Clostridium perfringens bacteriophage and coating process thereof

Abstract

The invention discloses a clostridium perfringens bacteriophage and a coating process thereof, the clostridium perfringens bacteriophage comprises a bacteriophage, the bacteriophage is clostridium perfringens bacteriophage CP1, and the preservation number is CCTCC NO: M20251192; through electron microscope observation, the clostridium perfringens bacteriophage CP1 is in a tadpole shape and can quickly and thoroughly kill clostridium perfringens, so that serositis of waterfowls, salpingitis of poultry and the like are effectively controlled, the production benefits of farms are improved, and the production cost is reduced. The genome of the clostridium perfringens bacteriophage CP1 does not contain virulence genes, lysogenic genes and drug-resistant genes and has high biological safety, the titer of the clostridium perfringens bacteriophage CP1 is only reduced by one order of magnitude when the clostridium perfringens bacteriophage CP1 is subjected to water bath at 50 DEG C for 24 hours, 10 < 4 > PFU / mL bacteriophage still survives when the clostridium perfringens bacteriophage CP1 is subjected to water bath at 60 DEG C for 48 hours, the clostridium perfringens bacteriophage CP1 has good thermal stability, meanwhile, the burst period of the bacteriophage CP1 is about 60 minutes, the explosion amount is 300; therefore, the bacteriophage CP1 has a relatively short incubation period and a relatively high outbreak amount.

Description

Technical Field

[0001] This invention belongs to the field of research and development technology of Clostridium perfringens bacteriophage, specifically relating to a Clostridium perfringens bacteriophage and its coating process. Background Technology

[0002] Clostridium perfringens is a Gram-positive bacterium that is a conditionally pathogenic bacterium commonly found in the gastrointestinal tract of healthy humans and animals. When the intestinal homeostasis is disrupted due to sudden changes in the external environment, changes in diet, or improper antibiotic treatment, Clostridium perfringens can increase the number of colonies and release toxins, thereby causing intestinal mucosal damage, tissue necrosis, and exacerbating inflammatory responses. In severe cases, it can lead to necrotizing enteritis or systemic infection.

[0003] Enterotoxemia caused by Clostridium perfringens is reported to be one of the most common diseases in ruminants worldwide, with a prevalence of 24.13%–100% in sheep and goats; in poultry, it causes necrotic enteritis with a mortality rate as high as 50%. Treatment with antibiotics not only results in poor prognoses but also leads to increasing drug resistance in animal-derived Clostridium perfringens, seriously impacting public health. Therefore, controlling the number of Clostridium perfringens is crucial to solving this disease. Clostridium perfringens can survive for extended periods in various environments, exhibiting strong adaptability, particularly in warm, humid soil, domestic and livestock wastewater, and feed. It can also survive and remain infectious in the intestines of dead animals for extended periods.

[0004] In theory, antibiotics are the first-line treatment, but to avoid antibiotic residues in meat and eggs and to ensure food safety, antibiotics should be avoided as much as possible. Therefore, there are currently no safe and residue-free drugs in farms to effectively control necrotic enteritis in poultry. Bacteriophages are a type of biological agent that can infect and kill bacteria. Compared with traditional antibiotics, bacteriophages have advantages such as high specificity, safety, and low resistance. Therefore, we need to provide a Clostridium perfringens bacteriophage and its coating process. Summary of the Invention

[0005] The purpose of this invention is to provide a Clostridium perfringens phage and its coating process, a virulent phage 24CP1 and its sustained-release formulation, which can effectively improve the stability of Clostridium perfringens phage, continuously, rapidly and thoroughly kill Clostridium perfringens, effectively control necrotic enteritis in poultry, and improve the production efficiency of farms. This addresses the problems mentioned in the background art, such as high antibiotic resistance and residue risk, lack of safe and residue-free control methods, poor stability of uncoated phages, low gastric passage rate, and insufficient long-term intestinal antibacterial ability.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a Clostridium perfringens phage, comprising:

[0007] The bacteriophage, specifically Clostridium perfringens phage CP1, is deposited at the China Center for Type Culture Collection on May 26, 2025, with accession number CCTCCNO: M20251192, and classified as Clostridium perfringens phage NNCP4P. Electron microscopy reveals that Clostridium perfringens phage CP1 is tadpole-shaped, with a polyhedral symmetrical head and a relatively long tail. The major diameter is 100–110 nm, the transverse diameter is 70–80 nm, the tail length is 120–130 nm, and the width is 15–20 nm. It possesses a contractile muscle sheath.

[0008] Preferably, the Clostridium perfringens phage CP1 has an outbreak period of approximately 60 minutes and an outbreak quantity of 300; thus, this phage CP1 has a short latency period and a high outbreak quantity.

[0009] Preferably, the Clostridium perfringens phage CP1 is unaffected at 40°C, maintains high activity after a 4-hour water bath at 50°C with virtually no change from its initial titer, and only decreases by one order of magnitude after a 2-hour water bath at 60°C. Furthermore, phages still survive after a 2-hour water bath at 70°C and a 1-hour water bath at 80°C. This demonstrates that Clostridium perfringens phage CP1 has excellent high-temperature resistance.

[0010] Preferably, after the Clostridium perfringens phage CP1 is treated under acidic conditions of pH=3 for 8 hours, although the titer is significantly reduced, the phage still has an activity of 105 pfu / mL. This shows that phage CP1 has strong acid resistance, suggesting that this phage may have a strong ability to resist gastric acid in animals, thus improving its clinical application effect in vivo.

[0011] Preferably, the Clostridium perfringens phage CP1 exhibits good tolerance to commonly used antiviral drugs, and after 72 hours of combined treatment with normal doses of ribavirin, astragalus polysaccharide, and Shuanghuanglian, it still retains at least 1×10⁻⁶ mmol / L. 7 The detection of phages at pfu / mL allows for their use in combination with the aforementioned antiviral drugs to achieve synergistic prevention and control of bacterial and viral diseases. Furthermore, the phage genome does not contain virulence genes, lysogenic genes, or drug resistance genes, and does not encode proteins that pose potential health risks, thus having no adverse effects on human and animal bodies.

[0012] A process for coating Clostridium perfringens phages includes the following steps:

[0013] Weigh trehalose and L-leucine at a mass ratio of 9:1, mix them and dissolve them in pure water to prepare a coating solution with a total solids concentration of 10 mg / mL.

[0014] The prepared coating solution was mixed with Clostridium perfringens phage CP1 suspension at a volume ratio of 1:50, and stirred for 30 min under suitable conditions to ensure that the phage and coating solution were fully combined.

[0015] The spray drying equipment was debugged and set with the inlet temperature at 130℃, the feed rate at 7.5mL / min, the atomized airflow at 742L / h, and the outlet temperature at 58±3℃.

[0016] The mixture of bacteriophage and coating solution is passed into a spray drying device with pre-set parameters for spray drying.

[0017] Collect the white powder (particle size 3-10μm) produced after spray drying, and seal it in a desiccator at 20±3℃ to obtain the coated Clostridium perfringens phage CP1 powder.

[0018] Preferably, the suitable stirring conditions are 25±2℃ and 150-200 r / min. A sterile, sealed container is used during stirring to avoid external microbial contamination and phage activity loss. Furthermore, the initial titer of the Clostridium perfringens phage CP1 suspension is not less than 1×10¹. 0 The concentration of PFU / mL was adjusted to ensure that the phage concentration in the mixed system met the requirements for subsequent coating and application.

[0019] Preferably, the spray drying process requires maintaining a sterile environment inside the equipment. Before drying, the equipment's feed pipe, atomization chamber, and collection chamber are sterilized with high-pressure steam at 121°C for 30 minutes. The collected white powder is required to have a moisture content of ≤3%. If the moisture content exceeds this range, the spray drying process must be repeated to ensure the stability and long-term preservation effect of the coated powder.

[0020] The application of a Clostridium perfringens bacteriophage in the preparation of drugs for the prevention or treatment of secondary bacterial infections in viral diseases.

[0021] The use of a Clostridium perfringens bacteriophage in the preparation of a drug for treating poultry diseases caused by Clostridium perfringens infection.

[0022] Technical effects and advantages of the present invention: The Clostridium perfringens bacteriophage and its coating process proposed in this invention have the following advantages compared with the prior art:

[0023] The Clostridium perfringens phage CP1 of this invention can rapidly and thoroughly kill Clostridium perfringens, effectively controlling serositis in waterfowl and oviductitis in poultry, thus improving the production efficiency of farms. The genome of Clostridium perfringens phage CP1 does not contain virulence genes, lysogenic genes, or drug resistance genes, exhibiting high biocompatibility. Furthermore, after a 24-hour water bath at 50°C, the titer of Clostridium perfringens phage CP1 only decreases by one order of magnitude, and after a 48-hour water bath at 60°C, 10⁴ PFU / mL of phage still survives, demonstrating good thermal stability. Simultaneously, the outbreak period of phage CP1 is approximately 60 minutes, with an outbreak quantity of 300. Therefore, this phage CP1 has a short latency period and a high outbreak quantity. Even after 8 hours of treatment under acidic conditions (pH=3), although the titer significantly decreases, the phage still retains 10⁵ PFU / mL of activity, indicating that phage CP1 has strong acid resistance.

[0024] The Clostridium perfringens phage CP1 of this invention was exposed to three antiviral drugs for 72 hours, and at least 1×107 pfu / mL of phage was still detected. This indicates that phage CP1 can coexist with some antiviral drugs for a period of time and has a strong ability to resist antiviral drugs. There was no significant difference in the titer in chicken serum after the first and last feeding, proving that ducks do not easily develop antibodies against it and are resistant to the immune clearance mechanism. It has good stability at 42°C and can effectively withstand the intestinal environment of poultry, thus exerting a good bactericidal effect.

[0025] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description and the drawings. Attached Figure Description

[0026] Figure 1 This is a flowchart of the steps of the present invention;

[0027] Figure 2 This is an electron micrograph of Clostridium perfringens phage CP1 of the present invention;

[0028] Figure 3 This is a photograph of the Clostridium perfringens phage CP1 plate culture of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention provides, for example Figure 1-3 The illustrated Clostridium perfringens bacteriophage comprises:

[0031] The bacteriophage, specifically Clostridium perfringens phage CP1, with accession number CCTCCNO: M20251192, was observed under an electron microscope. Clostridium perfringens phage CP1 is tadpole-shaped, with a polyhedral symmetrical head and a relatively long tail. Its major diameter is 100–110 nm, and its transverse diameter is 70–80 nm. The tail is 120–130 nm long and 15–20 nm wide, and possesses a contractile muscle sheath.

[0032] The Clostridium perfringens phage CP1 has an outbreak period of approximately 60 minutes and an outbreak quantity of 300; thus, this phage CP1 has a short latency period and a high outbreak quantity.

[0033] Specifically, the lysis characteristics of phage CP1 were determined using a one-step growth curve experiment: phage proliferation broth with a multiplicity of infection (MOI) of 10 was mixed with *Clostridium perfringens* in its logarithmic growth phase. After anaerobic incubation at 37°C for 5 min, unadsorbed phages were removed by centrifugation at 12000 r / min. The precipitate was then resuspended in preheated TSB medium at 37°C and anaerobically cultured at 37°C. Phage titers were measured every 10 min starting from time 0. The results showed that after phage CP1 infected the host bacteria, the titer remained stable at 1×10³ PFU / mL for the first 10 min, which was the latent period (approximately 10 min). Subsequently, the titer rapidly increased from 10 to 70 min, reaching 1×10³ PFU / mL at 70 min. 9 PFU / mL, the potency tended to stabilize and reached a peak of 3×10¹ after 80 min. 0 The burst period (approximately 60 minutes) is defined as the phase from the end of the incubation period to the point where the titer stabilizes. Based on the burst rate calculation formula (burst rate = phage titer at the end of lysis / host bacterial concentration at the initial stage of infection), the burst rate is 300, which is significantly higher than that of conventional Clostridium perfringens phage (average burst rate 100-200). It can proliferate rapidly and efficiently lyse host bacteria in a short period of time, quickly controlling the Clostridium perfringens load in the intestine.

[0034] The Clostridium perfringens phage CP1 was unaffected at 40°C and maintained high activity after a 4-hour water bath at 50°C, with little change from the initial titer. After a 2-hour water bath at 60°C, the activity decreased by only one order of magnitude. Furthermore, phages still survived after a 2-hour water bath at 70°C and a 1-hour water bath at 80°C. This indicates that Clostridium perfringens phage CP1 has good high-temperature resistance.

[0035] Specifically, the high-temperature resistance of bacteriophage CP1 was determined through temperature stability experiments: an initial titer of 1×10¹ was used. 0 One mL of pure phage CP1 culture medium (pfu / mL) was placed into sterile EP tubes and incubated in constant temperature water baths at 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 h, 2 h, 3 h, 4 h, and 24 h, respectively. After each time point, the EP tubes were immediately transferred to an ice bath for rapid cooling to terminate the continuous effect of temperature on phage activity. Subsequently, the phage titer of each group was determined using the double-layer plate method (the treated phage solution was serially diluted, mixed with Clostridium perfringens in the logarithmic phase, added to semi-solid TSB medium, poured onto TSA plates, and anaerobically incubated at 37℃ for 6 h before counting plaques). The experiment was repeated three times and the average value was taken.

[0036] After being exposed to acidic conditions at pH 3 for 8 hours, the Clostridium perfringens phage CP1 showed a significant decrease in titer, but still retained an activity of 105 pfu / mL. This indicates that phage CP1 has strong acid resistance, suggesting that this phage may have strong resistance to gastric acid in animals, thus enhancing its clinical application efficacy.

[0037] Specifically, the acid resistance of bacteriophage CP1 was determined through a pH stability experiment: First, sterile TSB medium with pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0 was prepared. 900 μL of each pH value medium was placed into sterile EP tubes and placed in a 25°C constant temperature water bath for 10 min to equilibrate. Then, 100 μL of a 1×10¹ initial titer was added to each EP tube. 0 PFU / mL pure culture medium of phage CP1 was mixed and allowed to stand at room temperature. Samples were taken after 1 h, 4 h, 8 h, 24 h and 96 h of incubation. The phage titer was determined by the double-layer plate method (the sample was serially diluted and mixed with the logarithmic phase of Clostridium perfringens culture, added to semi-solid TSB medium and poured onto TSA plates, and anaerobic incubated at 37℃ for 6 h before counting the plaques). The experiment was repeated 3 times and the average value was taken.

[0038] The Clostridium perfringens phage CP1 exhibits good tolerance to commonly used antiviral drugs. After 72 hours of co-treatment with normal doses of ribavirin, astragalus polysaccharide, and Shuanghuanglian, it still showed at least 1×10⁻⁶ mmol / L. 7 The detection of phages at pfu / mL allows for their use in combination with the aforementioned antiviral drugs to achieve synergistic prevention and control of bacterial and viral diseases. Furthermore, the phage genome does not contain virulence genes, lysogenic genes, or drug resistance genes, and does not encode proteins that pose potential health risks, thus having no adverse effects on human and animal bodies. Specifically, the initial valence is taken as 1×10¹ 0 Pure culture of phage CP1 (pfu / mL) and pure culture of control phage BP13 (preservation number CCTCCNO: MCCTCCNO: M20251192) were aliquoted into 50mL sterile centrifuge tubes, with three replicates per group. Ribavirin injection (final concentration 10mg / L), Astragalus polysaccharide oral solution (final concentration 20mg / L), and Shuanghuanglian oral solution (final concentration 15mg / L) were added to each centrifuge tube according to commonly used clinical treatment doses for livestock and poultry. Phage culture without added drugs served as a blank control. All samples were incubated at a constant temperature of 25℃. Samples were taken after 2h, 4h, 8h, 24h, 48h, and 72h. The phage titer was determined using the double-layer plate method (the sample was serially diluted and mixed with Clostridium perfringens in the logarithmic phase, added to semi-solid TSB medium and poured onto TSA plates, and anaerobic incubated at 37℃ for 6h before counting plaques).

[0040] The results showed that when bacteriophage CP1 coexisted with three antiviral drugs for 24 hours, its titer remained at 1×10⁻⁶. 9 above pfu / mL; after 48 hours of coexistence, the potency decreased to 1.4 × 10⁻⁶ pfu / mL. 8 ~2×10 8 pfu / mL; even after coexisting for 72 hours, it still has 1×10 7 ~2.5×10 7 Phages were detected at pfu / mL. However, after 24 hours of exposure to the three drugs, the titer of the control phage BP13 dropped to 1×10²~1.5×10³ pfu / mL, and its activity was completely undetectable after 48 hours of exposure, which fully demonstrates that CP1 has significant resistance to the above antiviral drugs.

[0041] A process for coating Clostridium perfringens phages includes the following steps:

[0042] Weigh trehalose and L-leucine at a mass ratio of 9:1, mix them and dissolve them in pure water to prepare a coating solution with a total solids concentration of 10 mg / mL.

[0043] The prepared coating solution was mixed with Clostridium perfringens phage CP1 suspension at a volume ratio of 1:50, and stirred for 30 min under suitable conditions to ensure that the phage and coating solution were fully combined.

[0044] The spray drying equipment was debugged and set with the inlet temperature at 130℃, the feed rate at 7.5mL / min, the atomized airflow at 742L / h, and the outlet temperature at 58±3℃.

[0045] The mixture of bacteriophage and coating solution is passed into a spray drying device with pre-set parameters for spray drying.

[0046] Collect the white powder (particle size 3-10μm) produced after spray drying, and seal it in a desiccator at 20±3℃ to obtain the coated Clostridium perfringens phage CP1 powder.

[0047] The suitable stirring conditions are 25±2℃ and 150-200 r / min. A sterile, sealed container is used during stirring to avoid external microbial contamination and phage activity loss. Furthermore, the initial titer of the Clostridium perfringens phage CP1 suspension is not less than 1×10¹. 0 PFU / mL, to ensure that the phage concentration in the mixed system meets the requirements for subsequent coating and application;

[0048] Specifically, the stirring temperature was set at 25±2℃ because this temperature range is the stable activity temperature of phage CP1 (below 20℃ will slow down the binding rate of phage to the coating solution, and above 28℃ may cause denaturation of some phage capsid proteins); the stirring speed was controlled at 150-200 r / min, which was verified through preliminary experiments: when the speed is below 150 r / min, the coating solution (trehalose-L-leucine solution) and the phage suspension are prone to stratification, resulting in insufficient mixing uniformity and some phages not being coated; when the speed is above 200 r / min, a large number of bubbles will be generated, and the impact force when the bubbles burst may damage the phage structure, resulting in a 10%-15% loss in titer. The stirring vessel is a sterile glass reactor with a sealed lid. Before use, it must be sterilized by high-pressure steam at 121℃ for 30 minutes and cooled before being added to the mixing system. During the stirring process, sterile nitrogen gas is continuously introduced (flow rate 50mL / min) to isolate the air and avoid contamination by other bacteria, and to prevent bubbles generated by stirring from adhering to the surface of the bacteriophage, thus further ensuring the activity of the bacteriophage. After stirring for 30 minutes under these conditions, the CP1 titer loss rate of the bacteriophage is ≤3%.

[0049] The spray drying process requires maintaining a sterile environment inside the equipment. Before drying, the equipment's feed pipe, atomization chamber, and collection chamber are sterilized with high-pressure steam at 121°C for 30 minutes. The collected white powder must be tested for moisture content, which must be ≤3%. If it exceeds this range, the spray drying process must be repeated to ensure the stability and long-term preservation effect of the coated powder.

[0050] Specifically, the feed line, atomization chamber, and collection chamber of spray drying equipment (such as centrifugal spray dryers) are critical areas where bacteriophages come into contact with the outside world. These areas require "full-process sterilization + aseptic verification" to ensure they are free from microbial contamination. First, close all valves on the equipment and introduce high-pressure saturated steam at 121℃ and 0.1MPa into the feed line, atomization chamber, and collection chamber for 30 minutes (this parameter effectively kills stubborn microorganisms such as bacterial spores and fungal spores; microbial challenge experiments have verified that the residual microorganisms in the equipment after sterilization are ≤1 CFU / 100cm²). After sterilization, purge the inside of the equipment with sterile compressed air (filtered through a 0.22μm membrane) for 30 minutes until the inner walls of the equipment are dry and the temperature drops below 50℃ to prevent residual condensate from diluting the mixture or affecting bacteriophage activity. During the drying process, the sterility of the equipment's inlet and outlet is continuously monitored: every 1 hour, 1 mL of the mixed solution is taken from the sampling port of the inlet pipeline and spread on a TSA plate (incubated at 37℃ for 24 hours); 0.1 g of powder sample is taken from the sampling port of the collection chamber, dissolved in 10 mL of sterile SM solution, and then spread on a TSA plate. If no colonies grow on all plates, it proves that the sterile environment inside the equipment is well maintained, which can ensure that the final coated powder is free from exogenous microbial contamination and avoid secondary infection after livestock and poultry consume it.

[0051] The application of a Clostridium perfringens bacteriophage in the preparation of drugs for the prevention or treatment of secondary bacterial infections in viral diseases.

[0052] The use of a Clostridium perfringens bacteriophage in the preparation of a drug for treating poultry diseases caused by Clostridium perfringens infection.

[0053] The CP1 phage coating process of Clostridium perfringens bacteriophage of the present invention can significantly improve the gastric passage rate of the phage and provide long-term sustained release in the intestine, with activity still detectable in the intestine 35 days after drug withdrawal.

[0054] The Clostridium perfringens phage CP1 described in this invention can be used as an effective ingredient in various environmental disinfection products, including but not limited to liquid immersion, spraying, and combined use with aqueous carriers, for disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities, or other environmental surfaces, effectively controlling the growth and activity of target bacteria. The liquid immersion and spraying methods include, but are not limited to, detergents, disinfectants, and stain removers; the aqueous carriers include, but are not limited to, phosphate buffer, TSB medium, LB medium, and chlorinated free water.

[0055] The product forms of the present invention may include, but are not limited to, application to the surface, mouth, rectum, pleura, and other parts of the host being treated by means of carrier delivery, concentrated injection, or drug immersion; as one embodiment, the carrier delivery forms include, but are not limited to, oral aqueous carriers, oral anhydrous carriers, cream preparations, etc.; the concentrated injection forms include, but are not limited to, vaccine injection, intrapleural injection, intravenous injection, etc.; the drug immersion forms include, but are not limited to, aerosols, rinsing agents, etc.

[0056] In the following examples:

[0057] The formula for TSB liquid culture medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, and 1000mL distilled water.

[0058] The formula for TSA solid culture medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar, and 1000mL distilled water.

[0059] TSA plates: TSA solid culture medium is sterilized and poured onto sterile plates, then cooled and solidified to make TSA plates;

[0060] The formula for TSB semi-solid agar medium is: 15g tryptone, 5g soybean peptone, 5g sodium chloride, 7g agar, and 1000mL distilled water.

[0061] The formula for blood agar plate culture medium is as follows: 10g peptone, 3g beef powder, 5g sodium chloride, 50mL defibrinated sheep blood, 15g agar, and 1000mL distilled water.

[0062] The SM solution formula is as follows: 8.5g sodium chloride, 2g magnesium sulfate, 50ml 1mol / L Tris-HCl, 0.25g gelatin, and 1000ml distilled water.

[0063] Example 1: Isolation and purification of Clostridium perfringens phage CP1

[0064] The source sample for isolating Clostridium perfringens phage CP1 in this invention was collected from sewage from a livestock farm in Tongling City, Anhui Province. After being filtered through double-layer filter paper, the sample was centrifuged at low speed and room temperature, and then the supernatant was filtered through a 0.22μm filter membrane.

[0065] Phage isolation: Take 10 mL of the filtered supernatant and add it to 10 mL of double-strength TSB medium, along with 1 mL of the phage host bacteria in logarithmic phase. Incubate anaerobicly at 37°C for 6 h. Centrifuge the culture at 8000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane. Take 0.5 mL of the phage host bacteria in logarithmic phase and add it to 5 mL of 40°C semi-solid TSB medium. Mix well and pour onto a TSA plate to prepare a double-layer plate containing the host bacteria. Take 10 μl of the filtered supernatant and drop it onto the solidified double-layer plate. Air dry under aseptic conditions and incubate anaerobicly at 37°C for 6 h to form phage spot dots.

[0066] Phage purification: Pick phage plaques and add them to 1 mL of SM buffer, shake for 1 min, and perform 10-fold serial dilutions. Add 0.5 mL of logarithmic-phase host bacteria to each of the 10², 10⁴, and 10⁶ dilutions, mix well, and let stand for 15 min. Then add 5 mL of 40°C semi-solid TSB medium and immediately pour it onto TSA plates. Shake well and let stand for 5 min to solidify. Incubate at 37°C for 6 h and observe to obtain bilayer plates containing single phage plaques. Pick single phage plaques and add them to 1 mL of SM buffer. Purify at least 3 times using the above method. Finally, pick single phage plaques of uniform morphology and size from the plates that formed the plaques (e.g., ...). Figure 2 The culture was placed in 50 mL of TSB medium containing 1 mL of logarithmic-phase host bacterial culture and incubated anaerobicly at 37 °C for 6 h. The culture was centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the purified phage solution, yielding Clostridium perfringensphage CP1 (accession number CCTCCNO: M20251192).

[0067] Example 2: Electron microscopic observation of Clostridium perfringens phage CP1

[0068] The purified phage solution prepared in Example 1 was observed under an electron microscope: 20 μL of sample was dropped onto a copper grid and allowed to precipitate naturally for 15 min. Excess liquid was absorbed from the side with filter paper. One drop of 2% phosphotungstic acid was added to the copper grid and stained for 10 min. The staining solution was then absorbed from the side with filter paper and dried before observation under an electron microscope.

[0069] The results are as follows Figure 2As shown, observation of the morphology of Clostridium perfringens phage CP1 under an electron microscope revealed that the phage has a polyhedral three-dimensional symmetrical head and a relatively long tail. The head diameter is approximately 50–55 nm; the tail length is approximately 20–30 nm, and the tail diameter is approximately 10–15 nm. It also has a contractile muscle sheath.

[0070] Example 3: Extraction and sequencing of the CP1 genome of Clostridium perfringens phage

[0071] Take 100 mL of the purified phage solution prepared in Example 1, add 20 μL of DNase I and 20 μL of RNase A at a concentration of 5 mg / mL in sequence, incubate at 37°C for 60 min, then add 5.84 g of NaCl, and place in an ice bath for 1 h after dissolution.

[0072] Centrifuge at 11,000 rpm for 10 min at 4℃, transfer the supernatant to a new centrifuge tube, add solid PEG8000 to make a final concentration of 10% (w / v), and incubate on ice for 1 h after the PEG8000 is completely dissolved.

[0073] Then, centrifuge at 11,000 rpm for 20 min at 4℃, add 1 mL of SM solution to resuspend the precipitate, and obtain the phage particle concentrate, which is stored at 4℃ for later use.

[0074] Example 6: Biological characteristics of Clostridium perfringens phage CP1

[0075] 6.1 Temperature stability of Clostridium perfringens phage CP1

[0076] Take 1 mL of pure phage CP1 culture medium with an initial titer of 1 × 10¹⁰ pfu / mL and place it in a sterile EP tube. Incubate the tube in a water bath at 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 h, 2 h, 3 h, 4 h, and 24 h, respectively. After the incubation period, remove the sample tube and immediately place it in an ice bath to cool. After appropriate dilution, determine the phage titer using the double-layer plate method. The experiment was repeated three times.

[0077] The results are shown in Table 3. Clostridium perfringens phage CP1 was unaffected at 40℃ and maintained high activity after a 4-hour water bath at 50℃, with little change from the initial titer. After a 2-hour water bath at 60℃, the activity decreased by only one order of magnitude. Furthermore, phages still survived after a 2-hour water bath at 70℃ and a 1-hour water bath at 80℃. This indicates that Clostridium perfringens phage CP1 has good high-temperature resistance.

[0078] Table 3. Potency of Clostridium perfringens phage CP1 at different temperatures.

[0079] 6.2 pH stability of Clostridium perfringens phage CP1

[0080] Add 900 μL of TSB medium at different pH values ​​(1-14) to sterile EP tubes and place them in a 25°C water bath. After temperature equilibration, add 100 μL of pure phage CP1 culture medium with an initial titer of 1×10¹⁰ PFU / mL and incubate at room temperature for 1 h. After the reaction time is complete, dilute the samples appropriately and determine the phage titer using the double-layer plate method. Repeat the above process at 4 h, 8 h, 24 h, and 96 h, for a total of 3 replicates.

[0081] The results are shown in Table 4. Clostridium perfringens phage CP1 showed no significant change in titer between pH 5 and pH 10, indicating good stability under neutral, slightly acidic, and slightly alkaline conditions. Under acidic conditions (pH=2) and alkaline conditions (pH=13), the phage titer decreased to 0 within one hour, indicating that the phage can tolerate pH 3-12. In particular, after 8 hours of treatment under acidic conditions (pH=3), although the titer decreased significantly, the phage still maintained an activity of 105 pfu / mL, demonstrating that phage CP1 has strong acid resistance. This suggests that the phage may have strong resistance to gastric acid in animals, potentially enhancing its clinical efficacy.

[0082] Table 4. Phage titers of Clostridium perfringens after different pH values ​​and reaction times.

[0083]

[0084] 6.3 One-step growth curve of Clostridium perfringens phage CP1

[0085] One mL each of phage proliferation medium with a multiplicity of infection (MOI) of 10 and fresh host bacteria proliferation medium were incubated anaerobically at 37°C for 5 min, centrifuged at 12000 rpm for 30 s, and the supernatant was discarded. An appropriate amount of TSB (37°C) medium was added, and the mixture was centrifuged at 12000 rpm for 1 min, and the supernatant was discarded. This process was repeated three times to remove unadsorbed host bacteria phage. 20 mL of TSB liquid was added, and the mixture was incubated anaerobically at 37°C. Samples were taken every 10 min starting from time 0, centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was collected. The phage titer was measured, and the experiment was repeated three times. A one-step growth curve was plotted with infection time (t) on the x-axis and phage titer (PFU / mL) on the y-axis to determine the phage latency and lysis period, and the lysis mass was calculated. Lysis mass = phage titer at the end of lysis / host bacteria concentration at the beginning of infection.

[0086] Table 5 shows that after phage CP1 infects the host bacteria, its titer remains basically unchanged within 10 minutes, stabilizing at 103 PFU / mL. This indicates that the incubation period of phage CP1 is approximately 10 minutes. Within 10–80 minutes after phage infection, the number of phages gradually increases, and the titer increase begins to stabilize at 80 minutes, at which point the titer can reach 3 × 10¹⁰ PFU / mL. This suggests that the outbreak period of phage CP1 is approximately 60 minutes, with an outbreak quantity of 300. Therefore, this phage CP1 strain has a relatively short incubation period and a relatively high outbreak quantity.

[0087] Table 5. One-step growth curve data of bacteriophage CP1

[0088] Example 7: Compatibility determination of Clostridium perfringens phage CP1 with antiviral drugs Phage CP1 (initial titer 1×10¹⁰ pfu / mL) and control phage BP13 (preservation number CCTCCNO: MCCTCCNO: M20251192, initial titer 1×10¹⁰ pfu / mL) were aliquoted into 50 mL sterile centrifuge tubes. Ribavirin injection, Astragalus polysaccharide oral solution, and Shuanghuanglian oral solution were added to the tubes respectively. The tubes were incubated at 25 °C for 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h. The phage titers were measured, and the results are shown in Table 6.

[0090] Table 6 shows that after 24 hours of coexistence with the antiviral drugs ribavirin injection, astragalus polysaccharide oral solution, and Shuanghuanglian oral solution, the titer of bacteriophage CP1 remained at 1×10⁹ pfu / mL. In contrast, after 24 hours of contact with the three antiviral drugs, the titer of another Escherichia coli bacteriophage, BP13, decreased significantly, with only 10²-10³ pfu / mL of bacteriophage remaining. After 48 hours of contact, BP13 bacteriophage was undetectable. However, after 72 hours of contact with the three antiviral drugs, bacteriophage CP1 still showed at least 1×10⁷ pfu / mL of bacteriophage. This indicates that bacteriophage CP1 can coexist with some antiviral drugs for a period of time and has a strong ability to resist antiviral drugs. This suggests that Clostridium perfringens bacteriophage CP1 can be used in combination with antiviral drugs in clinical practice to achieve simultaneous prevention and treatment of bacterial and viral diseases. Experiments show that compared to BP13, CP1 exhibits less titer loss when combined with antiviral drugs.

[0091] Table 6. Compatibility results of Clostridium perfringens phages CP1 and BP13 with antiviral drugs

[0092] Example 8: Immune clearance mechanism of Clostridium perfringens phage CP1 tolerance in the body

[0093] Even after long-term feeding with bacteriophages, high-titer bacteriophages can still be detected in the serum, indicating that the bacteriophages are not prone to producing antibodies and are resistant to the body's immune clearance mechanism.

[0094] Thirty healthy 2-week-old broiler chickens were randomly divided into three groups of 10 each. Group 1 was fed 1×10⁷ pfu of phage CP1 daily; Group 2 was fed 1×10⁷ pfu of phage BP13 daily; and Group 3 served as a control group with a normal diet. After the first phage feeding, 5 mL of blood was collected from the wing vein at 24, 48, and 72 hours after centrifugation, and the supernatant was used to determine the phage titer. Phage feeding continued for 60 days. After the last phage feeding, 5 mL of blood was collected from the wing vein at 24, 48, and 72 hours after centrifugation, and the supernatant was used to determine the phage titer. The average titer for each group was calculated. The results are shown in Table 7. There was no significant difference in serum CP1 titer between the first and last feedings, indicating that chickens do not readily develop antibodies against CP1, demonstrating a CP1 tolerance immune clearance mechanism. However, long-term feeding of BP13 can cause the body to produce antibodies that quickly eliminate it.

[0095] Table 7. Experimental results on the immune clearance capacity of Clostridium perfringens phages CP1 and BP13.

[0096]

[0097] Example 9: Clostridium perfringens phage CP1 exhibits long-term tolerance to visceral temperature environment.

[0098] Clostridium perfringens phage CP1 has long been resistant to 42℃ and can effectively withstand the internal temperature environment of chickens and laying hens.

[0099] 1 mL of pure phage CP1 and BP13 culture media, with an initial titer of 1 × 10⁹ pfu / mL, were placed in sterile EP tubes and incubated at 42 °C. Samples were collected at 7, 15, 30, 60, and 150 days, and after appropriate dilution, the phage titer was determined using the double-layer plate method. The experiment was repeated three times.

[0100] The results are shown in Table 8. After 150 days at 42℃, CP1 decreased by less than one order of magnitude, while BP13 decreased by two orders of magnitude. This proves that Clostridium perfringens phage CP1 has good stability at 42℃, can effectively withstand the intestinal environment of animals, and can exert a good bactericidal effect.

[0101] Table 8. Long-term stability of Clostridium perfringens phages CP1 and BP13 at 42℃

[0102] Example 10: Detection test for deletion of virulence genes or adverse genes in Clostridium perfringens phage CP1

[0103] In this embodiment, 103 virulence genes derived from lysogenic bacteriophages in pathogenic bacteria were selected, and the whole genome of Clostridium perfringens bacteriophage CP1 was determined and subjected to bioinformatics analysis.

[0104] In addition, the present invention also provides a terminal device. The Clostridium perfringens phage coating process involved in this embodiment is mainly applied in the terminal device, which can be a PC, a portable computer, a mobile terminal or other device with display and processing functions.

[0105] Specifically, the terminal device may include a processor (e.g., CPU), a communication bus, a user interface, a network interface, and memory. The communication bus is used to enable communication between these components; the user interface may include a display screen or an input unit such as a keyboard; the network interface may optionally include a standard wired interface or a wireless interface (such as a Wi-Fi interface); the memory may be high-speed RAM or stable non-volatile memory, such as disk storage, and may also optionally be a storage device independent of the aforementioned processor.

[0106] The memory stores a readable storage medium, which stores a Clostridium perfringens phage coating process program. The processor can call the Clostridium perfringens phage coating process program stored in the memory and execute the Clostridium perfringens phage coating process provided in this embodiment of the invention.

[0107] Understandably, a readable storage medium can be a tangible device capable of holding and storing instructions for use by an instruction execution device. A computer-readable storage medium can be, for example—but not limited to—an electrical storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination thereof. More specific examples (a non-exhaustive list) of computer-readable storage media include: portable computer disks, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable compact disc read-only memory (CD-ROM), digital multifunction disc (DVD), memory sticks, floppy disks, mechanical encoding devices, such as punch cards or recessed protrusions storing instructions thereon, and any suitable combination thereof. The computer-readable storage medium as used herein is not to be construed as a transient signal itself, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through waveguides or other transmission media (e.g., light pulses through fiber optic cables), or electrical signals transmitted through wires.

[0108] The computer-readable program instructions described herein can be downloaded from computer-readable storage media to various computing / processing devices, or downloaded via a network, such as the Internet, local area network, wide area network, and / or wireless network, to an external computer or external storage device. The network may include copper transmission cables, fiber optic transmission, wireless transmission, routers, firewalls, switches, gateway computers, and / or edge servers. A network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards them to the computer-readable storage media in the respective computing / processing device.

[0109] Computer program instructions used to perform operations of this disclosure may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, status setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Smalltalk, C++, etc., and conventional procedural programming languages ​​such as the "C" language or similar programming languages. The computer-readable program instructions may execute entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In cases involving a remote computer, the remote computer may be connected to the user's computer via any type of network—including a local area network (LAN) or a wide area network (WAN)—or may be connected to an external computer (e.g., via the Internet using an Internet service provider). In some embodiments, electronic circuitry, such as programmable logic circuitry, field-programmable gate arrays (FPGAs), or programmable logic arrays (PLAs), is personalized by utilizing the status information of the computer-readable program instructions to implement various aspects of this disclosure.

[0110] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Clostridium perfringens bacteriophage, characterized in that, The bacteriophage is Clostridium perfringens bacteriophage CP1, and the preservation number is CCTCC NO: M20251192. The Clostridium perfringens bacteriophage CP1 is tadpole-shaped under electron microscope observation, has a polyhedral head and a long tail, and has a long diameter of 100-110 nm, a transverse diameter of 70-80 nm, a tail length of 120-130 nm, and a width of 15-20 nm. The bacteriophage has a contractile sheath. The Clostridium perfringens bacteriophage CP1 has a burst period of about 60 min and a burst volume of 300. Therefore, the bacteriophage CP1 has a short latent period and a high burst volume.

2. A Clostridium perfringens bacteriophage according to claim 1, characterized in that: The Clostridium perfringens bacteriophage CP1 is not affected at 40 DEG C, still has high activity and basically no change in initial titer after water bath at 50 DEG C for 4 h, is reduced by only one order of magnitude after water bath at 60 DEG C for 2 h, and still has bacteriophage survival after water bath at 70 DEG C for 2 h and water bath at 80 DEG C for 1 h. Therefore, the Clostridium perfringens bacteriophage CP1 has good high-temperature resistance.

3. The Clostridium perfringens phage according to claim 1, characterized in that: Although the titer is obviously reduced, the bacteriophage still has activity of 105 pfu / mL after acting at an acidic condition of pH = 3 for 8 h. Therefore, the bacteriophage CP1 has strong acid resistance, and the bacteriophage has strong gastric acid resistance in the animal body, which improves the clinical application effect in the body.

4. The Clostridium perfringens phage according to claim 1, characterized in that: The method specifically comprises the following steps:

5. The Clostridium perfringens phage according to claim 1, characterized in that: The Clostridium perfringens bacteriophage CP1 has good tolerance to commonly used antiviral drugs, and at least 1x10 7 pfu / mL of the bacteriophage is detected after 72 hours of co-action with ribavirin, astragalus polysaccharide and Shuanghuanglian commonly used in normal doses, and the bacteriophage can be used in combination with the above-mentioned antiviral drugs to achieve the synergistic prevention and control of bacterial and viral diseases; and the bacteriophage genome does not contain virulence genes, lysogeny genes and drug resistance genes, and does not encode proteins that can cause potential health risks, and has no adverse effects on human and animal bodies.

6. A process for coating a Clostridium perfringens bacteriophage according to any one of claims 1 to 5, characterized in that, Trehalose and L-leucine are weighed according to a mass ratio of 9:1, mixed, and dissolved in pure water to prepare a coating liquid with a total solid concentration of 10 mg / mL; The prepared coating liquid and the Clostridium perfringens bacteriophage CP1 suspension are mixed at a volume ratio of 1:50, and the bacteriophage is fully combined with the coating liquid under suitable conditions for 30 min; The spray drying equipment is adjusted, the inlet temperature is set to 130 DEG C, the feeding rate is set to 7.5 mL / min, the atomizing airflow is set to 742 L / h, and the outlet temperature is controlled at 58 DEG C ± 3 DEG C; The mixed liquid in which the bacteriophage is fully combined with the coating liquid is introduced into the spray drying equipment with the set parameters for spray drying treatment; The white powder (particle size 3-10 um) produced after spray drying is collected and sealed and stored in a desiccator at 20 DEG C ± 3 DEG C to obtain the coated Clostridium perfringens bacteriophage CP1 powder. The spray drying process needs to maintain a sterile environment in the equipment. The feeding pipeline, atomizing chamber and collection chamber of the equipment are sterilized with 121 DEG C high-pressure steam for 30 min before drying. The moisture content of the collected white powder needs to be detected, and the moisture content is required to be ≤3%. If the moisture content exceeds the range, the spray drying process needs to be performed again to ensure the stability and long-term storage effect of the coated powder.

7. A process for coating a Clostridium perfringens bacteriophage according to claim 6, characterized in that: The suitable stirring condition is 25±2℃, 150-200r / min, and a sterile sealed container is used in the stirring process to avoid microbial contamination and loss of phage activity. The initial titer of the Clostridium perfringens bacteriophage CP1 suspension is not less than 1×10 0 PFU / mL, so that the phage concentration in the system after mixing meets the subsequent coating and application requirements.

8. The process for coating of Clostridium perfringens bacteriophage according to claim 6, wherein:

9. Use of a Clostridium perfringens bacteriophage in the preparation of a medicine for preventing or treating secondary bacterial infection of viral diseases.

10. Use of a Clostridium perfringens bacteriophage in the preparation of a medicine for treating poultry diseases caused by Clostridium perfringens disease. ​

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