RU2045960C1 - Vaccine for prevention of plague, infectious hepatitis, adenovirosis, parvoviral enteritis and canine leptospirosis - Google Patents

Abstract

FIELD: bioengineering, veterinary virology and microbiology. SUBSTANCE: this vaccine includes viral and bacterial antigens balanced in antigen and immunogenic aspects. It employs cultural virus suspension of Distemper canine strain "EPM" as plague antigen, inactivated cultural virus suspension of Mammaliadenovirus Adenovirus canis strain 2 "Ada" as adenovirus antigen inactivated cultural virus suspension of Virus panleucopenia felline strain "Gannibal" as parvovirus antigens, Canicola VGNKI-3 as antigens of leptospirs of serogroup, Icterohaemorrhagiae VGNKI-2 as antigens of leptospirs of serogroup, aluminum hydroxide and blood serum protein as adjuvant and gelatose and sorbitol solutions as stabilizer. EFFECT: disclosed vaccine allows highly intensifying immunity to both viral and bacterial components and minimizing material losses caused by above enumerated diseases. 3 tbl

Description

 The invention relates to biotechnology, in particular to means of specific prophylaxis of carnivore plague, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis of dogs (hexacanivac). Mono and associated vaccines are used to prevent these diseases in dogs.

 For the prevention of plague, infectious hepatitis, adenovirus infections and parvovirus enteritis in dogs, the Tetravac vaccine is used (auth. St. USSR, Application No. 4953741/13).

 However, this vaccine does not create immunity to leptospirosis, which is becoming more common among dogs and proceeds with a severe clinic and often animal death. The absence of leptospira in the vaccine requires additional vaccination of dogs with the polyvalent vaccine VGNKI against animal leptospirosis (ed. St. USSR N 828459, 1979) or concentrated vaccine against leptospirosis of animals (USSR patent application N 4890614, 1990). Both of these vaccines contain leptospira of four serological groups, two of which practically do not cause leptospirosis in dogs and are intended for the specific prevention of leptospirosis in animals of other species. In addition, the presence of 28-30% aluminum hydroxide in dogs in polyvalent vaccines causes undesirable side effects.

 The aim of the invention is a vaccine for the specific prophylaxis of carnivore plague, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis in dogs, characterized by high antigenic and immunogenic activity and harmlessness.

The goal is achieved in that the vaccine is balanced in antigenic and immunogenic respects viral and bacterial antigens: as the plague antigens, the viral culture suspension of the strain Distemper canine "EPM" with an infectious activity titer of 10 ³ -10 ⁴ TCD _{50 / 0.5 cm} ³ , as antigens adenovirus inactivated viral suspension culture strain Mammaliadenovirus adenovirus canis 2 "Hell" with a titer of infectious activity of 10 ^4.5 to 10 ⁵ TCD _{50 / cm} ^3, as antigens parvovirus inactivated viral suspension culture strain Virus panleucopenia felline "Gannib l "with a titer of infectious activity of 10 ⁴ to 10 ⁵ ID _{50 / cm} ^3, and activity in the RGA 1: 64-1: 256 Gai / cm ^3, as antigens from serogroup Leptospira Canicola strain VGNKI-3, as antigens from serogroup Leptospira strain Icterohaemorrhagiae VGNKI-2 in equal proportions at a concentration of 300-500 million cells / 0.2 cm ³ , as well as gelatose, sorbitol, Hanks solution, serum protein in the following ratio of components, vol.

Viral suspension of strain
Distemper canine "EPM" with
titer of infectious asset
10 ³ -10 ⁴ TCD _{50 / 0.5 cm} ³ 15.2-19.0
Inactivated viral
suspension of strain Mammali-
adenovirus Adenovirus canis 2
"Ada" with an infectious titer
activity 10 ^4.5 -10 ⁵
TCD _{50 / cm} ³ 11.8-15.0
Inactivated viral
suspension of strain Virus
panleucopenia felline "ganny-
ball "with an infectious titer
activity 10 ⁴ -10 ⁵ ID _{50 / cm} ³
and with activity in the RGA
1: 64-1: 256 GAU / cm ³ 24.3-30.4
A mixture of antigens from strains
leptospira serogroup Canicola
VGNKI-3 and Icterohaemorrhagiae
VGNKI-2, taken in equal
ratio at a concentration
tion 300-500 million cells /
/ 0.2 cm ³ phosphate buffer 9.2-10.6
Aluminum hydroxide 4.8-5.6
25% gelatine solution 8.5-11.3
50% sorbitol solution 2.8-3.8
Serum Protein 3.3-5.5
Distilled water 1.2-1.8
Hanks Solution Else
PRI me R 1. Get inactivated culture virus suspension of a strain of Mammaliadenovirus adenovirus canis 2 "Ada".

To grow a cell culture, a primary trypsinized cell culture of kidney cells of dogs of 1.3 months of age is used. As a growth medium, a medium containing 35-40% of 0.5% lactalbumin hydrolyzate in Hanks solution, 0.25% of 199 medium and Igla, 10% of blood serum of cattle and antibiotics (penicillin 100 U / cm ³ and streptomycin 10 mg per 1 cm ³ medium). As a supporting medium, a medium containing 30% 0.5% GLAE and 70% medium 199 is used.

In the preparation of the virus in cell culture flasks with the generated monolayer inoculated with a strain "Hell" at a dose 1,0-0,001 TCD _{50 / cm} ^3, then it is carried adsorption for 1 h at 37 ^C. After addition of the virus maintenance medium is incubated in a thermostat at 37 ^about With before the accumulation of the titer of infectious activity 10 ^4,5 -10 ⁵ TCD _{50 / cm} ³ . After three freezing at -20-40 ^{° C} and thawing at 37 ^C the resulting virus-containing culture suspension is poured into a container of 0.2% formalin were added to inactivate the virus.

 An inactivated culture virus suspension of the Virus panleucopenia felline Hannibal strain is obtained.

The primary trypsinized culture of the kitten’s kidney cells is infected by inoculating the mattresses with the native ultrafiltrate of the virus of the Hannibal strain with a titer in the RSA of at least 1: 16384 GAU / cm ³ and infectious activity for the culture of the kitten’s kidney cells 10 ⁴ -10 ⁵ ID _{50 / cm} ³ . The virus is diluted with Hanks' solution 1: 50-1: 100 and 5-7 cm ³ are added to each mattress. Incubation of the infected cell culture is performed at ³⁷ ° C in medium containing 26% medium "needle" and 34% GLAE in Hanks solution, 32% medium 199 in Hank's solution and 8% bovine serum. 9-10 th day mattresses cell culture frozen at -20-40 ° ^C. After thawing at room temperature is carried collecting virus-containing liquid. Plums of the virus-containing fluid are combined and the hemagglutinating activity in the RGA is determined, which should not be lower than 1: 64-1: 256 GAU / cm ³ . To inactivate the virus, 0.2% formalin is added to the total volume of viral collections.

 A culture viral suspension of the Distemper canine "EPM" strain is obtained.

The carnivore plague virus is grown 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% of cattle serum with antibiotics added. The virus is introduced into the cell suspension and grown rolernym method at ³⁷ ° C. After 4-6 days the growth medium was discarded and the cultivation was continued in maintenance medium consisting of 60-70% of the medium 199 and 30-40% 0.5% GLA in Earl's solution with antibiotics. After the full development of the CPP, the virus-containing culture fluid is drained, frozen at a temperature of minus 10-20 ^о С, thawed at room temperature and combined with a 1% drain (the infectious activity of the obtained plague of carnivores for cell culture of Japanese quail embryos should be 10 ³ -10 ⁴ TCD _{50 / 0.5 cm} ³ ).

Strains of leptospira serogroups Canicola VGNKI-3 and Icterohaemorrhagiae VGNKI-2 are grown separately on a buffer-serum medium until 70-100 million microbial cells are accumulated, formalin is added to a concentration of 0.24-0.26%. The leptospira suspension is mixed 1: 1, concentrated by ultrafiltration to 300-500 million / cm ^{3 of} microbial cells.

 A 3% alumina hydrate solution is added to the inactivated parvovirus component. The mixture was kept for 2 days at room temperature, after which 1/3 of the supernatant was removed and the same amount of inactivated adenoviral component, 3% alumina hydrate solution and inactivated leptospira suspension were added. The resulting mixture is thoroughly mixed and, after settling, the amount of residual formaldehyde is determined, to neutralize which, 2-3% of the total volume of sodium metabisulfite dissolved in distilled water is added to it. The total volume of the mixture was adjusted with Hanks solution to 100 liters.

A stabilizer for lyophilization is added to the combined mixture of the suspensions of the plague of carnivore virus, which is used as a 25% solution of gelatose and 50% solution of sorbitol, mix thoroughly on a shuttle machine and add antibiotics gentamicin, monomycin or kanamycin at a rate of 20 mg / cm ³ . The mixture is cooled to 5-10 ^about C for 5-7 hours, packaged in sterile glass bottles and lyophilized.

The associated vaccine is prepared by reconstituting a dry component containing a viral culture suspension of the Distemper canine "EPM" strain with an infectious activity titer of 10 ³ -10 ⁴ TCD _{50 / 0.5 cm} ³ in a liquid solution consisting of inactivated culture virus suspensions from Mammaliadenovirus Adenovirus strains canis 2 "Ada" and Virus panleucopenia felline "Hannibal" and bacterial suspension of Leptospira strains Canicola VGNKI-3 and Icterohaemorrhagiae VGNKI-2. Get three series of vaccines against carnivore plague, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis dogs (hexacanivac) in the following ratio of components, about.

series 1 series 2 series 3 Viral suspension of the strain Distemper canine "EPM" with a titer of infectious activity 10 ³ -10 ⁴ TCD _{50 / 0.5 cm} ³ 15.2 17.0 19.0
Inactivated virus suspension of the strain Mammaliadenovirus Adenovirus canis 2 "Ada" with a titer of infectious activity 10 ^4.5 -10 ⁵ TCD _{50 / cm} ³ 11.8 13.5 15.0
Inactivated viral suspension of the strain Virus panleucopenia felline "Hannibal" with a titer of infectious activity of 10 ⁴ -10 ⁵ ID _{50 / cm} ³ and with an activity in the RSA of 1: 64-1: 256 GAU / cm ³ 24.3 28 30.4
A mixture of antigens from Leptospira strains of the serogroup Canicola VGNKI-3 and Icterohaemorrhagiae VGNKI-2, taken in equal proportions at a concentration of 300-500 million cells / 0.2 cm ^{3 of} phosphate buffer 9.2 9.6 10.6
Aluminum hydroxide 4.8 5.2 5.6
25% solution
gelatose 8.5 9.5 11.3
50% sorbitol solution 2.8 3.2 3.8
Serum protein 3.3 4.4 5.5
Distilled water 1.2 1.5 1.8
Hanks Solution Else
PRI me R 2. Study the harmlessness of the vaccine against plague of carnivores, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis of dogs of three series on white mice weighing 14-16 g, guinea pigs weighing 300-350 g, puppies of dogs of two months of age, ferrets three months old, four-month-old minks with various doses of the vaccine. The total control results are presented in table. 1.

 The Hexacanivac vaccine is harmless to animals. Animals remained clinically healthy for 21 days after vaccination.

 PRI me R 3. Evaluation of the immunogenic activity of the vaccine against plague of carnivores, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis dogs "Hexacanivac" series 2 is carried out on puppies of dogs weighing up to 5 kg. Animals are vaccinated once and twice in various doses.

 The activity of the drug administered to animals is evaluated by studying the dynamics of the accumulation of specific antibodies in the blood serum of dogs, which are determined in the following serological reactions: in the neutralization reaction in the culture of Japanese quail cells to the carnivore plague virus, in the diffusion precipitation reaction in agar gel to carnivorous adenoviruses, in the inhibition reaction hemagglutination to the causative agent of parvovirus enteritis of dogs and in the reaction of microagglutination to the causative agents of leptospirosis. The results are presented in table. 2.

 From the table. 2 it follows that the Hexacanivac vaccine in puppies of dogs causes intense immunity to the carnivore plague virus, to adeno- and parvoviruses, as well as to leptospira.

 PRI me R 4. Determine the optimal dose of the vaccine "Hexacanivac" series 2 for puppies of dogs weighing up to 5 kg and dogs weighing over 5 kg. Animals are vaccinated with different doses of the vaccine once or twice with an interval of 7-10 days. The results are presented in table. 3.

The level of study of specific antibodies in the blood serum of dogs, as well as the severity of the post-vaccination reaction (Table 3) indicate that the optimal dose of the vaccine for puppies weighing up to 5 kg is a double administration of the vaccine in a dose of 1.2 cm ³ with an interval between injections 7-10 days, and for dogs weighing more than 5 kg, a single dose of 2.2 cm ³ .

Immunization of dogs is carried out 1 month before mating and not less than 14 days after immunization against other infections. Puppies are vaccinated from eight weeks of age. The vaccine is administered subcutaneously in the scapular region or intramuscularly from the intravenous surface of the thigh for dogs weighing up to 5 kg by 1.2 cm ³ , weighing more than 5 kg by 2.2 cm ³ . After 7-10 days, the puppies of the dogs are re-administered the same dose. Dogs aged 1 g or more are vaccinated once a year in the same doses.

 Using the proposed vaccine in veterinary practice will improve the effectiveness of ongoing measures for the prevention of infectious diseases of dogs.

Claims (1)

Vaccine for the prevention of plague, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis of dogs, including a viral suspension of the strain Distemper canine "EPM" with a titer of infectious activity 10 ³ 10 ⁴

inactivated viral suspension of the strain Mammaliadenovirus Adenovirus canis-2 "Ada" with a titer of infectious activity 10 ^{4 , 5} 10 ^{5 , 0}

inactivated virus suspension of the strain Virus panleucopenia felline "Hannibal" with a titer of infectious activity 10 ⁴ 10 ⁵

and with activity in RSA 1 64 1 256 GAU / cm ³ , a mixture of antigens from leptospira strains of serogroup Canocola VGNKI-3 and Icterohaemorrhagiae VGNKI-2, taken in equal proportions at a concentration of 300 500 million cells / 0.2 cm ³ phosphate buffer, hydroxide aluminum, 25% gelatin solution, 50% sorbitol solution, blood serum protein, distilled water, Hanks solution in the following ratio of components, vol. Viral suspension of strain Distemper canine "EPM" with a titer of infectious activity 10 ³ 10 ⁴

15.2 19.0
Inactivated viral suspension of the strain Mammaliadenovirus Adenovirus canis-2 "Ada" with a titer of infectious activity 10 ^{4 , 5} 10 ^{5 , 0}

11.8 15.0
Inactivated viral suspension of the strain Virus panleucopenia felline "Hannibal" with a titer of infectious activity 10 ⁴ 10 ⁵

and with activity in the RSA 1 64 1 256 GAU / cm ³ 24.3 30.4
A mixture of antigens from leptospira strains of serogroups Canicola VGNKI-3 and Icterohaemorrhagiae VGNKI-2, taken in equal proportions at a concentration of 300 500 million cells / 0.2 cm ³ phosphate buffer 9.2 10.6
Aluminum hydroxide 4.8 5.6
25% gelatin solution 8.5 11.3
50% sorbitol solution 2.8 3.8
Serum Protein 3.3 5.5
Distilled water 1.2 1.8
Hanks Solution Else

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