Method of production of vaccine for animal necrobacteriosis prevention and treatment, vaccine for animal necrobacteriosis prevention and treatment and method of animal necrobacteriosis prevention and treatment

FIELD: medicine.

SUBSTANCE: group of inventions concerns veterinary microbiology and immunology. Method of vaccine production includes cultivation of strain B.necrophomm "0-1" "ВИЭВ" on nutrient medium, inactivation with formalin, biomass isolation from culture fluid by centrifugation, centrifugate isolation and exotoxin ultrafiltration concentration to protein content 5.5-6.0 mg/ml (by Lowry). Exotoxin B.necrophomm of molecular weight 18000-20000 D is produced. It is added with formalin solution to end concentration 0.4% and inactivated within 15 days. Formalin inactivated exotoxin is mixed with oily adjuvant at the following component proportions (mass%): formalin inactivated exotoxin B.necrophorum - 60.0-67.0, oily adjuvant - 33.0-40.0. Then produced suspension is homogenised and end vaccine is packed up. A vaccine is injected intradermally in single or twice dosage 0.2-0.4 ml. Invention enables to simplify method of production of highly effective vaccine, to produce vaccine of reactogenity, as well as to lower vaccination dose and increase convenience of introduction.

EFFECT: development of effective method of necrobacteriosis vaccination in veterinary science.

10 cl, 4 ex

 

The invention relates to the field of veterinary Microbiology and immunology, and can be used for the prevention and treatment of microbacteria animals.

There is a method of making a vaccine for the prevention and treatment of microbacteria, including the allocation of local hearth epizootic cultures of microbacteria, its cultivation, inactivation, the separation of the biomass from the culture fluid by centrifugation, extraction of antigen from biomass by three consecutive freeze-thawing of cells and their homogenization followed by centrifugation and isolation of the supernatant protective antigen, representing the cytoplasmic fraction containing endotoxin, and parallel to this, the selection of exotoxin deposition of gelatin, the Association received antigens in the ratio of 1:3, re-adding inactivator formalin and connection with oil-lanolin adjuvant in a 1:1 ratio. Get the vaccine of the following composition: cytoplasmic fraction containing endo - and exotoxin, taken in the ratio 1:3 - 450-550 ml, formalin - 3,5-5,0 ml mineral oil - 381-497 ml, ml to 1 L. the Vaccine lead subcutaneously once, at a dose of 1-2 ml (see, for example, RU 2043773 C1, 20.09.1995).

The known method involves a preliminary selection of local strains for each of concr the private sector, it is impossible in industrial production. In addition, ethnologica phase separation of the bacterial suspension and the concentration of exotoxin and subcutaneous method of vaccine administration is very time consuming.

There is a method of making a vaccine for the prevention and treatment of microbacteria, including the use of strain B.necrophorum VIEW selected on the basis of the maximum immunogenicity and antigenic activity after cultivation which separate the biomass from the culture fluid by centrifugation, the separated biomass resuspended in physiological solution and the resulting suspension extracted endotoxin repeated freezing and thawing to the complete destruction of bacterial cells and separation from cellular detrita by centrifugation, and the culture fluid secrete an exotoxin, then mix the resulting endotoxin and exotoxin in equal volume ratios and add adjuvant based on mineral oil and lanolin. The resulting vaccine is injected subcutaneously twice at a dose of 2 ml, and then at a dose of 3 ml (see, for example, RU 2109519 C1, 27.04.1998).

There is a method of making a vaccine also ethnological and time-consuming, and two subcutaneous administration of the vaccine is also time-consuming and leads to the formation of granulomas at the injection site.

The known method projectors, the manufacture of a vaccine for the prevention and treatment of microbacteria, including the use of the modified strain F.necrophorum, after cultivation which secrete an exotoxin, and receive three types of exotoxin: "inactivated" (with the addition of formalin), "raw" and "paleoceanic", which is used as vaccines (see, for example, US 5455034 And 03.10.1995). The vaccine is also administered subcutaneously twice at a dose of 4.6-20 ml.

The known method is very time-consuming and laborious used doses are very high.

The present invention is to develop a method that allows to obtain highly immunogenic harmless vaccine, which you can enter the animal intradermally, and thus to reduce the dose of an administered drug.

The problem is solved by a method for manufacturing vaccines for the prevention and treatment of microbacteria includes the cultivation of the strain B.necrophorum "0-1" VIEW on a nutrient medium, inaktivirovanie formalin, the separation of the biomass from the culture fluid by centrifugation, separation of centrifugate and concentrated of exotoxin by ultrafiltration to a protein content of 5.5-6.0 mg/ml (Lowry), obtaining exotoxin with a molecular mass of 18000-20000 D., adding formalin to a final concentration of 0.4%, inaktivirovanie received exotoxin B.necrophorum within 15 days, mixing inactivated by formalin exotoxin with oil is a valuable adjuvant in the following components (in wt.%):

Inactivated by formalin exotoxin B.necrophorum60,0-67,0
oil adjuvant33,0-40,0,

the homogenization of the resulting suspension and packing of the finished vaccine.

The problem is solved also due to the fact that in the process of cultivation B.necrophorum through the medium periodically miss purified nitrogen concentration of exotoxin carry on ultrafiltration plant with hollow fibers having a pore size of 1.5 kDa, and after mixing inactivated by formalin exotoxin B.necrophorum with oil adjuvant homogenization resulting suspension is carried out on colloidal mill.

The problem is solved and due to the fact that is manufactured by the above method, the vaccine contains (in wt.%):

Inactivated by formalin exotoxin B.necrophorum60,0-67,0
oil adjuvant33,0-40,0

Suitable as an oil adjuvant use a light mineral oil or mineral oil HMD-6M.

The problem is solved also due to the fact that received the vaccine administered to the animals one, twice intradermally at a dose of 0.2-0.4 ml

It is expedient to introduce is accino using without needle injector. As without needle injector is advisable to use a injector B-7 or B-7M.

Suitable for the prevention and treatment of cattle in disadvantaged by nitrobacteria agriculture to administer the vaccine twice with an interval of 4-6 weeks and then once every 6 months at a dose of 0.4 ml

It is advisable to prevent microbacteria cattle to introduce the vaccine in conjunction with hyperimmune serum.

For the prevention and treatment of microbacteria deer it is expedient to introduce the vaccine intradermally a single dose of 0.2 ml.

The technical result of the claimed invention is a simplified method of manufacturing a highly effective vaccine with low reactogenicity, and reduced vaccination doses of the vaccine with increasing facilities of its introduction.

The invention is illustrated by the following examples.

Example 1.

Strain B.necrophorum "0-1" VIEW cultivated on medium Kitt-Tarozzi in bottles for 2 days at a temperature of 37°With, then subcultured into vials with the same environment. Grown macrovu sowing the seed is obtained in the bioreactor with a pre-sterilized nutrient medium, made on the basis of parivara of Hottinger, and cultured for 24-36 hours at 37°C. In the process of cultivation through Wednesday periodically miss purified nitrogen. OK what nanii growing culture checked for purity and optical concentration. Inactivate formalin to a final concentration of 0.4%. Grown inactivated culture is divided into biomass and the culture fluid by centrifugation. From the culture fluid is isolated and concentrated exotoxin on ultra-filtration plant with hollow fibers having a pore size of 1.5 kDa to protein content of 6.0 mg/ml (Lowry), then add formalin to a final concentration of 0.4% and incubated at 42°C for 15 days. Get exotoxin with a molecular mass of 18000-20000 Etc. For the manufacture of vaccines received an inactivated exotoxin mixed with oil adjuvant in the following ratio of components (wt.%): inactivated exotoxin - 60,0-67,0, oil adjuvant - 33,0-40,0. Then the vaccine is homogenized in a colloid mill and Packed. As oil adjuvant use a light mineral oil or mineral oil HMD-6M.

Example 2.

The vaccine obtained in example 1 and having the following composition (in wt.%) inactivated exotoxin - 65,0, oil adjuvant (light mineral oil) - 35,0 use in disadvantaged by nitrobacteria sector for the prevention and treatment of cattle.

The vaccine is administered twice with an interval of 5 weeks, then once every 6 months. at a dose of 0.4 ml intradermally using a needleless injector B-7. Su is th was vaccinated about 100 thousand sick and apparently healthy cattle in 25 regions of the Russian Federation.

Vaccine efficacy was 98±0,2%. The vaccine was no inflammation. The vaccine induces immunity within 6 months after vaccination.

Example 3.

To prevent microbacteria cattle injected hyperimmune serum, and then the vaccine obtained in example 1 and having the following composition: inactivated exotoxin - 63,0 wt.%, oil mineral oil adjuvant HMD-6M is 37.0 wt.%. The vaccine is administered twice with an interval of 6 weeks and then once every 6 months at a dose of 0.3 ml intradermally using a needleless injector B-7.

Inspection conducted on the farms of the Moscow region, showed that the vaccine together with hyperimmune serum helps eradicate the disease within 1-1,5 years.

Example 4.

Obtained in example 1 vaccine and having the following composition: inactivated exotoxin - 60,0 wt.%, oil adjuvant (light mineral oil) - 40.0 wt.% used for the prevention and treatment of microbacteria reindeer. Extensive production and testing of vaccines was conducted in deer farms of the Republic of Sakha (Yakutia). Was vaccinated about 110 thousand deer, incidence after vaccination was 1±0.4 percent. The vaccine was administered intradermally once using batigol the CSOs injector BI-7M dose of 0.2 ml.

The tests showed that the vaccine viacommunity, harmless, protect against disease microbacterium 98-99% vaccinated deer and creates immunity duration of 6 months.

Thus, the claimed invention allows to simplify the method of manufacturing a highly effective vaccine against microbacteria animals receiving the vaccine with low reactogenicity, and reduce vaccination vaccine dose and increase the convenience of its introduction.

1. A method of manufacturing a vaccine for the prevention and treatment of microbacteria animals, including cultivation of the strain B.necrophorum "0-1" VIEW on a nutrient medium, inaktivirovanie formalin, the separation of the biomass from the culture fluid by centrifugation, separation of centrifugate and concentration of exotoxin by ultrafiltration to a protein content of 5.5-6.0 mg/ml (Lowry), obtaining exotoxin B.necrophorum with a molecular mass of 18000-20000 D., adding formalin to a final concentration of 0.4%, inaktivirovanie received exotoxin within 15 days, mixing inactivated by formalin exotoxin with oil adjuvant at the following content, wt.%: inactivated by formalin exotoxin B.necrophorum - 60,0-67,0; oil adjuvant 33,0-40,0, the homogenization of the resulting suspension and packing of the finished vaccine.

2. The method according to claim 1, characterized in that h is in the process of cultivation B.necrophorum through the medium periodically miss purified nitrogen, the concentration of exotoxin carry on ultrafiltration plant with hollow fibers having a pore size of 1.5 KDa, and after mixing inactivated by formalin exotoxin B.necrophorum with oil adjuvant homogenization resulting suspension is carried out on colloidal mill.

3. Vaccine for the prevention and treatment of microbacteria animals produced by the method according to claim 1, containing, in wt.%: inactivated by formalin exotoxin B.necrophorum - 60,0-67,0; oil adjuvant 33,0-40,0.

4. The vaccine according to claim 3, characterized in that as an oil adjuvant contains a light mineral oil or mineral oil HMD-6M.

5. Method for the prevention and treatment of microbacteria animals, including the introduction of a vaccine, characterized in that the animals injected intradermally vaccine according to any one of p and 4 one - twice in a dose of 0.2-0.4 ml

6. The method according to claim 5, characterized in that the vaccine is injected with without needle injector.

7. The method according to claim 6, characterized in that as without needle injector use injector B-7 or B-7M.

8. The method according to claim 5, characterized in that for the prevention and treatment of cattle in disadvantaged by nitrobacteria sector vaccine administered intradermally at a dose of 0.4 ml twice with an interval of 4-6 weeks and then once every 6 months.

9. The method according to claim 5, characterized the eat, what's to prevent microbacteria cattle vaccine is administered in conjunction with hyperimmune serum.

10. The method according to claim 5, characterized in that for the prevention and treatment of microbacteria deer vaccine administered intradermally a single dose of 0.2 ml.



 

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3 ex, 1 tbl

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