Serological diagnosis method for identifying sheep brucellosis wild virus infection and artificial immunity

Abstract

The invention belongs to the field of brucellosis detection, and relates to a serological diagnosis method for identifying wild virus infection and artificial immunity of sheep brucellosis, which comprises the following steps: respectively diluting a brucellosis test tube agglutination test antigen, positive serum and negative serum with a PBS (Phosphate Buffer Solution), and dissolving dithiothreitol in the PBS to prepare a DTT (Dithiothreitol) diluent; the method comprises the following steps: respectively adjusting dilution degrees of each part of detected serum in two test tubes to 1: 12.5 and 1: 25 by using DTT diluent, and adding a dilution antigen to enable the dilution degrees in the two serum tubes to be 1: 25 and 1: 50 in sequence; respectively making positive serum control, negative serum control and antigen control, preparing a turbidimetric tube, and judging according to the degree of agglutination reaction. The method is convenient to operate, low in detection cost and capable of simply, efficiently and effectively regionally distributing the wild virus infection antibody and the vaccine immune antibody of the brucellosis.

Description

Technical Field

[0001] The invention relates to the field of brucellosis detection, in particular to a serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity in sheep. Background Art

[0002] Brucellosis is a zoonotic infectious disease caused by Brucella infection. It can infect pigs, cattle, sheep and other livestock. Once an outbreak occurs, it will not only have a serious impact on the livestock industry, but also threaten people's health. According to the relevant requirements of brucellosis purification, farms other than breeding farms need to use immune purification to carry out disease purification. In actual production, it is difficult to determine whether the sheep are immune during the live sheep trading process, and the conventional brucellosis laboratory detection methods such as tiger red plate and test tube agglutination test cannot effectively distinguish between wild virus infection antibodies and vaccine immune antibodies. Therefore, it is difficult to judge wild virus infection and immune antibodies, which brings great trouble to production.

[0003] There are two main aspects to the detection of brucellosis. On the one hand, it is mainly based on etiology, which mainly involves isolating and culturing Brucella in the tissues, biological fluids, and excrement of the target animals. In addition, the isolation and culture of Brucella is also the gold standard for diagnosing Brucella. However, etiological diagnosis is limited by laboratory functions, equipment requirements, long time periods, and operating techniques, and effective test results cannot be made in a timely manner. At the same time, it is difficult for livestock farms to operate normally. On the other hand, serological diagnosis is also the main measure for domestic control and prevention of epidemics, and it is also the main method for diagnosing brucellosis. At present, serological diagnosis mainly uses RBT, SAT, CFT, and ELISA.

[0004] Given that the specific antibodies IgG and IgM produced after natural infection and vaccine immunization have quantitative differences and also phase differences. Studies have shown that after human and animal immunization, IgM antibodies are produced about 5 days later, reaching a peak after 2 weeks, while IgG antibodies reach a peak at about 4 weeks and then gradually decline. IgG and IgM antibodies appear a few days after humans and animals are infected with wild brucellosis, and IgM antibodies decline rapidly. The peak period of IgG antibodies can last for 270 days, and it can still be maintained at the diagnostic level after 1 year. Therefore, the presence of serum antibody IgG is still an important indicator of the chronic brucellosis infection process. The determination of specific antibodies has important clinical diagnostic significance and is also an important indicator to distinguish between immunization and natural infection processes. Since CFT (complement fixation reaction) mainly checks IgG antibodies, it is often used for differential diagnosis between vaccine immunization and natural infection. However, the CFT technique is complicated and cumbersome to operate, and it is difficult to promote and apply it at the grassroots level. The Riv (Rivanol) test used in the United States can precipitate and eliminate the reaction caused by nonspecific proteins, and can also be used to identify antibodies for vaccination. The test is simple, easy, and economical in the serological diagnosis of chronic brucellosis patients, and is especially suitable for grassroots quarantine. The FAO / WHO Brucellosis Expert Committee pointed out that the Riv test can replace the more cumbersome CFT. The study by Chen Naichang et al. showed that after the serum was treated with Rivanol, IgM and serum albumin were precipitated and removed, and most of the IgG-like antibodies were retained. The test mainly measured the agglutination activity of IgG-like antibodies. If the serum antibody titer does not decrease significantly before and after treatment, it means that the serum contains IgG-like antibodies. If the serum titer decreases significantly after treatment, it means that the serum contains a large amount of IgM, which may be caused by vaccination. This study has certain reference significance in the identification of groups with natural infection or vaccine immunity.

[0005] In summary, the current Brucella gene-deficient live vaccine (M5-90Δ26 strain) or Brucella live vaccine (M5 strain) can be used for differential diagnosis by gene detection, but there are problems such as long detection process, high detection cost, and inconvenient detection. Therefore, those skilled in the art are in urgent need of developing a simpler serological diagnosis method for distinguishing wild-type infection of sheep brucellosis from artificial immunity. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to propose a serological diagnosis method for distinguishing wild-type brucellosis infection from artificial immunity in sheep, aiming to utilize the sulfhydryl group in DTT to destroy the disulfide bond (SS) in the macroglobulin molecule under certain conditions, thereby dissociating the 19S macromolecular antibody globulin with immune activity into 6.5-7S small-molecule antibody globulin without immune activity, but under this condition, the 7S small-molecule antibody globulin with immune activity cannot be decomposed, thereby achieving the purpose of differential diagnosis of wild-type brucellosis infection in sheep and artificial immunity.

[0007] To achieve one of the above objects, the present invention is implemented according to the following technical solutions:

[0008] The serological diagnosis method for distinguishing wild-type brucellosis infection from artificial immunity in sheep includes the following steps:

[0009] S1. The antigen, positive serum and negative serum of the brucellosis tube agglutination test were diluted 1:20 with 0.01 mol / L PBS solution to obtain 1:20 times diluted antigen, 1:20 times diluted positive serum and 1:20 times diluted negative serum respectively;

[0010] S2, dissolving dithiothreitol in PBS solution to prepare a DTT dilution solution with a concentration of 0.005 mol / L;

[0011] S3, adjust the dilution of each serum to 1:12.5 and 1:25 in two test tubes respectively with DTT diluent to obtain diluted serum tubes;

[0012] S4, add 0.5 mL of 1:20 diluted antigen to each serum tube diluted in step S3, and shake evenly, so that the dilution ratios in the two serum tubes of each serum tested become 1:25 and 1:50 respectively;

[0013] S5. For each serum sample tested, a positive serum control, a negative serum control and an antigen control are performed, wherein:

[0014] Negative serum control: The dilution and antigen addition methods of negative serum are the same as the dilution and antigen addition methods of the tested serum in steps S3 and S4;

[0015] Positive serum control: The positive serum must be diluted to the original titer, and the method of adding antigen is the same as the method of adding antigen to the tested serum in step S4;

[0016] Antigen control: Take 0.5 ml of 1:20 diluted antigen, add 0.5 ml of DTT diluent, and observe whether the antigen has self-agglutination phenomenon;

[0017] S6. Prepare turbidity tubes and make judgments based on the degree of agglutination reaction.

[0018] Preferably, in the PBS solution in step S1, the concentration of disodium hydrogen phosphate is 8.1 mmol / L, the concentration of sodium dihydrogen phosphate is 1.9 mmol / L, and the concentration of sodium chloride is 145 mmol / L.

[0019] Preferably, the test tube is a small test tube of 8-10 ml.

[0020] Preferably, step S3 includes the following steps:

[0021] Step S31, adding DTT diluent to two test tubes respectively, wherein 1.15 ml of DTT diluent is added to the first test tube, and 0.5 ml of DTT diluent is added to the second test tube;

[0022] Step S32, take 0.1 ml of the serum to be tested, add it to the first test tube, mix it thoroughly, then take out 0.25 ml of the mixed solution in the first test tube and discard it. Then the dilution of the serum in the first test tube is 1:12.5;

[0023] Step S33, aspirate 0.5 mL of the mixed solution in the first test tube and add it to the second test tube. After fully mixing, aspirate 0.5 mL of the mixed solution in the second test tube and discard it. Then, the dilution of the serum in the second test tube is 1:25.

[0024] Preferably, in both step S32 and step S33, a pipette is used to suck the serum to be tested and the corresponding mixed solution, and the same pipette is used to suck the serum and the corresponding mixed solution.

[0025] Preferably, in step S32 and step S33, the mixture is thoroughly mixed by blowing with a straw for 3-4 times.

[0026] Preferably, in step S6, preparing the turbidimetric tube comprises the following steps:

[0027] Step S61, diluting the 1:20 times diluted antigen with an equal amount of DTT diluent to obtain a 1:40 times diluted antigen solution;

[0028] Step S62: Take 5 small test tubes of 8-10 ml to make turbidimetric tubes No. 1-5, where:

[0029] Add 1.00 ml of DTT diluent to turbidimetric tube No. 1, and the clarity is regarded as 100%, which is recorded as ++++;

[0030] Add 0.25 ml of 1:40 diluted antigen solution and 0.75 ml of DTT diluent to turbidimetric tube No. 2. The clarity is considered to be 75% and is recorded as +++.

[0031] Add 0.50 ml of 1:40 diluted antigen solution and 0.50 ml of DTT diluent to turbidimetric tube No. 3. The clarity is considered to be 50% and is recorded as ++.

[0032] Add 0.75 ml of 1:40 diluted antigen solution and 0.25 ml of DTT diluent to turbidimetric tube No. 4. The clarity is considered to be 25% and is recorded as +.

[0033] Add 1.00 ml of 1:40 diluted antigen solution into No. 5 turbidimetric tube, the clarity is regarded as 0% and recorded as -;

[0034] Preferably, step S6 also includes the following steps:

[0035] After fully shaking all the test tubes, place them in a 37-38°C incubator. After waiting for 22-24 hours, use a turbidity tube to check and record the results. The highest dilution that shows more than 50% agglutination is the agglutination titer of the serum being tested.

[0036] Preferably, the degree of agglutination reaction in step S6 is determined according to the following criteria:

[0037] Refer to the turbidity tube and read according to the clarity of the upper liquid of each test tube.

[0038] ++++ means that the bacteria are completely agglutinated, 100% sinking, and the upper liquid is 100% clear;

[0039] +++ means that the bacteria are almost completely agglutinated and the upper liquid is 75% clear;

[0040] ++ indicates that bacterial agglutination is significant and the liquid is 50% clear;

[0041] + indicates that the agglutinate has precipitated and the liquid is 25% clear;

[0042] -Indicates that there is no precipitation of agglutinate and the liquid is uniformly turbid.

[0043] Preferably, in step S6, serum with an agglutination titer of 1:50 is judged as positive, and serum with an agglutination titer of 1:25 is judged as suspicious. Livestock with suspicious reactions are re-examined after 3 to 4 weeks, and if they are still suspicious during the re-examination, they are judged as positive.

[0044] The working principle of the present invention is that the use of a thiol compound (0.2 mol / L) of an appropriate concentration can destroy the disulfide bond of the macromolecular globulin IgM, causing it to lose its immunological activity, while IgG is not affected. Based on this principle, the agglutination reaction after the serum is treated with DTT (dimercaptoethanol) can distinguish between animals with natural infection or artificial immunization of brucellosis. The sulfhydryl group in DTT is used to destroy the disulfide bond (SS) in the macroglobulin molecule under certain conditions, thereby dissociating the immunologically active 19S macromolecular antibody globulin into a non-immunologically active 6.5-7S small molecule antibody globulin, but under this condition, the immunologically active 7S small molecule antibody globulin cannot be decomposed, thereby achieving the purpose of differential diagnosis.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The invention is easy to operate and has low detection cost, and can simply and efficiently distinguish wild-type Brucellosis virus infection antibodies from vaccine immunity antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a comparison chart of the results of non-immune positive sheep test tube agglutination and DTT test tube agglutination in the examples of the present invention.

[0048] Figure 2 This is a comparison chart of the results of immunopositive sheep test tube agglutination and DTT test tube agglutination in the examples of the present invention.

[0049] Figure 3 This is a comparison chart of the results of immunonegative sheep test tube agglutination and DTT test tube agglutination in the examples of the present invention.

[0050] Figure 4 It is a comparison chart of positive serum in the embodiment of the present invention.

[0051] Figure 5 It is a comparison chart of negative serum in the embodiment of the present invention.

[0052] Figure 6 This is an antigen control diagram in the examples of the present invention.

[0053] Figure 7 This is the record of the red bengal plate agglutination reaction in the embodiment of the present invention. DETAILED DESCRIPTION

[0054] The present invention is further described below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0055] The raw materials used in the present invention are purchased from conventional channels or prepared by existing technical methods.

[0056] Example

[0057] The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity of sheep proposed in this embodiment comprises the following steps:

[0058] S1. The brucellosis test tube agglutination test antigen, positive serum and negative serum were diluted 1:20 with 0.01 mol / L PBS solution to obtain 1:20 times diluted antigen, 1:20 times diluted positive serum and 1:20 times diluted negative serum respectively; wherein the brucellosis test tube agglutination test antigen, positive serum and negative serum were all produced and supplied by Harbin Biological Pharmaceutical Factory. In the PBS solution, the concentration of disodium hydrogen phosphate was 8.1 mmol / L, the concentration of sodium dihydrogen phosphate was 1.9 mmol / L, and the concentration of sodium chloride was 145 mmol / L.

[0059] S2. Dissolve dithiothreitol in PBS solution to prepare a DTT dilution solution with a concentration of 0.005 mol / L; the molecular weight of DTT is 154.3.

[0060] S3. Use DTT diluent to adjust the dilution of each serum to 1:12.5 and 1:25 in two 8-10ml test tubes respectively to obtain diluted serum tubes; the serum to be tested is the serum obtained by blood collection and separation according to the conventional method, and the serum must be fresh, without obvious protein floccules, hemolysis and corruption smell.

[0061] S4, add 0.5 mL of 1:20 diluted antigen to each serum tube diluted in step S3, and shake evenly, so that the dilution ratios in the two serum tubes of each serum tested become 1:25 and 1:50 respectively;

[0062] S5. For each serum sample tested, a positive serum control, a negative serum control and an antigen control are performed, wherein:

[0063] Negative serum control: The dilution and antigen addition methods of negative serum are the same as the dilution and antigen addition methods of the tested serum in steps S3 and S4;

[0064] Positive serum control: The positive serum must be diluted to the original titer, and the method of adding antigen is the same as the method of adding antigen to the tested serum in step S4;

[0065] Antigen control: Take 0.5 ml of 1:20 diluted antigen, add 0.5 ml of DTT diluent, and observe whether the antigen has self-agglutination phenomenon;

[0066] S6. Prepare turbidity tubes and make judgments based on the degree of agglutination reaction.

[0067] Wherein, step S3 includes the following steps:

[0068] Step S31, adding DTT diluent to two test tubes respectively, wherein 1.15 ml of DTT diluent is added to the first test tube, and 0.5 ml of DTT diluent is added to the second test tube;

[0069] Step S32, take 0.1 ml of the serum to be tested, add it to the first test tube, mix it thoroughly (by blowing and sucking 3-4 times with a pipette), then take out 0.25 ml of the mixed solution in the first test tube and discard it, then the dilution of the serum in the first test tube is 1:12.5;

[0070] Step S33, aspirate 0.5 mL of the mixed solution in the first test tube and add it to the second test tube. After fully mixing (using a pipette to blow 3-4 times), aspirate 0.5 mL of the mixed solution in the second test tube and discard it. The serum dilution in the second test tube is 1:25.

[0071] In this embodiment, in both step S32 and step S33, a pipette is used to suck the serum to be tested and the corresponding mixed solution, and the same pipette is used to suck the serum and the corresponding mixed solution.

[0072] In step S6, preparing the turbidity tube comprises the following steps:

[0073] Step S61, diluting the 1:20 times diluted antigen with an equal amount of DTT diluent to obtain a 1:40 times diluted antigen solution;

[0074] Step S62: Take 5 small test tubes of 8-10 ml to make turbidimetric tubes No. 1-5, where:

[0075] Add 1.00 ml of DTT diluent to turbidimetric tube No. 1, and the clarity is regarded as 100%, which is recorded as ++++;

[0076] Add 0.25 ml of 1:40 diluted antigen solution and 0.75 ml of DTT diluent to turbidimetric tube No. 2. The clarity is considered to be 75% and is recorded as +++.

[0077] Add 0.50 ml of 1:40 diluted antigen solution and 0.50 ml of DTT diluent to turbidimetric tube No. 3. The clarity is considered to be 50% and is recorded as ++.

[0078] Add 0.75 ml of 1:40 diluted antigen solution and 0.25 ml of DTT diluent to turbidimetric tube No. 4. The clarity is considered to be 25% and is recorded as +.

[0079] Add 1.00 ml of 1:40 diluted antigen solution into No. 5 turbidimetric tube, the clarity is regarded as 0% and recorded as -;

[0080] In this embodiment, step S6 also includes the following steps:

[0081] After fully shaking all the test tubes, place them in a 37-38°C incubator. After waiting for 22-24 hours, use a turbidity tube to check and record the results. The highest dilution that shows more than 50% agglutination is the agglutination titer of the serum being tested.

[0082] The degree of agglutination reaction in step S6 is determined according to the following criteria:

[0083] Refer to the turbidity tube and read according to the clarity of the upper liquid of each test tube.

[0084] ++++ means that the bacteria are completely agglutinated, 100% sinking, and the upper liquid is 100% clear;

[0085] +++ means that the bacteria are almost completely agglutinated and the upper liquid is 75% clear;

[0086] ++ indicates that bacterial agglutination is significant and the liquid is 50% clear;

[0087] + indicates that the agglutinate has precipitated and the liquid is 25% clear;

[0088] -Indicates that there is no precipitation of agglutinate and the liquid is uniformly turbid.

[0089] Result determination: Serum agglutination titer of 1:50 is judged as positive, and that of 1:25 is judged as suspicious. Livestock with suspicious reactions are re-examined after 3 to 4 weeks, and if they are still suspicious during the re-examination, they are judged as positive.

[0090] Comparative experiment:

[0091] From May 25 to June 15, 2024, a period of 20 days, 30 non-immune negative samples, 30 immune positive samples, and 12 non-immune positive samples were collected. Figure 1-7 As shown, the red tiger plate agglutination test, tube agglutination test, and DTT tube agglutination test were used for comparison. The results were as follows: for non-immune negative samples, all three methods were negative. For 12 non-immune positive samples, the red tiger plate agglutination reaction results were positive, the tube agglutination results were positive, and the DTT tube agglutination test results were positive. For 30 immune positive samples, the red tiger plate agglutination test results were positive, the tube agglutination test results were positive, and the DTT tube agglutination test results were negative.

[0092] Through the above comparative experiments, it is fully verified that the present invention can effectively identify antibodies infected by wild brucellosis virus and antibodies immune to vaccine.

[0093] The working principle of the present invention is that the use of a thiol compound (0.2 mol / L) of an appropriate concentration can destroy the disulfide bond of the macromolecular globulin IgM, causing it to lose its immunological activity, while IgG is not affected. Based on this principle, the agglutination reaction after the serum is treated with DTT (dimercaptoethanol) can distinguish between animals with natural infection or artificial immunization of brucellosis. The sulfhydryl group in DTT is used to destroy the disulfide bond (SS) in the macroglobulin molecule under certain conditions, thereby dissociating the immunologically active 19S macromolecular antibody globulin into a non-immunologically active 6.5-7S small molecule antibody globulin, but under this condition, the immunologically active 7S small molecule antibody globulin cannot be decomposed, thereby achieving the purpose of differential diagnosis.

[0094] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A serological diagnostic method for distinguishing wild-type brucellosis infection from artificial immunity in sheep, characterized in that: The steps include: S1. The antigen, positive serum and negative serum of the brucellosis tube agglutination test were diluted 1:20 with 0.01 mol / L PBS solution to obtain 1:20 times diluted antigen, 1:20 times diluted positive serum and 1:20 times diluted negative serum respectively; S2, dissolving dithiothreitol in PBS solution to prepare a DTT dilution solution with a concentration of 0.005 mol / L; S3, adjust the dilution of each serum to 1:12.5 and 1:25 in two test tubes respectively with DTT diluent to obtain diluted serum tubes; S4, add 0.5 mL of 1:20 diluted antigen to each serum tube diluted in step S3, and shake evenly, so that the dilution ratios in the two serum tubes of each serum tested become 1:25 and 1:50 respectively; S5. For each serum sample tested, a positive serum control, a negative serum control and an antigen control are performed, wherein: Negative serum control: The dilution and antigen addition methods of negative serum are the same as the dilution and antigen addition methods of the tested serum in steps S3 and S4; Positive serum control: The positive serum must be diluted to the original titer, and the method of adding antigen is the same as the method of adding antigen to the tested serum in step S4; Antigen control: Take 0.5 ml of 1:20 diluted antigen, add 0.5 ml of DTT diluent, and observe whether the antigen has self-agglutination phenomenon; S6. Prepare turbidity tubes and make judgments based on the degree of agglutination reaction.

2. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity of sheep according to claim 1, characterized in that: In the PBS solution in step S1, the concentration of disodium hydrogen phosphate is 8.1 mmol / L, the concentration of sodium dihydrogen phosphate is 1.9 mmol / L, and the concentration of sodium chloride is 145 mmol / L.

3. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity of sheep according to claim 1, characterized in that: The test tube is a small test tube of 8-10 ml.

4. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity of sheep according to claim 1, characterized in that: Step S3 includes the following steps: Step S31, adding DTT diluent to two test tubes respectively, wherein 1.15 ml of DTT diluent is added to the first test tube, and 0.5 ml of DTT diluent is added to the second test tube; Step S32, take 0.1 ml of the serum to be tested, add it to the first test tube, mix it thoroughly, then take out 0.25 ml of the mixed solution in the first test tube and discard it. Then the dilution of the serum in the first test tube is 1:12.5; Step S33, aspirate 0.5 mL of the mixed solution in the first test tube and add it to the second test tube. After fully mixing, aspirate 0.5 mL of the mixed solution in the second test tube and discard it. Then, the dilution of the serum in the second test tube is 1:

25.

5. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity of sheep according to claim 4, characterized in that: In both step S32 and step S33, a pipette is used to suck the serum to be tested and the corresponding mixed solution, and the same pipette is used to suck the serum and the corresponding mixed solution.

6. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity in sheep according to claim 5, characterized in that: In both step S32 and step S33, the mixture is mixed thoroughly and evenly by blowing with a straw 3-4 times.

7. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity in sheep according to claim 1, characterized in that: In step S6, preparing the turbidity tube comprises the following steps: Step S61, diluting the 1:20 times diluted antigen with an equal amount of DTT diluent to obtain a 1:40 times diluted antigen solution; Step S62: Take 5 small test tubes of 8-10 ml to make turbidimetric tubes No. 1-5, where: Add 1.00 ml of DTT diluent to turbidimetric tube No. 1, and the clarity is regarded as 100%, which is recorded as ++++; Add 0.25 ml of 1:40 diluted antigen solution and 0.75 ml of DTT diluent to turbidimetric tube No.

2. The clarity is considered to be 75% and is recorded as +++. Add 0.50 ml of 1:40 diluted antigen solution and 0.50 ml of DTT diluent to turbidimetric tube No.

3. The clarity is considered to be 50% and is recorded as ++. Add 0.75 ml of 1:40 diluted antigen solution and 0.25 ml of DTT diluent to turbidimetric tube No.

4. The clarity is considered to be 25% and is recorded as +. Add 1.00 ml of 1:40 diluted antigen solution into No. 5 turbidimetric tube, the clarity is regarded as 0% and recorded as -.

8. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity in sheep according to claim 7, characterized in that: Step S6 also includes the following steps: After fully shaking all the test tubes, place them in a 37-38°C incubator. After waiting for 22-24 hours, use a turbidity tube to check and record the results. The highest dilution that shows more than 50% agglutination is the agglutination titer of the serum being tested.

9. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity in sheep according to claim 8, characterized in that: The degree of agglutination reaction in step S6 is determined according to the following criteria: Refer to the turbidity tube and read according to the clarity of the upper liquid of each test tube. ++++ means that the bacteria are completely agglutinated, 100% sinking, and the upper liquid is 100% clear; +++ means that the bacteria are almost completely agglutinated and the upper liquid is 75% clear; ++ indicates that bacterial agglutination is significant and the liquid is 50% clear; + indicates that the agglutinate has precipitated and the liquid is 25% clear; -Indicates that there is no precipitation of agglutinate and the liquid is uniformly turbid.

10. The serological diagnosis method for distinguishing wild-type brucellosis infection and artificial immunity in sheep according to claim 9, characterized in that: In step S6, serum agglutination titer of 1:50 is judged as positive, and that of 1:25 is judged as suspicious. Livestock with suspicious reactions are re-examined after 3 to 4 weeks, and if they are still suspicious during the re-examination, they are judged as positive.

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