Method of manufacturing vaccine, associated against pseudomonosis and viral rabbit haemorrhagic disease

FIELD: medicine.

SUBSTANCE: invention relates to method of manufacturing vaccine, associated against pseudomonosis and viral rabbit haemorrhagic disease. Characterised method includes selection of affected organs from dead rabbits in the period of their disease from local epizootic focus, separation of pure cultures of disease causing agents, for which purpose separate growing of cultures Pseudomonas aeruginosa and virus of rabbit haemorrhagic disease if performed. To obtain causative agent of pseudomonosis, Pseudomonas aeruginosa culture is grown in beef-extract broth with addition of glucose. To obtain virus of haemorrhagic disease, rabbits are infected with virulent virus of rabbit haemorrhagic disease with infection activity not lower than 103 LD 50/cm3. Samples of dead rabbit's liver are taken, its milling and homogenisation performed. Inactivated cultures Pseudomonas aeruginosa and viral rabbit haemorrhagic disease are mixed in equal proportions Solution of aluminium hydroxide is introduced, mixed, packed and sealed.

EFFECT: claimed invention makes it possible to manufacture harmless, highly-immunogenic vaccine, stable in storage.

1 tbl

 

The invention relates to veterinary, in particular to a method of manufacturing a vaccine against associated pseudomonosis and viral haemorrhagic disease of rabbits and can be used in research and industrial institutions for the development and construction of the vaccine preparations, and also at the enterprises of biological industry.

A method of producing polyvalent vaccines against enteric infections (RF patent for the invention №2080875 from 18.03.92, MKI A61 K39/125, 39/135). This method of producing polyvalent vaccine includes the separate cultures of Salmonella typhi, Salmonella paratyphi B, Shigella Flexneria, Shigella Sonne, the separation of the biomass from the nutrient medium, the extraction of antigens is carried out with water-salt solution in totalrevenue in the presence of the antibiotic and melkorazdroblennyh glass for 1-1. 5 h, the extract is centrifuged, the purification is subjected to the supernatants and after mixing to get the desired product in the form of a mixture of cell walls containing O-antigens of cultures used, with flagella containing H-antigens, and soluble Vi-antigens of Salmonella. Technology is getting this vaccine is very complex and for the prevention of infectious diseases of rabbits is not recommended.

A method of producing associated vaccine for prevention in�of ECCI, caused by conditionally pathogenic bacteria (RF patent for the invention №2035189 from 02.01.86, MKI AC 39/125, 39/135). This method enables separate selection of protective antigens from Staphylococcus aureus strain No. B - 243 secrete cytoplasmic antigen from toxinproducing strain of Pseudomonas aeruginosa IEM No. PA - 7 receive toxoid Pseudomonas aeruginosa from clinical strains of Proteus mirabilis№6, 8, 40, 508, 1115, 4641 secrete soluble antigens by controlled proteolysis derived antigens mixed in a saline solution from the calculation of their content in 1 ml of vaccine 0.15-0.2 mg of protein, 15-30 µg protein 10-20 μg of protein, respectively, and the mixture was further added a commercial staphylococcal toxoid and aluminum hydroxide in the amount of 5-10 EU and 1.2 mg respectively. Technology for producing vaccines are very complex, to prevent rabbits not applicable.

A known method of manufacture of a vaccine against associated pseudomonosis and enterococcal infection nutria (patent RF №2388489, 10.05.2010), including selection of the affected organs from dead nutria from local epizootic foci, of which prepare the suspension, do the planting on differential diagnostic environment, isolated pure cultures of pathogens, separately grown cultures of Pseudomonas aeruginosa and Enterococcus faecalis in mesopatamia broth with the addition of glucose to 0.2% conc�Tracii with titer 4-5 billion microbial cells in 1 cm3, inactivate by adding formalin to 0.4 to 0.5% final concentration, incubated at 37°C for 72-96 h, mixed culture in equal proportions, then make a solution of aluminium hydroxide in the amount of 20% by volume, thoroughly mixed, Packed and sealed. The resulting vaccine is not used to prevent rabbits.

A method for manufacturing a bivalent vaccine against myxomatosis and viral hemorrhagic disease of rabbits (RF patent for the invention №2064304 from 27.07.96, MKI A61 To 39/275). This method involves the separate cultivation of vaccine virus myxomatosis of rabbits in cell culture of chicken fibroblasts activity is not below 104,0EID50/cm3, freeze-thawed, mixed with inactivated suspension of the liver of rabbits infected with virulent haemorrhagic disease of rabbits in the ratio 1:1, stabilize the mixture in a protective environment at a ratio of 10:1, Packed and lyophilizers after freezing at minus 55±5°C for 10±2 h by incubation of the material at the temperature from minus 50±5°C to 0°C for 31±1 h and then at 0°C to + 20±1°C steps and finally dried for 3±1 h. This method allows to obtain the bivalent vaccine against myxomatosis and viral hemorrhagic disease of rabbits. Technology �of Holocene this vaccine very difficult.

The technical result of the invention is to provide harmless, specific, vysokomernoy associated vaccine to protect rabbits against pseudomonosis and viral hemorrhagic disease, by introducing a new, inactivated pathogens, components, eliminating the negative and adverse effect.

The technical result is achieved in that in the method of manufacturing a vaccine associated against pseudomonosis and viral hemorrhagic disease of rabbits, including the selection of the affected organs from dead rabbits during the period of their disease Pseudomonas and viral haemorrhagic disease from local epizootic focus, the selection of pure cultures of pathogens, which are conducting separate cultures of Pseudomonas aeruginosa and haemorrhagic disease of rabbits, then to obtain the pathogen pseudomonosis culture of Pseudomonas aeruginosa grown in mesopatamia broth with the addition of glucose to 0.2% concentration with a titer of at least 4 billion microbial cells in 1 cm3within 12-24 hours at 37°C; to obtain hemorrhagic disease virus infect rabbits with virulent virus hemorrhagic disease of rabbits isolated from epizootic hearth with infectious activity of not less than 103LD50/cm3conduct the selection of liver cu fallen�face, its grinding and homogenization, the resulting mass was adjusted with saline to 10-15% suspension, then perform the extraction of the virus at a temperature of 4-6°C for 10-12 hours, decantation of the supernatant, add saline in the ratio 1:1, filtered, inactivate the culture by adding formalin 0.1 to 0.4% concentration and incubated at 37°C for 48-96 hours, then mix inactivated culture of Pseudomonas aeruginosa and viral hemorrhagic disease of rabbits in equal proportions, then make a solution of aluminium hydroxide in the amount of 20% by volume of the mixture, mix thoroughly, Packed and sealed.

The novelty of the claimed proposal due to the fact that unlike the known methods, conduct sampling of the affected organs from dead rabbits during the period of their disease Pseudomonas and viral haemorrhagic disease from local epizootic foci; separately grown cultures of Pseudomonas aeruginosa and haemorrhagic disease of rabbits; to obtain pathogen pseudomonosis culture of Pseudomonas aeruginosa grown in mesopatamia broth with the addition of glucose to 0.2% concentration with a titer of at least 4 billion microbial cells in 1 cm3within 12-24 hours at 37°C, to obtain hemorrhagic disease virus infect virulent hemorrhagic disease virus to�Alikov, isolated from epizootic hearth with infectious activity of not less than 103LD50/cm3conduct the selection of a liver from a fallen rabbit, which after grinding, homogenizing extracted the virus from 10-15% suspension of liver at a temperature of 4-6°C for 10-12 hours with occasional stirring, and after inactivation with formalin and extracts inactivated culture of Pseudomonas aeruginosa and haemorrhagic disease of rabbits mixed in equal proportions and make a solution of aluminum hydroxide.

Inactivated cultures of pathogens formalin to 0.1-0.4% final concentration at 37°C for 48-96 hours of pure and mixed culture in equal proportions, which allows to suppress the pathogenicity and to maintain a high immunogenicity against pseudomonosis and viral hemorrhagic disease of rabbits. The addition of aluminum hydroxide in the amount of 20% by volume of culture allows through 12-15 days after a single injection at a dose of 1 cm3rabbits vaccinated to ensure the formation of a busy immunity against the two infections.

The method provides for obtaining harmless, vysokomernoy specific vaccine to protect rabbits from pseudomonosis and viral hemorrhagic disease of rabbits.

According to the patent and scientific literature is not detected na�logical inventive combination of features, that allows to judge about the inventive step of the claimed technical solutions.

With regard to the criterion "industrial applicability", the components included in the vaccine, well-known and widely used in veterinary practice in the manufacture of inactivated vaccines: combined vaccine against myxomatosis and viral hemorrhagic disease of rabbits (one HUNDRED 00495549-0044-2007); the combined vaccine against pasteurellosis and viral hemorrhagic disease of rabbits (THE approved 30.09.94 G., manual for the production and control of this vaccine approved 27.05.94 G.); the vaccine against hemorrhagic disease of rabbits tissue inactivated aluminum hydroxide HUNDRED 00495549-0005-2006.

Methods of extraction and characterization of microorganisms of the genus Pseudomonas described in the "Workshop on veterinary Microbiology and immunology", authors: Kostenko T. S., Rodionova V. B., D. I. Skorodumov, M.: Kolos, 2001, p. 259-261; in the Handbook of Microbiological diagnostics of bacterial diseases of animals", authors: D. I. Skorodumov, Subbotin V. V., Sidorov M. A., Kostenko T. S., M.: "Painters", 2005, pp. 522-525.

Methods of extraction and characterization of viral hemorrhagic disease of rabbits described in "Methodological guidelines for laboratory diagnosis of viral haemorrhagic disease of rabbits", approved GUV of the state agricultural Committee of the USSR 2.12.88; in the book "Viral haemorrhagic disease of rabbits", authors: Shevchenko A. A., Vishnyakov I. F., Bakalov I. A., Vlasova T. A. M.: "Two Crowns", 1995, 48 p.; in the book "Diseases of rabbits", authors: Shevchenko A., Shevchenko, L. V., Litvinov A. M. M.: "Aquarium", 2002, pp. 42-57.

The essence of the proposed method lies in the fact that the first conduct sampling of the affected organs from dead rabbits during the period of their disease Pseudomonas and viral haemorrhagic disease from local epizootic focus. Then isolated pure cultures of pathogens, which are conducting separate cultures of Pseudomonas aeruginosa and haemorrhagic disease of rabbits. Next, to obtain the pathogen pseudomonosis culture of Pseudomonas aeruginosa grown in mesopatamia broth with the addition of glucose to 0.2% concentration with a titer of at least 4 billion microbial cells in 1 cm3within 12-24 hours at 37°C. To obtain hemorrhagic disease virus infect rabbits with virulent virus hemorrhagic disease of rabbits isolated from epizootic hearth with infectious activity of not less than 103LD50/cm3conduct the selection of a liver from a fallen rabbit, its grinding and homogenization, the resulting mass was adjusted with saline to 10-15% suspension. Then perform the extraction of the virus at a temperature of 4-6°C for 10-12 hours�, the decantation of the supernatant, add saline in the ratio 1:1, filtered. Inactivate the culture by adding formalin 0.1 to 0.4% concentration and incubated at 37°C for 48-96 hours. Then mix inactivated culture of Pseudomonas aeruginosa and viral hemorrhagic disease of rabbits in equal proportions. Then make a solution of aluminium hydroxide in the amount of 20% by volume of the mixture, thoroughly mixed, Packed and sealed.

An example of a specific implementation of the method of manufacturing a vaccine.

For the manufacture of a vaccine against associated pseudomonosis and viral hemorrhagic disease of rabbits conduct sampling of the affected organs from dead rabbits during the period of their disease Pseudomonas and viral haemorrhagic disease, of which prepare the suspension and conduct laboratory studies to obtain pure cultures of pathogens.

To determine the morphology and tinctorial properties of the pathogen pseudomonosis prepare brushstrokes-prints of the affected organs of dead nutria, paint them using gram. Microscopy preparations under oil immersion microscope system visible straight or curved sticks the size of 1.5-3.0 mm, housed singly, in pairs, in the form of chains, painted in pink color characteristic of Pseudomonas aeruginosa.

For some�management of cultural properties of the pathogen pseudomonosis make the crops selected culture of pathologic material in mesopotania broth in Petri dishes with mesopartner agar (MPA), MPA and blood a selective medium containing setitimer ammonium bromide, which others found not growing. After 24 hours incubation in thermostat at temperature 37°C see surface silvery-white film in the BCH, a characteristic sign and clouding of the environment. In Petri dishes with blood MPA see growth grayish, translucent colonies surrounded by a greenish zone of hemolysis, which is characteristic of Pseudomonas aeruginosa.

To produce viral raw materials in the manufacture of vaccines use not vaccinated against viral haemorrhagic disease of rabbits, weighing at least 1.5 kg, infect them with virulent virus hemorrhagic disease of rabbits isolated from epizootic hearth with infectious activity of 103LD50/ml3. 48-72 hours rabbits die from the fallen take the liver in plastic bags, frozen, crushed with scissors and on the homogenizer. The obtained raw material was adjusted with saline (pH 7.2) up to 10-15% suspension, then extracted with virus at a temperature of 4-6°C for 10-12 hours, stirring occasionally, decanted the supernatant, to the residue was added saline 1:1, mix. To free from cell debris filtration was performed through two layers of sterile gauze. Then the filtered biological raw materials poured into a capacious�you and spend inactivate making formalin 0.1 to 0.4% final concentration, incubated at 37°C for 48-96 hours with occasional stirring, then mixed inactivated antigens in equal proportions, make a solution of aluminium hydroxide in the amount of 20% by volume and thoroughly mixed, Packed and sealed.

Thus, the use for the manufacture of a vaccine against associated pseudomonosis and viral hemorrhagic disease of rabbits with pure cultures of pathogens Pseudomonas aeruginosa and viral hemorrhagic disease of rabbits, isolated from local epizootic focus, allows you to get specific, vysokogomogennogo a combined vaccine to protect against these two dangerous infectious diseases pseudomonosis and viral hemorrhagic disease of rabbits. Separate cultivation of pathogens allows to obtain the concentration of Pseudomonas aeruginosa for at least 4 billion in 1 cm3within 12-14 hours of incubation and haemorrhagic disease of rabbits with infectious activity of 103LD50/ml3. The use of formalin for inactivation at 0.1-0.4% final concentration allows for 48-96 hours at a temperature of 37°C to inhibit the pathogenicity and to maintain a high immunogenicity during 12 months of storage.

The vaccine prepared by the claimed method, has been tested. Rabbits were injected a single dose of 1 cm3poluchenno� vaccine and after 12-15 days in vaccinated rabbits the formation of a busy immunity against pseudomonosis and viral hemorrhagic disease of rabbits, lasting at least 9 months (observation period). After a single injection of rabbits intramuscularly does not cause vaccine-related complications.

The proposed method is simple to implement, affordable and is industrially applicable.

A method of manufacturing a vaccine associated against pseudomonosis and viral hemorrhagic disease of rabbits, including the selection of the affected organs from dead rabbits during the period of their disease Pseudomonas and viral haemorrhagic disease from local epizootic focus, the selection of pure cultures of pathogens, which are conducting separate cultures of Pseudomonas aeruginosa and haemorrhagic disease of rabbits, then to obtain the pathogen pseudomonosis culture of Pseudomonas aeruginosa grown in mesopatamia broth with the addition of glucose to 0.2% concentration with a titer of at least 4 billion microbial cells in 1 cm3within 12-24 hours at 37°C; to obtain hemorrhagic disease virus infect rabbits with virulent virus hemorrhagic disease of rabbits isolated from epizootic hearth with infectious activity of not less than 103 LD50/cm3conduct the selection of a liver from a fallen rabbit, its grinding and homogenization, the resulting mass was adjusted with saline to 10-15% suspension, then perform the extraction of the virus at a temperature of 4-6°C for 10-12 hours, decantation of the supernatant, add saline in the ratio 1:1, filtered, inactivate the culture by adding formalin 0.1 to 0.4% concentration and incubated at 37°C for 48-96 hours, then mix inactivated culture of Pseudomonas aeruginosa and viral hemorrhagic disease of rabbits in equal proportions, then make a solution of aluminium hydroxide in the amount of 20% by volume of the mixture, thoroughly mixed, Packed and sealed.



 

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32 cl, 33 dwg

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science and aims at the preventing pseudomonosis and rabbit viral haemorrhagic disease. As antigens, the vaccine contains a pure culture cell suspension of the pseudomonosis agent Pseudomonas aeruginosa and the rabbit viral haemorrhagic disease agent produced by sampling the involved organs of dead rabbits from a local epizootic centre. The suspension is prepared with the differential diagnostic media inoculated and pure agent cultures recovered. The growth procedure is performed separately in beef-extract broth of Pseudomonas aeruginosa with glucose in the concentration of microbial cells 4-5 bn in 1 cm3. The 10-15% rabbit liver suspension is prepared after being infected with the rabbit viral haemorrhagic disease, with an activity of not less than 103.0 LD50/cm3. This is mixed in equal ratios of formalin and aluminium hydroxide in the following proportions, wt %: pure culture cell suspension of the pseudomonosis agent Pseudomonas aeruginosa recovered from the local epizootic centre in the nutrient medium with a titre of 4-5 bn microbial cells in 1 cm3 38.0-41.5, the rabbit liver suspension with the rabbit haemorrhagic disease virus recovered from the local epizootic centre in normal saline with an activity of not less than 103.0-4.0 LD50/cm3 - 38.0-41.5, glucose - 2.0-1.0, formalin - 2.0-1.5, aluminium hydroxide - the rest.

EFFECT: using the declared invention enables higher specificity and efficacy of the associated pseudomonosis and rabbit viral haemorrhagic disease with no negative side effect on the animals.

1 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: from bred heifer the strain of bluetongue virus of serotype 14 is isolated, deposited in the collection of microorganisms under the State Scientific Institution of All-Russian Scientific Research Institute of Veterinary Virology and Microbiology of Russian Agricultural Academy under the number 3032. The strain is a new, previously unknown, isolated on the territory of the Russian Federation. The invention can be used in research institutes and enterprises of biological industry in the design of biological products, such as vaccines against bluetongue of serotype 14 and control of their immunogenicity, as well as the production of antigens for diagnostic sets.

EFFECT: improvement of the strain properties.

2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, virology and medicine. The first flaviviral particle comprises a psudoinfecting viral genome coding cis-active promoter elements required for the RNA replication, coat proteins and a complete kit of non-structural proteins of the flavivrus, and not coding capside proteins of the flavivirus. The second flaviviral particle comprises a complementary genome coding the cis-active promoter elements required for the RNA replication, a capside protein and a complete kit of proteins of the flavivrus, and not coding coat proteins. Since the genetic material of the flavivirus is distributed between two genomes, the flavivirus is replication-deficient and is not able to induce a disease, however it is able to induce an immune response. What is also described is a method for preparing this combination, a pharmaceutical composition and a method for using it. The invention can be used in medicine.

EFFECT: what is presented is a combination of the flaviviral particles.

13 cl, 18 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and concerns an adjuvant immunogenic composition containing meningococcal lipooligosaccharide (LOS) and serotype 14 pneumococcal capsular saccharide (CS14), wherein CS14 contains tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc, while LOS is free from tetrasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4Glc. The group of inventions also concerns a method for inducing the immune response in a mammal involving administering the above composition.

EFFECT: group of inventions provides the stronger immune response to CS14 A as compared to wild-type OMV containing LNnT.

15 cl, 4 dwg, 1 ex

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