Strain of bacteria pseudomonas aeruginosa № 9 for production of vaccine against pseudomonosis of pigs

FIELD: biotechnology.

SUBSTANCE: invention relates to a strain of the pathogen of pseudomonosis of pigs of the collection of Federal state budgetary institution "VGNKI", deposited under the name "Pseudomonas aeruginosa No.9" and the registration number "No.9-DEP", intended for production of a vaccine against pseudomonosis of pigs.

EFFECT: invention provides high immunogenic activity and the ability of production of the vaccine against pseudomonosis of pigs.

4 ex, 3 tbl

 

The invention relates to biotechnology and veterinary medicine and is intended for the production of vaccine preparations against pseudomonosis pigs.

Pseudomonas - an acute infectious disease of animals and poultry, characterized in young septic process, pulmonary, gastrointestinal tract and joints, and in adults of reproductive organs and breast cancer. The causative agent pseudomonosis is a gram negative aerobic bacterium Pseudomonas aeruginosa, which antigenic structure is divided into 20 serologic groups (Pseudomonas animals / I. A. Bolotsky, A. G. Shipitsin, A. K. Vasilev and others - M.: Kolos, 2010. - 223 p.).

Pseudomonas animals, including pigs, has been reported from all regions of Russia, regardless of the natural-geographical conditions. The vast majority of cases the causative agent pseudomonosis pigs is Pseudomonas aeruginosa serogroups O1, O3, O4, O6, O11, 019 (Prudakov S. V. Pseudomonas productive animals in the North Caucasus region / author. Diss. doctor. vet. Sciences. - Krasnodar, 2011. - S. 37). In these circumstances, for the prevention of disease was developed by a polyvalent vaccine against pseudomonosis farm animals (Pat. Of the Russian Federation No. 2308288), which as antigens contains a suspension of cells of pure cultures of Pseudomonas aeruginosa belonging to the 10 serogroups (O1, O2, O3, O4, O6, O10, O11, O13, O18, A19), p�obtained by selection of parenchymatous organs affected from local epizootic focus. A significant drawback of such a vaccine is to use to obtain non-characterized (random) strains with unknown immunogenic properties.

Known vaccine strain of the bacteria Pseudomonas aeruginosa 5142used for the manufacture of a vaccine against pseudomonosis fur-bearing animals (Pat. RF 2169193, prototype). The disadvantage of this strain is the fact that it focuses on the creation of a vaccine against pseudomonosis fur-bearing animals and vaccinated animals create immunity to the exotoxin A, but not to somatic O-antigen, which persists for a short time.

Technical solutions, in which was used a strain of Pseudomonas aeruginosa for vaccine pseudomonosis pigs with a given O-antigenic profile and immunogenity properties in the available scientific and patent literature was revealed.

Pseudomonas aeruginosa No. 9 selected from the fallen pig in 2003, in 2011, proposed as a vaccine strain, and in 2013 deposited in all-Russian collection of strains of microorganisms used in veterinary medicine and animal husbandry (Russia, 123022, Moscow, Zvenigorodskoe highway 5, the fgbi "VGNKI" under the registration number "No. 9-DEP".

Pseudomonas aeruginosa No. 9-CM is designed as a production strain for the manufacture of a vaccine against Pseudomonas�and pigs and stored in the FSUE "Armavir Biofactory".

The technical result from the use of the invention is to expand the Arsenal of strains of Pseudomonas aeruginosa, and having its long lasting immunogene properties and suitable for the manufacture of a highly effective vaccine preparations.

The technical result is achieved in that the strain of the pathogen pseudomonosis pigs, collection fsbi VGNKI deposited under the name of "Pseudomonas aeruginosa No. 9" and registration number "No. 9-DEP", intended for the manufacture of a vaccine against pseudomonosis pigs.

Pseudomonas aeruginosa No. 9-DEP is characterized by the following properties.

Cultural and morphological characteristics: gram-negative bacilli, size 2-3×0.5-0.8 microns, mobile, spores and capsules do not form, you'll be patinece. Grow on simple nutrient media with the formation of a blue-green pigment of the pyocyanin and mucus. On the surface of the meat-peptone broth gives the film a grayish-silver color, and on the surface of a meat-peptone agar - rough irregular shape of the colony.

Physiological and biochemical characteristics: mesophylous, aerobe, grows at temperatures from 18 to 42°C, but optimum growth temperature is 35-37°C. Oxidizes glucose, restores nitrates to nitrites, aged arginine, liquefies gelatin, roll milk, destroys the sheep red blood cells, forms citehr�oxidase.

Antigenic properties: agglutinated model 04 to serum titer of 1:1280.

Virulent properties: Causes the death of white mice by intraperitoneal injection of 2-3×106microbial cells.

Immunogenic properties of the double immunization of rabbits with formalin inactivated cells at a dose of 1-2×1010and adult pigs at a dose of 4-6×1010microbial cells 14 days after the last immunization serum of animals have antibodies in a titer of 1:512-1:1024. Protective antibody titre equal for a given strain 1:64, remains in immunized animals within 12 months.

The invention is illustrated by the following examples.

Example 1. Pseudomonas aeruginosa No. 9 is grown on broth of Law within 24-46 hours at 36-37°C with a pH of 7.2-7.4 and constant shaking at shuttel machine. After expiry of the incubation, the culture is checked for purity by gram staining, adjusted with physiological saline to a concentration of 10 billion microbial cells in 1 ml, and then inactivated with formalin, containing not less than 36% formaldehyde to a final concentration of 0.3-0.4% and incubated for 14 days at a temperature of cultivation. After the expiration of inactivation in culture make a 3% solution of aluminium hydroxide, Packed into vials and sealed. The resulting vaccine is a clear liquid yellow�ovato-greenish color with a whitish-grayish loose sediment, upon shaking forms a homogenous suspension.

Example 2. Obtained in example 1 the vaccine is tested for sterility and safety.

The sterility of the vaccine was monitored by the absence of growth when sown on medium Saburo (mushrooms), Endo (coliform bacteria), MPA (mesophilic aerobic bacteria), Citta-Tarazi (anaerobes). Growth on all the media was absent, indicating the sterility of the vaccine.

Safety check 3 white mice weighing 18-20 g, which the vaccine is administered subcutaneously in a volume of 0.3 ml and observe for 7 days. All experimental animals remained alive at the injection site of the pathological changes were made, demonstrating the harmlessness of the drug.

Example 3. Immunogenicity is assessed by protective capability in experimental Pseudomonas on white mice and the magnitude of accumulation titers of specific antibodies in immunized vaccine rabbits and pigs.

3 groups of white mice, 10 animals in each for 10 days before infection epidemic strain of Pseudomonas aeruginosa O4 immunized mice group 1 vaccine strain of Pseudomonas aeruginosa No. 9 in a dose of 0.1 ml subcutaneously; mice of group 2 vaccine from a strain of Pseudomonas aeruginosa 5142at a dose of 0.1 ml subcutaneously; mice of group 3 were injected with saline in a volume of 0.1 ml subcutaneously. 10 days after immunization, all mice infected with a pathogenic isolate of Pseudomons aeruginosa serogroup 04 at a dose of 100 million. microbial cells intraperitoneally. Within 14 days the mice were observed and made the registration of the fallen.

The results are shown in table 1, which shows that in the first group survived 80% of experimentally infected animals, which is 30% more than in the second group, animals which immunotherapies vaccine from epizootic strain. In the control group, where animals were administered saline, killed all mice. Therefore, a vaccine against pseudomonosis from a strain of Pseudomonas aeruginosa No. 9 has protected the greatest number of experimentally infected animals, and therefore it is more immunogenic than the vaccine prepared from a strain of Pseudomonas aeruginosa 5142.

Table 1 -
The protective effect of vaccines in experimental Pseudomonas due to Pseudomonas aeruginosa serogroup O4
GroupNumber of micePaloSurvivedThe protective effect of the vaccine, %
1. Was immunotherapies vaccine from a strain of Pseudomonas aeruginosa No. 9102880
. Was immunotherapies vaccine from a strain of Pseudomonas aeruginosa 5142105550
3. Control (administered saline)1010--

The immunogenicity of the strain Pseudomonas aeruginosa No. 9 was assessed by the ability to stimulate the production of specific antibodies in 10 rabbits and 10 adult pigs. For this immunize animals twice with an interval of 14 days rabbits at a dose of 1 ml per head, pigs - 5 ml per head. After 14, 30, 90, 180, 270 and 360 days after the last immunization in agglutination reaction determine the titers of antibodies to Pseudomonas aeruginosa serogroup O4.

The results are shown in table 2, which shows that the deposited strain Pseudomonas aeruginosa No. 9, intended for the manufacture of a vaccine against pseudomonosis pigs, has a more pronounced immunogenic properties than the vaccine from a strain of Pseudomonas aeruginosa 5142because the formation is 3-5 times higher level of specific antibodies. In addition, in the body immunized animals in the groups that used the vaccine from a strain of Pseudomonas aeruginosa No. 9, a protective level of antibodies to the somatic antigen O4 (1:64) were preserved in the Techa�s 12 months and in animals immunized with vaccine from strain Pseudomonas aeruginosa 5142- only 3-6 months. It follows that a vaccine prepared from a strain of Pseudomonas aeruginosa No. 9, provides a more lasting immunity than the vaccine from a strain of Pseudomonas aeruginosa 5142.

Table 2 -
Immunogenicity of vaccines against pseudomonosis
GroupThe titer of antibodies to the somatic antigen O4 Pseudomonas aeruginosa following immunization by days
143090180270360
Rabbits (n=10)
1. Was immunotherapies vaccine from a strain of Pseudomonas aeruginosa No. 91:819,21:1228,81:716,81:5121:232,81:64
2. Was immunotherapies vaccine from a strain of Pseudomonas aeruginosa 5142 1:273,11:341,31:of 204.81:641:161:8
Pigs (n=10)
1. Was immunotherapies vaccine from a strain of Pseudomonas aeruginosa No. 91:10241:1433,61:716,81:307,21:2561:83,2
2. Was immunotherapies vaccine from epidemic strain of Pseudomonas aeruginosa 51421:238,91 is 286.71:1281:44,81:81:2

Example 4. At pig-farm product, dysfunctional Pseudomonas, immunize 30 piglets 2 weeks of age obtained in example 1 vaccine strain of Pseudomonas aeruginosa No. 9 (experimental group) and 30 pigs of a similar age with vaccine from strain Pseudomonas aeruginosa 5142(positive control), 30 of piglets immunotherapies (negative control). Bacteriological studies revealed that the disease causes Pseudomonas aeruginosa serogroup O4. Farrowing�and experimental group and positive control group was immunotherapies by the same scheme: twice with an interval of 14 days intramuscularly with inner thigh at a dose of 1.0 and 1.5 ml. The observation of the animals is conducted within 120 days, noting all the cases and the deaths of piglets associated with Pseudomonas.

The test results are shown in table 3, from the materials of which it is seen that in the experimental group became infected with Pseudomonas 6 pigs, 1 of them fell. In the positive control group was ill 10 piglets, of which 3 Palo. In the negative control group ill 16 piglets, of which Palo - 7. Consequently, morbidity and mortality in the experimental group were respectively 20 and 3.3% in the positive control group is 33.3 and 10% in the negative control group is 50.3 and 23.3%. The safety of piglets in the experimental group was 96.7% in the positive control group - 90% in the negative control group is 76.7%.

30
Table 3 -
Prophylactic efficacy of a vaccine against pseudomonosis pigs from a strain of Pseudomonas aeruginosa No. 9
GroupThe number of pigs, subjected to immunizationSick piglets in the groupPalo piglets in the groupThe safety of piglets, %
1. Experienced, immunotherapies vaccine from a strain of Pseudomonas aeruginosa No. 96 (20%)1 (3,3%)96,7
2. Control, immunotherapies vaccine from a strain of Pseudomonas aeruginosa 51423010 (33,3%)3 (10%)90,0
3. Control, not immunotherapies3016 (50,3%)7 (23,3%)76,7

Thus, the vaccine is received in accordance with the present invention, namely from a strain of Pseudomonas aeruginosa No. 9, provides the greatest survival of animals in experimental and spontaneous Pseudomonas than the vaccine from a strain of Pseudomonas aeruginosa 5142because able more to stimulate the production and circulation in the body immunized animals of specific antibodies to the somatic antigen O4. Consequently, a strain of Pseudomonas aeruginosa No. 9 suitable for the manufacture of a vaccine against pseudomonosis pigs.

Strain of the pathogen pseudomonosis pigs, collection fsbi VGNKI deposited under the name of "Pseudomonas aeruginosa No. 9" and registration number "No. 9-DEP", intended for the manufacture of a vaccine against pseudomonosis pigs.



 

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