after 1h of action at 40 ℃ and 50 ℃; the titer is reduced by 2 orders of magnitude after 20min at 60 ℃, and the titer is reduced by 3 orders of magnitude after 1 h; the phage is inactivated at 70 deg.C and 80 deg.C for 20 min. The test results show that the phage vB _ PmuP _ PS02 can withstand high temperatures of 50 ℃ and below.

(VI) detection of the pH stability of the phages

Adding TSB broth 4.5ml with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) into sterile test tube, placing three tubes in water bath at 37 deg.C, and adding 500 μ l2.45 × 10 after temperature stabilization⁹PFU/ml phage proliferation liquid, mixing uniformly at 37 deg.C in water bath for 1h, 2h, 3 h. After the action is finished, adding a proper amount of HCl or NaOH into the mixed solution to ensure that the pH value of the mixed solution is about 7, and measuring the phage by a double-layer plate methodThe potency of (A).

As a result, the titer of the phage vB _ PmuP _ PS02 is almost unchanged or slightly reduced within the pH range of 5-11 and still remains at 10⁹The titer is reduced by 1 order of magnitude after 2 hours when the pH is more than PFU/ml and 4, so that the phage has a wide application range to the pH.

(seven) determination of the optimal multiplicity of infection (MOI) of bacteriophages

The Pasteurella phage vB _ PmuP _ PS02 and the host bacterium Pasteurella were propagated according to the conventional method, initial titer of phage and host bacterium concentration were determined, and phage vB _ PmuP _ PS02 and the host bacterium were appropriately diluted. 100 μ lvB _ PmuP _ PS02 and host bacteria were added to the TSB broth at a ratio of multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, respectively. The culture was shaken at 180rpm at 37 ℃ until the broth became clear, and the time to clear the broth was recorded. Centrifugation was carried out at 10000rpm for 5min, and the titer of phage was measured by the double-layer plate method, and the results are shown in Table 1.

TABLE 1 optimal multiplicity of infection (MOI) assay results for bacteriophages

As a result, it was found that the optimum multiplicity of infection of the phage was 0.01, under which the titer of the progeny phage produced by infecting the host bacterium with the phage was 8.97 × 10⁹PFU/ml, the phage titer was highest among 4 multiplicity of infection.

Example 2 determination of bacteriophage lysis Spectroscopy and in vitro lysis test

Lysis Spectroscopy determination of (A) phage vB _ PmuP _ PS02

(1) Lysis experiment of phage on swine pasteurella

In this example, 50 strains of Pasteurella suis from Shandong, Henan, Guangxi, Guangdong, etc. were selected as host bacteria for detecting the lysis spectrum of phage.

1. Testing of phage lysis profile: the method for determining the lysis spectrum of the phage by adopting a double-layer plate method comprises the following steps: a bacterial suspension of the host bacteria was prepared as in example 1. After 300. mu.l of the bacterial suspension and 300. mu.l of pasteurella phage are incubated at 37 ℃ for 5min, the mixture is added into upper agar (5% newborn calf serum is added) to prepare a double-layer plate, the double-layer plate is placed in a thermostat at 37 ℃ after the agar is solidified, the double-layer plate is inverted and cultured overnight to observe a cracking result, and the statistical result is shown in Table 2.

2. Detection of drug resistance of host bacteria: the drug resistance of 50 porcine pasteurella is detected by a drug sensitive paper sheet method, and the drug sensitive sheet comprises doxycycline, tetracycline, chloramphenicol, gentamicin, enrofloxacin, florfenicol, kanamycin and ofloxacin, and the result is shown in table 2 below.

3. Experimental results and analysis:

(1) from the results in Table 2, it is understood that 50 strains of swine pasteurella show different drug resistance characteristics, and the ratio of pasteurella having multiple drug resistance reaches 90%.

(2) After the lysis spectrum was measured using these 50 resistant Pasteurella as host bacteria, it was found that: the phage vB _ PmuP _ PS02 can crack 45 strains therein, the cracking rate reaches 90%, and the phage vB _ PmuP _ PS02 has excellent cracking performance on the swine pasteurella.

TABLE 2 lysis Profile of the Pasteurella phage vB _ PmuP _ PS02 against 50 strains of Pasteurella of porcine origin

(2) Lysis experiment of phage vB _ PmuP _ PS02 on other animal-derived Pasteurella multocida

A. The experimental method comprises the following steps: determination of Pasteurella phage vB _ PmuP _ PS02 the following different animal-derived Pasteurella were subjected to lysis profiling according to the experimental method described previously, and the results are shown in Table 3 below.

TABLE 3 lysis spectra of the Pasteurella phage vB _ PmuP _ PS02 against other sources of Pasteurella

B. Experimental results and analysis:

for 47 strains of pasteurella rabbit origin, 35 strains of phage vB _ PmuP _ PS02 can be lysed, and the lysis rate reaches 74.47%; for 25 strains of the fur-bearing animal source pasteurella, 21 strains of the phage vB _ PmuP _ PS02 can be cracked, and the cracking rate reaches 84%; for 12 strains of bovine pasteurella, 10 strains of phage vB _ PmuP _ PS02 can be lysed, and the lysis rate reaches 83.33%; for 13 strains of pasteurella avicularis, 10 strains of phage vB _ PmuP _ PS02 can be lysed, and the lysis rate reaches 76.92%. Therefore, the bacteriophage vB _ PmuP _ PS02 has excellent cracking performance on swine, rabbit, fur-bearing animal, cattle and poultry, wherein the highest cracking rate on swine pasteurella is achieved.

(3) Lysis profiling of phage compositions

A. The experimental method comprises the following steps: the lysis spectrum determination was carried out using a phage composition formed by compounding phages vB _ PmuP _ PS01 and vB _ PmuP _ PS02 in a ratio of 1:1, the experimental procedure being identical to that described previously.

TABLE 4 lysis profiles of Pasteurella phage compositions against different sources of Pasteurella

B. Experimental results and analysis:

for 50 porcine pasteurella, the phage composition was able to lyse 47 of the strains, with a lysis rate of 94%; for 47 strains of pasteurella rabbit origin, the phage composition can lyse 42 strains therein, and the lysis rate reaches 89.36%; for 25 strains of fur-bearing animal derived pasteurella, the phage composition can crack 22 strains therein, and the cracking rate reaches 88%; for 12 strains of bovine pasteurella, the phage composition can crack 10 strains of the 12 strains, and the cracking rate reaches 83.33%; for 13 strains of avian pasteurella, the phage composition was able to lyse 12 of them, with a lysis rate of 92.31%. Therefore, the phage composition has excellent cracking performance on pig sources, rabbit sources, fur animal sources, cattle sources and poultry sources. Compared with a single-strain bacteriophage, the bacteriophage composition has better cracking performance, and makes up for the defects of the single-strain bacteriophage.

(II) vB _ PmuP _ PS02 in vitro lysis test (OD method) on Pasteurella strain

1. The experimental method comprises the steps of setting 4 experimental groups and a control group to carry out a cracking experiment, adding pasteurella bacteria liquid and phage vB _ PmuP _ PS02 into a container in sequence according to a certain proportion in each experimental group, wherein the final concentration of pasteurella is 1.00 × 10⁹CFU/ml, the final concentration of phage in each of 4 experimental groups was 1.00 × 10⁹PFU/ml、1.00×10⁸PFU/ml、1.00×10⁷PFU/ml and 1.00 × 10⁶PFU/ml, the same amount of sterile broth as the phage in the experimental group, and the same amount and concentration of Pasteurella pasteurella as the experimental group were added to the control group. After mixing the bacterial liquid and the phage (5% newborn calf serum is added), the mixture is cultured by shaking in a shaker at 37 ℃ and 180 rpm. And measuring the OD value of the mixed solution at regular intervals until the mixed solution becomes clear, and simultaneously measuring the residual quantity of each group of bacteria acting at different times by a coating plate method.

2. The experimental results are as follows: from the experimental results of table 3, table 4 and fig. 5, it can be known that the lysis effect of the phage vB _ PmuP _ PS02 on the pasteurella strain is very good, the lysis efficiency of the 4 phage liquids with different concentrations on the pasteurella strain can reach more than 97.82%, and only the difference of the lysis time is that within 6h, the phage can achieve a very good killing effect on host bacteria at the four concentrations.

TABLE 3 change in OD value at each time period

TABLE 4 determination of the residual amounts of various groups of bacteria after the completion of OD value determination

Example 3 safety test of phages

Selecting 20 healthy SPF mice with the body weight of 20-22 g, dividing the mice into an experimental group and a control group, wherein the number of the mice in each group is half, and injecting purified phage proliferation solution (200 mu l10 mu l) into the abdominal cavity of the mice in the experimental group respectively⁹PFU/ml), mice in the experimental group were injected with the same amount of physiological saline (200 μ l) intraperitoneally for 3 days continuously, and changes in organs of the mice were observed by autopsy for 7 days.

The results show that the behavior of the mice of the experimental group and the control group is not abnormal and the death phenomenon does not occur; the autopsy finds that the liver, the lung, the heart, the spleen, the kidney and other organs of the mice in the group are normal, and has no obvious difference with the control group.

Example 4 testing of the therapeutic Effect of phages on Swine-derived Pasteurella after challenge

(I) detecting the treatment effect of the bacteriophage on pasteurellosis by oral feeding

1. The experimental method comprises the following steps: preparing 40 healthy SPF mice with the body weight of 20-22 g, dividing the mice into a test group and a pair in equal partsAnd (4) performing group control. According to half lethal dose ELD₅₀，2×10³CFU/mouse dose Each mouse group was intraperitoneally injected with Pasteurella ZS02, and after 6h of challenge, mice in the test group were orally administered with 500. mu.l of PBS diluted to 10⁹PFU/ml vB _ PmuP _ PS02 phage treatment, control mice each orally administered the same volume of PBS 1/d, observed for 1 week after 3 days of continuous administration, and each group of mice was scored for mortality.

2. The experimental results are as follows: after 1 week, the survival rate of the control group mice was only 30%, while the survival rate of the test group mice was as high as 85% after oral administration of the phage vB _ PmuP _ PS 02. This shows that oral administration of the bacteriophage vB _ PmuP _ PS02 can prevent and treat pasteurellosis of mice caused by swine pasteurella and has obvious effect.

(II) treatment test of Pasteurella by intraperitoneal injection

1. The experimental method comprises the following steps: 40 healthy SPF mice with the body weight of 20-22 g are divided into a test group and a control group, and the mice are male and female. According to half lethal dose ELD₅₀，2×10³CFU/P.intraperitoneal injection of Pasteurella in clinical isolation of pathogenic Pasteurella strain ZS02, after 6h of challenge, the test groups were administered 200. mu.l of 10 i.p. daily⁹PFU/ml vB _ PmuP _ PS02 phage treatment, control group each mice intraperitoneal injection of the same volume of PBS, 1/d, continuous 3 days after observation for 1 week, record the mice death.

2. The experimental results are as follows: the results showed that the survival rate of the mice in the control group was only 30%, while the survival rate of the mice in the test group was as high as 90% after the intraperitoneal injection of the phage vB _ PmuP _ PS 02. This shows that the intraperitoneal injection administration mode of the phage vB _ PmuP _ PS02 can prevent and treat mouse pasteurellosis caused by swine pasteurella and has obvious effect.

Example 5 bacteriophage in vivo prevention test against Pasteurella

1. The experimental method comprises the following steps: 40 healthy SPF mice with the body weight of 20-22 g are divided into a test group and a control group, and the mice are male and female. Experimental groups Each mouse was orally administered with 1ml 10⁹PFU/ml vB _ PmuP _ PS02, control group 1ml PBS/mouse orally, after continuous feeding for 3 days, experimental group and control group were according to 2 × 10³CFU/amount of Pasteurella intraperitoneally injected with the pathogenic strain ZS02 clinically, and after challenge, mice were recorded for 7 d. After 7d all mice were sacrificed for necropsy to observe symptoms and to isolate pasteurella for statistical morbidity.

2. The experimental results are as follows: after 7d observation, the mortality rate of the control group is 60 percent, and the mortality rate of the experimental group fed with the phage is 0; the lesion rate of the control group is as high as 80%, and the lesion rate of the experimental group fed with the phage is only 10%. This shows that the phage vB _ PmuP _ PS02 has a good effect of inhibiting and killing Pasteurella in mice.

Example 6 testing of the Sterilization of the bacteriophage vB _ PmuP _ PS02 on feed

1. The experimental method comprises the following steps: 20g of big pig feed is evenly divided into 2 parts, each part is 10g, and the 2 parts are spread on a sterilized plate to be respectively used as experimental objects of an experimental group and a control group. Respectively taking 1ml 10⁹Uniformly spraying the bacterial liquid of the Pasteurella pasteurella ZS02 at a concentration of CFU/ml on the surfaces of the feeds of the experimental group and the control group, and diluting 1ml of the bacterial liquid to 10 ml by PBS after drying⁹PFU/ml vB _ PmuP _ PS02 phage was sprayed uniformly on the feed surface of the experimental group, the same amount of PBS was sprayed correspondingly on the feed surface of the control group, the concentration of bacteria carried by the feed was determined by the coating plate method after 0h, 1h, 2h, 4h, 6h and 8h, respectively, and the concentration of phage carried by the feed was determined by the double-layer plate method.

TABLE 6 bacterial and phage content in experimental and control groups

2. The experimental result shows that the phage vB _ PmuP _ PS02 can survive in feed for more than 8h and the highest titer can reach 3.01 × 10⁸PFU/g, after spraying phage for 4-6h, the pasteurella content is greatly reduced, and the maximum content is reduced to 6 titer, which shows that phage vB _ PmuP _ PS02 has significant bactericidal effect on pasteurella carried on pig feed, and phage vB _ PmuP _ PS02 can be widely applied to disinfection and preservation of feed.

Example 7 decontamination of porcine Pasteurella by bacteriophage vB _ PmuP _ PS02

The experimental method comprises selecting a certain pig farm in Qingdao for experiment, wherein pasteurellosis occurs for multiple times in multiple pigsties of the pig farm, selecting weaned piglets of 1 month old, feeding bacteriophage in a feed adding manner at an amount of 5 × 10 in 20 columns of experimental group, 15 weaned piglets in each column, 20 columns of control group and 15 weaned piglets in each column, and feeding the bacteriophage in the experimental group⁷PFU/kg feed, 1 time per day, and 1 × 10⁶PFU/ml phage vB _ PmuP _ PS02 for pigsty disinfection 2 times per week, other according to the pig farm regulation normal feeding, medication, immunity; the control group was not fed with phage and the pig farm was disinfected, and the other operations were carried out normally according to the pig farm protocol, medication and immunization. The test group was continuously administered for 1 month, while the number of pasteurellosis-developing pigs in the test group and the control group was observed and recorded.

Experimental results and analysis: the number of the control group is 25, and the incidence rate is 8.33%; the number of diseases in the test group is 3, and the disease incidence is 1%; the incidence of disease in the test group was reduced by 7.3% compared to the control group, indicating that the use of phage significantly reduced the incidence of infection of swine with pasteurella. The results show that the bacteriophage vB _ PmuP _ PS02 can provide a novel biological agent or feed additive and environmental disinfectant for the purification of Pasteurella.

Example 8 therapeutic Effect of bacteriophage vB _ PmuP _ PS02 on porcine pasteurellosis

The experimental method comprises selecting a certain pig farm with pasteurellosis in Qingdao for experiment, selecting 20 weaned piglets of about 1 month old with pasteurellosis, 10 experimental groups and 10 control groups, and injecting bacteriophage into the experimental groups through muscle with dosage of 5 × 10⁷PFU/kg, 1 time per day, and 1 × 10⁶Sterilizing pigsty with PFU/ml phage vB _ PmuP _ PS02, 3 times per week, and feeding other pigs normally according to the pig farm protocol; the control group was treated intramuscularly with ceftiofur sodium at 3-5mg/kg 1 time per day without phage, and the others were fed normally according to the pig farm protocol. After 5 days of continuous medication, the clinical symptoms and death number of the sick pigs in the test group and the control group are observed and recorded.

The experimental results show that: the clinical symptoms of 8 piglets in the experimental group, such as asthma, cough and abdominal respiration, disappear and are obviously improved, 2 piglets die, the effective rate is 80%, the clinical symptoms of 6 piglets in the contrast antibiotic treatment group, such as asthma, cough and abdominal respiration, disappear and are obviously improved, 4 piglets die, and the effective rate is 60%. The effect of the phage treatment group is better than that of the antibiotic control group. The results show that the bacteriophage vB _ PmuP _ PS02 can provide a novel, safer and more effective biological agent for treating pasteurellosis.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A Pasteurella phage vB _ PmuP _ PS02 is characterized in that the preservation number is CGMCC No. 19972.

2. Use of the pasteuria phage vB _ PmuP _ PS02 according to claim 1 in the preparation of a medicament for the prevention and treatment of diseases infected by pasteuria.

3. The use according to claim 2, wherein the pasteurella infection disease comprises swine plague, progressive atrophic rhinitis in pigs.

4. A phage composition comprising the pasteuria phage vB _ PmuP _ PS02 of claim 1 and other phage.

5. The phage composition of claim 4, comprising Pasteurella phage vB _ PmuP _ PS01 and Pasteurella phage vB _ PmuP _ PS 02.

6. The phage composition of claim 4, further comprising one or more mutants of phage vB _ PmuP _ PS 02; the mutant has homology of not less than 90% with the corresponding phage and keeps basically the same bacteriostatic activity.

7. A bacteriophage pharmaceutical preparation comprising as an active ingredient pasteurella phage vB _ PmuP _ PS02 according to claim 1 or phage composition according to any one of claims 4 to 6.

8. A phage drug preparation according to claim 7, wherein said drug preparation is in the form of oral administration and injection.

9. The phage drug preparation according to claim 7, further comprising a pharmaceutically acceptable carrier in the form of solution, powder, lyophilized preparation or granule.

10. Pig feed additive, comprising the pasteurella phage vB p pu p PS02 of claim 1 or the phage composition of any one of claims 4 to 6, preferably wherein the concentration of each phage in the feed is at least 1 × 10⁸PFU/g。

11. A disinfectant, characterized in that the effective component comprises pasteurella phage vB _ PmuP _ PS02 according to claim 1 or phage composition according to any one of claims 4-6, and further comprises other active components for inhibiting or eliminating viruses and bacteria in the environment, preferably, the use concentration of phage is 1 × 10⁸PFU/ml or more.

12. The use of the disinfectant according to claim 11, wherein said disinfectant is used for pasteurisation of farming environments, including barns, silos, floors, walls, manure and bedding, by spraying, soaking.

13. A test kit comprising the Pasteurella phage of claim 1 or the phage composition of any of claims 4-6.

14. A biological bacteriostatic agent for disinfecting pork food, which is characterized by comprising the Pasteurella bacteriophages of claim 1 or the phage composition of any one of claims 4-6; the use method of the biological bacteriostatic agent comprises the following steps: the surface of the pork food product is soaked or sprayed with a biological bacteriostatic agent for disinfection, so as to inhibit the proliferation of pasteurella in the product processing or fresh-keeping process.