The way to obtain the drug on the basis of viral and bacterial strains from the local center for the preparation of the associated vaccine or polyvalent hyperimmune serum against diseases of cattle

 

(57) Abstract:

The invention is intended to receive vaccines or sera against viral and bacterial diseases of cattle. For the preparation of component "A", "B" and "C" of the drug from the local hearth select all the epizootic strains of pathogens of bacterial and viral origin. After identifying them separately cultivated. Viral strains grown on monolayers of cell cultures, such as MDBK. While adding a nutrient medium, such as a Needle, mA'am, to obtain the final concentration of each virus is not less than 1 to 106JRS/50 to 1 ml Drained vaccinated suspension of each strain in equal volumes, in total capacity. Mix and get component "A" of the drug. Part component "And" inactivate formalin at a final concentration of about 0.2-0.4 wt.%. Incubated 3-5 days at room temperature. Get component "B" of the drug. Bacterial strains cultivated in the mattresses on microbiological nutrient agar, for example RM-agar. Get the final concentration of each bacterial strain of not less than 1 to 1011cells/ml and Then collect them in equal volumes in the shared storage capacity with the use of a solution of er is less than 3-5 days at room temperature. Get component "B" of the drug. The invention allows to obtain a generic drug on the basis of local viral and bacterial strains broader spectrum of activity. 2 C.p. f-crystals.

The invention relates to the field of veterinary Microbiology and biotechnology, in particular the production of vaccines or sera against viral and bacterial diseases of cattle.

It is known that against many infectious diseases of cattle (cattle) is not currently developed vaccine and serum preparations (for example, against leukemia) and in some cases not identified by their agents. In connection with the foregoing, the development of associated vaccine or polyvalent hyperimmune serum against local viral and bacterial diseases of cattle is extremely important for veterinary medicine, animal production and health.

A known method of producing drug for the preparation of immunoglobulin "Polyangular", used in the treatment and prevention of viral infections, including separate growing culture of strains of parvovirus "Hercules" VGNKI N 29, adenovirus type-2 "Hell" VGNKI N 15 and distemper EPM (patent Application is comfort separately specified components (cultural strains) of the drug under the scheme: the first injection intramuscularly in the neck and croup in volume 10 - 100 cm3each antigen, the second injection is performed on day 7 in the region of the cervical lymph nodes in the amount of 30 to 90 cm3each antigen, the third and fourth injections take place respectively on the 22nd and 37th day intravenously in a volume of 80 - 120 cm3each antigen. On the 44th day to take blood from each group of horses, separate serum, sterilized and mixed in equal volumes. The mixture is then sera treated with PEG-6000 D and the resulting residue purified by the method of cascade filtration on nitrocellulose filters with a pore diameter of from 1.2 to 0.22 μm. Immunoglobulin "Polyangular" can be used for prophylactic and therapeutic purposes against parvovirus enteritis, adenoviral infections and the plague dogs.

However, polyvalent serum produced using product manufactured by this method can be effective in different regions, such as Russia, due to the fact that they were created on the basis of different antigenically viral strains isolated from animals in other regions of the country. In the preparation of polyvalent sera using product, made by the described method, the dilution of the specific anime viral strains. As a result, the use of such polyvalent sera requires the introduction of their sick animals in large quantities, thus increasing the stress on their immune system, and while receiving a weak therapeutic and prophylactic effects. In addition, the absence of the preparation of antibodies against pathogenic for animals bacteria restricts the range of validity of the immunoglobulin and needs in the treatment and prevention of animal diseases by the introduction of additional sera containing antibodies against bacterial antigens.

The closest technical solution (prototype) is a method of obtaining the drug (bacterial vaccines) against microbacteria cattle, including a preliminary allocation of the local hearth epizootic strain of microbacteria cattle, its identification, the time of the biomass in the culture of the pathogen microbiological method, the separation of the grown culture biomass and the culture fluid extraction of antigen from biomass and parallel to the deposition of an exotoxin from the culture fluid, the inactivation of antigens in formalin, the Association of inactivated antigens with subsequent addition of adjuvant-based mineral is>/P>The disadvantage of this method is the narrow range of action of the drug (vaccine), prepared on its basis, due to the use for the preparation of the drug only one epizootic cultures of nitrobacteria.

Object of the present invention is to provide such a method which would allow a generic drug on the basis of local viral and bacterial strains for the preparation of associated vaccine or polyvalent hyperimmune serum against diseases of cattle over a wide spectrum of action.

This task is solved in that in the method of producing the drug on the basis of local viral and bacterial strains for the preparation of associated vaccine or polyvalent hyperimmune serum against diseases of cattle, including a preliminary allocation of local home of one of the epizootic strains of disease, its identification, the time of the biomass in the culture of the pathogen microbiological method and, if necessary, the inactivation of the biomass of the culture of the pathogen chemical reagent, according to the invention for the preparation of component "A", "B" and "C" of the drug from double the toe after the identification of separately cultivated, and viral strains grown on monolayers of cell cultures, such as MDBK, particination culture of kidney cells calves, with the addition of a nutrient medium (such as a Needle MEM) to obtain the final concentration of each virus is not less than 1106JRS/50 to 1 ml with subsequent discharge vaccinated suspension of each strain in equal volumes, in total capacity when mixed with receiving component "A" of the drug, which inactivate formalin at a final concentration of about 0.2 - 0.4 wt.% and incubated 3-5 days at room temperature to obtain the component "B" of the drug, and bacterial strains cultivated in the mattresses on microbiological nutrient agar (for example agar of Hottinger, RM - agar) to obtain the final concentration of each bacterial strain is not less than 11011cells/ml, followed by their collection in equal volumes in the shared storage capacity with the use of a solution of Earl, the mixture of biomass strains of bacteria inactivate formalin at a final concentration of 0.2 - 0.4 wt.% subsequent incubation of at least 3-5 days at room temperature to obtain the component "B" of the drug.

Getting in drug conectivity actions prepared on the basis of the associated vaccine provides immunity) and hyperimmune serum obtained by immunization of horses with this drug (low titer).

For the preparation of associated vaccine use components "B" and "C" of the drug, which is mixed in equal proportions, and the resulting mixture was further added immunomodulator, for example, T-activin, or timaris, or Radostin, when the ratio of the immunomodulator and the mixture components "B" and "C" of the drug is not more than 1:10.

Immunomodulators are non-specific drugs for more effective action of the vaccine without the manifestation of adverse effects.

Radostin (VFS 42-245-7-94. Radostin for injection. Introduced from 02.03.95) - immunomodulator, dosage form dS-RNA killer strain of yeast Sac. cerevisiae, is in nikti BAS SRC VB "Vector", Novosibirsk. Obtained by enzymatic and mechanical destruction of the cell walls of yeast and extraction of dS-RNA fractionation solution of lithium chloride. The drug restores the immune activity, stimulates hematopoiesis.

T-activin (immunomodulator) preparation of polypeptide nature, derived from the thymus (thymic) cattle, standardized quantitative and functionality of the AET activity of T-killers (the Handbook. Drugs used in medicine. - M., 1989, - S. 370).

Timaris (immunomodulator) preparation of polypeptide nature, obtained by extraction from the thymus (thymic) cattle, restores immunological reactivity (regulates the amount and the ratio of T - and B-lymphocytes and their subpopulations, stimulates hematopoiesis (the Handbook. Drugs used in medicine. - M., 1989, - S. 381).

When the magnification ratio of the immunomodulator and components of the drug more than 1:10 showed a significant toxic effect of immunomodulators on the body of the vaccinated animal.

To obtain a polyvalent hyperimmune serum first produced in equal proportions of the mixture components "B" and "C", and in equal ratio mixture of components "a" and "b", the horse-producers subcutaneously in the neck region spend the first injection of the mixture components "B" and "C" of the drug in a volume of 10 - 20 ml; after 7 days the second injection of the mixture of components "a" and "b" of the drug in a volume of 40 to 50 ml and after another 7 days - the third injection of the mixture of components "a" and "b" of the drug in a volume of 150 - 200 ml; not less than 1.5 months spend again triple immunization in a similar way as was carried out through a sterile sealed system blood sampling up to 10 liters in the bottle and another 5-6 days are repeated blood sampling in the same volume; moreover, in each collected blood sample volume of 10 l impose no more than 300 ml of 10% aqueous solution of sodium citrate, then the material defend for 12 - 24 hours at room temperature and added to each blood sample volume of 10 l of 10 - 15 ml of 30% aqueous solution of calcium chloride, bottles of blood samples shaken and re-assert within 12 - 24 hours at room temperature before the formation of serum with subsequent discharge to a separate container.

The drug with three separate components (component "A" is a mixture of live viral strains, component B is a mixture of inactivated viral strains and the component "B" is a mixture of inactivated bacterial strains, including strains of pathogens derived from local focus allows you to prepare as associated vaccines, providing longer and more intense immunity and polyvalent hyperimmune serum broader spectrum of action and is more specific for cattle in this region, and thus operating more efficiently.

Example 1. The technology of reception of a preparation on the basis of viral and bacterial strains from local hearth

In JSC "Salair" Maslennikova district of the Novosibirsk region autumn is enemy (thermal response of 40.0 - 41,0oC) using the simple (for example agar of Hottinger, RM - agar, thioglycolate environment) and differential diagnostic microbiological environments (environment Altnickol, Endo, bismuth sulfite is selected E. coli and Salmonella. In addition, by conducting 4 passages on transplantable cell culture MDCK, viruses infectious bovine rhinotracheitis and parainfluenza 3.

Viruses produce on the culture of MDCK cells at a final concentration of not less than 1106JRS/50 to 1 ml of Viral biomass is mixed and divided into two parts, one of which (component "A") stored at -18oC in a separate container, and the other is mixed with formalin in a final concentration of 0.3 wt.% and incubated at room temperature for 3 to 5 days from receipt of the component "B" of the drug, which is also stored in a refrigerator at -18oC.

The obtained bacteria accumulating in the mattress on microbiological nutrient agar (RM-agar) and collected with a spatula and mortar Earl in a separate container (the final concentration of each bacterial species is thus 11011cells in 1 ml). Both types of bacteria are mixed together and the obtained mixture is added formaldehyde in the final con is Reparata, which has been stored in the refrigerator at +4oC.

Example 2. Technology of preparation of associated vaccine against diseases of cattle and study of the effectiveness of its action

For the preparation of associated vaccine use components "B" and "C" of the drug, which is mixed in equal proportions, and the resulting mixture was further added immunomodulator, for example timaris, in the amount of 1 ml per 10 ml of the mixture of components "B" and "C" of the drug. Received an associate vaccine research on specific sterility on the culture of MDCK cells and microbiological nutrient medium (RM - agar). In addition, this vaccine is examined for reactogenicity and safety on the response of the organism of laboratory animals (white mice weighing from 14 to 16 g and Guinea pigs weighing 200 - 250 g), which administered the vaccine subcutaneously in a volume of 0.2 ml and 0.5 ml, respectively). Thus prepared, the drug is a vaccine on the basis of local viral and bacterial strains for specific livestock farms (JSC Salair").

Before application of the drug in the household indicators of reactogenicity and safety evaluate the group of isolated calves, ispolzovanie was conducted during 14 - 20 days. The total time associated with the preparation of the final form of the vaccine was 1.5 months. After obtaining good results on isolated calves, vaccination is subjected twice pregnant dry cows for 2 and 1 month before calving and calves at the age of 25 to 30 days and 2 months.

To do this, hold the subcutaneous immunization of animals with the vaccine in the neck. Before use, the vaccine is dissolved in 50 ml of solvent (saline). 1 dose of each component of the drug is 2 ml primary vaccination and 6 ml when re-vaccination. In the result, the death of cattle in the economy decreased in 3 times in comparison with previous years, when this work was not carried out.

Example 3. Technology of production of polyvalent hyperimmune serum against diseases of cattle and study of the effectiveness of its action

To obtain a polyvalent hyperimmune serum first produced in equal proportions of the mixture components "B" and "C" and the mixture of components "a" and "b" of the proposed drug. Thus prepared mixture on the basis of local viral and bacterial strains for specific livestock farms (JSC Salair") used Osada: animal producers subcutaneously in the neck region spend the first injection of the mixture components "B" and "C" of the preparation of 10 - 20 ml After 7 days of holding a second injection of a mixture of components "a" and "b" of the drug in a volume of 40 to 50 ml and after another 7 days - the third injection of the mixture of components "a" and "b" of the drug in a volume of 150 to 200 ml and Then through at least 1.5 months after the last immunization the first cycle conduct a second round of immunization, similar to the first. After 8 to 9 days after the third (last) injection drug component of the second cycle of immunization in horses-producers carry a sterile sealed system blood sampling up to 10 liters in the bottle and another 5-6 days are repeated blood sampling in the same volume. Moreover, in each collected blood sample volume of 10 l enter 300 ml of 10% aqueous solution of sodium citrate. Then the material defend for 12 - 24 hours at room temperature and added to each blood sample volume of 10 l of 10 - 15 ml of 30% aqueous solution of calcium chloride. Bottles of blood samples shaken for foaming with fibrin and re-assert within 12 - 24 hours at room temperature before the formation of serum with subsequent discharge to a separate container.

Before using polyvalent serum household indicators of reactogenicity and safety assess skin (in a volume of 0.2 ml and 0.5 ml, respectively). Monitoring of laboratory and domestic animals should be performed within 14 to 20 days. The specific activity of antibodies to the virus of infectious bovine rhinotracheitis and parainfluenza in the serum was in the neutralization of 1:512 and 1:1024, and to E. coli and Salmonella in the reaction of precipitatio in the gel on Ouchterlony 1:32 to 1: 128. The total time associated with the preparation of the final form of the drug, is 3-4 months. After obtaining good results of the test serum is injected intramuscularly in an amount of 5 to 10 ml once sick calves in JSC "Salair and healthy with preventive purpose. In the result, the death of cattle in the economy decreased in 3 times in comparison with previous years, when this work was not carried out.

When using the drug, manufactured by the proposed method, it is possible to obtain serum containing antibodies to all pathogens in high titers equal to those obtained by immunization of animals with one or two microorganisms.

The widespread introduction of polyvalent hyperimmune sera against local viral and bacterial diseases of cattle are expected to repay enzootic foci of infectious diseases Credicard the risk of human infection, other domestic and wild animals and birds. This will create the opportunity to produce clean (referring to viral and bacterial contamination of meat and dairy products are high quality.

Industrial applicability. The invention can be used in veterinary medicine, applied Microbiology and Virology.

1. The way to obtain the drug on the basis of viral and bacterial strains from the local center for the preparation of the associated vaccine or polyvalent hyperimmune serum against diseases of cattle, including a preliminary allocation of the local hearth epizootic strains of pathogens, their identification, accumulation of biomass crops pathogens microbiological method and, if necessary, the inactivation obtained biomass chemical reagent, characterized in that for the preparation of component "A", "B" and "C" of the drug from the local hearth select all the epizootic strains of pathogens both bacterial and viral origin, which once identified separately cultivated, moreover, viral strains grown on monolayers of cell cultures, such as MDBK, with the addition of nutrient medium, e.g. the Levi vaccinated suspension of each strain in equal volumes, in total capacity when mixed with receiving component "A" of the drug, part of which inactivate formalin at a final concentration of about 0.2 - 0.4 wt.% and incubated for at least 3 - 5 days at room temperature to obtain the component "B" of the drug, and bacterial strains cultivated in the mattresses on microbiological nutrient agar, for example RM-agar, to obtain the final concentration of each bacterial strain of not less than 1 to 1011cells/ml, followed by their collection in equal volumes in the shared storage capacity with the use of a solution of Earl, the mixture of biomass strains of bacteria inactivate formalin at a final concentration of 0.2 - 0.4 wt.% followed by incubation for at least 3 - 5 days at room temperature to obtain the component "B" of the drug.

2. The method according to p. 1, characterized in that for the preparation of associated vaccine use components "B" and "C" of the drug, which is mixed in equal proportions, and the resulting mixture was further added immunomodulator, for example, T-activin, or timaris, or Radostin, when the ratio of the immunomodulator and the mixture components "B" and "C" of the drug is not more than 1 : 10.

3. The method according to p. 1, characterized in that to obtain a polyvalent hyperimmune serum first produced in equal proportions the mixture was put neck conduct the first injection of the mixture components "B" and "C" of the preparation of 10 - 20 ml in 7 days - the second injection of the mixture of components "a" and "b" of the drug in a volume of 40 to 50 ml and after another 7 days - the third injection of the mixture of components "a" and "b" of the drug in a volume of 150 - 200 ml, after not less than 1.5 months repeat a similar threefold introduction of the mixture components, after 8 to 9 days after the third (last) injection drug component in horses-producers carry a sterile sealed system blood sampling up to 10 l carboys and even after 5 to 6 days are repeated blood sampling in the same volume, and in each collected blood sample volume of 10 l impose no more than 300 ml of 10% aqueous solution of sodium citrate, then the material defend for 12 - 24 h at room temperature and added to each blood sample volume of 10 l of 10 - 15 ml of 30% aqueous solution of calcium chloride, bottles of blood samples shaken and re-assert within 12 - 24 h at room temperature before the formation of serum with subsequent discharge to a separate container.

 

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