A heat-resistant and acid-base gas-producing clostridium perfringens bacteriophage xhx-cp-18p and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a heat-resistant and acid-alkali-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 application also provides a bactericide product prepared from the bacteriophage. The bacteriophage has good acid-alkali resistance under the condition that pH is 3-13, and has heat resistance under the condition that temperature is 30-60 DEG C, so that the activity of the bacteriophage is ensured not to be lost. The bacteriophage has good lysis effect on Clostridium perfringens, has strong infection capacity, can infect more host cells with very small virus amount, and can effectively lyse A type and G type hosts, so that the bacteriophage can replace antibiotics, resist corrosion of gastric acid, and effectively treat host intestinal diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a heat-resistant and acid-alkali gas-producing Clostridium perfringens bacteriophage XHX-CP-18P and application thereof. BACKGROUND

[0002] Clostridium perfringens (Cp) is a gram-positive spore-forming bacillus that has long existed in nature. It is an anaerobic strain that appears as a thin straight rod under a microscope, with both ends being blunt and round. It is a serious conditional pathogenic bacterium. When the living environment of humans and animals is disturbed by external factors or the climate is variable, high-nutrient diet and antibiotics are used, Clostridium perfringens can produce more than 20 kinds of protein toxins or tissue-destroying enzymes, leading to diseases in the human or animal body. The pathogenic effect of Cp is mainly to produce various exotoxins and tissue-destroying enzymes. The currently discovered exotoxins are mainly alpha, beta, epsilon, iota, CPE and NetB. According to the carrying condition of the six toxins, Clostridium perfringens is divided into seven pathogenic types A-G, and all seven toxin types (A to G) of Clostridium perfringens carry and express alpha toxin. Type A bacteria are more serious toxin type bacteria, which can cause gas gangrene in the human or animal body, and can also cause enterotoxemia and gastrointestinal infectious diseases. The toxins in the gastrointestinal tract are absorbed into the body and circulate, thereby damaging other internal organs, seriously endangering human and animal public health safety and the development of modern poultry and livestock industry. G-type Clostridium perfringens is the main pathogenic bacterium of chicken necrotic enteritis, which is a new type of toxin strain discovered in recent years, and is prevalent abroad, but there are few reports on the prevalence of G-type Clostridium perfringens and whether it can cause chicken necrotic enteritis in China.

[0003] In recent years, the breeding strategy of "prevention first and treatment second" has been widely promoted. In order to promote animal growth and disease prevention, many breeding enterprises currently generally add antibiotics to feed to improve breeding efficiency. Although this method improves animal health to some extent, with the passage of time, the problem of antibiotic resistance is becoming more and more serious, and the emergence of drug-resistant bacteria seriously affects global public health.

[0004] Bacteriophages are viruses that can kill bacteria. Bacteriophages are the largest group of viruses known, with a number of about ten times that of bacteria. It is estimated that there are about 10 31 billion bacteriophages on earth. Compared with the broad-spectrum bacteriostasis of traditional antibiotics, bacteriophages have high specificity and only act on the corresponding host bacteria without affecting other intestinal flora. In addition, bacteriophages can co-evolve with host bacteria. As long as the host bacteria exist, bacteriophages can rely on the host bacteria for proliferation, realize automatic drug delivery, and automatically remove when the bacteria are lysed, without residual in the body.

[0005] At present, there are few products that can prevent and treat Clostridium perfringens resistant to heat and acid-base, so it is urgent to develop a bacteriophage product that can resist heat and strong acid and alkali, has a wide lysis spectrum, can prevent and treat, and has significant and stable genetic effects for treating Clostridium perfringens disease. SUMMARY

[0006] The present application aims at the deficiencies of the prior art, and provides a heat-resistant and acid-base-resistant Clostridium perfringens bacteriophage XHX-CP-18P and its application. The heat-resistant and acid-base-resistant Clostridium perfringens bacteriophage XHX-CP-18P provided by the present application can resist heat and strong acid and alkali, ensures that the activity of the bacteriophage will not be lost, can replace antibiotics, and can resist corrosion of gastric acid, so that the host intestinal diseases can be effectively treated.

[0007] The above-mentioned object of the present application is achieved by the following technical solutions: In a first aspect, the present application provides a heat-resistant and acid-base-resistant Clostridium perfringens bacteriophage XHX-CP-18P, which is isolated from Weifang, Shandong, China on June 6, 2024, and preserved in the China Center for Type Culture Collection (CCTCC) on March 17, 2025, with the address being Laojia Mountain, Baoyi Road, Wuchang District, Wuhan, Hubei Province, and the preservation number being CCTCC NO: M 2025492.

[0008] The titer of the bacteriophage XHX-CP-18P is as high as 6.0x10 10 PFU / mL after being cultured for 8h under the condition of M0I=0.000001. After being treated at 30-60℃ for 30min, the activity of the bacteriophage does not change significantly; the bacteriophage can still maintain its stability after being treated for 30min under the condition of pH being 3-13, and still has high activity in a strong acid and alkali environment, and still has high lysis effect on host bacteria. Compared with other existing bacteriophages, the bacteriophage has strong heat and acid-base resistance. Based on the above-mentioned excellent biological characteristics, the bacteriophage can adapt to harsh environments with high temperature, strong acid and strong alkali.

[0009] The bacteriophage has good lysis effect on Clostridium perfringens. Experiments prove that the bacteriophage can lyse host bacteria of A type, C type, D type, E type and G type. In the examples of the present application, the lysis rate of the bacteriophage on selected A type and G type Clostridium perfringens reaches 100%. This shows that the bacteriophage XHX-CP-18P of the present application has excellent lysis performance on A type and G type Clostridium perfringens, and has good application prospect in killing Clostridium perfringens in the environment and preventing and treating diseases caused by Clostridium perfringens.

[0010] The present application provides a bacteriophage composition comprising the heat-resistant and acid-base-resistant Clostridium perfringens bacteriophage as described above.

[0011] The application also provides the use of the heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage XHX-CP-18P or the bacteriophage composition in the preparation of a product for preventing or treating inflammation caused by Clostridium perfringens.

[0012] The term "prevention" herein refers to all behaviors of inhibiting or delaying the disease by administering the bacteriophage; and the term "treatment" herein refers to all behaviors of improving or ameliorating the disease by administering the bacteriophage.

[0013] The above-mentioned medicament can be used for preventing or treating Clostridium perfringens infection in humans and animals (chickens, pigs, cows, geese, ducks, turkeys, etc.).

[0014] The application provides a bacteriophage pharmaceutical preparation, wherein the effective component of the bacteriophage pharmaceutical preparation comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage or the bacteriophage composition.

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

[0016] The application provides a feed additive, wherein the feed additive comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage or the bacteriophage composition.

[0017] The application also provides a bactericide, wherein the bactericide comprises the heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage or the bacteriophage composition.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] (1) The heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage XHX-CP-18P provided by the application has good acid-alkali resistance under the condition of pH 3.0-13.0, can resist strong acid and strong alkali, and ensures that the activity of the bacteriophage will not be lost. The bacteriophage can replace antibiotics, resist corrosion of gastric acid, and effectively treat diseases of the host intestinal tract. The activity does not change significantly under the condition of 30-60℃ for 30 min, and the titer is kept at 10 10 pfu / mL, which ensures that the activity of the bacteriophage will not be lost under heat. The stability adjustment is provided for the bacteriophage to be made into a bactericide.

[0020] (2) The heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage XHX-CP-18P provided by the application can lyse hosts of A type and G type. G type Clostridium perfringens is the main pathogenic bacterium of chicken necrotic enteritis. The bacteriophage XHX-CP-18P can be used as a therapeutic drug for chicken necrotic enteritis due to the lytic effect on G type Clostridium perfringens, and is suitable for large-scale industrial production, thereby providing a bacteriophage source for industrial production of bacteriophages and prevention and treatment of diseases caused by Clostridium perfringens.

[0021] (3) The heat-resistant and acid-alkali-resistant Clostridium perfringens bacteriophage XHX-CP-18P provided by the application has a strong host infection ability, and the optimal infection multiple is 0.000001. A small amount of the bacteriophage can achieve a good infection effect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a Clostridium perfringens bacteriophage XHX-CP-18P plaque photograph;

[0023] Figure 2 is a temperature effect on the activity of the Clostridium perfringens bacteriophage XHX-CP-18P;

[0024] Figure 3 is a pH value effect on the activity of the Clostridium perfringens bacteriophage XHX-CP-18P;

[0025] Figure 4 is a Clostridium perfringens bacteriophage XHX-CP-18P optimal infection multiple diagram;

[0026] Figure 5 is a Clostridium perfringens bacteriophage XHX-CP-18P one-step growth curve diagram;

[0027] Figure 6 is a Clostridium perfringens bacteriophage XHX-CP-18P chick treatment survival curve diagram. DETAILED DESCRIPTION

[0028] The following examples are only used to more clearly illustrate the technical solutions of the application, and cannot be used to limit the protection scope of the application.

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

[0030] LB liquid medium (1L): 10g of proteose peptone, 5g of yeast powder, 10g of sodium chloride, add ddH2O to 1L, adjust the pH to 7.0, and then sterilize at 121℃ for 15min, and cool for standby.

[0031] 0.6% LB semi-solid medium (1 L): Tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar powder 6 g, ddH2O to 1 L, adjust pH to 7.0, sterilize at 121 °C for 15 min.

[0032] Trypticase-sulfite-cysteine agar base (TSC): Trypticase 15 g, soy peptone 5 g, yeast extract 5 g, sodium metabisulfite 1 g, ferric ammonium citrate 1 g, agar powder 20 g, ddH2O to 1 L, adjust pH to 7.6, sterilize at 121 °C for 15 min.

[0033] Meat broth medium: Tryptone 30 g, beef extract 3 g, yeast extract 5 g, soluble starch 2 g, glucose 3 g, NaH2PO4 5 g, ddH2O to 1 L, adjust pH to 7.0-7.4, add meat particles and liquid paraffin, sterilize at 121 °C for 15 min.

[0034] 2x TSB liquid medium: Tryptone 34 g, soy peptone 6 g, NaCl 10 g, K2HPO4 5 g, glucose 5 g, ddH2O to 1 L, adjust pH to 7.3, sterilize at 121 °C for 15 min.

[0035] TY solid medium: Tryptone 15 g, soy peptone 5 g, yeast extract 5 g, NaCl 5 g, agar powder 12 g, ddH2O to 1 L, adjust pH to 7.0, sterilize at 121 °C for 15 min.

[0036] SM solution (1 L): Weigh 6.055 g of Tris and dissolve in 20 mL of distilled water, adjust pH to 7.5 with concentrated HCl, dilute to 50 mL, then add 5.8 g of NaCl and 2 g of MgSO4, dissolve and dilute to 1 L, sterilize at 121 °C for 15 min.

[0037] Example 1

[0038] Isolation and identification of host Clostridium perfringens XHX-CP-18, including the following steps:

[0039] The intestinal tract of a dead chicken was sampled from a certain breeding farm in Shandong, and was streaked on a selective culture medium, trypticase-sulfite-cysteine agar (TSC), and was cultured overnight at 37°C in an anaerobic environment. Typical colonies were picked and streaked for 3-5 times until the colony morphology was consistent. Then, a single colony was picked and inoculated into 7 ml of bouillon medium, and was cultured at 37°C in an anaerobic incubator for 5-6 hours to the logarithmic phase, serving as a host bacteria suspension. After identification, it was determined to be pathogenic Clostridium perfringens, which was named XHX-CP18. The identified strain was streaked on a TSC plate culture medium plate and was cultured for 12 hours. All colonies were scraped with an inoculation ring into 60% glycerol liquid and was stored in a -80°C refrigerator.

[0040] Example 2

[0041] Isolation and identification of bacteriophage XHX-CP-18P, including the following steps:

[0042] (1) Sample treatment: The sewage sample of the present application was collected from the feces of a certain chicken farm in Shandong. 5g of feces was cultured in 2xTSB broth at 37°C, 200rpm for 12h, and then centrifuged at 4°C, 10000rpm for 10min. The supernatant was filtered with 0.45µm and 0.22µm filters, respectively, for standby use.

[0043] (2) Preparation of bacteriophage enrichment liquid: 0.2ml of bacterial suspension and 1ml of filtrate were added to 7mL bouillon medium, and were cultured overnight at 37°C in an anaerobic environment. Then, they were centrifuged at 10000rpm for 10min, and the supernatant was filtered through a 0.22µm filter. The filtrate was prepared for standby use.

[0044] (3) Bacteriophage isolation: Double-layer plate method was used to isolate bacteriophage. 0.1mL of host bacteria suspension was mixed evenly with 5mL of 0.6% LB semi-solid medium, and was plated on a TY solid plate. After solidification, 20µL of bacteriophage suspension was dropped on the central part of the plate. After natural air-drying, the plate was inverted and cultured overnight at 37°C in an anaerobic incubator. Then, whether there were plaques formed in the area where bacteriophage was added was observed. The transparent plaque was soaked in 0.5ml SM buffer at 4°C overnight, and was filtered through a 0.22µm filter to remove bacteria, thereby obtaining the bacteriophage. 0.1mL of bacteriophage and 0.1mL of bacterial suspension were added to 5mL of 0.6% LB semi-solid medium, and were mixed evenly. Then, they were plated on a TY solid plate, and were cultured overnight at 37°C in an anaerobic incubator. Then, a single transparent plaque with smooth edges was picked up in 0.5mL of SM buffer at 4°C overnight. After the bacteriophage was completely released, it was filtered through a 0.22µm filter to remove bacteria. The double-layer plate experiment was repeated according to the above steps until the entire plate was covered with bacteriophage plaques with uniform size and smooth edges, thereby obtaining a single bacteriophage. The diameter of the plaque formed by XHX-CP-18P in the double plate was 3mm-5mm, as shown in Figure 1. Figure 1The phage XHX-CP-18P preservation method: the phage proliferation liquid is mixed with 60% glycerol in a volume ratio of 1:1, and can be stored in a -80°C refrigerator or liquid nitrogen.

[0045] Example 3

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

[0047] Take 1 mL of the purified phage obtained in Example 2 (2.5 x 10 10 PFU / mL) in a 30°C, 40°C, 50°C, 60°C, 70°C, 80°C water bath for 30 min, and then measure the titer after cooling to analyze the temperature tolerance of the phage; use LB broth as the medium, and adjust the pH value (pH=2-14) with NaOH solution and HC1. Take 900 μL of LB broth with different pH values and mix with 100 μL of the phage, and then measure the titer in a 37°C water bath for 1 h to analyze the pH tolerance of the phage. The temperature detection results are shown in Figure 2 The phage XHX-CP-18P has no significant change in activity after being acted on at 30-60°C for 30 min, has a significant decrease in activity at 70°C, and has no phage survival after being acted on at 80°C for 30 min. The pH detection results are shown in Figure 3 The phage XHX-CP-18P can maintain good activity in the pH range of 3-13, can maintain a high titer, has a decrease in titer by only one order of magnitude at pH 13, has a decrease in titer by four orders of magnitude at pH 3, and has no detectable phage at pH 2.0 and 14.0. The decrease in titer of the phage is not obvious with the decrease or increase in pH value, indicating that the phage XHX-CP-18P can tolerate strong acid and strong base.

[0048] Example 4

[0049] The determination of the optimal multiplicity of infection of the phage XHX-CP-18P includes the following steps:

[0050] Centrifuge the bacterial liquid (absorbance A600mm=0.50) and resuspend 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, add 0.5 mL of the phage with different dilutions and 0.5 mL of the host bacterial liquid, add 4 mL of bouillon medium, and then culture anaerobically at 37°C for 8 h. Centrifuge at 8000 rpm for 5 min, filter bacteria with a 0.22 μm microporous filter, dilute appropriately, and then measure the phage titer by the double-layer plate method. The results are shown in Figure 4The optimal multiplicity of infection of the Clostridium perfringens bacteriophage XHX-CP-18P is 0.000001.

[0051] Example 5

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

[0053] After uniformly mixing the host bacteria culture prepared in Example 1 with the purified bacteriophage obtained in Example 2 at the optimal MOI ratio, incubating at 37°C for 10 min, centrifuging at 8000 rpm for 5 min, discarding the supernatant, resuspending the precipitate with 20 mL of the ground meat liquid medium, and quickly placing it in an anaerobic culture at 37°C, 500 μL is taken at intervals of 10 min within the range of 0-150 min, and the titer of the bacteriophage at each time point is determined by the double-layer plate method, and the determination is repeated three times at each time point to obtain the average value. The one-step growth curve of the bacteriophage is plotted with the titer of the bacteriophage as the vertical coordinate and the infection time as the horizontal coordinate, and the latent period, the burst period and the burst size of the bacteriophage are obtained. The one-step growth curve is shown in Figure 5 The latent period of the bacteriophage is about 20 min, the burst period is 30 min, and the burst size is 2900.

[0054] Example 6

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

[0056] The titer of the bacteriophage XHX-CP-18P obtained in Example 2 is adjusted to 10 10 PFU / mL for standby. The bacteriophage host spectrum is detected by the spot plaque method. 28 strains of hosts of different serotypes of Clostridium perfringens are randomly selected from the bacterial library, 0.1 mL of the Clostridium perfringens bacterial suspension is uniformly mixed with 5 mL of 0.6% LB semi-solid, and then double-layer plates are prepared, after air-drying, 10 μL of the bacteriophage droplet is taken and placed on the culture medium, and the plates are incubated at 37°C anaerobically for 12-16 h, and whether the plaques appear is observed. If the plaques appear, it indicates that the bacteriophage has a lytic effect on the strain, and if the plaques do not appear, it is recorded as “-”.

[0057] As shown in Table 1, the bacteriophage XHX-CP-18P has a lytic effect on 57 strains of 65 strains of Clostridium perfringens selected in the experiment, and the Clostridium perfringens is isolated from the laboratory of the applicant and is chicken-derived. The lytic rate of the bacteriophage on the selected A-type and G-type Clostridium perfringens reaches 100%. This indicates that the bacteriophage XHX-CP-18P of the present application has excellent lytic performance on A-type and G-type Clostridium perfringens, and the results are shown in Table 1.

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

[0059]

[0060] Example 7

[0061] The chick treatment test of Clostridium perfringens bacteriophage XHX-CP-18P includes the following steps:

[0062] 90 healthy 7-day-old chicks were randomly divided into 3 groups, 30 in each group, group A was blank control, group B was challenge group, and group C was treatment group. Group B and group C were orally administered Clostridium perfringens XHX-CP-18 bacteriophage liquid (concentration about 1 x 10 8 CFU / mL, 2 mL per chick). Group A and group B normally drank pure water, and group C drank water containing bacteriophage XHX-CP-18P (concentration about 200 CFU / mL) for treatment for 1 h, and then changed to pure water.

[0063] During the test period, the growth and death of the chickens were observed and recorded. From the beginning of the challenge, the death of the chicks was observed and counted every day for 7 days. On the 7th day after treatment, 10 chicks were randomly selected from each group for autopsy to observe intestinal lesions and count the lesion scores. The scoring system was based on a standard of 0-6 points, 0 points (no obvious damage), 1 point (thinning or fragility of intestinal wall), 2 points (1-5 necrotic foci), 3 points (6-15 necrotic foci), 4 points (16 or more than 16 necrotic foci), 5 points (2-3 cm long patchy necrosis), and 6 points (large diffuse necrosis).

[0064] As shown in Table 2, the control group had the lowest lesion score. After Clostridium perfringens challenge, the lesion scores of the challenge group and the treatment group increased. After bacteriophage treatment, the lesion score of the treatment group was significantly lower than that of the challenge group (P < 0.05), indicating that the bacteriophage had good therapeutic effect on necrotic enteritis caused by Clostridium perfringens in broilers. Figure 6 Table 2 Lesion score table

[0065]

[0066] Note: The same row with different lowercase letters indicates a significant difference (P < 0.05).

[0067]

[0068] ​Example 8

[0069] The treatment of Clostridium perfringens bacteriophage XHX-CP-18P on necrotic enteritis of yellow-feathered broilers includes the following steps:

[0070] In a certain yellow-feathered broiler farm in Shandong, there is a chicken house with 10,000 chickens. The infected chickens show depression, disheveled feathers, loss of appetite, and even death, which also causes persistent infection in the farm. Autopsy shows that the small intestine is the main lesion site, with small intestine hemorrhagic necrosis or pneumatosis; the small intestine is thickened, and the internal inflation phenomenon can be observed, and there are blood clots in the intestinal contents; the intestinal wall is congested, with varying numbers of bleeding points; the intestinal mucosa can be thickened due to edema; or thinned due to intestinal epithelial erosion; sometimes the intestinal mucosa is covered with a yellow-green pseudomembrane.

[0071] The bacteriophage XHX-CP-18P fermentation liquor (effective concentration 10 7 PFU / mL) is freeze-dried to form a freeze-dried powder, which is added to the feed to feed the chicken group. According to 200g / ton of feed, the bacteriophage is used 24h later, and the diarrhea symptoms are significantly reduced. After 48h, the diarrhea phenomenon basically disappears, the feed intake increases, the spirit becomes good, and the chicken group is cured after 72h.

[0072] The experiment proves that the bacteriophage of the present application can treat Clostridium perfringens disease in practical application, and the effect is obvious, and the resistance is replaced by resistance, green and no residue. By adding Clostridium perfringens bacteriophage XHX-CP-18P in the feed, yellow-feathered broiler necrotic enteritis can be effectively treated, and intestinal pathogenic bacteria colonization can be inhibited, which provides an effective solution for antibiotic-free breeding.

[0073] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should be considered as the protection scope of the present application.

Claims

1. A heat and acid-base resistant Clostridium perfringens bacteriophage XHX-CP-18P, characterized by, 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 was classified as Clostridium perfringens phage ( Clostridium perfringens bacteriophage The Clostridium perfringens phage XHX-CP-18P was treated for 30 min at a pH of 3.0-13.0, and the titer was maintained at 10 10 pfu / mL; and the lysis rate of the Clostridium perfringens phage XHX-CP-18P on type A and type G Clostridium perfringens reached 100%.

2. A bacteriophage XHX-CP-18P composition, characterized in that, The heat-resistant and acid-base resistant Clostridium perfringens bacteriophage XHX-CP-18P as claimed in claim 1.

3. Use of the heat-resistant and acid-base resistant Clostridium perfringens bacteriophage XHX-CP-18P as claimed in claim 1 or the bacteriophage XHX-CP-18P composition as claimed in claim 2 in the preparation of a product for preventing and / or treating inflammation caused by Clostridium perfringens.

4. A pharmaceutical preparation of the bacteriophage XHX-CP-18P, characterized in that, The active ingredient of the bacteriophage XHX-CP-18P pharmaceutical preparation comprises the heat-resistant and acid-base resistant Clostridium perfringens bacteriophage XHX-CP-18P as claimed in claim 1 or the bacteriophage XHX-CP-18P composition as claimed in claim 2.

5. The bacteriophage XHX-CP-18P pharmaceutical preparation of claim 4, wherein, The bacteriophage XHX-CP-18P pharmaceutical preparation further comprises a pharmaceutically acceptable carrier; the dosage form of the bacteriophage XHX-CP-18P pharmaceutical preparation comprises 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-base resistant Clostridium perfringens bacteriophage XHX-CP-18P as claimed in claim 1 or the bacteriophage XHX-CP-18P composition as claimed in claim 2.

7. A bactericide characterized by comprising: The bactericide comprises the heat-resistant and acid-base resistant Clostridium perfringens bacteriophage XHX-CP-18P as claimed in claim 1 or the bacteriophage XHX-CP-18P composition as claimed in claim 2.

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