CS267937B1 - Vital attenuated vaccine against parvovirosis,distemper,infective laryngotracheitis,infective hepatitis and parainfluenza 2 and method of its preparation - Google Patents

Abstract

The solution concerns live attenuated vaccines against parvovirosis, distemper, infectious laryngotracheitide, infectious hepatitide and parainfluanze 2 and treatment its preparation for cell cultures. vaccine is intended for protective vaccination of dogs and anti-carnivorous fur animals infections. The vaccine is lyophilized and characterized in that it comprises at 2 ml of at least 103 TKID-- parvovirus particles dogs, strain CPVA BN 80/82 CAPM V-290, W5 TKIDjq distemper virus particles. strain CBV-F-BN 10/83 CAPM V-289, 10ž5 TKID-θ infectious laryngotracheitis virus particles Tribe_CADV-2 H-S / 85 MDCK40 CAPM V- -320 and 104 'of TKID-n particles of parainfluenza virus 2, strain CPIV-2 BN4 / 85/32 CAPM V-321 adapted to cell cultures. Infectious laryngotracheitis virus strain is propagated on a stable cell line MEAT in the rain-essential essential medium Eagle and parainfluenza virus strain 2 on the cell line FE and MDCK in minimal the essential medium of Eagle's medium. After end culturing the viral materials mixed with lyophilization medium and freeze-drying medium and lyophilized.

Description

The invention relates to a live attenuated vaccine against parvovirus, canine distemper, infectious laryngotracheitis, infectious hepatitis and parainftuenza Z, and to a process for its preparation in cell cultures.

In addition to the well-known causes of viral canine diseases such as rabies, canine distemper and infectious hepatitis, which are reliably controlled by effective attenuated vaccines, there are a number of Russians that cause common gastrointestinal and respiratory diseases. These include, in particular: canine parvovirus, a causative agent of acute intestinal disease mainly affecting pups, infectious laryngotracheitis virus and parainfluenza virus of 2 dogs, which alone or in conjunction with the bacterial germ Bordetella Bronchiseptica cause mass respiratory disease / Kennel respiratory syndrome / Kennel. In 1978, Appel defined the pathogenicity of canine parvovirus (CPV 2) and identified it as the cause of an epizootic of intestinal disease that first appeared in 1978 in the United States. The disease caused by this virus is clinically manifested by anorexia, malaise, vomiting and diarrhea, which can be bloody. The animals have a fever and a hernatological examination usually shows a reduced number of leukocytes. The cardiac form of the disease affects only puppies and is characterized by a rapid and very short course and subsequent death. The disease causes significant losses in dog breeding, with death accounting for 20 to 40% of the total number of animals. The disease affects all age groups of animals, but especially puppies, and the onset is rapid and the course is very rapid.

Canine adenovirus serotype 2 (CADV-2), isolated and identified by DITSCHFIELDON, 1962, is serologically, antigenically, different from canine infectious hepatitis virus (ICH) in its tropism and clinical manifestation of the disease.

Hemaglutinating parainfluenza virus 2 (CPIV-2), affecting the respiratory tract, isolated BINN in 1967. Due to its antigenic similarity to the simian virus SV5, it was designated SV5- like canine parainfluenza virus.

Diseases caused by these viruses have the character of infectious laryngotracheitis, tracheobronchitis, inflammation of the nasopharynx and lung with clinical manifestations of rhinitis, spontaneous cough caused by narrowing of bronchial patency and pulmonary hyperventilation accompanied by bronchial lymphatic enlargement. The pathological-anatomical picture is characterized by infiltration of submocosis by lymphocytes, plasma cells and neutrophils, proliferative interstitial pneumonia, necrotization of bronchi and bronchiolitis. In CAOV-2 virus infection, intronuclear inclusions located in the epithelial cells of the nasal and pharyngeal mucosa are detected.

The specific prophylaxis of these viral diseases, which pose a serious health problem in dog breeding, is based on passive immunization by administration of hyperimmune sera or immune serum globulins, but active implanization is most commonly used.

R Inactivated and live monovalent and combination vaccines / CANDUR op R R have been developed worldwide

-P, HULTIMUN 7, NOBI VAC-SH, ENDURACELL 7 - tiSR, GALAXY -VI - USA /. First live canine parvovirus vaccine, Vet.Rec. No. 17, 1984, 114 no. 11, p.255> Stettner, W. - Protective effects at the dog, Vienna. TierSrztliche Monatsschrift, 72, 1985, Heft 5, s.178-182. - Charlier, P. - Florent, G. - Gill, M.A. - and company. Kleintierpraxis 30 1985, 45-49.

And Ke3 by various viral strains in vaccines is a considerable diversity in general it is desired that a single vaccine dose contains at least 10 ^ ^'9 and 10 ° ^3z TKXD50 of each virus.

The close antigenic relationship between infectious laryngrotacheitis viruses (CADV 2) and infectious hepatitis (CADV 1 Rubart's disease) was confirmed by Fa irchild, G. A. - Cohen, 0., Serologic study of a Canine Adenovirus / Toronto KZblbZI infection in dogs. Am.J .Vet.Res.,

CS 267937 Θ1

Vol.30 No. 6, 1968 p. 923-928, and Appel, M. - Bistner, S.I. - Menegus, M. - a spot. Pathogenicity of low virulence strainsoof two canine adenovirus types, Am.J.Res., 34 1973, p.543-550. Therefore, vaccination of susceptible animals with attenuated strains of infectious laryngotracheitis virus induces reliable protection against natural or experimental infection with CAffV-1 strains via serum antibodies. Emmery, J.B. - House, J.A. - Brown, W.R. - Cross-protective immunity to canine adenovirus type 2 by canine adenovirus type 1 vyccination, Am. J.Vet. Res., Vol.39 No.11 1978 p.1778-1783. As early as 1972, Marusyk pointed out that vaccination with infectious laryngotracheitis virus reliably protects against infection with infectious hepatitis virus, but, conversely, vaccination with infectious hepatitis virus did not protect animals from infectious virus infection. Harusical, R .G. two canine adenoviruses, Canadian J.Microbiol, Vol.18 1972 p-817-823.

Due to the general incidence of canine parovirus and the increasing number of respiratory diseases, while eliminating the effect of canine complex viruses, we set ourselves the goal of obtaining or isolating the corresponding virus strains with the prospect of using them for vaccine preparation. Individual isolates of parainfluenza 2 viruses, infectious laryngotracheitis and parvovirus were identified, cloned, confirmed for purity and electronically optical. After completion of attenuation of individual viruses on various types of cell cultures, belief in their harmlessness in susceptible animals and on the basis of literature data Lynna, R, C., How to control canine infectious trachobronchitis through vaccination, Vet.Med., June 1986 p.544-550 , and i. sue has begun the development of a live attenuated vaccine against parvovirus, canine distemper, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2.

Appel, M.-Carmichael, L.E. - Rcbson, D.S., Canine adenovirus type 2 induced immunity to two canine adenoviruses in pups with maternal antibody, Am.J .Vet. Res., 36 1975 p.1199-1202, and Powell, K.W., Use of canine adenovirus type 2 vaccine to control kennel cough syndrome. Vet.Med.Small Anim. Clin., 74, 1979, pp.801-804.

A live attenuated vaccine against parvovirus, canine distemper, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2 was prepared, lyophilized, containing 2 ml of at least 10 TKIïjq canine distemper virus particles, strain COV-F-BN 10/83 CAPM V-289; 10 ^d '

TKXP and 10 particles of Infectious Laryngotracheitis Virus, CAPV 2 strain H-5/85 M0CK40 camp v-320;

TKIDjq of 2 dog parainfluenza virus particles, strain CPIV-2 BN4 / 85/32 CAMP V-321.

Canine parvovirus virus strain designated CPVA-BN 80/82 and collections. no. CAPM v-290 was adapted to cell cultures of the stable feline FE line. Minimally essentially Eagle's medium enriched in bovine serum in which the virus was propagated at 36 to 38 ° C for 4 to 8 days was used as growth medium. After making distinct cytopathic changes, the viral material was mixed with the lyophilization medium to contain at least 10 ΤΚΙΟ ^ θ · .ml. Said virus strain was obtained from a flood of virulent frizz isolated from infectious materials by serial passages on a cat line marked FE. Homogeneity of the virus population and stability of immunobiological properties were achieved by isolating the line by the six-fold borderline dilution method.

Canine distemper strain with the designation COV-F-BN 10/83 and collection no. CAPM V-289 was adapted to primary or secondary cultures prepared from smoking embryos and to cultures of the stable VEVO monkey ladvin cell line. As a growth medium, bovine serum-enriched Eagle's medium was minimally essential, in which the virus was propagated at 36 to 38 ° C for 24 to 48 hours, i. until a monolayer is formed on the culture surface. The growth medium was then changed to Eagle's minimally essential maintenance medium in which the vaccine virus was produced at 36-38 ° C for 5-9 days. After making clear cytopathic changes, the viral material was mixed with lyophilized medium to contain at least 10 ΤΚΙΟ ^ θ .ml.

The canine distemper virus vaccine strain was bred from canine distemper virus, the Onderstepoort strain, attenuated by serial passages on chorioallantoic membranes of chicken embryos, adhered to cell cultures prepared from smoking embryos and cell cultures of a stable VEVO line, followed by high virus selection. immunizing activity with very good pasting properties on said tissue types with the development of significant cytopathic changes suggesting viral propagation e.

The infectious laryngotracheitis virus strain designated CADV-2 H-5/85-MDCK40 with collection number CAPM V-320 was adapted to cultures of a stable canine kidney cell line MDCK. In the propagation of infectious laryngotracheitis virus, MDCK cell cultures deprived of growth medium were infected with said vaccine strain, at a multiplicity of infection of 0.1 to 0.01 and after adsorption of the virus to the cells they were irradiated with minimal essential medium according to Eagle. Infected cultures were infected at 36 to 38 ° C for 3 to 5 days. After making distinct cytopathic changes, the viral material was mixed with lyophilized starting medium to contain at least 10 'ΤΚίΟ, -θ. ml.

Infectious laryngotracheitis virus is a virulent strain of foreign origin obtained in the form of a lyophilisate of infectious tissue fluids in passage 5 on cell cultures. Attenuation of the virus, homogeneity of the virus population and stability of immunobiological properties was achieved in passage 35 on b.1. MDCK. To prepare the vaccine, the virus used in passage 40 was attenuated on this type of tissue and defined at our workplace.

The 2 canine paranfluenza virus strain designated CPIV-2 BN4 / 85/32 and collection number CAPM V-321 was adapted to cultures of the stable canine ladle cell line MDCK and to cultures of the stable feline FE line. As a growth medium, bovine serum-enriched Eagle's medium in which the virus was propagated at 36 to 38 ° C for 4 to 7 days was used at least essentially according to Eagle's medium. Virus growth on 1. FE is accompanied by marked cytopathic changes in the cellular gameteer. The infectious tissue fluids obtained contain 16 to 128 haemagglutination units in 0.05 ml and the infectious titer is 10 to 10 ⁷ ' ⁵ TKID-n. ml ^-1 . At the end of the incubation, the viral material was mixed with lyophilization ^3U 45-1 medium to contain a minimum of 10 ' ΤΚΙ0 > ml. Primoisolate propagated in the amniotic cavities of 10 day old chicken embryos from SPF eggs, we adapted to embryonic cell cultures with stable cell unions FE and MDCK. The homogeneity of the virus population and the stability of the immunobiological properties of the attenuated strain were achieved by the borderline dilution method in the 32nd tissue passage.

Canine parvovirus virus strains CPVA-BN 80/82 CAPM V-290, canine CDV-F-BN 10/83 CAPM V-289, infectious laryngotracheitis CADV-2 H-5/85 MDCK4Q CAPM V-320 and parainfluenza 2 CPIV-2 BN4 / 85/32 CAPM V-321 are stored in the Czechoslovak Collection of Microorganisms at the Research Institute of Veterinary Medicine in Brno, Hudcova 60.

Vaccine production procedure:

The method of vaccine production is based on the fact that said vaccine strains, harmless for all age categories of dogs and carnivorous fur animals by any application, are propagated on defined cell substrates grown on glass, plastic, cultured stationary or in rotated bottles / rollers / .

κ or microcarriers.

Method of infection of FE cells with canine parvovirus vaccine virus:

A cell suspension with a density of 5 to 10.10 cells. ml in growth medium is mixed with vaccine virus at a multiplicity of infection of 0.0001 to 0.001. The virus-cell mixture is incubated at 36-38 ° C for 30-60 minutes. The virus-cell mixture is then diluted in growth medium, which is at least essentially Eagle's medium with an sH of 6.8 to 7.3 and a preparation of five to nine weight percent of bovine serum of the indicated quality.

It is also possible to add an inoculum of vaccine virus with a multiplicity of infection of 0.0001 to 0.001 to the cell suspension in growth medium and to pour the mixture of virus and cells into a culture vessel.

The cell suspension thus prepared for the preparation of the vaccine is cultured on glass, plastic, stationary method or in rotating bottles (rollers), optionally on microcarriers, at 36 to 38 ° C for 4 to 8 days.

Infection of adherent cells on the culture surface is also possible - after four to six hours of incubation of FE cells at 36 to 38 ° C. Vaccine virus inoculum is added to the growth medium at a multiplicity of infection of 0.0001 to 0.001. Incubation is continued at 36 to 38 ° C for 4 to 8 days. As in point A and B, virus replication is accompanied by significant cytopathic changes.

Method of infection of cells of chicken embryonic fibroblasts KEB and cells of the VĚRO line with canine distemper virus:

KEB or VĚRO cell cultures are infected with a canine distemper vaccine strain at a multiplicity of infection of at least 0.005. Cells grown on the culture surface are infected for 24 to 48 hours by changing the growth medium to the maintenance medium to which the infectious inoculum is added. The maintenance medium is of the same composition as the growth cell for each cell type, but does not contain serum and has an adjusted sodium bicarbonate content of 0.1 to 0.2% by weight.

Alternatively, the mixture of virus and cells is allowed to incubate for 60 to 120 minutes at 37 ° C, then the cell suspension is diluted in growth medium to contain 4 to 8. 10 ⁶ . • Even cells in growth medium.

It is also possible to add the infectious inoculum to a suspension of cells in growth medium and pour it into a culture vessel without prior incubation. After 24 to 48 hours, the growth medium is changed, without rinsing the monolayer cells for maintenance, and the incubation is continued under the same conditions. After detecting the first cytopathic changes (usually 72 to 96 hours), the medium is changed at 24-hour intervals, lyophilized medium is added and stored frozen until the combined vaccine is prepared before lyophilization, or the culture is allowed to continue for more than 50 percent of the monolayer cells are affected by virus-specific changes and the virus suspension is collected only once.

Method of infection of MDCK cells with infectious laryngotracheitis vaccine virus:

Cell cultures are infected with a vaccine strain of infectious laryngotracheitis virus at a multiplicity of infection of 0.1 to 0.001. 24-48 hour cell cultures grown on the culture surface are infected by removing the growth medium and, after 30-60 minutes of adsorption of the virus to the cells at 36-38 ° C, maintenance medium is added. The maintenance medium is of the same composition as the growth medium but does not contain serum. Incubation is continued at 36 to 38 ° C for 3 to 5 days.

It is also possible to add the infectious inoculum to a suspension of cells in growth medium and

CS 267937 31 poured into a culture vessel without prior incubation. After 24 to 48 hours, the paste medium is changed, without rinsing the monolayer cells for maintenance, and the incubation is continued under the same conditions for 3 to 5 days. In both cases, the culture is allowed to proceed as long as more than 80 percent of the monolayer cells are affected by virus-specific changes.

Method of infection of FE cells or MDCK cells with parainfluenza vaccine virus in 2 dogs:

A suspension of FE cells or MDCK cells with a density of 5 to 10.10 ⁶ ml ^-1 in growth medium is mixed with the vaccine virus at a multiplicity of infection of 0.001 to 0.01. The virus-cell mixture is incubated at 36-38 ° C for 30-60 minutes, then diluted in growth medium, which in both cell lines is at least Eagle's medium at pH 6.8-7.3 and the addition of five to ten parts by weight. percent bovine serum.

It is also possible to add an inoculum of vaccine virus with a multiplicity of infection of 0.001 to 0.01 to the cell suspension in growth medium and to pour it immediately into the culture vessel. Incubation is continued at 36 to 38 ° C for 4 to 7 days. The growth of the virus on the FE line is accompanied by the development of marked cytopathic changes in the cell monolayer.

At the end of the culture, the infectious fluids of the individual viruses are mixed with the lyophilization medium in a ratio of 50 to 75 weight percent of the virus to 50 to 25 weight percent of the lyophilized medium and stored at -15 to -20 ° C until lyophilization. The lyophilization medium can be added just before assembly and lyophilization of the vaccine, while maintaining the same weight ratios. For the preparation of a vaccine is freeze lyophilizate PO 4 -1 infection employing a fluid containing at least 10 TKID_ _n. ml of CPVA-BN virus particles 4 ^{5 in} X

80/82 CAMP V-290, 10 ΤΚΙΟ ^ θ CDV-F-BN virus particles 10/83 CAPM V-289, 10 TKID ₅₀ CADV-2 Virus particles H-5/85 MDCK40 CAMP V-320 and 10 ⁵ ΤΚΙΟ ^ θ CPIV-2 BN4 / 85/32 CAMP V-321 virus particles, bacteriologically sterile. The virus suspensions thus prepared are mixed in a ratio of 0.5 parvovirus diet + 1 canine distemper virus diet + 1 infectious thyngotracheitis virus diet + 1 parainfluenza virus 2 diet with lyophilisate medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin but -, or another source of protein, dextran or gelatin / BOVARNICK, MR - MILLER, JC - SNYDER, JC, The influence of certain salts, ammonoacids, sugaes, and proteins on the stability of rickettsiae. J. Bacteriot., 59, 1950,: 509-522) are dispersed in the form of a homogenate into ampoules or vials, lyophilized, sealed airtight and stored at refrigerator temperature. The vaccine prepared according to the above procedure contains in a single application dose at least 10% of the rabies virus, 10 ^{2% of the} infectious laryngotracheitis virus and 10 ⁴ % of the parainfluenza virus 2, which represents one immunization dose.

Example 1

The way. cultivation:

FE, MDCK, VĚRO cell suspensions prepared by proteolytic enzymes and chicken embryonic cell cell suspension (KEB), prepared by proteolytic enzymes from 11-day-old chicken embryos in growth media containing a defined amount of amino acids and thymines with the addition of 5 weight percent serum in cultures KEB and VĚRO and 10 weight percent of tissue culture serum in FE and MDCK cultures are cultured on glass in a stationary manner and in suspension.

Example 2

Method of infection of cell cultures with canine parvovirus vaccine virus:

Cell cultures prepared and grown according to point 1 are infected with CPVA-BN 80/82 vaccine virus at a multiplicity of infection of at least 0.001 in the following ways:

a / Suspension of FE line cells with a density of 10.10 ⁶ cells. ml ” ¹ in growth medium is mixed with vaccine virus at a multiplicity of infection of 0.001. The virus-cell mixture is incubated at 37 ° C for 30 minutes. The virus-cell mixture is then diluted in growth medium, which is at least essentially Eagle's medium, pH 7.1, with the addition of 10 weight percent bovine serum.

-1 b / Oo suspension of FE line cells with density 5.10. ml in growth medium, an inoculum of vaccine virus with a multiplicity of infection of 0.001 is added and the infected suspension is immediately poured into a culture vessel. The cell suspension thus prepared to prepare the virus for action was cultured in Roux flasks at 37 ° C for 6 days. .

c / Infection of adherent cells on the culture surface after culturing FE cells at 37 ° C for six hours, an inoculum of vaccine virus is added to the growth medium at a multiplicity of infection of 0.001. Incubation is continued at 37 ° C for 6 days. As in a) and b), virus replication is accompanied by significant cytopathic changes.

Vaccine virus is produced in growth medium on FE cell cultures grown according to point 1 and infected according to point 2. After extensive cytopathic changes determined microscopically, the culture is terminated and lyophilization medium in a ratio of 50% by weight is added directly to the culture medium with propagated virus. percent virus suspension and 50 weight percent lyophilization medium. The mixture is stored in culture vessels in a frozen state.

Example 3

Method of infection of cell cultures with canine distemper vaccine virus:

The vaccine virus inoculum in which the virus is stabilized by the addition of 10 weight percent dimethyl sulfoxide is stored frozen at -70 ° C.

Cell cultures prepared and grown in accordance with point 1 shall be infected with at least 0.005 in the following ways:

a / Infection of grown KEB monolayer cells:

After 24 hours of growth, the growth medium is removed from the cell monolayer, and the cell culture is infected with an inoculum added to the maintenance medium to replace the growth medium.

-1 b / The cell suspension in growth medium with a density of 10.10 cells is mixed with the inoculum of the vaccine virus and the mixture is incubated at 37 ° C for 30 minutes. contained 4.10. ml of cells in 1 ml and cultured at 37 ° C for 48 hours, i. until the formation of clamped monolayer cells on the culture surface. This is followed by replacement of the growth medium with maintenance medium.

-1 c / Oo of KEB cell suspension in growth medium and density 4.10. ml inoculum of vaccine virus is added. The suspension is immediately poured into a culture vessel and incubated at 37 ° C for 24 hours. i.e. until the formation of monolayer cells. This is followed by replacement of the growth medium with maintenance medium.

The vaccine virus is produced in a maintenance medium which, in its basic composition, is the same as the Eagle's minimal essential growth medium, except that it does not contain serum and the concentration of pellet bicarbonate is up to 0.30% by weight. Virus from cell cultures grown in accordance with point 1 and infected with point 3 is obtained by the following methods:

CS 267937 81 rJ The maintenance medium is left unchanged on the cultures until extensive cytopathic changes determined microscopically. To the virus maintenance medium, lyophilization medium in a ratio of 2 diets of virus suspension and one part of lyophilization medium is added directly to the culture vessels. The mixture is further stored in culture vessels in a frozen state.

b / After the first signs of specific cytopathic changes appear, the maintenance medium is decanted and the same amount of maintenance medium is poured into the culture vessels. The procedure is repeated at 24-hour intervals until an extensive cytopathic effect is created, at which point the final treatment is carried out according to a). The lyophilization medium is added to the virus suspension obtained by repeated changes of maintenance medium immediately after decantation and further stored frozen. '

Example 4

Method of infection of cell cultures with infectious laryngotracheitis vaccine virus:

Cell cultures prepared and grown according to point 1 are infected with CADV-2 H-5/85 MDCK40 vaccine virus at a multiplicity of infection of at least 0.01 as follows:

a / Infection of grown MDCK monolayer cells: After growth for 48 hours at 37 ° C, the growth medium is removed from the growth medium, which is at least essentially Eagle's medium, and the cell culture is infected by adsorbing the virus to the cells for 60 minutes at 37 ° C. Maintenance medium, which is of the same composition as the growth medium but does not contain serum, is then added.

-1 b / Into a cell suspension in growth medium with a density of 4.10. ml inoculum of vaccine virus is added. The suspension is immediately poured into a culture vessel and incubated at 37 ° C for 48 hours, i. until the formation of monolayer cells. The paste medium is then changed to maintenance medium and the incubation is continued at 37 ° C for 2 days. In both cases, virus replication is accompanied by marked cytopathic changes in the cell monolayer. Vaccine virus is produced in growth medium on MDCK cell cultures grown according to point 1 and infected according to point 4. After extensive cytopathic changes have occurred, the culture is terminated and lyophilization medium in a ratio of 2 parts of virus is added directly to the culture medium with propagated virus. suspension and one part of lyophilization medium. The mixture is further stored in culture vessels in a frozen state.

Example 5

Method of infection of FE and MDCK cells with parainfluenza vaccine 2 virus:

Cell cultures prepared and grown according to point 1 are infected with CPIV-2 BN4 / 85/32 vaccine virus at a multiplicity of infection of at least 0.001 in the following ways:

*} a / Suspension of FE line cells with density 10.10. ml in growth medium is mixed with vaccine virus at a multiplicity of infection of 0.01. The virus-cell mixture is incubated at 37 ° C for 30 minutes. The virus-cell mixture is then diluted in growth medium, which is at least essentially Eagle's medium and 5 weight percent bovine serum.

5-1 b / To a suspension of MDCK cells with a density of 5.10. ml of growth medium is then added to a 1 [mu] m vaccine virus with a multiplicity of infection of 0.01 and the infected suspension is immediately poured into a culture vessel. The cell suspension thus prepared for virus preparation was cultured at 37 ° C for 5 days. The multiplication of the virus on FE cells is accompanied by marked cytopathic changes in the cell monolayer.

The virus virus is produced in growth medium on FE and MOCK cell cultures grown according to point 1 and infected according to point 5. At the end of the culture, lyophilization medium in a ratio of 2 parts of virus suspension and one part of the virus suspension is added directly to the culture medium. part of the lyophilization medium. The mixture is further stored in culture vessels in a frozen state.

Example 6

Preparation of the combined vaccine:

^{Infectious fluids containing at least 10 4} TKIOqn are used to prepare the lyophilized vaccine. ml ^-1 particles of virus CPVA-BN80 / 82, 10 ⁴ TKID „n. ml “ ¹ particles of CDV ⁵ X ^{3 U} 5 -1

-F-BN 10/83, 10 TKID _5Q CADV-2 virus particles H-5/85 MDCK40 and 10 ΤΚΙΟ ^ θ. ml of CPIV-2 BN4 / 85/32 virus particles; bacteriologically sterile. The virus suspensions thus prepared are mixed in a ratio of 0.5 diets of parvovirus and 1 part of canine distemper virus and 1 part of infectious laryngotracheitis virus and 1 part of para influenza virus 2. Homogenized virus material with the required content of infectious units is adjusted, lyophilized, sealed and stored at refrigerator temperature.

The vaccine prepared according to the above procedure contains at least 3? WITH

TKIïjg canine parvovirus, 10 ΤΚΐ0 ₅ θ canine distemper virus, 10 'TKID infectious laryngotracheitis virus and 10 ⁴ ' ⁵ TKIOjg parainfluenza virus 2 in a single vaccination dose.

Prove the efficacy of the combined vaccine:

Comparison of the efficacy of live attenuated parvovirus, canine distemper vaccine, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2 with the efficacy of monovalent parvovirus vaccine, canine distemper vaccine and bivalent vaccine of infectious lavine and infectious lavine vaccine 1. The values shown graphically represent the average titers of heme-inhibitory (HI) antibodies against parvovirus, infectious laryngotracheitis virus and parainfluenza virus 2, and virus-neutralizing antibodies (VN) against canine distemper virus. It is clear from the presented results that, after vaccination with the polyvalent vaccine, the values of the Specific Peibodies were obtained at the same level or higher than after the application of mono. resp. bivalent vaccines. The induction of such an immune response has not been anticipated and has not been established.

The results of Table 1 show that neither vaccinated nor control animals responded to infection with a field strain of canine parvovirus with a detectable medical condition. These findings are consistent with McAdaragh, J.P. - Eustis, S.L. - Nelson, D.Tj - a spol ,, Experimental infection of conventional doge with vanine parvovirs. A. J.Vet.Res., Vol 43 No.4, 1982 p.693-696; Pollock, R.V.H. - Carmichael, L.E., Dog response to inactivated canine parvovirus and feline panleukopenia virus vaccines. Cornell Vet.1982, 72, 16-35 et al., That infection of susceptible animals with field strains of canine parvovirus remains without demonstrable clinical manifestation. After the infection! All vaccinated animals remained healthy with virulent canine distemper virus, with one of the two control animals dying and the other showing characteristic signs of canine distemper. The result of challenge of animals vaccinated with parainfluenza virus 2 is characteristic of infection of conventional animals with this virus, the pathogenicity of which is determined by the involvement of other infectious agents in the development of the disease. With the protection of vaccinated animals infected with infectious laryngotracheitis virus, both controls responded to pre-existing but overt airway disease.

All vaccinated animals infected with the infectious hepatitis virus also remained clinically healthy, reflecting the close antigenic relationships between the two viruses and the cross-protection of animals vaccinated with the infectious laryngotracheitis virus against infection and in the infectious hepatitis virus. Both unvaccinated controls responded to the infection by developing clinical signs characteristic of Rubatth's disease (infectious hepatitis in dogs).

ANIMALS VIRUS INFECTION POÍET ANIMAL THE RESULTS without wedges, at characters clinically ill death CPV 3 3 0 0 CDV 3 3 0 0 Va kc i no- CPIV-2 3 3 0 0 waved CADV-2 3 3 0 0 CADV-1 3 3 0 0 CPV 2 2 0 0 CDV 2 e 1 1 Checks CPIV-2 2 1 1 0 CAĎV-2 2 0 2 0 - CADV-1 2 0 2 0

Tab. no. 1

Claims (5)

Russian infectious hepatitis. Both non-vaccinated controls responded to infection with the development of clinical pathogens characteristic of Rubatth's disease / infectious hepatitis. ANIMAL INFECTION VIRUS VIRUS WORLD RESULTS without wedges. for signs of clinically ill mortality CPV 3 3 0 0 CDV 3 3 0 0 Va kc i no-CPIV-2 3 3 0 0 net CADV-2 3 3 0 0 CADV-1 3 3 0 0 CPV 2 2 0 0 CDV 2 e 1 1 CPIV-2 checks 2 1 1 0 CADV-2 2 0 2 0 - CADV-1 2 0 2 0 Tab. No. 1 OF THE INVENTION A live attenuated parvovirus vaccine comprising the CPVA-BN 80/82 V-290 strain in a quantity of 103 TKIDj-g, a clot containing 103e, -θ, infectious, CDV-F-BN 10/83 CAPM V-289 strain laryngotracheitis, infectious hepatitis and parainfluenza 2, characterized in that it contains in 2 ml parvovirus strain of dogs CPVA-BN 80/82 CAPM V-290 of at least 103 TCIDSO, distemper virus strain CDV-F-BN 10/83 CAPM 289 at at least 10 3, θ, infectious laryngotracheitis virus strain CADV-2 H-5 / 85MDCK 40 CAPM V-320 at least ΙΟ ^ ® ΤΚΙΟ ^ θ and parainfluenza 2 strain CPIV-2BN4 / 85/32 CAPM V-321 in an amount of at least 10 < 4 > 2. A live attenuated vaccine according to claim 1, wherein the lyophilization medium is 25 to 50 weight percent based on the weight of the entire vaccine. 3. A live attenuated vaccine according to claim 1, wherein the lyophilization medium comprises sucrose, phosphate buffer, sodium glutamate, or potassium, bovine albumin or another source of protein and gelatin or dextran. 4. A method for producing a vaccine according to item 1 by culturing parvovirus CPVA-BN 80/82 CAPM V-290a of a canine distemper virus CDV-F BN 10/83 CAPM V-289, characterized in that the cultures of parvovirus and canine distemper virus are mixed with the infectious virus culture medium. larynthistracheid CADV-2 H 5/85 MDCK40 CAPM V-320 obtained by propagation on stable MDCK cell lines after 30-60 min infection of 24-48 hour monolayer in malignant essential medium of Eagle at baseline 36-38 ° C for 3-6 days, until cytopathic changes were formed, mixed with lyophilization medium to a content of at least 10 3 TIDID. ml-1 and with the culture of parallel dog 2 CP1V-2 BN4 / 85 / 32CApM V-321 virus obtained by propagation on stable FE and MOCK phlegm in the immortal essential medium of eagle and pH 6.8 to 7.3 enriched in bovine serum at 36 to 38 ° C for 4-8 days supplemented with lyophilized medium to a minimum of 103 TKID / g. . and the mixture of the viruses is lyophilized 5. The method of manufacture of claim 4, wherein the lyophilization medium is added to the viral suspensions immediately after recovery and the mixture is stored frozen until freeze-dried or lyophilized immediately. 2 drawings