Heat-resistant and acid-base-resistant clostridium perfringens bacteriophage XHX-CP-18P and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a heat-resistant and acid-base-resistant clostridium perfringens bacteriophage XHX-CP-18P and application thereof, the bacteriophage is preserved in the China Center for Type Culture Collection (CCTCC) on March 17, 2025, and the preservation number is CCTCC NO: M 2025492. The invention also provides a bactericide product prepared from the bacteriophage. The bacteriophage has good acid and alkali resistance under the condition that the pH is 3-13, has heat resistance under the condition that the temperature is 30-60 DEG C, and ensures that the activity of the bacteriophage is not lost. The bacteriophage has a good lysis effect on clostridium perfringens, is strong in infection ability, can infect more host cells with a very small amount of viruses, can effectively lyse A-type and G-type hosts, and can replace antibiotics to resist corrosion of gastric acid, so that intestinal diseases of the hosts are effectively treated.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a heat-resistant and acid-alkali-resistant Clostridium perfringens phage XHX-CP-18P and an application thereof. Background Art

[0002] Clostridium perfringens (Cp) is a Gram-positive bacillus, a long-standing anaerobic strain found in nature. Microscopically, it appears as a thin, straight rod with blunt ends. It is a serious opportunistic pathogen. When the living environment of humans and animals is disturbed by external factors such as a changing climate, a high-nutrient diet, and the use of antibiotics, C. perfringens can produce more than twenty protein toxins or tissue-destructive enzymes, causing disease in humans and animals. C. perfringens' pathogenicity is primarily due to the production of various exotoxins and tissue-destructive enzymes. Currently, the main pathogenic exotoxins identified are α, β, ε, ι, CPE, and NetB. C. perfringens is divided into seven pathotypes (A, G, and G) based on the presence of these six toxins. All seven toxin types (A through G) of C. perfringens carry and express the α toxin. Type A bacteria are particularly harmful, causing emphysema in human or animal tissues, enterotoxemia, and gastrointestinal infectious diseases. Toxins in the gastrointestinal tract are absorbed and circulate throughout the body, damaging other internal organs and severely endangering human and animal public health and the development of the modern poultry industry. Type G Clostridium perfringens is the primary pathogen of necrotic enteritis in chickens. A newly discovered toxin-producing strain, G, is widespread internationally. However, limited research has been conducted in China on its prevalence and potential for necrotic enteritis in chickens.

[0003] In recent years, the "prevention first, treatment second" farming strategy has been widely promoted. To promote animal growth and prevent disease, many farming companies are now adding antibiotics to feed to improve profitability. While this approach has improved animal health to some extent, the problem of antibiotic resistance has become increasingly serious over time, and the emergence of drug-resistant bacteria has seriously impacted global public health.

[0004] Bacteriophages are viruses that can kill bacteria. Bacteriophages are the largest known group of viruses, with an estimated population of about 10 billion on Earth. 31 Compared to the broad-spectrum antibacterial properties of traditional antibiotics, phages are highly specific, acting only against their host bacteria and leaving other intestinal flora unaffected. Furthermore, phages can co-evolve with their host bacteria. As long as the host bacteria are present, phages can rely on them for proliferation, enabling self-administration. When the bacteria lyse, the phages are automatically eliminated, leaving no residue in the body.

[0005] Currently, there are very few products that can prevent and treat heat-resistant and acid-alkali-resistant Clostridium perfringens. Therefore, it is urgent to develop a phage product that is heat-resistant and resistant to strong acids and alkalis, has a wide lysis spectrum, has both preventive and therapeutic effects, and has significant effects and stable genetics for the treatment of Clostridium perfringens disease. Summary of the Invention

[0006] The present invention aims to address the shortcomings of existing technologies by providing a heat- and acid-resistant Clostridium perfringens bacteriophage XHX-CP-18P and its applications. The heat- and acid-resistant Clostridium perfringens bacteriophage XHX-CP-18P provided by the present invention is heat- and acid-resistant, strong acid and alkali resistant, ensuring that the phage's activity is not lost. It can replace antibiotics, resist gastric acid corrosion, and effectively treat intestinal diseases in the host.

[0007] The above-mentioned objects of the present invention are achieved through the following technical solutions: In the first aspect, the present invention provides a heat-resistant and acid-alkali resistant Clostridium perfringens phage XHX-CP-18P, which was isolated from Weifang, Shandong, China on June 6, 2024, and deposited in the China Center for Type Culture Collection (CCTCC) on March 17, 2025. The deposit address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2025492.

[0008] The titer of the phage XHX-CP-18P was as high as 6.0×10 10 PFU / mL. The phage showed no significant change in activity after exposure to temperatures between 30 and 60°C for 30 minutes. It also maintained stability after treatment at a pH of 3-13 for 30 minutes, exhibiting high activity in strong acid and alkaline environments and maintaining a high lytic effect on host bacteria. Compared to other existing phages, it exhibited superior heat and acid-base resistance. Based on these excellent biological characteristics, the phage is capable of adapting to harsh environments with high temperatures, strong acids, and strong bases.

[0009] This bacteriophage has a good lytic effect on Clostridium perfringens. Experiments have shown that it can lyse hosts with serotypes A, C, D, E, and G. In the examples of the present invention, the lysis rate for selected serotypes A and G of Clostridium perfringens reached 100%. This shows that the bacteriophage XHX-CP-18P of the present invention has excellent lytic performance against both serotypes A and G of Clostridium perfringens, and has great application prospects in killing Clostridium perfringens in the environment and preventing and treating diseases caused by Clostridium perfringens.

[0010] The present invention provides a phage composition comprising the heat-resistant and acid-alkali-resistant Clostridium perfringens phage described above.

[0011] The present invention also provides use of the heat- and acid- and alkali-resistant Clostridium perfringens phage XHX-CP-18P or the phage composition described above in preparing a product for preventing or treating inflammatory reactions caused by Clostridium perfringens.

[0012] The term "prevention" herein refers to all actions including suppressing or delaying the disease by administering the bacteriophage; the term "treatment" herein refers to all actions including improving or ameliorating the disease by administering the bacteriophage.

[0013] The above-mentioned drugs can be used to prevent or treat Clostridium perfringens infections in humans and animals (chickens, pigs, cattle, geese, ducks, turkeys, etc.).

[0014] The present invention provides a phage pharmaceutical preparation, wherein the active ingredient of the phage pharmaceutical preparation comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens phage or phage composition as described above.

[0015] Furthermore, the phage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier; the dosage form of the phage pharmaceutical preparation includes one or more of a solution, a powder, a gel, a granule, an emulsion, a suspension and a lyophilized agent.

[0016] The present invention provides a feed additive, comprising the heat-resistant and acid-alkali-resistant Clostridium perfringens phage or phage composition as described above.

[0017] The present invention also provides a fungicide, which comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens phage or phage composition described above.

[0018] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0019] (1) The present invention provides a heat-resistant and acid-base resistant Clostridium perfringens phage XHX-CP-18P, which has good acid and alkali resistance under pH conditions of 3.0-13.0 and can withstand strong acids and alkalis, ensuring that the activity of the phage will not be lost. The phage can replace antibiotics, resist the corrosion of gastric acid, and effectively treat intestinal diseases of the host. The activity does not change significantly after treatment at a temperature of 30-60°C for 30 minutes, and the titer remains at 10 10 pfu / mL, ensuring that the phage activity is not lost under heat, and providing stability regulation for phage-made fungicides.

[0020] (2) The present invention provides a heat-resistant and acid-base resistant Clostridium perfringens phage XHX-CP-18P, which can lyse hosts with type A and type G serotypes. Type G Clostridium perfringens is the main pathogen of necrotic enteritis in chickens. Due to its lysis effect on type G Clostridium perfringens, phage XHX-CP-18P can be used as a therapeutic drug for necrotic enteritis in chickens. It is suitable for large-scale industrial production and provides a source of phage for industrial production and for the prevention and treatment of diseases caused by Clostridium perfringens.

[0021] (3) The present invention provides a heat-resistant and acid-alkali-resistant Clostridium perfringens phage XHX-CP-18P, which has a strong host infection ability and an optimal infection multiplicity of 0.000001. Only a very small amount is needed to achieve a good infection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the plaque of Clostridium perfringens phage XHX-CP-18P;

[0023] Figure 2 This is a schematic diagram showing the effect of temperature on the activity of Clostridium perfringens phage XHX-CP-18P;

[0024] Figure 3 This is a schematic diagram showing the effect of pH on the activity of Clostridium perfringens phage XHX-CP-18P;

[0025] Figure 4 This is the optimal infection multiplicity graph of Clostridium perfringens phage XHX-CP-18P;

[0026] Figure 5 This is a one-step growth curve of Clostridium perfringens phage XHX-CP-18P;

[0027] Figure 6 This is a survival curve of chicks treated with Clostridium perfringens phage XHX-CP-18P. DETAILED DESCRIPTION

[0028] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0029] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0030] LB liquid medium (1 L): 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, add ddH2O to 1 L, adjust the pH to 7.0, autoclave at 121°C for 15 min, and cool for later use.

[0031] 0.6% LB semi-solid medium (1 L): 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 6 g of agar powder, add ddH2O to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 15 min before use.

[0032] Tryptic-sulfite-cycloserine agar base (TSC): 15 g tryptic, 5 g soytone, 5 g yeast extract, 1 g sodium metabisulfite, 1 g ammonium ferric citrate, 20 g agar powder, add ddH2O to 1 L, adjust the pH to 7.6, and autoclave at 121°C for 15 min before use.

[0033] Cooked meat granule broth culture medium: 30g peptone, 3g beef extract powder, 5g yeast extract powder, 2g soluble starch, 3g glucose, 5g sodium dihydrogen phosphate, add ddH2O to 1L, adjust the pH to 7.0-7.4, add cooked meat granules and liquid paraffin, and sterilize at 121℃ for 15min before use.

[0034] 2×TSB liquid medium: 34 g tryptone, 6 g soy peptone, 10 g sodium chloride, 5 g dipotassium hydrogen phosphate, 5 g glucose, add ddH2O to 1 L, adjust the pH to 7.3, and sterilize by autoclaving at 121°C for 15 min before use.

[0035] TY solid medium: 15 g tryptone, 5 g soy peptone, 5 g yeast extract powder, 5 g sodium chloride, 12 g agar powder, add ddH2O to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 15 min before use.

[0036] SM solution (1 L): Weigh 6.055 g of Tris and dissolve it in 20 mL of distilled water. Adjust the pH to 7.5 with concentrated hydrochloric acid and make the volume up to 50 mL. Then, add 5.8 g of NaCl and 2 g of MgSO4, dissolve them, and make the volume up to 1 L. Sterilize by autoclaving at 121°C for 15 min.

[0037] Example 1

[0038] The isolation and identification of the host Clostridium perfringens XHX-CP-18 includes the following steps:

[0039] Samples were collected from the intestines of dead chickens at an affected farm in Shandong Province using aseptic techniques. The intestines were streaked onto tryptic-sulfite-cycloserine agar (TSC) based selective medium and incubated overnight at 37°C under anaerobic conditions. Representative colonies were selected and streaked and purified 3-5 times until the colonies were uniform in morphology. A single colony was then inoculated into 7 ml of cooked meat granule broth and incubated in an anaerobic incubator at 37°C for 5-6 hours until the logarithmic phase. This suspension was then used as the host bacterial suspension. The strain was identified as pathogenic Clostridium perfringens and designated XHX-CP18. The identified strain was streaked onto TSC plates and incubated for 12 hours. All colonies were scraped with an inoculating loop into 60% glycerol and stored at -80°C.

[0040] Example 2

[0041] The isolation and identification of bacteriophage XHX-CP-18P includes the following steps:

[0042] (1) Sample processing: The sewage samples of the present invention were collected from a chicken farm in Shandong Province. 5 g of feces were cultured in 2×TSB broth at 37°C and 200 rpm for 12 h, and then centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was filtered with 0.45 μm and 0.22 μm filter membranes respectively for later use.

[0043] (2) Preparation of phage enrichment solution: 0.2 ml of bacterial suspension and 1 ml of filtrate were added to 7 ml of cooked meat broth medium, cultured anaerobically at 37 °C overnight, then centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 µm filter. The filtrate was set aside.

[0044] (3) Phage isolation: Phage isolation was performed using the double-layer plate method. 0.1 mL of host bacterial suspension was mixed evenly with 5 mL of 0.6% LB semi-solid medium and then spread on a TY solid plate. After solidification, 20 µL of phage suspension was dripped onto the center of the plate. After drying naturally, the plate was inverted and incubated overnight at 37°C in an anaerobic incubator. The phage drop area was observed for plaque formation. The transparent plaque was placed in 0.5 mL of SM buffer and soaked overnight at 4°C. The phage was sterilized by filtration using a 0.22 µm filter. Take 0.1mL of phage and 0.1mL of bacterial suspension, add 5mL of 0.6% LB semi-solid medium and mix evenly, spread on TY solid plate, place in 37℃ anaerobic incubator for overnight culture, pick a single transparent spot with smooth edges and add it to 0.5mL SM buffer, store at 4℃ overnight, wait for the phage to be completely released, and filter sterilize with a 0.22µm filter. Repeat the double-layer plate experiment according to this step until the entire plate is covered with plaques of uniform size and smooth edges, and a single phage is obtained. The diameter of the plaques formed by XHX-CP-18P on the double plate is 3mm-5mm. Figure 1Storage method for bacteriophage XHX-CP-18P: Mix the phage proliferation solution with 60% glycerol at a volume ratio of 1:1 and store in a -80°C freezer or in liquid nitrogen.

[0045] Example 3

[0046] The temperature and acid-base tolerance experiment of bacteriophage XHX-CP-18P includes the following steps:

[0047] Take 1 mL of the purified phage obtained in Example 2 (2.5×10 10 PFU / mL) were placed in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 30 minutes, and the sample was cooled and then its titer was measured to analyze the temperature tolerance of the phage; LB broth was used as the medium, and the pH value was adjusted with NaOH solution and HCl (pH = 2-14). 900μL of LB broth with different pH values ​​was mixed with 100μL of phage, and the titer was measured in a water bath at 37℃ for 1 hour to analyze the pH tolerance of the phage. The temperature test results are as follows Figure 2 As shown in the figure, the activity of phage XHX-CP-18P did not change significantly after being exposed to 30-60℃ for 30 minutes; the activity decreased significantly at 70℃; and no phage survived after being exposed to 80℃ for 30 minutes. Figure 3 As shown, phage XHX-CP-18P maintained good activity and high titer in the pH range of 3-13. At pH 13, the phage titer decreased by only one order of magnitude; at pH 3, the titer decreased by four orders of magnitude, and no phage was detected at pH 2.0 and 14.0. The phage titer did not decrease significantly with decreasing or increasing pH, indicating that XHX-CP-18P can tolerate strong acids and bases.

[0048] Example 4

[0049] Determination of the optimal multiplicity of infection of bacteriophage XHX-CP-18P includes the following steps:

[0050] Take the bacterial solution (absorbance A600mm=0.50) and centrifuge it, then resuspend it to 10 8 CFU / mL. According to MOI-100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001, 0.0000001, 0.5 mL of phage with different dilutions was added and mixed with 0.5 mL of host bacterial solution. Then 4 mL of cooked meat broth was added. After anaerobically incubating at 37°C for 8 hours, the mixture was centrifuged at 8000 rpm for 5 minutes, filtered and sterilized with a 0.22 μm microporous filter, and then appropriately diluted. The phage titer was determined by the double-layer plate method. The results are shown in the figure. Figure 4The results showed that the optimal infection multiplicity of Clostridium perfringens phage XHX-CP-18P was 0.000001.

[0051] Example 5

[0052] The one-step growth curve determination of bacteriophage XHX-CP-18P includes the following steps:

[0053] After uniformly mixing the host bacterial culture prepared in Example 1 and the purified phage obtained in Example 2 at the optimal MOI ratio, the mixture was incubated at 37°C for 10 min, centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with 20 mL of cooked meat pellet liquid culture medium. The mixture was quickly placed in 37°C for anaerobic culture. Within the range of 0 to 150 min, 500 μL was taken at intervals of 10 min, and the titer of the phage at each time point was determined by the double-layer plate method. The titer was repeated 3 times at each time point to obtain the average value. The one-step growth curve of the phage was drawn with the phage titer as the vertical axis and the infection time as the horizontal axis to obtain the incubation period, outbreak period, and outbreak volume of the phage. The results of the one-step growth curve are shown in Figure 2. Figure 5 As shown, the incubation period of the infected host bacteria is about 20 minutes, the outbreak period is 30 minutes, and the outbreak volume is 2900.

[0054] Example 6

[0055] Host spectrum analysis of bacteriophage XHX-CP-18P includes the following steps:

[0056] The titer of phage XHX-CP-18P obtained in Example 2 was adjusted to 10 10 The PFU / mL is reserved. The phage host spectrum is detected using the dot plaque method. 28 strains of Clostridium perfringens with different serotypes were randomly selected from the bacterial library. 0.1 mL of the host Clostridium perfringens suspension was mixed evenly with 5 mL of 0.6% LB semi-solid medium. The mixture was then spread on a double-layer plate. After drying, 10 μL of the phage solution was dropped onto the culture medium. The culture was incubated anaerobically at 37°C for 12-16 hours. The appearance of plaques was observed. If so, the phage had a lytic effect on the strain. If plaques were produced, the result was marked as "+", otherwise, "-".

[0057] As shown in Table 1, bacteriophage XHX-CP-18P was able to lyse 57 of the 65 strains of Clostridium perfringens selected in the experiment. These strains were isolated in the applicant's laboratory and were all from chickens. Its lysis rate against both type A and type G C. perfringens strains reached 100%. This demonstrates that bacteriophage XHX-CP-18P of the present invention has excellent lysis performance against both type A and type G C. perfringens. The results are shown in Table 1.

[0058] Table 1 Host spectrum analysis of bacteriophage XHX-CP-18P

[0059]

[0060] Example 7

[0061] The chicken treatment trial of Clostridium perfringens phage XHX-CP-18P includes the following steps:

[0062] Ninety healthy chicks aged 7 days were randomly divided into three groups, with 30 chicks in each group. Group A was the blank control, group B was the challenge group, and group C was the treatment group. Groups B and C were orally administered with Clostridium perfringens XHX-CP-18 liquid (concentration of about 1×10 8 Groups A and B drank purified water normally, while group C drank water containing bacteriophage XHX-CP-18P (concentration of about 200 CFU / mL) for 1 hour before being replaced with purified water.

[0063] During the trial, the growth, morbidity, and mortality of the chickens were observed and recorded. Starting from the challenge, chick mortality was recorded daily for 7 consecutive days. On the 7th day after treatment, 10 chickens were randomly selected from each group for autopsy to observe intestinal lesions and calculate the lesion score. The scoring system ranged from 0 to 6, with 0 (no obvious damage), 1 (thinning or brittle intestinal wall), 2 (1 to 5 necrotic foci), 3 (6 to 15 necrotic foci), 4 (16 or more necrotic foci), 5 (2 to 3 cm long patchy necrosis), and 6 (large, diffuse necrosis).

[0064] like Figure 6 As shown in the results, after the experimental chicks were infected with the host, the challenge group experienced severe mortality. Within 7 days, 16 chicks died, with a mortality rate of 53.33%. In the control group, 3 chicks died within 7 days, with a mortality rate of 10%. In the treatment group, 4 chicks died within 7 days, with a mortality rate of 15.38%. This experiment demonstrated that bacteriophage XHX-CP-18P has a significant protective effect against broiler mortality caused by Clostridium perfringens. As shown in Table 2, the control group had the lowest lesion score. After challenge with C. perfringens, the lesion scores in both the challenge and treatment groups increased. After phage treatment, the lesion score in the treatment group was significantly lower than that in the challenge group (P < 0.05). These results indicate that phage has a significant therapeutic effect against necrotic enteritis caused by C. perfringens in broiler chickens.

[0065] Table 2 Lesion scoring table

[0066]

[0067] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0068] Example 8

[0069] The treatment of necrotic enteritis in yellow-feathered broiler chickens with Clostridium perfringens phage XHX-CP-18P comprises the following steps:

[0070] At a yellow-feathered broiler farm in Shandong province, a single shed housed 10,000 birds. Infected birds exhibited depression, ruffled feathers, loss of appetite, and even death, leading to persistent infection within the farm. Autopsies revealed the small intestine as the primary site of lesions, with hemorrhage, necrosis, or bloating. The small intestine became thicker, with internal inflation visible upon dissection, and blood clots were present within the intestinal contents. The intestinal wall became congested, with varying numbers of hemorrhages visible. The intestinal mucosa may thicken due to edema or thin due to intestinal epithelial erosion. Sometimes, a yellow-green pseudomembrane may be found covering the intestinal mucosa.

[0071] The phage XHX-CP-18P fermentation liquid (effective concentration 10 7 PFU / mL), freeze-dried into a freeze-dried powder, and fed to the chickens. Adding 200g / ton of feed, the phage significantly alleviated diarrhea 24 hours after application, and essentially disappeared 48 hours later. Feed intake increased, and the chickens' spirits improved. After 72 hours, the chickens recovered.

[0072] This experiment demonstrates that the phage of this invention can effectively treat Clostridium perfringens disease in practical applications, with significant efficacy, reducing antibiotic use and replacing antibiotics, and is green and residue-free. By adding the Clostridium perfringens phage XHX-CP-18P to feed, it can effectively treat necrotic enteritis in yellow-feathered broiler chickens and inhibit the colonization of pathogens in the intestine, providing an effective solution for antibiotic-free farming.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A heat-resistant and acid- and alkali-resistant Clostridium perfringens phage XHX-CP-18P, characterized in that: The Clostridium perfringens phage XHX-CP-18P was deposited in the China Center for Type Culture Collection on March 17, 2025, with a deposit number of CCTCC NO: M 2025492, and is classified as Clostridium perfringens bacteriophage.

2. A bacteriophage XHX-CP-18P composition, characterized in that: The invention comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens phage XHX-CP-18P as claimed in claim 1.

3. Use of the heat- and acid- and alkali-resistant Clostridium perfringens phage XHX-CP-18P according to claim 1 or the phage XHX-CP-18P composition according to claim 2 in the preparation of a product for preventing and / or treating inflammatory reactions caused by Clostridium perfringens.

4. A bacteriophage XHX-CP-18P pharmaceutical preparation, characterized in that: The active ingredient of the bacteriophage XHX-CP-18P pharmaceutical preparation includes the heat- and acid- and alkali-resistant Clostridium perfringens phage XHX-CP-18P according to claim 1 or the bacteriophage XHX-CP-18P composition according to claim 2.

5. The bacteriophage XHX-CP-18P pharmaceutical preparation according to claim 4, characterized in that The bacteriophage XHX-CP-18P pharmaceutical preparation further comprises a pharmaceutically acceptable carrier; the dosage form of the bacteriophage XHX-CP-18P pharmaceutical preparation includes one or more of a solution, a powder, a gel, a granule, an emulsion, a suspension and a lyophilized agent.

6. A feed additive, characterized in that The feed additive comprises the heat-resistant and acid- and alkali-resistant Clostridium perfringens bacteriophage XHX-CP-18P according to claim 1 or the bacteriophage XHX-CP-18P composition according to claim 2.

7. A fungicide, characterized in that: The bactericide comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage XHX-CP-18P according to claim 1 or the bacteriophage XHX-CP-18P composition according to claim 2.

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