Attenuated strains of Brucella brucellosis and their applications, vaccines, antigens and reagent kits

Abstract

This invention relates to the field of veterinary biological products, specifically to a live attenuated strain of Brucella brucellosis RB39, its applications, vaccines, antigens, and kits. The live attenuated Brucella brucellosis RB39 of this invention can be used to prepare a highly safe vaccine that can be injected into pregnant animals and provides good immunoprotection. Furthermore, this vaccine is suitable for immunization of multiple animal species. In addition, based on the live attenuated Brucella brucellosis RB39 of this invention, an antigen can be prepared to identify Brucella brucellosis antibodies, effectively distinguishing between vaccine immunization and natural infection, providing a scientific basis for brucellosis prevention and control.

Description

Technical Field

[0001] This invention relates to the field of veterinary products, and more particularly to a crude Brucella attenuated strain and its applications, vaccines, antigens and reagent kits. Background Technology

[0002] Brucellosis, caused by Brucella bacteria, is a zoonotic disease characterized by abortion and fever, seriously threatening the lives and health of humans and various animals. Human infection primarily results from contact with infected animals and their contaminated animal products. Therefore, immunization of animals with Brucella vaccines in high-prevalence areas is a globally recognized effective measure for controlling brucellosis. Most current veterinary brucellosis vaccines are made from attenuated strains of Brucella glabrata. Their biggest drawback is their relatively high virulence, making them unsuitable for immunizing pregnant animals. Furthermore, the antibodies produced after vaccination cannot be distinguished from those produced by clinically infected, virulent strains of Brucella glabrata. This leads to animals being mistakenly identified as sources of infection and culled, or positive animals within a group being mistakenly identified as immunized and not culled, resulting in the continued presence of the infectious source and hindering disease eradication. Currently, the commonly used method is to use genetically engineered marker vaccines to differentiate between clinical infection and vaccine immunization, but this is costly and complex. These shortcomings limit the use and promotion of brucellosis vaccines.

[0003] Attenuated strains of Brucella brucellosis (roughly brucea) are safer than attenuated strains of Brucella sclerotium (smoothly brucea). After injection into animals, they stimulate the production of rough-type antibodies, which can effectively distinguish them from the smooth-type antibodies produced by clinically infected virulent strains of Brucella. This is a major direction in the research of novel brucellosis vaccines. Currently, the only widely approved and used rough-type vaccine strain globally is RB51. This vaccine was obtained by American scientists in the 1990s through rifampicin mutagenesis and has good immunogenicity. It has been used in the United States and several Latin American countries. However, because rifampicin is an effective antibiotic for treating brucellosis in humans, and because this vaccine is derived from a virulent strain, although its virulence is somewhat reduced, injection into pregnant animals can still cause adverse reactions such as abortion. Therefore, the safety of this vaccine remains controversial. Furthermore, existing rough-type vaccines are only applicable to specific animal species and cannot comprehensively cover all susceptible animals, limiting their widespread application. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a crude attenuated strain of Brucella that is highly safe, does not cause adverse reactions such as miscarriage, is applicable to a variety of animals, and can stimulate the body to produce specific antibodies, making it easy to distinguish between natural infection and vaccine immunization, thereby effectively controlling the spread of brucellosis and expanding the application scope of vaccines.

[0005] Another technical problem to be solved by the present invention is to provide an application of a rough, attenuated strain of Brucella in the preparation of Brucella vaccines or in the preparation of antigens for detecting Brucella antibodies.

[0006] Another technical problem that this invention aims to solve is to provide a Brucella vaccine that is highly safe, can be injected into pregnant animals for immunization, and is applicable to multiple animal species.

[0007] The technical problem that this invention also needs to solve is to provide an antigen that is highly specific and can effectively distinguish between natural infection and vaccine immunization.

[0008] Another technical problem that this invention aims to solve is to provide a reagent kit that can effectively distinguish between natural infection and vaccine immunization.

[0009] To address the aforementioned technical problems, as a first aspect of this invention, the present invention provides a rough type, B. abortus, attenuated strain RB39, which was deposited on February 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 46377. Its biological classification is attenuated strain of B. abortus. The address of the CGMCC is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Specifically, strain RB39 is a stable, rough-type attenuated Brucella strain obtained by passaged smooth-type Brucella a19 (CVCC 70202) for 39 generations in TSB medium at pH 5.5, followed by 65 generations in TSB medium at pH 4.5, and finally 35 generations in mice. Its colonies were identified as 100% rough-type by crystal violet staining and identified as Brucella abortus by AMOS-PCR. This strain RB39 not only has high safety but also significant immunogenicity, making it suitable for various animals such as cattle, sheep, and pigs. It effectively stimulates the production of specific antibodies, facilitating the differentiation between natural infection and vaccine-immunized status, and significantly improving the control of brucellosis.

[0011] As a second aspect of this invention, the application of the aforementioned crude Brucella attenuated strain RB39 in the preparation of a Brucella vaccine is disclosed. This vaccine exhibits high safety and strong immunogenicity; when used in pregnant animals, it does not induce adverse reactions such as miscarriage, ensuring the safety of both mother and fetus. Furthermore, this vaccine is suitable for multiple animal species (including but not limited to mice, guinea pigs, cattle, goats, sheep, and pigs), significantly enhancing the protective effect of the vaccine and effectively controlling the spread of brucellosis.

[0012] As a third aspect of the present invention, the present invention provides a Brucella vaccine comprising the aforementioned crude Brucella attenuated strain RB39. Specifically, the Brucella vaccine can be prepared by inactivating the aforementioned crude Brucella attenuated strain RB39 and adding excipients. The excipients of the Brucella vaccine include, but are not limited to, adjuvants, stabilizers, preservatives, diluents, and antioxidants.

[0013] Preferably, in some embodiments, the vaccine includes a lyophilization protectant, which may include, but is not limited to, PEG, gelatin, lactose, glycine, etc. The lyophilization protectant can prevent damage to the activity of the bacterial strain during the lyophilization process, ensuring vaccine stability and further improving the actual application effect of the vaccine.

[0014] Specifically, using the Brucella vaccine of this invention, cattle are given one dose (8.0 × 10⁻⁶). 10 CFU ~ 1.6 × 10 11 CFU), sheep at 1 / 4 head (2.0 x 10) 10 CFU ~ 4.0 × 10 10 Subcutaneous injection of CFU (carnitine hydroxybenzoate) into pregnant animals did not cause abortion.

[0015] As a fourth aspect of the present invention, the present invention also provides the application of the above-mentioned crude Brucella attenuated strain RB39 in the preparation of an antigen for detecting Brucella antibodies, wherein the Brucella antibodies are produced after animals are immunized with the Brucella vaccine as described above; through the detection, vaccine immunization and natural infection can be distinguished, improving diagnostic accuracy, assisting in the precise prevention and control of brucellosis, and ensuring the healthy development of animal husbandry.

[0016] Specifically, the detection is a serological test, which is simple and efficient. More specifically, it can be the RBT method, SAT method, CFT method, or ELISA method, but is not limited to these. More preferably, the detection is the RBT method, which is simple to operate, can be quickly completed under field conditions, and provides accurate and reliable results. It is suitable for large-scale screening and can quickly and effectively distinguish between natural infection and vaccine immunization, providing a scientific basis for the formulation of brucellosis control strategies.

[0017] As a fifth aspect of the present invention, the present invention also provides an antigen for detecting Brucella antibodies produced in animals immunized with the above-mentioned Brucella vaccine, which is obtained by culturing and inactivating the above-mentioned attenuated Brucella strain RB39.

[0018] Specifically, depending on the detection method, the antigens mentioned above require further processing. For example, when using the RBT method, the inactivated antigen needs to be stained; when using the CFT method, the antigen needs to be broken down, LPS extracted, and purified; when using the ELISA method, the antigen needs to be broken down, LPS extracted, and coated. It should be noted that those skilled in the art can optimize the antigens according to the specific detection method to ensure the accuracy and reliability of the detection results.

[0019] Preferably, in some embodiments, the antigen is used for the RBT method to detect Brucella antibodies, and its preparation method includes:

[0020] The above-mentioned attenuated Brucella strain RB39 was prepared into a bacterial solution using physiological saline and then inactivated.

[0021] Add red cinnabar dye to the inactivated bacterial solution at a volume ratio of 0.5:100 to obtain stained bacterial cells;

[0022] The stained bacterial cells were reselected to the preset concentration using Tris buffer to obtain the final product.

[0023] More preferably, the above-mentioned attenuated Brucella brucellosis strain RB39 was prepared into a concentrated bacterial suspension using physiological saline. After inactivation, 4% red erythromycin was added at a volume ratio of 0.5% for staining. The stained bacterial cells were resuspended in Tris buffer at a certain ratio, and then diluted 1:3, 1:4, 1:5, and 1:6 times with Tris buffer. 30 μL of each of the diluted solutions were mixed with 10 IU / mL and 5 IU / mL Brucella brucellosis strain RB39 positive sera, and the results were observed within 4 minutes. The antigen dilution that resulted in a "+" reaction at 10 IU / mL and a "-" reaction at 5 IU / mL was the antigen concentration used.

[0024] As a sixth aspect of the invention, the invention also provides a kit for detecting Brucella antibodies, comprising the antigens described above.

[0025] Implementing this invention has the following beneficial effects:

[0026] 1. This invention develops a crude Brucella attenuated strain RB39 and its vaccine, which can be subcutaneously injected into pregnant animals without causing abortion, overcoming the defect of existing brucellosis vaccines that cannot be injected into pregnant animals; in addition, this vaccine is applicable to the immunization of multiple species of animals. Specifically, after immunizing mice, guinea pigs, cattle, goats and sheep with a certain dose of the vaccine of this invention, it can provide no less than 70% protection.

[0027] 2. This invention develops an antigen for the differential diagnosis of RB39 vaccine immunization and natural infection. Based on this antigen, antibodies produced by vaccine immunization and antibodies produced by natural infection can be quickly distinguished by serological detection methods. This solves the technical problem that it is impossible to perform differential diagnosis between vaccine immunization and natural infection after immunizing animals with existing brucellosis vaccines, and provides a scientific basis for brucellosis prevention and control. Attached Figure Description

[0028] Figure 1 This is an AMOS-PCR electrophoresis image of Brucella rubella strain RB39;

[0029] Figure 2 This is a genome circle diagram of Brucella RB39 strain;

[0030] Figure 3 This is a graph showing the change in antibody levels in cattle after immunization with Brucella brucellosis strain RB39;

[0031] Figure 4 This is a graph showing the changes in antibody levels in sheep after immunization with Brucella RB39 strain. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. It should be noted that, unless otherwise specified, the reagents used in the examples can be purchased commercially. Example: Induction and domestication of RB39 strain

[0033] 1.1 Induction under pH 5.5 acidic conditions: Smooth Brucella strain A19 (CVCC 70202, purchased from commercial Brucella live vaccine A19) was continuously passaged on TSB medium at pH 5.5. Every three passages, the strain was plated onto TSA plates using the limiting dilution method and incubated at 37°C for 3 days. After single colonies grew, the colonies on the plates were stained with crystal violet. Colonies with the most pronounced crystal violet staining (rough colonies stained, smooth colonies did not stain) were selected for further passage. If no crystal violet-positive colonies appeared, they were randomly selected. This method was used for a total of 39 passages, resulting in a crystal violet staining rate of over 20% for rough colonies.

[0034] 1.2 Induction under pH 4.5 acidic conditions: The rough colonies selected in 1.1 were passaged on TSB medium at pH 4.5. Every 5 passages, the colonies were plated onto TSA plates using the limiting dilution method and incubated at 37°C for 3 days. After single colonies grew, the colonies on the plates were stained with crystal violet. The colonies with the most obvious crystal violet staining were selected for further passage. After 65 consecutive passages, the positive rate of rough colonies stained with crystal violet reached over 90%.

[0035] 1.3 In vivo passage and domestication in mice: The rough-type colonies selected in 1.2 were processed at a rate of 1.0 × 10⁻⁶. 8 One BALB / c mouse was subcutaneously injected into the groin at a dose of CFU / mouse. Seven days later, the mice were euthanized, and the spleen tissue was homogenized and spread onto TSA plates using the limiting dilution method. The plates were incubated at 37°C for 3 days until single colonies appeared. The colonies on the plates were then stained with crystal violet. Colonies with the most pronounced crystal violet staining were selected for further passage. After 25 consecutive passages, the positive rate of coarse-type colonies stained with crystal violet reached 100%. Subsequently, the cells were passaged in mice for another 10 passages (a total of 139 passages). The positive rate of coarse-type colonies isolated in all 10 passages was 100%. The genetically stable coarse-type Brucella obtained was named strain RB39.

[0036] Example 2: Identification of Brucella RB39 strain

[0037] 2.1 Morphological and biochemical characteristics: Gram-negative cocci, without spores or capsules. Gram-negative; positive for hydrogen sulfide.

[0038] 2.2 Culture Characteristics: It grows well on tryptic soybean agar (TSA) medium. When streaked onto the above medium plates and incubated at 37°C for 3 days, 100% of the colonies are coarse-type. In liquid media such as TSB, it grows uniformly, but the medium is turbid and opaque.

[0039] 2.3 Variation test: Consistent with the characteristics of a rough colony. 100% of colonies were stained using the crystal violet staining method; precipitation was observed in the heat agglutination test; agglutination was observed in the acridine yellow agglutination test.

[0040] 2.4 Serological characteristics: The antigen prepared from RB39 culture did not agglutinate with positive serum of Brucella smootha, but agglutinated with positive serum of Brucella rougha.

[0041] 2.5 PCR identification: Identification was performed using the AMOS-PCR method, and the following four primers were synthesized (see Table 1).

[0042] Table 1 AMOS-PCR Primers

[0043]

[0044] Genomic DNA from RB39 was extracted using a commercial bacterial genomic extraction kit and used as a template. In a 20 μL reaction system, 10 μL of 2×PCR mix, 1 μL each of the four primers, 1 μL of template DNA, and 5 μL of DEPC-treated water were added. After mixing, PCR was performed. Simultaneously, a Brucella abortus A19 primer set was prepared (corresponding primer F). abortus Brucella medullaris M5 (CVCC18, purchased from commercial Brucella live vaccine M5, corresponding primer F)melitensis Brucella suis S2 (CVCC 70502, purchased from commercial Brucella live vaccine S2, corresponding primer F) suis The study included a nucleic acid control group and a blank control group without added nucleic acid. The PCR reaction program was as follows: 95℃ for 5 min, followed by 35 cycles of 95℃ for 30 s, 54℃ for 30 s, and 72℃ for 1 min; a final extension at 72℃ for 10 min. The amplified products were identified by electrophoresis on a 1.5% agarose gel. RB39 should amplify two specific PCR bands, 178 bp and 498 bp in size, indicating *Brucella bovis*. Figure 1 As shown.

[0045] Example 3: Whole genome sequencing of strain RB39

[0046] Whole-genome sequencing and sequence assembly were performed by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The sequencing workflow is shown in the Nanopore library construction flowchart. After genome assembly, the results showed that the whole genome length of RB39 was 3282094 bp, and its genome size is shown in the RB39 genome circle. Figure 2 As shown.

[0047] Example 4: Preparation of crude Brucella live vaccine (RB39 strain)

[0048] 4.1 Preparation of Seeds for Seedling Production

[0049] The bacterial strain was streaked onto tryptic soy agar (TSA) and incubated at 37°C for 3 days. After visual inspection for purity, an appropriate amount of peptone water (pH 6.5–7.0) was added to wash off the bacterial growth. The culture was then inoculated onto tryptic soy broth (TSB) at a ratio of 10% and incubated at 37°C for 2 days. After passing the purity test, the culture was stored at 2–8°C for no more than 30 days.

[0050] 4.2 Preparation of bacterial culture for seedling production

[0051] Add 0.01% (v / v) of antifoaming agent to the culture medium. After sterilization, inoculate the seed culture at 1% (v / v) of the culture medium volume. Ferment at 37°C for 36 hours, gradually increasing the aeration rate during the culture. Add 50% glucose solution as needed to control the pH of the culture medium to 7.2±0.1, with each addition being approximately 1% (v / v) of the total culture medium volume. After the culture is completed, perform purity testing and viable cell count.

[0052] 4.3 Concentration of bacterial culture

[0053] After the bacterial culture passed the purity test, add 0.8% sodium carboxymethyl cellulose (CMC) solution at 1 / 8 (V / V) of the total volume of the culture medium, mix well, and let the bacterial cells stand for 48 hours to precipitate. Then, remove the precipitated bacterial cells for use in the purity test and viable cell count.

[0054] 4.4 Seedling Distribution and Packaging

[0055] The qualified bacterial solution was mixed evenly with a freeze-drying protectant containing 7% sucrose and 10% skim milk powder at a ratio of 1:7 (V / V). Dispense 8.0 × 10⁸ doses per head. 10 CFU ~ 1.6 × 10 10 CFU is dispensed in quantitative quantities.

[0056] 4.5 freeze-dried

[0057] After dispensing, the product is quickly freeze-dried according to the freeze-drying curve. The steps are as follows: 1°C is introduced into the freeze-drying chamber; the temperature is lowered to -40°C or below at a rate of 1°C per minute, and maintained for approximately 4 hours; the temperature of the plates is controlled at -15°C, and maintained for 16–18 hours; the temperature is increased by 10°C every 2 hours, and maintained at 25°C for 2 hours before being removed from the chamber. The entire process takes approximately 36 hours.

[0058] Example 5: Finished product testing of crude Brucella live vaccine (RB39 strain)

[0059] 5.1 Appearance: Sponge-like loose clumps, easily separated from the bottle wall, and dissolves rapidly after adding diluent.

[0060] 5.2 Purity test: The test was conducted according to the current method of the Chinese Veterinary Pharmacopoeia, and the result was pure.

[0061] 5.3 Variation test: Dilute with peptone water to 1000 CFU / mL, take 100 μL of diluted bacterial solution and spread it evenly on TSA plate, incubate at 37℃ for 3 days, and examine with crystal violet staining method. 100% of the colonies are rough type.

[0062] 5.4 Viable Bacterial Count: Dilute the vaccine with peptone water and inoculate it onto TSA plates for viable bacterial count. The viable bacterial count per dose is 8.0 × 10⁻⁶. 10 CFU ~ 1.6 × 10 11 CFU. Unless otherwise specified, one dose in the subsequent embodiments of this invention is 8.0 × 10⁻⁶. 10 CFU ~ 1.6 × 10 11 CFU.

[0063] 5.5 Safety Testing: Dilute the vaccine to a concentration of 5.0 × 10⁻⁶ live bacteria per 1.0 mL. 9 CFU was administered subcutaneously to five mice weighing 18–20 g, with 0.2 mL per mouse. All mice should survive within six days.

[0064] 5.6 Residual moisture content determination: The determination was carried out in accordance with the current Chinese Veterinary Pharmacopoeia and met the requirements.

[0065] 5.7 Vacuum degree determination: The determination was carried out in accordance with the current Chinese Veterinary Pharmacopoeia and met the requirements.

[0066] Example 6: Safety test of crude Brucella live vaccine (RB39 strain)

[0067] 6.1 Mouse safety test: Dilute the RB39 vaccine to 1 / 20 head dose per 1.0 mL, and subcutaneously inject 0.25 mL into 5 mice weighing 18-22g. All mice should be healthy and alive within 6 days.

[0068] 6.2 Guinea Pig Safety Test: RB39 vaccine was diluted with physiological saline to a concentration of 1 / 80 dose per 1.0 mL. Five female Hartley guinea pigs weighing 350–400 g were subcutaneously injected in the groin with 1 mL of the solution each. After 14–15 days, the guinea pigs were euthanized, their spleens were collected, weighed, and prepared as an emulsion. This emulsion was inoculated onto TSA agar plates. The bacterial count in the spleen was calculated based on the number of colonies grown. The bacterial count per 1 g of spleen should not exceed 2.0 × 10⁻⁶. 5 CFU.

[0069] 6.3 Safety trial in pregnant cows: The RB39 vaccine was diluted to 1 dose / mL and subcutaneously injected into 5 adult cows each at 3, 5, and 7 months of pregnancy, at 1 dose / cow. The cows were clinically observed for 30 days, and pregnancy and calving events were continuously recorded. No abortion or other adverse reactions occurred in the pregnant cows, and calving was normal.

[0070] 6.4 Safety test in pregnant sheep: The RB39 vaccine was diluted to a dose of 1 / 4 head per 1.0 mL. Ten adult goats and ten sheep, each in their second and fifth months of pregnancy, were subcutaneously injected with 1.0 mL per goat. Clinical observation was conducted for 30 days, and pregnancy and lambing outcomes were continuously recorded. No abortion or other adverse reactions occurred in the pregnant sheep, and lambing was normal.

[0071] Example 7: Efficacy test of crude Brucella live vaccine (RB39 strain)

[0072] 7.1 Mouse efficacy test: The RB39 vaccine was diluted with physiological saline to a concentration of 1 / 80 head dose per 1.0 mL. Ten mice weighing 18–22 g were injected subcutaneously into the groin of each mouse with 0.1 mL of the diluted vaccine. After 30 days, the mice were injected subcutaneously into the groin with 100 CFU / mouse of the virulent strain of Brucella mesenteriae M28 (CVCC70003, purchased from the National Veterinary Microbiology Culture Collection Center). After 30–35 days of observation, the mice were euthanized, and their spleens were collected for bacterial culture. The results are shown in Table 2. The table shows that 70% (7 / 10) of the mice did not exhibit the virulent strain.

[0073] Table 2 Results of mouse efficacy test

[0074]

[0075] 7.2 Guinea Pig Efficacy Test: The RB39 vaccine was diluted with physiological saline to a concentration of 1 / 20 dose per 1.0 mL. Ten guinea pigs weighing 350–400 g were injected subcutaneously into the groin of each with 1.0 mL of the diluted vaccine. After 30 days, a booster immunization was administered via the same dose and route. 40–60 days after the second immunization, the guinea pigs were injected subcutaneously into the groin with 1–3 infectious doses (10–30 CFU of live bacteria) of the virulent strain of Brucella mesenteriae M28. After 30–35 days of observation, the guinea pigs were euthanized, and their spleens were collected for bacterial culture. The results are shown in Table 3. As can be seen from the table, 70% (7 / 10) of the guinea pigs did not show the virulent strain.

[0076] Table 3 Results of Guinea Pig Efficacy Test

[0077]

[0078]

[0079] 7.3 Bovine efficacy test: The RB39 vaccine was diluted with physiological saline to a concentration of 1 dose per 1.0 mL. Five adult cattle were subcutaneously injected with the vaccine at a dose of 1 dose per head. A booster immunization was administered 30 days later via the same route and at the same dose. Ninety days after the second immunization, a dose of 2.0 × 10⁻⁶ was administered. 7 CFU / head of virulent Brucella melioides M28 strain from sheep was subcutaneously injected. After 45 days of observation, the animals were euthanized, and their spleens were harvested for bacterial culture. An A19 control group and a blank control group were established. The A19 control group consisted of 5 adult cattle subcutaneously injected with a 1 / 60 dose as per the product instructions. Ninety days post-immunization, the cattle were treated with 2.0 × 10⁻⁶ doses. 7 Sheep were challenged with a highly virulent strain of Brucella M28 via subcutaneous injection (CFU / head). After 45 days of observation, the animals were euthanized, and their spleens were collected for bacterial culture. The results are shown in Table 4. Similar to the A19 vaccine, two immunizations with the RB39 strain resulted in 80% (4 / 5) of the cattle being free of the virulent strain.

[0080] Table 4 Results of the Bovine Efficacy Test

[0081]

[0082] 7.4 Efficacy test in sheep: RB39 vaccine was diluted with physiological saline to a concentration of 1 / 4 head per 1.0 mL. Five adult sheep and five goats were subcutaneously injected with 1.0 mL per animal. A booster immunization was administered 30 days later via the same dose and route. Ninety days after the second immunization, a dose of 1.0 × 10⁻⁶ was administered. 10 Sheep were challenged with a virulent strain of Brucella M28 (CFU / head) via subcutaneous injection. After 45 days of observation, they were euthanized, and their spleens were harvested for bacterial culture. An M5 control group and a blank control group were also established. The M5 control group was administered a 1-dose subcutaneous injection to 5 adult sheep and 5 goats, as per the instructions. 90 days post-immunization, they were treated with a 1.0 × 10⁻⁶ dose. 10Sheep were challenged with a highly virulent strain of Brucella M28 by subcutaneous injection (CFU / sheep), observed for 45 days, and then euthanized. Spleens were collected for bacterial culture. The results are shown in Table 5. After two immunizations with the RB39 strain vaccine, similar to the M5 vaccine, 80% (4 / 5) of the sheep were free of the virulent strain.

[0083] Table 5 Results of the sheep efficacy test

[0084]

[0085]

[0086] 7.5 Monitoring of antibody levels in immunized cattle and sheep: For cattle and sheep that underwent efficacy tests as described in 7.3 and 7.4, blood samples were collected every 15 days from the first immunization until challenge. Blood samples were also collected from the control group at the same time, and serum was separated. A 1.0 × 10⁻⁶ antibody level was used. 10 Inactivated RB39, A19, and M5 bacterial suspensions (CFU / mL) were sonicated at 350W for 40 minutes and centrifuged. The supernatant of lysed bacterial protein was collected and diluted to a protein concentration of 10 μg / mL with PBS buffer. 100 μL per well was coated onto ELISA plates and incubated at 2–8°C for 16 hours. The plates were washed once with PBST buffer, and 2% bovine serum albumin was added to each well. The plates were blocked at 2–8°C for 24 hours, and then washed three times with PBST buffer. Serum samples from different immunization groups were added to the corresponding antigen-coated ELISA plates. Serum samples from the blank control group were added to all plates coated with different antigens as a negative control. The reaction was carried out at 37°C for 30 minutes. Add 100 μL of HRP-labeled rabbit anti-bovine IgG or HRP-labeled mouse anti-sheep IgG to each well, incubate at 37°C for 30 min, wash three times with PBST washing buffer, add 100 μL of substrate chromogenic solution to each well, incubate in the dark for 15 min, add 50 μL of stop solution to each well, read the OD value at 450 nm using an ELISA reader, and calculate the sample S / N value (i.e., the OD of the immune sample). 450nm / Negative samples from the blank control group OD 450nm ), the results are shown Figures 3-4 The results showed that after the initial immunization of cattle and sheep with the RB39 vaccine, antibody levels were high 15 days after immunization, then decreased until 30 days after immunization, and reached their highest level 15 days after the second immunization (i.e., 45 days after the initial immunization), followed by a gradual decline. In contrast, after immunization of cattle and sheep with A19 and M5 vaccines, antibody levels reached their highest level 30 to 45 days after immunization, and then declined, with a general trend consistent with that of the RB39 strain. Moreover, the antibody titers produced were not significantly different from those of the RB39 strain (P > 0.05).

[0087] Example 8: Preparation of antigen for the red agglutination test of Brucella RB39 strain (rough type) on Brassica juncea agglutination plate.

[0088] 8.1 Preparation of positive serum from Brucella rubella strain RB39: Brucella rubella strain RB39 was inoculated into TSA medium and cultured at 37°C for 3 days. The culture was then harvested and prepared into 4×10⁻⁶ sera. 10 The bacterial suspension at CFU / mL was inactivated in an 80°C water bath for 2 hours. Healthy brucellosis antibody-negative cattle were immunized with the inactivated antigen by subcutaneous injection of 4 mL per cow in the neck. One month after immunization, a double dose was administered as a booster immunization. Two weeks after the booster immunization, blood was collected for tube agglutination testing with the corresponding antigen, and the agglutination titer was determined to be no less than 1:100. Blood was collected venously, serum was separated, and filtered through a 0.22 μm filter for sterilization. Positive serum was diluted with commercial fetal bovine serum (Shuangru Biotechnology, catalog number S711-001S) to a tube agglutination titer of 1:100 (i.e., 100 IU / mL). ProClin 300 was added to a final concentration of 0.1%, and the solution was aseptically aliquoted and stored below -15°C.

[0089] 8.2 Preparation of red iodine staining antigen for Brucella brucellosis strain RB39: Brucella brucellosis strain RB39 was inoculated into TSA medium and cultured at 37°C for 3 days. The culture was then harvested and OD was prepared. 600nm The bacterial suspension was concentrated to 1.5% and inactivated in an 80°C water bath for 2 hours. 4% red erythromycin dye was added to the inactivated suspension at a volume ratio of 0.5%, and the mixture was stirred thoroughly for 30 minutes. After centrifugation at 8000 rpm for 20 minutes, the supernatant was discarded. The precipitate was resuspended in 4 mL of Tris buffer per 1 g of weight, stirred thoroughly for 30 minutes, and then standardized.

[0090] 8.3 Standardization of the antigen for the Brassica raphe RB39 strain rose tincture plate agglutination test: The rose tincture antigen was diluted with Tris buffer at ratios of 1:3, 1:4, 1:5, and 1:6. Positive serum of Brassica raphe RB39 strain was diluted with Tris buffer to 10 IU / mL and 5 IU / mL. 0.03 mL of each serum dilution was used to perform a plate agglutination reaction with an equal volume of the antigen at each dilution, and the results were observed within 4 minutes. Negative serum and positive serum of Brucella sclerotium were used as controls. The antigen dilution at which positive serum of Brassica raphe RB39 strain showed a "+" reaction at 10 IU / mL and a "-" reaction at 5 IU / mL was the antigen concentration used. The results are shown in Table 6. Based on the results in Table 6, the antigen diluted with Tris buffer to the used concentration (1:4) is the antigen for the Brassica raphe RB39 strain rose tincture plate agglutination test.

[0091] Table 6. Results of antigen labeling in the red agglutination test of Brucella rhabdominis strain RB39 on Brassin Plate.

[0092]

[0093] Note: ++: There are obvious aggregated particles, and the liquid is slightly transparent; +: Slight aggregates are visible, and the liquid is cloudy; -: No aggregates are visible, and the liquid is uniformly cloudy.

[0094] Example 9: Differential Diagnostic Test of Animals Immunized with Rough Brucella Live Vaccine (RB39 Strain)

[0095] 9.1 Immunization: Dilute RB39 vaccine with physiological saline to a dose of 1.0 mL per head, and subcutaneously inject 5 adult cattle (1 dose / cattle) as the immunization group; simultaneously, a non-immunized control group (5 cattle / group) was set up. Dilute RB39 vaccine with physiological saline to a dose of 1 / 4 head, and subcutaneously inject 5 adult sheep and 5 adult goats (1.0 mL / goat) as the immunization group; simultaneously, a non-immunized control group (5 sheep / group) was set up.

[0096] 9.2 Blood Collection: 14 days post-immunization, blood was collected from all immunized and control animals to separate serum. Simultaneously, serum was collected from cattle, goats, and sheep farms that had not been vaccinated against brucellosis. Five serum samples from each farm that tested positive for both the red rostrum plate agglutination test (smooth type) and complement fixation test were selected as clinically positive serum.

[0097] 9.3 Differential Diagnostic Tests: Serum from animals immunized with RB39 vaccine, negative serum from the non-immunized control group, and positive serum from clinical infection were all tested using the Rose Bengal agglutination test antigen of Brucella RB39 strain and the Rose Bengal agglutination test antigen of smooth brucellosis. The results are shown in Table 7. Serum from cattle, goats, and sheep immunized with Brucella RB39 live vaccine showed positive reactions (+) to both the Brucella RB39 strain Rose Bengal agglutination test antigen and negative reactions (-) to both the smooth brucellosis agglutination test antigen. Serum from cattle, goats, and sheep clinically infected animals showed negative reactions (-) to both the Brucella RB39 strain Rose Bengal agglutination test antigen and positive reactions (+) to both the smooth brucellosis agglutination test antigen. Serum from the non-immunized control group showed negative reactions (-) to both antigens. Therefore, serum antibodies from clinical RB39 vaccine immunization and clinical infection can be distinguished, enabling differential diagnosis between Brucella immunity and infection.

[0098] Table 7. Differential Diagnostic Results of the Red Tiger Plate Agglutination Test

[0099]

[0100]

[0101] Note: +: positive, indicating agglutination reaction; -: negative, indicating no reaction.

[0102] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A rough-type Brucella abortus attenuated strain RB39, which was deposited with the China General Microbiological Culture Collection Center on February 17, 2025, and has the accession number CGMCC No.46377. 2.Use of the rough-type Brucella abortus attenuated strain RB39 of claim 1 in the preparation of a Brucella vaccine.

3. A Brucella vaccine, characterized in that, The rough-type Brucella abortus attenuated strain RB39, which was deposited with the China General Microbiological Culture Collection Center on February 17, 2025, and has the accession number CGMCC No.46377.

4. The Brucella vaccine of claim 3, wherein the Brucella vaccine is a Brucella abortus vaccine. The freeze-drying protective agent is also included. 5.Use of the rough-type Brucella abortus attenuated strain RB39 of claim 1 in the preparation of an antigen for detecting Brucella antibodies produced after animals are immunized with the Brucella vaccine of claim 3 or 4. The detection can distinguish between vaccine immunization and natural infection.

6. The use according to claim 5, wherein the compound is ###0002### The detection uses the RBT method, the SAT method, the CFT method or the ELISA method.

7. An antigen for detecting Brucella antibodies produced by an animal immunized with a Brucella vaccine according to claim 3 or 4, characterized in that, The antigen is obtained by culturing and inactivating the rough-type Brucella abortus attenuated strain RB39 of claim 1.

8. The antigen of claim 7, wherein The antigen is used for detecting Brucella antibodies by the RBT method, and the preparation method comprises the following steps: The rough-type Brucella abortus attenuated strain RB39 of claim 1 is made into a bacterial solution with normal saline, and is inactivated. Tiger red dye is added to the inactivated bacterial solution in a volume ratio of 0.5:100 to obtain dyed bacterial bodies. The dyed bacterial bodies are reselected with Tris buffer to a preset concentration.

9. A kit for detecting antibodies to Brucella, characterized in that, The antigen of claim 7 or 8 is included.

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