RU2005490C1 - Method of carnivores plague prophylaxis - Google Patents

Abstract

FIELD: virology. SUBSTANCE: for prophylaxis of carnivores plague lyophilized vaccine against carnivores plague is rehydrated with mink enteritis virus inactivated with formalin with titer 10^4,0-4,5 ID₅₀/ml containing sodium metabisulfite (final concentration is 2,8- 4,3 g/l). Solvent is added to vaccine at equal ratio. Administration is carried out to animal of 8-10 weeks age by a single dose for 24 h after vaccine rehydration. EFFECT: improved method of prophylaxis. 5 tbl

Description

 The invention relates to veterinary virology and can be used at the enterprises of the biological industry for the production of means for the specific prevention of viral enteritis and carnivorous plague.

 Viral enteritis is an acute specific disease of minks, characterized by inflammatory necrotic lesions of the gastrointestinal tract, mesenteric lymph nodes and spleen; it affects minks of all ages, but most often young growth, the waste among which is from 10 to 50%.

 Carnivore plague is also an acute contagious disease of carnivores, including minks, characterized by fever, catarrh of the mucous membranes of the respiratory and digestive tracts, damage to the nervous system and skin; It affects representatives of the canine and marten families of all ages, but the young animals are most susceptible, among which the waste is up to 70-80%.

 These diseases are one of the main reasons for young growth at an early age, causing great economic damage.

 Currently, immunization of fur animals is carried out separately.

There is a method of obtaining a vaccine and immunization against carnivore plague, according to which young animals aged 8-10 weeks are administered a parenteral vaccine containing a non-virulent plague virus adapted in a culture of Japanese quail cells at a dose of 100-200 TCD ₅₀ .

 Also known is a method of producing a vaccine and immunization against mink viral enteritis. In the period after the creation of immunity against carnivore plague, i.e., at the age of 12-14 weeks, young minks are given an inactivated vaccine obtained on the basis of a virus grown in a primary trypsinized culture of kitten's kidney cells in a nutrient medium containing Igloo and serum cattle.

 Known methods of immunization against plague and viral enteritis have several disadvantages.

 With a separate method of immunizing puppies vaccinated against plague at the age of 8-10 weeks, they are unprotected against viral enteritis. To create a tense immunity against viral enteritis, an additional 4-5 weeks is required after vaccination of animals against plague.

 Due to the fact that puppies from the age of 7 weeks have an increased susceptibility to viral enteritis, among them there is a significant departure when the enteritis virus enters.

 The aim of the invention is to increase the immunogenicity of the plague vaccine and the creation of intense immunity at an earlier age in young mink against both diseases.

This goal is achieved by the fact that to the enteric component with a titer of virus 10 ^4.0 -10 ^4.5 ID ₅₀ / ml add sodium metabisulfite in a final concentration of 2.8-4.3 g / l and use it as a solvent for the plague component ( with a titer of 10 ^2.8 -10 ^4.3 TCD ₅₀ / ml) in a 1: 1 ratio. A ready-to-use vaccine is used to immunize minks aged 8-10 weeks. Vaccination is carried out within 2 hours from the moment of mixing in a dose of 1 ml. intramuscularly with a vaccine dose of plague virus 10 ^2.0 -10 ^3.0 TCD ₅₀ / ml and antigen of viral enteritis 10 ^4.0 -10 ^4.5 ID ₅₀ / ml

 PRI me R 1. The manufacture of the plague component.

 An attenuated strain of the carnivore plague virus is grown.

 Use the attenuated strain "EPM" of the virus of carnivore, deposited under the number "10-76", which is obtained by repeated passages of the wild virus in cell cultures of animals and humans.

To make the plague component, the virus is grown on a culture of primary trypsinized cells of Japanese quail embryos in a nutrient medium containing a lactalbumin hydrolyzate solution or medium 199 and cattle serum. The resulting virus-containing liquid with the vaccine strain of the virus is lyophilized to preserve for a long time without loss of activity of the live vaccine virus in the presence of 4.5-5% by weight of sorbitol and 1.4-6% by weight of gelatine. Plagued components are stored at + 4-6 ^{° C} for 1 year. The virus titer in a dry vaccine is 10 ^2.8 -10 ^4.3 TCD ₅₀ / ml.

 PRI me R 2. The manufacture of enteric component.

The enteric component of the associated vaccine is prepared by growing in a primary trypsinized culture of kidney cells of a kitten of 2-6 months of age of a virulent strain of mink enteritis virus (strain "Rodniki", deposited at N "P-1631"). After trypsinization cells into suspension contribute ultrafiltrate native mink enteritis virus "P" and cultivated in a nutrient medium consisting of medium 199 and bovine serum at 37 ^{° C} for 9-11 days. Upon reaching a hemagglutination titer of at least 1: 16 - 1: 64 GAU / ml, the first discharge of the culture fluid is carried out, a support medium is added to the cell monolayer and incubated again for 3-4 days. The first draining the culture fluid was stored at 2-4 ° ^C. After this period the contents of mattresses frozen at -20 ^{° C,} then thawed. All viral collections are combined and the titer of the virus is determined, which should be not less than 10 ⁴ ID ₅₀ / ml according to the RGA. The mink enteritis culture virus is inactivated with a formaldehyde solution in a ratio of 0.1% formalin to the volume of the virus. Inactivation is conducted at 37 ^{° C} for 20 hours. After viral inactivation charge checked for sterility by plating on MPA, MPB, MPPB under mineral oil in Sabouraud medium, and then make a 3% aluminum hydroxide solution in a ratio of 10-15% for the volume of viral collection, mix thoroughly and pack.

 PRI me R 3. Preparation of the associated vaccine against plague and viral enteritis (table. 1).

To the virus-containing material from Example 2 with an RGA titer of 10 ^4.0 - 10 ^4.5 ID ₅₀ / ml, a 20% sodium metabisulfite solution at a final concentration of 0.8 is added on a phosphate-buffered solution; 1.4; 2.0; 2.8; 3.2; 4.0; 4.3; 5.0 and 5.8 g / l. To 1 ml of each of the obtained samples with different contents of sodium metabisulfite in enteric vaccine, 1 ml of culture fluid containing the plague vaccine virus in a titer of 10 ^3.8 TCD ₅₀ / ml is added.

In the table. 1 shows that when the concentration of sodium metabisulfite is 2.0 or less, plague virus titers decrease from 10 ^2.3 to 10 ^1.3 TCD ₅₀ / ml. Also from the table. 1 shows that the plague virus in the highest titers receive (stored) in a mixture at final concentrations of sodium metabisulfite equal to 2.8; 3.2; 4.0 and 4.3 g / l. A further increase in the concentration of sodium metabisulfite does not significantly affect the titers of the plague virus. A decrease in sodium metabisulfite leads to a decrease in the titer of the plague virus.

 Thus, the metabisulfite content equal to 2.8-4.3 g / l can be considered optimal.

PRI me R 4. The safety of the plague virus in a mixed vaccine with different duration of contact with an enteric solvent
The material obtained in example 3 is kept at room temperature for various periods of time. From the table. 2 shows that when the content of sodium metabisulfite taken in the working concentration for 2 hours, the plague virus titers are stored in values sufficient to create intense immunity. Longer exposure of the mixture leads to a decrease in the titers of the vaccine virus of the plague.

AT
PRI me R 5. The effect of shelf life on the immunogenicity of the plague component in a mixed vaccine
This determination is carried out in biological tests on minks with infection of vaccinated and control animals with a virulent strain of the plague virus; in the studied blood sera of vaccinated and control animals, the level of anti-plague antibodies titers in the neutralization reaction (pH) of the cytopathic effect (CPD) in the cell culture was determined. The state of immunity in minks was determined 1 month after vaccination with the associated drug.

 From the table. Figure 3 shows that during the storage period of the mixed vaccine for 2 hours out of 5 vaccines after infection with the plague virus, all 5 burrows remained alive. At the same time, antibody titers were equal to an average of 1: 32. An increase in the shelf life of the mixture of viral components for more than 2 hours reduces the immunogenic effect of the drug, which leads to an increase in the number of dead animals after control of the plague virus and a sharp decrease in the titer of anti-plague antibodies in the blood serum of the vaccinated .

 PRI me R 6. Control of the immunogenicity of an associated vaccine.

Evaluation of the protective effect of the vaccine obtained in the previous examples is carried out on mink puppies older than 8-10 weeks. Before vaccination, a mixture is prepared comprising plague and enteric components in titers of 3.8 TCD ₅₀ / ml and 4.0 ID ₅₀ / ml, respectively, mixing them in equal proportions. For this, the batch of mixed vaccine obtained is administered intramuscularly to 10 non-immune burrows in a volume of 1 ml. 4 weeks after immunization, 5 immunized and 5 control (non-immune) minks are infected with a virulent carnivorous plague virus (Snyder Hill strain) in a dose of 10 ³ LD ₅₀ . The other 5 immunized and 5 control (non-immune) minks are infected with enteritis virus, the Rodniki strain at a dose of 10 ⁴ ID ₅₀ / mink.

 For comparison, 5 mink vaccines are administered against the plague from the EPM strain, and 5 other mink vaccines are administered against the viral enteritis from the Rodniki strain. After 1, 3, 6, 9 and 12 months, serum samples were taken from all immunized and control animals to determine antibody titers. The results are presented in table. 4 and 5.

 During the first 6 months, antibody titers in blood serum samples during immunization by the proposed method were significantly higher compared to immunization with monovaccine.

 As can be seen from the table. 4 anti-plague antibodies are detected in sufficient titers 12 months after vaccination with both associated and monovaccine.

 As can be seen from the table. 5, anti-enteric antibodies are constantly present in serum samples in sufficient titers 12 months after vaccination with both associated and monovaccines. In control, unvaccinated minks, antibodies appeared after 3 months in lower titres than in vaccinated minks, as a result of spontaneous passage of weakly virulent strains of mink enteritis in animals.

 Feasibility study.

 In a wide production test in Norwegian economies of the proposed method for the prevention of plague and viral enteritis with the test vaccine, not a single case of immune breakthrough was noted.

 During 1987-89 4.5 million doses of the modified enteric vaccine (solvent for the dry vaccine against carnivorous plague) were produced in the bio-workshop and sold in more than 50 animal farms in various regions of the country. Immunization of minks with this drug showed itself only on the positive side. The proposed vaccine creates intense immunity for at least 12 months, allows 2 or more times to reduce material and labor costs in its manufacture and use, creates immunity against both infections in a shorter time, and also allows you to increase labor productivity, saving labor by the farm. This significantly reduces the cost of the preventive treatment of thousands of livestock mink fur farms. (56) Copyright certificate of the USSR N 1221787, cl. A 61 K 39/12, 1980.

 U.S. Patent No. 4,224,412, cl. C 12 N 7/00, 1980.

Claims (1)

METHOD FOR PREVENTING PLATIVITY OF PLATIVES, including rehydration of a lyophilized vaccine against carnivores with a solvent followed by administration to animals, characterized in that the formalin inactivated mink enteritis virus with a titer of 10 ^4.0 - 10 ^4.5 ID ₅₀ / ml with the addition of sodium metabisulfite in a final concentration of 2.8 - 4.3 g / l, the solvent is added to the vaccine in equal proportions, and administration is performed to animals aged 8 to 10 weeks once for 2 hours from the moment of rehydration of the vaccine.

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