A serological method for differentiating brucella a19-delta virb12 vaccine-immunized animals from infected animals
The NH-AGID method was used to distinguish between animals immunized with Brucella A19-△VirB12 vaccine and infected animals, solving the problem of identification that cannot be solved in existing technologies and achieving precise control and eradication of brucellosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-22
- Publication Date
- 2026-03-31
AI Technical Summary
Current technology cannot effectively distinguish between animals immunized with Brucella A19-△VirB12 vaccine and infected animals, which increases the difficulty of Brucella prevention and control and affects the Brucella eradication process.
Serological detection was performed using the NH-AGID method. Brucella hapten agar diffusion test plates were used, and S-LPS and NH mixed antigen and standard serum were added. Combined with incubation and precipitation line analysis, the vaccine-immunized animals and infected animals were distinguished.
It enables accurate differential diagnosis between animals immunized with A19-△VirB12 vaccine and infected animals, effectively blocking the source of brucellosis infection and supporting precise prevention and control of brucellosis.
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Figure CN116183902B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of veterinary vaccine detection technology, and in particular to a serological method for differentiating between animals immunized with Brucella A19-△VirB12 vaccine and infected animals. Background Technology
[0002] Brucellosis is a serious zoonotic infectious disease caused by infection with bacteria of the genus Brucella (Brucella spp.), posing a significant threat to livestock production and human public health. It is prevalent worldwide. Preventing and eradicating brucellosis has become a primary task in building national and regional public health security defense systems. Carrier animals are the main source of infection for other animals and humans. Therefore, curbing the rising trend of brucellosis in livestock at its source is a crucial prerequisite for controlling human brucellosis.
[0003] In brucellosis-endemic areas, a control strategy primarily based on vaccination, supplemented by quarantine and culling, is adopted. The brucellosis vaccine is a live attenuated vaccine, posing a potential risk of infection in both humans and animals. In brucellosis-immunized areas, the sources of infection mainly include two aspects: naturally infected animals and vaccine-infected animals. For example, in areas immunized with the A19-△VirB12 strain of Brucella vaccine, after cattle were vaccinated with the A19-△VirB12 vaccine according to the vaccine instructions, approximately 80% of the animals showed detectable A19-△VirB12 vaccine antibodies within 30 days, while approximately 85% of the animals showed undetectable antibody levels after 180 days. Antibodies in 15% of the animals can persist in the body for more than one year, severely interfering with the legally mandated serological testing for brucellosis twice a year.
[0004] Due to individual animal variability, the duration of antibody retention in the animal body varies. Brucellosis vaccine is a live attenuated vaccine, which is pathogenic to both humans and animals. Therefore, animals with persistently positive antibodies also possess vaccine-prevented antibodies. These animals can shed the bacteria through urine, feces, or other means, posing infection and disease risks to the ecological environment and livestock farmers. The indirect enzyme-linked immunosorbent assay (ELISA) established according to the Xinjiang local standard (DB 65 / T 4495—2022) has solved the problem of differentiating naturally infected brucellosis animals from those immunized with the A19-ΔVirB12 vaccine. However, there is currently no serological method to identify brucellosis A19-ΔVirB12 vaccine-infected animals, thus delaying the eradication and purification process of brucellosis in animals.
[0005] Given the current pressure and status of brucellosis prevention and control in livestock, establishing a serological method to distinguish between animals immunized with Brucella A19-ΔVirB12 vaccine and infected animals is of great practical significance. Summary of the Invention
[0006] The purpose of this invention is to provide a serological method for distinguishing between animals immunized with Brucella A19-ΔVirB12 vaccine and infected animals, which solves the problem of being unable to distinguish between animals immunized with Brucella A19-ΔVirB12 vaccine and animals infected with the vaccine, thus helping to achieve precise prevention and control of brucellosis in animals and accelerating the process of brucellosis prevention and control.
[0007] To achieve the above objectives, the present invention provides the following solution:
[0008] This invention provides a serological detection method for bovine brucellosis A19-ΔVirB12 vaccine, including a step of antibody identification of the serum using the NH-AGID method.
[0009] Furthermore, the NH-AGID method is performed using Brucella hapten agar diffusion test plates; the Brucella hapten agar diffusion test plates have an antigen well in the center, six sample wells on the side, and an NH antigen-antibody complex precipitation line.
[0010] Furthermore, the serological testing method specifically includes the following steps:
[0011] (1) Add S-LPS and NH mixed antigen to the central antigen well of the Brucella hapten agar diffusion test plate;
[0012] (2) Add brucellosis standard positive serum to one of the sample wells on the side of the Brucella hapten agar diffusion test plate;
[0013] (3) Add brucellosis standard negative serum to one of the sample wells on the side of the Brucella hapten agar diffusion test plate;
[0014] (4) Add the serum sample to be tested to the remaining sample wells on the other side of the Brucella hapten agar diffusion test plate;
[0015] (5) After the liquid in the sample well is absorbed, invert the agar plate into the humidified box and incubate for 16h to 48h.
[0016] Furthermore, the determination method is as follows: if no NH antigen-antibody complex precipitation line appears during the test, it is an animal immunized with A19-ΔVirB12 vaccine; if an NH antigen-antibody complex precipitation line appears, it is an animal infected with Brucella.
[0017] Furthermore, the amount of S-LPS, the NH mixed antigen, the brucellosis standard positive serum, the brucellosis standard negative serum, and the serum of the sample to be tested added is 15 μL.
[0018] Furthermore, in step (5), the incubation temperature is 25°C.
[0019] The present invention discloses the following technical effects:
[0020] This invention provides a serological method for differentiating between animals vaccinated with Brucella A19-ΔVirB12 vaccine and infected animals. The NH-AGID serological method can differentiate between A19-ΔVirB12 vaccine immune antibodies and infection antibodies, which helps in identifying brucellosis vaccine-infected animals. The application of the NH-AGID method fully improves the animal brucellosis A19-ΔVirB12 vaccine prevention and control technology system, and supplements the methods for differentiating between brucellosis-infected animals and vaccine-vaccinated animals in production. This method fundamentally solves the problem of comprehensively and promptly eliminating brucellosis sources in livestock herds, effectively blocking the spread and diffusion of brucellosis outbreaks, and providing technical support for the precise prevention and control of bovine brucellosis. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 A schematic diagram of the Brucella NH-agar diffusion test; where "+" represents the standard positive serum control well; "-" represents the standard negative serum control well; "Ag" represents the wells where S-LPS and NH antigen are added; "1-4" represent the wells where the sample to be tested is added, where "well 1" is Brucella antibody negative, "well 2" and "well 3" are Brucella infection antibody positive, and "well 4" is Brucella vaccine immune antibody positive. Detailed Implementation
[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0028] Example 1
[0029] 1. Select 35 calves aged 3-6 months that are brucellosis negative and administer A19-ΔVirB12 vaccine subcutaneously at the standard dose according to the vaccine instructions.
[0030] 2. Whole blood was collected on days 15, 30, 60, 90, 120, 150, and 180 post-immunization, and serum was separated for later use. Bovine serum samples with positive RBT serological results were selected according to the Rose Bengal Plate Test (RBT) method in the national standard GB18646-2018.
[0031] 3. RBT test results showed that the brucellosis antibody positivity rate in calves vaccinated with A19-ΔVirB12 reached 88.6% at 30 days and 11.4% at 180 days. This indicates that nearly 90% of animals lost their A19-ΔVirB12 immune antibodies 6 months after immunization.
[0032] 4. Collect 165 RBT serologically positive bovine serum samples and detect them using the NH-AGID method.
[0033] The specific steps of the NH-AGID method are as follows:
[0034] 4.1 Place the Brucella hapten agar diffusion test plate at room temperature (23°C). A schematic diagram of this plate is shown below. Figure 1 As shown;
[0035] 4.2 Add 15 μL of a mixture of S-LPS and NH antigen to the center antigen well (Ag) of the agar plate;
[0036] 4.3 Add 15 μL of brucellosis standard positive serum to one well on the side of an agar plate and mark it with a "+" sign;
[0037] 4.4 Add 15 μL of brucellosis standard negative serum to the side of the agar plate near the positive sample well and mark it with "-";
[0038] 4.5 Add 15 μL of the positive bovine serum sample from step 4 to the remaining 4 sample wells on the side of the agar plate, and record the numbers;
[0039] 4.6 After the liquid in the sample well has been aspirated, invert the agar plate into the humidified box and incubate it in a constant temperature incubator at 25°C.
[0040] 5. Determination of test results
[0041] 5.1 Judgment Method
[0042] After incubation for 48 hours (16-48 hours is also acceptable), remove the agar plates and read the results using an oblique light source. Using black paper as a base, observe the presence, location, and number of precipitation lines between the antigen wells in the center of the plate and the serum wells to be tested from different refractive angles.
[0043] 5.2 Conditions for the Experiment to be Successful
[0044] The test is valid when an LPS precipitation line and an NH precipitation line appear between the positive control serum well (+) and the central antigen well (Ag) (as shown in Appendix A for the positive control serum wells), while no precipitation line appears between the negative control serum well (-) and the central antigen well (Ag) (as shown in Appendix A for the negative control serum wells); otherwise, the test should be repeated.
[0045] 6. Result Determination
[0046] 6.1 When two precipitation lines of LPS and NH or only one precipitation line of NH appear between the tested serum well and the central antigen well (Ag), the tested serum is determined to be positive for Brucella infection antibody, indicating that the animal is a Brucella infection positive animal and that Brucella in the animal's body is in the active or shed bacteria phase.
[0047] 6.2 When only one LPS precipitation line appears between the tested serum well and the central antigen well (Ag), the tested serum is determined to be positive for Brucella vaccine immune antibodies, indicating that the animal is a Brucella-immunized animal.
[0048] If no precipitation line appears between the tested serum well and the central antigen well (Ag), the tested serum is determined to be Brucella antibody negative, indicating that the animal is a Brucella negative animal.
[0049] 7. Experimental results showed that within 15 to 150 days post-immunization with the A19-ΔVirB12 vaccine, the NH-AGID method did not produce NH antigen-antibody complex precipitation lines for A19-ΔVirB12 vaccine antibodies, indicating a zero NH-AGID positivity rate. This result demonstrates that the NH-AGID method is negative for A19-ΔVirB12 vaccine antibodies and can differentiate between A19-ΔVirB12 vaccine antibodies and infection antibodies. At 180 days post-immunization, NH antigen-antibody complex precipitation lines appeared in the immune serum of two cattle, suggesting that these two cattle were diagnosed as vaccine-infected animals and were in the vaccine shedding period. These animals should be promptly isolated or removed from the herd.
[0050] 8. The above experimental data demonstrate that the positive rate of the NH-AGID method is 5.71%. The specificity of NH antigen for detecting A19-ΔVirB12 vaccine immune antibodies reaches 94.29%. The NH-AGID method can distinguish between animals immunized with A19-ΔVirB12 vaccine and those infected with vaccine antibodies; the results are shown in Table 1.
[0051] Table 1. Results of subcutaneous brucellosis A19-ΔVirB12 vaccination in calves
[0052]
[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. Use of S-LPS and NH mixed antigens for the preparation of a reagent for differentiating between A19-ΔVirB12 vaccine immune antibodies and infection antibodies, characterized in that, The NH-AGID method is used to distinguish A19-ΔVirB12 vaccine immunized animals and vaccine infected animals.
2. Use according to claim 1, characterized in that, The NH-AGID method uses a Brucella hapten agar diffusion test plate; the plate has an antigen hole in the center, six sample holes on the side, and an NH antigen antibody complex precipitation line.
3. Use according to claim 2, characterized in that, The NH-AGID method specifically includes the following steps: (1) Add S-LPS and NH mixed antigen to the center antigen hole of the Brucella hapten agar diffusion test plate; (2) Add brucellosis standard positive serum to one of the side sample holes of the Brucella hapten agar diffusion test plate; (3) Add brucellosis standard negative serum to one of the side sample holes of the Brucella hapten agar diffusion test plate; (4) Add the sample serum to be tested to the remaining side sample holes of the Brucella hapten agar diffusion test plate; (5) After the liquid in the sample hole is absorbed, the agar plate is inverted in a wet box and incubated for 16-48 hours.
4. Use according to claim 2, characterized in that, If the NH antigen antibody complex precipitation line does not appear during detection, it is an antibody produced by A19-ΔVirB12 vaccine immunized animals, and if the NH antigen antibody complex precipitation line appears, it is an antibody produced by animals infected with A19-ΔVirB12 vaccine.
5. Use according to claim 3, characterized in that, The addition amount of the S-LPS, the NH mixed antigen, the brucellosis standard positive serum, the brucellosis standard negative serum, and the sample serum to be tested is 15 μL.
6. Use according to claim 3, characterized in that, In step (5), the temperature of the incubation is 25°C.