CN118165943A - Low-temperature-resistant clostridium perfringens bacteriophage RDPCP23040 and application thereof in environmental disinfection - Google Patents

Abstract

The invention discloses a low-temperature-resistant clostridium perfringens bacteriophage (Clostridium perfringens bacteriophage) RDPCP23040 and application thereof in environmental disinfection, wherein the bacteriophage is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 45583 in the year of 2023 and the month of 12. The present invention provides a phage composition comprising clostridium perfringens phage RDPCP23040 as described above. The invention also provides the application of the clostridium perfringens bacteriophage RDPCP23040 or the phage composition in preparing a product for preventing and/or treating poultry diseases caused by clostridium perfringens. The invention also provides an application of the clostridium perfringens or the bacillus thereof in preparing products for killing or inhibiting clostridium perfringens or the bacillus thereof. The clostridium perfringens bacteriophage RDPCP23040 provided by the invention can keep the bioactivity and stability at low temperature, can effectively prevent and control diseases caused by clostridium perfringens, has a good killing effect on clostridium perfringens, is environment-friendly, and has a good application prospect.

Description

Low-temperature-resistant clostridium perfringens bacteriophage RDPCP23040 and application thereof in environmental disinfection

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a low-temperature-resistant clostridium perfringens bacteriophage RDPCP and 23040 and application thereof in environmental disinfection.

Background

Clostridium perfringens (clostridium perfringens) is an anaerobic bacillus and gram-positive. Originally isolated from a highly rotten cadaveric vascular tissue by the U.S. pathologist w.h.welchii et al, one is named by his name and is also known as clostridium welchii. The academic name has been revised many times later due to the change in international bacterial naming regulations and is ultimately more named clostridium perfringens. The length of the bacterial cells is 3-9 mu m, the width is 0.6-2.4 mu m, the both ends of the bacterial cells are rounded to be thick rod-shaped, and the edges of the bacterial cells are straight. The cell morphology is not fixed, and tends to be spherical at the early stage of cell growth, while tadpoles, filaments and grains are often present at the end of growth due to exhaustion of nutrient components of the medium. The mirror may be arranged singly or in short chains, and sometimes in pairs.

The bacterium is capable of producing a variety of strong exotoxins, currently known as α, β, ε, γ, η, δ, iota, θ, K, λ, μ, and v twelve. Of these, the four exotoxins of alpha, beta, epsilon and iota are all proteins, and have enzymatic activity and antigenicity. They are the most predominant lethal and genotyping toxins. Clostridium perfringens can be divided into, depending on the main exotoxin secreted: A-E5 toxin types, each of which can cause morbidity in animals and humans to varying degrees.

Phage, also known as bacterial viruses, are microorganisms that complete their proliferation in bacteria and release themselves into the environment by lysing the bacteria. Compared with antibiotic treatment, phage therapy has the advantages of low metabolic efficiency, low minimum bacterial killing dosage, high specificity, high sterilizing effect, high screening speed and the like.

In recent years, the research on phage has been greatly developed, and the application research range of phage is wide at home and abroad, including the aspects of prevention and treatment of human diseases, prevention and treatment of animal diseases, removal of pathogenic bacteria in food, prevention and treatment of bacterial infection of crops, and lysis of bacterial biofilm. Although phage application is promising, it is not neglected that phages often have a narrow host spectrum, and that one phage usually only lyses one bacterium.

Clinically, the basic approach to the treatment of clostridium perfringens is to use antibiotics. In recent years, with the massive use of antibacterial drugs, particularly in China, antibiotics are added into feed for a long time as feed additives, so that pathogenic bacteria generate drug resistance, the drug resistance spectrum is continuously expanded, the transmission speed is also faster and faster, and the health and public health safety of people are seriously threatened.

At present, a low temperature resistant phage product which can effectively prevent and treat clostridium perfringens and kill bacterial spores and is suitable for environmental disinfection does not appear, so that a phage product which can resist low temperature, has the advantages of preventing and treating, has a good spore killing effect, is stable in inheritance and can be suitable for environmental disinfection is developed to prevent and treat clostridium perfringens.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and provides a low-temperature-resistant clostridium perfringens bacteriophage RDPCP and 23040 and application thereof in environmental disinfection. The clostridium perfringens bacteriophage RDPCP23040 provided by the invention can be used for preparing products for preventing and/or treating poultry diseases caused by clostridium perfringens, and can be used for preparing products for killing or inhibiting clostridium perfringens or spores thereof. In particular, the phage preparation for preventing and treating poultry diseases caused by clostridium perfringens can be prepared, and the disinfectant for killing or inhibiting clostridium perfringens can be prepared. The bacteriophage and its bacteriophage composition are safe and have no side effect, and are used in treating clostridium perfringens infection.

The above object of the present invention is achieved by the following technical scheme:

In a first aspect, the invention provides clostridium perfringens bacteriophage (Clostridiumperfringens bacteriophage) RDPCP23040 isolated from the cecum of diseased birds from a disease farm in Shandong, which has been deposited at the China general microbiological culture Collection center, month 06 and 12 of 2023, at the following deposit address: the preservation number of the Qingyang area North Star Xili No. 1 and 3 of Beijing is CGMCC No.45583. Clostridium perfringens CP-20032 used in the experiments of the present invention was isolated from the disease chicken cecum in some disease farm in the Shandong province and deposited with clostridium perfringens phage RDPCP23040 of the present invention.

Observed by electron microscopy: the phage RDPCP23040 has a polyhedral, stereospecific head, surrounding nucleic acid, about 42nm in diameter, a tail of about 31nm in length, a tail sheath, and a neck connecting the head and tail. The phage were identified according to the International Commission on classification of viruses (ICTV) and classified as having the order of the Rheumatoid, brevibacteriaceae.

The phage RDPCP23040 was cultured for 8h under MOI=0.01, its titer was as high as 9.01X10. 10 ⁸ pfu/mL.

The phage RDPCP23040 can maintain good activity in pH5.0-11.0, and the phage can maintain high potency.

The phage RDPCP23040 always maintains higher potency in the process of passage for 30 times, and no obvious fluctuation appears, which indicates that the phage can be stably inherited and is suitable for large-scale industrial production.

The phage RDPCP23040 has better cracking effect on clostridium perfringens, and experiments prove that the phage has high cracking rate of 100% on clostridium perfringens, and can crack a host of various serotypes A, B, C, D and E.

The phage RDPCP23040 is placed in an environment of 4 ℃ for 7 days, always keeps higher titer, has no obvious fluctuation, and can be stably stored at low temperature.

Clostridium perfringens spores (hereinafter referred to as "gas producing spores") are a kind of microorganism dormancy formed under environmental stress (such as low temperature, dryness or nutrient deficiency, etc.), and have extremely strong stress resistance to various stress factors (such as high temperature, high pressure, toxic chemicals and radiation), are not easy to kill, germinate and produce toxicity under suitable conditions, have extremely strong pathogenicity, and are often difficult to effectively kill by conventional disinfection treatment and most phage. The phage RDPCP23040 treated by the invention has obvious inactivation phenomenon, and experimental results show that the phage can reduce the number of the gas-producing spores by 3 orders of magnitude, which indicates that the phage has good killing effect on the gas-producing spores, and has good application prospects in the aspects of food safety, clinical medical treatment, biological control and the like.

In a second aspect, based on the same inventive concept, the present invention provides a phage composition characterized by comprising clostridium perfringens phage RDPCP23040 as described above.

In a third aspect, the invention also provides the use of clostridium perfringens bacteriophage RDPCP23040 as described above or a bacteriophage composition as described above for the preparation of a product for the prevention and/or treatment of a disease in poultry caused by clostridium perfringens, based on the same inventive concept.

In a fourth aspect, the invention also provides the use of clostridium perfringens bacteriophage RDPCP23040 as described above or according to a bacteriophage composition as described above for the preparation of a product for killing or inhibiting clostridium perfringens or spores thereof, based on the same inventive concept.

In a third aspect, based on the same inventive concept, the present invention also provides a phage pharmaceutical preparation, the active ingredient comprises clostridium perfringens phage RDPCP23040 as described above or phage composition as described above.

Preferably, the clostridium perfringens bacteriophage RDPCP23040 is at a concentration of at least 10 ⁸ pfu/mL.

In a fourth aspect, the invention also provides a disinfectant comprising clostridium perfringens bacteriophage RDPCP23040 as described above or a bacteriophage composition as described above as an active ingredient, based on the same inventive concept.

Preferably, the clostridium perfringens bacteriophage RDPCP23040 is used at a concentration of at least 10 ⁸ pfu/mL.

Preferably, the disinfectant can disinfect clostridium perfringens from an avian farming environment by spraying and soaking.

Preferably, the poultry breeding environment is poultry in vivo, poultry body surface, poultry breeding place on the ground, poultry breeding place air, feed, drinking water and breeding appliances, including but not limited to chicken coops, chicken coops ground, waterline, trough, breeding personnel walking ground, tap water discharge port, sewage discharge port and the like.

The beneficial effects of the invention are as follows:

1. The clostridium perfringens bacteriophage RDPCP23040 provided by the invention is placed for 7 days in an environment of 4 ℃ and always keeps higher titer, no obvious fluctuation appears, and the bacteriophage can be stably stored at low temperature, which indicates that the bacteriophage can keep the bioactivity and stability of the bacteriophage at low temperature, and the low temperature resistant characteristic enables the bacteriophage to effectively prevent and control diseases caused by clostridium perfringens in a low temperature environment.

2. The clostridium perfringens bacteriophage RDPCP and 23040 provided by the invention has a good effect of killing the gas producing spores, is environment-friendly, and has a good application prospect.

3. The clostridium perfringens bacteriophage RDPCP23040 provided by the invention is obtained from nature, can be inherited stably, is suitable for large-scale industrial production, and the phage pharmaceutical preparation or disinfectant prepared from the phage not only can reduce the cost, but also has the advantages of green and safety. In particular, the prepared disinfectant can be applied to disinfection of poultry cultivation low-temperature environment, and is beneficial to purifying cultivation environment.

Drawings

FIG. 1 is an electron micrograph of phage RDPCP 23040;

FIG. 2 is a one-step growth curve of phage RDPCP 23040;

FIG. 3 is a graph of the results of a pH stability test of phage RDPCP 23040;

FIG. 4 is a plot of phage RDPCP23040 versus temperature sensitivity;

FIG. 5 is a graph of the results of a genetic stability test of phage RDPCP 23040;

FIG. 6 is a graph showing the results of a low temperature resistance test of phage RDPCP 23040;

FIG. 7 is a graph showing the effect of phage RDPCP23040 on spores.

Detailed Description

The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the present invention, clostridium perfringens bacteriophage RDPCP23040 or simply "phage RDPCP23040" or "RDPCP23040".

Example 1 isolation and characterization of Clostridium perfringens CP-20032 as a host

Sampling from a disease-causing farm in Shandong province, taking diseased chicken cecum through aseptic operation, marking out on a selective culture medium, culturing for 18-24 hours in an anaerobic environment at 37 ℃, wherein bacteria form dome-shaped colonies on a blood agar culture medium, and the colonies are smooth in surface, semitransparent in radius of 1-2.5mm and neat in edge; grass green colonies with double-layer hemolysis rings around are selected on a blood agar basal plate (5% sheep blood plus 1% glucose), inoculated on a pancreatic month indicator-sulfite-cycloserine agar foundation (TSC), placed in an anaerobic environment and cultured for 18-24 hours at 45 ℃; the typical colony is picked up and purified for 3 to 5 times continuously until the colony morphology is consistent, then single colony is picked up and inoculated into 5mL of liquid thioglycolate (FTG) broth culture medium, and the culture is carried out for 12 hours in an anaerobic incubator at 37 ℃ to obtain even turbid bacterial suspension. And then, determining the bacterial strain as pathogenic clostridium perfringens through 16sRNA molecular identification and serotype identification, namely, naming one bacterial strain as CP-20032, streaking the identified bacterial strain on a blood agar basal plate culture medium plate, culturing for 12 hours, scraping all bacterial colonies into 60% glycerol-FTG liquid by using an inoculating loop, and storing in a refrigerator at the temperature of minus 80 ℃.

Example 2 isolation and identification of phage RDPCP23040

(1) And (3) treating the disease materials: taking sick chickens from a farm, dissecting and reserving cecum, taking the cecum content by aseptic operation, adding 2mL of sterilized FTG liquid, fully soaking the cecum content, centrifuging at 10000rpm for 30min, taking supernatant, and filtering the supernatant with a 0.22 mu m filter to reserve;

(2) Preparing phage enrichment liquid: adding 0.2mL of bacterial suspension and 1mL of filtrate into 5mL of FTG broth, culturing at 37 ℃ in an anaerobic box for overnight, centrifuging at 10000rpm for 5min, and filtering the supernatant with a 0.22 mu m filter to obtain filtrate for later use;

(3) Phage isolation: separating phage by double-layer plate method, mixing 0.1mL of phage enrichment filtrate and 0.2mL of host clostridium perfringens bacterial suspension uniformly, placing in 37 ℃ water bath for 10min, paving double plates, placing in an anaerobic incubator at 37 ℃ for culturing for 6-8h, picking up transparent spots, soaking in 1mL of SM buffer solution at 4 ℃ for 12h, taking 0.1mL of leachate and 0.2mL of bacterial suspension in 37 ℃ water bath for 10min, paving double plates, culturing in an anaerobic incubator at 37 ℃ for 4-6h for purification, purifying for 5 times according to the steps to obtain phage monomer, and naming the phage as RDPCP23040, wherein the diameter of the plaque formed in the double plates is 1-3 mm.

Phage RDPCP23040 preservation method: mixing phage multiplication solution with 60% glycerol at volume ratio of 1:1, and storing in-80deg.C refrigerator or liquid nitrogen.

Example 3 Electron microscopic observations of phage RDPCP23040

20 Mu L of liquid containing crude phage particles is dripped on a copper mesh, natural precipitation is carried out for 15min, excessive liquid is sucked off from the side face by using filter paper, a drop of 2% phosphotungstic acid (PTA) is added on the copper mesh for dyeing phage for 10min, then the dyeing liquid is sucked off from the side face by using filter paper, and after a sample is dried, the shape of phage is observed by using an electron microscope, as shown in figure 1.

As a result, phage RDPCP23040 has a polyhedral, stereosymmetrical head, surrounding nucleic acid, about 42nm in diameter, a tail of about 31nm in length, a tail sheath, and a neck connecting the head and tail. According to the ninth report of the International taxonomic organization of viruses, the phage were classified as having the order of the Rhinoceroviridae.

The phage is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 2023, 06 and 12 days, and the preservation address is: the collection number is CGMCC No.45583 and is classified and named as phage in the Chaoyang area North Star Xili No. 1 and 3 of Beijing city.

Example 4 ORF prediction and functional annotation of the Whole genome of phage RDPCP23040

Concentrating the high-purity phage solution, and performing full genome second-generation sequencing assembly by adopting a second-generation Illumina Hiseq sequencing technology.

Phage RDPCP23040 was sequenced from the whole genome. The newly sequenced genomes were used for coding gene prediction using GENEMARKS software. And (3) respectively comparing the protein sequences of the predicted genes with Nr and Swiss-Prot database rows blastp, so as to obtain annotation information of the predicted genes. And performing blastp comparison with eggNOG databases to obtain COG annotation results corresponding to the genes, and performing functional classification on the proteins according to the COG annotation results. The possible virulence genes and drug resistance genes are checked one by one through an online website Virulence Factor Database, VFDB (http:// www.mgc.ac.cn/VFs/main. Htm) and CARD (https:// CARD. Mcmaster. Ca /), and finally the function annotation of the phage is completed. The phage whole genome assembly results are shown in Table 1.

TABLE 1 statistical Table of phage genome assembly results

Phage genome ORF analysis and functional annotation are as follows, phage gene information statistics are shown in table 2.

TABLE 2 statistical Table of phage Gene information

The amino acid sequence of the phage RDPCP23040 genomic ORF protein was compared with the amino acid sequences in the nr and swiss databases, 36 open reading frames were predicted, the average length was 758bp, the potential function of 13 proteins was predicted by functional homology, and the remaining 23 proteins were not similar to known phage or bacterial proteins. Phage RDPCP23040 are categorized into 3 parts by protein function: cell wall/membrane/envelope biogenesis (ORF 6, ORF18, ORF 24), DNA replication and regulation related genes (ORF 3, ORF21, ORF 36) and phage structure related proteins (ORF 5, ORF12, ORF18, ORF23, ORF29, ORF31, ORF 32). Wherein ORF24 encodes a peptidoglycan hydrolase: N-acetylmuramyl-L-alanine amidase (N-acetylmuramoyl-L-ALANINE AMIDASE) capable of specifically cleaving the amide bond between the lactoyl group of muramic acid and the alpha-amino group of L-alanine. Phage RDPCP23040 contains no drug resistance genes and no virulence genes, and no lysogenic related genes.

Example 5 determination of optimal multiplicity of infection for phage RDPCP23040

1. The experimental method comprises the following steps:

Phage proliferation solution and host were added to FTG broth at a ratio of multiplicity of infection of 100, 10, 1, 0.1, 0.01, 0.001 and the total volume of the culture system was ensured to be the same. After 8h of culture in an anaerobic box at 37 ℃, the culture was centrifuged at 12000r/min for 5min at normal temperature, and the supernatant was spread on double plates to determine the titer. See in particular table 3.

TABLE 3 determination of optimal multiplicity of infection

2. Experimental results and analysis:

As can be seen from Table 3, when the multiplicity of infection was 0.01, the proliferation solution was clear after 8 hours of culture, and the titer was the highest, which indicates that the optimal multiplicity of infection of RDPCP23040 was 0.01, and the corresponding highest titer was 9.01X10. 10 ⁸ pfu/mL.

Example 6 determination of one-step growth curve of phage RDPCP23040

1. The experimental method comprises the following steps:

The clostridium perfringens CP-20032 and phage RDPCP23040 are inoculated according to the ratio of optimal infection complex, water bath is carried out for 10min at 37 ℃, then centrifugation is carried out for 5min at 12000r/min at normal temperature, supernatant is discarded, free phage which is not adsorbed on the host is removed, and the culture medium is washed twice by FTG broth. The pellet was resuspended in FTG broth at 37 ℃ and rapidly placed in a shaking table at 37 ℃ for 200r/min for shaking culture and timing. Sampling and counting every 5min for the first 30min, counting every 10min for 30-100min, counting every 30min for 100-300min, taking time as an abscissa, taking a phage titer logarithmic value as an ordinate, drawing a growth curve, obtaining a incubation period and a lysis period of phage, and calculating average lysis amount. Average lysis = phage titer at end of burst/host concentration at initial stage of infection. Average lysis = phage titer at end of burst/host concentration at initial stage of infection. The results are shown in FIG. 2.

2. Experimental results and analysis:

As can be seen from FIG. 2, there is no significant change in potency within 40min after infection of the host bacteria by the phage, indicating a incubation period of about 40min, a significant rise in potency of the phage within 40-100min after infection, and a subsequent stabilization, indicating a phage lysis period of about 60min. Through calculation, the phage lysis amount is about 186 PFU/infected cell, which shows that phage has strong lysis and replication capacity.

Example 7 determination of the pH stability of phage RDPCP23040

1. The experimental method comprises the following steps:

Preparing an exponential phase host bacterial liquid of 10 ⁵ cfu/mL, and adjusting the concentration of the phage liquid according to the optimal infection complex. The pH values of the FTG liquid culture mediums are respectively adjusted to be 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 and 13, phage liquid is taken and added into equal volumes of the FTG liquid culture mediums with different pH values to be uniformly mixed, a culture box is placed at 37 ℃ for 2 hours, 100 mu L of mixed liquid is taken and mixed with equal volumes of bacterial liquid, the titer is measured through a double-layer plate method, and three pH values are parallel. And drawing a phage pH stability curve by taking the pH value as an abscissa and the logarithmic value of phage titer as an ordinate, and determining the pH range of the highest point. The results are shown in FIG. 3.

2. Experimental results and analysis:

as can be seen from FIG. 3, phage RDPCP23040 maintains good activity in the pH range of 5.0-9.0, phage maintains high titers, and phage titers drop more at pH less than 5 or greater than 9.0. Phage RDPCP23040 was shown to be tolerant of a degree of weak acids and bases, although not tolerant of strong acids and bases.

Example 8 determination of optimal growth temperature for phage RDPCP23040

1. The experimental method comprises the following steps:

Preparing 10 ⁵ cfu/mL of exponential phase host bacterial liquid, diluting phage stock solution to a synthetic concentration according to the optimal infection complex number, mixing 100 mu L with an equal volume of bacterial liquid, pouring into a double-layer plate, respectively placing the plates at 30 ℃,40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ for culturing for 30min and 60min, taking out phage liquid every 5min after 30min, cooling to room temperature, and measuring phage titer by a double-layer plate method, wherein three temperatures are parallel. And drawing a phage-to-temperature sensitivity curve by taking the temperature as an abscissa and taking the logarithmic value of phage titer as an ordinate, and determining the temperature range of phage RDPCP and 23040. The results are shown in FIG. 4.

2. Experimental results and analysis:

As can be seen from FIG. 4, the phage RDPCP23040 can maintain 80% of its activity at below 60℃and can maintain over 30min of its activity at 70℃and can decrease rapidly in the environment above 80℃indicating that the phage can withstand a certain high temperature and cannot withstand extremely high temperatures.

Example 9 determination of the genetic stability of phage RDPCP23040

1. The experimental method comprises the following steps:

RDPCP23040 was serially passaged 30 times, equal amounts of RDPCP23040 and CP-20032 were added to each culture medium, each culture time was 6 hours, and then the plates were inverted and titers were determined. The results are shown in FIG. 5.

2. Experimental results and analysis:

as can be seen from FIG. 5, phage RDPCP23040 always maintains higher titers during 30 passages, and no obvious fluctuation appears, which indicates that the phage can be stably inherited.

Example 10 phage lysis spectroscopies

1. The experimental method comprises the following steps:

Phage host profile was detected by spot method. Randomly selecting host 40 strains of clostridium perfringens with different serotypes from a bacterial library, respectively coating 100 mu L of bacterial liquid to be detected on a culture medium of anaerobic jerky Shang Guti, after airing, absorbing 2 drops of 10 mu L of phage liquid on the culture medium, carrying out anaerobic inversion culture at 37 ℃ for 12-16 hours, observing whether plaque appears, and if so, indicating that the phage has a lysis effect on the strain. The results are shown in Table 4.

2. Experimental results and analysis:

as can be seen from Table 4, phage RDPCP23040 had a lytic effect on 40 strains of the 40 Clostridium perfringens strains selected in the test, with a lytic rate of 100%, indicating a broader lytic spectrum of the phage.

TABLE 4 phage RDPCP23040 cleavage spectra

-Representing an inability to cleave; + represents cleavable.

As can be seen from Table 4, the phage has a better lysis effect on clostridium perfringens, can lyse hosts of various serotypes, has a wider lysis spectrum, can replace antibiotics, and is environment-friendly.

Example 11 Low temperature resistance test of phage RDPCP23040

1. The experimental method comprises the following steps:

Taking 10 ⁸ pfu/mL of phage fermentation broth, respectively placing in the environments of 4 ℃,0 ℃,5 ℃ below zero and 10 ℃ below zero for 7 days, taking out phage liquid every 24 hours, measuring phage titer by a double-layer plate method, and recording the result. The results are shown in FIG. 6.

2. Experimental results and analysis:

As can be seen from fig. 6, phage RDPCP23040 was left in the 4 ℃ environment for 7 days, always maintaining higher titers, without significant fluctuations; the preparation method comprises the following steps of standing for 7 days at the temperature of 0 ℃ and reducing the titer by 2 orders of magnitude; the preparation method comprises the following steps of standing for 7 days at the temperature of-5 ℃, wherein the titer is reduced by 3 orders of magnitude; the potency was reduced by 4 orders of magnitude after 7 days in a-10 ℃ environment. As can be seen from FIG. 6, the phage has good stability in low-temperature storage, and can be stably stored for one week without inactivation even in an environment of about-10 ℃.

EXAMPLE 12 killing effect of phage RDPCP23040 on Clostridium perfringens spores

1. The experimental method comprises the following steps:

in the first step, spore suspension preparation:

(1) Clostridium perfringens CP-20032 was inoculated into tryptone glucose yeast extract broth (TPGY) and placed in an anaerobic jar for anaerobic cultivation for 24 hours.

(2) 0.1ML clostridium perfringens CP-20032 bacterial liquid in the culture medium is inoculated into 10mL of freshly prepared liquid thioglycolate culture medium (FTG), activated for 20min in a water bath at 75 ℃, cooled and then subjected to anaerobic culture for 18h. Then 1ml of the FTG which is just cultivated is sucked into a new 1 branch of liquid thioglycolate culture medium, anaerobic cultivation is carried out for 4 hours, and the steps are repeated for 1 time.

(3) The cells in FTG were then inoculated into freshly prepared strong medium of danken (Duncanstrong, DS). Anaerobic culturing in an anaerobic tank for 3 days. Centrifuging cultured Clostridium perfringens spores, centrifuging 10000g at 4deg.C for 10min, and repeatedly centrifuging for 3 times. The centrifuged spores were stored in sterile water and stored in a 4 ℃ refrigerator.

(4) The spore content was determined by optical microscopy counting. Firstly, 10mL of culture solution is smeared on a glass slide, dried and fixed, then 3-5 drops of malachite green staining solution are added, the staining solution is heated to heat, but not boiled, after the staining solution is heated for 4-5 min, the staining solution is poured out and washed, and spores are observed under an optical microscope.

(5) The initial amount of spores after preparation was about 1X 10 ⁵ cfu/mL.

Second, phage RDPCP23040 was validated for spore killing:

(1) Dividing the prepared spores into three groups, wherein group A takes 5mL of spores and 1mL of normal temperature mixing treatment for 48 hours, group B is negative control, group C takes 5mL of spores and 1mL of phage fermentation liquor for 48 hours;

(2) After 48h of treatment, respectively transferring the 3 groups of mixed solutions into fresh FTG culture medium for anaerobic culture, and observing the growth condition of spores;

(3) And (3) respectively carrying out gradient dilution on the 3 groups of mixed solutions after treatment, counting in a TSC flat-plate culture medium, and counting results. The results are shown in FIG. 7.

2. Experimental results and analysis:

The growth results of FTG show that the group A normal saline group and the group B negative control group grow well, uniform bacterial liquid can be formed, the group C phage treatment group grows flocculent, uniform bacterial liquid cannot be formed, as can be seen from FIG. 7, spores treated by phage have obvious inactivation phenomenon, and the counting results show that the phage can reduce the survival number of spores by 3 orders of magnitude, so that the phage has good killing effect on the gas-producing spores, and is friendly to the environment and has good application prospect.

Example 13 use of phage RDPCP23040 in therapy

1. The experimental method comprises the following steps:

The test is carried out on a certain layer farm in the Shandong province, and the chicken farm has 8000 layers raised in one chicken house, and the chicken house has the advantages of reduced egg production, inappetence, rough feathers, diarrhea and little death and panning. The section of the dead and panning chicken is examined, and the intestinal canal is filled with gas and accompanied by mucus, and the swelling and congestion of the intestinal canal are visible. The henhouse death and disease-elutriation feed was sent to a laboratory and the results showed clostridium perfringens infection.

Phage RDPCP23040 broth (titer 10 ⁸ pfu/mL) was waterline fed to the chicken flock. After the phage is used for 24 hours continuously for 3 days, the diarrhea symptom is obviously relieved, after the phage is used for 48 hours, the diarrhea phenomenon is basically eliminated, the feed intake is increased, the egg laying is improved to some extent, the spirit is good, after the phage is used for 72 hours, the egg laying is recovered, and the chicken flock is healed.

2. Experimental results and analysis:

The large group of experiments prove that the phage has better effect of treating clostridium perfringens disease in practical application, realizes anti-replacement resistance reduction, and is green and has no residue.

Example 14 use of phage RDPCP23040 for disinfection of poultry housing environments

1. The experimental method comprises the following steps:

In a certain farm in Shandong, a chicken house is provided with 5000 chickens, the feed intake of the chicken flock is reduced, feathers are fluffy, and water sample and thin manure are pulled to be accompanied by mucus. The chicken dying of illness is dissected and examined, the intestinal canal is engorged, the swelling is several times, and the internal flatulence is obviously accompanied with mucus.

Sampling and detecting the chicken coop, and respectively carrying out sample collection and laboratory detection on the chicken coop, the chicken coop ground, the waterline, the trough and the like, wherein the results show that clostridium perfringens and spores thereof can be detected by the chicken coop, the chicken coop ground, the waterline, the trough and the like.

Phage RDPCP23040 broth (titer 10 ⁸ pfu/mL) was fed to the waterline for treatment. Meanwhile, phage fermentation liquid (titer 10 ⁸ pfu/mL) is used for spraying on the ground of the henhouse, the henhouse and the trough respectively, and the phage liquid is a green biological agent, has no stimulation to organisms, can be used for sterilizing chickens in the henhouse once a day, is used for 3 days continuously, and samples and detects the henhouse environment after 3 days.

2. Experimental results and analysis:

As a result, it was found that clostridium perfringens was removed from each environment of the chicken house, and the presence of viable bacteria could not be detected. The experiment shows that the phage RDPCP23040 has a good therapeutic effect on clostridium perfringens of chicken flocks, has a good bactericidal effect on the environment of a chicken house, can kill pathogenic bacteria of the chicken, and can kill spores generated by clostridium perfringens, thereby having a good application prospect.

Example 15 application of phage RDPCP23040 in a cultivation Environment

1. The experimental method comprises the following steps:

In a certain farm in the Shandong region, clostridium perfringens is detected in a breeding henhouse, and the surrounding environment outside the henhouse of the farm is sampled and detected. The sampling place comprises a walking area of the breeding personnel outside the henhouse, tap water, a sewage discharge port and other environments around the henhouse. Firstly, collecting samples for aseptic operation at each place, and sending the samples to a normal laboratory for detection; and secondly, analyzing the detection result, and making a reasonable disinfection scheme for the detection result which possibly appears.

2. Experimental results and analysis:

the detection result shows that clostridium perfringens is also detected in the surrounding environment of the chicken house, which shows that the environment of the chicken house is polluted by pathogenic bacteria, thereby causing enteritis of the laying hens. Phage sterilization was performed on the surrounding of the farm, phage RDPCP23040 fermentation broth (titer 10 ⁸ pfu/mL) was used, spray sterilization was performed on different environmental areas, each day, for 5 days, and the sterile areas were re-sampled and re-inspected after 5 days. The recheck results show that clostridium perfringens is not detected in the environment of the farm.

The experiment shows that the phage fermentation broth can be used for treating clostridium perfringens disease of poultry, can be applied to disinfection of surrounding environment of a farm, eliminates harmful bacteria, purifies the cultivation environment, and is beneficial to healthy development of poultry cultivation industry.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (10)

1. Clostridium perfringens bacteriophage (Clostridium perfringens bacteriophage) RDPCP23040, which is characterized in that clostridium perfringens bacteriophage is preserved in China general microbiological culture Collection center (CGMCC No. 45583) at 2023, month 06 and 12.

2. A phage composition comprising clostridium perfringens phage RDPCP23040 according to claim 1.

3. Use of clostridium perfringens bacteriophage RDPCP23040 according to claim 1 or of a bacteriophage composition according to claim 2 for the preparation of a product for the prevention and/or treatment of diseases in poultry caused by clostridium perfringens.

4. Use of clostridium perfringens bacteriophage RDPCP23040 according to claim 1 or a bacteriophage composition according to claim 2 for the preparation of a product for killing or inhibiting clostridium perfringens or spores thereof.

5. A phage pharmaceutical formulation, wherein the active ingredient comprises clostridium perfringens phage RDPCP23040 of claim 1 or a phage composition of claim 2.

6. The phage pharmaceutical formulation of claim 5, wherein the clostridium perfringens phage RDPCP23040 is at a concentration of at least 10 ⁸ pfu/mL.

7. A disinfectant comprising the clostridium perfringens bacteriophage RDPCP23040 of claim 1 or the bacteriophage composition of claim 2 as an active ingredient.

8. The disinfectant according to claim 7, wherein the clostridium perfringens bacteriophage RDPCP, 23040 is used at a concentration of at least 10 ⁸ pfu/mL.

9. Use of a disinfectant according to claim 7 or 8 for environmental disinfection, wherein the disinfectant is used for disinfection of clostridium perfringens in an avian farming environment by spraying, soaking.

10. The use according to claim 9, wherein the poultry farming environment is poultry in vivo, poultry on surface, poultry farm on ground, poultry farm air, feed, drinking water and feeding appliances.