The method of making emulsion vaccine protivopedikuleznoy

 

(57) Abstract:

The invention is intended for veterinary Microbiology and biotehnologii. Industrial strains of Pasteurella multocida serotypes a, D and cultivated in a nutrient medium containing broth in Hottinger with the addition of 10-15% serum of cattle, pH of 7.6-7.8 and figure amine nitrogen 200-250 mg%. The obtained bacterial mass is subjected to inactivation by aminoethylethanolamine (AAAI). AAAI contribute to the bacterial suspension to a concentration of 0.5-4%. The mixture is incubated for 12-16 h at 37-38°C. after inactivation of the resulting antigen contribute to Saharsa-gelatin medium. After that, the antigen is combined with an oil adjuvant at a ratio of 3:7, respectively. The invention increases the immunogenic activity and specific vaccine safety. 4 C. p. F.-ly, 4 PL.

The invention relates to the field of veterinary Microbiology and can be used in the development and production of specific prevention of pasteurellosis animals and birds.

Pasteurellosis, haemorrhagic septicaemia, is an infectious disease of animals and birds, as well as human characterizing septicemic phenomena and hemorrhagic inflammatory processes lizblaine is a still small oval-shaped gram-negative bacterium of 0.25-0.5 µm wide and 0.5-2 μm in length. Pasteurella causing disease in different species of animals do not differ in their cultural-morphological and enzymatic properties, but the pathogenicity of their most high for the type of animal from which they are selected. The source of the pathogen is sick and recovered animals. Infection occurs most often through the respiratory tract. The bird was infected most often when Slavyani infected corpses.

In our country pasteurellosis of agricultural animals and birds up to the present time it is widely distributed and causes agriculture significant damage. An important place among the control measures pasteurellosis is vaccination. For the prevention of pasteurellosis know the use of inactivated adsorbed and emulsion vaccines, which are not without drawbacks. The experience of industrial production of inactivated vaccine against pasteurellosis has several decades, however, still the problem of matching funds for the inactivation of pasteurellosis and modes of inactivation, which allows to maximally preserve the native structure of bacteria, continues to be relevant.

A known method of manufacture of sorbed protivopedikuleznogo a 10% solution of aluminum alum and inactivation 0,16-0,18% formalin for 6 days (ed. mon. USSR N 94044, 30 N 6, 17.02.51 year).

The lack of vaccines obtained by this method is the short duration of immunity in vaccinated livestock, while vaccinations are performed twice in relatively high doses, especially for calves: 50 ml - first inoculation, 10 ml - second vaccination.

Also known various methods of inactivation of viruses in the production of vaccines against viral diseases, including the effects on a living virus ethylenimine or its derivatives (patent Germany N 2309329, NCI 30 N 6, 29.08.74 G. ; US patent N 3318775, NCI 424-89, 09.05.67,; patent Germany N 1924303, NCI 30 N 6, 17.12.70,; patent USSR N 594771, A 61 K 39/12, C 12 N 7/04, 07.07.93 year).

The main disadvantage of using ethylenimine lies in its high toxicity and it requires the most stringent precautions.

Disadvantages derived etilenimina (acetylethylenediamine, ethylethylenediamine, dimer etilenimina) consist in the fact that their concentrations used for inactivation of viruses, not suitable for the inactivation of pasteurellosis.

The closest present invention, the set of essential characteristics is a method of making emulsion protivopedikuleznoy vaccinea inactivated antigen with an oil adjuvant (ed. mon. USSR N 1839092, A 61 K 39/102, 39/39, 30.12.93 year).

The disadvantage of the prototype method is that the resulting product has a relatively low immunogenic activity. This is due to the harsh chemical effects of formaldehyde on protein antigens, their partial denaturation, leading to a decrease in their antigenic and immunogenic.

The task of the invention was to find a new chemical reagent to inactivate pasteurellosis and development on its basis of preparation method of emulsion vaccine against pasteurellosis animals and birds with high immunogenic activity and specific security.

The technical result from the use of the invention is to improve immunogenic and specific safety of the drug by maintaining the original properties of the protein antigens of pasteurellosis in inactivation.

This technical result is achieved by the invention, is characterized by the following set of features:

1) a method of manufacturing the emulsion protivopedikuleznoy vaccine;

2) obtaining a suspension culture of pasteurellosis, at least one serotype;

3) inactivation is received from the applications suspension in an effective quantity;

6) AAAI contribute to the bacterial suspension to a concentration of 0.5-4%;

7) inactivation of conduct for 12-16 hours at 37-38oC;

8) the deposition of bacterial antigen by centrifugation;

9) breeding bacterial antigen stabilizing environment;

10) as a stabilizing environment of the use of sacharose gelatin environment;

11) connection bacterial antigen with an oil adjuvant;

12) bacterial antigen and an oil adjuvant combined in the ratio of 4: 6-2:8;

13) bacterial antigen and an oil adjuvant combined in a ratio of preferably 3:7.

The present invention includes the following set of essential features that provide technical result, in all cases, which sought legal protection:

1) preparation method of emulsion protivopedikuleznoy vaccine;

2) obtaining a suspension culture of pasteurellosis, at least one serotype;

3) inactivation of the obtained culture;

4) as inactivant use AAAI;

5) AAAI contribute in bacterial suspension in an effective quantity;

6) breeding bacterial antigen stabilizing environment;

7) connection bacterial antigen a particular form of execution or specific conditions of its use:

1) AAAI contribute to the bacterial suspension to a concentration in the range of 0.5-4%;

2) inactivation of conduct for 12-16 hours at 37-38oC;

3) the deposition of bacterial antigen by centrifugation;

4) as a stabilizing environment of the use of the sugar-gelatin medium;

5) bacterial antigen and an oil adjuvant combined in the ratios 4:6-2:8;

6) bacterial antigen and an oil adjuvant combined in a ratio of preferably 3:7.

Features of the invention, describing the proposed method and the matching with the characteristics of the prototype, including a generic term that reflects the assignment are:

1) a method of manufacturing the emulsion protivopedikuleznoy vaccine;

2) obtaining a suspension culture of pasteurellosis, at least one serotype;

3) inactivation of the obtained culture;

4) connection of bacterial antigen with an oil adjuvant.

Compared with the method of the prototype of the salient features of the invention are:

1) as inactivant use AAAI;

2) AAAI contribute in bacterial suspension in an effective quantity;

3) after inactivation of bacterial antigen diluted in stabilizirawe the specific form of execution, or special conditions of use:

1) AAAI contribute to the bacterial suspension to a concentration in the range of 0.5-4%;

2) inactivation of conduct for 12-16 hours at 37-38oC;

3) bacterial antigen precipitated by centrifugation;

4) as a stabilizing environment of the use of the sugar-gelatin medium;

5) bacterial antigen and an oil adjuvant combined in the ratio of 4: 6-2:8;

6) bacterial antigen and an oil adjuvant combined in a ratio of preferably 3:7.

Through the use of the proposed method obtained emulsion protivopostavleny vaccine with compared to prototype a higher immunogenic activity and specific security.

The achievement of the technical result from the use of the proposed method is explained by the fact that inactivation of pasteurellosis use AAAI that destroying the DNA of the infectious agent, does not change its protein structure and retains its original antigenic properties and immunogenic activity.

In addition, to save antigen from destruction during storage after inactivation of it diluted in a stabilizing environment, which use the sugar-gelatin smeo-technical information and identification of sources containing information about the analogues of the proposed method allowed us to establish that the applicant had not found the source, which is characterized by signs, identical to all the essential features of the proposed method. The definition from the list of identified unique prototype, as the most similar set of features analogue, has allowed to establish the essential towards perceived by the applicant to the technical result of the distinctive features in the proposed method, set forth in the claims.

Therefore, the proposed method meets the condition of patentability "novelty."

To verify compliance of the proposed method to the condition of patentability "inventive step" by the applicant conducted an additional search of the known solutions to identify topics included in the characterizing part of the claims. In the search result set as follows.

Know the use of AEEI for the inactivation of the FMD virus (RF patent N 594771, A 61 K 39/12, C 12 N 7/04, 07.07.73 year). For inactivation of pasteurellosis AAAI used by the authors for the first time. AAAI inactivates Pasteurella also by the reaction of the first porjadka FMD, not acceptable for inactivation of pasteurellosis. For inactivation of pasteurellosis authors proposed to use AEEI in a concentration of 0.5-4%, mostly 1-2%, which goes far beyond the concentration values AAAI used for the inactivation of the FMD virus. This is the essential difference between the proposed method of the invention by the patent RF N 594771.

Another key feature of the proposed method lies in the fact that immediately after inactivation, the authors proposed to dissolve the resulting antigen in stabilizing the environment in which it is advisable to use the sugar-gelatin mixture. The necessity of this operation is due to the fact that the authors of the results of the research showed that during storage antigen is quickly destroyed by the action of enzymes, losing its antigenic properties and immunogenic activity. Breeding pasterilezom antigen saharso gelatin environment can prevent its destruction and stored indefinitely prior to use in emulsion vaccine. Operation breeding pasterilezom antigen in stabilizing the environment proposed by the authors for the first time in the manufacture of inactivated emulsion vaccine against pasteurellosis the obvious way from the prior art, set out in the relevant section of the description (not identified solutions that have the signs consistent with the distinctive features of the proposed method), and revealed no effect provided the essential features of the proposed method transforms to achieve a technical result. Therefore, the proposed method meets the condition of patentability "inventive step".

The essence of the proposed method is illustrated by examples of its execution, which do not limit the scope of the invention.

Example 1.

For the preparation of a series of vaccine use 3 capsules with dry crop production strains Pasterella multocida serotypes A, D and B. In each ampoule make a 2-3 cm3the nutrient medium. As the nutrient medium using a broth in Hottinger with a pH of 7.6-7.8 and figure amine nitrogen 200-250 mg%. The obtained uniform suspension of pasteurellosis each serotype in the amount of 0.3-0.5 cm3sow 2-3 bottle of capacity of 100 cm3containing 40-50 cm3the nutrient medium. At the same time produce control the sowing of each crop in tubes with MPA, MPB, MPB under paraffin oil. Crops grown for 12-16 hours at 37-38oC. In ryut microscopically. To obtain a culture of the second generation pure culture of the first generation of each serotype seeded at 5-15 cm3each serotype in a nutrient medium in three-, five - or desyatiletiye bottles with the contents of the environment 1/2 volume bottles with simultaneous control of seed tube with MPA, MPB, MPB under vaseline oil and bacteriological Cup with MPA.

Matrix culture of the second generation in the quantity required for planting in the reactor is grown in thermostat for 6-8 hours at 37-38oC with 2-3 shuffle culture within 1-2 minutes. From the grown culture make the crops in tubes with nutrient media in Petri dishes with MPA, spend microscopy and determine the concentration of microbial cells, which must be not less than 109CFU/cm3. To obtain bacterial mass matrix culture of pasteurellosis each serotype contribute in a separate reactor with nutrient medium. Matrix culture make in the amount of 5-10% of the working volume of the reactor. The cultivation is carried out at 37-38oC and a pH of 7.4 to 7.7 for 12 hours to obtain a suspension with a concentration of microbial cells 2-41010CFU/cm3. For pH control use solutions of Na2HPO4and NaH2oC. after inactivation of the antigen leave at room temperature under stationary conditions for 18-24 hours. Control of completeness of inactivation is carried out by seeding the contents of the reactor broth and agar for Hottinger and incubation at 37-38oC for 18-24 hours. Growth of bacterial microflora on nutrient media should not be. The resulting antigen clear of ballast proteins and AAAI, and concentrated by precipitation on flow-through centrifuge. Concentrate antigen resuspending in Saharsa-gelatin medium to a concentration of 10-1210CFU/cm3optical standard gisk named after. Lytvyn. After that, the antigen is combined with an oil adjuvant. Thus, the amount of the aqueous phase containing the antigen, is by volume of 30% and an oil adjuvant - 70%. For the manufacture of emulsion vaccines use of the colloid mill. The resulting vaccine is an emulsion belelieu detachment of mineral oil over not stratifying homogeneous emulsion. After thorough shaking, the vaccine takes the form of a homogeneous mass.

Example 2.

Bacteriological control of sterility derived vaccine is as follows. Samples of the vaccine were seeded in serum broth and agar, liquid and dense environment Saburo, MPA, MPB - 3 test tubes at MPB - 2 tubes and 2 bottles, Wednesday Endo - 3 cups. To identify the aerobes are sown in 0.5 cm3vaccines in one tube and 2 cm3in one bottle, and for the identification of anaerobes, respectively at 1 and 5 cm3. Tubes, bacteriological cups, bottles crops in all environments, except Saburo, incubated at (37,00,5)oC, on the environment Saburo if (21,00,5)oC for 7 days. After a specified period of time do change, except crop MPA, Wednesday Endo, agar Saburo and serum agar. Subcultured samples on the same nutrient medium and in the same amounts as at sowing. Secondary crops can withstand 7 days. At the same time they control the sterility of nutrient media. The results of primary and secondary crops are estimated by microscopic examination of crops. Microbial growth on nutrient media is missing.

Example 3.

Counter the varali 6 Guinea pigs and 12 white mice. The drug was administered subcutaneously Guinea pigs in the groin 1 and 2 cm3, white mice in the back in the order of 0.5 cm3. Observation of the animals were carried out over 10 days. All animals remained alive.

To check the reactogenicity of the drug was administered intramuscularly 2 chickens - 5 cm3in breast muscle and two piglets - 10 cm3behind the ear. 14 days after administration of the vaccine in piglets on the injection of the drug remained local changes in tissue sealing, a value from 1 to 3 cm3Seal homogeneous, without softening, painless. At the opening of piglets in the thickness of the muscular layer of the detected light yellow granuloma thick consistency.

Example 4.

For testing immunogenic received vaccines on white mice used 10 animals for each serotype. The vaccine was administered 30 white mice subcutaneously in the dorsal area in the amount of 0.1 cm3(1.5 billion M. K.) once. After 21 days after immunization 10 vaccinated and 5 intact white mice were infected subcutaneously pre-titrated with a lethal dose of pasteurellosis respective serotype. Serotype B was administered at a dose of 3-5 W.m.to. the white mouse (0.5 cm320-hour bouillon Kul is within 24 hours. Vaccinated remained alive for 10 days of observation after the death of control.

Serotype A was administered at a dose of 10-20 W.m.to. the white mouse (0.2 cm320-hour broth culture at dilution 10-7stacked 1 billion W.m.to. 1 cm3the nutrient medium). Control mice fell within 24-36 hours. Vaccinated remained alive for 10 days of observation after the death of control.

Serotype D was administered at a dose of 300 W.m.to. the white mouse (0.3 cm320-hour broth culture at dilution 10-6stacked 1 billion W.m.to. 1 cm3the nutrient medium). Control mice fell within 6 days. Vaccinated remained alive for 10 days of observation after the death of the last control of the white mouse. In zaklucheniye evaluation of vaccines quantitative method.

The results of a comparative study of the effectiveness of protivopostavleny vaccines made from serotypes A, B, and D, obtained using different inactivated, on white mice are presented in table. 1.

Example 5.

For testing immunogenic derived vaccine in rabbits used in 4 animals for each serotype. The vaccine was administered 12 rabbits intramuscularly in the thigh at about the movie were infected intramuscularly in the thigh pre-titrated with a lethal dose of pasteurellosis respective serotype.

Serotype B was administered at a dose of 50 g.m.to. one rabbit (0.5 cm320-hour broth culture at dilution 10-8stacked 1 billion W.m.to. 1 cm3the nutrient medium). Control animals fell within 24 hours. Vaccinated remained alive for 10 days of observation after the death of control.

Serotype A was administered at a dose of 50 g.m.to. one rabbit (0.5 cm320-hour broth culture at dilution 10-8stacked 1 billion W.m.to. 1 cm3the nutrient medium). Control animals fell within 24-36 hours. Vaccinated remained alive for 10 days of observation after the death of control.

Serotype D was administered at a dose of 500 g.m.to. one rabbit (0.5 cm320-hour broth culture at dilution 10-8stacked 1 billion W.m.to. 1 cm3the nutrient medium). Control animals fell within 6-7 days. Vaccinated remained alive for 10 days of observation after the death of control.

The results of a comparative study of the effectiveness of protivopostavleny vaccines made from serotypes A, B, and D, obtained using different inactivated, quantitative method rabbits are presented in table. 2.

Example 6.

and, immunized against pasteurellosis a single administration of 0.5 cm3vaccine subcutaneously in the lower third of the neck, and 5 chickens 21 days of age immunized against pasteurellosis single introduction 0.3 cm3vaccine (W 4.5 billion.m.K.) subcutaneously in the lower third of the neck. One month after immunization experimental and 4 control chickens were infected intramuscularly pre-titrated with a lethal dose of pasteurellosis serotype A: 11 goals at a dose of 1000 mouse LD50(0.25 cm320-hour broth culture at dilution 10-8stacked 2 billion W.m.to. 1 cm3nutrient medium, which amounted to 5000 W.m.to. pasteurellosis) for experimental and control chickens and 2000 mouse LD50(0.5 cm320-hour broth culture at dilution 10-8stacked 2 billion W.m.to. 1 cm3nutrient medium, which amounted to 10000 W. M. K. pasteurellosis) for 5 experienced chick. Control chickens fell for 68-72 hours with bacteriological confirmation of pasteurellosis. Experimental chickens were alive during the 10 days of observation after the death of the last control.

The results of a comparative study of the effectiveness of protivopostavleny vaccines made from serotype A, obtained from the pores 7.

Immunogenic activity of the vaccine in pigs was determined by direct infection of vaccinated pigs (privigna dose 2 cm3, intramuscular injection, 1 animal on the serotype). After 21 days after vaccination animals infected 20-hour broth cultures of pasteurellosis: serotype B - 0.3 cm3, breeding 10-4; serotype A - 0.5 cm3; serotype D - 7 cm3.

Control animals fell and experienced survived for 10 days of observation after the death of control.

Example 8.

Produced experiments on the comparative study of the effectiveness of inactivated protivopostavleny vaccines, obtained using different inactivated, pigs in conditions of PSC "Ukraine" Ivanovo district, Kherson region of Ukraine. Experiments were carried out on 2-month-old pigs during rearing within 3 months. Tests were conducted on the reproductive pig farms. The results of the tests are presented in table. 4.

Thus, the above information shows the implementation of the use of the present invention, the following cumulative conditions:

the method of making emulsion protivopedikuleznoy vaccine, wopmay Microbiology and biotechnology;

for the present invention in the form as it is described in the independent claim, confirmed the possibility of its implementation using the steps described in the application or known before the priority date tools and techniques;

- when using the proposed method achieved technical result provided by the task of invention.

Therefore, the present invention meets the condition of patentability "industrial property".

Sources of information

1. Auth. mon. USSR N 94044, 30 N 6, 17.02.51,

2. Patent Germany N 2309329, NCI 30 N 6, 29.08.74,

3. U.S. patent N 3318775, 424-89, 09.05.67,

4. Patent Germany N 1924303, NCI 30 N 6, 17.12.70,

5. Patent USSR N 594771, A 61 K 39/12, C 12 N 7/04, 07.07.93,

6. Auth. mon.USSR N 1839092, A 61 K 39/102, 39/39, 30.12.93, (prototype).

7. Emelianenko P. A., Koval, C., Kudlay D., and other Veterinary Microbiology. M.: Kolos, 1982, S. 177-180.

8. Radchuk N. A., Dunaev, Century, Kolychev N. M. and other Veterinary Microbiology and immunology. M: Agropromizdat, 1991, S. 217-219.

9. Yartsev M. J. development of the technology of vaccine against pasteurellosis animals and birds. Veterinary medicine, 1996, N 2, S. 17-19.

10. Dusuk P. C., Belk Is

11. Azarian, S. L. study of the reactogenicity and immunogenicity protivopostavleny vaccines with different adjuvants on laboratory animals. In Proc. of the scientific. works "Diagnosis, prevention and control measures of infectious and invasive diseases of agricultural animals and birds in the North Caucasus". Novocherkassk, 1990, S. 52-59.

12. Dusuk P. C., Azarian, S. L., Kim, C. D. and others study of the reactogenicity and immunogenic protivopostavleny vaccines for farm animals. In Proc. of the scientific. works "Diagnosis, prevention and control measures of infectious and invasive diseases of agricultural animals and birds in the North Caucasus". Novocherkassk, 1990, S. 59-63.

13. Azarian, S. L. Finding oil emulsified protivopostavleny vaccines. In Proc. of the scientific. works "Diagnosis, prevention and control measures of infectious and invasive diseases of agricultural animals and birds in the North Caucasus". Novocherkassk, 1990, S. 64-69.

14. Auth. mon. USSR N 1566533, A 61 K 39/102, 15.11.94,

15. RF patent N 2050161, A 61 K 39/102, A 61 B 10/00, 20.12.95,

16. RF patent N 2071662, A 61 K 39/295, 39/102, 39/125, 10.01.97,

17. RF patent N 2099083, A 61 K 39/116, 39/09, 39/102, C 12 N 1/20, 20.12.97,

18. RF patent N 2099084, A 9/102, C 12 N 1/20, 5/00, 27.04.99,

1. The method of making emulsion protivopedikuleznoy vaccines, including the production of suspension culture of pasteurellosis, at least one serotype, inactivation of the obtained culture and connection bacterial antigen with an oil adjuvant, characterized in that as inactivant use aminoethylethanolamine, which is made in suspension culture to a concentration of 0.5 - 4%, and after inactivation of the resulting antigen contribute in stabilizing the environment.

2. The method according to p. 1, characterized in that the inactivation lead for 12 - 16 h at 37 - 38oC.

3. The method according to p. 1, characterized in that after the inactivation of bacterial antigen precipitated by centrifugation.

4. The method according to p. 1, characterized in that as a stabilizing environment of the use of the sugar-gelatin medium.

5. The method according to p. 1, characterized in that the antigen and an oil adjuvant combined in the ratio of 3:7.

 

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