A method of manufacturing a vaccine against pasteurellosis animals and birds

 

(57) Abstract:

The invention relates to biotechnology and can be used in industrial production of live and inactivated vaccine against pasteurellosis animals and birds. The technical problem of the invention is the replacement of food raw meat as the basis of the nutrient medium on the hydrolysate of waste meat production in the manufacture of a vaccine against pasteurellosis of agricultural animals and birds. The method consists in the following: deep cultivation of pasteurellosis is carried out in a cheaper nutrient medium on the basis of the hydrolyzate of the dry protein concentrate, regulation of pH, partial pressure of dissolved in the culture liquid oxygen and fractional flow of glucose. The resulting culture is concentrated and Packed into vials and lyophilizer (live vaccines) or inactivate, mixed with adjuvant and Packed into vials (inactivated vaccines) or used to prepare multivalent and complex vaccines. The method allows to reduce the cost of vaccine, to improve stability and standardization of the biological properties of drugs. table 1.

The invention relates to agricultural production and can be ispolnenii animals and birds.

Known methods for producing vaccines against pasteurellosis of agricultural animals and birds (1, 2).

Existing methods for the production of vaccine against pasteurellosis of agricultural animals and birds have a serious drawback: the cultivation of the microorganisms is carried out in an environment based on expensive and scarce food protein - meat hydrolysate on Hottinger; preparations made with the use of this hydrolysate, do not have sufficient stability, custom, production costs are high.

An object of the invention is the replacement of food raw meat as the basis of the nutrient medium on the hydrolysate of waste meat production in the manufacture of a vaccine against pasteurellosis of agricultural animals and birds to ensure the stability of the biological properties of drugs and reduce their costs.

It is due to the fact that periodic controlled cultivation of pasteurellosis is carried out in a cheaper nutrient medium on the basis of the dry protein concentrate (SBC) (3), containing an extract of fodder yeast (FS 42-386 SU 91), sodium chloride (GOST 4233-77), sodium phosphate disubstituted (GOST Modact processing special meat-and-bone broth, allows you to get cheap and standard product with high protein content (up to 95%), providing a stable high accumulation of cultivated microorganisms. In hydrolysate SBC contains 27 amino acids compared to 18 amino acids in meat preware. Cultivation of pasteurellosis is carried out with the regulation of pH, partial pressure of dissolved in the culture liquid oxygen (pO2), redox potential (eH) and fractional supply of glucose. The resulting culture is concentrated and Packed into vials and liofilizarea (live vaccines) or inactivate, mixed with adjuvant and Packed into vials (inactivated vaccines).

The method is as follows. In a sterile fermenter download nutrient medium on the basis of the hydrolyzate SBC, prepared according to the following recipe, wt.%:

The hydrolysate of the dry protein concentrate (dry matter) - 4,5-6,0

Extract fodder yeast - 0,2-0,8

Sodium chloride - 0,1-0,5

Sodium phosphate disubstituted - 0,3-1,3

Distilled water - Rest

Nutrient medium in the fermentor inoculated with culture of one of the production strains of pasteurellosis (Pasteurella multocida strains - "AB", "K", A-1231,-681, D-T-80 and within 6-8 h in accordance with known control algorithm process: regulation of pH, partial pressure dissolved in the culture liquid oxygen (pO2), redox potential (eH) and fractional supply of glucose (1).

The resulting culture is concentrated and Packed into vials (live vaccines) or inactivate, mixed with adjuvant and Packed into vials (inactivated vaccines). Examples of the method

Example 1. A method of manufacturing a vaccine with a minimum of parameter values. For the cultivation of pasteurellosis use liquid nutrient medium on the basis of the hydrolyzate SBC, prepared according to the following recipe, wt.%:

The hydrolysate of the dry protein concentrate (dry matter) - 4,5

The fodder yeast extract and 0.2

Sodium chloride - 0,1

Sodium phosphate disubstituted - 0,3

Distilled water - Rest

Nutrient medium contains 150,0 mg% amino nitrogen and has a pH of 7.8 to 8.0. Before planting the pH of the nutrient medium was adjusted to 8.2 and 8.6 10% solution of sodium hydroxide. The environment is seeded with a culture of pasteurellosis (Pasteurella multocida), grown in liquid rigorous environment of similar composition, and cultivated at a temperature of (371)oC for 6-8 h

Seeded culture is kept on the level of saturation dissolved in pivilege potential (eH) minus 150-30 mV. Then include the stirrer and aeration to maintain pO2level (155)%. The air flow rate is gradually increased up to 1.5 volume min volume environment (culture). the pH of the culture within 2-3 h is reduced to 8.0, and then include the regulation of pH using the automatic document feeder 10% solution of sodium hydroxide and stabilize the pH at this level until the end of cultivation. At the same time increase pO2$ (355%%).

In the glucose solution: after 1.5 and 3 hours serves 0.3% glucose solution in terms of the volume of the culture. In the future, glucose serves the same doses while limiting growth, characterized by cessation of reducing the pH of the culture fluid, and a significant increase pO2with constant aeration. With abundant pricing serves sterile antifoam - propanol B-400 is based 0,001-0,003%.

The accumulation of biomass in 1 cm3environment is 10.2 billion microbial cells (M. K. ). The resulting culture inactivate add adjuvant and Packed into vials (monovalent) or mixed with other inactivated cultures of pasteurellosis or other microorganisms, add adjuvant and Packed into vials (polyvalent vaccine). The culture of other strains of pasteurellosis get a similar way. Vacci the EAN. The method is carried out similarly to example 1, except for the cultivation of pasteurellosis use liquid nutrient medium on the basis of the hydrolyzate SBC, prepared according to the following recipe, wt.%:

The hydrolysate of the dry protein concentrate (dry matter) - 5,25

Extract fodder yeast - 0,50

Sodium chloride - 0,30

Sodium phosphate disubstituted - 0,80

Distilled water - Rest

Nutrient medium contains 175,0 mg% amino nitrogen. The environment is seeded with a culture of pasteurellosis grown in liquid nutrient medium of the same composition, and cultured in accordance with known control algorithm process. The accumulation of biomass in 1 cm3environment is 21.6 billion m K. the Obtained culture inactivate add adjuvant and Packed into vials (monovalent) or mixed with other inactivated cultures of pasteurellosis or other microorganisms, add adjuvant and Packed into vials (or polyvalent complex vaccine). The culture of other strains of pasteurellosis get a similar way. The vaccine is stable physico-chemical and biological properties, including safety and immunogenicity.

Example 3. A method of manufacturing a vaccine with max the use of liquid nutrient medium on the basis of the hydrolyzate SBC, prepared according to the following recipe, wt.%:

The hydrolysate of the dry protein concentrate (dry matter) - 6,0

Extract fodder yeast - 0,8

Sodium chloride and 0.5

Sodium phosphate disubstituted - 1,3

Distilled water - Rest

Nutrient medium contains 200.0 mg% amino nitrogen. The environment is seeded with a culture of pasteurellosis grown in liquid nutrient medium of the same composition, and cultured in accordance with known control algorithm process. The accumulation of biomass in 1 cm3environment is 14.1 billion m K. the Obtained culture inactivate add adjuvant and Packed into vials (monovalent) or mixed with other inactivated cultures of pasteurellosis or other microorganisms, add adjuvant and Packed into vials (or polyvalent complex vaccine). The culture of other strains of pasteurellosis get a similar way. The vaccine is harmless and immunogen.

Example 4. A method of manufacturing a vaccine with values below the minimum. The method is carried out similarly to example 1, except for the cultivation of pasteurellosis use liquid nutrient medium on the basis of the hydrolyzate SBC, prepared according to the following recipe, wt.%:
BR> Sodium chloride - 0,05

Sodium phosphate disubstituted - 0,20

Distilled water - Rest

Nutrient medium contains 100.0 mg% amino nitrogen. The environment is seeded with a culture of pasteurellosis grown in liquid nutrient medium of the same composition, and cultured in accordance with known control algorithm process. The accumulation of biomass in 1 cm3environment is 5.3 billion m K. the obtained culture was observed polymorphism.

Example 5. A method of manufacturing a vaccine with values above the maximum. The method is carried out similarly to example 1, except for the cultivation of pasteurellosis use liquid nutrient medium on the basis of the hydrolyzate SBC, prepared according to the following recipe, wt.%:

The hydrolysate of the dry protein concentrate (dry matter) - 6,5

Extract fodder yeast - 0,9

Sodium chloride - 0,6

Sodium phosphate disubstituted - 1,4

Distilled water - Rest

Nutrient medium contains 217,0 mg% amino nitrogen. The environment is seeded with a culture of pasteurellosis grown in liquid nutrient medium of the same composition, and cultured in accordance with known control algorithm process. The accumulation of biomass is practical options making multivalent vaccine against pasteurellosis animals and birds listed in the table.

Similar results were obtained in the manufacture of other inactivated and live mono-and polyvalent complex vaccines.

The advantages of the proposed method before known.

Used in the manufacture of live and inactivated vaccine against pasteurellosis of agricultural animals and birds of the nutrient medium on the basis of the hydrolyzate SBC allows to obtain stable physico-chemical and biological properties of drugs, not inferior to the safety and the immunogenicity of vaccines produced using meat hydrolysates.

The use of environment-based hydrolysate SBC reduces the cost of vaccines due to the exclusion from the process expensive food raw meat.

Sources of information taken into account:

1. RF patent N 1566533 And 61 To 39/102, 03.06.1988.

2. Instructions for the manufacture and control of the polyvalent vaccine emulsified against pasteurellosis pigs, approved by the Chief veterinary Department Ministry of Agriculture of the USSR on November 13, 1991

3. RF patent N 2015673, A 23 K 1/00, 1/10, 31.07.92.

A method of manufacturing a vaccine against pasteurellosis animals and birds, including seeding vaccinat yeast sodium chloride, sodium phosphate disubstituted and distilled water, cultivation, followed by concentration and lyophilization or inactivation of bacterial suspension and mixed with adjuvant, characterized in that the liquid nutrient medium is used as the protein product of animal origin hydrolyzed dry protein concentrate, yeast extract - the extract of fodder yeast, and the components of the environment are taken in the following ratio, wt.%:

The hydrolysate of the dry protein concentrate (dry matter) - 4,5 - 6,0

Extract fodder yeast - 0,2 - 0,8

Sodium chloride - 0,1 - 0,5

Sodium phosphate disubstituted - 0,3 - 1,3

Distilled water - the Rest

 

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