Preparation process of high-specificity salmonella typhimurium plate agglutination antigen for pullorum disease

Abstract

The invention discloses a high-specificity pullorum disease salmonella gallinarum plate agglutination antigen preparation process, which comprises the following steps: respectively resuscitating pullorum disease salmonella C79-1 and C79-7 strains to prepare seed solutions, inoculating a suitable culture medium, carrying out fermentation culture according to a specific process, carrying out centrifugal collection on thalli after inactivation, and carrying out freeze drying to obtain the high-specificity pullorum disease salmonella gallinarum plate agglutination antigen. The preparation method comprises the following steps: carrying out high pressure at 121 DEG C for 2 hours, washing for multiple times, removing protein antigen components which are easily subjected to cross reaction with escherichia coli or enterobacteriaceae bacteria which are commonly used in other environments, adjusting the C79-1 strain and the C79-7 strain to proper bacterium concentration, adding Tween-20, crystal violet and glycerol, and fully homogenizing to obtain the salmonella typhimurium plate agglutination antigen for pullorum disease. The antigen has higher specificity, and is more accurate when being used for purifying and detecting the pullorum disease; according to the fermentation process, the bacterial yield is 4 times of that of common fermentation and 2 times of that of solid culture, ethanol precipitation is not needed, and the production cost is lower; tween-20 is added, so that the surface tension of the antigen is increased, the problem of difficulty in uniform coating during use is solved, and a new and better diagnostic reagent is provided for purification of pullorum disease.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary diagnosis, and particularly relates to a preparation process of a Salmonella pullorum and Salmonella gallinarum plate agglutination antigen with high specificity. Background Art

[0002] Salmonella pullorum and Salmonella gallinarum diseases are foodborne zoonotic infectious diseases mainly infecting poultry caused by Salmonella pullorum. It mainly causes septicemia in chicks and turkeys, resulting in death. Adult chickens are infected but do not get sick and are important sources of infection. The pathogens of the two Salmonella diseases can be vertically transmitted through eggs. This disease not only causes serious harm to the production performance of animals, but also endangers human health through the foodborne route, thus causing serious public health problems. At present, the domestic prevention and control strategy for this disease is population purification. Therefore, establishing a rapid and accurate diagnostic method is of great significance for preventing and controlling and eliminating this disease.

[0003] The Salmonella pullorum and Salmonella gallinarum plate agglutination test is currently the most commonly used method for purifying Salmonella pullorum. This method is simple and rapid, can be directly used for the detection of blood samples, and is suitable for clinical screening. However, the traditional polyvalent stained plate agglutination test antigen for Salmonella pullorum and Salmonella gallinarum is prepared according to the process in the 2000 Edition of the Rules for Veterinary Biological Products of the People's Republic of China. The strains used are the standard type C79-1 and the variant type C79-7 Salmonella pullorum and Salmonella gallinarum. The freeze-dried bacterial strains are inoculated into ordinary broth and cultured at 37°C for 18 - 24 hours, and then inoculated onto ordinary agar plates and cultured at 37°C for 18 - 24 hours to obtain first-level seeds; the first-level seeds are respectively inoculated onto ordinary agar slants or ordinary agar small flat flasks and cultured at 37°C for 18 - 24 hours to obtain second-level seeds; the second-level seeds are respectively made into bacterial suspensions with physiological saline or can be directly used to streak and inoculate the second-level seeds onto thiosulfate agar flat flasks and cultured at 37°C for 44 - 48 hours. The agglutinated water in the flat flasks is aspirated, and 20 - 30 ml of phosphate buffer saline (0.07 mo1 / L, pH 7.0 - 7.2) containing 2% formaldehyde solution is added to each bottle to wash down the culture, and it is collected in a glass bottle with glass beads. After shaking to break the bacterial clumps, it is inactivated at 37°C for 48 hours; then the bacterial solution is filtered through a sterilized cotton gauze funnel into a large glass bottle, about twice the amount of 95% ethanol is added, and it is precipitated for more than 3 days. The supernatant is discarded, the precipitate is centrifuged again, the supernatant is discarded, and the centrifuged precipitate is suspended with phosphate buffer saline (0.07 moL / L, pH 7.0 - 7.2) containing 1% formaldehyde solution. The main disadvantages of this process are: (1) The preparation process uses solid flat flasks for culture. To prepare 3000 bottles (15 ml / bottle) of antigen, about 1200 flat flasks are required. Each flat flask needs to use an inoculation loop to streak and inoculate the bacterial strain. Completing this work requires 6 professional personnel to work simultaneously for 5 hours, washing and harvesting the bacterial cells requires 4 professional personnel to work simultaneously for 5 hours, and precipitating the bacterial cells consumes about 1.2×10 5ml; Its antigen culture efficiency is low, the pollution risk is high, the between-batch stability is not ideal, and at the same time, a large amount of absolute ethanol is required, resulting in high production and waste liquid treatment costs; (2) The obtained antigen contains a large amount of K antigen and H antigen, which is prone to cross-react with Escherichia coli or other Enterobacteriaceae bacteria infections in the environment, resulting in disadvantages such as unstable sensitivity and specificity of the prepared antigen, causing troubles in the purification process of pullorum disease.

[0004] Chinese Patent CN113265358 A (Salmonella pullorum and Mycoplasma synoviae double plate agglutination antigen and its preparation method and application), the method for preparing Salmonella pullorum C79-1 antigen is specifically as follows: (1) Preparation of seed bacterial solution: After resuscitating and propagating the Salmonella pullorum C79-1 strain in TSB liquid medium, inoculate it on TSA solid plate medium, culture at 37 °C for 20 h, then wash the bacterial lawn with Martin liquid medium, after expanding the culture, inoculate it into a fermenter containing Martin broth medium for fermentation culture according to a volume ratio of 5.0%; The solid plate medium is made by adding 1.5% purified agar powder to the liquid medium; (2) Bacterial culture: After 18 h of high-density fermentation culture at 37 °C, harvest the bacterial solution, perform viable count, add formaldehyde to a final concentration of 0.3% by volume, and inactivate at 37 °C for 24 h; (3) Antigen preparation: Add 2-fold volume of 95% ethanol by volume to the inactivated bacterial solution, precipitate for 3 d, discard part of the supernatant, centrifuge the remaining part at 8000 r / min for 10 min to discard the supernatant, and resuspend the precipitate with 1% formaldehyde physiological saline, and adjust the bacterial solution concentration to 1.0×10 10 CFU / mL. This process uses the conventional fermentation method to culture bacteria, which can reduce the pollution risk to a certain extent, but the bacterial yield is low, and it cannot solve the defects of high cost caused by using ethanol to precipitate bacteria and unstable sensitivity and specificity of the obtained antigen.

[0005] In addition, due to the fact that the existing culture medium contains animal-derived components, the oil content of some culture media is high or the blood lipid of the screened chickens is high, it is sometimes difficult to coat the antigen evenly during use, which affects the detection results and efficiency. Summary of the Invention

[0006] The object of the present invention is to provide a preparation process for a highly specific Salmonella pullorum and Salmonella gallinarum pullorum plate agglutination antigen, which process reduces the production cost and pollution risk, improves the specificity of diagnosis, makes the diagnosis more accurate, and provides a new and better diagnostic reagent for the purification of pullorum disease.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A preparation process for a highly specific Salmonella pullorum and Salmonella gallinarum pullorum plate agglutination antigen, comprising the following steps:

[0009] (1) Preparation of primary seeds

[0010] The standard Salmonella pullorum strain C79-1 and the variant Salmonella pullorum strain C79-7 were respectively inoculated on a common agar plate for cultivation; colonies on the common agar plate were selected and inoculated on a common agar slant medium for cultivation to obtain primary seeds; the preservation numbers of the C79-1 strain and the C79-7 strain are CVCC79201 and CVCC79201 respectively;

[0011] (2) Propagation of secondary seeds

[0012] The primary seeds were inoculated in a common broth medium for cultivation to obtain secondary seeds;

[0013] (3) High-density fermentation culture of antigen

[0014] The secondary seeds were fermented and cultured in a fermenter, the bacterial liquid was harvested, and formaldehyde with a final concentration of 0.2% was added to inactivate for 12 h, and the bacterial liquid was harvested;

[0015] (4) Bacterial body treatment

[0016] The bacterial liquid harvested in step (3) was centrifuged at 8000 rpm for 20 min, the precipitate was collected, resuspended with PBS (0.01 mol / L, pH value 7.0 - 7.2), placed at 121 °C under high pressure for 2 h, centrifuged according to the above protocol to collect the precipitate, washed with PBS until the supernatant was clear, and the bacterial body was resuspended with PBS containing 2% formaldehyde (0.01 mol / L, pH value 7.0 - 7.2), and the concentration of the bacterial liquid was adjusted to 5×10 10 CFU / ml;

[0017] (5) Preparation of antigen

[0018] The bacterial suspensions of the standard Salmonella pullorum strain C79-1 and the variant Salmonella pullorum strain C79-7 were respectively diluted to different concentrations, and 50 μl of each was respectively used for a plate agglutination test with an equal volume of the standard type / variant positive serum national standard product containing 0.5 IU. The concentration of the bacterial liquid showing no less than 50% agglutination was used as the concentration for preparing the antigen.

[0019] During the fermentation in step (3), the pH value was controlled at 7.0 - 8.0 with ammonia water.

[0020] After 12 h of fermentation in step (3), 40% glucose was added according to 5% of the volume of the fermentation broth, and then glucose was supplemented once every 4 h.

[0021] In step (3), inoculate the secondary seeds at a ratio of 2% of the total amount of the fermentation broth and perform fermentation culture in a fermenter containing a common broth medium at 37°C. During fermentation, control the dissolved oxygen > 40%, control the pH value at 7.0 - 8.0 with ammonia water. After 12 hours of fermentation, add 40% glucose at a ratio of 5% of the volume of the fermentation broth, and then add 40% glucose at a ratio of 5% of the volume of the fermentation broth every 4 hours. When the pH value starts to rise during fermentation, add formaldehyde with a final concentration of 0.2% to inactivate for 12 hours, and harvest the bacterial liquid.

[0022] In the antigen prepared in step (5), the final concentration of crystal violet is 3%, the final concentration of glycerol is 10%, and the final concentration of Tween - 20 is 1%.

[0023] In step (5), first place crystal violet and PBS in a homogenizer and homogenize at 7000 rpm for 3 - 4 minutes, add glycerol and Tween - 20, continue to homogenize for 4 minutes, finally add the bacterial liquid, and homogenize at 2000 rpm for 2 minutes, then quantitatively sub - pack to prepare the antigen.

[0024] The present invention has the following beneficial effects: (1) In the treatment of the bacterial body, the high - pressure sterilization method is used to remove the K antigen and H antigen that are prone to cross - react with Escherichia coli or other Enterobacteriaceae bacteria infections in the environment. Since the purification of Salmonella gallinarum mainly targets Salmonella groups with serotypes O9 and O12, removing the K antigen and H antigen does not affect the purification and improves the specificity and accuracy of diagnosis; (2) Ethanol is not used in the treatment of the bacterial body, reducing the use of organic reagents and lowering the production cost and the cost of waste liquid treatment; (3) The bacterial body production adopts a specific high - density fermentation process. By adding glucose to supplement the carbon source required for bacterial metabolism and using ammonia water instead of NaOH to adjust the pH value, the salt content of the culture medium is reduced, and the antigen yield can reach 2.7×10 10 CFU / ml, and the yield is more than 4 times that of conventional fermentation and 2 times that of solid culture; (4) In the antigen configuration, the component of Tween - 20 is added. It has been verified that it does not affect the test results, but increases the surface tension of the antigen, solving the influence of high oil content in the culture medium or high chicken blood lipid on the test results. Description of the Drawings

[0025] Figure 1 For the detection results of hyperlipidemic serum with antigen added with Tween - 20 and antigen without added Tween - 20;

[0026] Figure 2 For the specific test results;

[0027] Figure 3 For the judgment standard of the antigen detection results of the Salmonella pullorum - typhimurium plate agglutination test. Detailed Embodiments

[0028] For the convenience of those skilled in the art, the present invention will be further described below in conjunction with embodiments. The content mentioned in the embodiments does not limit the present invention.

[0029] I. Preparation Process of Salmonella Pullorum-Gallinarum Plate Agglutination Test Antigen

[0030] (1) Preparation of Primary Seeds

[0031] Open the freeze-dried CVCC79201 (C79-1 strain) and CVCC79201 (C79-7 strain) bacterial strains. After dissolving them with normal saline respectively, streak inoculate them on a common agar plate and culture at 37°C for 18 - 24 h to observe the bacterial morphology and purity. Select the smooth, moist, slightly raised, semi-transparent and round colonies on the common agar plate, inoculate several tubes of common agar slant medium, and culture at 37°C for 24 h. After pure inspection, serological detection, specificity inspection and biochemical property inspection, those meeting the requirements are used as primary seeds.

[0032] (2) Propagation of Secondary Seeds

[0033] Inoculate the common agar slant culture (primary seeds) into 500 ml of common broth medium and culture at 37°C for 12 h. After passing the pure inspection, it is used as secondary seeds.

[0034] (3) High-Density Fermentation Culture of Antigen

[0035] Prepare the dry powder of common broth medium in the fermenter according to the instructions and sterilize it under high pressure. Inoculate the secondary seeds at a ratio of 2% of the total volume of the fermentation broth and culture at 37°C. During fermentation, control the dissolved oxygen > 40%, control the pH value at 7.0 - 8.0 with ammonia water. After fermenting for 12 h, add 40% glucose at a ratio of 5% of the volume of the fermentation broth, and then add glucose once every 4 h. When the pH value begins to rise during fermentation, add formaldehyde with a final concentration of 0.2% to inactivate for 12 h, and harvest the bacterial liquid.

[0036] Result: At the end of fermentation, the OD of bacteria 600nm can reach 24, that is, the viable bacteria count before inactivation is about 2.7×10 10 CFU / ml.

[0037] (4) Bacterial Cell Treatment

[0038] Centrifuge the bacterial solution in step (3) at 8000 rpm for 20 min, collect the precipitate, resuspend it with PBS (0.01 mol / L, pH 7.0 - 7.2), place it in a high-pressure sterilizer at 121 °C for 2 h, centrifuge and collect the precipitate according to the above protocol, continue to wash it with PBS three times until the supernatant is clear, resuspend the bacterial cells with PBS containing 2% formaldehyde (0.01 mol / L, pH 7.0 - 7.2), filter the collected bacterial solution through eight layers of gauze, collect it in a large glass bottle, filter it through a No. 2 sulfuric acid filter, and adjust the concentration of the bacterial solution to 5×10 10 CFU / ml.

[0039] (5) Preparation of antigen

[0040] Dilute the bacterial suspensions of the standard Salmonella pullorum strain C79-1 and the variant Salmonella pullorum strain C79-7 into 1.0×10 10 CFU / ml, 1.2×10 10 CFU / ml, 1.5×10 10 CFU / ml, 1.7×10 10 CFU / ml, 2.0×10 10 CFU / ml, five concentrations, and perform plate agglutination tests with 50 μl of each concentration and an equal volume of the standard type / variant positive serum national standard product containing 0.5 IU respectively. The concentration of the bacterial solution at which no less than 50% agglutination occurs is used as the concentration for preparing the antigen.

[0041] Adjust the standard Salmonella pullorum strain C79-1 and the variant Salmonella pullorum strain C79-7 to the above-determined concentrations of each bacterial solution in 1000 ml. The final concentration of crystal violet is 3%, the final concentration of glycerol is 10%, the final concentration of Tween-20 is 1%, and make up to 1000 ml with PBS containing 1% formaldehyde (0.07 mol / L, pH 7.0 - 7.2). First, place crystal violet and PBS in a homogenizer and homogenize at 7000 rpm for 3 - 4 min, add glycerol and Tween-20, continue to homogenize for 4 min, finally add the bacterial solution, and homogenize at 2000 rpm for 2 min, then quantitatively dispense to prepare the antigen.

[0042] Results: The final concentration of the standard antigen is 1.5×10 10 CFU / ml, and the final concentration of the variant antigen is 1.2×10 10 CFU / ml.

[0043] After adding Tween-20 in the preparation of the antigen, the serum and antigen are easier to mix during use, and the result judgment is more accurate. Fifteen samples were detected, and the results showed that the judgment results were consistent, but it was easier to observe after adding Tween-20, saving the detection time.

[0044] The hyperlipidemic sera were detected using the antigen with Tween-20 added and the antigen without Tween-20 added respectively, and the results are as Figure 1 shown. The left side is the detection result of the serum without adding Tween-20, where the antigen and serum are unevenly mixed and coated. The right side is the detection result of the antigen prepared by the new process. The serum and antigen are easily mixed and coated, and the detection result is easier to observe.

[0045] II. Inspection of the prepared pullorum-typhoid salmonella plate agglutination test antigen for chickens

[0046] 1. Sterility test

[0047] It is carried out in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia".

[0048] 2. Titer determination

[0049] Using 50ul of the antigen respectively for plate agglutination test with equal volumes of the standard type and variant type positive serum national standards of 10IU / ml. When 50% agglutination appears within 2 minutes, it is preliminarily determined that the test is qualified.

[0050] 3. Specificity test

[0051] The antigen prepared by the process of the present invention (hereinafter referred to as: the antigen of this process), the antigen prepared in accordance with the "Regulations for Veterinary Biological Products of the People's Republic of China" in 2000 edition (hereinafter referred to as: the antigen of the original process) and the iELISA method were respectively used to detect 120 samples of grandparent stock chicken sera, pullorum-negative sera and chicken Escherichia coli positive sera collected clinically.

[0052] Table 1 Results of specificity test

[0053]

[0054] The results show that: 42 positive samples of pullorum-typhoid salmonella were detected by the antigen of the original process, 35 positive samples of pullorum-typhoid salmonella were detected by the antigen of this process, and 36 positive samples of pullorum-typhoid salmonella were detected by the iELISA method. The detection result of the antigen of this process is basically consistent with the iELISA detection result based on the O antigen; the detection results of the antigen of this process, the antigen of the original process and iELISA for pullorum-negative sera are all negative; the detection result of the antigen of this process for chicken Escherichia coli positive sera is negative, while the detection by the antigen of the original process is weakly positive. Figure 2 On the left side in the figure is the result of detecting Escherichia coli positive serum by the antigen of this process, which is judged to be negative. On the right side is the result of detecting Escherichia coli positive serum by the antigen of the original process, which is judged to be positive. It shows that when the antigen of the original process is used for detecting pullorum-typhoid salmonella, it will cross-react with Escherichia coli and show false positive. However, the antigen of this process is not interfered by Escherichia coli. The antigen prepared by this process has better specificity and is more accurate for pullorum purification detection.

[0055] III. Application and determination

[0056] This antigen is used for the detection of Salmonella pullorum and Salmonella gallinarum in chicken flocks in chicken farms. The detection objects are serum or freshly collected blood. Mix 30 μl of the antigen with 30 μl of the sample to be tested thoroughly, and determine the result within 2 minutes. Those with more than 50% agglutination are judged as positive. Figure 3 From left to right are ++++, +++, ++, +, -, respectively. Among them, ++++: large agglutination clumps appear, 100% of the antigen is agglutinated, and the bottom liquid is clear;

[0057] +++: obvious agglutination clumps appear, 75% - 99% of the antigen is agglutinated, and the bottom liquid is slightly turbid;

[0058] ++: significant fine agglutination particles appear, 50% - 74% of the antigen is agglutinated, and the bottom liquid is turbid;

[0059] +: a small amount of agglutination particles appear, 25% - 49% of the antigen is agglutinated, and the bottom liquid is turbid;

[0060] -: no agglutination particles.

[0061] IV. Comparison of high-density fermentation process, conventional fermentation and solid culture

[0062] 1. Solid culture

[0063] Inoculate the freeze-dried strains of Salmonella pullorum C79-1 and C79-7 into ordinary broth, incubate at 37 °C for 18 - 24 hours, then inoculate into ordinary agar plates, and incubate at 37 °C for 18 - 24 hours to obtain primary seeds; inoculate the primary seeds into ordinary agar small flat flasks respectively, incubate at 37 °C for 18 - 24 hours to obtain secondary seeds; make the secondary seeds into bacterial suspensions with normal saline respectively or directly use the secondary seeds to streak inoculate into thiosulfate agar flat flasks, and incubate at 37 °C for 44 - 48 hours.

[0064] 2. Conventional fermentation

[0065] After the strains of Salmonella pullorum C79-1 and C79-7 are resuscitated and propagated in TSB liquid medium, inoculate them into TSA solid plate medium, incubate at 37 °C for 20 h, then wash the bacterial lawn with Martin liquid medium, after enlarged culture, inoculate into a fermenter containing Martin broth medium for fermentation culture at a volume ratio of 5.0%; after fermentation culture at 37 °C for 18 h, harvest the bacterial liquid.

[0066] 3. High-density fermentation

[0067] Prepare the dry powder of ordinary broth medium in a fermenter according to the instructions and autoclave it. Inoculate the secondary seeds at a ratio of 2% of the total volume of the fermentation broth and culture at 37°C. During fermentation, control the dissolved oxygen > 40%, control the pH value at 7.0 - 8.0 with ammonia water. After 12 h of fermentation, add 40% glucose at a ratio of 5% of the volume of the fermentation broth, and then replenish glucose once every 4 h. When the pH value starts to rise during fermentation, add formaldehyde with a final concentration of 0.2% to inactivate for 12 h, and harvest the bacterial liquid.

[0068] 4. Comparison of Bacterial Yields under Different Processes

[0069] Calculated based on the yield of culturing strain C79 - 1 in 10000 mL of medium, the bacterial yields are shown in the following table (the cost of solid medium includes the dry powder medium, preparation and post - treatment of flat flasks, and the cost of liquid medium includes the dry powder of the medium, preparation and costs before and after tank preparation). The high - density fermentation culture process in this application is used for cell production. Glucose is added to supplement the carbon source required for bacterial metabolism, and ammonia water is used instead of NaOH to adjust the pH value, reducing the salt content of the medium. The antigen yield, that is, the antigen concentration, can reach 2.7×10 10 CFU / ml, and the yield is 4 times that of conventional fermentation and 2 times that of solid culture. The specific results are shown in Table 2.

[0070] Table 2 Comparison of Yields and Costs of Producing Antigens by Different Culture Methods

[0071]

[0072] The present invention improves the production process of the antigen for the poly - valent stained plate agglutination test for Pullorum disease and typhoid fever in chickens, reduces the production cost and pollution risk, and improves the specificity and sensitivity of diagnosis, making the diagnosis more accurate; no ethanol precipitation is required, reducing the cost and environmental protection pressure; adding tween - 20 increases the surface tension of the antigen and solves the problem of difficult uniform coating during use, providing a new and better diagnostic reagent for the purification of Pullorum disease.

[0073] The above - mentioned embodiments are only preferred embodiments cited to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A process for preparing a highly specific Salmonella pullorum plate agglutination antigen, characterized in that: The following steps are involved: (1) Preparation of primary seeds Standard Salmonella pullorum C79-1 strain and variant Salmonella pullorum C79-7 strain were inoculated on common agar plates for culture respectively; colonies on the agar plates were selected to be inoculated on common agar slant medium for culture to obtain primary seeds; the deposit numbers of C79-1 strain and C79-7 strain were CVCC79201 and CVCC79201 respectively; (2) Propagation of secondary seeds The first-level seeds are inoculated into ordinary broth medium to obtain the second-level seeds; (3) Antigen high-density fermentation culture The secondary seeds were fermented and cultured in a fermentation tank, the bacterial liquid was harvested, and formaldehyde with a final concentration of 0.2% was added to inactivate for 12 hours, and the bacterial liquid was harvested; (4) Bacteria treatment The bacterial solution harvested in step (3) was centrifuged at 8000 rpm for 20 min, the precipitate was collected, resuspended with PBS (0.01 mol / L, pH 7.0-7.2), placed in 121°C high pressure for 2 h, and centrifuged to collect the precipitate according to the above scheme, washed with PBS (0.01 mol / L, pH 7.0-7.2) until the supernatant was clear, and the bacterial solution was resuspended with PBS (0.01 mol / L, pH 7.0-7.2) containing 2% formaldehyde to adjust the bacterial solution concentration to 5×10 10 CFU / ml; (5) Preparation of Antigen The bacterial suspensions of standard Salmonella pullorum C79-1 strain and variant Salmonella pullorum C79-7 strain were diluted to different concentrations, and 50ul of each was used for plate agglutination test with an equal amount of standard / variant positive serum national standard containing 0.5IU. The bacterial suspension concentration that showed no less than 50% agglutination was used as the concentration for preparing the antigen.

2. A process for preparing a highly specific Salmonella pullorum plate agglutination antigen according to claim 1, characterized in that: During the fermentation in step (3), the pH value is controlled at 7.0-8.0 using aqueous ammonia.

3. A process for preparing a highly specific Salmonella pullorum plate agglutination antigen according to claim 1, characterized in that: In the step (3), after fermentation for 12 hours, 40% glucose is added at a ratio of 5% of the volume of the fermentation liquid, and then glucose is added once every 4 hours at a ratio of 5% of the volume of the fermentation liquid.

4. The process for preparing a highly specific Salmonella pullorum plate agglutination antigen according to claim 1, characterized in that: In the step (3), the secondary seeds are inoculated at a ratio of 2% of the total amount of the fermentation liquid and fermented in a fermenter containing a common broth medium at 37° C. During the fermentation, the dissolved oxygen is controlled to be greater than 40%, and the pH value is controlled at 7.0-8.0 with ammonia water. After fermentation for 12 hours, 40% glucose is added at a ratio of 5% of the volume of the fermentation liquid, and glucose is added once every 4 hours. When the pH value begins to rise during fermentation, formaldehyde with a final concentration of 0.2% is added for inactivation for 12 hours, and the bacterial liquid is harvested.

5. A process for preparing a highly specific Salmonella pullorum plate agglutination antigen according to any one of claims 1 to 4, characterized in that: The final concentration of crystal violet in the antigen prepared in step (5) is 3%, the final concentration of glycerol is 10%, and the final concentration of Tween-20 is 1%.

6. A process for preparing a highly specific Salmonella pullorum plate agglutination antigen according to claim 5, characterized in that: In the step (5), crystal violet and PBS are first placed in a homogenizer and homogenized at 7000 rpm for 3-4 minutes, glycerol and Tween-20 are added, and the homogenization is continued for 4 minutes, and finally the bacterial solution is added, homogenized at 2000 rpm for 2 minutes, and quantitatively packaged to prepare the antigen.

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