RU2753410C2 - Method for obtaining toxoid vaccine against escherichiosis of animals - Google Patents

Abstract

FIELD: pharmaceutics; veterinary medicine.SUBSTANCE: method for obtaining a vaccine against escherichiosis includes the selection of epizootic strains of Escherichia coli with genes of thermolabile, thermostable and shiga-like toxins, their cultivation in a nutrient broth at a temperature of 37°C for 6-7 days with daily double mixing and further inactivation with formalin to its final concentration of 0.4% for 14 days, followed by mixing in equal volumes and separating the bacterial mass using sterilizing filtration. Then adjuvants are added, as which a preparation of pyrogenal is used containing 100 mcg/ml of the active substance, and 3% aqueous solution of polyacrylic acid, which are introduced into a cell-free culture medium containing thermolabile, thermostable and shiga-like toxins of Escherichia coli, alternately, followed by mixing until a homogeneous mixture is obtained and at the following ratio of components, wt.%: a solution of pyrogenal containing 100 mcg/ml of the active substance is 9-11; 3% aqueous solution of polyacrylic acid is 9-11; cell-free culture medium containing thermolabile, thermostable, and shiga-like Escherichia coli toxins is up to 100.EFFECT: use of this invention makes it possible to obtain a vaccine, the injection of which provides a more intense and prolonged specific immunity against Escherichia infection.1 cl, 7 ex, 5 tbl

Description

The invention relates to biotechnology, in particular to methods of designing vaccines, and can be used in institutions involved in the creation of vaccine preparations for the prevention of Escherichiosis in animals.

It has been established that effective means of specific prevention of escherichiosis in animals are vaccines containing inactivated exotoxins of pathogenic Escherichia coli (Biological preparations for specific prevention and therapy of escherichiosis in animals / M.K. Pirozhkov // Diss. Doctor of Veterinary Sciences. - Moscow . 2002. - 298 p.). Currently, there are studies on the use of biological products, which include three types of toxins of pathogenic Escherichia at once: heat-labile (LT) and thermostable (ST) and shiga-like (STX) toxins, both in pure form and in combination with adjuvants (Karaev Y.M. Immunogenic and protective properties of Escherichiosis toxoid: dis .... Candidate of Veterinary Sciences / Ya.M. Karaev - Krasnodar: KubGAU, 2009. - 132 pp .; Tishchenko AS Influence of adjuvants on immunogenic properties of Escherichiosis toxoid: dis. ... Candidate of Veterinary Sciences / AS Tishchenko. - Krasnodar: KubSAU, 2011. - 124 p.). However, these studies found that Escherichiosis toxoid stimulates the cellular and humoral links of immunity in the first day after the administration of drugs, and the use of adjuvants, although it enhances the immune response, but for a short time. Given these circumstances, it is necessary to look for new ways to increase the immunogenic activity of vaccine preparations based on inactivated E. coli toxins. Known vaccine against Escherichiosis of animals (RF patent No. 2043771, CL A61K 39/108, 1995) made from somatic - Q78, Q141; protein adhesive - K88, K99, 987P, F41; capsular polysaccharide antigens - K80. K87, and also includes TL and TS-toxoid. Currently, this vaccine has undergone modernization by adding strains of Escherichia and the inclusion, in addition to TL and TS, of shiga-like toxins (VT1 and VT2 toxoids) of Escherichia coli, inactivated with formalin in an amount of 0.4% with the addition of aluminum hydrate gel in an amount of 25% B as an adjuvant. 1 cm ^{3 of the} vaccine contains 10 billion microbial bodies of Escherichia. Commercial name - Vaccine against animal Escherichiosis Verokolivak K88, K99, 987P, F41, TL-, TC-, VT1 and VT2 toxoids (manufacturing organization of FSUE Armavir Biofabrika, Russia) (Vaccine prophylaxis of Escherichiosis of farm animals // http: // www .armbio.info / verokolivak.html; Vaccine against Escherichiosis in animals // http://www.armbio.info/catalog/1006-3).

The disadvantage of this improved vaccine is that it contains a very large amount of ballast antigens in the form of whole E. coli cells and an insoluble mineral adjuvant, and this negatively affects the formation of immune defense in animals, as well as the development of side reactions and complications in vaccinated animals. ... In addition, the method of obtaining such a vaccine is very complicated, since it requires separate cultivation of 18 strains of Escherichia.

It is known to use pyrogenal and polyacrylic acid separately as adjuvants to enhance the immunogenicity of Escherichiosis toxoid containing inactivated toxins (TL, TS, STX) E. coli (Terekhov, V.I. Terekhov, Ya.M. Karaev, A.S. Tishchenko, A.V. Ivanov // Tr. / KubSAU. - 2009. - Issue No. 1 (part 1) Series: Veterinary sciences. - P. 100- 102 .; Tishchenko A.S. et al. Effect of bacterial polysaccharide and polyelectrolyte on the immunogenic properties of Escherichiosis toxoid // Young Scientist. - 2016. - No. 20 (124). - P. 113-115; Tishchenko A.S., Terekhov V.I., Serdyuchenko I.V. Immunogenicity of escherichiosis toxoid when used with pyrogenal and polyacrylic acid // Scientific support of the agro-industrial complex: collection of articles based on the materials of the X All-Russian conference of young scientists. - Krasnodar: KubGAU, 2017 - С . 287-288.).

The disadvantage of known studies is that Escherichiosis toxoid using pyrogenal as an adjuvant and Escherichiosis toxoid using polyacrylic acid as an adjuvant had low immunogenic properties, which negatively affected the formation and duration of post-vaccination protection in animals against E. coli toxins. In addition, white rats and rabbits were the biological model in these studies, and it is not known how the vaccine components will manifest themselves in calves and piglets.

It is known the use of adjuvants, including polyacrylic acid and pyrogenal, in combination with Escherichiosis toxoid in experiments on pregnant cows and pregnant sows (Tishchenko A.S. Influence of adjuvants on the immunogenic properties of Escherichiosis toxoid: dis ... Candidate of Veterinary Science / A.S. Tishchenko. - Krasnodar: KubSAU, 2011. - 124 p .; Tishchenko, AS Evaluation of the humoral immune response in pregnant sows immunized with Escherichiosis toxoid in combination with adjuvants / AS Tishchenko, VI Terekhov // Tr. / KubSAU. - 2011. - Issue No. 2 (29). - P. 144-147; Terekhov, V.I. S. Tishchenko // Veterinary medicine of the Kuban. - 2011. - No. 3. - P. 19-21; Tishchenko A.S., Novikova E.N., Vinokurova D.P., Kiyashchenko A.A., Kremyanskiy V.V. The spread of escherichiosis in piglets and the method of its specific prevention / Polythematic network electronic scientific journal of the Kuban State Agrarian University theta. - 2018. - No. 137. - S. 220-229), and also determined the preventive efficacy of these drugs
(Tishchenko, A.S. Prophylactic efficacy of escherichiosis toxoid in escherichiosis of piglets and calves / A.S. Tishchenko, V.I. scientific-practical conf. / KubSAU. - Krasnodar, 2010. - pp. 380-381).

The disadvantage of known studies is that Escherichiosis toxoid, using separately pyrogenal and polyacrylic acid as adjuvants, had a short immune response, which affected the effectiveness of specific prophylaxis of Escherichiosis. In addition, the adjuvant components used with Escherichiosis toxoid separately, in general, have weak immunogenicity and may exhibit rectogenicity.

The closest in technical essence is a method for producing Escherichia toxoid (patent No. 2432174 dated 10/27/2011, IPC A61K 39/108, C12N 1/20, A61P 1/00), in which epizootic strains of Escherichia coli with genes are selected and separately cultivated thermolabile, thermostable and shiga-like toxins, in a nutrient broth at 37 ° C for 6-7 days with two daily stirring. Inactivation of cultures is carried out by adding formalin to a concentration of not more than 0.4%, at a temperature of 37 ° C for 14 days with two daily stirring. After that, cultures are selected and mixed in equal proportions, the bacterial mass is separated using sterilizing filtration and a complex preparation is obtained.

The disadvantage of this method is the lack of adjuvants, and as a consequence, the low immunogenic activity of the drug.

The technical result of the invention is to increase the effectiveness of specific prophylaxis of Escherichiosis in calves and piglets, increase the immunogenicity, harmlessness and safety of the vaccine.

The technical result is achieved by the fact that in the method of obtaining a vaccine against Escherichiosis of animals, including the selection of epizootic strains of Escherichia coli possessing genes of heat-labile, thermostable and shiga-like toxins, their cultivation in a nutrient broth at a temperature of 37 ° C for 6-7 days with daily double stirring , then they are inactivated with formalin to its final concentration of 0.4% for 14 days with double daily stirring, then cultures are selected and mixed in equal proportions, according to the invention, adjuvants are added, which are used as a pyrogenal solution containing 100 μg / ml of active substances and a 3% solution of polyacrylic acid, which are introduced into a cell-free culture medium containing thermolabile, thermostable and shiga-like toxins of Escherichia coli alternately, followed by stirring until a homogeneous mixture is obtained, with the following ratio of components, wt. %:

pyrogenal solution containing 100 μg / ml of active ingredient 9-11 3% aqueous solution of polyacrylic acid 9-11 cell-free culture medium containing inactivated heat-labile, thermostable and shiga-like toxins Escherichia coli up to 100.

The novelty of the technical solution lies in the fact that due to the use of cell-free cultures of epizootic strains of Escherichia coli, producing thermolabile, thermostable and shiga-like toxins inactivated with formalin, with the combined use of adjuvants: solutions of pyrogenal and polyacrylic acid, an increase in the immunogenic properties of the vaccine and the effectiveness of corpuscles of prophylaxis of Escherichiosis in and piglets, as well as the safety of the biological product due to the absence of post-vaccination reactions after application.

In the patent and scientific and technical literature, a similar set of features has not been found, which makes it possible to judge the inventive level of the proposed proposal.

The proposed method for obtaining a vaccine meets the criterion of "industrial applicability" because it is easily reproducible, does not require complex technological equipment and conditions.

The method of obtaining toxoid vaccine against Escherichiosis of animals is as follows.

The initial stages of obtaining Escherichiosis toxoid were carried out according to the RF patent for invention No. 2432174 dated October 27, 2011, IPC A61K 39/108, C12N 1/20, A61P 1/00.

Preliminarily, from the epizootic strains of Escherichia coli, strains with genes for heat-labile, heat-stable and shiga-like toxins were selected. For this, the test systems "AmpliSens E. coli-tox" (Central Research Institute of Epidemiology, Ministry of Health of the Russian Federation, Moscow) were used. Confirmation of toxin formation on an artificial nutrient medium was carried out using a biotest on ciliates-stylonichia (RF patent for invention 2262529 dated 20.10.2005, IPC C12N 1/10, C12Q 1/10). According to the known patent, to test the survival of ciliates, a culture of E. coli grown in Hottinger's broth with an incubation period of 1-3 days was used, then the medium with the ciliates was mixed with the broth culture of E. coli in a ratio of 3: 1. If exotoxins are present in the culture medium, the number of dead ciliates should be at least 70-80%, while the number of dead ciliates in the control sample that does not contain the toxin should not exceed 5%.

The selected strains were individually inoculated into tubes with 10 ml of nutrient broth for the accumulation of E. coli toxins, which includes acidic blood hydrolyzate (9-11%), baker's yeast autolysate (9-11%), peptone (0.9-1, 1%), naria chloride (0.4-0.6%), disubstituted sodium phosphate (0.05-0.15%) and potassium chloride (0.01-0.03%) (RF patent for invention 2342425 dated 27.12.2008, IPC C12N 1/20, C12Q 1/04). Then they were placed in a thermostat and cultured at 37 ° C for 6-8 h until a slight turbidity appeared, indicating the growth of microorganisms. Then, the resulting cultures were transferred into flasks with 200-300 ml of a similar nutrient broth and incubated at 37 ° C for: producing heat-labile and heat-stable ones - 6 days, and those producing shiga-like toxin - 7 days. The flasks were shaken twice daily. Before the end of incubation, 10 ml of culture fluid was taken from each flask, centrifuged at 10 thousand rpm for 30 min, and the presence of toxins was determined by biotesting on infusoria.

In the presence of toxins, formalin was added to the remaining cultures to a concentration of 0.3-0.4%. Cultures were inactivated for 14 days at 37 ° C with double daily stirring. After completion of inactivation, equal volumes of cultures were combined, thus obtaining a complex preparation containing 3 types of antigens. Using sterilizing filtration, the microbial mass was separated from the cultivation medium, thus obtaining a cell-free Escherichia coli culture medium (Escherichiosis antigenic toxoid component). Then, the following adjuvants were added to the cell-free Escherichia coli culture medium: the pharmaceutical preparation pyrogenal (lipopolysaccharide isolated from the microbial cells of Salmonella typhi, manufactured by the N.F. ml of which contains 100 μg of active ingredient)) and 3%> aqueous solution of polyacrylic acid (produced by OOO Khimdirect service, Moscow) with the following ratio of components, wt. %:

pyrogenal at 0.01%) concentration 9-11 3%> aqueous solution of polyacrylic acid 9-11 cell-free culture medium containing inactivated heat-labile, thermostable and shiga-like toxins Escherichia coli up to 100.

The effectiveness of the toxoid vaccine against Escherichiosis depends on the properties of the selected epizootic strains of Escherichia coli, the sequence of preparation and the percentage of the components included in its composition.

The quantitative values of the introduction of adjuvants were selected experimentally, taking into account the immunogenic activity of the toxoid vaccine. With the introduction of pyrogenal and polyacrylic acid in a volume of 8%, the immunogenic activity increased, but still was not sufficient for long-term preservation of post-vaccination immunity. With the introduction of adjuvants in the amount of 12%, the increase in the production of specific antibodies was more significant than with their introduction in the amount of 9-11%, and the economic costs increased. In addition, following an increase in the administration of polyacrylic acid, the risk of developing a local inflammatory reaction due to tissue irritation increased.

The resulting vaccine is packed in 100 ml sterile vials, sealed and checked for sterility and harmlessness. In appearance, the finished vaccine is a clear, light straw-colored liquid without sediment and impurities. Shelf life in a dry and dark room at a temperature of 4-10 ° C - 1 year.

The resulting toxoid vaccine was tested for sterility and harmlessness.

At control inoculations of toxoid on MPA, MPB, Kitta-Tarozzi, Saburo, Endo medium, the growth of bacterial and fungal flora was absent, which indicated the sterility of the preparation.

With the intraperitoneal injection of toxoid in a dose of 0.3 ml to white mice weighing 20-22 g, they did not notice inhibition and death within 10 days, which is an indicator of the harmlessness of the drug.

To prove the effectiveness of the proposed toxoid vaccine, studies were carried out on white rats, calves and piglets. A satisfactory result was obtained by optimizing the addition of pyrogenal and polyacrylic acid, which is demonstrated by the following experiments.

Experience 1 - in which the results of studies carried out on white rats were presented.

There were formed 4 groups of white rats, 6 goals in each, which were injected intramuscularly once with 0.15 ml of the drug, in the first group, Escherichiosis toxoid without adjuvant (EA) was used, in the second - Escherichiosis toxoid with pyrogenal (EA + LPS), in the third - Escherichiosis toxoid with polyacrylic acid (EA + PAA), in quaternary - Escherichiosis toxoid with pyrogenal and polyacrylic acid (EA + LPS + PAA). After 7, 14, 21, 60, 90 and 120 days in the reaction of indirect hemagglutination, the titer of antibodies to E. coli toxins was determined, the results of the experiment are shown in table 1.

From the data in the table it can be seen that after the introduction of toxoid in animals of all groups, antibodies are produced for 7 days. However, in groups 2 and 3, where it was injected with adjuvants, the level of antibodies was 1.7 times higher than in animals to which the toxoid was injected without pyrogenal and polyacrylic acid, and in animals of group 4, the level of antibodies was 2 times higher. On the following days, in animals of group 1, the level of antibodies decreased, but in animals of groups 2, 3 and 4, it increased until day 14, exceeding the indicators of group 1 by 1.9, 1.8 and 2.4 times, respectively. Subsequently, in all groups, a gradual decrease in antibodies by day 120 was established to a value of 1.2 ± 0.7, 2.0 ± 0.6, 1.8 ± 0.7, and 3.6 ± 0.4 log2, respectively. In animals of group 4, the level of antitoxic antibodies remained the highest even after 120 days after immunization and was sufficient for a protective effect against E. coli toxins.

Experience 2 - the results of studies carried out on calves and piglets are presented.

For research, the toxoid vaccine was inoculated to pregnant cows and heifers twice subcutaneously. The first time in 30, and the second time 15 days before calving in a dose of 5 and 10 ml, respectively. The calves were also vaccinated twice, the first time at the age of 15-20 days, and the second after 10-14 days at a dose of 1 and 2 ml, respectively.

Pregnant sows were injected intramuscularly twice, the first time in 30 days, and the second time 20 days before farrowing in a dose of 5 ml; piglets were vaccinated for the first time at the age of 15-17 days, and the second time at the age of 28-30 days at a dose of 0.25 and 0.5 ml, respectively.

The use of the toxoid vaccine makes it possible to protect calves and piglets from Escherichiosis infection for at least 6 months (observation period).

There are five examples (1-5) of the dependence of the effectiveness of the toxoid vaccine against Escherichiosis of calves and pigs on the ratio of the claimed components per 100 ml of vaccine and two examples (6, 7) demonstrating the effectiveness of using the claimed invention.

For examples 1-5, epizootic strains of Escherichia coli were used, possessing genes for heat-labile, thermostable and shiga-like toxins, they were cultivated in a nutrient broth at 37 ° C for 6-7 days with double daily stirring, then they were inactivated with formalin to its final concentration 0.4% for 14 days with double daily stirring, after which the cultures were selected and mixed in equal proportions, the bacterial mass was separated using sterilizing filtration to obtain a cell-free culture medium, into which adjuvants were introduced alternately with subsequent stirring with the following volumetric amounts and percentage:

Example 1. With the following ratio of components, mass. %:

pyrogenal at 0.01% concentration eight 3%> aqueous solution of polyacrylic acid eight cell-free culture medium containing Escherichia coli toxins up to 100.

With this ratio of components, the toxoid-vaccine has a weak protective activity and protects against Escherichiosis infection caused by pathogenic Escherichia coli calves in 75%, and piglets in 72% of cases.

Example 2. With the following ratio of components, mass. %:

pyrogenal at 0.01%) concentration nine 3%> aqueous solution of polyacrylic acid nine cell-free culture medium containing Escherichia coli toxins up to 100.

With this ratio of components, the toxoid-vaccine protects against Escherichia infection caused by pathogenic Escherichia coli in 86%, and piglets in 82% of cases.

Example 3. With the following ratio of components, mass. %:

pyrogenal at 0.01% concentration ten 3% aqueous solution of polyacrylic acid ten cell-free culture medium containing Escherichia coli toxins up to 100.

With this ratio of components, the toxoid-vaccine protects against Escherichia infection caused by pathogenic Escherichia coli in 92%, and piglets in 87% of cases.

Example 4. With the following ratio of components, mass. %:

pyrogenal at 0.01% concentration eleven 3% aqueous solution of polyacrylic acid eleven cell-free culture medium containing Escherichia coli toxins up to 100.

With this ratio of components, the toxoid-vaccine protects against Escherichiosis infection caused by pathogenic Escherichia coli in 94%, and piglets in 90% of cases.

Example 5. With the following ratio of components, mass. %:

pyrogenal at 0.01% concentration 12 3% aqueous solution of polyacrylic acid 12 cell-free culture medium containing Escherichia coli toxins up to 100.

With this ratio of components, the toxoid-vaccine protects against Escherichiosis infection caused by pathogenic Escherichia coli in 84%, and piglets in 80% of cases. At the injection site, some animals, especially piglets, developed dense painful swellings and an increase in body temperature.

Confirmation of the effectiveness of the use of toxoid vaccine for the prevention of Escherichiosis in calves and piglets is given in examples 6 and 7.

Example 6 - shows the results of studies on the use of toxoid vaccine for the prevention of Escherichiosis in calves.

In the farm of the Krasnodar Territory, unsuccessful in calf Escherichiosis, a production test of the claimed toxoid vaccine was carried out. According to the veterinary laboratory, diarrhea in young cattle in this farm is caused by pathogenic strains of E. coli. The effectiveness of the vaccine made according to the claimed method was compared with the effectiveness of the prototype; for this, 3 groups of pregnant cows, 25 heads each, were formed.

The first group of cows was immunized with the claimed toxoid vaccine the first time 30 days before calving at a dose of 5 ml, the second time 15 days before calving at a dose of 10 ml. The calves obtained from these cows were also immunized with the claimed toxoid vaccine for the first time at the age of 15-20 days at a dose of 1 ml, and the second time - 10-15 days later at a dose of 2 ml. The vaccine was injected subcutaneously into the upper third of the neck.

The second group of cows and calves obtained from them were immunized with Escherichiosis toxoid with pyrogenal according to the scheme and in doses similar to those in the first group.

The third group of cows and calves obtained from them were immunized with Escherichiosis toxoid with polyacrylic acid according to the scheme and in doses similar to those in the first group.

The fourth group of cows and the calves obtained from them were immunized with Escherichiosis toxoid without adjuvants according to the scheme and in doses similar to those in the first group.

The fifth group of cows was a control one; they and the calves obtained from them were not immunized.

The effectiveness of the vaccines was assessed when the calves reached 3 months of age and transferred their other corps to group housing. The evaluation criteria were morbidity and mortality rates.

The results of the experiment are reflected in table 2, from the materials of which, it can be seen that in the first group, Escherichiosis infection was registered in 2 calves, while their diarrhea was stopped within 3 days, and on the 5th day the calves recovered completely. Thus, the prophylactic efficacy of the claimed vaccine was 92%.

In the 2nd group, Escherichiosis infection was registered in 5 calves, and in one calf it was very difficult and ended in the death of the animal. The preventive effectiveness of the analogue was 80%.

In the third group, Escherichiosis infection was registered in 6 calves, in two calves it was very difficult and ended in the death of animals. The preventive effectiveness of the analogue was 76%.

In the fourth group, Escherichiosis infection was registered in 8 calves, of which in three calves it was very difficult and ended in the death of animals. The preventive efficiency of the prototype was 68%.

In the fifth group of 25 calves, 18 animals fell ill, of which 5 died. Thus, in group 3, the morbidity was 72%, and the mortality rate was 27.8%, which indicates a significant severity of the disease.

Consequently, the use of toxoid vaccine against Escherichiosis, obtained by the claimed method, made it possible to more effectively prevent the disease of calves with Escherichiosis infection. The incidence of calves vaccinated with the claimed vaccine was 8%, and the safety rate was 100%, while when using prototype 1, the incidence rate of calves was 20%, and the safety rate of 96%, prototype 2 - incidence rate 24%, safety rate 92%, prototype 3 - incidence rate 32%, safety 88%. In the control group, the incidence of acute intestinal infection in calves was 12%, and the survival rate was 80%.

The immunogenic properties of Escherichiosis toxoid were studied in calves obtained from vaccinated pregnant cows. To determine the presence of antitoxic antibodies in the diffuse precipitation reaction, blood serum was collected 7, 14, 28, and 56 days after their last vaccination.

The effect of the use of toxoid vaccines on the antibody titer in calves is presented in Table 3.

From the materials of table 3 it can be seen that the immunization of cows and calves by the claimed method provides a more intense and long-lasting specific immunity against Escherichiosis infection. This is evidenced by the number of antibodies to E. coli toxins in calves, which in animals of the first group is 2-16 times more than in animals from the second and third groups, and 8-32 times more than in animals of the fourth and fifth groups.

Example 7. Substantiation of the effectiveness of the use of toxoid vaccine for the prevention of escherichiosis in piglets.

In the farm, long-term dysfunctional for Escherichiosis, piglets formed 5 groups of pregnant sows, 5 animals in each.

In the first group of sows and piglets obtained from them were immunized with the proposed vaccine. Sows were immunized for the first time 30 days before farrowing at a dose of 5 ml, the second time - 20 days before at a dose of 5 ml intramuscularly in the area of the base of the auricle. Piglets were immunized twice, the first time at the age of 15-17 days at a dose of 0.25 ml, and the second time at the age of 28-30 days at a dose of 0.5 days intramuscularly in the area of the inner thigh.

The second group of sows and the piglets obtained from them were immunized with Escherichiosis toxoid with pyrogenal according to the scheme and in doses similar to those in the first group.

The third group of sows and the piglets obtained from them were immunized with Escherichiosis toxoid with polyacrylic acid according to the scheme and in doses similar to those in the first group.

The fourth group of sows and the piglets obtained from them were immunized with Escherichiosis toxoid without adjuvants according to the scheme and in doses similar to those in the first group.

The fifth group of sows was the control, they and the piglets obtained from them were not immunized.

The piglets were clinically observed for 90 days, the number of sick and dead animals was taken into account. The results of the study are presented in table 3. From the materials of the table it can be seen that in the 1st group from 5 sows 53 piglets were obtained, of which 8 heads fell ill and 2 died. The morbidity in the group was 15.1%, mortality 3.8% ... Consequently, the prophylactic efficacy of the proposed method was 84.9%, with the safety of piglets in the group being 96.2%.

In the 2nd group, where sows were immunized with toxoid with pyrogenal, 52 piglets were born, of which 12 fell ill and 4 died. The morbidity in this group was 23.1%, mortality 7.7%, preventive efficacy 78.9%, with preservation 92.3%.

In the 3rd group, 54 piglets were received, of which 14 got sick, 6 died. The incidence rate was 25.9%, the mortality rate was 11.1%, the preventive efficacy was 74.1%, and the safety was 88.8%.

In the fourth group, where sows were vaccinated with toxoid without adjuvants, 53 piglets were born, 20 of them got sick, 8 died. The incidence rate was 37.7%, the mortality rate was 15.1%, the preventive efficacy was 62.3%, and the safety was 84.9%.

In the 5th group, 53 piglets were received, of which 28 fell ill, 9 died. The incidence rate was 52.8%, the mortality rate was 17%, and the survival rate was 83%.

Consequently, the use of toxoid vaccine against Escherichiosis, obtained by the claimed method, made it possible to more effectively prevent the disease of piglets with Escherichiosis infection. At the same time, the incidence decreased in comparison with prototype 1 by 8%, prototype 2 by 10.8%, prototype 3 by 22.6%, and in comparison with the negative control troupe by 37.7%, while the mortality rate decreased by 2-4 , 5 times, which indicates the high efficiency of the claimed toxoid vaccine.

The immunogenic properties of Escherichiosis toxoid were studied in piglets obtained from vaccinated pregnant sows. To determine the presence of antitoxic antibodies in the diffuse precipitation reaction, blood serum was collected 7, 14, 28, and 56 days after their last vaccination.

The effect of the use of toxoid vaccines on the antibody titer in piglets is presented in Table 5.

From the materials of table 5 it can be seen that the immunization of sows and piglets by the claimed method provides a more intense and long-lasting specific immunity against Escherichiosis infection. This is evidenced by the amount of antibodies to E. coli toxins in piglets, which are 2-32 times higher in animals of the first group than in animals from other groups.

Claims (2)

A method of obtaining a vaccine against escherichiosis of animals, including the selection of epizootic strains of Escherichia coli with genes of heat-labile, thermostable and shiga-like toxins, their cultivation in a nutrient broth at a temperature of 37 ° C for 6-7 days with daily double stirring, then inactivating them with formalin until it a final concentration of 0.4% for 14 days, followed by mixing in equal volumes and separating the bacterial mass using sterilizing filtration, characterized in that adjuvants are added, which are pyrogenal solution containing 100 μg / ml of active ingredient, and 3% an aqueous solution of polyacrylic acid, which is introduced into a cell-free culture medium containing thermolabile, thermostable and shiga-like toxins of Escherichia coli, alternately with subsequent stirring until a homogeneous mixture is obtained and at the following ratio of components, wt%: pyrogenal solution containing 100 μg / ml active substance 9-11 3% aqueous solution of polyacrylic acid 9-11 cell-free culture medium containing thermolabile, thermostable and shig-like Escherichia coli toxins up to 100