Swine-derived A-type clostridium perfringens and application thereof in vaccine preparation

Abstract

The invention belongs to the field of biology, and discloses swine A type clostridium perfringens SP20 capable of secreting and expressing high-level alpha toxin, which is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M 2025386. The strain has strong pathogenicity to medium and large pigs, and can be used for establishment of a pig challenge model and efficacy evaluation of a pig A-type clostridium perfringens vaccine. The invention further discloses a vaccine for preventing intestinal flatulence and sudden death of medium and large pigs caused by the A-type clostridium perfringens. The vaccine comprises the alpha toxin generated by the SP20 strain. The invention fills the blank of the disease model of intestinal flatulence and sudden death of middle and large pigs, and is beneficial to control of disease infection and reduction of economic loss of breeding industry.

Description

Technical Field

[0001] The present invention relates to a highly virulent strain of Clostridium perfringens type A from pigs and its application in the preparation of an alpha-toxin vaccine, belonging to the biological field. Background Art

[0002] Clostridium perfringens ( Clostridium perfringensClostridium perfringens is a type of Gram-positive bacillus. According to the types and combinations of α, β, ι, and ε toxins it produces, it can be divided into types A, B, C, D, E, F, G, H, etc. Among them, Clostridium perfringens type A only produces α toxin among α, β, ι, and ε toxins. Clostridium perfringens type A is widely distributed in natural environments such as soil and water and can be detected in the intestines of various animals, including common livestock such as pigs, horses, sheep, rabbits, etc. (Camargo A et.al,. Intra-species diversity of Clostridium perfringens: A diverse genetic repertoire reveals its pathogenic potential. Front Microbiol. 2022 Jul 22;13:952081.). Although Clostridium perfringens type A has a wide range of sources, its typing is only based on the fact that its genome contains the α toxin-encoding gene but does not contain the β, ι, and ε toxin-encoding genes. The genomes of different Clostridium perfringens type A strains vary greatly. Among the 8 Clostridium perfringens type A strains with publicly available whole-genome sequences from different sources in GenBank, the core genes are 1,807, which is only 61.2% of the average gene number of 2,951 per strain (Hassan K A et al., Genomic analyses of Clostridium perfringens isolates from five toxinotypes[J].Research in Microbiology, 2015.DOI:10.1016 / j.resmic.2014.10.003.). Moreover, in addition to secreting α toxin (the basis for toxin typing), Clostridium perfringens type A may also secrete toxins such as CPB2, PFO, and δ toxin (Svobodová et al., Nontyping virulence factors of Clostridium perfringens[J].Acta Veterinaria Brno, 2024, 93(1); Navarro M A et al., The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model[J].mSphere, 2020, 5(3).). In addition to the known toxins, Clostridium perfringens type A also has other virulence factors, such as nanJ and nagJ.

[0003] From the perspective of pathogenicity, Clostridium perfringens type A can infect hosts through mechanical trauma or the fecal-oral route, causing gas gangrene in humans, yellow lamb disease in sheep, diarrhea and necrotic enteritis in neonatal piglets, necrotic enteritis in chickens, and Clostridium welchii disease in rabbits, etc. Whole-genome nucleotide polymorphisms further reveal that strains from different sources and pathogenic types are on different evolutionary branches (Geier R R et. al, Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors[J]. Frontiers in Microbiology, 2021, 12.). In short, except for only secreting the alpha toxin among the alpha, beta, iota, and epsilon toxins, Clostridium perfringens type A from different species and sources vary greatly in terms of genomic composition, virulence factors, secreted toxin types, and pathogenicity. Generally, Clostridium perfringens type A from a specific source is pathogenic to a specific species.

[0004] In veterinary clinical practice, sudden death of medium and large pigs is often observed, accompanied by intestinal flatulence. Bacteriological detection suspects that Clostridium perfringens type A is one of the pathogenic bacteria, but no typical cases, that is, intestinal flatulence and sudden death of medium and large pigs, have been reported to be replicated in the laboratory. Due to the rapid course and acute onset of flatulence and sudden death in medium and large pigs clinically, the therapeutic effect of antibiotics is often very poor. Immunoprevention is one of the most important means to control infectious diseases. Currently, only the inactivated vaccine of Clostridium perfringens type A is used to prevent diarrhea in piglets. The whole-bacterium inactivated vaccine has a high antigen protein content and large side effects, and the genomic differences among different Clostridium perfringens type A are very large, and the cross-protection effect is not ideal. Therefore, there is an urgent need to develop a vaccine specifically targeting sudden death and intestinal flatulence in medium and large pigs to reduce the economic losses caused by sudden death of pigs.

[0005] Alpha toxin is one of the most important virulence factors of Clostridium perfringens type A, which can cause host hemolysis, platelet aggregation, vasoconstriction, and intestinal damage. The toxoid formed after detoxifying alpha toxin can be used as a vaccine antigen. However, the yield of alpha toxin in Clostridium perfringens type A is relatively low, generally 10 - 100 MLD / mL. The carboxyl-terminal domain of the alpha toxin expressed by genetic engineering (such as amino acids 251 - 379) has shown good immune evaluation effects in mice, but its immunogenicity has not been evaluated in pigs (Ferreira M R et al, Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines. Toxins (Basel). 2016 Nov 21;8(11):340.). More importantly, in veterinary clinical practice, sudden deaths of medium and large pigs caused by Clostridium perfringens type A result in significant economic losses. However, classic cases have not been replicated in the laboratory yet, and it is impossible to directly evaluate the effects of various types of Clostridium perfringens type A vaccines in pigs.

[0006] Therefore, isolating Clostridium perfringens type A from diseased pig materials, replicating typical clinical symptoms including intestinal flatulence and sudden death, establishing a model, and obtaining a pig-derived Clostridium perfringens type A strain that highly secretes alpha toxin are the keys to developing vaccines to prevent intestinal flatulence and sudden death in medium and large pigs. Summary of the Invention

[0007] An object of the present invention is to provide a pig-derived Clostridium perfringens type A strain that can secrete and express high levels of alpha toxin and has strong pathogenicity to medium and large pigs. This strain can be used to establish a challenge model for sudden death and intestinal flatulence in pigs and to prepare an alpha toxin vaccine.

[0008] To achieve the above object, the applicant isolated a strain of Clostridium perfringens from the intestine of a fattening pig that had sudden death due to flatulence in a pig farm in Enshi, Hubei. After identification by PCR and Western Blot, this strain was identified as Clostridium perfringens type A and named strain SP20. Further biological activity assays showed that strain SP20 highly expressed alpha toxin, the content of alpha toxin in its culture supernatant was not less than 120 MLD / mL, this strain could maintain a long time in the growth plateau phase, and obvious intestinal flatulence and sudden death occurred after oral challenge of medium and large pigs weighing 30 - 90 kg, indicating strong pathogenicity.

[0009] The strain is classified and named as Clostridium perfringens type A SP20, which is deposited in the China Center for Type Culture Collection (CCTCC) in Wuhan, Hubei Province, China. The deposit date is March 6, 2025, and the deposit number is CCTCC NO: M 2025386.

[0010] The second object of the present invention is to provide a vaccine for preventing porcine intestinal flatulence and sudden death caused by Clostridium perfringens type A and a preparation method thereof.

[0011] The vaccine provided by the present invention comprises the following components: (a) α-toxin produced by the SP20 strain; (b) Adjuvant; (c) Immune enhancer.

[0012] Among them, the adjuvant is selected from one or more of aluminum hydroxide adjuvant, aqueous adjuvant or oil-in-water adjuvant, and preferably aluminum hydroxide adjuvant at 2 - 4 mg / dose.

[0013] Among them, the immune enhancer is selected from one or more of CpG oligonucleotides, Poly(I:C) or monophosphatide A, and preferably CpG at 10 - 30 μg / dose.

[0014] Among them, the dose of the α-toxin is 30 - 400 MLD / dose, and preferably 100 - 400 MLD / dose.

[0015] The preparation method provided by the present invention comprises the following steps: (a) Culturing the SP20 bacteria in a fermenter, controlling the stirring speed at 20 - 100 rpm, the temperature at 34 - 37°C, and the pH at 6.0 - 7.0; (b) Collecting the culture supernatant, ultrafiltrating and washing and filtering it through a 5 - 100 kD membrane package; (c) Detoxifying the concentrated α-toxin; (d) Mixing the detoxified α-toxin with the adjuvant and the immune enhancer to prepare a vaccine preparation.

[0016] Among them, the fermentation medium comprises 10 g / L of tryptone, 10 g / L of beef extract powder, 5 g / L of beef liver extract powder, 5 g / L of yeast extract powder, 5 g / L of glucose and 5 g / L of sodium chloride.

[0017] Among them, the cut-off molecular weight of the membrane package is 30 kD.

[0018] The above-mentioned strain was cultured until the late logarithmic growth phase. The content of alpha toxin in the culture supernatant was determined by the method specified in the Chinese Veterinary Pharmacopoeia, reaching 120 MLD / mL. After concentration by 10 times, the content was 1030 MLD / mL, and the recovery rate was approximately 85.7%.

[0019] The immune effect of the vaccine was evaluated in pigs. The results showed that the Clostridium perfringens type A toxoid vaccine provided by the present invention could induce complete immune protection in pigs and resist the lethal attack of Clostridium perfringens type A on pigs.

[0020] The beneficial effects of the present invention include: 1. The strain provided by the present invention fills the gap in the morbidity models of intestinal flatulence and sudden death in medium and large pigs caused by Clostridium perfringens type A challenge globally, laying a foundation for the development of related drugs; 2. The toxoid vaccine provided by the present invention can prevent intestinal flatulence and sudden death in pigs caused by Clostridium perfringens type A, which is beneficial to controlling disease transmission and reducing economic losses in the aquaculture industry; 3. The toxoid vaccine provided by the present invention has controllable material sources and low preparation costs, and is suitable for large-scale industrial production. Description of the Drawings

[0021] Figure 1 . PCR identification of suspected Clostridium perfringens type A. Lanes 1-1 to 1-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP01; lanes 2-1 to 2-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP02; lanes 3-1 to 3-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP03; lanes 4-1 to 4-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP14; lanes 5-1 to 5-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP15; lanes 6-1 to 6-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP17; lanes 7-1 to 7-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP18; lanes 8-1 to 8-4 are the electrophoresis of the amplification products of the alpha, beta, epsilon, and iota toxin-encoding genes of strain SP20.

[0022] Figure 2 . Immunoblotting was used to detect the expression of alpha toxin in the supernatants of different suspected Clostridium perfringens type A strains.

[0023] Figure 3 . Growth and toxin secretion curves of Clostridium perfringens type A. A, strain SP20; B, strain C57-1. The square markers on the curve represent log (CFU / ml), and the circular markers represent MLD / mL.

[0024] Figure 4 . Type A Clostridium perfringens challenge of 30 - 40 kg pigs and gross pathological lesions during necropsy. A, Appearance of blank control pigs; B, Necropsy of blank control group; C, Appearance of pigs after challenge with C57 - 1 strain, without obvious abnormalities; D, Necropsy of pigs after challenge with C57 - 1 strain, without obvious lesions; E, Appearance of suddenly dead pigs after challenge with SP20 strain, showing abdominal distension and purple skin in the part close to the ground; F, Necropsy of suddenly dead pigs after challenge with SP20 strain, showing intestinal distension and organ corruption.

[0025] Figure 5 . Type A Clostridium perfringens challenge of 70 - 90 kg pigs and gross pathological lesions during necropsy. A, Appearance of blank control pigs; B, Necropsy of blank control group; C, Appearance of pigs after challenge with C57 - 1 strain, without obvious abnormalities; D, Necropsy of pigs after challenge with C57 - 1 strain, without obvious lesions; E, Appearance of suddenly dead pigs after challenge with SP20 strain, showing abdominal distension; F, Necropsy of suddenly dead pigs after challenge with SP20 strain, showing intestinal distension and organ corruption.

[0026] Figure 6 . Comparison of the recovery rates of α - toxin by ultrafiltration concentration with different membrane pore sizes. The left figure shows the SDS - PAGE electrophoresis analysis of the protein band distribution after ultrafiltration concentration with different membrane pore sizes. M, Protein Marker; Lane 1, Supernatant original sample; Lane 2, Concentrated 10 - fold by 100 kD membrane package; Lane 3, Concentrated 10 - fold by 50 kD membrane package; Lane 4, Concentrated 10 - fold by 30 kD membrane package; Lane 5, Concentrated 10 - fold by 10 kD membrane package; Lane 6, Concentrated 10 - fold by 5 kD membrane package. The right figure shows the immunoblot detection of the α - toxin content after ultrafiltration concentration with different membrane pore sizes. Lane 1, Supernatant original sample; Lane 2, Concentrated 10 - fold by 100 kD membrane package; Lane 3, Concentrated 10 - fold by 50 kD membrane package; Lane 4, Concentrated 10 - fold by 30 kD membrane package; Lane 5, Concentrated 10 - fold by 10 kD membrane package; Lane 6, Concentrated 10 - fold by 5 kD membrane package. Detailed implementation manners

[0027] The present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the described specific embodiments are only used to explain the present invention, rather than to limit the present invention. In addition, for the experimental operations involved in the embodiments, the parts not specifically described in the text are all conventional operations in the art, and those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant instructions, manuals, etc. before the filing date of this invention application for implementation.

[0028] Example 1: Screening and identification of Type A Clostridium perfringens 1.1 PCR identification Intestinal contents from sudden death pigs with intestinal flatulence from Jiangsu, Yunnan, Guizhou, Hubei and other places were streaked on TSN solid medium and anaerobically cultured at 37 °C for about 46 h. Single colonies were picked from the plates of 8 different sources of diseased tissue streaks for PCR identification. The results showed that the single colonies isolated from 6 of the diseased materials could amplify the gene encoding α-toxin, while the genes encoding β, ε, and ι toxins were not detected. It was determined that the 7 isolated strains, SP01, SP02, SP03, SP14, SP15, SP17, and SP20, were Clostridium perfringens type A ( Figure 1 ). In addition to using single PCR for amplification, the PCR primers and amplification program were referred to the literature (Miao Xipeng, et al. Establishment and preliminary application of multiplex PCR detection method for Clostridium perfringens [J]. Chinese Journal of Veterinary Science, 2022(008):042.).

[0029] 1.2 Identification of α-toxin secreted by Clostridium perfringens type A by Western Blot The supernatant of the Clostridium perfringens type A bacterial solution was collected after centrifugation at 8000 r / min for 10 min and then subjected to SDS-PAGE electrophoresis. After completion, it was fixed in the order of negative electrode, gauze, filter paper, protein gel, NC membrane, filter paper, gauze, and positive electrode, placed in the electrophoresis tank and filled with transfer buffer. Transfer the membrane with a current of 200 mA for 2 h. Then, 5% skim milk prepared with PBST was used as the blocking solution, and the NC membrane was incubated in the blocking solution at room temperature for 1 h. The blocked NC membrane was rinsed 3 times with PBST, 5 min each time. The α-toxin antibody was diluted 1000 times with 5% skim milk and incubated at room temperature for 1 h. After incubation with the primary antibody, the NC membrane was rinsed 3 times with PBST, 5 min each time. The goat anti-rabbit secondary antibody was diluted 1000 times with 5% skim milk and incubated at room temperature for 1 h; the NC membrane was rinsed 3 times with PBST, 5 min each time. 1 mL of 1.5 mol / L (pH 8.9) Tris-HCl was added to 9 mL of pure water, 50 μL of 44 mg / mL luminol, 22 μL of 15 mg / mL p-coumaric acid solution, and 5 μL of 30% H2O2 were mixed evenly. The NC membrane was placed in the prepared developing solution, photographed with software after developing for 10 seconds, and the size of the α-toxin band was about 42 kD. The results showed that the standard strain C57-1 strain (CVCC number 37), SP02, SP03, SP17, and SP20 strains had obvious α-toxin bands near 42 kD ( Figure 2 ).

[0030] 1.3 Bioactivity determination of the toxin secreted in the culture supernatant of Clostridium perfringens type A strains The preserved bacterial strain was inoculated into 1 tube of culture medium and cultured statically at 37°C. The revived bacterial strain was inoculated into 250 mL of Clostridium liquid medium at a ratio of 1% and cultured statically at 37°C for 16 h. 0.2 mL of the bacterial culture supernatant was taken by centrifugation and serially diluted 2-fold for tail vein injection into 2 mice, and observed for 24 h. The dose that caused the death of a group of 2 mice within 24 h was defined as the minimum lethal dose (MLD). The results showed that there were significant differences in the toxin expression levels in the supernatants of different strains. The toxin content in the supernatant of the reference strain C57-1 was 10 MLD / mL, and the others isolated ranged from <5 MLD to 120 MLD, as shown in Table 1 below. In summary, we isolated 4 strains of Clostridium perfringens type A from pigs that could detect α-toxin, and the SP20 strain had the highest toxin production level.

[0031] Table 1. α-Toxin production levels of different Clostridium perfringens type A

[0032] 1.4 Determination of the growth and toxin secretion curves of Clostridium perfringens type A strain SP20 To further study the characteristics of Clostridium perfringens type A strain SP20 isolated from a pig farm in Enshi, Hubei, we determined the growth curve and toxin secretion curve of strain SP20, with the standard strain C57-1 as a control. First, 0.1 ml of the preserved bacterial strain was taken and resuspended in the bacterial suspension and inoculated into 1 tube of culture medium and cultured statically at 37°C. The revived bacterial strain was inoculated into 250 mL of Clostridium liquid medium at a ratio of 1% and cultured statically at 37°C for 16 h. Eight bottles of culture medium, 250 ml each, were prepared and inoculated at 1% into fresh culture medium and cultured statically at 37°C. One bottle was taken out at 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, and 24 h of culture respectively, sampled and serially diluted for plating to count viable bacteria, and at the same time the supernatant was taken to determine the MLD for mice. The results showed that after inoculation, strain SP20 entered the logarithmic growth phase at 4 h and continued until 12 h, then entered the plateau phase and continued until 16 h, and then entered the decline phase; the toxin began to be secreted from the logarithmic growth phase and reached the peak of 120 MLD / mL at the plateau phase, and then remained unchanged. After inoculation, strain C57-1 entered the logarithmic growth phase at 4 h and continued until 10 h, then entered the decline phase and remained for 4 h to 14 h; the toxin began to be secreted from the logarithmic growth phase, reached the peak of 10 MLD / mL, and then remained unchanged ( Figure 3 ). Generally speaking, the total number of bacteria in both strain SP20 and strain C57-1 could reach more than 10 8 CFU / mL during the culture process, but the level of α-toxin in the supernatant of strain SP20 was significantly higher than that of the standard strain C57-1.

[0033] 1.5 Pathogenicity test of Clostridium perfringens type A strain SP20 on pigs Take the SP20 strain and C57-1 strain bacterial solutions in the late logarithmic growth phase, and take 10 - 50 mL each to orally challenge commercial pigs weighing 30 - 40 kg and 70 - 90 kg. Five pigs are orally administered in each group, and each pig is orally challenged with 30 - 50 mL of the bacterial solution. Observe once every 4 h after challenge for 72 h, and record the death situation of the pigs. Immediately dissect the dead pigs to observe organ lesions, especially whether the intestines are distended. The control group is euthanized and dissected at 72 h as a control. The results show that after challenge with each dose of the C57-1 strain, the pigs had no obvious clinical symptoms and no obvious lesions were found during autopsy, showing no significant difference from the control group; while after challenge with the SP20 strain in pigs weighing 30 - 40 kg and 70 - 90 kg, sudden death occurred, and intestinal distension and severe organ corruption were visible during autopsy, indicating strong pathogenicity to medium and large pigs (Table 2, Figures 4 - 5 ) Table 2. Determination of the minimum lethal dose of Clostridium perfringens type A for pigs of different weights

[0034] Example 2: Fermentation expression and purification of α-toxin of Clostridium perfringens type A SP20 strain The SP20 strain was resuspended with 1 mL of sterile 0.01 mol / L PBS. Take 0.1 mL and inoculate it into a test tube containing the medium, and incubate at 37°C for 16 h as the primary seed. Take 2 mL of the primary seed and inoculate it into 220 mL of the medium, and incubate for 10 h as the secondary seed. After inoculating the seeds into the fermenter at 5 - 10%, set the stirring speed to 20 rpm - 100 rpm, control the pH at 6.0 - 7.0, and the culture temperature at 34°C - 37°C. Supplement 5 M sodium hydroxide to maintain the pH, and connect the 20% - 80% glucose feeding bottle and the sodium hydroxide solution feeding bottle to the fermenter and stir evenly to automatically adjust the pH value. Take 10 ml of samples every hour and detect the OD 600 value. When the OD 600 value increases to more than 8, the culture is terminated. The results show that high levels of α-toxin can be obtained at 20 rpm - 100 rpm, 34°C - 37°C, and pH 6.0 - 7.0. The optimal conditions are 20 rpm, 36°C, and pH 6.5; followed by 20 rpm, 34°C, and pH 6.0 (Table 3).

[0035] Fermentation medium: Tryptone 10 g / L, Beef extract powder 10 g / L, Beef liver extract powder 5 g / L, Yeast extract powder 5 g / L, Glucose 5 g / L, Sodium chloride 5 g / L.

[0036] Table 3. Fermentation growth and toxin secretion curves of Clostridium perfringens type A SP20 strain under different conditions

[0037] 4 L of the supernatant harvested under the optimal fermentation conditions was concentrated using a POOL 0.1 m 2 5 - 100 kD membrane package and washed and filtered with 2 L of PBS in an equal volume. Finally, it was concentrated 10 times, and the samples during the washing and filtering process were collected for SDS - PAGE, WB, and toxin content detection and analysis. The results showed that after concentrating 10 times with a 30 kD membrane package, the total amount of miscellaneous proteins was less, and the content of immunoblotting α - toxin was higher ( Figure 6 ), and the content of α - toxin determined by bioactivity assay was 1030 MLD / mL, with a recovery rate of about 85.7%.

[0038] Example 3: Vaccine preparation and immunization The detoxified α - toxin containing 30 - 400 MLD, adjuvants (aluminum hydroxide adjuvant, aqueous adjuvant, or oil - in - water adjuvant), and immune enhancers (CpG, Poly(I:C), or monophosphatide A) were formulated into a vaccine of 2 mL / dose according to the vaccine group in Table 4. Each vaccine was used to immunize 5 piglets of about 20 kg. After 21 days, the same vaccine was used for booster immunization once; while the control group was immunized with 2 mL of sterile PBS, and the rest of the feeding management was the same as that of the immunized group. Blood was collected before each immunization or challenge to separate serum for detecting the neutralization titer. 14 days after the second immunization, the immunized group and the control group were simultaneously challenged orally with 1×10 10 CFU of Clostridium perfringens type A strain SP20. The results showed that 4 / 5 pigs in the challenged control group died suddenly, accompanied by intestinal flatulence. 3 / 5 - 5 / 5 pigs survived in each immunized group after challenge, and some surviving pigs showed shortness of breath after challenge but all recovered within 24 h after challenge. Vaccines containing 100 MLD and above of antigen, aluminum hydroxide adjuvant, aqueous adjuvant, or oil - in - water adjuvant, and immune enhancers CpG, Poly(I:C), or monophosphatide A could achieve a survival rate of ≥80% after two - dose lethal challenge.

[0039] Table 4. Results of Clostridium perfringens type A challenge test after pig immunization

[0040] The above - mentioned examples are only preferred examples of the present invention, and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Clostridium perfringens type A strain of porcine origin, deposited with the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2025386, which can produce α-toxin at no less than 120 MLD / mL and can cause intestinal flatulence and sudden death in pigs through oral challenge.

2. Use of the Clostridium perfringens type A strain of porcine origin according to claim 1 in the preparation of a vaccine for preventing intestinal flatulence and sudden death in pigs caused by Clostridium perfringens type A.

3. A vaccine for preventing porcine intestinal flatulence and sudden death caused by Clostridium perfringens type A, characterized in that The vaccine comprises the following components: (a) α-toxin produced by the Clostridium perfringens type A strain of porcine origin according to claim 1; (b) an adjuvant; (c) an immune enhancer.

4. The vaccine according to claim 3, wherein: The adjuvant is selected from one or more of aluminum hydroxide adjuvant, aqueous adjuvant or oil-in-water adjuvant.

5. The vaccine according to claim 3, characterized in that: The immune enhancer is selected from one or more of CpG oligonucleotides, Poly(I:C) or monophosphoryl lipid A.

6. The vaccine according to claim 3, wherein: The dose of the α-toxin is 100 - 400 MLD per dose.

7. A method for preparing the vaccine according to any one of claims 3-6, characterized in that, It includes the following steps: (a) Culturing the Clostridium perfringens type A strain of porcine origin according to claim 1 in a fermenter, controlling the stirring speed at 20 - 100 rpm, the temperature at 34 - 37 °C, and the pH at 6.0 - 7.0; (b) Collecting the culture supernatant, ultrafiltrating and diafiltrating it through a 5 - 100 kD membrane package; (c) Detoxifying the concentrated α-toxin; (d) Mixing the detoxified α-toxin with the adjuvant and the immune enhancer to prepare a vaccine preparation.

8. The preparation method according to claim 7, characterized in that: The fermentation medium comprises 10 g / L of tryptone, 10 g / L of beef extract powder, 5 g / L of beef liver extract powder, 5 g / L of yeast extract powder, 5 g / L of glucose and 5 g / L of sodium chloride.

9. The preparation method according to claim 7, characterized in that: The cut-off molecular weight of the membrane package is 30 kD.

Images