Way of reception of brucellous antigen from strain brucella abortus 19 for manufacturing of uniform brucellous antigen for aa (agglutination assay), cfr (complement fixation reaction) and lcfr (long-term complement fixation reaction), brucellous antigen for rose bengal assay (rba) and brucellous antigen for ring test (rt) with milk, way of manufacturing of brucellous diagnostic serum and diagnostic sets

FIELD: medicine, veterinary science.

SUBSTANCE: group of inventions concerns veterinary science and biotechnology area. The way of reception of a brucellous antigen from the Brucella abortus 19 strain for manufacturing of a uniform brucellous antigen for AA, CFR and LCFR includes reception of an inoculum and propagation of brucellas in deep conditions on a liquid nutrient medium with regulation of level of partial pressure of oxygen dissolved in a culture liquid throughout all propagation process, separation and concentration of evolved bacteriemic cells using ultrafiltration and their inactivation using warming. Thus brucellas propagation in deep conditions on a liquid nutrient medium with regulation of level of partial pressure of oxygen dissolved in a culture liquid throughout all propagation process is performed in a following regimen: temperature 37±0.5°C, pH 6.9-7.2 aeration from 1 hour till 3 hour - 20-25 pO2, from 3 hour till 6 hour - 25-40 pO2, from 6 hour till 15 hour - 40-45 pO2 and from 15 hour till 16-18 hour - 30-35 pO2. Separation and concentration of the evolved bacteriemic cells by ultrafiltration is carried out with use of membranous cartridges. The concentrate of an antigen received by given way is used also for reception of an antigen for Rose Bengal Assay (RBA), for reception of a brucellous antigen for ring test (RT) with milk and reception of diagnostic brucellous serum which, together with blood serum of healthy livestock, make a part of sets for brucellosis diagnostics. The group of inventions allows to raise efficiency of process of brucellosis diagnostics and to improve control at serologic examination of animals.

EFFECT: rising of efficiency of process of brucellosis diagnostics and improvement of control at serologic examination of animals

22 cl, 11 ex

 

Group of inventions relates to the fields of veterinary medicine and biotechnology.

A method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of a single Brucella antigen for RA, PCK and RDS, including the cultivation of the causative agent of brucellosis on the solid nutrient medium in the quarters (see, for example, kas'yanov A.N. and other uniform Brucella antigen for PA, PCK and RDSC, veterinary medicine, 1973, No. 10, p.50-52).

The method requires the use of expensive, difficult to manufacture a dense nutrient medium, large quantities of glassware, when it is used there is a possibility of contact with live culture of Brucella.

A method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of a single Brucella antigen for RA, PCK and RDS, including the production of seed material on the solid nutrient medium, deep cultivation of the strain Br.abortus 19 in a liquid nutrient medium, concentration and purification by ultrafiltration installation and inactivation of the antigen in the bioreactor at a temperature of 80°C for one hour (see, for example, RU 2085212 C1, 27.07.1997). This method is time-consuming, because provides for seed on a dense nutrient medium and the concentration of the bacterial mass in two steps.

A method of obtaining antigen d is I the serological diagnosis of brucellosis, according to which, for the manufacture of antigen used vaccine Brucella abortus strain 19 Brucella bacteriophage (see, for example, SU 1713592 And 23.02.1992). Obtained in this method, the antigen is active, has a high sensitivity, but the method of its production is complex, requires a large consumption of bacterial material, presence of specific Brucella bacteriophage, the operation of centrifuging the suspension dangerous for staff.

The closest is a method for Brucella antigen of Brucella abortus strain 19 for the manufacture of a single Brucella antigen for PA, PCK and RDS, including the production of inoculum and cultivation of the causative agent of brucellosis in deep conditions on liquid nutrient medium with the regulation of the level of partial pressure of dissolved in the culture liquid oxygen throughout the process of cultivation, separation and preconcentration grown bacterial cells by ultrafiltration on the fibers and their inaktivirovanie heating (see, for example, RU 2163141 C1, 20.02.2001). However, this method is time-consuming, because the stage of cultivation of the causative agent of brucellosis is 19-21 hour, the concentration step of bacterial cell - 5-7 hours, and during ultrafiltration frequently a gap fibers, resulting in a loss of bacterial mass and its Contamines is tion by other organisms.

A method of obtaining Brucella antigen for rose Bengal test (BPO), including staining of antigen Brucella rose Bengal (see, e.g., Veterinary drugs. The Handbook. M.: Kolos. Str-187).

A method of obtaining Brucella antigen for the ring response (CR) with milk, including staining of antigen Brucella hematoxylin blue or tetrazolium in cherry red color (see, e.g., Veterinary drugs. The Handbook. M.: Kolos. Str-185).

The task of the group of inventions in terms of method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of a single Brucella antigen for RA, RSK and RDSC, Brucella antigen for BPO, Brucella antigen for CU with milk is unification and simplification of the technology and reduce the time of receipt of antigen for drugs: a single Brucella antigen for RA, RSK and RDSC, Brucella antigen for BPO, Brucella antigen for CU with milk, enhancing quality and improving the culture of its production.

The task in terms of method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of a single Brucella antigen for RA, RSK and RDS is solved in that in the method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of a single brucellosis ant the gene for RA, RAC and RDS, including the production of inoculum and cultivation of the causative agent of brucellosis in deep conditions on liquid nutrient medium with the regulation of the level of partial pressure of dissolved in the culture liquid oxygen throughout the process of cultivation, separation and preconcentration grown bacterial cells by ultrafiltration and inaktivirovanie heating, according to the invention the cultivation of the causative agent of brucellosis in deep conditions on liquid nutrient medium with the regulation of the level of partial pressure of dissolved in the culture liquid oxygen throughout the entire cultivation process carried out in the following mode: temperature 37±0.5°C, pH 6.9 to 7.2, aeration is from 1 to 3 hours - 20-25 Rho2from 3 to 6 hours 25-40 pO2from 6 to 15 hours 40-45 pO2and from 15 to 16 to 18 hours 30-35 pO2and the separation and preconcentration grown bacterial cells carry out ultrafiltration using a membrane cassettes.

The problem is solved also by the fact that the concentration of inoculum is 3.5-5.0 mlgl/cm3.

The problem is solved also by the fact that inaktivirovanie microbial cells by heating is carried out after the completion of their cultivation in a fermenter at a more benign antigen mode: a temperature of 70±1°C, duration - 1 hour.

The problem is solved by Thu the cultivation of the causative agent of brucellosis in deep conditions is carried out for 16-18 hours.

The problem is solved also by the fact that when the concentration using membrane cassettes limit the detention of 20 KD.

The problem is solved also by the fact that when the concentration using membrane cartridge with filter holder ASF-007.

The problem is solved by the concentrating is carried out for 2-2,5 hours.

The problem is solved also by the fact that after concentrating the resulting antigen resuspending sterile 1%fenrisians isotonic to approx 250-300 mlgl/cm3.

The task in terms of method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of brucellar antigen for rose Bengal test (BPO) is solved in that in the method of obtaining Brucella antigen for deferrals, including staining concentrate antigen Bengal pink, according to the invention using the concentrate antigen, obtained as described above.

The problem is solved also by the fact that rose Bengal before staining titrated in a water bath at 40°C, and staining of the antigen is carried out at the same temperature for one hour.

The task in terms of method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of brucellar antigen for the ring response (CR) with milk is solved in that in the method of producing brucellose the antigen ring for reaction with milk, including staining resuspending bacterial mass of the antigen tetrazolium chloride or with hematoxylin, according to the invention using the antigen obtained by the method described above.

The technical result obtained by the claimed group of inventions is increasing accumulation of Brucella reduction of the time of cultivation, increase agglutinating properties by reducing the concentration of microbial cells in a final product, reducing the concentration of bacterial cells and the elimination of contamination by other organisms in the concentration process, and the elimination of staff contact with live culture of Brucella during concentration by ultrafiltration installation and reduce the time staining Brucella antigen for deferrals.

Known principal way to obtain monospecific sera obtained by immunization of rabbits with intravenous single doses of antigen from strain Br.abortus dose containing 109CFU in 1 ml Then 5-7 days after immunization the animals bleed when the titer of serum agglutination of not less than 1:1280, followed by absorption of the serum with Bakassi strain heterologous species of Brucella, incubated at 37°C for 2 h, recover by centrifugation, dissolved in finalcolor R is the target, Packed and stored at 4°C (see, for example, Corbel M.J. et al., Methods for the Identification of Brucella. Ministry of Agriculture, Floweriest and Food. Research Section. Central veterinary Lab. New. Ham. Weighbridge. Surry RT, 153, 13B, 1978).

The disadvantage of this method is small, the sensitivity of the resulting serum.

A method of obtaining diagnostic brucellosis agglutinins serum, such as the immunization of rabbits-producers of live virulent cells .abortus 146 and B.melitensis And-297. Rabbits of the Chinchilla breed weight 2.5-3.0 kg subjected to immunization suspension of living cells in a 0.85%solution of sodium chloride 48-hour agar culture of Brucella. Animals injected subcutaneously three times at four points back along the spine of 0.5, 0.75 and 1.0 ml of a mixture of equal amounts of crops with an equal volume of complete adjuvant's adjuvant at weekly intervals. Two weeks after the final injection of antigen is injected subcutaneously in the region of the inner upper third of the thigh 1.0 ml of the mixture of cultures in a 0.85%solution of sodium chloride. 14 days after immunization of rabbits with totally Deplete. Serum inactivate by heating and can add thimerosal sodium (see, for example, RU 2005781 C1, 15.01.1994).

When using live virulent culture requires strict anti-epidemic regime, the method is tedious and long.

The closest is a method for watering the Lenten diagnostic brucellosis serum which are subjected to immunization cattle aged from 2 to 5-6 years of use non-castrated bulls-producers. Prior to the immunization of animals kept for 1 month in quarantine, followed by Grund-immunization by a single injection of 10 ml of Brucella antigen subcutaneously, and after 1 month of start of immunization. Immunization is carried out in 2-4 cycles with intervals of 20-30 days. Within one cycle spend 5 daily injections of antigen with volumes of 10, 15, 20, 30, 40 ml At the end of the cycle of immunization on the 10th day after the last injection of antigen take a control blood sample for setting the agglutination reaction. In the presence of antibody titer in the serum of the blood of at least 1:3200 spend bloodletting. For immunization of an animal used antigens, inactivated by heating at 60°C for 2 hours followed by the addition of carbolic acid and curing at 37°C for 48 hours. Antigens are suspended cultures .abortus 544, .melitensis 16-M and .suis 1330 (see, for example, Serum diagnostic brucellosis agglutinating. TU-316-82 from 01.06.1982, appr. The SE for the production of bacterial and viral preparations, USSR Ministry of health,).

The disadvantages of the method are the length, the complexity of the process, epidemic risk associated with the use of virulent Brucella cultures, and high is I the cost of the final product.

The invention in part of the way to obtain serum Brucella diagnostic is to reduce the complexity of the process of its production and increased activity of serum.

The task in terms of method of obtaining serum Brucella diagnostic is solved in that in the method of obtaining serum Brucella diagnostic, including hyperimmunization animal-producers Brucella antigen with subsequent bloodletting, Department of whey, its preservation, the test for sterility, the activity and specificity and packaging, is solved by the fact that according to the invention hyperimmunization animal-producers carry out antigen (living culture of Brucella)obtained by the method described above.

As animal producers use oxen.

Hyperimmunization animal-producers spend antigen made from the vaccine strain .abortus 19, subcutaneously in the region of the middle third of the neck according to the following scheme: day 1 - enter 5 cm3antigen (75 mlgl), 10-day - enter the 8 cm3antigen (120 mlgl), 20-21 day - enter 10 cm3antigen (150 mlgl), 27-28 day conduct trial krovavaya, and on 30-31-day - carry out a production bloodletting.

The blood is maintained at a temperature of 37-38°C for 2-3 hours, and then placed in Jolo is ilnik at 2-8°C for 3-5 days, then separate the serum.

The resulting serum preserved with 5%phenol solution in isotonic saline solution at a ratio of 1:10.

The resulting serum preserved by adding 4% of dry boric acid, then heated in a water bath at a temperature of 54-56°C for 40 minutes with constant stirring, and then placed in the refrigerator at 2-8°C for 10 days.

Serum should be sterile, to have a title in RA is not less than 1:1000, and the DNC is not lower than 1:20.

After filling serum implement lyophilization.

Technical result achieved when using the inventive method, is that significantly reduced life cycle hyperimmunization animals, simplified way to obtain serum and significantly reduced its cost by increasing the activity. The method eliminates the possibility of contamination of personnel.

The feature sets for the diagnosis of brucellosis in the prior art is unknown.

The invention in terms of feature sets for the diagnosis of brucellosis in animals is the improved control of the serological study of animal brucellosis.

Set of components for the diagnosis of animal brucellosis in PA, PCK and RDSK according to the claimed invention contains the antigen Brucella one for RA, PCK and GSK obtained by the above method, when the crank brucellosis diagnostic dried, obtained by the above method, and the serum of healthy cattle dried.

Set of components for the diagnosis of brucellosis in animals in the rose Bengal test (BPO) according to the claimed invention contains the antigen Brucella for BPO obtained by the above method, the serum Brucella diagnostic dried, obtained by the above method, and the serum of healthy cattle dried.

Set of components for the diagnosis of brucellosis in animals in the annular reaction (KR) with milk according to the claimed invention contains the antigen Brucella for CU obtained by the above method, and serum Brucella diagnostic dried, obtained by the above method.

The technical result of the group of inventions in terms of sets is the fact that the claimed equipment sets can unify serological diagnosis of brucellosis in animals and to control the accuracy of staging serological reactions in all veterinary laboratories in the country.

The group of inventions is characterized by the following examples.

Example 1.

For obtaining seed production and cultivation of Brucella use sterile nutrient medium on the basis of parivara X is ttinger, containing, vol.%: parivar of Hottinger - 25, glycerin - 2, glucose - 1,5, sodium chloride and 0.5, yeast autolysate native - 3,4, water up to 100. For obtaining seed pure culture of Brucella abortus strain 19 sow in microfilter with this nutrient medium. The concentration of inoculum is 3.5 mlgl/cm3. Cultivated for 24 hours prior to the accumulation of 35 mlgl/cm3.

Industrial cultivation is carried out in-depth method in the bioreactor in a nutrient medium of the same composition. Under cultivation the pH is maintained within the range of 6.9 and a temperature in the range of 36.5°C. the Level of partial pressure of dissolved in the culture liquid oxygen is: 1 to 3 hours 20 Rho2from 3 to 6 hours - 25 pO2from 6 to 15 hours 40 Rho2and 15 to 16 hours - 30 pO2. Time of cultivation is 16 hours, accumulation of cells is 70 mlgl/cm3. After culturing the bacterial suspension concentrate by ultrafiltration using a membrane cassettes limit the detention of 20 KD. Time of concentration is 2.5 hours. Concentrate in the bioreactor resuspending sterile 1%fenrisians isotonic to 250 mlgl/cm3and inactivate by heating in the following mode: a temperature of 70±1°C, duration - 1 hour.

Inactivated bacterial suspension drain the t in sterile bottles and stored in the refrigerator.

The obtained concentrate bacterial suspension used for the manufacture of a single Brucella antigen for PA, PCK and GSK, as well as to obtain Brucella antigen for rose Bengal test (BPO) and for Brucella antigen for the ring response (CR) with milk.

Example 2.

The antigen is produced analogously to example 1, but for obtaining seed production and cultivation of Brucella use sterile nutrient medium on the basis of parivara of Hottinger containing, vol.%: parivar of Hottinger - 20, glycerin - 1, glucose - 1,0, sodium chloride and 0.5, yeast autolysate native - 3,0, water up to 100. For obtaining seed pure culture of Brucella abortus strain 19 sow in microfilter with this nutrient medium. The concentration of inoculum is 5.0 mlgl/cm3. Cultivate 26 hours prior to the accumulation of 45 mlgl/cm3.

Industrial cultivation is carried out in-depth method in the bioreactor in a nutrient medium of the same composition. Under cultivation the pH is maintained within the range of 7.2 and a temperature in the range of 37.5°C. the Level of partial pressure of dissolved in the culture liquid oxygen is: from 1 to 3 hours - 25 pO2from 3 to 6 hours 40 pO2from 6 to 15 hours - 45 pO2and with 15 to 18 hours 35 pO2. Time of cultivation is 18 hours, estimates the group of cells is 75 mltm/cm 3. After culturing the bacterial suspension concentrate by ultrafiltration using a membrane cartridges with filter holder ASF-007. Time of concentration - 2.0 hours. Concentrate in the bioreactor resuspending sterile 1%fenrisians isotonic to 300 mlgl/cm3that inactivate by heating in the following mode: a temperature of 70±1°C, duration - 1 hour.

Inactivated bacterial suspension is poured into sterile bottles and stored in the refrigerator.

The obtained concentrate bacterial suspension used for the manufacture of a single Brucella antigen for PA, PCK and GSK, as well as to obtain Brucella antigen for rose Bengal test (BPO) and for Brucella antigen for the ring response (CR) with milk.

Example 3.

The activity and specificity obtained in examples 1 and 2 antigen is determined during its titration in RA with a standard serum sample against Brucella abortus. The proposed antigen containing fewer cells (17,3-17,5 mlgl/cm3)gives the reaction intensity 2 cross with a serum dilution of 1:500 (final dilution 1:1000), indicating its compliance with the International standard.

Example 4.

Getting Brucella antigen for rose Bengal test (BPO).

To obtain brucellosis and is of Tigana for rose Bengal test (BPO) concentrate antigen, manufactured according to example 1 or example 2, is placed in biomedical. Bengal pink titrated in a water bath at 40°C. Staining of the antigen is carried out at the same temperature. The result is fast, within hours of full bright and uniform staining of the bacterial cell.

Example 5.

The activity and specificity obtained according to example 4 of the antigen is determined during its titration in BPO with a standard serum sample against Brucella abortus. The proposed antigen containing fewer cells (95 mlgl/cm3) gives the reaction intensity in the 4 cross with serum at a dilution of 1:10 containing 100 ME, 1-2 cross with serum at a dilution of 1:20 (50 ME) and a negative reaction with the serum in a dilution of 1:35 (28,5 ME), which indicates its standard.

Example 6.

Getting Brucella antigen for the ring response (CR) with milk.

To obtain Brucella antigen for the ring response (CR) with milk resuspending bacterial mass antigen, produced according to example 1 or example 2, stained with hematoxylin blue or tetrazolium in cherry red color. The activity and specificity obtained in this example, the antigen is determined during its titration with 10 samples fresh fresh or chilled bovine milk containing 2 cm340, 20 and 10 Meantime. The proposed antigen contains fewer cells (95-100 mlgl/cm3), which accounts for its higher activity in comparison with the known diagnosticum.

Example 7.

Obtaining serum Brucella diagnostic.

To obtain serum Brucella diagnostic spend hyperimmunization animal-producers antigen (living culture of Brucella)obtained in example 1 or example 2. As producers use oxen. Hyperimmunization animals spend subcutaneously in the region of the middle third of the neck according to the following scheme: day 1 - enter 5 cm3antigen (75 mlgl), 10th day -

8 cm3(120 mlgl), 20-day - 10 cm3(150 mlgl). On the 27th day conduct trial krovavaya, and at 30 days, if a sufficient level of antibodies in RA, implement the production bloodletting. The blood is incubated at 37°C for 3 hours, and then placed in the refrigerator at 2°C for 3 days, then separate the serum.

The resulting serum preserved with 5%solution of phenol in saline solution in a ratio of 1:10.

The serum is sterile, has a title in RA 1:1000, and the DNC is not lower than 1:20.

Ready serum Packed and lyophilizers.

Example 8.

Obtaining serum Brucella diagnostic carried out analogously to example 7, but hyperimmunization performed subcutaneously in the region of the middle third of the neck according to the following scheme: day 1 - enter 5 cm3antigen (75 mlgl), 10-day - enter the 8 cm3antigen (120 mlgl), 21-day - enter 10 cm3antigen (150 mlgl). On the 28th day conduct of the trial bloodletting, and on the 31st day perform production bloodletting. The blood was kept at 38°C for 2 hours, and then placed in a refrigerator at 8°C for 5 days, after which separate the serum. The resulting serum preserved by adding 4% of dry boric acid, then heated in a water bath at 50°C for 40 minutes with constant stirring, and then placed in a refrigerator at a temperature of 5±3°C for 10 days.

The serum is sterile, has a title in RA 1:1000, and the DNC is not lower than 1:20.

Ready serum Packed and lyophilizers.

Example 9.

Set of components for the diagnosis of animal brucellosis in PA, PCK and GSK.

The kit includes the following components:

- Brucella antigen common to RA, PCK, GSK obtained in examples 1-2,

serum Brucella diagnostic dried, obtained in examples 7-8,

serum negative, representing a lyophilized serum of healthy cattle.

Antigen Brucella common reveals in these serological reactions of specific antibodies in the serum of patients with Brucella is zoom immunized animals or agglutinogens protivopoloznymi vaccines.

Serum Brucella diagnostic contains specific Brucella antibodies, giving a positive serological reactions with Brucella antigen.

The serum is negative, representing the serum of healthy cattle, does not contain specific Brucella antibodies and does not give serological reactions with Brucella antigen.

Both serums are the control, confirming the specificity of the reaction and the correctness of the technology of its production.

Example 10.

Set of components for the diagnosis of animal brucellosis in BPO (rose Bengal test).

The kit includes the following components:

- Brucella antigen for BPO obtained in examples 4-5;

serum Brucella diagnostic dried, obtained in examples 7-8;

serum negative, representing a lyophilized serum of healthy cattle.

The set of components used for serological rapid diagnosis of brucellosis in unimmunized Brucella vaccines animals by the method of plate agglutination reaction with the serum.

Antigen Brucella, painted Bengal pink, reveals plate agglutination reaction of specific antibodies in the serum of patients with brucellosis LM is now.

Serum Brucella diagnostic contains specific Brucella antibodies, giving a positive serological reactions with Brucella antigen.

The serum is negative, representing the serum of healthy cattle, does not contain specific Brucella antibodies and does not give serological reactions with Brucella antigen.

Both serums are the control, confirming the specificity of the plate agglutination reaction and the correctness of the technology of its production.

Example 11.

Set of components for the diagnosis of brucellosis in animals in the annular reaction (KR) with milk.

The set of components used to study milk in the annular reaction to determine the welfare of cattle (farms) cattle brucellosis (Buffalo) and for the rapid diagnosis of brucellosis in the sale of milk in the markets.

The set consists of the following components:

- Brucella antigen for CU obtained in example 6.

serum Brucella diagnostic dried, obtained in examples 7-8.

The Brucella antigen for CU with milk identifies the method annular reaction of specific antibodies contained in the milk of patients with brucellosis in animals.

Serum Brucella diagnostic contains specific brucellosis EN is the body, giving positive ring react with Brucella antigen.

This kit allows you to control the specificity of the rapid method and the correctness of the given reaction.

As shown in the above examples, the technical result achieved in the process of implementing the claimed group of inventions is to increase the accumulation of Brucella reduction of the time of cultivation, increasing agglutinating properties by reducing the concentration of bacterial cells in the finished antigen due to the proposed mode of submerged cultivation, and reduce the time of concentration of bacterial cells with 5-7 hours to 2-2,5 hours and the elimination of contamination by other organisms in the concentration process by replacing the hollow fiber membrane cartridge and exceptions rupture of fibres during the filtration and elimination of staff contact with live culture of Brucella during concentration by ultrafiltration installation for the expense of carrying out their inactivation after culturing in the fermenter.

The proposed method of staining Brucella antigen for BPO allows you to shorten the process from four days to two hours, and the proposed technology hyperimmunization producers can improve the activity of serum brucellin the th diagnostic in RA up to 1000 IU of antibody in 1 ml The use of bundles of serum in the lyophilized form allows twice as long to maintain their activity, and the introduction of the kits antigen, positive and negative serum helps to improve the control serological study of animal brucellosis and to unify the serological diagnosis of brucellosis.

Thus, the task is solved.

1. The method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of a single Brucella antigen for RA, RSK and RDS, including the production of inoculum and cultivation of Brucella in deep conditions on liquid nutrient medium with the regulation of the level of partial pressure of dissolved in the culture liquid oxygen throughout the process of cultivation, separation and preconcentration grown bacterial cells by ultrafiltration and inaktivirovanie by heating, characterized in that the cultivation of Brucella in deep conditions on liquid nutrient medium with the regulation of the level of partial pressure of dissolved in the culture liquid oxygen throughout the entire cultivation process carried out in the following mode: temperature 37±0,5°C, pH 6.9 to 7.2, aeration 1 to 3 h - 20-25 pO2from 3 to 6 h 25-40 pO2from 6 to 15 h for 40-45 pO2and with 15 16-18 h 30-35 Rho2and from the bookmark and the concentration of the grown bacterial cells carry out ultrafiltration using a membrane cassettes.

2. The method of obtaining Brucella antigen according to claim 1, characterized in that the concentration of inoculum is 3.5-5.0 mlgl/cm3.

3. The method of obtaining Brucella antigen according to claim 1, characterized in that inaktivirovanie microbial cells by heating is carried out after the completion of their cultivation in a fermenter in the following mode: temperature 70±1°C, time 1 hour

4. The method of obtaining Brucella antigen according to claim 1, characterized in that the cultivation of Brucella in deep conditions is carried out for 16-18 hours

5. The method of obtaining Brucella antigen according to claim 1, characterized in that when the concentration of bacterial mass with the use of membrane cassettes limit the detention of 20 KD.

6. The method of obtaining Brucella antigen according to claim 5, characterized in that when the concentration of bacterial mass with the use of membrane cartridge with filter holder ASF-007.

7. The method of obtaining Brucella antigen according to claim 1, wherein the concentration is carried out for 2-2,5 hours

8. The method of obtaining Brucella antigen according to claim 1, characterized in that after concentrating the resulting antigen resuspending sterile 1%fenrisians isotonic to approx 250-300 mlgl/cm3.

9. The method of obtaining Brucella antigen of Brucella abortus strain 19 for whom izgotovlenie Brucella antigen for rose Bengal test (BPO), including staining concentrate antigen Bengal pink, characterized in that use concentrate antigen, obtained according to any one of claims 1 to 7.

10. The method of obtaining Brucella antigen according to claim 9, characterized in that the Bengal rose before staining titrated in a water bath at 40°C, and staining of the antigen is carried out at the same temperature for one hour.

11. The method of obtaining Brucella antigen of Brucella abortus strain 19 for the manufacture of brucellar antigen for the ring response (CR) with milk, including staining resuspending bacterial mass of the antigen tetrazolium chloride or with hematoxylin, characterized in that use antigen, obtained according to any one of claims 1 to 8.

12. The method of obtaining serum Brucella diagnostic, including hyperimmunization animal-producers Brucella antigen with subsequent bloodletting, Department of serum, canning and packaging, test for sterility, the activity and specificity, characterized in that hyperimmunization animal-producers carry out antigen obtained by the method described above.

13. The method of obtaining serum Brucella diagnostic indicated in paragraph 12, characterized in that as animal producers use oxen.

14. A method of obtaining a serum b is Wallasey diagnostic indicated in paragraph 12, characterized in that hyperimmunization animal-producers spend subcutaneously in the region of the middle third of the neck according to the following scheme: 1 day enter 5 cm3antigen (75 mlgl), 10-day - 8 cm3(120 mlgl), 20-21 day - 10 cm3(150 mlgl), 27-28 day conduct trial krovavaya, and on the 30-31 day perform production bloodletting.

15. The method of obtaining serum Brucella diagnostic software 14, characterized in that the blood is maintained at a temperature of 37-38°C for 2-3 h and then placed in the refrigerator at 2-8°C for 3-5 days, then separate the serum.

16. The method of obtaining serum Brucella diagnostic indicated in paragraph 15, characterized in that the whey can 5%phenol solution in isotonic saline solution at a ratio of 1:10.

17. The method of obtaining serum Brucella diagnostic indicated in paragraph 15, characterized in that the whey can by adding 4% of dry boric acid, then heated in a water bath at 50°C for 40 min with constant stirring, and then placed in the refrigerator at 2-8°C for 10 days.

18. The method of obtaining serum Brucella diagnostic indicated in paragraph 12, wherein the serum is sterile titers in RA is not less than 1:1000, and the DNC is not below 1:20.

19. The method of obtaining serum is rosellini diagnostic indicated in paragraph 12, characterized in that after filling serum lyophilizer.

20. Diagnostic kit for the diagnosis of animal brucellosis in RA, RSK and RDSC, characterized in that it contains the antigen Brucella one for RA, RSK and GSK obtained according to any one of claims 1 to 8, serum Brucella diagnostic dried, obtained according to any one of PP-19, and the serum of healthy cattle dried.

21. Diagnostic kit for the diagnosis of animal brucellosis in BPO, characterized in that it contains the antigen Brucella for BPO obtained according to any one of p-10, serum Brucella diagnostic dried, obtained according to any one of PP-19, and the serum of healthy cattle dried.

22. Diagnostic kit for the diagnosis of animal brucellosis in Kyrgyzstan with milk, characterized in that it contains the antigen Brucella for CU obtained in paragraph 11 and serum Brucella diagnostic dried, obtained according to any one of PP-19.



 

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15 cl, 11 tbl

FIELD: veterinary.

SUBSTANCE: medication contains metronidazole, tilosin, emulsifier, distilled water and forming base. As forming base medication contains water-oily emulsion of vitamins E and C with following ratio of components, wt %: tilosin - 23-25, metronidazole - 12-15, oily concentrate of vitamin E - 18-20, water solution of vitamin C - 18-20, emulsifier - 0.8-1.0 and distilled water - the remaining part.

EFFECT: medication has prolonged action, low toxicity and high therapeutic efficiency.

4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention concerns area of medical products, in particular to a tablet for preventive maintenance or treatment of bacteriemic diseases at the animals, containing from 20 to 45 wt % of enrofloxacin, from 18 to 35 wt % of lactose, from 5 to 10 wt % of microcrystallic cellulose and from 5 to 20 wt % of meat aromatisers. Besides the invention concerns a way of reception of the specified tablet.

EFFECT: maintenance of optimum mechanical properties of tablets.

2 cl, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention claims application of Xidiphone as bactericide medium and method of bactericide Xidiphone effect in prevention and treatment of inflammatory periodontium and peri-implant tissue diseases. Expressed dosage-dependent effect of Xidiphone is demostrated for Staphylococcus epidermidis and Esherichia coli cultures representing primary complex origin factor of inflammatory periodontium and peri-implant tissue diseases.

EFFECT: novel application of Xidiphone drug (INN ethydronic acid) and respective method of treatment and prevention of inflammatory periodontium and peri-implant tissue diseases.

2 cl, 1 tbl

FIELD: veterinary.

SUBSTANCE: medication for treatment of mammary glands inflammation and traumas in cows contains as active component ketoprofen, and as base silicon-organic glycerohydrogel with composition: Si(C3H7O3)4·xC3H8O3·yH2O, where 3≤x≤10, 20≤y≤40 with dynamic viscosity 4.8-28.5 Pa.s. (20±0.5°C), with component ratio, wt %: ketoprofen 2; silicon-organic glycerohydrogel - the remaining part, medication being prepared by mechanical mixing of components.

EFFECT: invention ensures increase of treatment efficiency.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biotechnology and concerns antigen-containing liposome preparation process. Substance of the invention includes mixing the dissolved H and D fractions of pseudocholera agent antigens, dissolving the neutral lipids in chloroform, blending the mixed dissolved antigen and lipids, preparing the liposomes by phase-inversion evaporation, removing unenclosed material vesicles by dialysis thereby using phosphate buffer 0.01 M, pH 7.2. The prepared liposomes contain 89-93% H and D fractions of pseudocholera agent antigens of size 600-800 nm.

EFFECT: advantage of the invention consists in liposome preparation of high load of pseudocholera antigen.

2 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to gynecology, and concerns remedy for treating bacterial vaginosis, which contains fermented mare's milk of 2-3 day ageing with proteinase activity 475.5 units, obtained using microorganism Lactobacterium bulgaricum and yeasts of genus Torula, and includes lactic acid, ascorbic acid, panthotenic acid, riboflavin, thiamine, vitamin B 12. Course of treatment is 10 days.

EFFECT: invention ensures fast vagina sanation and recovery of its normal microflora.

2 cl, 1 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: claimed group of inventions relate to medicine, in particular to endocrinology and concerns treatment of infectious foot lesions in diabetes. For this purpose oxazolidinone antibiotic linezoide is introduced in efficient amount. Linezolide is able to penetrate into affected with diabetes tissues and accumulate in them in efficient amounts.

EFFECT: treatment of infection caused by antibiotic-resistant strains of microorganisms.

30 cl, 2 dwg, 3 ex, 4 tbl

FIELD: pharmacology, veterinary medicine.

SUBSTANCE: invention is related to the field of veterinary. Preparation consists of the following components, wt %: Gentamicin sulfate - 3.5-4.5, Dioxidin - 0.5-1.5, Disodium salt ethylene diamine -N,N,N',N'-of tetraacetic acid 2-hydrate - 0.01, Water for injections - up to 100. Prior to application preparation is heated up to 37-38°C and injected intramuscularly daily twice in dose of 0.1 ml/kg of body weight for 3-5 days, in case of serious run of disease - for 5-7 days.

EFFECT: increased efficiency of colibacillosis treatment, fast recovery of sick animals, reduced consumption of active substances and their side effect at organism.

10 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method of preparing diagnostic agglutinating serum for pathogenic Yersinia strains involves hyperimmunisation of rabbits with antigen representing 0.4-0.6% formalin inactivated antigen (pYV+) of Yersinia enterocolitica My 03R strain into auricular cranial vein. Immunisation of rabbits with said antigen is fourfold in dosage as follows: 290-310 million kl/ml, 490-510 million kl/ml, 0.99-1.1 billion kl/ml and 1.9-2.1 billion kl/ml, respectively with dosage interval 6-7 days. Further the producer is examined for immunogenic properties. Serum separated from the sampled blood is preserved.

EFFECT: method ensures preparation of high-quality agglutinating serum used in yersiniosis diagnostics in animals.

3 ex

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary science. The method includes preparing an antigen from inactivated and concentrated Pasterella multocida serotypes A, B and D, Haemophilus pleuropneumoniae serotypes I and II, Streptococcus suis serotype II (Castel strain) and administering initial immunisation and hyperimmunisation with increasing antigen doses followed by blood taking and end product recovery. Oxen are used as producers and hyperimmunisation is administered in 7 to 10 intramuscular injections with a total dose of 1800-2400 billion microbial bodies of each antigen at 2-3 days' intervals. Initial immunisation is administered hypodermically to different body areas two times with 10-15 billion microbial bodies injected at first and 20-25 billion microbial bodies injected in 14 days plus 10 cm3 aluminium hydroxide.

EFFECT: method is simple and time-saving and yields 3,2-3,5 times more serum.

2 tbl, 4 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology can be used for development of specific colibacillosis diagnostic aids. According to the invention the method covers producing antibody erythrocytic colibacillosis diagnosticum and provides carrier processing with antibodies to Escherichia antigens being adhesive antigens extracted from Escherichia cultures with phosphate-carbamide buffer 1.8-2.0 M of pH 7.2-7.4 at temperature 40-45°C during 25-30 min. The culture fluid containing Escherichia cell culture and phosphate-carbamide buffer are taken in mass ratio 1:0.4-0.6 respectively. The supernatant from the extraction is heated up at 65-68°C within 25-30 min. Gamma globulin fractions are used as serum antibodies for carrier processing.

EFFECT: method allows for high-quality end product due to improved sensitivity and specificity.

3 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology, can be used for development of specific diagnostic aids. According to the invention the method covers producing antibody erythrocytic pasteurellosis diagnosticum by carrier processing with serum antibodies of Pasteurella antigens being capsular antigens extracted from Pasteurella cultures with 2.0-2.5% sodium chloride brine at 40-42°C during 30-40 min, with centrifuging the extract, separating the supernatant thereafter warmed up at 65-70°C during 15-30 min followed with sterilisation filtration. The serum antibodies for making diagnosticum are gamma globulin fraction or G or M immunoglobulins.

EFFECT: application of the method allows for high-quality end product due to improved sensitivity and specificity.

2 cl, 9 ex

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns preparations, the antibody-containing person, applied to treatment of microbacterial infections. The essence of the invention includes the preparations containing human antibodies IgG either secretory IgA, or their admixture, specific to antigens Mycobacterium tuberculosis and the vaccine of Calmette and Guerin in concentration from 40 to 160 mg/ml and suitable pharmaceutical medium.

EFFECT: advantage of the invention consists in scope expansion.

5 cl, 2 ex, 3 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: substance of the invention involves production of drug product of human antianthrax intravenous immunoglobulin representing 5.0±0.5% protein fraction containing 100% IgG separated from blood plasma of donors tested for absence of human immunodeficiency virus and hepatitis antibodies and immunised with combined anthrax vaccine.

EFFECT: prolonged immunity maintenance.

1 tbl

Neisseria antigens // 2347813

FIELD: chemistry; biochemistry.

SUBSTANCE: invented here are proteins of the meningococcus bacteria Neisseria meningitides (mainly strain B), with immunogenic properties. The proteins have defined amino acid sequences, presented in the description, and are coded with corresponding nucleotide sequences. Description is also given of an antibody, specific to the indicated meningococcus proteins. These proteins, coding their nucleotide sequences, as well as the specific antibody, can be used as an active ingredient in compositions for treating or preventing infection caused by Neisseria meningitides. The presented proteins can be used as antigens for making effective vaccines, immunogenic compositions.

EFFECT: obtaining proteins, used as ingredients for making effective vaccines, immunogenic compositions.

11 cl, 2 tbl, 104 ex

FIELD: veterinary microbiology and biotechnology.

SUBSTANCE: method consists in hyperimmunization of bulls producers with polyvalent antigen followed by sampling blood, separating serum and preserving it, said polyvalent antigen being the one containing cultures of causative agents of colibacteriosis (escherichiosis), salmonellosis, and klebsiellosis and the following proportions of cultures being engaged (in 1 L physiological solution): colibacteriosis causative agents culture 5×1012-6×1012, salmonellosis causative agents culture 5×1012-6×1012, klebsiellosis causative agents culture 5×1012-6×1012, formalin 2.9-3.2 ml.

EFFECT: increased therapeutical and prophylactic properties.

3 cl, 4 tbl, 5 ex

FIELD: veterinary science.

SUBSTANCE: method involves the combined method of treatment of sick animals including using conventional medicinal agents and 10% solution of specific anti-chlamydium immunoglobulins applied on genital organ mucosa tissues in the dose 0.2 ml/kg of animal mass but not above 30 ml per a head. Method is effective in treatment of chlamydium endometritis, vaginitis and balanoposthitis in animals.

EFFECT: enhanced effectiveness of treatment.

2 ex

FIELD: molecular biology, veterinary.

SUBSTANCE: invention proposes isolated DNA sequence (variants) encoding Ehrlichia canis protein of size 30 kDa. Also, invention proposes vector comprising such sequence, recombinant Ehrlichia canis 28 kDa protein encoded by this sequence, a cell-host comprising this sequence, a method for preparing the protein, immunoreactive antibody specific to this protein and a method for inhibition of Ehrlichia canis infection in subject. Recombinant protein of size 28 kDa from Ehrlichia canis shows immune reactivity with respect to serum against Ehrlichia canis. Proposed group of inventions can be used in development of vaccines and serodiagnosticum that shows high effectiveness for prophylaxis of diseases and for carrying out the serodiagnosis.

EFFECT: improved preparing method, valuable medicinal and veterinary properties of protein.

19 cl, 17 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria. The immunogene compositions containing a transferrin-binding fiber and Hsf, and a way of their reception are offered. It is shown, that the combination of these two antigens synergically influences on production of antibodies with high activity in the analysis of bactericidal Serum. The composition can be used in vaccines against Gram-negative bacteria, including Neisseria meningitides, Neisseria gonorrhoeae.

EFFECT: creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria.

56 cl, 10 ex, 1 dwg

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