RU2248806C1 - Alive vaccine against porcine colibacteriosis - Google Patents

Abstract

FIELD: veterinary microbiology and biotechnology.

SUBSTANCE: the suggested alive vaccine is being the suspension containing a solvent and an antigen out of E.coli strain as bacterial mass of colony-forming microbial cells. The vaccine contains physiological solution as the solvent and an antigen out of E.coli strain "B-5" as the antigen at the content of 2.4 x 10⁸ - 2.4 x 10⁹ microbial cells/1 cu. cm physiological solution. The suggested alive vaccine is of highly immunogenic properties and safe.

EFFECT: higher efficiency of application.

5 ex, 3 tbl

Description

The invention relates to veterinary microbiology and biotechnology and can be used to obtain live vaccines against colibacteriosis of farm animals.

Colibacteriosis (Escherichiosis) - an infectious disease caused by enteropathogenic Escherichia, is widespread in many countries of the world and causes significant economic damage to livestock due to the high mortality of calves and piglets.

Known vaccine against colibacteriosis of farm animals (see RF patent No. 1149466, class A 61 K 39/108, publ. 10.12.95), containing Escherichia antigens, formalin-based preservative, adjuvant, Hottinger broth and physiological saline. The vaccine is made on the basis of strains producing thermolabile and thermostable toxins, has pronounced antigenic properties, a wide range of immunogenicity.

However, the effectiveness of this vaccine is low, since it, being inactivated, loses its immunogenicity upon inactivation. In addition, the manufacture of this vaccine requires considerable labor, and it is expensive, since it involves the use of several strains.

A live vaccine against colibacteriosis of farm animals for intramuscular administration is also known (see Wilson RA, Jotila JW Experimental neonatal colibacillosis caws. Serological studies // J. Infection and Jmmunity. - 1976. - V.13. - No. 1 - P.92) containing broth culture of strain “B-44” (E. coli 0.9: K: H) with a content of 3 × 10 ¹⁰ microbial cells in cm ³ and an adjuvant. The vaccine is administered once subcutaneously, and the second - intramuscularly.

However, the virulence of this vaccine is quite high. In addition, the vaccine is a multicomponent system, since it includes an adjuvant, and the need for a two-time administration increases labor costs.

Closest to the claimed is a live vaccine against colibacteriosis of piglets (see Nagy LK, Phogal BS The effect of colostrums on the adhesion of Escherihia coli to the small intenstine of the pig.// Research in Veterinery sience. - 1976 - v.21. - p.303-308) containing a suspension of strain 0149: K91: K88, while the suspension contains 1 × 10 ¹⁰ colony-forming microbial cells in cm ³ . The strain is resuspended in casein acid hydrolyzate. The vaccine is intended for oral immunization of newborn piglets.

However, this vaccine has residual virulence. In addition, the use of casein hydrolyzate can lead to allergic reactions, distraction of the immune system to an additional irritant, as well as the appearance of an inflammatory process at the injection site.

The invention is aimed at solving the problem of creating a safe living vaccine against colibacteriosis of pigs for intramuscular administration with highly immunogenic properties.

To solve the problem, a live vaccine against pig colibacteriosis, which is a suspension containing a solvent and antigen from E. coli strain in the form of a bacterial mass of colony-forming microbial cells, according to the invention contains physiological saline as a solvent, and antigen from strain E as an antigen .coli “B-5” with a content of 2.4 × 10 ⁸ -2.4 × 10 ⁹ microbial cells in 1 cm ^{3 of} physiological saline.

The sources of patent and scientific literature known to the authors do not describe avirulent live vaccines against colibacteriosis of pigs with highly immunogenic properties due to the use of antigen from E. coli strain “B-5”, which is a bacterial mass with a content of 2.4 × 10 ⁸ - 2.4 × 10 ⁹ microbial cells in cm ³ .

The foregoing allows us to conclude that there is a criterion of "inventive step" in the claimed invention.

The strain intended for the manufacture of the vaccine is characterized by the following properties.

Cultural and morphological characters.

Cultivation is carried out first on meat and peptone broth (MPB), then on meat and peptone agar (MPA) at a temperature of 37 ° C for 18 to 48 hours under aerobic conditions. At the same time, colonies grow diffusely on the MPB, forming an intense haze and easily breaking sediment, on the MPA, initially S-shaped colonies are smooth, convex, shiny with smooth edges. Subsequently, the formation of transitional from S- to R-shaped colonies and dry with uneven edges of the R-shaped colonies (up to 50%) is possible. On the Endo agar, dark red colonies are formed with a metallic sheen.

Enzymatic properties.

The microorganism has mobility, exhibits catalase activity, gives a positive reaction with methylroth, produces indole, ferments glucose with gas formation, as well as lactose, arabinose, mannitol, maltose, xylose, sorbitol, dulcite.

Conditions and composition of media for toxin formation: Hottinger broth.

Antigenic properties.

The product synthesized by the strain Escherichia coli B-5 is a thermolabile exotoxin that is not susceptible to neutralization with polyvalent antitoxic serum against colibacteriosis (Escherichiosis) of farm animals.

The molecular genetic feature of the strain is that the tracks of the strain in both S- and R-forms have a replicon of about 0.3 kV in size, in this replicon there is a gene encoding the B-subunit of the thermolabile toxin (LT), which determines the immunogenic activity of the strain. LT structural genes are additionally integrated into the chromosome of the microbial cell. In the cells of the strain there is a replicon that carries genetic information of regulatory properties necessary for the synthesis of LT products. The strain has an immunogenic epitope, but there is no replicon responsible for the reactogenicity of Escherichia.

Sensitivity to antibiotics (growth retardation zone in mm): polymyxin - 11, streptomycin - 25, chloramphenicol - 25, rifampicin -9, carbenicillin - 15; not sensitive to: oxacillin, doxycycline, lincomycin, tetracycline, oleandomycin, ampicillin, penicillin.

Virulent properties.

Intraperitoneal administration of the strain culture at a concentration of 5 · 10 ⁸ m.k./cm ³ and a volume of 0.5 ml of death of white mice does not cause. LD ₅₀ - 1.2 · 10 ¹⁰ m.k./cm ³ . The death of white mice occurs within 24 hours.

Determination of enterotoxigenic properties of the strain.

To determine the toxin formation, the strain was cultured in the nutritional composition of Mundeli for 20 hours. The resulting broth culture was divided into toxin-containing material and Bakmass by sterilizing filtration. The culture fluid devoid of the bacterial component served as a material containing E. coli exotoxin.

Temperature resistance was determined by heating the toxin-containing material for 10 minutes at 100 ° С and then introducing it into white mice.

The determination of TL-toxin was carried out using: heated toxin-containing material, not heated and Mundeli culture medium as a negative control.

The cultivation of the strain can be carried out, for example, on the developed and patented Saratov NIVS (patent No. 2142505) nutrient medium of the following composition, wt.%: Aminokrov 5-20; peptone 0.6-1.5; ammonium chloride 0.08-0.18; sodium chloride 0.09-0.11; casein hydrolyzate 9-20; nutrient medium “Igla” 52-61; distilled water - the rest. On this medium, cultivation is carried out for 7 days at a temperature of 37 ° C, then the suspension is centrifuged at 6000 rpm for 30 minutes. The supernatant was an enterotoxigenic material.

Example 1

A method of manufacturing a live vaccine against colibacteriosis of pigs is as follows.

The strain is cultured. For this, E. coli strain “B-5” is seeded in test tubes, first on meat and peptone broth (MPB), then on meat and peptone agar (MPA) at a temperature of 37 ° C for 18 hours under aerobic conditions.

The resulting bacterial mass is washed off with physiological saline and a suspension is obtained with a content of microbial cells of 1.2 × 10 ¹⁰ in 1 cm ^{3 of} solution. Then, by adding to the resulting suspension of physiological saline, the content of microbial cells is adjusted to 2.4 × 10 ⁸ -2.4 × 10 ⁹ in 1 cm ^{3 of} physiological solution.

In this case, a suspension with a content of microbial cells of 2.4 × 10 ⁸ in 1 cm ^{3 is} used for vaccination of piglets, and with a content of microbial cells of 2.4 × 10 ⁹ in 1 cm ³ for vaccination of sows.

Example 2. Evaluation of the immunogenic properties of the vaccine was carried out by determining the titer of specific antibodies in the blood serum of rabbits immunized with the claimed vaccine.

For this, an agglutination reaction (RA) was set with the blood sera of immunized and intact rabbits. Serum dilutions were performed in serological tubes starting from 1:25 to 1: 6400 in a volume of 1 ml. 2 drops of antigen were added to each serum dilution, 10 billion microbial suspensions of E. coli (live and inactivated) were used as this. The sera were mixed with antigen by shaking the tubes, after which they were placed in a thermostat and kept at first for 4 hours at 37 ° C, and then 18-24 hours at room temperature. The reaction was taken into account by the degree of enlightenment of the sera and the formation of conglomerate (precipitate in the form of an “umbrella”) at the bottom of each tube. The result of determining the titer of specific antibodies in the blood serum of immunized and intact rabbits is presented in table 1, where the nature of agglutination is presented as follows:

++++ - complete agglutination,

+++ - almost complete agglutination,

++ - incomplete agglutination,

+ - slight agglutination,

- lack of agglutination.

Table 1 Serum Antibody titer 1:25 1:50 1: 100 1: 200 1: 400 1: 800 1: 1600 1: 3200 1: 6400 WITH LIVE CULTURE Experienced ++++ ++++ ++++ ++++ +++ ++ + + + Control ++++ +++ ++ + + + + + - WITH INACTIVATED CULTURE Experienced ++++ ++++ ++++ ++++ +++ ++ + + + Control ++++ +++ ++ + + + + + -

Thus, a single administration of a live vaccine provides an increase in the level of immunity in relation to H- (flagellar) and O- (somatic) antigens in comparison with the control by 8 times.

Example 3. To assess the intensity of immunity used two groups of white mice. The first group of mice (10 animals) was subjected to a single immunization with a culture of E. coli strain B-5, the second group of mice (10 animals), which were injected with saline in the same doses, was a control. After 14 days, both groups of animals were infected with an intraperitoneal virulent culture of Escherichia with a concentration of 5 × 10 ⁸ microbial cells in 1 cm ^{3 of} physiological saline in a dose of 0.5 ml. The research results are presented in table 2, from which it follows that the safety of immunized animals is 100%, in the control group all animals died.

table 2 Groups of white mice Number of goals Fallen, goals Survived Goals Immunized 10 0 10 Control 10 10 0

Example 4

To prove the avirulence of the vaccine, it was administered to mice at a concentration of 5 × 10 ⁸ microbial bodies. The death of mice did not occur.

Example 5

The vaccine dose was tested both for sows and piglets. Sows received a vaccine with a concentration of 2.4 × 10 ⁹ microbial cells in 1 cm ³ and piglets with a concentration of 2.4 × 10 ⁸ in 1 cm ^{3 of} physiological saline.

For this, one group of sows in an amount of 7 animals was injected with the vaccine intramuscularly at a dose of 2 ml, and the other group in an amount of 6 animals was given a dose of 3 ml. As a result of the experiments, a dose of 2 ml was selected, which provided the most effective protection of animals and their safety - 98.5%, while the born pigs did not get colibacillosis. At the injection site, no inflammatory reactions were recorded in animals. The introduction of the vaccine to sows in a dose of 3 ml did not have a particular effect on the effectiveness of vaccination.

Similarly, the vaccine was tested at a concentration of 2.4 × 10 ⁸ on piglets. The research result is presented in table 3, from which it follows that the safety of piglets in the experimental group is 6.9% more than in the control. In this case, the best efficiency was achieved with the introduction of a vaccine in a dose of 2 ml.

Table 3 Name of animal groups Number of pigs Survived Goals % safety Experienced 254 250 98.4 Control 249 228 91.5

Extensive production testing of the vaccine was carried out on sows and separately on pigs.

The vaccine was administered to 4783 sows intramuscularly at a dose of 2 ml once on 104-105 days of gestation. In the control group of animals (in the amount of 852 heads), generally accepted methods for the prevention of colibacteriosis were used. The safety of suckling piglets from vaccinated sows in the experimental group was 8.7% higher than in the control.

The introduction of the vaccine to 5194 piglets at the age of 2-7 days intramuscularly once in a dose of 2 ml showed an increase in safety by 10.8% compared with the control group (in the amount of 7148 piglets), which were prevented by the methods adopted in the farms.

Thus, the claimed vaccine is highly immunogenic, does not have reactogenicity, allergenicity, does not cause inflammatory reactions at the injection site and provides protection for piglets from colibacteriosis.

Claims (1)

Live vaccine against pig colibacteriosis, which is a suspension containing a solvent and an antigen from E. coli strain in the form of a bacterial mass of colony-forming microbial cells, characterized in that it contains physiological saline as a solvent, and an antigen from E. coli strain as an antigen "B-5" with a content of 2.4 × 10 ⁸ -2.4 × 10 ⁹ microbial cells in 1 cm ³ physiological saline.