CN116286674A - Riemerella anatipestifer phage vB_RanS_PT24, phage composition and application thereof - Google Patents

Riemerella anatipestifer phage vB_RanS_PT24, phage composition and application thereof Download PDF

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CN116286674A
CN116286674A CN202310170863.2A CN202310170863A CN116286674A CN 116286674 A CN116286674 A CN 116286674A CN 202310170863 A CN202310170863 A CN 202310170863A CN 116286674 A CN116286674 A CN 116286674A
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phage
riemerella anatipestifer
rans
composition
riemerella
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张召佐
孙虎芝
闫艳新
李柯汝
张宁
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Qingdao Phagepharm Bio Tech Co ltd
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Abstract

The application relates to the field of microorganisms, and particularly discloses a riemerella anatipestifer phage vB_RanS_PT24, a phage composition thereof and application thereof; the riemerella anatipestifer phage is named as riemerella anatipestifer phage vB_RanS_PT24, and the preservation number is CGMCC No.23814; the riemerella anatipestifer phage provided by the application has strong and specific cracking capability on the riemerella anatipestifer, and can effectively prevent and treat diseases caused by riemerella anatipestifer infection; and secondly, the phage and the phage composition thereof can be used as active ingredients for preparing medicines, disinfectants, feed additives, kits and the like for preventing and treating riemerella anatipestifer, can be widely applied to various links in the waterfowl cultivation process and the product processing production process, can quickly, efficiently and safely kill riemerella anatipestifer, and can effectively reduce the disease incidence rate and the death rate of waterfowl.

Description

Riemerella anatipestifer phage vB_RanS_PT24, phage composition and application thereof
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to a riemerella anatipestifer phage vB_RanS_PT24, a phage composition thereof and application thereof.
Background
Riemerella Anatipestifer (RA) infects various poultry such as ducks, geese, chickens and turkeys and wild birds, and has reports of bacteria of normal and healthy ducks, pigs, aquatic fishes and the like. Riemerella anatipestifer is a gram-negative small bacillus with the size of 0.2-0.4 mu m, often exists singly or in pairs, sometimes also takes the shape of filaments, has no flagella, is subjected to bipolar enrichment by Rayleigh staining, and has capsules by Indian ink staining. Riemerella anatipestifer is transmitted through polluted feed, drinking water, spray, dust and the like through respiratory tract, digestive tract, damaged skin and other ways, and can be developed all the year round, and the highest incidence and death rate are achieved in seasons with low air temperature and high humidity.
Riemerella anatipestifer disease, also called duck serositis, duck septicemia and the like, is characterized in that 14-49 days old gosling and young goose are taken as susceptible groups, and the death rate is about 40%, even more than 90% at most. Part of farms can adopt a goose and duck polyculture mode, so that the infection risk of riemerella anatipestifer is further increased, and meanwhile, other diseases such as colibacillosis, barbites, duck viral hepatitis and the like can promote the occurrence of the disease. The disease is frequently seen in group morbidity, a large number of gosling and duckling are simultaneously ill, sudden death cases exist below 14 days old, and no obvious symptoms exist before death; the diseased geese and ducks often show symptoms of mental depression, appetite reduction, long-term lying, slow movement, outlier and the like, and partial diseased geese are accompanied with cough and nasal discharge, saggy wings, drowsiness and weak constitution, mucus, serous fluid and even purulent secretion appear at the parts of eyes, noses and the like, so that feathers around eyesockets are adhered and nasal cavity endocrine are accumulated to influence the normal breathing of the diseased geese; a few geese develop necks, have gentle head twitches, land with the mouth and neurological symptoms, and die rapidly. Due to the high incidence and mortality of the disease, it has become one of the major bacterial infections that jeopardize the breeding industry in various countries.
Antibiotics are the first choice for preventing and treating riemerella anatipestifer, but due to the long-term wide and unreasonable use of antibacterial drugs, the multi-drug resistance of the bacteria is increasingly serious, and the drug-resistant strain not only causes huge loss to the breeding industry, but also is transmitted to human beings through means such as food chains and the like, thereby threatening the health of human bodies and food safety. At present, the development speed of antibiotics is far from the speed of drug resistance increase, so that an antibiotic substitute is urgently needed to solve the problem.
The phage is a virus capable of efficiently and specifically adsorbing and lysing bacteria, and has the advantages of strong specificity, wide distribution, short research and development time and the like compared with antibiotics, and is widely applied to the treatment of bacterial diseases at present. In the treatment and prevention processes, the phage only aims at the corresponding host bacteria, so that the intestinal flora balance of the animal organism is not destroyed, and no side effect is generated. At present, no phage product can effectively prevent and treat the riemerella anatipestifer disease of geese. Therefore, research and development of a phage preparation which has both prevention and treatment effects and is obvious and safe to use is urgent for preventing and treating the riemerella anatipestifer disease of geese.
Disclosure of Invention
The application aims to provide a phage capable of effectively preventing and treating riemerella anserinaceus diseases and a treatment method of a composition of phage. The phage and the composition thereof provided by the application have strong cracking capability on the riemerella anatipestifer, can inhibit overgrowth of the riemerella anatipestifer, and can effectively treat the riemerella anatipestifer of geese.
In order to achieve the above purpose, the present application adopts the following technical scheme:
in a first aspect, the present application provides a riemerella anatipestifer bacteriophage, which adopts the following technical scheme:
the phage was isolated from Duck feathers; the riemerella anatipestifer phage vB_RanS_PT24 is preserved in China general microbiological culture Collection center (CGMCC) at the 4 th month 26 of 2022, and the preservation address is the North Chen Xili No. 1, 3 of the Korean region of Beijing, and the preservation number is CGMCC No.23814.
Observed under an electron microscope: the riemerella anatipestifer phage vB_RanS_PT24 has a strong cracking effect on riemerella anatipestifer of goose origin and also has a certain cracking effect on riemerella anatipestifer of duck origin. In the application, the bacteriophage is a long-tail bacteriophage from the aspect of an electron microscope, the head is about 65-70 nm long, the head is 55-60 nm wide, and the tail is about 210-220 nm long.
In a second aspect, the present application provides a phage composition, using the following technical scheme:
a phage composition comprising riemerella anatipestifer phage vb_rans_pt24. The phage composition can be compounded with other phages through the riemerella anatipestifer phage vB_RanS_PT24, has strong cracking power on goose-derived and duck-derived riemerella anatipestifer strains, and is used for preparing various products for preventing and treating riemerella anatipestifer diseases.
In a third aspect, the present application further provides an application of the riemerella anatipestifer phage or phage composition in preparing a medicament for preventing and/or treating riemerella anatipestifer infection diseases, which adopts the following technical scheme:
the application of the riemerella anatipestifer phage or phage composition in preparing medicines for preventing and/or treating riemerella anatipestifer infection diseases of ducks and geese is provided.
Preferably, the riemerella anatipestifer is selected from duck source and goose source. Riemerella anatipestifer of goose origin and duck origin mentioned in the above summary of the invention contains a plurality of serotypes including, but not limited to, type I and type II.
The phage application method comprises the following steps: the riemerella anatipestifer phage or the phage composition or the processed medicament thereof is used as a therapeutic drug to be added into the feed of the waterfowl with diseases (namely mixed feed), or is directly infused or is added into drinking water, or is injected into muscle (such as pectoral muscle) or is injected into respiratory tract (such as spray administration), and the operation of the application mode is simple and the application is strong.
In a fourth aspect, the present application further provides a phage pharmaceutical formulation, which adopts the following technical scheme:
a phage pharmaceutical preparation contains effective components of Riemerella anatipestifer phage and/or composition containing the phage.
Preferably, the phage pharmaceutical formulation also includes other ingredients with synergistic effects.
Preferably, the phage pharmaceutical formulation also includes phage of other specific pathogenic bacteria.
Preferably, the phage pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include saline, sterile water, ringer's solution, buffered saline, albumin infusion, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. They may be used alone or in any combination thereof.
Preferably, other conventional additives, such as antioxidants, buffers, bacteriostats, and the like, may be added.
The phage drug preparation is in the form of oral administration, injection or respiratory tract administration. Preferably, an oral dosage form is employed.
The dosage forms of the pharmaceutical preparation are solutions, powders, gels, granules or freeze-drying agents.
The riemerella anatipestifer phage preparation can be prepared into powder, water aqua or freeze-dried agent, and can be administered in modes of injection, oral administration, spraying and the like, so that the riemerella anatipestifer in animals can be rapidly and effectively cracked.
In a fifth aspect, the present application further provides a feed additive, which adopts the following technical scheme:
a feed additive comprising a riemerella anatipestifer bacteriophage and/or a bacteriophage composition comprising the bacteriophage; the duck infectious serositis can be prevented or treated by mixing with waterfowl feed and feeding.
Preferably, the content of each bacteriophage in the feed additive is at least 1X 10 8 PFU/g。
In a sixth aspect, the present application further provides a disinfectant, which adopts the following technical scheme:
the disinfectant mainly comprises the riemerella anatipestifer bacteriophage and/or a composition containing the bacteriophage as active ingredients.
Preferably, the phage titer is 1X 10 8 PFU/mL or more.
The environmental disinfectants described above also contain other active ingredients or other adjuvants for the inhibition or elimination of bacteria in the environment.
The phage and the phage composition thereof can be used as active ingredients for preparing medicines, disinfectants, feed additives, kits and the like for preventing and treating riemerella anatipestifer, can be widely applied to various links in the waterfowl cultivation process and the product processing and production process, can quickly, efficiently and safely kill pathogenic riemerella anatipestifer and even drug-resistant riemerella anatipestifer, and can effectively reduce the disease incidence and death rate of waterfowl.
The application method of the disinfectant comprises the following steps: the disinfectant is used in slaughterhouses, waterfowl product processing workshops, appliances and culture environments, and prevents the pollution of riemerella anatipestifer in the environments.
In a seventh aspect, the present application further provides a detection kit, which adopts the following technical scheme:
a test kit comprising a riemerella anatipestifer bacteriophage or phage composition as described above. The kit is used for detecting the specific infected riemerella anatipestifer or preventing and controlling diseases caused by the infection of the host riemerella anatipestifer.
The application has the following beneficial effects:
1. the riemerella anatipestifer phage provided by the application has strong and specific cracking capability on the riemerella anatipestifer, and can effectively prevent and treat diseases caused by riemerella anatipestifer infection; and secondly, the phage and the phage composition thereof can be used as active ingredients for preparing medicines and disinfectants for preventing and treating riemerella anatipestifer, and can quickly, efficiently and safely kill the riemerella anatipestifer.
2. The riemerella anatipestifer bacteriophage provided by the application has the advantages of no toxic or side effect, high safety, no residue, no influence on animal intestinal flora balance, better effect on the treatment of riemerella anatipestifer, and provision of a green novel biological agent for the treatment of riemerella anatipestifer in the waterfowl industry.
Drawings
FIG. 1 is a plaque picture of phage vB_RanS_PT 24;
FIG. 2 is an electron microscope picture of phage vB_RanS_PT 24;
FIG. 3 is a picture of the thermostability of phage vB_RanS_PT 24;
FIG. 4 is a picture of the pH stability of phage vB_RanS_PT24.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application. In the present application, unless otherwise specified, the equipment and materials used, etc., are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
In the following examples of the present invention,
TSA plate formulation: 15.0g of tryptone, 5.0g of soybean peptone, 5.0g of sodium chloride, 15.0g of agar, pH 7.3.+ -. 0.2 (25 ℃).
TSB broth formulation: 17.0g of tryptone, 3.0g of soybean peptone, 5.0g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose and pH 7.3+/-0.2.
SM buffer formulation: 5.89g of sodium chloride, 2g of magnesium sulfate, 50mL of 1MTris-HCL (pH 7.5) and 5mL of 2% gelatin.
EXAMPLE 1 isolation culture and biological Properties of phages
Sample collection
Samples such as manure samples, padding, sewage, fur and the like are collected from different duck farms in a hillock.
Resuscitating culture of strain and preparation of bacterial suspension
The frozen stock solution of Riemerella anatipestifer is streaked on a TSA flat plate (added with calf serum with the volume concentration of 2 percent) by using a sterilization inoculation loop, single colonies are separated, and the single colonies are cultured in a candle jar at 37 ℃ for 24 to 48 hours. Single colonies were picked and inoculated into 5mLTSB broth (added with 5% by volume of calf serum) and cultured in an air shaker at 37℃for 20h with shaking at 220rpm to give a single bacterial suspension.
Isolation and purification of phage (II)
The collected sample was added with an appropriate amount of sterile PBS, shaken at 37℃and 170rpm for 60min, centrifuged at 11000rpm for 5min, and the supernatant was filtered through a 0.45 μm bacterial filter for further use.
Adding each strain of riemerella anatipestifer into each 50mL centrifuge tube according to the total volume of 25%, adding the supernatant (supernatant after filtration of a bacterial filter) subjected to centrifugal filtration according to the total volume of 25%, adding a 2 XSSC culture medium (added with calf serum with the volume concentration of 5%) according to the total volume of 50%, uniformly stirring, standing for culturing overnight at 37 ℃, centrifuging at 11000rpm for 5min, and filtering and sterilizing a bacterial filter with the volume of 0.45 mu m for later use; repeating the above steps, mixing the filtrate with host bacteria, incubating at 37deg.C for 15min, spreading double-layer plate, solidifying, and culturing in a candle jar at 37deg.C overnight.
If phage exist, transparent and regular round plaques are formed on the culture medium, namely plaques. The single plaque is picked up, leached in 1mLSM buffer solution in an air oscillator at 37 ℃ for 60min, placed in a refrigerator at 4 ℃ for standing for 3h, centrifuged at 11000rpm for 5min, the supernatant is taken out and then the single plaque is obtained by a double-layer flat plate method, and the steps are repeated for 3-5 times until a round transparent plaque with clear boundary and plaque diameter of about 0.5mm is formed, as shown in figure 1.
(III) proliferation and potency determination of phages
100 mu L of host bacteria and phage spot-picking leaching liquid are added into 5mL of TSB broth (added with calf serum with volume concentration of 5 percent), and cultured for 5-8 hours under the condition of 170rpm of an air oscillator at 37 ℃ until the mixed liquid becomes clear, thus obtaining phage proliferation liquid. Phage proliferation was diluted 10-fold, and phage titers were determined by double-layer plate method, 3 replicates were made for each dilution.
The results show that: counting 3 parallel samples, and calculating the titer to be about 2 multiplied by 10 9 PFU/mL~4×10 9 PFU/mL。
(IV) Transmission Electron microscopy for observing the morphology of phage
Taking a height of more than 1×10 9 20. Mu.L of the PFU/mL phage sample (phage multiplication solution) was dropped on a microporous copper mesh, and the solution was precipitated for 15min, and the excess solution was sucked off with filter paper. 15 mu L of phosphotungstic acid (PTA) with the volume concentration of 2% is dripped on a copper wire, the copper wire is dyed for 5min, redundant dye liquor is sucked by filter paper, and the copper wire is observed by a transmission electron microscope and photographed after being dried.
The results show that: the head of the phage is about 65-70 nm long, the head is 55-60 nm wide, the tail of the phage is about 210-220 nm long, and the phage electron microscope diagram is shown in figure 2; according to the definition of the International Commission on viral Classification (ICTV), the morphology of phage PT24 corresponds to the characteristics of the long-tailed phage family, belonging to the long-tailed phage family, and this phage was designated vB_RanS_PT24.
Detection of the thermal stability of phage
Will be 3.37X10 9 The PFU/mL multiplication solution of phage vB_RanS_PT24 is respectively subjected to the action for 30min in water bath at 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ at each temperature, and two parallel groups are arranged at each temperature. Phage titers were determined by double-layer plate method.
As shown in FIG. 3, phage vB_RanS_PT24 substantially maintains primary activity after 30min of action at 40-55deg.C; after 30min at 60 ℃, phage titers decreased near 1 titer. The test results demonstrate that phage vB_RanS_PT24 can withstand high temperatures of 55℃or below.
Detection of pH stability of phage
Taking 1.5mL sterile centrifuge tubes, adding 900 μL SM buffer solution with different pH values (4, 5, 6, 7, 8, 9, 10, 11) into each tube, placing into a water bath at 37deg.C, and adding 100 μL 3.37X10 to each tube after temperature is stabilized 9 PFU/mL phage proliferation solution, mixing well, and performing water bath at 37 ℃ for 1h. After completion of the action, 200. Mu.L of the mixture was aspirated from each centrifuge tube, and the mixture was added to a 1.5mL centrifuge tube containing 800. Mu.L of LSM buffer, and the titers of phages after different pH treatments were determined by the double-layer plate method.
As shown in fig. 4, the titer of phage vb_rans_pt24 was hardly changed or slightly decreased in the range of pH5 to 10, the titer of phage vb_rans_pt24 was decreased by 1 titer at ph=4, and the titer of phage vb_rans_pt24 was decreased by 2 titers at ph=11, which suggests that phage vb_rans_pt24 can withstand certain acidic and basic environments.
Determination of optimal multiplicity of infection (MOI) of phage (seven)
The riemerella anatipestifer phage vB_RanS_PT24 and the host bacteria riemerella anatipestifer T05 are proliferated according to a conventional method, the initial titer of the phage and the concentration of the host bacteria are measured, and the phage vB_RanS_PT24 and the host bacteria are diluted appropriately. 200. Mu.L of phage vB_RanS_PT24 and host bacteria T05 were added to TSB broth (added to 5% by volume calf serum) at a ratio of multiplicity of infection of 10, 1, 0.1, 0.01. Shaking culture was carried out at 37℃and 220rpm until the liquid became clear, and the time for the liquid to become clear was recorded. The phage titer was determined by double-layer plate method by centrifugation at 11000rpm for 5min, and the results are shown in Table 1.
TABLE 1 determination of the optimal multiplicity of infection of phage vB_RanS_PT24
Figure BDA0004098120220000061
The test results show that the titer of progeny phage produced by phage infection of the host bacteria at a multiplicity of infection of 0.1 is 5.14X10 9 PFU/mL, phage titers were highest among 4 multiplicity of infection.
Example 2: genomic analysis of phages
Extracting genome of riemerella anatipestifer phage vB_RanS_PT24, performing whole genome sequencing, and analyzing the sequence: the genome size of phage vB_RanS_PT24 is 43704bp, the proportion of A, G, C, T bases in the genome sequence is 36.92%, 18.66%, 16.15%, 28.27%, and the G+C% content is 34.81%, respectively.
The phage vB_RanS_PT24 genome contains 79 Open Reading Frames (ORFs) by RAST predictive analysis; the 79 ORFs of phage vb_rans_pt24 containing 11 known encoded functional proteins and the remaining ORFs are hypothetical proteins were aligned using the online tools BLASTp and the Conserved Domain Database (CDD), respectively.
Phage vb_rans_pt24 in the genome: the sequence of the recombinase/integrase (recombase/integerase) gene is shown in SEQ ID NO. 1, and the highly conserved terminal enzyme large subunit (terminase large subunit) gene sequence is shown in SEQ ID NO. 2. Specific information on the above genes is shown in Table 2 below.
TABLE 2 information table of the vB_RanS_PT24 Gene sequence of phages
Figure BDA0004098120220000071
Example 3: determination of phage cleavage spectra
The method for determining the splitting spectrum of the phage by adopting a double-layer flat plate method comprises the following steps: a bacterial suspension of 120 different host bacteria (60 duck-origin bacteria and 60 goose-origin bacteria among the 120 Riemerella anatipestifer) was obtained as in example 1. After 100. Mu.L of each host bacterium suspension was incubated with 100. Mu.L of Riemerella anatipestifer phage vB_RanS_PT24 at 37℃for 15min, a double-layer plate was prepared by adding the suspension to the upper layer agar (adding calf serum at 5% by volume), and after the agar solidified, the plates were placed in an incubator at 37℃for inversion culture overnight to observe the lysis results, and the results are shown in Table 3.
TABLE 3 phage lysis spectra of Riemerella anatipestifer
Figure BDA0004098120220000081
Note that: +: splitting, wherein the soft agar plate method has transparent plaque; -: not cleaved.
As can be seen from Table 3, the 120 Riemerella anatipestifer is used as a host bacterium for determination of a lysis spectrum, and 95 strains of phage vB_RanS_PT24 can be lysed, so that the lysis rate reaches 79.16%; aiming at 60 duck-origin riemerella anatipestifer cracking 43 strains, the cracking rate is 71.67%; aiming at 60 goose-source riemerella anatipestifer cracking 52 strains, the cracking rate is 86.67%.
The phage vB-RanS-PT 24 has a good cracking effect on riemerella anatipestifer, especially riemerella anatipestifer of goose origin, can quickly, efficiently and safely kill pathogenic riemerella anatipestifer and even drug-resistant riemerella anatipestifer, effectively reduces the disease incidence and death rate of waterfowl, and improves the economic benefit of farms.
Example 4: safety test of phage
Selecting 20 healthy BALB/C mice with weight of 18-20 g, dividing the mice into an experimental group and a control group (10 mice in each group), and respectively filling phage proliferation liquid (300 mu L, 10) into 2 mice 9 PFU/mL) and TSB broth (300. Mu.L) were filled continuously for 7 days, the behavior of the mice was observed, and then the changes in the tissue organs of the mice were observed by dissection.
The mice in the experimental group and the control group have no abnormal behaviors, and the split examination shows that: the liver, lung, heart, spleen, kidney and other organs are normal, and have no obvious difference with the control group. This demonstrates that phages are safe for the test mice.
Example 5: phage disinfection test
Diluting the serum type II Riemerella anatipestifer clinical isolate GT47 bacterial liquid to 1X 10 4 CFU/mL. The control group was first diluted 1mL to 1X 10 4 CFU/mL bacterial liquid is evenly sprayed at 1m 2 Then evenly spraying an equal volume of TSB broth; test groups were first diluted 1mL to 1X 10 4 CFU/mL bacterial liquid is evenly sprayed at 1m 2 Then 1 x 10 will be 8 PFU/mL phage vB_RanS_PT24 are sprayed evenly on the ground, 2 replicates per group. Every 1h, sampling on the ground with the same area, detecting the number of ground host bacteria by using a plate counting method, and detecting for 6h, wherein the result is shown in Table 4.
Table 4 disinfection test results
Figure BDA0004098120220000091
As can be seen from Table 4, after the phage vB_RanS_PT24 was used to disinfect the floor contaminated with Riemerella anatipestifer, the plate count results showed that the number of Riemerella anatipestifer began to decrease after 1h and the number of Riemerella anatipestifer decreased to 10 after 2h 3 About CFU, the number of the ground riemerella anatipestifer is reduced to about 10CFU after 3 hours, the riemerella anatipestifer can not be detected on the ground after 4 hours, and the number of the ground riemerella anatipestifer in the control group is slightly increased. The phage vB_RanS_PT24 can effectively kill the riemerella anatipestifer in the environment.
Example 6: phage test for preventing riemerella anatipestifer disease of goose and duck
The weight of 20 healthy 3-week-old open-eye geese with similar weights was selected and equally divided into a test group and a control group (10 in each group). Test group 1mL of 1 x 10 per goose oral liquid 9 PFU/mL of riemerella anatipestifer phage vB_RanS_PT24 was used for prevention, and the control group was orally administered an equal amount of TSB broth each; after phage administration for 2h, 0.5mL of a clinically isolated strain of Riemerella anatipestifer GT47 of serotype II (5X 10) was subcutaneously injected into the gooseneck of each of the test and control groups 8 CFU/person), the goose status was observed, the death of the goose person was recorded, and the observation was continued for 7 days. After 7 days, all geese were dissected and examined for pathological changes of tissue and organs.
The results show that: within 7 days, the death rate of the eye-open geese of the prevention test group is 2 and 20 percent; 9 control groups die, the death rate is 90%, and the protection rate of phage reaches 80%, which shows that the phage can effectively prevent the occurrence of Riemerella anserinaceus disease.
Example 7: phage toxicity-counteracting treatment test for riemerella anatipestifer of geese
The 20 healthy 3-week-old geese with similar body weight were selected and divided into a treatment group and a control group (10 each). Two groups of geese were injected subcutaneously in their necks with 0.5mL of Riemerella anatipestifer clinical isolate GT47 (5X 10) of serotype II 8 CFU /) 2h after treatment, the geese of the treatment group were injected with 1mL of 1 x 10 per day into the pectoral muscle 9 PFU/mL of riemerella anatipestifer phage vB_RanS_PT24 is treated, the control group is injected with equal amount of TSB broth into pectoral muscle every day for 1 time/day for 3 days, the status of goose is observed, the death condition of goose is recorded, and 7 days after stopping the drug is observed.
The results show that: the treatment group only died 3 geese, the mortality rate was 30%, the control group only died 9 geese, the mortality rate was 90%, and the protection rate of the riemerella anatipestifer phage vB_RanS_PT24 was 70%. The result shows that the phage injection in the chest muscle can effectively treat the Riemerella anatipestifer disease of the goose.
The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims (9)

1. The riemerella anatipestifer phage is characterized by being named as riemerella anatipestifer phage vB_RanS_PT24 and having a preservation number of CGMCC NO.23814.
2. A phage composition comprising the riemerella anatipestifer phage vb_rans_pt24 of claim 1.
3. Use of a phage of riemerella anatipestifer according to claim 1 or a phage composition according to claim 2 for the preparation of a medicament for the prevention and/or treatment of riemerella anatipestifer infection diseases.
4. A phage pharmaceutical formulation characterized in that: comprising the riemerella anatipestifer bacteriophage of claim 1 and/or the bacteriophage composition of claim 2.
5. The phage pharmaceutical formulation of claim 4, wherein: the phage pharmaceutical formulation also includes a pharmaceutically acceptable carrier.
6. The phage pharmaceutical formulation of claim 5, wherein: the phage drug preparation is in the form of oral administration, injection or respiratory tract administration.
7. A feed additive comprising the phage of riemerella anatipestifer of claim 1 and/or the phage composition of claim 2; the phage content in the feed additive is at least 10 8 PFU/g。
8. A disinfectant comprising the riemerella anatipestifer bacteriophage of claim 1 and/or the bacteriophage composition of claim 2; phage titer in disinfectant was 1X 10 8 PFU/mL or more.
9. A test kit comprising the phage of riemerella anatipestifer of claim 1 and/or the phage composition of claim 2.
CN202310170863.2A 2023-02-27 2023-02-27 Riemerella anatipestifer phage vB_RanS_PT24, phage composition and application thereof Pending CN116286674A (en)

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