CN116218707B - Rough sheep brucella attenuated strain, vaccine and application thereof - Google Patents
Rough sheep brucella attenuated strain, vaccine and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biological products for animals, in particular to a brucella crudus attenuated strain for sheep and a vaccine and application thereof. The brucella is a naturally separated brucella crudus attenuated strain of the sheep species, is named as RM101, and has a preservation number of: CGMCC No.25728. The brucellosis live vaccine prepared from the brucellosis RM101 strain of the rough sheep has good safety, immunogenicity and efficacy, can be used for immunity of pregnant animals, does not cause abortion, and overcomes the defect that the conventional vaccine cannot be used for pregnant animals. The vaccine of the invention can realize differential diagnosis through a micro-agglutination test, and solves the technical problem that the existing cloth disease vaccine can not realize differential diagnosis of vaccine immunity and wild bacterial infection clinically.
Description
Technical Field
The invention relates to the field of biological products for animals, in particular to a brucella crudus attenuated strain for sheep and a vaccine and application thereof.
Background
Brucellosis, also known as wavy heat, mediterranean heat or maltese heat, is an infectious disease of both humans and animals. All people can be infected, have no age or sex difference, and can be transmitted by directly or indirectly contacting infected animals or products thereof. After the brucellosis infection of the human beings, the disease course is long, the repeated attack is carried out, the disease is not healed for a long time, and the serious people lose labor force; abortion and sterility occur when livestock are infected, which causes serious harm to animal husbandry production and human health.
Brucella has multiple organism species, such as sheep species, cattle species, pig species, canine species, sheep epididymis species, sarin mouse species, etc., and is respectively sheep species, cattle species and pig species according to the intensity of infected people.
The world brucellosis prevention and control practice repeatedly proves that the well-controlled animal brucellosis is the most economical and effective strategy for preventing and controlling the human brucellosis, and vaccine immunity is the only choice for effective control under the condition of serious brucellosis epidemic. Since the beginning of the 20 th century, researchers have not abandoned developing a suitable vaccine for the control of brucellosis, but to date no more ideal brucellosis vaccine has been obtained.
The low safety is always the pain point of the current brucellosis vaccine. At present, brucellosis vaccines used in China mainly comprise sheep species M5 strain, pig species S2 strain and cattle species A19 strain, and mainly comprise sheep species Rev.1, cattle species S19 and cattle species RB51 strain internationally, none of the brucellosis vaccines can immunize pregnant animals through an injection method, and the brucellosis vaccines have different degrees of infection risks for human beings. Because animal pregnancy is difficult for herdsman to master, the wide application of brucellosis vaccines is limited. Only the problem of injection for immunizing pregnant animals is solved, so that the popularization and the use and the immunity density of brucellosis vaccines in China can be ensured, and the immune protection effect is achieved.
In addition, the inability of brucellosis to perform differential diagnosis is another important factor limiting the wide application of brucellosis vaccines. Several publications internationally report that Brucella gene-deleted vaccine, the deleted gene is usually a non-major antigen gene, the amount of antibody produced in serum is very small, even no detectable antibody can be produced, and clinical differential diagnosis is difficult to realize. The existing vaccine is smooth brucella strain, and the infected wild bacteria are smooth brucella strain, so that the development of safe, efficient and identifiable brucella virus strain is an urgent strategic subject.
Disclosure of Invention
In order to solve the technical problems, the invention provides a brucella crudus attenuated strain.
The brucella strain of the rough sheep is named as RM101 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 9 and 16 of 2022, and the preservation address is: the preservation number of the Beijing city Chaoyang area North Chen Xili No. 1 and 3 is: cgmccno.25728.
The invention also provides application of the brucella minor strain in preparing medicines for preventing and treating diseases caused by brucella.
The invention also provides the application of the brucellosis ragmentosa attenuated strain in preparation of brucellosis vaccines.
The invention also provides application of the brucella ovis attenuated strain in preparation of a reagent for differential diagnosis of diseases caused by brucella ovis.
The invention also provides a live vaccine prepared from the brucella ragmitis attenuated strain.
The invention also provides a freeze-dried live vaccine prepared from the rough brucella ovis attenuated strain, which is characterized by being prepared by freeze-drying the rough brucella ovis attenuated strain and a freeze-drying protective agent according to the proportion of 1:1-7:1.
Further, the freeze-drying protective agent is prepared from gelatin and sucrose according to the proportion of 1:2-1:5.
The invention also provides an inactivated agglutination antigen containing the brucella crudus attenuated strain.
Compared with the prior art, the invention has the beneficial effects that:
the Brucella strain provided by the invention is separated from lymph nodes of sheep and is a natural separation attenuated strain. The brucella strain is identified as sheep species by using a Bruce-ladder method, colony crystal violet staining is determined to be rough, the whole genome size is 3.36Mb, the brucella strain is a weak strain which can be used for vaccine preparation, and experiments show that the brucella strain has toxicity weaker than that of the existing brucella weak strain and has good safety and effectiveness.
The brucellosis live vaccine prepared by the brucellosis strain can prevent brucellosis of cattle and sheep, can be used for injection immunization of pregnant animals, and solves the major defects that the existing brucellosis vaccine cannot be used for pregnant animals and is unsafe for the pregnant animals.
The brucellosis live vaccine prepared by the brucellosis strain can realize clinical differential diagnosis, and solves the worldwide scientific problem that the existing vaccine can not realize clinical differential diagnosis, so that the control can not be accurately and scientifically realized. Serum generated after animals are immunized by the vaccine and serum generated by wild brucella infection can be identified by a microagglutination experiment, so that clinical differential diagnosis is realized.
Drawings
FIG. 1 is a Bruce-ladder PCR electrophoresis diagram, wherein M is Marker 2000;1, brucella melitensis M111 control; isolate B.melitensis 101; negative control;
FIG. 2 shows the results of crystal violet staining of colonies, wherein, A.Sus domestica strain S2 (smooth); B. sheep RM101 strain (rough);
FIG. 3 is a genomic circle map of RM101, wherein A.chromosomal I; B. genome circle map of chromosome II. The outermost circle is a mark of genome size, and each scale is 5kb; the second and third circles are genes on the positive and negative strands of the genome, respectively, with different colors representing different COG functional classifications; the fourth circle is a repeated sequence; the fifth circle is tRNA and rRNA, blue is tRNA, purple is rRNA; the sixth circle is GC content, the light yellow part shows that the GC content of the region is higher than the average GC content of the genome, the higher the peak value is, the larger the difference from the average GC content is, and the blue part shows that the GC content of the region is lower than the average GC content of the genome; the innermost ring is GC-skew, dark gray represents a region with G content larger than C, and red represents a region with C content larger than G;
FIG. 4 is a diagram showing the results of differential diagnosis micro agglutination assay, wherein T2 is a schematic diagram of T1, A, B, C, D rows of agglutination antigens added per well in T1 are coarse agglutination antigens, and the final dilutions of the corresponding 1, 2, 3, 4 columns of agglutination antigens are 1:50, 1:100, 1:200, and 1:400; E. f, G, H the agglutination antigens added in four rows per well are smooth antigens, and the final dilutions of the corresponding 1, 2, 3, 4 columns of agglutination antigens are 1:50, 1:100, 1:200, and 1:400; a, B, C, D, E, F, G, H is a rough positive serum, a rough negative serum, a smooth positive serum, a smooth negative serum, a rough positive serum, a rough negative serum, a smooth positive serum, and a smooth negative serum, respectively, when applied.
FIG. 5 is a graph of gram and Kelvin staining results, wherein A. Kelvin staining is a graph; B. gram staining;
FIG. 6 is a flow chart of the Nanopore library creation.
FIG. 7 is a diagram showing the appearance after lyophilization
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention; reagents used in the examples, if not indicated, were commercially available; the brucellosis live vaccine S2 strain and A19 strain are Chongqing Australian biological products limited company products; m5 is Tiankang biopharmaceutical Co., ltd; the Brucella M111 strain and the Brucella M28 strain are saved for Beijing livestock veterinary research institute of China academy of agricultural sciences.
EXAMPLE 1 isolation and identification of brucella ovis RM101 strain
The biological and biochemical properties of RM101 strain were verified by a series of isolation and identification studies. After the RM101 strain is separated from lymph nodes, the RM101 strain is identified by tests such as gram and kohlrabi staining, serological specificity tests, crystal violet staining, bruce-ladder PCR, toxicity measurement of mice and guinea pigs, and the result shows that the RM101 strain is a rough strain of Brucella melitensis attenuated strain. The strain is preserved by China general microbiological culture Collection center (China center) for 9 months and 16 days in 2022, and the preservation number is: CGMCC No.25728.
1.1 separation
Extracting nucleic acid from sheep lymph tissue of slaughterhouse in Shandong, detecting Brucella infection during high throughput sequencing analysis, grinding the preserved lymph tissue, homogenizing, spreading with rag-type Brucella selective medium plate, 100 ul/plate, and placing in 5% CO 2 Culturing in a constant temperature incubator at 37 ℃ for 5 days. The dewdrop-shaped colonies with neat, round and raised edges on the plate are taken and then streaked and cultured on a new TSA plate. This strain was designated as RM101 strain.
1.2 dyeing discrimination
Gram staining was performed on single colonies growing on the plates, the strains were gram-negative, and the kohl staining was red, as shown in the gram and kohl staining results of fig. 5.
1.3 Biochemical identification
The results of measurement of the general biochemical characteristics of RM101 strain are shown in Table 1. Independent of CO 2 ，H 2 S test positive, can grow on a culture medium containing sulfur and alkali reddish.
TABLE 1 Biochemical Properties of RM101 strain
1.4 serological specificity
The antigen is prepared from culture, and has obvious agglutination reaction with R factor serum and no agglutination reaction with A and M factor serum. After 14 days from infection of guinea pigs with the culture, the collected serum was negative for plate agglutination assay with smooth antigens such as S2, M5, A19, etc., and positive for plate agglutination antigen with M111 antigen, as shown in Table 2. Test tube agglutination assays were performed simultaneously with guinea pig serum antibody titers ranging from 1:80 to 1:320.
Table 2 agglutination of guinea pig antisera of RM101 strain with plates of different Brucella antigens
1.5 species specificity
The Bruce-ladder amplification products were identified by electrophoresis on a 1.5% agarose gel. According to the designed primer, the brucella ovis should have 6 bands of 152bp, 450bp, 587bp, 774bp, 1071bp and 1682 bp. The PCR result of the strain shows 6 specific bands with the sizes of 152bp, 450bp, 587bp, 774bp, 1071bp and 1682bp (shown in the Bruce-ladder PCR electrophoresis of FIG. 1) which accord with the characteristics of Brucella melitensis.
1.6 crystal violet staining of colonies
The colony is dyed with crystal violet, 100% of the colonies are dyed with different colors such as red, blue and purple, the edges are neat, round and drop-out, and the colonies are judged to be coarse-type colonies. As shown in FIG. 2, the results of the crystal violet staining of the colonies are shown.
1.7 toxicity assay
Balb/c mice at 6 weeks of age were immunized and spleen-borne condition was measured, resulting in RM101 strain having a duration (RT 50) of 4.2.+ -. 1.2 weeks in mice, control strain M111 of 6.9.+ -. 1.3 weeks, and S2 of 5.2.+ -. 1.1 weeks. The toxicity of the RM101 strain is lower than that of the attenuated strain M111 and the existing weakest vaccine strain S2, namely the RM101 strain has stronger safety than that of the S2 vaccine strain.
Example 2 safety and effectiveness verification of RM101 strain
2.1 safety test verification
2.1.1 safety of mice
1.0×10 9 The RM101 strain of CFU infects 10 mice subcutaneously in groin, and the mice all show reduced feeding on the first day and return to normal by the next day without other adverse reactions. Proved by safety and effectiveness tests, the result proves that RM101 is an excellent vaccine candidate strain.
2.1.2 spleen content of guinea pigs
At 1X 10 9 CFU/dose of female guinea pigs of 350-400 g 14 days after infection, the measured spleen bacterial load range was 5X 10 3 CFU/g～3.0×10 4 CFU/g meets the requirements of biological preparation for animals of the people's republic of ChinaThe requirement of the product code (2000 edition) on the seed virulence of Brucella vaccine strain, namely the bacterial content of spleen per gram is less than or equal to 2.0X10 5 CFU, based on spleen-containing biomass, verified that RM101 strain was a good vaccine candidate.
2.2 test of effectiveness
From the analysis of the results of 2 independent challenge tests, RM101 strain was 2.5X10 strain 9 ～3.0×10 9 After CFU/dose single immunization, there was no less than about 70% protective effect on M28; has obvious effect of enhancing immunity, and can provide protection of not less than 90% for the guinea pigs with M28 toxicity after secondary immunity. In 2 experiments, the virus can be separated from spleens of all guinea pigs in a control group, and the control is established. Demonstrating that RM101 has better immunogenicity and efficacy.
EXAMPLE 3RM101 strain whole genome sequencing
Complete gene sequencing and sequence assembly were performed by Baimeike biotechnology Co., ltd, and the sequencing flow is shown in the Nanopore library-building flow chart of FIG. 6. After genome assembly, the result shows that the whole genome length of RM101 is 3361521bp, and the genome size is shown in the genome circle diagram of RM101 in FIG. 3.
EXAMPLE 4 study of the ratio of RM101 strain to lyoprotectant
Preparing gelatin and sucrose freeze-drying protective agent according to the weight-volume ratio, namely 12% gelatin and 40% sucrose, sterilizing at 116 ℃ for 30 minutes after preparation, uniformly mixing RM101 strain and gelatin and sucrose protective agent according to the ratio of 7:1, 7:3 and 1:1 respectively, and then subpackaging and freeze-drying.
Lyophilization was performed using lyophilization profiles of-40℃for 5h, -15℃for 12h, -5℃for 2h, and 26℃for 5 h.
After freeze-drying, the content is in a milky loose lump, is easy to separate from the bottle wall, and is quickly dissolved after the diluent is added. As shown in the appearance profile after lyophilization in fig. 7.
The freeze-dried samples are respectively placed at the temperature of minus 15 ℃, and viable bacteria count is detected in sampling mode at the 1 st, 3 rd, 5 th, 7 th, 9 th, 11 th, 13 th and 15 th months, and the result shows that the effect of the RM101 strain and the gelatin sucrose protectant is best when the ratio is 1:1, and the samples can be stored for 12 months at the temperature of minus 15 ℃. The viable count results are shown in Table 3.
TABLE 3 viable count
Example 5 detection of the final product of a brucellosis live vaccine (Rough, RM101 strain)
After the cultured RM101 strain bacterial liquid and the heat-resistant freeze-drying protective agent are freeze-dried, cheng Bulu bacterial live vaccine (coarse strain, RM101 strain) is prepared, the live vaccine is subjected to finished product inspection according to Chinese animal pharmacopoeia (2015 edition three), and various inspection results show that the prepared bacterial live vaccine (coarse strain, RM101 strain) is qualified.
5.1 Properties
Is in milky loose agglomerate, is easy to separate from the bottle wall, and is dissolved rapidly after being added with diluent.
5.2 pure test
The test was performed according to the method of pure test in annex 3306 of the Chinese animal pharmacopoeia (three 2015 edition), and the results were pure.
5.3 mutation test
Sampling, diluting vaccine properly, inoculating pancreatic proteinAgar plates were incubated at 37℃for 4 days, and examined by Brucella colony crystal violet staining (appendix 3301 of Chinese animal pharmacopoeia (2015, three)), 100% of the colonies were stained as coarse colonies.
5.4 viable count
Diluting vaccine with peptone water, inoculating tryptic proteinThe agar plate is used for viable count (appendix 3405 of Chinese animal pharmacopoeia (2015 edition three parts)), and the viable count of each part is not less than 8.0X10 10 CFU。
5.5 safety inspection
The vaccine is diluted to contain 0.1 parts per 1ml, 5 mice with the weight of 18-20 g are injected subcutaneously in the abdomen, 0.25ml each, and all the mice are healthy after 6 days of observation.
5.6 residual moisture determination
The result is 2.2% and lower than 4% according to the annex 3204 of Chinese animal pharmacopoeia (2015 edition three parts), which meets the regulations.
5.7 vacuum measurement
The purple shimmer appears according to the measurement of annex 3103 of Chinese animal pharmacopoeia (2015 edition three parts) and meets the regulations.
EXAMPLE 6 preparation of agglutination antigen for differential diagnosis
6.1 cultivation of bacterial liquid
RM101 and S2 were inoculated into TSA square plates, respectively, incubated in an incubator at 37℃for 4 days, colonies were then washed with physiological saline, and filtered with 4 layers of gauze. Taking a small amount of bacterial liquid for smear microscopic examination, and carrying out heat agglutination and acridine Huang Ningji test simultaneously to obtain a negative reaction.
6.2 inactivation and concentration
Inactivating the bacterial liquid in 6.1 in 70-80 deg.c water bath for 1 hr, cooling, centrifuging to discard supernatant, re-suspending the precipitate bacterial liquid with 0.5% physiological phenol salt solution to make S2 and RM101 bacterial liquid concentration 20 times as high as that of the agglutination antigen stock solution, and storing at 2-8 deg.c.
6.3 standardization of
The concentrated bacterial solutions were diluted as shown in Table 4. Brucellosis agglutination test positive serum national standard each 1ml of lyophilized serum was dissolved in 100ml of 0.5% phenol biological brine in proportion, diluted 1:100, and further diluted as per table 5:
TABLE 4 dilution comparison Table of concentrated bacterial liquids
TABLE 5 dilution comparison Table of concentrated bacterial liquids
A disposable 96-well plate is used, and 7 rows and 5 columns are taken. From left, 50ul of each 1:300 serum diluent was added to each well of row 1, and 50ul of each 1:400, 1:500, 1:600, 1:700 serum diluent was added to each well of rows 2, 3, 4, 5 in order. Then 50ul of each 1:20 dilution liquid of the concentrated bacterial liquid is added from the 1 st row of each hole, 50ul of each 1:24 dilution liquid, 1:28 dilution liquid, 1:32 dilution liquid, 1:36 dilution liquid and 1:40 dilution liquid are sequentially added from the 2 nd row, 3 rd row, 4 th row, 5 th row and 6 th row of each hole, and 50ul of each reference antigen application liquid (namely, 1:20 times dilution liquid of the reference antigen stock solution) is added from the 7 th row of each tube. After mixing each tube of antigen and serum dilutions uniformly, the reaction was observed at 37℃for 24 hours, with a dilution of the concentrated bacteria solution of 1:24 and a dilution of the serum of 1:1000 being optimal.
The above-mentioned 96-well plate operation was repeated with a clean test tube, and the amount of each liquid added was increased to 0.5ml while preparing a turbidimetric tube. The results obtained by the measurement are shown in Table 6:
the antigen stock solution is 20 times thicker than the application solution, so 1ml of the concentrated bacterium solution of RM101 and S2 is respectively taken, and 0.5 percent of phenol physiological saline is added to 1.2ml of the concentrated bacterium solution of RM and S2, thus obtaining the antigen stock solution.
TABLE 6 agglutination test results of different concentrations of antigen on positive serum national standards
EXAMPLE 7 application of Brucella live vaccine (Rough form, RM101 strain) to sheep
7.1 immunization
Taking 10 sheep with negative Brucella antigen and antibody, dividing into two groups of 5 Brucella live vaccine (coarse strain, RM101 strain) with 1 st group for immunization, and subcutaneously immunizing neck for 2×10 10 CFU/alone, group 2 was not immunized as a control.
7.2 observation and blood sampling
The sheep in all immune groups and control groups have good mental state, normal feeding, normal body temperature and no swelling at injection sites after continuous observation for 14 days after immunization. Serum was collected and separated weekly at 1 to 12 weeks after immunization, and the serum was assayed by a microagglomeration method.
7.3 differential diagnosis and serum antibody agglutination test
The antigen stock solutions of RM101 strain and S2 strain prepared in example 6 were used to detect the collected sheep serum.
The 7.3.1RM101 strain and S2 strain antigen stock solution were diluted 1:20-fold to give antigen stock solution.
7.3.2 sheep serum to be tested was diluted 12.5 fold, 25 fold, 50 fold, 100 fold and 200 fold respectively, while negative and positive serum controls were set.
7.3.3RM101 strain and S2 strain are respectively mixed with serum to be detected diluted by different multiples uniformly to be 50ul, added into a 96-well plate, incubated for 24 hours at 36-37 ℃ and observed.
7.3.4 differential diagnosis and agglutination test results
Sheep serum of the immunoblots vaccine (coarse, RM101 strain) reacted with the smooth S2 strain antigen, but reacted with the RM101 strain antigen, and the results are shown in table 7. Thus, the clinical vaccine immunity and wild fungus infection serum antibodies can be distinguished, the differential diagnosis is realized, and the agglutination titer of the serum antibodies is quantified.
TABLE 7 sheep serum antibody agglutination reaction
Note that: n represents no reaction
EXAMPLE 8 use of Brucella live vaccine (Rough form, RM101 strain) on pregnant sheep
8.1 immunization
Taking 6 sheep pregnant for 2-3 months, dividing into 2 groups of 3 sheep, immunizing Brucella live vaccine (coarse strain, RM101 strain) of group 1, and subcutaneously immunizing neck of 2X10 10 CFU/alone, group 2 was not immunized as a control.
8.2 observations
Farrowing is continuously observed after immunization, and the immune group and the control group have good mental state, normal ingestion, normal body temperature and no abortion phenomenon during pregnancy. After parturition, the lambs were normal, and the lambs were observed to grow for 1 month and normal.
8.3 conclusion
The brucella live vaccine (coarse strain, RM101 strain) can be used for pregnant sheep by injection immunization, and is safe without abortion.
EXAMPLE 9 use of Brucella live vaccine (Rough form, RM101 strain) on beef cattle
9.1 immunization
Taking 10 cattle of 1-2 years old, dividing into two groups, 5 groups each, immunizing Brucella live vaccine of group 1 (coarse strain, RM101 strain), and subcutaneously immunizing neck 8×10 10 CFU/head, group 2, no immunization as control.
9.2 observation and blood sampling
After immunization, the cattle in all the immunized groups and the control group are observed continuously for 14 days, and have good mental state, normal ingestion, normal body temperature and no swelling phenomenon at injection sites. Serum was collected and separated weekly at 1 to 12 weeks after immunization, and the serum was assayed by a microagglomeration method.
9.3 microagglutination assay detection antibodies
The antigen stock solutions of RM101 strain and S2 strain prepared in example 6 were used to detect the collected bovine serum.
The 9.3.1RM101 strain and S2 strain antigen stock solution were diluted 1:20-fold to give antigen stock solution.
9.3.2 bovine serum to be tested is diluted 12.5 times, 25 times, 50 times, 100 times and 200 times respectively, and negative and positive serum controls are arranged at the same time.
9.3.3RM101 strain and S2 strain are respectively mixed with serum to be detected diluted by different multiples uniformly to be 50ul, added into a 96-well plate, incubated for 24 hours at 36-37 ℃ and observed.
9.3.4 differential diagnosis and serum antibody agglutination assays
Bovine serum of the immunoblotter vaccine (coarse, RM101 strain) reacted with the smooth S2 strain antigen, but reacted with the RM101 strain antigen, and the results are shown in table 8. Serum generated after the cattle is immunized by the brucellosis vaccine (rough type, RM101 strain) can be used for distinguishing clinical vaccine immunization and wild fungus infection serum antibodies by a microagglutination test, so that differential diagnosis is realized, and the agglutination titer of the serum antibodies is quantified.
TABLE 8 bovine serum antibody agglutination reaction
Note that: n represents no reaction
EXAMPLE 10 application of Brucella live vaccine (Rough form, RM101 strain) to cows
10.1 immunization
Taking 6 cows in milk producing period, dividing into two groups, 3 groups each, immunizing Brucella live vaccine (coarse strain, RM101 strain) of group 1, and subcutaneously immunizing neck 8×10 10 CFU/head, group 2, no immunization as control.
10.2 observations and detection
After immunization, the milk cows are observed to have a transient increase in body temperature, good mental state and normal ingestion. Serum was collected and separated weekly at 1 to 12 weeks after immunization, and the serum was assayed by a microagglomeration method. Milk production was recorded 3 days before and daily after immunization, up to 30 days after immunization.
10.3 differential diagnosis and serum antibody agglutination test
The procedure is as described in example 9 at 9.3, and the results obtained are shown in Table 9. Serum produced after the brucellosis vaccine (coarse type, RM101 strain) is immunized on the dairy cows can be used for distinguishing clinical immunity from wild fungus infection by a microagglutination test, so that differential diagnosis is realized, and meanwhile, the agglutination titer of serum antibodies is quantified.
TABLE 9 agglutination of milk serum antibodies
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Note that: n represents no reaction
10.4 milk yield Effect
The average milk yield of the 2 groups is 18.3kg and 20.8kg respectively 3 days before immunization, the average milk yield of the immunized group is 15.4kg at the 1 st week after immunization, and the average milk yield of the control group is 19.1kg. The average milk yields of the immunized groups were 17.6, 18.5 and 17.9kg at weeks 2, 3 and 4 after immunization, respectively, while the average milk yields of the control groups were 19.2, 20.6 and 20.1kg. The results show that when cows are immunized with a cloth disease vaccine (coarse, RM101 strain), the initial milk yield is affected, but after week 2, recovery is achieved.
EXAMPLE 11 use of Brucella live vaccine (Rough form, RM101 strain) on pregnant cattle
11.1 immunization
Taking 6 cattle with gestation age of 2-3 months, dividing into 2 groups of 3 cattle, immunizing Brucella live vaccine (coarse strain, RM101 strain) of group 1, and subcutaneously immunizing neck 8×10 10 CFU/head, group 2, no immunization as control.
11.2 observations
Farrowing is continuously observed after immunization, and the immune group and the control group have good mental state, normal ingestion, normal body temperature and no abortion phenomenon during pregnancy. After delivery, the calves are normal, the calves are observed to grow normally until 1 month.
11.3 conclusion
The brucella live vaccine (rough, RM101 strain) can be used for pregnant cattle, and is safe without abortion.
Claims (8)
1. A brucella (Brucella Melitensis) attenuated strain of the species brucella, characterized by a deposit number of: CGMCC No.25728.
2. The use of the attenuated strain of brucella ovis according to claim 1 for the preparation of a medicament for preventing a disease caused by brucella or for the preparation of a reagent for diagnosing a disease caused by brucella.
3. Use of a brucellosis minor strain of brucellosis ovis according to claim 1 in the preparation of a brucellosis vaccine.
4. The use of the brucella minor strain of claim 1 in the preparation of a reagent for differential diagnosis of diseases caused by brucella.
5. A live vaccine made from the brucella ragmitis attenuated strain of claim 1.
6. A lyophilized live vaccine prepared from the brucella crudus attenuated strain of claim 1, wherein the brucella crudus attenuated strain and the lyoprotectant are lyophilized at a ratio of 1:1-7:1.
7. The freeze-dried live vaccine according to claim 6, wherein the freeze-dried protective agent is prepared from gelatin and sucrose according to a ratio of 1:2-1:5.
8. An inactivated agglutination antigen comprising the brucella minor strain of claim 1.
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