RU2325183C1 - Production method of animal colibacillosis vaccine - Google Patents

Abstract

FIELD: veterinary.

SUBSTANCE: escherichia coli cells containing adhesive gene K 88 ab, K 88 ac, K 88 ad, K 99, F-41, Att-25, 987P are cultivated in liquid nutrient medium to produce vaccine. Physical, chemical and biological indicators are investigated in the process of bacterial bulk growth. In addition, activity of fimbrial adhesin in passive hemagglutination of cellular bacterial bulk is determined. Further cultivation is terminated when fimbrial adhesins titers values are K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P equal to: 1:2048-4096; 1:1024-2048; 1:256-512; 1:1024-2056; 1:1024-2056; 1:32-64; 1:256-512, accordingly. Biological bulk is then concentrated and spinned at 3000-5000 rev/min for 20-25 minutes to separate culture broth from cellular bacterial bulk. Then vaccine is inactivate.

EFFECT: improvement of target product quality.

2 tbl, 5 ex

Description

The invention relates to veterinary microbiology and biotechnology, can be used in the development of means of specific prophylaxis, in particular for obtaining a vaccine against escherichiosis of animals.

A known method of obtaining a vaccine against animal escherichiosis, including the cultivation of Escherichia in a liquid nutrient medium, followed by inactivation (RF Patent No. 2043771, "Vaccine against animal Escherichiosis", Bull. No. 26, 09/20/1995, MKI AK61K 39/108).

A disadvantage of the known vaccine is the low quality of the target product, detected with low antigenic and immunogenic activity of Escherichia strains containing adhesive antibodies, insufficient immunogenicity due to incompleteness of the antigenic composition of the components of the vaccine, namely the lack of immunity against strains with Att-25 adhesive antigens, and also the presence of highly toxic strains of serogroups 078, 0141, etc., designed to create immunity to O-antigens, causes a high content of ballast substances, a reactogen spine and sensitization of animals.

The objective of the claimed technical solution is to improve the quality of the target product by expanding its antigenic spectrum and enriching it with the main protective adhesive antibodies, increasing their antigenic and immunogenic activity, which cause the formation of antibodies that interfere with the adsorption of enteropathogenic Escherichia in the small intestine of animals and inhibit the main processes of development of coli infection (Escherichiosis), and also reduces the non-specific load on the immune system of animals and reduces reactogenic mb.

The problem is solved in a method of obtaining a vaccine against escherichiosis of animals, including the cultivation of Escherichia in a liquid nutrient medium, the study of physico-chemical and biological indicators in the process of growth of the bacterial mass, followed by the termination of the cultivation of Escherichia, their concentration and inactivation, characterized in that they cultivate Escherichia containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, during the cultivation of Escherichia, the activity of fimbrial adhesins is additionally determined in the reaction of passive hemagglutination of the bacterial cell mass, then the cultivation is terminated at titers of Escherichia fimbrial adhesins containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, equal to 1 : 2048-4096; 1: 1024-2048; 1: 256-512; 1: 1024-2056; 1: 1024-2056; 1: 32-64; 1: 256-512, respectively.

The problem is also solved by the fact that the culture fluid is freed from the bacterial cell mass by centrifugation at 3000-5000 rpm for 20-25 minutes.

It is known to obtain and use fibrous adhesins to obtain antisera (US patent No. 4769240, CL A61K 39/02, 1988, RF patent No. 2077337, A61K 39/02. Bull. 11, 04/20/1997).

In the patent and scientific and technical literature, technical solutions are not known containing signs similar to those claimed, i.e. the proposal meets the criterion of "novelty."

The proposal is feasible in laboratory and industrial conditions, aimed at solving a real technical problem, i.e. the proposal is “industrially applicable”.

Existing vaccines against escherichiosis in animals are made from the bacterial mass of Escherichia, grown without taking into account the protective characteristics of pathogen cell components that are functionally related to pathogenicity factors. Reactive cultivation of Escherichia to the stationary phase of bacterial growth is carried out without taking into account the determination of the maximum level of specific activity, the preservation of this activity and the distribution of protective fimbrial adhesins in the growth medium. The cultivation process is carried out in an uncontrolled mode of pathogenicity factors. We first established that when obtaining the maximum yield of biomass, as is currently customary in the industrial production of a vaccine against animal escherichiosis, the quality of antigenic material decreases, because upon reaching a certain developmental phase of the culture under the influence of the cell’s own enzymes during its aging, destructive changes occur in part of the components of fimbrial adhesins, which leads to the loss of immunologically active material and is unacceptable in the production of an effective vaccine against escherichiosis of animals. In addition, we found that the cultivation of Escherichia, containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, allows to obtain a vaccine that exactly coincides with epizootic strains of the causative agent of animal colibacteriosis.

For the first time, a method for producing a vaccine against escherichiosis of animals has been proposed, in which instead of determining the criterion for the number of Escherichia cells by physicochemical and biological indicators, as well as by assessing the content of adhesive antigens, the maximum level of antigen production is determined by the level of activity of Escherichia fimbrial adhesins in passive hemagglutination reaction in fluid freed from the cellular back mass, which allows you to get a non-obvious positive effect - improving the quality of the target Ukta and acceleration method, i.e. the proposal meets the criteria of "novelty" and "inventive step".

The method is illustrated in the following examples.

Example 1

Seven production strains of E. coli containing adhesive antigens K 88 ab, K 88 as, K 88 ad, K99, F-41, Att-25, 987P (strains No. 729, 661/16, 798 were selected for the vaccine against escherichiosis in animals). , 957, 688/19, 4 and 745), which are grown in Hottinger broth containing amine nitrogen - 150 mg%, tryptophan - 50 mg% at pH 7.8. Each strain is grown separately. In each culture, in the process of its growth, the activity of Escherichia fimbrial adhesins is determined, for which purpose the culture fluid is separated from the bacterial mass in a culture sample by centrifugation at 3000 rpm for 20 minutes. The bacterial mass is resuspended in 2 M phosphate-urea buffer with a pH of 7.4, the cell concentration is adjusted to 100 billion microbial cells in 1 ml and heated at a temperature of 60 ° C for 20 minutes. Next, a suspension of microbial bodies is cooled to 5 ° C and centrifuged at 13000 rpm for 20 minutes. The obtained supernatant is used to determine the activity of fimbrial adhesins in the passive hemagglutination reaction. After 5 hours of cultivation with titers of Escherichia fimbrial adhesins containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, equal to 1: 2048; 1: 1024; 1: 256; 1: 1024; 1: 1024; 1:32; 1: 256, respectively, the cultivation of the above strains is stopped. The bacterial mass is separated by centrifugation at 3000 rpm for 20 min, which is resuspended in 2 M phosphate-urea buffer with a pH of 7.4, the cell concentration is adjusted to 100 billion microbial bodies in 1 ml and heated at a temperature of 60 ° C for 20 minutes. Next, a suspension of microbial bodies is cooled to 5 ° C and centrifuged at 10,000 rpm for 20 minutes. The obtained supernatants of each strain in equal parts are mixed, canned with formalin in a final concentration of 0.3% and used to obtain the target product (composition 1).

Example 2

Seven production strains of E. coli containing adhesive antigens K 88 ab, K 88 as, K 88 ad, K99, F-41, Att-25, 987P (strains No. 729, 661/16, 798 were selected for the vaccine against escherichiosis in animals). , 957, 688/19, 4 and 745, which are grown in Hottinger broth containing amine nitrogen - 150 mg%, tryptophan - 50 mg% at pH 7.8. Each strain is grown separately. In each culture, the activity is determined during its growth. Escherichia fimbrial adhesins, for which purpose the culture fluid from the bacterium is separated by centrifugation at 3000 rpm for 20 minutes Bacterial mass is resuspended in 2.1 M phosphate-urea buffer with a pH of 7.3, the cell concentration is adjusted to 120 billion microbial cells in 1 ml and heated at a temperature of 65 ° C for 25 minutes, then the suspension of microbial bodies is cooled to 4 ° C and centrifuged at 15000 rpm for 25 minutes, the resulting supernatant is used to determine the activity of fimbrial adhesins in the reaction of passive hemagglutination. After 6 hours of cultivation with titers of Escherichia fimbrial adhesins containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, equal to 1: 4096; 1: 2048; 1: 512; 1: 2056; 1: 2056; 1:64; 1: 512, respectively, the cultivation of the above strains is stopped. The bacterial mass is separated by centrifugation at 3000 rpm for 20 min, which is resuspended in 2.1 M phosphate-urea buffer with a pH of 7.3, the cell concentration is adjusted to 120 billion microbial bodies in 1 ml and heated at 65 ° C. for 25 minutes. Next, a suspension of microbial bodies is cooled to 4 ° C and centrifuged at 15,000 rpm for 25 minutes. The obtained supernatants of each strain in equal parts are mixed, preserved with formalin in a final concentration of 0.5% and used to obtain the target product (composition 2).

Example 3

Seven production strains of E. coli containing adhesive antigens K 88 ab, K 88 as, K 88 ad, K99, F-41, Att-25, 987P (strains No. 729, 661/16, 798 were selected for the vaccine against escherichiosis in animals). , 957, 688/19, 4 and 745, which are grown in Hottinger broth containing amine nitrogen - 150 mg%, tryptophan - 50 mg% at pH 7.8. Each strain is grown separately. In each culture, the activity is determined during its growth. Escherichia fimbrial adhesins, for which purpose the culture fluid from the bacterium is separated by centrifugation at 3000 rpm for 20 minutes Bacterial mass is resuspended in 2.05 M phosphate-urea buffer with a pH of 7.35, the cell concentration is adjusted to 110 billion microbial cells in 1 ml and heated at a temperature of 62.5 ° C for 22.5 minutes. a suspension of microbial bodies is cooled to 3 ° C and centrifuged at 10,000 rpm for 22 minutes, the resulting supernatant is used to determine the activity of fimbrial adhesins in the reaction of passive hemagglutination. After 6 hours of cultivation with titers of Escherichia fimbrial adhesins containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, equal to 1: 4096; 1: 2048; 1: 512; 1: 2056; 1: 2056; 1:64; 1: 512, respectively, the cultivation of the above strains is stopped. The bacterial mass is separated by centrifugation at 3000 rpm for 20 min, which is resuspended in 1.9 M phosphate-urea buffer with a pH of 7.35, the cell concentration is adjusted to 110 billion microbial cells in 1 ml and heated at a temperature of 62 ° C. for 22 minutes. Next, a suspension of microbial bodies is cooled to 3 ° C and centrifuged at 13000 rpm for 20 minutes. The obtained supernatants of each strain in equal parts are mixed, preserved with formalin in a final concentration of 0.4% and used to obtain the target product (composition 3).

Example 4

Used the resulting compositions as follows. 4 groups of 100 pregnant sows are formed, selected according to the principle of analogues. Each animal of the experimental groups was administered a vaccine of compositions 1-3 at the rate of 2.5 ml. Each animal in the control group was injected with the composition of the prototype at the rate of 6 ml. Table 1 shows the technical data of the vaccines obtained according to the known (prototype) and proposed (formulations 1-3) technical solutions.

Table 1 Indicators Composition 1 Composition 2 Composition 3 Prototype Vaccinated pregnant sows one hundred one hundred one hundred one hundred The number of pigs obtained from pregnant sows 1250 1280 1309 1230 The number of pigs with Escherichiosis 156 135 122 386 The number of pigs that died from Escherichiosis thirty 28 25 85 Livestock safety 97.6 97.8 98 68

As shown by the results of experimental studies, the proposed vaccine against escherichiosis of animals can increase the safety of livestock of farm animals by 29.6-30%.

Example 5

Used the resulting compositions as follows. 4 groups of 50 pregnant cows are formed, selected according to the principle of analogues. Each animal of the experimental groups was administered a vaccine of compositions 1-3 at the rate of 5.0 ml. Each animal in the control group was injected with the composition of the prototype at the rate of 12 ml. Table 2 shows the technical data of the vaccines obtained according to the known (prototype) and proposed (formulations 1-3) technical solutions.

table 2 Indicators Composition 1 Composition 2 Composition 3 Prototype Vaccinated pregnant cows fifty fifty fifty fifty The number of calves obtained from pregnant cows 47 46 48 45 The number of calves with Escherichiosis 10 9 8 21 The number of calves that died from Escherichiosis 3 3 2 6 Livestock safety 93.6 93,4 95.8 86.6

As shown by the results of experimental studies, the proposed vaccine against escherichiosis of animals can increase the safety of the livestock of farm animals by 6.8-9.2%.

As shown by the results of experimental studies, the proposed vaccine against escherichiosis of animals can increase the safety of livestock of farm animals by 6.8-30%, and also reduces the time for receiving a vaccine against escherichiosis of animals by 18-42 hours, since the cultivation time according to the known method is 24-48 hours (in the proposed method - 5-6 hours).

Claims (1)

A method of obtaining a vaccine against Escherichia coli animals, including the cultivation of Escherichia coli cells in a liquid nutrient medium, the study of physico-chemical, biological indicators during the growth of the bacterial mass, followed by the termination of the cultivation of Escherichia coli cells, their concentration and inactivation, characterized in that Escherichia coli cells are cultured containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, during the cultivation of Escherichia coli cells, the activity of fimbrial adhesins in p reactions of passive hemagglutination of the bacterial cell mass, then termination of cultivation is carried out at titer of fimbrial adhesins of Escherichia coli cells containing adhesive antigens K 88 ab, K 88 ac, K 88 ad, K99, F-41, Att-25, 987P, equal to 1 : 2048-4096; 1: 1024-2048; 1: 256-512; 1: 1024-2056; 1: 1024-2056; 1: 32-64; 1: 256-512, respectively, and at the concentration stage, the culture fluid is freed from the bacterial cell mass by centrifugation at 3000-5000 rpm for 20-25 minutes.