Establishment and application of A-type clostridium perfringens indirect hemagglutination detection method
An antigen-sensitized red blood cell suspension was prepared by using Clostridium perfringens exotoxin solution of Clostridium perfringens, combined with indirect hemocloxation test method, a highly efficient and specific type Clostridium perfringens vaccine suitable for the grassroots was established. The effectiveness detection method solves the problems of complex and high cost in the prior art detection method.
Patent Information
- Application Number
- CN202410894712.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-04
- Publication Date
- 2025-01-07
AI Technical Summary
The existing efficacy detection methods of C. perfringens vaccines such as serum neutralization method and immune toxicity method are complex in operation, high in cost, and are not suitable for temporary ranch testing, and lack simple and efficient indirect hemoclotting detection methods.
The antigen-sensitized red blood cell suspension was prepared by aldehyde and tanning treatment, combined with indirect hemoclox assay, the highest serum titer of positive serum was determined to establish sensitivity. , Indirect hemagglutination test method with high specificity is used for vaccine efficacy testing.
It realizes the efficacy detection of C. perfringens vaccine with simple operation, low cost and intuitive results. It is suitable for grassroots promotion, replacing the traditional immune and poison-attacking method and serum neutralization method, and has high sensitivity and specificity.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of veterinary biological product testing, and in particular to the establishment and application of an indirect hemagglutination detection method for type A Clostridium perfringens. Background Art
[0002] Clostridium perfringens, also known as Clostridium perfringens, exists in the intestines of many healthy animals. When feeding and environmental conditions change, it can cause Clostridium perfringens disease in livestock and poultry. Once the disease occurs, it will lead to rapid death of livestock and poultry. The course of the disease is extremely short, and they often die before they are effectively treated. Since the discovery of Clostridium perfringens, a disease with a short course, rapid onset, rapid death, and high mortality (Clostridium perfringens disease) has appeared in livestock and poultry in various regions. The symptoms of the disease are sometimes not obvious, and livestock and poultry often begin to die before the disease is treated, causing immeasurable economic losses to the animal husbandry industry.
[0003] Therefore, the disease is mainly prevented by vaccines. At present, the main methods for testing the efficacy of animal immunization vaccines are serum neutralization method and immune challenge method. Among them, the serum neutralization method requires a large number of test animals and has high requirements on the operator's skills. It requires a clean animal test room and is not suitable for temporary inspection samples in pastures. The immune challenge method requires target animals for testing, which has high economic costs and is inconvenient to operate.
[0004] The indirect hemagglutination test (IHA) is a classic method in serological testing. After continuous improvement, it has been widely used to detect serum antibodies of various pathogens. Compared with the serum neutralization method, the indirect hemagglutination test has a simple operation process, saves time and experimental animals, and has low cost.
[0005] Therefore, how to establish an indirect hemagglutination test method that can be used for testing the efficacy of Clostridium perfringens type A vaccine has become a technical problem that needs to be solved urgently in this field. Summary of the invention
[0006] First, the present invention provides a method for preparing an antigen-sensitized red blood cell suspension, comprising: An exotoxin solution is prepared by using type A Clostridium perfringens to obtain an immunogen; the deposit number of type A Clostridium perfringens is CGMCC No.14880; The red blood cell suspension is mixed with glutaraldehyde and then aldehyde-treated for 2 to 4 hours to obtain a aldehyde-treated red blood cell suspension; the aldehyde-treated red blood cell suspension is then tanned to obtain a aldehyde-tanned red blood cell suspension; The immunogen is mixed with the aldehyde-tannic acid erythrocyte suspension to prepare an antigen-sensitized erythrocyte suspension.
[0007] In the art, when preparing sensitized red blood cells, the immunogen usually used is a purified protein, but due to the complex purification method and harsh purification conditions, it is not easy to purify the protein that meets the requirements of sensitized red blood cells. The present invention finds that the exotoxin solution prepared by the above-mentioned type A Clostridium perfringens can be directly used as an immunogen to sensitize red blood cells, eliminating the protein purification step.
[0008] Furthermore, the present invention has found that when the exotoxin solution of type A Clostridium perfringens is used as an immunogen to sensitize red blood cells, it is necessary to control the hydroformylation time to 2 to 4 hours during the hydroformylation process of red blood cells. If the time is too long, all the indirect hemagglutination test holes will self-coagulate, resulting in false positives. If the time is too short, more red blood cells will be broken and the storage time of the hydroformylated red blood cells will be shortened.
[0009] More preferably, the hydroformylation time is 2.5 to 3 hours.
[0010] Preferably, the hydroformylation temperature is 4°C to 25°C.
[0011] In some embodiments, the type A Clostridium perfringens is inoculated into an anaerobic meat liver and peptic ulcer digestion broth medium, and the bacterial liquid is harvested after static culture at 36-37°C for 4-5 hours, filtered with a 0.22 μm filter membrane after centrifugation, and the filtrate is concentrated 18-22 times with a 4-6KD hollow fiber to obtain the exotoxin solution (immunogen).
[0012] In some embodiments, after the immunogen is prepared, it is diluted into different titers and then injected into mice, and the minimum lethal dose for mice is obtained based on the lethality of mice.
[0013] Preferably, the toxicity of the immunogen is 620-630 mice minimum lethal dose (MLD) per milliliter.
[0014] In some embodiments, the red blood cells in the red blood cell suspension are derived from sheep blood.
[0015] In some embodiments, the concentration of the suspension of formaldehyded red blood cells is 4% to 6%.
[0016] In some embodiments, the concentration of the aldehyde-tanned red blood cell suspension is 4% to 6%.
[0017] In some embodiments, the concentration of the antigen-sensitized red blood cell suspension is 1.5% to 2.5%.
[0018] In some embodiments, positive serum and negative serum are used to determine the antigen sensitization unit, and the minimum amount of antigen-sensitized red blood cells that show the highest titer of agglutination with the corresponding positive serum is regarded as one sensitization unit, and the antigen-sensitized red blood cells have no self-agglutination or rupture phenomenon.
[0019] In some embodiments, the immunogen containing 1 to 2 sensitizing units is mixed with a 4% to 6% aldehyde-tannic acid erythrocyte suspension to prepare an antigen-sensitized erythrocyte suspension.
[0020] Furthermore, the present invention provides an antigen-sensitized red blood cell suspension prepared by any of the above schemes.
[0021] Furthermore, the present invention provides a method for establishing an indirect hemagglutination test method for Clostridium perfringens type A, comprising: The immunogen of claim 1 with different exotoxin contents is used to sensitize the aldehyded-tannic red blood cell suspension, the highest serum titer of the same type A Clostridium perfringens positive serum is measured, the antigen dilution corresponding to the highest serum titer is determined, and an indirect hemagglutination test method for type A Clostridium perfringens is established.
[0022] In some embodiments, the method for preparing the Clostridium perfringens type A positive serum comprises: An exotoxin solution is prepared using Clostridium perfringens type A, and then the exotoxin solution is inactivated and detoxified to obtain a Clostridium perfringens type A toxoid solution, and then the Clostridium perfringens type A toxoid solution is prepared into an inactivated Clostridium perfringens toxoid vaccine; and then the inactivated vaccine is used to immunize animals, and the serum is separated to obtain the Clostridium perfringens type A positive serum.
[0023] Preferably, aluminum hydroxide gel is used as an adjuvant to prepare the inactivated vaccine of Clostridium perfringens type A toxoid.
[0024] Furthermore, the present invention provides the use of the antigen-sensitized red blood cell suspension or the establishment method in the detection of serum neutralizing antibody titer.
[0025] In some embodiments, the use comprises: The type A Clostridium perfringens exotoxin solution is prepared into an antigen solution with an antigen dilution corresponding to the highest serum titer, sensitized to the aldehyded-tannic red blood cell suspension, and then the serum to be tested is added for reaction, and the serum neutralizing antibody titer is determined based on the red blood cell agglutination situation.
[0026] In some embodiments, the reaction is carried out at room temperature for 45 to 60 minutes; Or the reaction is carried out at 35-39° C. for 30-45 min.
[0027] In some embodiments, the antigen dilution corresponding to the highest serum titer is 140-160 mouse minimum lethal dose toxin / mL.
[0028] In some embodiments, the indirect hemagglutination titer (nlog2) is calculated as:
[0029] Furthermore, the present invention provides the antigen-sensitized red blood cell suspension, or the establishment method, or the application in the efficacy test of Clostridium perfringens type A vaccine.
[0030] In some embodiments, the efficacy of the Clostridium perfringens type A vaccine is tested using the Clostridium perfringens type A indirect hemagglutination test after quantitatively determining the correlation between the indirect hemagglutination titer and the serum neutralizing antibody titer.
[0031] In some embodiments, the neutralizing antibody titer is greater than or equal to 6 and the serum indirect hemagglutination antibody titer is greater than or equal to 5.
[0032] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts a type A Clostridium perfringens exotoxin solution as an immunogen, takes the minimum lethal dose of the type A Clostridium perfringens exotoxin antigen as a basis, and establishes an indirect hemagglutination test method for type A Clostridium perfringens with good sensitivity, specificity and compliance rate by screening the optimal dilution of the antigen in the indirect hemagglutination test. The test method is used to detect the indirect hemagglutination titer of the serum of vaccine-immunized animals, and can replace the immune challenge method and the serum neutralization method in the efficacy test of the type A Clostridium perfringens vaccine.
[0033] The method of the present invention has strong specificity, good sensitivity and high compliance rate; it is also simple to operate, does not require special equipment, and the result judgment is intuitive and visible to the naked eye. It can be detected quickly and is suitable for promotion in grassroots work. DETAILED DESCRIPTION
[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the present invention will be clearly and completely described below in combination with the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0035] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.
[0036] Example 1: Preparation of Clostridium perfringens exotoxin antigen type A
[0037] 1. Preparation of immunogen
[0038] The type A Clostridium perfringens strain (CNM0708 strain, preserved in the Microbiological Center of China Microorganism Culture Collection Administration, with the collection number of CGMCC No.14880) was inoculated with anaerobic meat liver soup medium (Table 1), and cultured at 36-37°C for 12-16 hours to resuscitate the strain; the revived strain liquid was subcultured and propagated, and 4L anaerobic meat liver peptic digestion soup medium (Table 2) was inoculated at 2%-4%, and the strain liquid was harvested after being cultured at 36-37°C for 4-5 hours; the strain liquid was centrifuged at 3000rpm / min for 20min, filtered with a 0.22μm filter membrane, and the centrifuged supernatant was concentrated 20 times with a 5KD hollow fiber to obtain the type A Clostridium perfringens exotoxin solution.
[0039] Table 1 Composition of anaerobic liver soup medium
[0040] Table 2 Composition of anaerobic meat liver and stomach enzyme digestion soup medium
[0041] The above-mentioned type A Clostridium perfringens exotoxin was diluted to the required titer with 1% peptone water according to the dilution route shown in Table 3 below. Using 16-20g KM mice, each titer of toxin solution was injected into the tail vein of the mice at an injection dose of 0.2mL / mouse × 5 mice. After 24h of observation, the minimum lethal dose for mice was determined to be 0.0016mL, that is, 625 mice per milliliter contained a lethal dose (MLD).
[0042] Table 3 Toxin dilution pathways
[0043] Example 2: Preparation of sheep red blood cells and sensitization treatment
[0044] 1. Hydroformylation
[0045] The newly collected ram blood (containing 50% Aldrich solution) was washed five times with PBS solution at 2000rpm / min for 10min, and the last time was centrifuged for 30min, and a 5% red blood cell suspension was prepared with PBS solution. 2.5% glutaraldehyde stored at 2~8℃ was mixed with 5% red blood cell suspension at a ratio of 1:5 (v / v), and aldehyded for 2h on a shaker at room temperature (25℃) at a speed of 120rpm / min. It was washed five times with PBS solution at 2000rpm / min for 2min, and then washed five times with sterile deionized water at 2000rpm / min for 2min, and the last time was centrifuged for 30min. According to the hematocrit, a 5% aldehyded red blood cell suspension was prepared with deionized water and stored at 4℃ for use. It can be stored for 4 to 7 months.
[0046] 2. Tannic acid
[0047] Mix equal amounts of freshly prepared 1:20000 tannic acid solution and 5% aldehyded red blood cell suspension, incubate in a 37°C water bath for 20 min, mix every 5 min, wash three times with PBS solution by centrifugation at 2000 rpm / min for 2 min, and prepare 5% aldehyded-tannic red blood cell suspension. Store at 4°C for antigen sensitization.
[0048] 3. Antigen sensitization unit determination
[0049] After the antigen was diluted to the optimal concentration, it was mixed with 5% aldehyded-tannic red blood cells in a volume ratio of 1:1, placed in a 37°C water bath for 45 min, mixed every 5 minutes, washed twice with PBS solution (diluent) containing 1% healthy rabbit serum, and prepared into a 2% antigen-sensitized red blood cell suspension with PBS solution containing 1% healthy rabbit serum (prepared immediately before use). It was measured with positive serum and negative serum. The minimum amount of antigen-sensitized red blood cells that showed the highest titer of agglutination with the corresponding positive serum was regarded as one sensitized unit, and the antigen-sensitized red blood cells showed no self-agglutination or rupture.
[0050] 4. Preparation of antigen-sensitized red blood cells
[0051] Add 1 part of 5% aldehyded-tanned red blood cell suspension to 1 part of diluted antigen (containing 1-2 sensitizing units), mix well, place in a 37°C water bath for 45 min, mix every 5 minutes, wash twice with PBS solution (diluent) containing 1% healthy rabbit serum, and use PBS solution containing 1% healthy rabbit serum to prepare 2% antigen-sensitized red blood cell suspension for experiment.
[0052] Example 3: Determination of positive and negative sera for Clostridium perfringens type A
[0053] Preparation of positive and negative serum for Clostridium perfringens type A: The type A Clostridium perfringens strain (CNM0708 strain, preserved in the Microbiological Center of China Microorganism Culture Collection Administration, with the collection number of CGMCC No.14880) was inoculated with anaerobic meat liver soup culture medium, and cultured at 36-37°C for 12-16 hours to resuscitate the strain; the revived strain liquid was subcultured and propagated, and 4L anaerobic meat liver peptic enzyme digestion soup culture medium was inoculated at 2%-4%, and the strain liquid was harvested after being cultured at 36-37°C for 4-5 hours; the strain liquid was centrifuged at 3000rpm / min for 20min, filtered with a 0.22μm filter, and the centrifuged supernatant was concentrated 20 times with a 5KD hollow fiber to obtain a type A Clostridium perfringens exotoxin solution.
[0054] Add formaldehyde solution (formaldehyde concentration is 40%) with a volume ratio of 0.5% to the exotoxin solution to the type A Clostridium perfringens exotoxin solution, inactivate and detoxify at 37°C, shake at least twice a day, inactivate and detoxify for 48 to 72 hours, and obtain the type A Clostridium perfringens toxin solution. Prepare the vaccine according to the ratio of type A Clostridium perfringens toxin solution to aluminum hydroxide gel at a ratio of 4:1 to obtain the type A Clostridium perfringens toxin inactivated vaccine.
[0055] Two test cattle were divided into two groups. One group was vaccinated with type A Clostridium perfringens toxoid inactivated vaccine, and the other group was vaccinated with sterile saline, 2 mL / head, injected intramuscularly into the hind limbs. After 14 days, a booster immunization was performed with the same dose. 21 days after the booster immunization, the serum of the test cattle in each immunized group was collected, namely the positive serum and the negative serum, respectively.
[0056] Example 4: Determination of the antigen dilution corresponding to the highest serum titer
[0057] The test was conducted using Clostridium perfringens type A exotoxin as an antigen. The toxin was diluted 2-fold to sensitize aldehyde-tanned erythrocytes, sensitized in a water bath at 37°C for 45 minutes, washed twice with 0.04 mol / L pH7.2 PBS containing 1% rabbit serum, and then used this dilution to prepare a 2% sensitized erythrocyte suspension. Then, the positive serum with a neutralizing antibody titer of 20 was used for detection. The results are shown in Table 4. When the sensitizing antigen concentration was 156.25 mice minimum lethal dose (MLD) toxin, the positive serum hemagglutination titer was the highest, and erythrocyte autoagglutination occurred with the increase of antigen concentration. Therefore, the optimal concentration of antigen sensitization was 156.25 mice minimum lethal dose (MLD) toxin, and the results were stable after repeated tests.
[0058] Table 4 Optimal concentration test for antigen sensitization
[0059] Example 5: Evaluation of the specificity, sensitivity and compliance of the established method
[0060] 1. Specificity evaluation
[0061] The rabbit high immune serum of Clostridium putrefaciens (CSA), type B and type C Clostridium (CPB), type D Clostridium (CPC) and type C Clostridium (ETX) was tested at the optimal sensitizing concentration. When the agglutination intensity was "++" or above, it was judged as positive and the reaction results were recorded as shown in Table 5.
[0062] Table 5 Specificity test
[0063] The method was used to detect CSA, CPB, CPC and ETX rabbit immune serum, and the results were all negative. The results of CPA positive bovine serum and CPA rabbit immune serum were positive, indicating that the method has good specificity.
[0064] 2. Sensitivity evaluation
[0065] The established method was used to detect CPA-positive bovine serum. When the positive serum was diluted to 1:256, the test result was still positive, and the antibody titer results at different dilution concentrations were linearly correlated, indicating that the method of the present invention has good sensitivity. The test results are shown in Table 6.
[0066] Table 6 Sensitivity test results
[0067] 3. Compliance rate evaluation
[0068] 39 serum samples from cattle used for product potency testing in the project team's laboratory were tested, and the indirect hemagglutination antibody titers of sera with neutralizing antibody titers greater than or equal to 6 were all greater than or equal to 5. At the same time, 32 serum samples were tested using two batches of sensitized red blood cells, and the results of the two tests were consistent (13 / 32), with a difference of 1 titer (14 / 32) and a difference of 2 titers (5 / 32). The results are shown in Table 7, indicating that the detection error was within 1 titer (27 / 32, 84.4%) and within 2 titers (32 / 32, 100%).
[0069] Table 7 Correlation test between hemagglutination antibody titer and neutralizing serum titer
[0070] Example 6: Clinical sample testing
[0071] The results of clinical sample testing using the above batch of Clostridium perfringens type A indirect hemagglutination detection reagent prepared in the above embodiment are as follows: 1. Antibody test results of Ningxia Dingshengxiang Dairy Farm Professional Cooperative
[0072] Indirect hemagglutination titer test and serum neutralizing antibody titer test were conducted on Ningxia Dingshengxiang Dairy Farming Professional Cooperative. A total of 20 sera were collected. The indirect hemagglutination antibody titer of sera with neutralizing antibody titer greater than or equal to 6 was greater than or equal to 5. The detailed results are shown in Table 8.
[0073] Table 8 Ningxia Dingshengxiang Dairy Farming Professional Cooperative
[0074] 2. Test results of Ningxia Changhongkang Ranch
[0075] Indirect hemagglutination titer test and serum neutralizing antibody titer test were conducted on Ningxia Changhongkang Ranch. A total of 16 sera were collected. The indirect hemagglutination antibody titer of sera with neutralizing antibody titer greater than or equal to 6 was greater than or equal to 5. The test results are shown in Tables 9 and 10.
[0076] Table 9 Ningxia Changhongkang Ranch (I)
[0077] Table 10 Ningxia Changhongkang Ranch (II)
[0078] 3. Test results of Yuzhong Ruifeng Ranch
[0079] Indirect hemagglutination titer test and serum neutralizing antibody titer test were conducted on Yuzhong Ruifeng Ranch. A total of 10 sera were collected. The indirect hemagglutination antibody titer of sera with neutralizing antibody titer greater than or equal to 6 was greater than or equal to 5. The test results are shown in Table 11.
[0080] Table 11 Yuzhong Ruifeng Ranch
[0081] Example 7
[0082] In this example, sheep red blood cells were prepared and sensitized using the same method as in Example 2, except that the hydroformylation time was 3 h.
[0083] The results showed that red blood cells turned into chocolate color during the hydroformylation process. After antigen sensitization, the blank control of the indirect hemagglutination test was precipitated. For positive agglutination, the titer of red blood cells hydroformylated for 3 hours was the highest.
[0084] Comparative Example 1
[0085] In this comparative example, sheep red blood cells were prepared and sensitized in the same manner as in Example 2, except that the hydroformylation time was 1 h.
[0086] The results showed that the color of red blood cells remained basically unchanged, but after antigen sensitization, the red blood cells ruptured, self-coagulated and their storage time was significantly shortened.
[0087] Comparative Example 2
[0088] In this comparative example, sheep red blood cells were prepared and sensitized in the same manner as in Example 2, except that the hydroformylation time was 7 h.
[0089] The results showed that the color of red blood cells changed to chocolate after 7 h of aldehyde treatment, but after antigen sensitization, the blank control and test wells were all self-agglutinated in the indirect hemagglutination test.
[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing an antigen-sensitized red blood cell suspension, characterized in that: include: An exotoxin solution is prepared by using type A Clostridium perfringens to obtain an immunogen; the deposit number of type A Clostridium perfringens is CGMCC No.14880; The red blood cell suspension is mixed with glutaraldehyde and then aldehyde-treated for 2 to 4 hours to obtain a aldehyde-treated red blood cell suspension; the aldehyde-treated red blood cell suspension is then tanned to obtain a aldehyde-tanned red blood cell suspension; The immunogen is mixed with the aldehyde-tannic acid erythrocyte suspension to prepare an antigen-sensitized erythrocyte suspension.
2. The preparation method according to claim 1, characterized in that: The concentration of the antigen-sensitized red blood cell suspension is 1.5% to 2.5%.
3. An antigen-sensitized red blood cell suspension, characterized in that: It is prepared by the preparation method described in claim 1 or 2.
4. A method for establishing an indirect hemagglutination test for Clostridium perfringens type A, characterized in that: include: The immunogen of claim 1 with different exotoxin contents is used to sensitize the aldehyded-tannic red blood cell suspension, the highest serum titer of the same type A Clostridium perfringens positive serum is measured, the antigen dilution corresponding to the highest serum titer is determined, and an indirect hemagglutination test method for type A Clostridium perfringens is established.
5. The establishment method according to claim 4, characterized in that: The method for preparing the type A Clostridium perfringens positive serum comprises: An exotoxin solution is prepared using Clostridium perfringens type A, and then the exotoxin solution is inactivated and detoxified to obtain a Clostridium perfringens type A toxoid solution, and then the Clostridium perfringens type A toxoid solution is prepared into an inactivated Clostridium perfringens toxoid vaccine; and then the inactivated vaccine is used to immunize animals, and the serum is separated to obtain the Clostridium perfringens type A positive serum.
6. Use of the preparation method according to claim 1 or 2, or the antigen-sensitized red blood cell suspension according to claim 3, or the establishment method according to claim 4 or 5 in the detection of serum neutralizing antibody titer.
7. The use according to claim 6, characterized in that: include: The type A Clostridium perfringens exotoxin solution is prepared into an antigen solution with an antigen dilution corresponding to the highest serum titer, sensitized to the aldehyded-tannic red blood cell suspension, and then the serum to be tested is added for reaction, and the serum neutralizing antibody titer is determined based on the red blood cell agglutination situation.
8. The use according to claim 7, characterized in that: The reaction conditions are as follows: reacting at room temperature for 45 to 60 minutes; Or the reaction is carried out at 35-39° C. for 30-45 min.
9. The use according to claim 7, characterized in that: The antigen dilution corresponding to the highest serum titer is 140-160 mice minimum lethal dose toxin / mL.
10. Use of the preparation method according to claim 1 or 2, or the antigen-sensitized red blood cell suspension according to claim 3, or the establishment method according to claim 4 or 5, or any one of claims 6 to 9 in the efficacy test of Clostridium perfringens type A vaccine.