Rough brucella attenuated strain as well as application, vaccine, antigen and kit thereof

Abstract

The invention relates to the field of veterinary biological products, in particular to a rough brucella attenuated strain RB39 as well as application, a vaccine, an antigen and a kit thereof. The rough brucella attenuated strain RB39 provided by the invention can be used for preparing a high-safety vaccine, and the vaccine can be injected to immunize pregnant animals and provide good immune protection. And the vaccine is applicable to immunization of animals of multiple species. Besides, based on the rough brucella attenuated strain RB39, an antigen for identifying the rough brucella antibody can be prepared, vaccine immunity and natural infection are effectively distinguished, and a scientific basis is provided for brucellosis prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of veterinary products, and in particular to a rough Brucella abortus strain, its application, vaccine, antigen and kit. Background Art

[0002] Brucellosis (referred to as "Brucellosis") is a zoonotic disease characterized by abortion and fever caused by Brucella, which seriously threatens the lives and health of humans and various animals. Human infection is mainly caused by contact with diseased animals and their contaminated animal-derived products. Therefore, vaccination with animal Brucella vaccines in areas with high prevalence of Brucellosis is an effective measure recognized globally to control Brucellosis. Most current veterinary Brucella vaccines are made from smooth Brucella abortus strains, and their biggest drawback is that the virulence is relatively strong and they cannot be used for injection immunization of pregnant animals; after vaccination, smooth antibodies are produced, which cannot be distinguished from the antibodies produced by clinical infection with smooth virulent strains. This results in animals being considered as the source of infection and being wrongly killed; or positive animals in the group are considered as vaccinated animals and cannot be culled from the group, leading to the continuous existence of the source of infection and the inability to complete the purification of the disease. Currently, the commonly used method is to use genetically engineered marker vaccines to distinguish between clinical infection and vaccine immunization, but the cost is high and the operation is complex. The above defects have restricted the use and promotion of Brucella vaccines.

[0003] The rough Brucella abortus strain has higher safety compared to the smooth Brucella abortus strain. After injection into animals, it stimulates the body to produce rough antibodies, which can be effectively distinguished from the smooth antibodies produced by clinical infection with smooth virulent strains. It is the main direction of research on new Brucella vaccines. Currently, the only rough vaccine strain approved for marketing and widely used globally is RB51. This vaccine was obtained by rifampicin mutagenesis by American scientists in the 1990s and has good immunogenicity. It has been used in many countries in the United States and Latin America. However, since rifampicin is an effective antibiotic for the treatment of human Brucellosis and this vaccine is induced from a virulent strain, although the virulence has been weakened, injection immunization of pregnant animals still causes adverse reactions such as abortion. Therefore, the safety of this vaccine has always been controversial. In addition, the existing rough vaccines are only applicable to specific animal species and cannot comprehensively cover all susceptible animals, which limits their wide application. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a rough Brucella abortus strain, which has high safety, does not cause adverse reactions such as abortion, is applicable to a variety of animals, and can stimulate the body to produce specific antibodies, facilitating the distinction between natural infection and vaccine immunization, thereby effectively controlling the spread of Brucellosis and expanding the application scope of the vaccine.

[0005] The technical problem to be further solved by the present invention is to provide an application of a rough-type Brucella attenuated strain in the preparation of a Brucella vaccine or an antigen for detecting Brucella antibodies.

[0006] The technical problem to be further solved by the present invention is to provide a Brucella vaccine with high safety, which can be used for injection immunization of pregnant animals and is applicable to animals of multiple species.

[0007] The technical problem to be further solved by the present invention is to provide an antigen with strong specificity, which can effectively distinguish natural infection from vaccine immunization.

[0008] The technical problem to be further solved by the present invention is to provide a kit, which can effectively distinguish natural infection from vaccine immunization.

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

[0010] Specifically, the strain RB39 is a stable rough-type Brucella attenuated strain obtained by continuously passaging the smooth-type Brucella attenuated strain A19 (CVCC 70202) 39 times in TSB medium with a pH of 5.5, then continuously passaging 65 times in TSB medium with a pH of 4.5, and finally passaging 35 times in mice. Its colonies were identified as 100% rough type by crystal violet staining method and identified as Brucella abortus by AMOS-PCR. The strain RB39 not only has high safety but also has significant immune efficacy, is applicable to various animals such as cattle, sheep, pigs, etc., can effectively stimulate the body to produce specific antibodies, is convenient for distinguishing natural infection from vaccine immunization status, and significantly improves the prevention and control effect of brucellosis.

[0011] As the second aspect of the present invention, the present invention discloses the application of the above-mentioned rough-type Brucella attenuated strain RB39 in the preparation of a Brucella vaccine. This vaccine has high safety and strong immune efficacy. When applied in pregnant animals, it will not cause adverse reactions such as miscarriage, ensuring the safety of the mother and fetus. And this vaccine is applicable to animals of multiple species (including but not limited to mice, guinea pigs, cattle, goats, sheep, pigs), can significantly improve the protection effect of the vaccine, and effectively prevent and control the spread of brucellosis.

[0012] As the third aspect of the present invention, the present invention provides a Brucella vaccine, which comprises the rough-type Brucella attenuated strain RB39 described above. Specifically, the Brucella vaccine can be prepared by inactivating the above-mentioned rough-type Brucella attenuated strain RB39 and adding excipients. Among them, the excipients of the Brucella vaccine include adjuvants, stabilizers, preservatives, diluents, antioxidants, etc., but are not limited thereto.

[0013] Preferably, in some embodiments, the vaccine includes a lyoprotectant, which may include PEG, gelatin, lactose, glycine, etc., but is not limited thereto. The lyoprotectant can avoid damaging the activity of the strain during the freeze-drying process, ensure the stability of the vaccine, and further improve the actual application effect of the vaccine.

[0014] Specifically, when using the Brucella vaccine of the present invention, cattle are subcutaneously immunized with a dose of 1 dose (8.0×10 10 CFU~1.6×10 11 CFU), and sheep are subcutaneously immunized with a dose of 1 / 4 dose (2.0×10 10 CFU~4.0×10 10 CFU) to immunize pregnant animals, which does not cause abortion.

[0015] As the fourth aspect of the present invention, the present invention also provides the application of the above-mentioned rough-type Brucella attenuated strain RB39 in the preparation of an antigen for detecting Brucella antibodies, wherein the Brucella antibodies are produced after immunizing animals with the Brucella vaccine as described above; through the detection, vaccine immunization and natural infection can be distinguished, the diagnostic accuracy can be improved, the precise prevention and control of brucellosis can be facilitated, and the healthy development of the livestock industry can be guaranteed.

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

[0017] As the fifth aspect of the present invention, the present invention also provides an antigen, which is used to detect the Brucella antibodies produced by immunizing animals with the above-mentioned Brucella vaccine, and is obtained by culturing and inactivating the above-mentioned rough-type Brucella attenuated strain RB39.

[0018] Specifically, depending on the different detection methods, the above-mentioned antigen also needs to be processed. For example, when using the RBT method, the inactivated antigen needs to be stained; when using the CFT method, operations such as breaking the antigen, extracting LPS, and purifying are required; when using the ELISA method, the antigen needs to be broken, LPS extracted, and coated. It should be noted that those skilled in the art can perform corresponding optimization processing on the antigen 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 detecting Brucella antibodies by the RBT method, and its preparation method includes:

[0020] The above-mentioned attenuated Brucella strain RB39 is made into a bacterial solution with normal saline and inactivated;

[0021] According to a volume ratio of 0.5:100, Rose Bengal dye is added to the inactivated bacterial solution for staining to obtain stained bacterial cells;

[0022] The stained bacterial cells are resuspended with Tris buffer to a preset concentration, and that is obtained.

[0023] More preferably, the above-mentioned rough-type Brucella attenuated strain RB39 is made into a concentrated bacterial solution with normal saline, and after inactivation, 4% Rose Bengal dye is added for staining according to a volume ratio of 0.5%. The stained bacterial cells are resuspended with Tris buffer in a certain proportion, and then diluted 1:3, 1:4, 1:5, 1:6 times with Tris buffer. 30 μL each of the positive sera of rough-type Brucella strain RB39 at 10 IU / mL and 5 IU / mL are taken and mixed evenly, and the results are observed within 4 minutes. The antigen dilution that can cause the positive sera of rough-type Brucella strain RB39 to show a "+" reaction at 10 IU / mL; and a "-" reaction at 5 IU / mL is the antigen usage concentration.

[0024] As the sixth aspect of the present invention, the present invention also provides a kit for detecting Brucella antibodies, which includes the above-mentioned antigen.

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

[0026] 1. The present invention has developed a rough-type Brucella attenuated strain RB39 and its vaccine, which can be subcutaneously injected to immunize pregnant animals without causing abortion, overcoming the defect that existing Brucella vaccines cannot be injected to immunize pregnant animals; in addition, this vaccine can be applied to the immunization of multiple species of animals. Specifically, after the vaccine of the present invention is immunized in mice, guinea pigs, cattle, goats, and sheep at a certain dose, it can provide a protective efficacy of not less than 70%.

[0027] 2. The present invention has developed an antigen for differentiating diagnosis between RB39 vaccine immunization and natural infection. Based on this antigen, antibodies produced by vaccine immunization and those produced by natural infection can be quickly distinguished through serological detection means, solving the technical problem that it is impossible to conduct differential diagnosis between vaccine immunization and natural infection after immunizing animals with existing brucellosis vaccines, and providing a scientific basis for brucellosis prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the AMOS-PCR electrophoresis map of rough Brucella strain RB39;

[0029] Figure 2 is the genomic circular map of rough Brucella strain RB39;

[0030] Figure 3 is the graph of the change in antibody level after immunizing cattle with rough Brucella strain RB39;

[0031] Figure 4 is the graph of the change in antibody level after immunizing sheep with rough Brucella strain RB39. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. It should be noted that all reagents used in the examples can be purchased from commercial channels without special instructions. Induction and domestication of strain RB39 in the examples

[0033] 1.1 Induction under acidic conditions with a pH value of 5.5: The smooth Brucella strain A19 (CVCC 70202, purchased from the commercial live brucellosis vaccine A19) was continuously passaged on TSB medium with a pH value of 5.5. After every 3 passages, TSA plates were coated by the limited dilution method and cultured at 37°C for 3 days. After single colonies grew out, the colonies on the plates were stained with crystal violet. The colonies with the most obvious degree of crystal violet staining (rough colonies are stained, smooth colonies are not stained) were selected for continuous passage. If no colonies with positive crystal violet staining appeared, colonies were randomly picked. In this way, a total of 39 passages were carried out, and the result was that the positive rate of rough colonies with crystal violet staining reached more than 20%.

[0034] 1.2 Induction under acidic conditions with a pH value of 4.5: The rough colonies picked in 1.1 were continuously passaged on TSB medium with a pH value of 4.5. After every 5 passages, TSA plates were coated by the limited dilution method and cultured at 37°C for 3 days. After single colonies grew out, the colonies on the plates were stained with crystal violet. The colonies with the most obvious degree of crystal violet staining were selected for continuous passage. After 65 consecutive passages, the result was that the positive rate of rough colonies with crystal violet staining reached more than 90%.

[0035] 1.3 Subculture and domestication in mice: The rough colonies picked in 1.2 were subcutaneously injected into the groin of 1 BALB / c mouse at a dose of 1.0×10 8 CFU / mouse. After 7 days, the mouse was sacrificed, and the spleen tissue was homogenized. Then, the homogenate was spread on TSA plates by the limiting dilution method and cultured at 37°C for 3 days. After single colonies grew out, the colonies on the plates were stained with crystal violet. The colonies with the most obvious crystal violet staining were selected for continuous passage. After 25 consecutive passages, the positive rate of rough colonies stained with crystal violet reached 100%. Subsequently, it was passaged in mice for another 10 generations (a total of 139 passages). As a result, the positive rate of rough colonies isolated in 10 consecutive generations was 100%. The genetically stable rough Brucella strain obtained was named RB39 strain.

[0036] Example 2 Identification of rough Brucella strain RB39

[0037] 2.1 Morphological and biochemical characteristics: Gram-negative coccobacilli, without spores and capsules. Gram staining is negative; hydrogen sulfide test is positive.

[0038] 2.2 Culture characteristics: It grows well on tryptic soy agar medium (TSA). Streak on the above medium plate and culture at 37°C for 3 days. 100% of the colonies belong to the rough type. It grows evenly, turbidly, and opaquely in liquid media such as TSB.

[0039] 2.3 Variation inspection: It conforms to the characteristics of rough colonies. 100% of the colonies are stained by the crystal violet staining method for colonies; precipitation appears in the heat agglutination test; agglutination appears in the acridine yellow agglutination test.

[0040] 2.4 Serological characteristics: Using the RB39 culture as an antigen, no agglutination occurs with the positive serum of smooth Brucella, and agglutination occurs with the positive serum of rough Brucella.

[0041] 2.5 PCR identification: Identification was carried out by the AMOS-PCR method, and the following 4 primers were synthesized (see Table 1).

[0042] Table 1 AMOS-PCR primers

[0043]

[0044] Using a commercial bacterial genomic DNA extraction kit to extract RB39 genomic DNA as a template, in a 20 μL reaction system, add 10 μL 2×PCR mix, 1 μL of each of the 4 primers, 1 μL of template DNA, and 5 μL of DEPC-treated water. After mixing, perform PCR reaction. At the same time, set up Brucella abortus strain A19 (the corresponding primer is F abortus ), Brucella melitensis strain M5 (CVCC18, purchased from the commercial live Brucella vaccine M5, the corresponding primer is Fmelitensis ) Nucleic acid control group of Brucella suis S2 (CVCC 70502, purchased from commercial live Brucella vaccine S2, corresponding primer is F suis ), and a blank control group without added nucleic acid. PCR reaction procedure: After 5 min at 95 °C, perform 35 cycles of 30 s at 95 °C, 30 s at 54 °C, and 1 min at 72 °C; extend at 72 °C for 10 min. The amplified product was identified by electrophoresis on a 1.5% agarose gel. For RB39, two specific PCR bands should be amplified, with sizes of 178 bp and 498 bp respectively, which are Brucella abortus, as Figure 1 shown.

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

[0046] The whole-genome sequencing and sequence assembly were completed by the Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, and its sequencing process is as shown in the Nanopore library construction flow chart. After the genome assembly was completed, the results showed that the full-genome length of RB39 was 3282094 bp, and its genome size is as shown in the genome circle of RB39 Figure 2 shown.

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

[0048] 4.1 Preparation of seed for vaccine production

[0049] The strain was streaked and transplanted onto tryptic soy agar medium (TSA) and cultured at 37 °C for 3 d. After visual inspection for purity, an appropriate amount of peptone water (pH 6.5 - 7.0) was added to wash down the bacterial lawn, and then inoculated into tryptic soy broth medium (TSB) at a ratio of 10%. After culturing at 37 °C for 2 d and passing the purity test, it was stored at 2 - 8 °C for no more than 30 days.

[0050] 4.2 Preparation of bacterial liquid for vaccine production

[0051] Add 0.01% (V / V) antifoaming agent according to the amount of medium. After sterilization, inoculate the seed bacterial liquid at 1% (V / V) of the amount of medium, and ferment and culture at 37 °C for 36 hours. During the culture process, gradually increase the ventilation volume, and add 50% glucose solution as needed to control the pH value of the culture solution to 7.2 ± 0.1, with each addition amount being approximately 1% (V / V) of the total amount of medium. After the culture is completed, perform purity test and viable count.

[0052] 4.3 Concentration of bacterial liquid

[0053] After the purity test of the bacterial liquid is qualified, add 0.8% sodium carboxymethylcellulose (CMC) solution at 1 / 8 (V / V) of the total volume of the medium, mix well, and let the bacterial cells settle for 48 hours. Then, aspirate the precipitated bacterial cells for standby, and perform purity test and viable count.

[0054] 4.4 Bacterial strain preparation and sub-packaging

[0055] For the qualified bacterial liquid after inspection, mix the bacterial liquid with the lyophilization protectant containing 7% sucrose and 10% skim milk powder evenly at a ratio of 1:7 (V / V). Conduct quantitative sub-packaging at 8.0×10 10 CFU~1.6×10 10 CFU per dose.

[0056] 4.5 Lyophilization

[0057] After sub-packaging, conduct freeze-drying rapidly according to the freeze-drying curve. The steps are as follows: Put into the drying chamber at 20°C; cool down to -40°C or below at a rate of 1°C per minute and maintain for about 4 hours; control the shelf temperature at -15°C and maintain for 16 - 18 hours; raise the temperature by 10°C every 2 hours and maintain at 25°C for 2 hours, then take out of the drying chamber. The whole process takes about 36 hours.

[0058] Example 5 Finished product inspection of live rough Brucella vaccine (RB39 strain)

[0059] 5.1 Character: Sponge-like loose mass, easy to separate from the bottle wall, and dissolves rapidly after adding the diluent.

[0060] 5.2 Purity inspection: Conduct the inspection according to the method in the current "Chinese Veterinary Pharmacopoeia", and the result is pure.

[0061] 5.3 Variation inspection: Dilute with peptone water to 1000CFU / mL, take 100μL of the diluted bacterial liquid and spread it evenly on the TSA plate, incubate at 37°C for 3 days, and check by the colony crystal violet staining method. 100% are rough colonies.

[0062] 5.4 Viable bacteria count: Dilute the vaccine with peptone water, inoculate on the TSA plate for viable bacteria count. The viable bacteria count per dose is 8.0×10 10 CFU~1.6×10 11 CFU. Unless otherwise specified, 1 dose in the subsequent examples of the present invention is 8.0×10 10 CFU~1.6×10 11 CFU.

[0063] 5.5 Safety inspection: Dilute the vaccine to contain 5.0×10 9 CFU viable bacteria per 1.0mL, subcutaneously inject 5 mice weighing 18 - 20g, 0.2mL for each mouse, and all should be healthy and alive within 6 days.

[0064] 5.6 Residual moisture determination: Conduct the determination according to the current "Chinese Veterinary Pharmacopoeia", and it meets the requirements.

[0065] 5.7 Vacuum degree determination: Conduct the determination according to the current "Chinese Veterinary Pharmacopoeia", and it meets the requirements.

[0066] Example 6 Safety Test of Live Rough Brucella Vaccine (Strain RB39)

[0067] 6.1 Safety test on mice: Dilute the RB39 vaccine to contain 1 / 20 dose per 1.0 mL, and subcutaneously inject 5 mice weighing 18 - 22 g, 0.25 mL for each. All of them should be healthy and alive within 6 days.

[0068] 6.2 Safety test on guinea pigs: Dilute the RB39 vaccine with normal saline to contain 1 / 80 dose per 1.0 mL, and subcutaneously inject 5 female Hartley guinea pigs weighing 350 - 400 g in the groin, 1 mL for each. After 14 - 15 days, sacrifice them, take the spleens, weigh them together, make emulsions, inoculate TSA culture medium plates, and calculate the bacterial content in the guinea pig spleens according to the number of colonies grown. The bacterial content per 1 g of spleen should not exceed 2.0×10 5 CFU.

[0069] 6.3 Safety test on pregnant cows: Dilute the RB39 vaccine to 1 dose / mL, and subcutaneously inoculate 5 adult cows each at 3 months, 5 months and 7 months of pregnancy, 1 dose per cow. Clinically observe for 30 days and continuously record the pregnancy and parturition conditions. There are no adverse reactions such as abortion in pregnant cows and the parturition is normal.

[0070] 6.4 Safety test on pregnant sheep: Dilute the RB39 vaccine to contain 1 / 4 dose per 1.0 mL, and subcutaneously inoculate 10 adult goats and sheep each at 2 months and 5 months of pregnancy, 1.0 mL per sheep. Clinically observe for 30 days and continuously record the pregnancy and lambing conditions. There are no adverse reactions such as abortion in pregnant sheep and the lambing is normal.

[0071] Example 7 Efficacy Test of Live Rough Brucella Vaccine (Strain RB39)

[0072] 7.1 Efficacy test on mice: Dilute the RB39 vaccine with normal saline to contain 1 / 80 dose per 1.0 mL, and use 10 mice weighing 18 - 22 g. Subcutaneously inject 0.1 mL of the diluted vaccine into the groin of each mouse. After 30 days, subcutaneously inject 100 CFU / mouse of the virulent strain of Brucella melitensis M28 (CVCC70003, purchased from the National Veterinary Microbial Culture Collection Center) into the groin of the mice. Observe for 30 - 35 days and then sacrifice them. Take the spleens for bacterial culture examination. The results are shown in Table 2. It can be seen from the table that no virulent bacteria appear in 70% (7 / 10) of the mice.

[0073] Table 2 Results of Efficacy Test on Mice

[0074]

[0075] 7.2 Guinea pig potency test: Dilute the RB39 vaccine with normal saline to contain 1 / 20 dose per 1.0 mL. Use 10 guinea pigs weighing 350 - 400 g, and subcutaneously inject 1.0 mL of the diluted vaccine into the groin of each guinea pig. After 30 days, boost the immunization once with the same dose and route. 40 - 60 days after the second immunization, subcutaneously inject the guinea pigs in the groin with a virulent strain of Brucella melitensis M28 at 1 - 3 infectious doses (10 - 30 CFU viable bacteria). Observe for 30 - 35 days and then sacrifice the animals. Take the spleen for bacterial culture examination. The results are shown in Table 3. It can be seen from the table that 70% (7 / 10) of the guinea pigs showed no virulent bacteria.

[0076] Table 3 Results of Guinea Pig Potency Test

[0077]

[0078]

[0079] 7.3 Bovine potency test: Dilute the RB39 vaccine with normal saline to contain 1 dose per 1.0 mL. Subcutaneously inject 5 adult cattle, 1 dose per head. After 30 days, boost the immunization once with the same dose and route. 90 days after the second immunization, challenge the cattle subcutaneously with a virulent strain of Brucella melitensis M28 at 2.0×10 7 CFU / head. Observe for 45 days and then sacrifice the animals. Take the spleen for bacterial culture examination. At the same time, set up A19 control group and blank control group. In the A19 control group, subcutaneously inject 5 adult cattle at 1 / 60 dose according to the instructions. 90 days after immunization, challenge the cattle subcutaneously with a virulent strain of Brucella melitensis M28 at 2.0×10 7 CFU / head. Observe for 45 days and then sacrifice the animals. Take the spleen for bacterial culture examination. The results are shown in Table 4. After two immunizations with the RB39 strain vaccine, like the A19 vaccine, 80% (4 / 5) of the cattle showed no virulent bacteria.

[0080] Table 4 Results of Bovine Potency Test

[0081]

[0082] 7.4 Ovine potency test: Dilute the RB39 vaccine with normal saline to contain 1 / 4 dose per 1.0 mL. Subcutaneously inject 5 adult sheep and 5 adult goats respectively, 1.0 mL per animal. After 30 days, boost the immunization once with the same dose and route. 90 days after the second immunization, challenge the animals subcutaneously with a virulent strain of Brucella melitensis M28 at 1.0×10 10 CFU / animal. Observe for 45 days and then sacrifice the animals. Take the spleen for bacterial culture examination. At the same time, set up M5 control group and blank control group. In the M5 control group, subcutaneously inject 5 adult sheep and 5 adult goats respectively at 1 dose according to the instructions. 90 days after immunization, challenge the animals subcutaneously with a virulent strain of Brucella melitensis M28 at 1.0×10 10The virulent strain M28 of Brucella melitensis was subcutaneously injected for challenge. After 45 days of observation, the animals were sacrificed, and the spleens were taken for bacterial culture examination. The results are shown in Table 5. After two immunizations with the RB39 strain vaccine, like the M5 vaccine, 80% (4 / 5) of the sheep had no virulent bacteria detected.

[0083] Table 5 Results of the potency test in sheep

[0084]

[0085]

[0086] 7.5 Monitoring of antibody levels in immunized cattle and sheep: For the cattle and sheep subjected to the potency test in 7.3 and 7.4, blood was collected every 15 days from the first immunization until before challenge. Blood was also collected from the control group at the same time, and the sera were separated. Inactivated RB39 bacterial solution, A19 bacterial solution, and M5 bacterial solution at a concentration of 1.0×10 10 CFU / mL were sonicated at a power of 350 W for 40 minutes and then centrifuged. The supernatant of the lysed bacterial protein was taken, and the protein supernatant was diluted to a protein concentration of 10 μg / mL with PBS buffer to coat the ELISA plate, 100 μL per well, and placed at 2 - 8°C for 16 h. The plate was washed once with PBST washing solution, 2% bovine serum albumin was added to each well, and it was blocked at 2 - 8°C for 24 h. After washing 3 times with PBST washing solution, it was ready for use. Serum samples from different immunized groups were added to the corresponding antigen-coated enzyme-linked immunosorbent assay (ELISA) plates, and serum samples from the blank control group were added to all antigen-coated plates as negative controls. The reaction was carried out at 37°C for 30 min. 100 μL of the working concentration of HRP-labeled rabbit anti-bovine IgG or HRP-labeled mouse anti-sheep IgG was added to each well, and the reaction was carried out at 37°C for 30 min. The plate was washed 3 times with PBST washing solution, 100 μL of the substrate chromogenic solution was added to each well, and it was developed colorimetrically in the dark for 15 min. 50 μL of the stop solution was added to each well, and the OD value was read at 450 nm with an enzyme-linked reader, and the S / N value of the sample (i.e., OD of the immunized sample 450nm / OD of the negative sample in the blank control group 450nm ) was calculated. The results are shown in Figures 3 to 4 . The results showed that after the first immunization of cattle and sheep with the RB39 vaccine, the antibody level was relatively high 15 days after immunization, then decreased until 30 days after immunization, reached the highest level 15 days after the second immunization (i.e., 45 days after the first immunization), and then showed a gradually decreasing trend; while after injection immunization of cattle and sheep with A19 and M5, the antibody reached the highest level 30 - 45 days after immunization and then decreased. The basic trend was consistent with that of the RB39 strain, and there was no significant difference in the antibody titer produced compared with the RB39 strain (P > 0.05).

[0087] Example 8 Preparation of the Rose Bengal Plate Agglutination Test Antigen of the Rough Brucella Strain RB39

[0088] 8.1 Preparation of positive serum of Brucella rough strain RB39: Inoculate Brucella rough strain RB39 into TSA medium and culture at 37 °C for 3 days. Harvest the culture and prepare a bacterial suspension with a concentration of 4×10 10 CFU / mL. Inactivate it in a water bath at 80 °C for 2 hours. Immunize healthy cattle negative for Brucella antibodies with the inactivated antigen, and inject 4 mL subcutaneously into the neck of each cattle. One month after immunization, boost the immunization once with double the dose. Collect blood 2 weeks after boost immunization, and perform a tube agglutination test with the corresponding antigen to determine that the agglutination titer is not lower than 1:100. Collect blood from the vein, separate the serum, filter and sterilize it with a 0.22 μm filter, dilute the positive serum to a tube agglutination titer of 1:100 (i.e., 100 IU / mL) with commercial fetal bovine serum (Shuangru Biology, product number S711-001S), add ProClin 300 to a final concentration of 0.1%, dispense it aseptically, and store it at -15 °C or below.

[0089] 8.2 Preparation of Rose Bengal staining antigen of Brucella rough strain RB39: Inoculate Brucella rough strain RB39 into TSA medium and culture at 37 °C for 3 days. Harvest the culture and prepare a concentrated bacterial suspension with an OD 600nm of 1.5. Inactivate it in a water bath at 80 °C for 2 hours. Add 4% Rose Bengal dye to the inactivated bacterial suspension according to a volume ratio of 0.5%, stir well for 30 minutes, centrifuge at 8000 rmp for 20 minutes, and discard the supernatant. Resuspend the precipitate by adding 4 mL of Tris buffer per 1 g of weight, stir well for 30 minutes, and standardize it.

[0090] 8.3 Standardization of Rose Bengal plate agglutination test antigen of Brucella rough strain RB39: Dilute the Rose Bengal staining antigen 1:3, 1:4, 1:5, and 1:6 times with Tris buffer. Dilute the positive serum of Brucella rough strain RB39 to 10 IU / mL and 5 IU / mL with Tris buffer. Take 0.03 mL of sera with different dilutions and perform a plate agglutination reaction with an equal volume of staining antigen with different dilutions, and observe the results within 4 minutes. At the same time, set negative serum and positive serum of smooth Brucella as controls. When the positive serum of Brucella rough strain RB39 shows a "+" reaction at 10 IU / mL and a "-" reaction at 5 IU / mL, the antigen dilution is the antigen use concentration, and the results are shown in Table 6. According to the results in Table 6, dilute the staining antigen to the use concentration (1:4) with Tris buffer to obtain the Rose Bengal plate agglutination test antigen of Brucella rough strain RB39.

[0091] Table 6 Results of standardization of Rose Bengal plate agglutination test antigen of Brucella rough strain RB39

[0092]

[0093] Note: ++: Obvious agglutination particles, slightly transparent liquid; +: Slight agglutination visible, turbid liquid; -: No agglutination, evenly turbid liquid.

[0094] Example 9 Differential Diagnosis Test after Immunizing Animals with Live Rough Brucella Vaccine (Strain RB39)

[0095] 9.1 Immunization: Dilute the RB39 vaccine with normal saline to contain 1 dose per 1.0 mL, and subcutaneously inject 5 adult cattle at a dose of 1 dose / head as the immunized group; at the same time, set up non-immunized controls, 5 heads / group. Dilute the RB39 vaccine with normal saline to contain 1 / 4 dose per 1.0 mL, and subcutaneously inject 5 adult sheep and 5 goats at a dose of 1.0 mL / head as the immunized group; at the same time, set up non-immunized controls, 5 heads / group.

[0096] 9.2 Blood Sampling: 14 days after immunization, collect blood from all animals in the immunized group and the control group to separate serum. At the same time, collect animal sera from cattle, goats, and sheep farms that have not been immunized with Brucella vaccine, and select 5 serum samples each that are detected as positive by both the Rose Bengal Plate Agglutination Test (smooth type) and the Complement Fixation Test as clinically infected positive sera.

[0097] 9.3 Differential Diagnosis Test: Perform plate agglutination tests on the sera of animals immunized with the RB39 vaccine, the negative sera of the non-immunized control group, and the clinically infected positive sera using the Rose Bengal Plate Agglutination Test antigen of rough Brucella strain RB39 and the Rose Bengal Plate Agglutination Test antigen of smooth type. The results are shown in Table 7. The sera of animals in the groups immunized with live rough Brucella vaccine (strain RB39) in cattle, goats, and sheep all showed positive reactions (+) with the Rose Bengal Plate Agglutination Test antigen of rough Brucella strain RB39 and no reactions (-) with the Rose Bengal Plate Agglutination Test antigen of smooth type; the sera of animals in the clinically infected groups of cattle, goats, and sheep all showed no reactions (-) with the Rose Bengal Plate Agglutination Test antigen of rough Brucella strain RB39 and positive reactions (+) with the Rose Bengal Plate Agglutination Test antigen of smooth type; the sera of the non-immunized control group animals showed no reactions (-) with both antigens. Therefore, it is possible to distinguish the serum antibodies of clinical RB39 vaccine immunization and clinical infection, and achieve the differential diagnosis of Brucella immunization and infection.

[0098] Table 7 Results of Differential Diagnosis by Rose Bengal Plate Agglutination Test

[0099]

[0100]

[0101] Note: +: Positive, with agglutination reaction; -: Negative, no reaction.

[0102] The above are the preferred embodiments of the invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A rough Brucella attenuated strain RB39, which was deposited in the General Microbiological Culture Collection Center of China General Microbiological Culture Collection Administration on February 17, 2025, with the deposit number CGMCC 46377.

2. The rough Brucella attenuated strain RB39 as claimed in claim 1, characterized in that The colonies were identified as 100% rough type by crystal violet staining and Brucella bovis by AMOS-PCR.

3. Application of rough Brucella attenuated strain RB39 as claimed in claim 1 or 2 in the preparation of Brucella vaccine.

4. A Brucella vaccine, characterized in that Including the rough Brucella attenuated strain RB39, which was deposited in the China General Microbiological Culture Collection Center on February 17, 2025, with the deposit number CGMCC46377.

5. Brucella vaccine as claimed in claim 4, characterized in that, Lyoprotectants are also included.

6. Use of the rough Brucella attenuated strain RB39 as claimed in claim 1 or 2 in the preparation of an antigen for detecting Brucella antibodies, wherein the Brucella antibodies are produced by immunizing an animal with the Brucella vaccine as claimed in claim 4 or 5; Through this test, vaccine immunity can be distinguished from natural infection.

7. The use according to claim 6, characterized in that The detection adopts RBT method, SAT method, CFT method or ELISA method.

8. An antigen for detecting Brucella antibodies produced by animals immunized with the Brucella vaccine according to claim 4 or 5, characterized in that: The rough Brucella attenuated strain RB39 is obtained by culturing and inactivating the rough Brucella attenuated strain RB39 as claimed in claim 1 or 2.

9. The antigen according to claim 8, characterized in that The antigen is used for detecting Brucella antibodies by RBT method, and its preparation method comprises: The Brucella attenuated strain RB39 as claimed in claim 1 or 2 is prepared into a bacterial solution with physiological saline to inactivate the bacterial solution; Adding red bengal dye to the inactivated bacterial solution at a volume ratio of 0.5:100 to obtain stained bacterial cells; The stained bacteria are reselected to a preset concentration using Tris buffer.

10. A kit for detecting Brucella antibodies, characterized in that, Comprising the antigen as claimed in claim 8 or 9.

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