A strain of Clostridium perfringens type A from pigs and its application in vaccine preparation

Abstract

This invention belongs to the field of biology and discloses a porcine Clostridium perfringens strain SP20 that secretes and expresses high levels of α-toxin. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025386. This bacterium exhibits strong pathogenicity in medium and large-sized pigs and can be used to establish porcine challenge models and evaluate the efficacy of porcine Clostridium perfringens type A vaccines. This invention also discloses a vaccine for preventing intestinal bloating and sudden death in medium and large-sized pigs caused by Clostridium perfringens type A. This vaccine contains α-toxin produced by the aforementioned SP20 strain. This invention fills the gap in the development of pathogenic models for intestinal bloating and sudden death in medium and large-sized pigs, which is beneficial for controlling disease transmission and reducing economic losses in the livestock industry.

Description

Technical Field

[0001] This invention relates to a highly virulent strain of Clostridium perfringens type A from pigs and its application in the preparation of α-toxin vaccines, belonging to the field of biology. Background Technology

[0002] Clostridium perfringens ( Clostridium perfringensClostridium perfringens (C.) is a group of Gram-positive bacilli. Based on the types and combinations of α, β, ι, and ε toxins they produce, they can be classified into types A, B, C, D, E, F, G, and H. Type A *C. perfringens* produces only the α toxin, one of the α, β, ι, and ε toxins. Type A *C. perfringens* 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, and rabbits (Camargo A et al., Intra-species diversity of *C. perfringens*: A diverse genetic repertoire reveals its pathogenic potential. Front Microbiol. 2022 Jul 22;13:952081.). While Type A *C. perfringens* has a wide range of sources, its typing is based solely on the presence of α toxin-coding genes in its genome, without the presence of β, ι, or ε toxin-coding genes. The genomes of different Clostridium perfringens types A are highly diverse. Among the eight Clostridium perfringens type A strains with published whole genome sequences from different sources in GenBank, there are 1807 core genes, which is only 61.2% of the average number of genes (2951) of each strain (Hassan KA 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 MA 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).). Besides the known toxins, Clostridium perfringens type A also has other virulence factors, such as nanJ and nagJ.

[0003] From a pathogenicity perspective, *Clostridium perfringens* type A can infect hosts through mechanical trauma or the fecal-oral route, causing emphysematous gangrene in humans, yellow lamb disease in sheep, diarrhea and necrotic enteritis in newborn piglets, necrotic enteritis in chickens, and clostridial infection in rabbits. Genome-wide nucleotide polymorphisms further reveal that strains from different sources and with different pathogenic types are on different evolutionary branches (Geier RR et al, Comparative Genomics of *Clostridium perfringens* Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors[J]. Frontiers in Microbiology, 2021, 12.). In summary, *Clostridium perfringens* type A from different species differs greatly in genomic composition, virulence factors, types of secreted toxins, and pathogenicity, except that they only secrete α-toxin among α, β, ι, and ε toxins. Often, *Clostridium perfringens* type A from a specific source is pathogenic to a specific species.

[0004] Sudden death in medium and large-sized pigs, accompanied by intestinal bloating, is frequently observed in veterinary clinics. Bacteriological testing suggests that *Clostridium perfringens* type A is one of the pathogens, but typical cases of intestinal bloating and sudden death in medium and large-sized pigs have not yet been reported in the laboratory. Because the onset of bloating and sudden death in medium and large-sized pigs is rapid and acute, antibiotic treatment is often ineffective. Immunoprophylaxis is one of the most important means of controlling infectious diseases. Currently, only inactivated vaccines against *Clostridium perfringens* type A are used to prevent diarrhea in piglets. Whole-cell inactivated vaccines have high antigen protein content, significant side effects, and the genomes of different *Clostridium perfringens* types are highly different, resulting in unsatisfactory cross-protection. Therefore, there is an urgent need to develop specific vaccines against sudden death and intestinal bloating in medium and large-sized pigs to reduce the economic losses caused by sudden death in 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 detoxification of alpha toxin can be used as a vaccine antigen, but the yield of Clostridium perfringens type A alpha toxin is low, generally 10-100 MLD / mL; the C-terminal domain of the alpha toxin expressed by genetic engineering (such as amino acids 251-379) has good immunogenicity in mice, but its immunogenicity has not been evaluated in pigs (Ferreira MRet. 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 death of medium and large pigs caused by Clostridium perfringens type A results in significant economic losses, but the laboratory has not yet been able to replicate classic cases, making it impossible to directly evaluate the effectiveness of various types of Clostridium perfringens type A vaccines in pigs.

[0006] Therefore, isolating Clostridium perfringens type A from diseased pig samples, replicating typical clinical symptoms including intestinal bloating and sudden death, establishing a model, and obtaining porcine Clostridium perfringens type A with high levels of alpha toxin secretion are key to developing vaccines to prevent intestinal bloating and sudden death in medium and large pigs. Summary of the Invention

[0007] One object of the present invention is to provide a porcine Clostridium perfringens type A strain that can secrete and express high levels of α-toxin and has strong pathogenicity in medium and large pigs. This strain can be used to establish pig sudden death and intestinal bloating challenge models and to prepare α-toxin vaccines.

[0008] To achieve the above objectives, the applicant isolated a strain of *Clostridium perfringens* from the intestines of fattening pigs that died suddenly from bloating at a pig farm in Enshi, Hubei Province. PCR and Western blotting identified the strain as *Clostridium perfringens* type A, and it was named strain SP20. Further bioactivity assays showed that strain SP20 expressed high levels of α-toxin, with the α-toxin content in its culture supernatant not less than 120 MLD / mL. This strain could maintain its growth plateau phase for a relatively long time, and after oral challenge to medium-sized and large pigs weighing 30–90 kg, it produced significant intestinal bloating and sudden death, demonstrating strong pathogenicity.

[0009] The strain was classified and named Clostridium perfringens type A SP20, and was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, Hubei Province, China, on March 6, 2025, with accession number CCTCC NO: M 2025386.

[0010] The second objective of this invention is to provide a vaccine for preventing bloating and sudden death in pigs caused by Clostridium perfringens type A, and a method for preparing the same.

[0011] The vaccine provided by this invention comprises the following components:

[0012] (a) The α-toxin produced by the SP20 strain;

[0013] (b) Adjuvant;

[0014] (c) Immune enhancers.

[0015] The adjuvant is selected from one or more of aluminum hydroxide adjuvant, aqueous adjuvant, or oil-in-water adjuvant, preferably 2-4 mg / dose of aluminum hydroxide adjuvant.

[0016] The immune enhancer is selected from one or more of CpG oligonucleotides, Poly(I:C) or monophospholipid A, preferably CpG 10~30 μg / dose.

[0017] The dosage of the α-toxin is 30-400 MLD / dose, preferably 100-400 MLD / dose.

[0018] The preparation method provided by this invention includes the following steps:

[0019] (a) Cultivate the SP20 bacteria in a fermenter, controlling the stirring speed at 20-100 rpm, the temperature at 34-37℃, and the pH at 6.0-7.0;

[0020] (b) Collect the culture supernatant, concentrate it by ultrafiltration through a 5-100 kD membrane and wash the filter;

[0021] (c) Detoxification treatment of the concentrated α-toxin;

[0022] (d) The detoxified α-toxin is mixed with adjuvants and immune enhancers to prepare a vaccine formulation.

[0023] The fermentation medium contains 10 g / L tryptone, 10 g / L beef extract, 5 g / L beef liver extract, 5 g / L yeast extract, 5 g / L glucose, and 5 g / L sodium chloride.

[0024] The membrane has a molecular weight cutoff of 30 kD.

[0025] The strain was cultured to the late logarithmic growth stage, and the α-toxin content in the culture supernatant was determined to be up to 120 MLD / mL using the method specified in the Chinese Veterinary Pharmacopoeia. After being concentrated 10 times, the content was 1030 MLD / mL, with a recovery rate of approximately 85.7%.

[0026] The immunization efficacy of the vaccine was evaluated in pigs, and the results showed that the Clostridium perfringens type A toxoid vaccine provided by the present invention can induce complete immune protection in pigs and resist the lethal attack of Clostridium perfringens type A on pigs.

[0027] The beneficial effects of the present invention include:

[0028] 1. The strain provided by this invention fills the gap in the global model of intestinal bloating and sudden death in medium and large pigs caused by Clostridium perfringens type A challenge, laying the foundation for the development of related drugs;

[0029] 2. The toxoid vaccine provided by this invention can prevent intestinal bloating and sudden death in pigs caused by Clostridium perfringens type A, which is beneficial for controlling disease transmission and reducing economic losses in the livestock industry;

[0030] 3. The toxoid vaccine provided by this invention has controllable material sources and low preparation costs, making it suitable for large-scale industrial production. Attached Figure Description

[0031] Figure 1 PCR identification of suspected Clostridium perfringens type A. Lanes 1-1 to 1-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP01, respectively; lanes 2-1 to 2-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP02, respectively; lanes 3-1 to 3-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP03, respectively; lanes 4-1 to 4-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP14, respectively; lanes 5-1 to 5-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP15, respectively; lanes 6-1 to 6-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP17, respectively; lanes 7-1 to 7-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP18, respectively; and lanes 8-1 to 8-4 are for electrophoresis of amplified products of the α, β, ε, and ι toxin-coding genes of strain SP20, respectively.

[0032] Figure 2 Immunoblotting was used to detect the expression of α-toxin in the supernatant of different suspected Clostridium perfringens type A strains.

[0033] Figure 3Growth and toxin secretion curves of Clostridium perfringens type A. A, SP20 strain; B, C57-1 strain. Squares on the curves are labeled as log (CFU / ml), and circles are labeled as MLD / mL.

[0034] Figure 4 . 30-40 kg pigs challenged with Clostridium perfringens type A and observed lesions upon necropsy. A, Appearance of blank control pigs; B, Dissection of blank control group; C, Appearance of pigs challenged with C57-1 strain, no obvious abnormalities; D, Dissection of pigs challenged with C57-1 strain, no obvious lesions; E, Appearance of pigs that died suddenly after challenged with SP20 strain, abdominal distension and purplish discoloration of the skin near the ground are visible; F, Dissection of pigs that died suddenly after challenged with SP20 strain, intestinal distension and organ putrefaction are visible.

[0035] Figure 5 Clostridium perfringens type A challenged 70-90 kg pigs and observed lesions during necropsy. A, Appearance of blank control pigs; B, Dissection of blank control group; C, Appearance of pigs challenged with C57-1 strain, no obvious abnormalities; D, Dissection of pigs challenged with C57-1 strain, no obvious lesions; E, Appearance of pigs that died suddenly after challenged with SP20 strain, abdominal distension is visible; F, Dissection of pigs that died suddenly after challenged with SP20 strain, intestinal distension and organ putrefaction are visible.

[0036] Figure 6 Comparison of α-toxin recovery rates after ultrafiltration concentration using membranes with different pore sizes. The left figure shows the protein band distribution after ultrafiltration concentration using SDS-PAGE electrophoresis analysis of different membrane pore sizes. M represents the protein marker; lane 1: original supernatant; lane 2: 100 kD membrane concentrate (10-fold concentration); lane 3: 50 kD membrane concentrate (10-fold concentration); lane 4: 30 kD membrane concentrate (10-fold concentration); lane 5: 10 kD membrane concentrate (10-fold concentration); lane 6: 5 kD membrane concentrate (10-fold concentration). The right figure shows the α-toxin content after ultrafiltration concentration using immunoblotting of different membrane pore sizes. Lane 1: original supernatant; lane 2: 100 kD membrane concentrate (10-fold concentration); lane 3: 50 kD membrane concentrate (10-fold concentration); lane 4: 30 kD membrane concentrate (10-fold concentration); lane 5: 10 kD membrane concentrate (10-fold concentration); lane 6: 5 kD membrane concentrate (10-fold concentration). Detailed Implementation

[0037] The present invention will be described in detail below with reference to specific embodiments. It should be noted that the specific embodiments described are only for explaining the present invention and are not intended to limit the present invention. Furthermore, the experimental operations involved in the embodiments, unless otherwise specified in the text, are all conventional operations in the art, and those skilled in the art can implement them by referring to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention.

[0038] Example 1: Screening and identification of Clostridium perfringens type A

[0039] 1.1 PCR identification

[0040] Intestinal contents from pigs that died suddenly with intestinal bloating, collected from Jiangsu, Yunnan, Guizhou, and Hubei provinces, were streaked onto TSN solid medium and anaerobically cultured at 37°C for approximately 46 hours. Single colonies were picked from eight streaked tissue samples from different sources for PCR identification. The results showed that single colonies isolated from six of the samples amplified the gene encoding α-toxin, while the genes encoding β, ε, and ι toxins were not detected. Seven isolated strains, SP01, SP02, SP03, SP14, SP15, SP17, and SP20, were identified as Clostridium perfringens type A. Figure 1 Except for the use of singleton PCR for amplification, the PCR primers and amplification procedures were performed in accordance with the literature (Miao Xipeng et al. Establishment and preliminary application of multiplex PCR detection method for Clostridium perfringens [J]. Chinese Journal of Veterinary Medicine, 2022(008):042.).

[0041] 1.2 Western Blot identification of α-toxin secreted by Clostridium perfringens type A

[0042] After centrifuging the Clostridium perfringens type A bacterial culture at 8000 r / min for 10 min, the supernatant was collected and subjected to SDS-PAGE electrophoresis. After completion, the membrane was fixed in the following order: negative electrode, gauze, filter paper, protein gel, NC membrane, filter paper, gauze, positive electrode, and placed in the electrophoresis tank, which was filled with transfer buffer. Transfer was performed at 200 mA for 2 h. Then, 5% skim milk was prepared with PBST as blocking buffer, and the NC membrane was incubated in the blocking buffer at room temperature for 1 h. The blocked NC membrane was washed three times with PBST for 5 min each time. The α-toxin antibody was diluted 1000-fold with 5% skim milk and incubated at room temperature for 1 h. After primary antibody incubation, the NC membrane was washed three times with PBST for 5 min each time. The goat anti-rabbit secondary antibody was diluted 1000-fold with 5% skim milk and incubated at room temperature for 1 h. The NC membrane was then washed three times with PBST for 5 min each time. Pipette 1 mL of 1.5 mol / L (pH 8.9) Tris-HCl into 9 mL of pure water, add 50 μL of 44 mg / ml luminol, 22 μL of 15 mg / ml p-coumaricacid solution, and 5 μL of 30% H2O2. Mix well. Place the NC membrane in the prepared colorimetric solution and develop for 10 seconds. Take a picture using software. The α-toxin band size is approximately 42 kD. The results show that standard strain C57-1 (CVCC number 37), SP02, SP03, SP17, and SP20 all have obvious α-toxin bands around 42 kD. Figure 2 ).

[0043] 1.3 Determination of the bioactivity of secreted toxins in the culture supernatant of Clostridium perfringens strain type A

[0044] The preserved bacterial strain was inoculated into one culture medium and incubated statically at 37°C. The revived bacterial strain was then inoculated at a 1% ratio into 250 mL of Clostridium liquid culture medium and incubated statically at 37°C for 16 h. 0.2 mL of the bacterial culture supernatant was centrifuged and serially diluted twofold before being injected intravenously into two mice, with observation for 24 h. The dose that caused death in two mice within 24 hours was defined as the minimum lethal dose (MLD). Results showed significant differences in toxin expression levels in the supernatant among different strains. The toxin content in the supernatant of the reference strain C57-1 was 10 MLD / mL, while the levels of the other isolates ranged from <5 MLD to 120 MLD, as detailed in Table 1 below. In summary, we isolated four strains of Clostridium perfringens type A from pigs that showed detectable α-toxin, with strain SP20 exhibiting the highest toxin production level.

[0045] Table 1. Alpha toxin levels in different Clostridium perfringens types A

[0046]

[0047] 1.4 Determination of growth and toxin secretion curves of Clostridium perfringens strain SP20 (type A)

[0048] To further investigate the characteristics of Clostridium perfringens type A strain SP20 isolated from a pig farm in Enshi, Hubei Province, we determined its growth and toxin secretion curves, using the standard strain C57-1 as a control. First, 0.1 ml of the preserved bacterial suspension was inoculated into one culture medium and incubated statically at 37°C. The revived bacterial strain was then inoculated at a 1% ratio into 250 mL of Clostridium liquid culture medium and incubated statically at 37°C for 16 h. Eight 250 ml bottles of culture medium were prepared, each inoculated at a 1% ratio into fresh culture medium and incubated statically at 37°C. One bottle was taken at 4 h, 6 h, 8 h, 10 h, 12 h, 16 h, and 24 h of incubation, and samples were serially diluted and plated for viable cell count. Simultaneously, the supernatant was used to determine the MLD (metastatic disease effect) in mice. The results showed that SP20 entered the logarithmic growth phase 4 hours after inoculation and lasted until 12 hours, then entered the plateau phase and lasted until 16 hours, before entering the apoptosis phase; toxin secretion began in the logarithmic growth phase, reaching a peak of 120 MLD / mL at the plateau phase, and then remained constant. C57-1 entered the logarithmic growth phase 4 hours after inoculation and lasted until 10 hours, then entered the apoptosis phase and remained constant for 4 to 14 hours; toxin secretion began in the logarithmic growth phase, reaching a peak of 10 MLD / mL, and then remained constant. Figure 3 In general, the total bacterial count of both SP20 and C57-1 strains reached 10⁻⁶ during culture. 8The levels of CFU / mL were above 100, but the α-toxin level in the supernatant of SP20 strain was significantly higher than that of the standard strain C57-1.

[0049] 1.5 Pathogenicity test of Clostridium perfringens strain SP20 in pigs

[0050] Late logarithmic growth stage bacterial suspensions of SP20 and C57-1 were taken, and 10-50 mL of each was orally administered to commercial pigs weighing 30-40 kg and 70-90 kg. Five pigs were administered the suspension to each group. Each pig was orally administered 30-50 mL of the bacterial suspension. Pigs were observed every 4 hours for 72 hours after challenge, and mortality was recorded. Dead pigs were immediately dissected to observe organ lesions, especially intestinal bloating. The control group was euthanized and dissected after 72 hours as a control. Results showed that pigs challenged with C57-1 at all doses showed no obvious clinical symptoms, and no obvious lesions were observed upon dissection, with no significant difference from the control group. However, pigs challenged with SP20 at 30-40 kg and 70-90 kg showed sudden death, and dissection revealed intestinal bloating and severe organ putrefaction, indicating strong pathogenicity in medium and large-sized pigs (Table 2). Figures 4-5 );

[0051] Table 2. Minimum lethal dose of Clostridium perfringens type A in pigs of different weights

[0052]

[0053] Example 2: Fermentation expression and purification of α-toxin from Clostridium perfringens strain SP20 (type A)

[0054] SP20 strain was reconstituted in 1 mL of sterile 0.01 mol / L PBS. 0.1 mL of this solution was inoculated into a test tube containing culture medium and incubated at 37°C for 16 h as the primary seed culture. 2 mL of the primary seed culture was then inoculated into 220 mL of culture medium and incubated for 10 h as the secondary seed culture. After inoculating the seed culture at 5-10% concentration in the fermenter, the stirring speed was set to 20-100 rpm, the pH was maintained at 6.0-7.0, and the incubation temperature was 34-37°C. 5 M sodium hydroxide was added to maintain the pH. A 20%-80% glucose feed bottle and a sodium hydroxide solution feed bottle were connected to the fermenter and stirred thoroughly, with the pH automatically adjusted. A 10 mL sample was taken every hour to measure the OD value. 600 Value, when OD 600 The culture was terminated when the pH value increased to above 8. The results showed that high levels of α-toxin could be obtained at 20 rpm to 100 rpm, 34℃ to 37℃, and pH 6.0 to 7.0. The optimal conditions were 20 rpm, 36℃, and pH 6.5; followed by 20 rpm, 34℃, and pH 6.0 (Table 3).

[0055] Fermentation medium: 10 g / L tryptone, 10 g / L beef extract, 5 g / L beef liver extract, 5 g / L yeast extract, 5 g / L glucose, and 5 g / L sodium chloride.

[0056] Table 3. Fermentation growth and toxin secretion curves of Clostridium perfringens strain SP20 under different conditions.

[0057]

[0058] 4 L of the supernatant harvested under optimal fermentation conditions was used in a 0.1 mL POOL container. 2 5–100 kD membrane packs were concentrated and washed with an equal volume of 2 L PBS, resulting in a final concentration of 10-fold. Samples from the washing process were collected for SDS-PAGE, Western blotting, and toxin content analysis. Results showed that a 30 kD membrane pack, after 10-fold concentration, contained less total protein and had a higher content of immunoblot α-toxin. Figure 6 The bioactivity assay showed that the α-toxin content was 1030 MLD / mL, with a recovery rate of approximately 85.7%.

[0059] Example 3: Vaccine Preparation and Immunization

[0060] A vaccine containing 30-400 MLD detoxified α-toxin, adjuvants (aluminum hydroxide adjuvant, aqueous adjuvant, or oil-in-water adjuvant), and immunostimulants (CpG, Poly(I:C), or monophospholipid A) was prepared into 2 mL / dosage doses according to the vaccine groups in Table 4. Each vaccine was used to immunize 5 piglets weighing approximately 20 kg each, followed by a booster immunization with the same vaccine 21 days later. The control group was immunized with 2 mL of sterile PBS, and all other feeding and management practices were the same as the immunized group. Blood was collected before each immunization or challenge to separate serum and determine the neutralizing titer. 14 days after the second immunization, both the immunized and control groups were simultaneously challenged orally with 1×10⁻⁶ oz. 10 CFU strain of Clostridium perfringens type A, SP20. Results showed that 4 / 5 of the pigs in the challenge control group died suddenly, accompanied by intestinal bloating. In each immunization group, 3 / 5 to 5 / 5 survived after challenge; some surviving pigs experienced shortness of breath after challenge, but all recovered within 24 hours. The vaccine containing 100 MLD or higher of antigen, aluminum hydroxide adjuvant, aqueous adjuvant, or oil-in-water adjuvant, and immunostimulants CpG, Poly(I:C), or monophospholipid A, resulted in a ≥80% survival rate after two lethal challenges.

[0061] Table 4. Results of Clostridium perfringens type A challenge test in pigs after immunization

[0062]

[0063]

[0064] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the principles and rules of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Clostridium perfringens type A from pigs, deposited at the China Center for Type Culture Collection (CCTCCNO: M 2025386), wherein the strain is capable of producing α-toxin of not less than 120 MLD / mL and can induce intestinal bloating and sudden death in pigs by oral challenge.

2. The application of the porcine Clostridium perfringens type A as described in claim 1 in the preparation of a vaccine, wherein the vaccine is used to prevent intestinal bloating and sudden death in pigs caused by Clostridium perfringens type A.

3. The application as described in claim 2, characterized in that, The method for preparing the vaccine includes the following steps: (a) The porcine type A Clostridium perfringens of claim 1 is cultured in a fermenter, with the stirring speed controlled at 20-100 rpm, the temperature at 34-37°C, and the pH at 6.0-7.0; (b) Collect the culture supernatant, concentrate it by ultrafiltration through a 5-100 kD membrane and wash the filter; (c) Detoxification treatment of the concentrated α-toxin; (d) The detoxified α-toxin is mixed with adjuvants and immune enhancers to prepare a vaccine formulation.

4. The application as described in claim 3, characterized in that: The fermentation medium contains 10 g / L tryptone, 10 g / L beef extract, 5 g / L beef liver extract, 5 g / L yeast extract, 5 g / L glucose, and 5 g / L sodium chloride.

5. The application as described in claim 3, characterized in that: The membrane has a molecular weight cutoff of 30 kD.

6. The application as described in claim 3, characterized in that: The adjuvant is selected from one or more of aluminum hydroxide adjuvants, aqueous adjuvants, or oil-in-water adjuvants.

7. The application as described in claim 3, characterized in that: The immune enhancer is selected from one or more of CpG oligonucleotides, Poly(I:C), or monophospholipid A.

8. The application as described in claim 3, characterized in that: The dosage of the α-toxin is 100-400 MLD / dose.

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