RU1066074C - Pseudomonose vaccine for fur-bearing animals, mainly, minks and method of producing the same and method of its prophylaxis - Google Patents

Description

 The invention relates to veterinary microbiology, in particular to methods for producing vaccines used for the prevention of animal pseudomonosis.

 Pseudomoniasis is a widespread infectious disease of farm animals, birds, fur animals. The most malignant pseudomonosis occurs in fur animals of minks and Arctic foxes. It manifests itself enzootically, accompanied by hemorrhagic inflammation and pulmonary edema. The death of mink from pseudomonosis in the focus of infection can reach more than 50% of the total number of animals.

 The causative agent of pseudomonosis has serotypic variability. It is known that the four serotypes of pseudomonosis pathogens are most prevalent (5,6,8 and 11 serotypes according to the Hubs classification).

 A vaccine preparation is known that contains the antigen of the causative agent of pseudomonosis of the third serotype (according to the Hubs classification) and protease and elastase enzymes. However, the manufacturing method of this drug is complicated and time-consuming.

 Also known is an inactivated monovalent formulquas alum vaccine against pseudomonosis of fur animals, containing the antigens of the pseudomonas Pseudomonas aeruginosa N 202 of the sixth serotype, formalin, adjuvant, a ten percent solution of potassium alum and physiological saline.

A method of producing inactivated monovalent vaccine formolkvastsovoy pseudomonosis fur animals, comprising cultivation of a strain of Pseudomonas aeruginosa N 202 sixth serotype in a liquid nutrient medium, inactivating the bacterial mass of 0.3% formalin for 5 days at a temperature of 35-37 ^{° C} and adding adjuvant (8% ten percent solution of potassium alum.

 A known method for the prevention of pseudomonosis of fur-bearing animals, which consists in a single intramuscular injection of an inactivated monovalent formoliquot vaccine to minks aged 3 months and older at a dose of 3 ml.

The disadvantages of the inactivated monovalent formolvac vaccine against pseudomonosis of fur animals, as well as the method of its manufacture and the method of prevention of pseudomonosis are as follows:
the known vaccine promotes the formation of immunity to the disease caused by the causative agent of pseudomoniasis of one sixth serotype, and does not prevent the pseudomonosis caused by pathogens of other serotypes;
the production strain of Pseudomonas aeruginosa N 202 of the sixth serotype used in the vaccine has unstable virulent and immunogenic properties.

 The aim of the invention is to expand the spectrum of action, increasing immunogenicity and reducing the reactogenicity of the vaccine.

The goal in the vaccine against pseudomonosis of fur animals, mainly minks, is achieved by the fact that it contains a mixture of four strains of four serotypes of the pseudomonas Pseudomonas aeruginosa N 40 of the sixth serotype, Preudomonas aeruginosa N 48 of the eleventh serotype, Pseudomonas aeruginosa N 63 of the eighth serotype and as antigens N 77 of the fifth serotype in equal amounts with the following ratio of components per 1 liter of vaccine: Ps.aeruginosa pseudomonad antigen N 40 of the sixth serotype, N 48 of the eleventh serotype, N 63 of the eighth serotype, N 77 fifth serotype, mikr. 1,9˙10 ¹² -2,2˙10 ¹² alu- minum hydroxide, 2.8-3.0 g Formalin, 3.0-3.2 ml broth Hottin- ger 200-300 Fiziologiches- solution cue Other
The goal in the method of obtaining a vaccine against pseudomonosis of fur-bearing animals, mainly minks, is achieved by separately growing strains of Ps.aeruginosa N 40, N 48, N 63 and N 77, inactivating the bacterial mass with formalin for 15-17 days, mixing the components in the following ratio per 1 liter of vaccine: Antigens of the pseudo-monad Ps.aeruginosa N 40 of the sixth serotype, N 48 of the eleventh serotype, N 63 of the eighth serotype, N 77 of the fifth serotype, micr. Cl 1,9˙10 ¹² -2,2˙10 ¹² alu- minum hydroxide, 2.8-3.0 g Formalin, 3.0-3.2 ml broth Hottin- ger, 200-300 ml Saline Other
The goal is also achieved in a method for the prevention of pseudomonosis of fur animals, mainly minks, by a single intramuscular injection of a vaccine containing as antigens a mixture of four serotypes of the pseudomonas Pseudomonas aeruginosa N 40 of the sixth serotype, Pseudomonas aeruginosa N 48 of the eleventh serotype, Pseudomonas aeruginosa N 63 of the eight and eight .aeruginosa N 77 of the fifth serotype in equal amounts with the following ratio of components per 1 liter of vaccine: Ps. pseudomonad antigens Ps.aeugginosa N 40 of the sixth serotype, N 48 of the eleventh serotype, N 63 of the eighth gray Ipa, N of 77 five-serotype mikr.kl. 1.9˙10 ¹² -2.2˙10 ¹² Aluminum hydroxide, g 2.8-3.0 Formalin, ml 3.0-3.2 Hottinger broth, ml 200-300 Physiological solution The rest, is administered at a dose of 1.5-2.0 ml.

PRI me R 1. Strains Ps.aeruginosa N 40 (laboratory N 381-11), Pseudomonas aeruginosa N 48 (laboratory N 1), Ps.aeruginosa N 77 (laboratory NAM) and Ps.aeruginosa N 63 (laboratory N 1677) are sown to obtain matrovodnogo seed separately 0.5 L each in 200.0 ml bottles containing 80-100 ml of MPB (pH 7.2-7.4). At the same time, for the control check for purity, the same strains are seeded on MPA. Cultivation is carried out at 36-38 ^about C.

 Cultures of 8-10 hour growth are checked for purity by microscopy of Gram stained smears, serological properties are checked with monoreceptor O-agglutinating serums.

Proven cultures from vials are each seeded separately in bottles with Hottinger broth (20-30 ml of culture per 4-5 l of broth). In parallel, they again sow on MPA and MPB to check for cleanliness. The cultures are grown for 18-24 hours at a temperature of 36-38 ^about C, periodically mixed.

The resulting matrovuyu rasplodku each strain was inoculated into a separate reactor in an amount of 8-10% by volume of the medium and grown for 24-36 hours at a temperature of 36-38 ^{° C,} continuous aeration and stirring. In the growth process, samples are taken every 2-3 hours to determine the concentration of microbial cells, pH and purity of the culture. To improve the conditions for the growth of microbes and regulate the pH of the medium when increasing it to more than 7.4, a sterile 40% glucose solution is added to a concentration of 0.1-0.15% of the total volume.

 Growth is stopped when the concentration of microbial cells no longer rises or rises slightly. At the end of the cultivation, the concentration of microbial bodies, pH and the purity of the grown culture are determined.

The bacterial mass thus obtained in each reactor is diluted with sterile saline to a concentration of 2 billion microbial bodies in 1 ml, and then canned by adding formalin to a concentration of 0.3-0.32% of the total volume. Inactivation of cultures with formalin is carried out for 15-17 days at a temperature of 37-38 ^o C. In the process of inactivation of the culture is mixed for 5 minutes 1-2 times a day.

 Two billion suspensions of inactivated microbial cells of strains N 40, N 48, N 63 and N 77 are mixed in equal amounts in a separate reactor. Aluminum hydroxide was added to the mixture of cultures at the rate of 2.8-3.0 g per 1 liter of water, the pH of the vaccine was adjusted to 7.2-7.4 and checked for sterility.

 The sterility-tested vaccine is packaged with periodic stirring in vials that are closed with rubber stoppers, rolled in metal caps and labeled.

 The table shows the composition of the vaccine.

The vaccine was allowed to stand for 14 days at a temperature of 16-20 ^{° C,} then tested for sterility, harmlessness and immunogenic activity.

 The sterility of the vaccine is checked by seeding from each control vial of 0.1-0.2 ml in tubes with MPA, MPB, Saburo medium and MPPB under paraffin oil. After one day of cultivation, 1-2 ml passages are made from test tubes with MPB on MPPB under vaseline oil and MPB in 200.0-milliliter bottles containing 80.0-100.0 ml of medium. Crops are cultivated: primary 10 days, and secondary 8 days. Growth on culture media should not be.

 The safety of the multivalent vaccine against pseudomonosis of fur animals was studied in experiments on guinea pigs (300 pieces), thorzofret (200 animals) and minks (100 animals).

 Guinea pigs weighing 300-350 g of the vaccine were injected subcutaneously into the back at a dose of 3-4 ml (6-8 billion microbial cells), thorsophrenic and mink-intramuscularly at a dose of 5-6 ml (10-12 billion microbial cells) 2.5-3.0 ml in the right and left hind limbs. The results of the studies showed that the vaccine is harmless, there were no clinical signs of the disease in vaccinated animals.

PRI me R 2. To test the immunogenic properties of the vaccine, studies were conducted on 300 thorzofret and 300 mink. The minimum immunizing dose of IMD _{50 of the} multivalent vaccine for minks and thorzofreets is 63-125 million microbial cells of the pathogen pseudomonosis of each serotype included in the vaccine, the optimal immunizing dose of 250-500 million microbial cells of the pathogen of each serotype.

 Studies have shown that a multivalent vaccine is highly immunogenic. The introduction of the drug creates in animals a tense immunity to all pathogens. The duration of immunity is 6-8 months. In vaccinated animals, antibodies are detected in the blood serum on the 6-8th day after administration, the maximum titer of antibodies is observed after 12-18 days and is 1: 200-1: 400.

 Polyvalent formol-hydroxide-aluminum vaccine against pseudomonosis of fur-bearing animals from Pseudomonas aeruginosa strains of the 5th, 6th, 8th and 11th serotypes is used for prophylactic purposes in farms unsuccessful for this disease, and it is necessary in case of pseudomonosis among fur-bearing animals.

Vials with the vaccine are thoroughly shaken before use and during vaccination, and in the cold season they are heated in a water bath to a temperature of 36-37 ^o C.

 The vaccine is administered once intramuscularly from the inner surface of the thigh in compliance with aseptic rules for minks from 3 months of age and older in doses of 1.5-2.0 ml.

A vaccine suitable for use within 12 months from the date of production, provided it is stored in a dark, dry place at a temperature of 4-15 ° ^C.

 The proposed vaccine, the method for its production, as well as the method for the prevention of pseudomoniasis using the proposed vaccine have significant advantages over the known technical solutions.

 The strains used for the manufacture of the vaccine are characterized by high growth energy, expressed antigenic and immunogenic properties, provide the manufacture of a drug that can prevent mink pseudomonosis in any region of the Soviet Union.

 The manufacturing technology of the vaccine according to the proposed method allows for 24-36 hours to obtain the accumulation of bacterial mass up to 18-20 billion microbial bodies in 1 ml of medium and to produce 10 times more doses of polyvalent formol-hydroxide-aluminum vaccine than the known monovalent vaccine.

 The proposed vaccine in minks creates immunity of high tension and duration after a single injection of the drug, equally to all serotypes, while the known vaccine creates immunity only to the sixth serotype, but it is not effective against other common serotypes of the pseudomonosis pathogen.

Claims (1)

1. The vaccine against pseudomonosis of fur animals, mainly minks, containing pseudomonas antigens in a liquid nutrient medium, formalin, adjuvant and physiological saline, characterized in that, in order to expand the spectrum of action and increase the immunogenicity of the vaccine, it contains a mixture of four strains of four serotype pseudomonas Pseudomonas acruginosa N 40 of the sixth serotype, Pseudomonas acruginosa N 48 of the eleventh serotype, Pseudomonas acruginosa N 63 of the eighth serotype and Pseudomonas acruginosa N 77 of the fifth serotype in equal amounts in the following ratio and components per 1 liter of vaccine:
Antigens Pseudomonas Ps. acruginosa N 40 of the sixth serotype, N 48 of the eleventh serotype, N 63 of the eighth serotype, N 77 of the fifth serotype, microcl. 1.9 · 10 ^{1 2} 2.2 · 10 ^{1 2}
Aluminum hydroxide, g 2.8 3.0
Formalin, ml 3.0 3.2
Hottinger broth, ml 200 300
Saline
2. A method of obtaining a vaccine against pseudomonosis of fur animals, mainly minks, including growing bacteria, inactivating the bacterial mass, adding an adjuvant, mixing the components and packaging the preparation, characterized in that, in order to increase the immunogenicity and expand the spectrum of the vaccine, strains of Pseudomonas acruginosa are separately grown N 40 of the sixth serotype, Pseudomonas acruginosa N 48 of the eleventh serotype, Pseudomonas acruginosa N 63 of the eighth serotype and Pseudomonas acruginosa N 77 of the fifth serotype, forms are inactivated ling 17 for 15 days, and the mixing of the components is performed in the following proportions per 1 liter of vaccine:
Antigens Pseudomonas Ps. acruginosa N 40 of the sixth serotype, N 48 of the eleventh serotype, N 63 of the eighth serotype, N 77 of the fifth serotype, microcl. 1.9 · 10 ^{1 2} 2.2 · 10 ^{1 2}
Aluminum hydroxide, g 2.8 3.0
Formalin, ml 3.0 3.2
Hottinger broth, ml 200 300
Saline
3. A method for the prevention of pseudomonosis of fur animals, mainly minks, comprising a single intramuscular injection of a vaccine, characterized in that, in order to reduce reactogenicity, a vaccine containing as antigens a mixture of four strains of four serotypes of the pseudomonas Pseudomonas acruginosa N 40 of the sixth serotype, Pseudomonas acruginosa N 48 of the eleventh serotype, Pseudomonas acruginosa N 63 of the eighth serotype and Pseudomonas acruginosa N 77 of the fifth serotype in equal amounts in the following ratio of components per 1 liter of vaccine:
Antigens Pseudomonas Ps. acruginosa N 40 of the sixth serotype, N 48 of the eleventh serotype, N 63 of the eighth serotype, N 77 of the fifth serotype, microcl. 1.9 · 10 ^{1 2} 2.2 · 10 ^{1 2}
Aluminum hydroxide, g 2.8 3.0
Formalin, ml 3.0 3.2
Hottinger broth, ml 200 300
Saline
administered in doses of 1.5 to 2.0 ml.

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