RU2344832C1 - Vaccine drug against colibacteriosis - Google Patents

Abstract

FIELD: medicine; biotechnologies.

SUBSTANCE: vaccine drug includes deactivated Escherichia coli bacteria of strain No 389 (078) and auxiliary substances. Additionally the drug includes aminoethylethyleneimine and liposome-forming mix as auxiliary substance at the following component rate in fluid form, wt %: aminoethylethyleneimine - 0.5-3; liposome-forming mix - 7.3-15; suspension of deactivated Escherichia coli bacteria of serotype 078 - 82.0-91.5.

EFFECT: harmless for fowl, enhanced antigenic and immunogenic activity.

3 cl, 4 tbl, 6 ex

Description

The invention relates to the field of veterinary medicine, in particular to poultry farming, in particular to vaccines against infectious diseases, namely against colibacteriosis.

Colibacteriosis (colisepticemia, coli infection, escherichiosis) is an enzootic septic disease of various species of poultry (mainly chickens, ducks, geese) caused by pathogenic types of E. coli of various serological groups. Clinically, colibacteriosis is manifested by depression, loss of appetite, diarrhea, shortness of breath, wheezing, or termination of oviposition. With postmortem autopsy, pericarditis, perihepatitis, aerosacculitis, salpengitis, ovaryitis, peritonitis are noted. The disease causes significant economic damage to poultry farming.

Currently, antibiotics, sulfonamide, nitrofuran and various complex preparations are widely used for the prevention and treatment of infectious diseases. In particular, for the prevention and treatment of colibacteriosis, oxytetracycline, tetracycline, biovit, levomycetin, tetrachloride, sulfagin, phthazine, biofusol and several other drugs are recommended (Veterinary preparations: Handbook / Comp. L.P. Malanin et al. M., Agropromizdat, 1988 , p.5-33; Romanov EA Biological veterinary preparations in Russia: vaccines, serums, diagnostics: Reference book. - Publisher: K .: RUTEN, 2005, p.636).

The disadvantages of most drugs are low efficiency, especially for solving preventive problems, a large number of contraindications, high cost of drugs.

In the prevention of colibacillosis, the main role is played by the strict implementation of a set of organizational, economic, zoohygienic and veterinary and sanitary measures, however, specific prevention plays an important role. In this case, the most effective way to combat veterinary infections is to vaccinate the livestock of the infected herd.

Currently, a significant number of live and inactivated vaccines are known, usually mono- or divalent, designed to prevent certain infectious diseases, such as Newcastle disease, avian reovirus, etc. (EP 0100752; EP 0044920; US 4515777; US 4302444; N.G. Rybalsky, B.C. Romanova and other Vaccines as an object of the invention, M., VNIIGPE, 1988, pp. 110-229). More convenient multivalent vaccine preparations that protect the livestock simultaneously from several viral diseases have not yet been widely used, because the creation of such drugs is very time-consuming due to the need to take into account the interaction of both the vaccine components themselves and the antibodies that arise in the body of the vaccinated animal or bird when they are introduced.

In particular, a vaccine against colibacteriosis of birds (SU 1792330) is known, obtained by culturing a strain of Escherichia coli (E. coli), followed by inactivation of the cells with acetone and isolating the resulting pellet. The disadvantage of the vaccine is its low efficiency and the high probability of infection of birds by surviving bacterial cells.

The closest to the claimed vaccine in terms of composition and achieved effect is an inactivated vaccine (TU 10-19.68-89), which is a finely divided white suspension containing formalin-inactivated bacteria Escherichia coli serotype 078 in a mixture with an adjuvant based on aluminum hydroxide (GOA formolvaccine). Vaccination is carried out by a single injection into the pectoral or femoral muscle in a volume of 0.5 ml to all healthy birds aged 28-32 days.

The disadvantages of this vaccine are not sufficiently high antigenic and immunogenic activity, as well as the possibility of inflammatory reactions at the site of its administration with the subsequent development of granulomas and abscesses.

The technical problem solved in the framework of the present invention was the creation of a more effective vaccine against colibacteriosis.

The technical result was achieved by creating a vaccine, which is a composition containing in liquid form the following ingredients, wt.%:

aminoethylethyleneimine (AEEI) 0.5-3.0; liposome forming mixture (VOC) 7.3-15.0; suspension of inactivated bacteria E. coli serotype 078 82.0-91.5.

Additionally, the preparation intended for further drying may contain chloroform or another solvent.

The resulting vaccine preparation is a suspension of liposomes containing a mixture of inactivated antigen for the bacterium Escherichia coli strain No. 389 (078) with aminoethylethyleneimine enclosed in a liposomal vesicle. Instead of E. coli strain No. 389 (078), another E. coli strain causing colibacteriosis can also be used.

The proposed new vaccine was based on the assumption that, when treating bacterial cells with aminoethylethylenimine, it is possible, on the one hand, to exclude the presence of live microorganisms in the vaccine, and, on the other hand, to ensure an increase in the content of antigen suspension due to cell destruction, which increases its effectiveness. At the same time, the introduction of liposomes into the composition ensures the placement of the active material in the vesicle, which is destroyed directly in the animal’s body, which ensures the maximum concentration of antigen in the injection zone and, accordingly, the increased production of antibodies by the body of the vaccinated bird.

The use of AEEI in a concentration of less than 0.5 wt.% Reduces the effectiveness of the vaccine and increases the risk of the presence of live bacteria in it, provides an increase in the concentration of more than 3%, without affecting the concentration of antigen, makes the vaccine more toxic.

The nature of the LIS used does not significantly affect antibody production. So, both individual phospholipids and their mixtures obtained from plant, animal or biotechnological raw materials were commercially available as LIS; a mixture of phospholipids and dry fillers (sugars, polyols, salts) according to the technology described in RU 2130771, or a mixture containing phospholipids with 10-12% sugars and 1-3% water-soluble polymers previously used in the authors patent (RU 2169581, RU 012968). The nature of the liposomes used has almost no effect on antibody production.

The content of LIS in a concentration of more than 15 wt.% Increases the viscosity of the drug, in a concentration of less than 7.3% does not ensure the completeness of the antigen in the vesicle and impairs its storage stability.

Vaccination of birds using the inventive vaccine consists in the introduction of the aforementioned liposomal vaccine into the body of the bird parenterally, intramuscularly in the pectoral or femoral muscle.

As a rule, the indication for the use of the vaccine is the lack of economy on the above infections. All clinically healthy birds are vaccinated at the age of 28-32 days. The drug is administered intramuscularly in the pectoral or femoral muscle at a dose of 0.5 ml per 1 head.

The essence and practical applicability of the claimed invention are illustrated by the following examples.

EXAMPLE 1. The vaccine was prepared according to the following procedure. 0.45 L of a suspension of cells of Escherichia coli strain No. 389 (078) with a concentration of 2 × 10 ¹⁰ CFU / cm ³ obtained from VGNKI was mixed at a temperature of 37 ° C under continuous stirring with 50 ml of 20% AEEI solution for 20 min. The resulting preparation contained antigens and did not contain viable bacterial cells.

20 ml of a preparation containing antigens was mixed with 2.0 g of total phospholipids of plant lecithin, 0.5 g of butyloxyanisole and 0.5 g of lactose. The mixture was homogenized for 10 minutes at a rotation speed of 700g and a mixture temperature of 22-24 ° C.

The liquid vaccine in 1 ml contained 1.5 × 10 ¹⁰ cells of Escherichia coli strain No. 389 (078); 0.002 g AEEI, 0.14 g VOC.

Example 2. The vaccine was prepared according to the following method. 0.475 l of a suspension of cells of Escherichia coli strain No. 389 (078) with a concentration of 2 × 10 ¹⁰ CFU / cm ³ obtained from VGNKI, was mixed at a temperature of 37 ° C in continuous mixing with 25 ml of a 20% solution of AEEI for 20 minutes The resulting preparation contained antigens and did not contain viable cells.

20 ml of a suspension containing antigens, 1.2 g of total phospholipids from sunflower seeds, 0.8 g of sorbitol and 5.0 g of chloroform were placed in a hermetically sealed container with a volume of 30 cm, at a temperature of 37 ° C. Drying was carried out under vacuum for 1 hour, at a temperature of 37 ° C and constant stirring at a speed of 50 rpm The result was 2.1 g of dry fine powder with a moisture content of 0.7%, containing 2 × 10 ¹¹ cells of Escherichia coli strain No. 389 (078); 0.2 g AEEI, 1.8 g VOC.

For vaccination, 0.5 g of the liposome preparation was dissolved in 10 ml of distilled water.

Example 3. The vaccine was prepared according to the following method. 1.0 l of a suspension of cells of Escherichia coli strain No. 389 (078) with a concentration of 2 × 10 ¹⁰ CFU / cm ³ obtained from VGNKI, was mixed at a temperature of 37 ° C in continuous mixing with 150 ml of a 20% solution of AEEI for 20 minutes The resulting preparation contained antigen and did not contain viable bacteria.

20 ml of a preparation containing antigens was mixed with 1.0 g of total phospholipids from sunflower seeds, 0.6 g of sorbitol and 5.0 g of chloroform in a hermetically sealed container with a volume of 150 cm, at a temperature of 37 ° C. The container was closed, air was pumped out until a vacuum of 0.3 atm was reached, and the mixture was kept for 1 hour at a temperature of 37 ° C with constant stirring at a speed of 50 rpm. The result was 15 ml of concentrate containing 2.0 g of liposomal vesicles.

To 100 volumes of a concentrate of liposome antigen vesicles, 5 volumes of a 20% aqueous gelatin solution and 50 volumes of a 20% sucrose solution were added. The resulting mixture was thoroughly mixed and poured into pallets with a layer thickness of 3.0-10.0 mm, closed with lids and transferred to the shelves of the sublimation chamber. The material was frozen to a temperature of -30-40 ° C and kept at this temperature for 2-3 hours, after which the chamber was evacuated at 120-130 mm Hg. Subsequently, the shelves were heated to + 24 ° C at a speed of 4 deg / h. The total drying time was 42-46 hours. For vaccination, 0.5 g of the liposome preparation was dissolved in 10 ml of distilled water.

EXAMPLE 4. The vaccine was prepared according to the method of example 1. 0.43 l of a suspension of cells of Escherichia coli strain No. 389 (078) with a concentration of 2 × 10 ¹⁰ CFU / cm ³ obtained from VGNKI, was mixed at a temperature of 37 ° C with constant stirring with 70 ml 25% AEEI solution for 20 minutes The resulting preparation contained antigens and did not contain viable bacterial cells.

20 ml of a preparation containing antigens was mixed with 2.3 g of total phospholipids of plant lecithin, 0.6 g of butyloxyanisole and 0.6 g of lactose. The mixture was homogenized for 10 minutes at a rotation speed of 700 g and a mixture temperature of 22-24 ° C, after which it was used for vaccination.

EXAMPLE 5. The vaccine was prepared as described in Example 1. 0.489 l cell suspension Escherichia coli strain №389 (078) with a concentration of 2 × ¹⁰ October CFU / cm _^3, obtained from VGNKI were mixed at 37 ° C in a constant stirring with 11 ml 25% AEEI solution for 20 minutes The resulting preparation contained antigens and did not contain viable bacterial cells.

20 ml of a preparation containing antigens was mixed with 1.2 g of total phospholipids of plant lecithin, 0.3 g of butyloxyanisole and 0.3 g of lactose. The mixture was homogenized for 10 minutes at a rotation speed of 700 g and a mixture temperature of 22-24 ° C, after which it was used for vaccination.

The composition of the obtained vaccine preparations (without taking into account its changes during drying) is shown in table 1.

Table 1 The composition of liquid vaccines Example The content of ingredients in the liquid mixture, wt.% AEEI VOC Suspension The content of E. coli cells one 1.8 13.8 84,4 1.5 × 10 ¹⁰ 2 0.6 * (0.9) 7.5 * (10.7) 74.0 * (88.4) 1.4 × 10 ¹⁰ 3 2.0 * (2.4) 6.0 * (7.3) 73.2 * (90.3) 1.3 × 10 ¹⁰ four 3.0 15.0 82.0 1.5 × 10 ¹⁰ 5 0.5 8.0 91.5 1.5 × 10 ¹⁰ * concentration taking into account the presence of chloroform in the mixture

Example 6. The vaccine was used to prevent colibacillosis of birds in the experimental base of the St. Petersburg State Academy of Veterinary Medicine. The drug was administered intramuscularly into the pectoral or femoral muscle at a dose of 0.5 ml per head.

The vaccine was tested for safety of antigenic and immunogenic activity. The intensity of post-vaccination immunity is determined 7, 28, 45, 60, 90 days after vaccination in the agglutination reaction. The test results obtained are presented in tables 2-4.

table 2 Assessing the injection site of the colibacteriosis vaccine of Example 1 10 days after the injection. №№ Vaccine Research results Degree of defeat Vaccine traces Granulomas Swelling one Liposomal vaccine light Not found Not found twenty 2 GOA Formol vaccine average 60 Not found 40

Table 3 Evaluation of the injection site of the colibacteriosis vaccine of Example 1 90 days after the injection. №№ Vaccine Visible changes at the injection sites,% Degree of defeat No visible changes Does not mean. qty. vaccines Fibrin one Liposomal vaccine light not found not found not found 2 GOA Formol vaccine average 40 60 not found

Table 4. The level of specific antibodies in the blood serum of vaccinated chickens (antibody titers in log ₂ (M ± m)). №№ Groups Research days after immunization 7 28 45 60 90 one Liposomal vaccine according to approx. one 6.1 ± 0.2 9.5 ± 0.3 9.9 ± 0.2 9.8 ± 0.1 9.8 ± 0.2 2 Liposomal vaccine according to note 2 5.8 ± 0.2 9.6 ± 0.3 9.8 ± 0.2 9.6 ± 0.1 9.6 ± 0.2 3 Liposomal vaccine according to note 3 6.0 ± 0.2 9.3 ± 0.3 9.6 ± 0.2 9.1 ± 0.1 9.1 ± 0.2 four Liposomal vaccine according to note 4 6.0 ± 0.2 9.7 ± 0.3 9.7 ± 0.2 9.6 ± 0.3 9.3 ± 0.4 5 Liposomal vaccine according to note 5 5.9 ± 0.2 9.0 ± 0.4 9.2 ± 0.2 9.0 ± 0.1 9.1 ± 0.2 6 GOA formol vaccine 5.4 ± 0.5 7.7 ± 0.5 7.4 ± 0.5 7.2 ± 0.4 6.8 ± 0.3 7 The control not found not found not found 0.5 ± 0.1 0.7 ± 0.1

As follows from the above results, the vaccine preparation is harmless to birds, has a pronounced antigenic and immunogenic activity.

Claims (3)

1. A vaccine preparation against colibacteriosis, containing in its composition inactivated bacteria Escherichia coli strain No. 389 (078) and excipients, characterized in that it additionally contains aminoethylethyleneimine, and as excipients, a liposome-forming mixture in the following ratio of ingredients in liquid form, wt .%:
aminoethylethyleneimine 0.5-3 liposome forming mixture 7.3-15 inactivated bacteria suspension Escherichia coli serotype 078 82.0-91.5 2. The vaccine against colibacteriosis according to claim 1, characterized in that as a liposome-forming mixture contains a mixture of phospholipids and excipients. 3. The vaccine against colibacteriosis according to claim 1, characterized in that as a liposome-forming mixture contains a mixture of phospholipids, sugars and water-soluble polymers.