RU2194530C1 - Associated vaccine for preventing diseases in furred animals - Google Patents

Abstract

FIELD: veterinary biotechnology, virology, microbiology. SUBSTANCE: the suggested associated vaccine contains antigenously and immunogenously balanced viral and bacterial antigens: as an antigen for carnivorous plague virus - a culture viral suspension of alive attenuated strain Distemper canine "EPM", as salmonellosis antigens - alive attenuated strains: Salm. typhimurium N3, Salm. cholerae suis N9, Salm. dublin N6, as an antigen of carnivorous adenovirus - a formalin- inactivated and aluminum hydroxide-deposited culture viral suspension of Adenovirus canis - 2 "Ada" strain. Associated vaccine, also, contains a sorbitol-gelatinous stabilizer and a Hanks' solution. Application of associated vaccine under production conditions enables to develop high-intensity immunity in animals during a short period of time to the agents of plague, adenoviral infections and salmonellosis, minimize material damage in fur farms caused by the above-mentioned infections. EFFECT: higher efficiency of prophylaxis. 5 ex, 7 tbl

Description

 The invention relates to the field of veterinary biotechnology, in particular to an associated vaccine for the prevention of plague, adenoviral infections and salmonellosis of fur animals.

 Currently, the simultaneous immunization of fur animals against plague, adenovirus infections and salmonellosis is relevant.

 For the prevention of diseases of fur animals, associated vaccines are used, including the plague virus antigen from the Distemper canine strain "EPM" [1, 2, 3, 4], the inactivated antigen of the pathogen of the viral enteritis from the Virus enteritis lutreo larum strain "Rodniki - 1631" [1, 2 ], antigen of botulinum toxoid type C adsorbed on aluminum hydroxide - the toxoid of the strain Clostridium botulinum 59 [1], inactivated antigens of the pathogen pseudomonosis from strains of Pseu-domonas aeruginosa DN-0577, 381-P-06 N40, 1677-08 N63 [2], a mixture of Salmonella antigens of three strains of Salmonella typhimurium N3, Salmonella cholerae suis N9 and Salmonel la dublin N6 [3], adenovirus antigen from the Adenovirus canis - 2 Ada strain [4] adsorbed on formalin and adsorbed onto aluminum hydroxide, a sorbitol-gelatin stabilizer and Hanks solution [1, 2, 3, 4].

 The use of known associated vaccines does not allow achieving the instantaneous desired effect, since the absence of an adenoviral antigen [3] and salmonella in the first case [1, 2, 4] in the vaccine requires additional measures for vaccination of animals with monovaccines, and this extends the time of immunization of animals contributes to stress and complications.

 Combining the associated vaccines with monovaccines in a single syringe for simultaneous immunization leads to the inactivation of live attenuated strains of the plague and salmonella with formaldehyde, which are part of the inactivated vaccines against adenoviral infections and to the inactivation of salmonella by antibiotics used in the manufacture of antiviral vaccines. With an unbalanced ratio of introduced antigens, vaccination efficiency is insufficient due to their competitive effect on the animal’s immune system.

 The aim of the invention is an associated vaccine for the prevention of plague, adenoviral infections and salmonellosis in fur animals, which has high immunogenic activity, safety, with a high protective effect.

The goal is achieved by the fact that the vaccine includes antigen-balanced and immunogenic-balanced viral antigens (attenuated strain of Distemper canine "EPM", inactivated strain of Adenovirus canis - 2 "Ada") and additional bacterial antigens (a mixture of antigens of three strains of Salmonella: Salmonella typhimurium N3, Salmonella cholerae suis N9 and Salmonella dublin N6, taken in equal proportions, at a concentration of 10 billion cubic meters / cm ³ ), with the following ratio of components vol.%:
Plague virus antigen from strain Distemper canine "EPM"12.5-15.0;
Formalin inactivated and adsorbed onto aluminum hydroxide adenovirus antigen from strain Adenovirus canis - 2 "Ada"0.15-0.30;
A mixture of Salmonella antigens of three strains: Salmonella typhimurium N3, Salmonella cholerae suis N9 and Salmonella dublin N6, taken in equal proportions at a concentration of 10 billion m3 / cm ³ 12.5-15.0;
Sorbitol-gelatin stabilizer 20.0-25.0;
Hanks' solution - the rest.

The invention is illustrated by the following examples:
Example 1

Carnivore plague virus antigen is obtained from Distemper canine strain "EPM" in a primary trypsinized cell culture of Japanese quail embryos in a growth medium consisting of 0.5% GLA in Hanks solution, 25-30% of 199 and 8-10% serum cattle with the addition of antibiotics (gentamicin 0.1 mg per 1 cm ³ medium). The virus is introduced into the cell suspension and grown by roller method at 37 ^o C. After 46 hours, the growth medium is drained, and cultivation is continued in a support medium, including 60-70% of medium 199 and 30-40% of 0.5% GLA in Earl's solution with the addition of antibiotics (gentamicin 0.1 mg per 1 cm ³ medium). After the full development of the CPP, the virus-containing culture fluid is drained, frozen at a temperature of minus 10-20 ^o C, thawed at room temperature.

Strains of Salmonella Salm. typhimurium N3, Salm. cholerae suis N9, Salm. dublin N6 is grown separately in separate reactors on the Hottinger broth for 10-12 hours at a temperature of 37 + 0.5 ^o With constant stirring and continuous aeration. Cultivation is stopped at the beginning of the stationary phase of growth, when the concentration of microbes ceases to increase markedly. The grown cultures are cooled to a temperature of 6-8 ^o C and subjected to verification. Then they are concentrated on a supercentrifuge type SGO 100/750 or OTP-101K. The bacterial mass is diluted with sterile saline to a concentration of 10 billion microbial cells in 1 cm ³ according to the optical standard GISK them. Tarasevich and mix thoroughly.

Bacterial masses Salm. typhimurium N3, Salm. cholerae suis N9, Salm. dublin N6, diluted to a concentration of 10 billion microbial cells in 1 cm ^{3 are} combined in equal volumes and thoroughly mixed.

To the mixed bacterial mass of salmonella antigens are added the antigen of the plague of carnivores, containing at least 10 ³ - 10 ⁴ TCD _{50 / 0.5 cm} ³
After thorough mixing of the antigens, a sorbitol-gelatin stabilizer is added in an amount of 10-25% and mixed again.

 The resulting mass is packaged with continuous stirring in sterile ampoules or vials and lyophilized.

Adenovirus antigen is obtained from the strain Adenovirus canis - 2 "Ada" on the primary cell culture of a dog’s puppy kidney. As a growth medium, a medium containing lactoalbumin hydrolyzate is used - 50%, Igla medium with glutamine - 20%, medium 199 - 20%, bovine serum - 10% and antibiotics (gentamicin 0.1 mg per 1 cm ^{3 of} medium) .

The cell culture is infected on the formed monolayer. As a support, a medium is used that contains the same components as the growth, but without the addition of serum. After the full development of the CPP, the virus-containing culture dishes are frozen at a temperature of minus 10-20 ^o C, thawed at room temperature. The culture fluid is drained. Viral collection with a titer of 10 ^4.5 TCD _{50/0 cm} ³ to 10 ^5.5 TCD _{50/0 cm} ^{3 is} inactivated with formalin (0.2% of the total volume) and 1 part 3-6% is added to 9 parts of the inactivated virus aluminum hydroxide. Residual formaldehyde and antibiotics are eliminated by settling aluminum hydroxide with an adenoviral antigen adsorbed on it and replacing the supernatant of the preparation with a 2-3-fold Hanks solution. The resulting antigen is packaged in sterile vials.

 The associated vaccine is obtained by diluting a dry component containing a plague virus antigen and a mixture of salmonella antigens with an equal volume of a liquid component containing an adenovirus antigen.

 Three series of the associated vaccine are obtained for the prevention of plague, adenoviral infections and salmonellosis of fur animals (Table 1).

 The finished vaccine is a yellowish-pink liquid in appearance with a loose precipitate that easily breaks when shaken into a homogeneous suspension.

 Example 2

 The safety of the associated vaccine is tested on laboratory animals (guinea pigs 300-400 g and mice 14-16 g) and naturally susceptible animals (arctic foxes, foxes, thorzofret, dogs 2-4 months of age).

 The vaccine is administered to experimental animals in the appropriate dose (see table 2) and observation is carried out for 21 days.

 Table 2 shows that the vaccine is harmless. During the period of observation of the inflammatory reaction at the injection site, no cases of animal disease are noted.

 Example 3

 Evaluation of the immunological effectiveness of the vaccine is carried out by the method of experimental infection of vaccinated susceptible animals (thorzofret, guinea pigs, dogs) with virulent strains of pathogens of carnivorous plague, salmonellosis and adenovirus carnivore hepatitis.

The immunogenicity of the vaccine against carnivorous plague is monitored on thorzofret. Experimental animals are inoculated with an associated vaccine against plague, salmonellosis and infectious hepatitis carnivores intramuscularly at a dose of 1 cm ³ . Infection is carried out 21 days after vaccination with a virulent strain of the plague virus Sneider Hyll at a dose of 10 ³ LD _{50 / cm} ³ . The results of the experiment are presented in table 3. As can be seen from the table, the associated vaccine causes the formation of a stable immunity to the virus of carnivorous plague.

The immunogenic activity of the associated drug in relation to carnivore adenoviruses is tested in an acute experiment on dogs. Dog puppies are vaccinated intramuscularly or subcutaneously with 1 cm ³ . 21 days after the administration of the associated vaccine, the carnivorous infectious hepatitis causative agent is infected. The strain VGNKI administered intraperitoneally at 10 ² ID _{50 / cm} ³ (see table 4).

 As can be seen from table 4, the associated vaccine causes the formation of stable immunity to carnivorous adenovirus.

The immunogenicity of the salmonellosis vaccine is monitored in guinea pigs. The associated vaccine is administered to guinea pigs subcutaneously in the groin at a dose of 1 cm ³ . Control infection of experimental animals is carried out 21 days after immunization with virulent strains (see table 5) of the corresponding pathogens in a dose of 1.5 billion microbial bodies.

 From table 5 it is seen that in guinea pigs with the introduction of the associated vaccine, a certain immunological resistance is formed to control infection with virulent strains of salmonellosis pathogen cultures. The results of the experiments indicate that the most effective and appropriate for use is the vaccine series 2, 3.

 Example 4

Antigenic activity of the associated vaccine is determined in experiments on 2-4 month old foxes. Animals are vaccinated intramuscularly or subcutaneously at 1 cm ³ and after 7, 14 and 21 days blood is taken to determine the level of vaccine-induced humoral antibodies.

 Antibodies to the carnivore plague virus are determined in the RNGA using the erythrocyte diagnostic kit for detecting antibodies to the carnivore plague virus (TU 9384-001-00008064-96), to carnivorous adenoviruses in the rtga with the Ada strain of type 2 dog adenovirus, to salmonella in RA with homologous antigens.

 The vaccine has antigenic activity, since the titer of antibodies in the blood serum of vaccinated arctic foxes to the carnivore plague virus in RNGA, to carnivorous adenovirus in rtga and to salmonella in RA increases by 2 - 4 or more times.

 According to table 6, it is seen that an increase in the level of antibodies to all antigens included in the vaccine is most pronounced two weeks after vaccination.

Example 5
The associated vaccine for the prevention of plague, adenoviral infections and salmonellosis is administered to fur animals intramuscularly or subcutaneously at a dose of 1 cm ³ . Young animals are vaccinated twice with an interval of 10-14 days starting from 50-60 days of age. Adult animals of the main herd are vaccinated once in December - January, before rutting.

 Within 1 month of observing the inflammatory reaction at the injection site, there is no decrease in appetite, cases of illness and death of animals.

 The species composition and number of fur animals from the main herd vaccinated in 2000 with the associated vaccine against plague, adenovirus infections and salmonellosis of fur animals in the Vyatka fur farm is shown in Table 7.

 The use of the “Associated vaccine for the prevention of plague, adenoviral infections and salmonellosis of fur-bearing animals” under production conditions will make it possible in a short time to create high immunity in animals against the causative agents of plague, adenovirus infections and salmonellosis, and to minimize the material damage caused to animals by these infections.

Sources of information
1. RU 1615919 A1, 05.24.1989.

 2. SU 1792548 A3, 04/11/1990.

 3. RU 2166329 C1, 02/11/1999.

 4. RU 2030917 C1, 06/28/1991 - prototype.

Claims (1)

Associated vaccine for the prevention of diseases of fur animals, including the plague virus antigen from the strain Distemper canine "EPM", formalin inactivated and adsorbed on aluminum hydroxide adenovirus antigen from the strain Adenovirus canis-2 "Ada", sorbitol-gelatin stabilizer and Hanks solution, characterized in that it additionally contains a mixture of Salmonella antigens of the three Salm strains. Typhimurium N3, Salm. Cholerae suis N9, Salm. Dublin N6, taken in equal proportions at a concentration of 10 billion m. K. / cm ³ , with the following ratio of components, vol. %:
Plague virus antigen from Distemper canine strain "EPM" - 12.5-15.0
Formalin inactivated and adsorbed on aluminum hydroxide adenovirus antigen from strain Adenovirus canis-2 "Ada" - 0.15-0.30
A mixture of Salmonella antigens of three Salm strains. Typhimurium N3, Salm. Cholerae suis N9 and Salm. Dublin N6, taken in equal proportions at a concentration of 10 billion m. K. / cm ³ - 12.5-15.0
Sorbitol-gelatin stabilizer - 20.0-25.0
Hanks Solution - Else

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