RU2159127C1 - Vaccine for preventing pseudomonosis of fur-bearing animals - Google Patents

Abstract

FIELD: veterinary medicine. SUBSTANCE: vaccine has mixture of antigens belonging to Ps. Aeruginosa strains number 40 from the sixth serovar, number 63 from the eighth serovar, number 77 from the fifth serovar, number 110 from the second serovar, number 278 from the third serovar. The strains are taken in optimum proportions in concentration of 6-8 mlr units in 1 cm³ of Hottinger broth, 33.33-75% in volume. The vaccine contains required quantity of aluminum hydroxide, formaldehyde solution and physiological salt solution. The PSEUDOPOLYVAC vaccine is completely harmless for foxes and polar foxes. Geometric mean of agglutinin titers in polar fox and fox blood serum is 1: 220-1: 380. The vaccine is applied in optimum time and in optimum doses. EFFECT: enhanced effectiveness in preventing fox and polar fox pseudomonosis. 1 tbl

Description

 The invention relates to veterinary biotechnology, in particular, to means of specific prophylaxis of pseudomonosis of fur animals. Pseudomonosis of fur-bearing animals is a contagious infectious disease that leads to the death of 50% or more animals in the focus of infection.

 For the prevention of this disease, biologics based on the causative agent of pseudomonosis of the fifth, sixth, eighth and eleventh serovars have been developed and are used according to the International Classification.

So, are known:
vaccine against pseudomonosis of minks, which is based on the causative agent of pseudomonosis of the third serovar and enzymes: protease and elastase [1];
vaccine against pseudomonosis of fur animals, containing the antigen of the causative agent of pseudomonosis of the sixth serovar, formalin, potassium alum and physiological saline [2], etc.

 However, these biological products do not provide the appropriate preventive effect, since a mixed course of infection is more likely to occur, while they to a small extent protect against the infection of arctic foxes and foxes and are intended mainly for the prevention of pseudomonosis of minks.

 Also known is a vaccine against pseudomonosis of fur-bearing animals, mainly minks, containing a mixture of antigens from the Ps strain. aeruginosa N 40 of the sixth serovar, N 48 of the eleventh serovar, N 63 of the eighth serovar and N 77 of the fifth serovar, aluminum hydroxide, formalin, Hottinger broth and physiological saline [3].

 This vaccine does not provide foxes and foxes with the development of reliable immunity, does not protect them from reinfection with pseudomonas pathogens of other serological variants.

 The aim of the invention is to increase the immunological activity of the target product for arctic foxes and foxes.

This goal was achieved in that it additionally contains antigens from strains of Ps. aeruginosa VGNKI N 110 of the second serovar and N 278 of the third serovar, taken in a mixture in an effective ratio, at a concentration of 6-8 billion microbial cells, in 1 cm ^{3 of} Hottinger broth, in the following ratio of components, vol.%:
A mixture of antigens from strains of Ps. aeruginosa VGNKI N 40 of the sixth serovar, N 63 of the eighth serovar, N 110 of the second serovar, N 77 of the fifth serovar and N 278 of the third serovar, taken in an effective ratio at a concentration of 6-8 billion m.k. in 1 cm ³ Hottinger broth - 33.33-75.00
Aluminum hydroxide - 0.30-0.35
Formalin - 0.32-0.34
Saline - Rest
For the manufacture of vaccines using new strains of Ps. aeruginosu second and third serovars. Strains Ps. aeruginosu are deposited in the collection of microorganisms of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and have registration numbers 110-02 and 278-0Z, respectively.

 Strain Ps. aeruginosa N 110 (lab. Ainazhi) of the second serovar isolated from pathological material of the fox in 1985

 Strain Ps. aeruginosa N 278 (lab. Arctic fox) of the third serovar isolated from pathological material of the fallen Arctic fox in 1987

 Strains Ps. aeruginosa VGNKI N 110-02 and VGNKI N 278-03 are characterized by the following features and properties.

Morphological features
Microscopy of smears of broth cultures indicates Gram / negative movable sticks with slightly rounded edges 0.8-1.2 mmk long and 0.6-0.8 mmk wide, located singly or in pairs in stored cultures or in the form of short chains in freshly passaged cultures . Spore and capsules do not form.

Cultural properties
On dense nutrient media: MPA, Hottinger and Marten agar, strain cultures grow in the form of round mucous colonies with a grayish tint, with the formation of a pronounced blue-green zone (strain N 110-02) or yellow-green zone (N 278-03). When cultured on liquid nutrient media: BCH, Hottinger and Marten broth, the formation of a gray-white surface film is characteristic with staining after 14-18 hours of the medium in blue-green color (culture strain N 110-02) or yellow-green color (strain culture N 278-03)
Enzymatic properties
The strain cultures ferment glucose without gas formation, peptone milk, possess hemolytic, catalase, gelatinase and lecithinase activity.

 Need for oxygen, strict aerobes.

Antigenic and serological properties
Culture strains have flagellar "H" and somatic "O" antigens. When identifying with serotype serums according to the International Classification, strain N 110 refers to the 02 serological variant and strain N 278 to the 03 serological variant. On the introduction of formalin inactivated cultures of strains (Ps. Aeruginosa in the body of arctic foxes, foxes and minks, agglutinins antibodies are formed in titers of 1: 180-1: 320.

Virulent properties
LD ₅₀ culture of strain Ps. aeruginosa VGNKI N 110-02 for non-linear mice weighing 14-16 g is 38.9 mln.m.k., and the culture of strain Ps. aeruginosa VGNKI N 278-03 22.3 mln.m. For guinea pigs weighing 300-350 g - 135 and 269 million m. respectively.

Immunogenic properties
The daily culture of Ps strain inactivated by 0.3% formalin. aeruginosa N 110-02 protects against infection with 50% of guinea pigs weighing 300-350 g at a dose of 33.8 million m.k., and the culture of the strain Ps. aeruginosa N 278-03 at a dose of 38.9 million m.k. Minimum immunizing dose of polyvalent antigen based on Ps strains. aeruginosa N 110, 278, 77, 63, 40 for guinea pigs weighing 300-350 g body is 19.9 million m.

 The introduction of a vaccine based on a mixture of antigens from strains of Ps. aeruginosa N 40, 63, 77, 110 and 278 foxes, arctic foxes, minks induces in 98% of animals after 6 days the formation of stable immunity lasting at least 12 months (observation period).

 The invention is illustrated by the following examples.

Example 1. Matrovye seed crop cultures of strains of Ps. aeruginosa VGNKI N 40 of the sixth serovar, N 63 of the eighth serovar, N 77 of the fifth serovar, N 110 of the second serovar and N 278 of the third cepouapa are grown in separate reactors in the Hottinger broth at 36.5-37.5 ^o C to a maximum bacterial mass accumulation of 12- 18 billion m. in 1 cm ³ . At the end of cultivation using physiological saline, the concentration of the bacterial mass of each strain is reduced to 6-8 billion m.k./cm ³ , formalin is added at the rate of 0.32-0.34% of the volume and inactivated for 12-14 days at 6.5-37.5 ^o C. After inactivation, the suspension of each strain in an effective ratio is mixed in a separate reactor, add 0.30-0.35% of the volume of aluminum hydroxide and adjust the pH of the biological product to 7.2-7.4, check on sterility and packaged.

 The finished vaccine is a homogeneous suspension of a grayish-white color. When settling on the bottom of the bottle, a grayish-whitish precipitate forms, which easily breaks when the bottle is shaken.

The vaccine is kept for 14 days at 16-20 ^o C, and then again checked for sterility, as well as for harmlessness and immunogenicity.

Example 2. Explore the harmlessness of the vaccine against pseudomonosis of fur animals, prepared according to example 1, containing in its composition the following components, vol.%:
A mixture of antigens from strains of Ps. aeruginosa VGNKI N40 of the sixth serovar, N 63 of the eighth serovar N 77 of the fifth serovar, N 110 of the second serovar and N 278 of the third serovar, taken in an effective ratio, at a concentration of 8 billion m in 1 cm ³ Hottinger broth - 75.00
Aluminum hydroxide - 0.30
Formalin - 0.32
Saline - Rest
Guinea pigs in the amount of 270 animals with a live weight of 300-350 g inject the vaccine subcutaneously into the back at a dose of 3.0-3.5 cm ³ , Arctic foxes (120 animals) and foxes (75 animals) intramuscularly at a dose of 5-6 cm ³ (2.5-3.0 cm ³ ) in the right and left hind limbs.

 The study showed that the vaccine is harmless to animals. As a result of observation for 10 days in animals there were no deviations from the normal physiological state. All animals remained alive.

Example 3. The activity obtained for example 1 vaccine against pseudomonosis of fur animals in comparison with the known vaccine (prototype) is tested on guinea pigs (235 goals.) Live weight 300-350 g. For this purpose, guinea pigs of experimental groups, once, intramuscularly administered pre-titrated dose of the vaccine containing the following components, vol. %:
A mixture of antigens from strain Ps. aeruginosa VGNKI N 40 of the sixth serovar, N 63 of the eighth serovar, N 77 of the fifth serovar, N 1110 of the second serovar, taken in an effective ratio, at a concentration of 7 billion microbial cells in 1 cm ^{3 of} Hottinger broth - 40.00
Aluminum hydroxide - 0.33
Formalin - 0.33
Saline - Rest
A control group of guinea pigs (30 animals) was administered a similar vaccine against pseudomonosis of fur animals, which contains antigens from Ps strains. aeruginosa VGNKI N40 of the sixth serovar, N 48 of the eleventh serovar, N 63 of the eighth serovar and N 77 of the fifth serovar (prototype).

 After 7-10 days, blood is taken from 50% of the vaccinated animals and the serum is examined in RA with homologous strains of the pseudomonosis causative agent included in the vaccine.

 The results are presented in the table.

 From the above data it is seen that the activity of the proposed vaccine is significantly higher than the activity of the known vaccine, despite the same amount of antigen in the dose of each drug.

Example 4. Check the immunogenic properties of the vaccine against pseudomonosis of fur animals, prepared according to example 1, containing in its composition the following components, vol.%:
A mixture of antigens from strain Ps. aeruginosa N 40 of the sixth serovar, N 63 of the eighth serovar, N 77 of the fifth serovar, 110 of the second serovar and N 278 of the third serovar, taken in an effective ratio, at a concentration of 6 billion microbial cells in 1 cm ^{3 of} Hottinger broth - 33.33
Aluminum hydroxide - 0.35
Formalin - 0.34
Saline - Rest
Guinea pigs (675 animals) with a live weight of 300-350 g are vaccinated with a live vaccine of the indicated composition once, intramuscularly in doses of 0.01; 0.05; 0.1; 0.5 cm ³ foxes and foxes are immunized intramuscularly at a dose of 1.0 and 1.5 cm ^3, respectively. Control animals are not given a vaccine. 14 days after immunization, the vaccinated and control guinea pigs are infected with a titrated dose of cultures of Ps strains. aeruginosa VGNKI N40 of the sixth serovar, N 63 of the eighth serovar, N 77 of the fifth serovar, N 110 of the second serovar and N 278 of the third serovar.

Installed 100% immunological protection of vaccinated guinea pigs in the death of 95% of unvaccinated guinea pigs from the causative agent of pseudomonosis. Death among the vaccinated livestock of arctic foxes and foxes has also not been established. In foxes vaccinated in doses of 1.0 cm ³ and foxes vaccinated in doses of 1.5 cm ³ , the presence of agglutinins in the titer of 1: 220-1: 380 was established in the blood serum.

Example 3. The vaccine against pseudomonosis of fur-bearing animals of the composition (according to example 4) is used for prophylactic purposes when immunizing female foxes and foxes (a total of 1,500 animals) in fur farms unsuccessful for pseudomonosis. The vaccine is administered once, intramuscularly from the inside of the thigh at a dose of 1.0-1.5 cm ³ , foxes at a dose of 1.5-2.0 cm ³ . The control group (1) of animals (500 animals. Arctic foxes and foxes) is administered the known vaccine (according to the prototype) in the same doses. Some animals are not vaccinated (control II - 503 female polar foxes and foxes). Vaccinated and control animals are under clinical and epizootological observation during the year. The study found that in the group of foxes and foxes vaccinated with the proposed vaccine, there were no cases of complications and a breakthrough of immunity. In groups of animals vaccinated with a vaccine (according to the prototype), abortions of pseudomonous etiology were noted in 18 cases. Among the unvaccinated animals in the control group, there were 38 cases of abortion and resorption of the fetus in the first half of pregnancy.

 Using the proposed vaccine in veterinary practice will improve the effectiveness of ongoing measures to combat pseudomonosis of foxes and foxes.

Sources of information
1. US patent N 4120950, CL. 424-87, 1978.

 2. USSR copyright certificate N 575889, cl. C 12 K 5/00, 1972.

 3. Copyright certificate of the USSR N 1066074, cl. A 61 K 39/104, 1982 (prototype).

Claims (1)

Vaccine against pseudomonosis of fur animals, mainly arctic foxes and foxes, containing antigens from the strains of Pseudomonas aeruginosa VGNKI N 40 of the sixth serovar, N 63 of the eighth serovar, N 77 of the fifth serovar in Hottinger broth, aluminum hydroxide, formalin and physiological saline, characterized in that it additionally It contains antigens from strains of Pseudomonas aeruginosa VGNKI second serovar N 110 and N 278 of the third serovar taken in a mixture in an effective ratio at a concentration of 6 - 8 billion microbial cells per 1 cm ³ of broth with the following ratio Hottinger COMPONENT. ntov, vol.%:
A mixture of antigens from strains of Pseudomonas aeruginosa VGNKI N 40 of the sixth serovar, N 63 of the eighth serovar, N 77 of the fifth serovar, N 110 of the second serovar and N 278 of the third serovar, taken in an effective ratio at a concentration of 6-8 billion microbial cells in 1 cm ^{3 of} broth Hottinger - 33.33 - 75.00
Aluminum hydroxide - 0.30 - 0.35
Formalin - 0.32 - 0.34
Saline - Rest

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