UA7023U - Method for preparing emulsion vaccine against pasteurella - Google Patents

Abstract

The method for preparing the emulsion vaccine against Pasteurella comprises the production of Pasteurella suspension culture, its inactivation, and addition of the oil adjuvant. The domestic Pasteurella strains are used, namely Pasteurella multocida No. 3 var. D, No. 12 var. A, No. 31 var. B. The bacterial cells are inactivated by dimeromethyleimine added to the culture at a concentration 4-5%, pH 7.2-7.6. Upon inactivation, the antigen is added to the stabilizing medium containing the oil adjuvant.

Description

Description of the invention

The useful model relates to the field of veterinary microbiology, epizootology and biotechnology and can be used in the development and production of means of specific prevention of factor (endogenous) pasteurellosis in young animals of rural areas. animals Pasteurellosis (lat. RazietsgePyoviv) is a zoonotic infectious disease of animals and birds, as well as humans, characterized by septicemic phenomena and hemorrhagic inflammatory processes of the mucous membranes of the respiratory tract and intestines, pneumonia, pleuropneumonia, and edema. The infectious process proceeds according to the classic or factor type. In the first case, the infectious 70 process occurs on potential hosts in the form of hemorrhagic septicemia; in the second - on obligate hosts according to the type of factor disease and has the form of enzootic infectious pneumonia of pasteurellosis etiology.

The causative agent of the disease is a stationary small oval gram-negative bacterium 0.25-0.5 µm wide and 0.5-2 µm long. Pasteurella, which cause diseases in different species of animals, do not differ in their cultural, morphological and enzymatic properties, but their pathogenicity is highest for the 12 species of animals from which they are isolated. The source of the causative agent of infection is sick and diseased animals. Infection occurs most often through the respiratory tract. In our country, factorial (endogenous) pasteurellosis of young farm animals in the form of enzootic infectious pneumonia is widespread and causes considerable damage to agriculture. Vaccine prophylaxis occupies an important place among the measures to combat pasteurellosis. There is a known method of producing a sorbed anti-pasteurellosis vaccine, which includes suspension cultivation of production strains of Pasteurella, precipitation of the obtained culture with a solution of aluminum alum 1096 and inactivation with 0.16-0.18956 formalin for 6 days. (AS USSR Mo94044, cl. ZONb, 17.02.51

The disadvantage of the vaccine obtained by this method is the short-term immunity of vaccinated farm animals.

A known method of inactivating viruses in the production of vaccines against viral diseases in which ethyleneimine is used as an inactivator (AS USSR Mo1594771 AbЭ1КЗ39/12, 12М7/04, 07.07.93).

The disadvantage of using ethyleneimine is its high toxicity, and also the disadvantage is that the concentrations of ethyleneimine used for inactivating viruses are not suitable for inactivating Pasteurella.

The closest to the proposed useful model, in terms of the set of essential features, is the method of manufacturing She emulsion anti-pasteurellosis vaccine, which includes obtaining a suspension of the pathogen, its inactivation with a 0.5-490 He solution of acetylethyleneimine and the subsequent connection of the inactivated antigen with an oil adjuvant ( patent of the Russian Federation

M2162339, cl. Ab61K39/102, A6E11I 2/16, C12M1/00, 04/17/2000|. The disadvantage of the method is that the obtained drug has relatively low immunogenic activity when used in Ukraine. This is due to the inconsistency of antigenic determinants of production strains of Russian origin, epizootic variants circulating in Ukraine, which leads to a decrease in their antigenic and immunogenic activity. This solution can be a prototype.

The basis of a useful model is the task of developing a process for the production of an emulsion "anti-pasteurella vaccine, which includes obtaining a suspension of a pasteurella culture, inactivating the obtained 8 culture and combining a bacterial antigen with an oil adjuvant by using as immunogens the strains of 50 pasteurellas: Ravzietsgeia tiyosida MoZ serovar D, Mo12 serovar A, Mo31 serovar B, which are inactivated by a dimer with ethyleneimine (DEI), which is added to the culture suspension to a concentration in the range of 4-595 with a corrected pH of 7.2-7.6,

After inactivation, the obtained antigen is introduced into a stabilizing medium and combined with an oil adjuvant to ensure the process of manufacturing an emulsion anti-pasteurellosis vaccine. The technical result of using the cinnamon model is to increase the immunogenic activity and specificity of the drug by selecting domestic (regional) strains and preserving the original properties of pasteurized proteins and antigens during inactivation. av | The specified technical result is achieved by creating a useful model, characterized by the following set of features: o 1) the process of manufacturing an emulsion vaccine against factor (endogenous) pasteurellosis of the young of rural areas. se" 20 animals; 2) obtaining a culture suspension of pasteurella, at least one serovar from domestic strains;

C) inactivation of the obtained culture; 4) dimerethyleneimine (DE) is used as an inactivant; 5) DEI are introduced into the bacterial suspension in an effective amount;

SS o5 6) WHERE! added to the bacterial suspension to a concentration of 4-595 with adjusted hydrochloric acid to a pH of 7.2-7.6; 7) inactivation is carried out for 12-16 hours at 37-389C; 8) deposition of bacterial antigen by centrifugation; 9) dilution of the bacterial antigen with a stabilizing medium; 10) sucrose-gelatin medium is used as a stabilizing medium; 60 11) combination of bacterial antigen with oil adjuvant; 12) bacterial antigen and oil adjuvant are combined in a ratio of 3:7-1:1.

Thanks to the use of the proposed process, a vaccine was obtained against factor (endogenous) pasteurellosis of the young of the rural area. animal emulsion inactivated, which has, in comparison with the prototype, higher immunogenic activity and specific safety. because the achievement of the technical result from the use of the proposed process is due to the fact that highly immunogenic epizootic strains of domestic origin were used: Ravigeigeia thiosida Mo3 serovar D,

Mo12 serovar A, Mo31 serovar B.

In addition, the vaccine is intended for the prevention of enzootic infectious pneumonia of calves and piglets of pasteurellosis etiology (factorial infection).

Therefore, the proposed process meets the conditions of patentability and the "novelty" criterion.

The process of making an emulsion vaccine is carried out as follows.

Example 1

For the preparation of a series of vaccines, Z ampoules with a dry culture of the production strains of Raziecshgeiia 70 tiyosides of serovars A, O and B were used. In each ampoule, 2-3 cm? nutrient medium. Hottinger's broth with a pH of 7.2-7.6 and an amino nitrogen index of 200-250mg9o was used as a nutrient medium. 2-3 vials with a capacity of 100 cm3, containing 40-50 cm? nutrient medium.

At the same time, control sowings of each culture were made in test tubes with MPA, MPB, MPPB under petroleum jelly. 15 Crops were cultivated for 12-16 hours at 37-389С. As a result, the culture of the first generation of pasteurella of each serovar was obtained. Cultures of the first generation were studied microscopically. To obtain a culture of the second generation, a pure culture of the first generation of each serovar was sown in 5-15 cm3 of each serovar on a nutrient medium in three-, five-, or ten-liter bottles with a medium content of 1/2 the volume of the cell with simultaneous control sowing in test tubes with MPA, MPB, MPPB under vaseline oil and in 20 bacteriological cups with MPA. The matrix culture of the second generation in the amount required for seeding in the reactor was grown in a thermostat for 6-8 hours at 37-389C with 2-3 stirrings of the culture for 1-2 minutes. From the grown culture, inoculations were made in test tubes with nutrient media, Petri dishes with MPA, microscopy was performed and the concentration of microbial cells was determined, which should be at least 109 CFU/cm. To obtain the bacterial mass, the pasteurized matrix culture of each serovar was introduced into a separate reactor with 25 nutrient medium. The matrix culture was introduced in a volume of 5-1095 of the working volume of the reactor. /Ш-

Cultivation was carried out at 37-3892C and pH 7.4-7.7 for 12 hours until a suspension with a concentration of microbial cells of 2-4x109 CFU/cm3 was obtained. To adjust the pH, solutions of MaoHRO and ManoroO»u were used. Grown Pasteurella cultures of each serovar were brought to a single dose of the lowest concentration with the help of a buffered physiological solution, mixed in equal proportions, and pumped into a sterile reactor. The resulting bacterial mixture was subjected to inactivation, DEI. For this, DE was added to the bacterial mixture! to a concentration of 4-595, and inactivated for up to 12 hours under stirring conditions at 50-80 rpm and a temperature of 37-382C. At the end of the inactivation, the antigen was left at room temperature in stationary conditions for 18-24 hours. Control of the completeness of inactivation was carried out by sowing the contents of the reactor on broth and Hottinger agar and incubation -

Зз5 were seeded at 37-382С for 18-24 hours.

Bacterial microflora should not grow on nutrient media. The obtained antigen was purified from ballast proteins and DEI, and also concentrated by sedimentation in a flow centrifuge. The antigen concentrate was resuspended in a sucrose-gelatin medium to a concentration of 10-12 CFU/cm? on 18 of the optical standard DISK named after Tarasovich. After that, the antigen was combined with an oil adjuvant. At the same time, the amount of the aqueous phase containing the antigen was 3095, and the oil adjuvant was 7095. Colloid mills were used for the production of the emulsion vaccine. a The resulting vaccine is a white emulsion with a yellowish tinge, with a slightly viscous consistency, "» pH 7.2-7.6. With long-term storage, a slight delamination of mineral oil is possible over a non-layered homogeneous emulsion. With thorough shaking, the vaccine acquires the appearance of a homogeneous mass. 45 Example 2 Bacteriological control of the sterility of the received vaccine was carried out as follows. Samples of the vaccine were sown on serum broth and agar, liquid and dense medium Sabouraud, MLA, MPB - from the tube, on

MPPB - 2 test tubes and 2 vials each, Endo medium - 3 cups each. For the detection of aerobes, 0.5 cm of the vaccine was sown in one test tube and 2 cm3 in one vial, and for the detection of anaerobes, 1 and 5 cm of the vaccine were sown, respectively. Sabouro, kept in a thermostat at (37.0--0.5)2С, on Sabouro medium at (21.04-0.5)С for 7 days. After the end of the specified period, reseeding was done, with the exception of sowing on MPA, Endo's medium, Sabouraud's agar, and serum agar.

Samples were transplanted to the same nutrient media in the same volumes as during sowing. Secondary crops were kept for 7 days. At the same time, control of the sterility of the nutrient media was carried out. The results of the primary and secondary

55 crops were evaluated by microscopic examination of the crops. There was no growth of microorganisms on nutrient media.

Example C

Control of harmlessness and reactogenicity of the received vaccine was carried out in the following way. The harmlessness of the vaccines was tested on 6 guinea pigs and 12 white mice. The drug was administered subcutaneously to 60 guinea pigs in the groin area of 1 and 2 cm, to white mice in the back area in a volume of 0.5 cm3. Animals were observed for days. All animals remained alive. To check the reactogenicity, the drug was administered intramuscularly to two calves and two piglets by 10 cm Z. 14 days after the injection of the vaccine, local changes in the form of tissue compaction, ranging from 1 to 3 cm Z, remained in the animals at the injection site. The compactions are uniform, without softening, painless. At the autopsy of calves and piglets in the thickness of the muscle layer, b5 light-yellow granulomas of a dense consistency were found.

Example 4

To test the immunogenic activity of the restrained vaccine on white mice, 10 animals for each serovar were used. The vaccine was administered to 30 white mice subcutaneously in the back region in the amount of 0.000 ml (1.5 billion micrograms) once. 21 days after immunization, 10 vaccinated and 5 intact white mice were infected subcutaneously with a previously titrated lethal dose of Pasteurella of the corresponding serovar. Serovar B was administered at a dose of 3-Bzh.m.k. per white mouse (0.5 cm3 of a 20-hour broth culture in a dilution of 109 with the accumulation of Tmlrd.j.m.c. in 1 cm3 of nutrient medium. Control mice died within 24 hours. The vaccinated remained alive during 10 days of observation after the death of the control. Serovar A was administered at a dose of 10-20 mcg per white mouse 70. (0.2 cm3 of a 20-hour broth culture in a dilution of 107 with the accumulation of tmnrd. mcg per cm of nutrient medium. Control mice died within 24-36 hours. The vaccinated remained alive during 10 days of observation after the death of the control. Serovar OO was administered at a dose of 300 mg.m.c. to a white mouse (0.3 cm in a 20-hour broth culture in a dilution of 109 with the accumulation of Imlrd.j.m.c. in cm From nutrient) medium.

Control mice fell during the day. The vaccinated remained alive for 10 days, observation after 72 death of the last control white mouse. In the conclusion, the vaccine was assessed using a quantitative method.

Example 5

Four animals for each serovar were used to test the immunogenic activity of the received vaccine on rabbits. The vaccine was administered to 12 rabbits intramuscularly in the thigh in a volume of 0.5 cm Z (7.Bmlrd.j.m.c.) once. 21 days after immunization, 4 vaccinated and 2 intact rabbits were infected intramuscularly in the thigh with a previously titrated lethal dose of Pasteurella of the corresponding serovar. Serovar B was administered at a dose of BOzh.m.k. per rabbit (0.5 cm of a 20-hour broth culture in dilution 103 with accumulation of 1 Tmlrn.j.m.k. in the nutrient medium). Control animals died within 24 hours. The vaccinated remained alive for 10 days of observation after the death of the control. Serovar A was administered at a dose of 50 g.m.c. ov per one rabbit (0.5 cm? of a 20-hour broth culture in a dilution of 103 with the accumulation of tmlrd.j.m.k. in 1 cm3 of nutrient medium). Control animals died within 24-36 hours. The vaccinated remained dead for 10 days of observation after the death of the control. Serovar Y was administered at a dose of 500 mg. per rabbit (0.5 cm? of a 20-hour broth culture in a dilution of 10 with the accumulation of imlrd.j.m.c. in cm From the nutrient medium). Control animals died within 6-7 days. The vaccinated remained alive during 10 days of observation after the death of the control. high school

Example 6

The immunogenic activity of the vaccine on calves and piglets was determined by direct infection of vaccinated (av) animals (inoculation dose 2 cm 3, intramuscularly). 21 days after vaccination, the animals are infected with M 24-hour broth cultures of Pasteurella: serovar B - 0,Zsmu; serovar A - 0.5 cm?; serovar O - 10 cm.

Z Control animals died, and vaccinated animals remained alive during 10 days of observation after the death of the control. "

Example 7

A study was conducted on the comparative study of the effectiveness of inactivated anti-pasteurellosis vaccines, obtained on the basis of domestic production strains of pasteurella, on calves and piglets in the conditions of the "Ukraine" and "Russia" KSPs of the Ivanivka District of the Kherson Region and the Komunar State Farm and the "Pryhorodniy" KSP of Ukraine. The experience was conducted on 1-3-month-old animals, the observation period was 6 months. Studies have confirmed the high immunogenicity of the anti-pasteurellosis vaccine from domestic strains.

Thus, thanks to the use of the proposed method, a vaccine was obtained against factor (endogenous) pasteurellosis of the young of rural areas. animal emulsion is inactivated and has higher - 45 immunogenic activity and specific safety. at

Claims (5)

The formula of the invention is named) p» 50 1. The method of manufacturing an emulsion anti-pasteurellosis vaccine, which includes obtaining a suspension of a pasteurella culture, inactivating the obtained culture and combining a bacterial antigen with an oil adjuvant, which differs in that domestic strains of pasteurella are used as immunogens: Ravzietsgeiia tyPioside Mo Z serovar D, Mo 12 serovar A, Mo 31 serovar B, which inactivate dimeromethyleneimine (DEI), which is added to the culture suspension to a concentration of 4-5,956 at pH 7.2-7.6, and after inactivation the antigen obtained contribute to C; o55 stabilizing medium and combined with an oil adjuvant. 2. The method according to claim 1, which differs in that the inactivation is carried out for 12-16 hours at 37-3820. C. The method according to claim 1, which differs in that after inactivation, the bacterial antigen is precipitated by centrifugation. 4. The method according to claim 1, which differs in that a 60% sucrose-gelatin medium is used as a stabilizing medium. 5. The method according to claim 1, which differs in that the antigen and oil adjuvant are combined in a ratio of 3:7-1:1. Official bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2005, M 6, 15.06.2005. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine.