CS267937B1 - Live attenuated vaccine against parvovirus, distemper, infectious laryngotracheitis, infectious hepatitis, and parainfluenza 2 - Google Patents

Abstract

The solution relates to a live attenuated vaccine against parvovirus, distemper, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2 and a method for its preparation in cell cultures. The vaccine is intended for protective vaccination of dogs and carnivorous fur animals against the above infections. The vaccine is lyophilized and is characterized by containing in 2 ml at least 103 TKID-- particles of canine parvovirus, strain CPVA BN 80/82 CAPM V-290, W5 TKIDjq particles of distemper virus. strain CBV-F-BN 10/83 CAPM V-289, 105 TKID-θ particles of infectious laryngotracheitis virus, strain CADV-2 H-S/85 MDCK40 CAPM V- -320 and 104' TKID-n particles of parainfluenza virus 2, strain CPIV-2 BN4/85/32 CAPM V-321 adapted to cell cultures. The infectious laryngotracheitis virus strain is propagated on a stable MDC cell line in Eagle's minimum essential medium and the parainfluenza virus strain 2 on FE and MDCK cell lines in Eagle's minimum essential medium. After completion of the cultivation, the viral materials are mixed with the lyophilization medium and the lyophilization medium and lyophilized.

Description

CS 267937 B1 1

The present invention relates to a live attenuated vaccine against parvovirus, distemper, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2, and a method for its preparation by cell cultures.

In addition to well-known viral diseases in dogs such as fever, canine disease and infectious hepatitis, reliably controlled by effective attenuated vaccines, there are a number of Russians who are the causative agents of frequent gastrointestinal and respiratory diseases, including: parvovirus dogs , an acute intestinal tract inflicted predominantly on juveniles, infectious laryngotracheitis virus, and parainfluenza virus 2 dogs, which alone or in conjunction with the BordetellaBronchiseptica bacterial germ cause mass respiratory disease / Kennel cough syndrome /. parvovirus dog (CPV 2) and described it as a gut epidemic epidemic that first appeared in the US in 1978. The virus-induced disease is clinically manifested by appetite, malaise, vomiting by diarrhea that may be bloody. fever and in the hematologic The cardiovascular form of the disease affects favorably pups and is characterized by a rapid and very short course and subsequent death. The disease causes significant losses in dog breeds, with mortality of 20 to 40% of the total number of animals. The disease affects all age groups of animals, but especially puppies, with the onset being rapid and very fast.

The adenoviruses of serotype 2 / CAOV-2, isolated and identified by DITSCHFIELDOM, 1962, are serologically, antlgeneously, by their tropism and clinical manifestations of disease different from the infectious hepatitis virus (ICH).

The parainfluenza 2 / CPIV-2 / haemagglutinating virus, affecting the respiratory tract, isolated BINN in 1967. Due to its antigenic similarity to monkey SV5, the SV5-like canine parainfluenza virus was designated.

The diseases caused by these viruses have the character of infectious laryngotracheitis, tracheobronchitis, inflammation of the nasopharynx and pluc with clinical manifestations of rhinitis, spontaneous cough due to narrowing of bronchial patency and perverse hyperventilation mediated by bronchial lymph node elevation. The pathological-anatomical image is characterized by submocosis infiltration by lymphocytes, plasma cells and neutrophils, proliferative interstitial pneumonia, necrosis of bronchi and bronchioli. In CAOV-2 infection, intraternal inclusions located in the epithelium of the nasal and pharyngeal mucosa are detected.

The specific prophylaxis of said viral diseases, which is a serious health problem in the breed, is based on passive immunization with the administration of hyperimmune sera or immune serum globulins, but active implants are most commonly used. Inactivated and live monovalent and combined vaccines have been developed in the world. HULTIHUNR7, N0BIR VAC-8H, ENDURACELLR7 - tiSR, GALAXYR-VI - USA /. First live canineparvovirus vaccine, Vet.Rec. 17, 1984, 114. 11, p.255> Stettner, W. - Schutzimpfungenbeim Kund, Wiener. TierSrztliche Nonatsschrift, 72, 1985, Heft 5, p.178-182., Langzei-tuntersuchung zur ImmunogenitSt eines Kombinationimpfstoffes fOr Hunde, Zygraich, N. - Charlier,?. - Florent, G. - Gill, M.A. - a spol. Kleintierpraxis 30 1985, 45-49.

While there is considerable variation in the proportion of individual virus strains in the vaccine, one vaccination dose is generally required to contain at least 10 ^ 3 aj103 / θΤΚΧ05θ from each virus.

Close antigenic affinity of infectious laryngrotacheitis / CADV 2 / and infectious hepatitis / CADV 1 Rubartt's disease / confirmed by Fa irchild, G. A. - Cohen, D., Sero-gia study of Canine Adenovirus / Toronta MblbZI infection 1n dogs, Am.J.Vet.Res., 2 CS 267937 81

Vol.30 No. 6, 1968 pp. 923-928, and ΑρρβΙ, Η. - Bistner, S.I. - Menegus, M. - a spol. Pathogenicity of low virulence strain of two canine adenovirus types, Am.J.Res., 34 1973, p.543-550. Therefore, vaccination of susceptible vet with attenuated strains of infectious laryngotracheitis virus elicits reliable protection against natural or experimental infection with CAffV-1 strains via serum antibodies. Emmers, J.B. - House, J.A. - Brown, W.R. - Cross-protective Immunity to canine adenovirus type 2 by canine adenovirus type1 vyccination, Am. J.Vet. Res., Vol.39 No.11 1978 p.1778-1783. As early as 1972, Marusyk pointed to the fact that vaccination with infectious laryngotracheitis virus reliably protects against infection by Infectious hepatitis virus, but, on the contrary, vaccination with infectious hepatitis virus does not protect animals from disease caused by the infectious laryn-gotracheitldy virus Harusyk, R, G., Comparison of the immunological properties of two canineadenoviruses, Canadian J.Hicrobiol, Vol. 18 1972 p-817-823.

Due to the general incidence of parovirosis in dogs and the increasing number of respiratory pains, excluding the effects of distemper complexes, we decided to recover or isolate the corresponding virus strains with the prospect of using them for vaccine preparation. Individual isolates of parainfluenza 2, infectious laryngotracheitis and parvovirus virus have been identified, cloned, confirmed purity and electronoptically shown by chemical, serological, and logical methods. After closure of the individual viruses on a variety of cell cultures, believing their innocuousness on susceptible animals and on the basis of lteral data, Lynna, R, C., How to con- trol can infectious trachobronchitis through vaccination, Vet.Med., June 1986 p. 544-550, and others have developed a live attenuated vaccine against parvovirosis, psinitis, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2.

High risk of developing post-vaccination reactions characterized by uveitld, interstitial pneumonia, or focal interstitial nephritis after vaccinated vaccinations with CADV strains of infectious hepatitis virus, was reported by Carmichael, L.E. - Rcbson, DS, Canine adenovirus type 2 immunity to two cani-ne adenoviruses in pups with maternal antibody, Am.J.Vet.Res., 36 1975 p.1199-1202, a? Owell, KW, Use of canine adenovirus type 2 vaccine to control kennel cough syndrome.Vet.Med.Srnall Anim. Clin., 74, 1979, p.801-804.

A live attenuated vaccine against parvovirus, distemper, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2 was prepared, lyophilized, containing 2/5 2 ml at least 10 µl of distemper virus particles, strain CDV-F-BN 10 / 83 CAPM V-289; 10TKXPrQ virus particles Infectious laryngotracheitis, strain CADV 2 H-5/85 MDCK40 CAMP v-320a 10 '5 parainfluenza virus particles 2 dogs, strain CPIV-2 BN4 / 85/32 CAMP V-321.

Parvovirus virus strain of dogs designated CPVA-BN 80/82 and collection. CAPM V-290bol adapted to cultures of stable feline Union FE cells. As a growth medium, at least esendal bovine serum enriched medium was used, in which the virus was propagated at 36-38 ° C for 4-8 days. Upon formation of distinct cytopathic changes, the viral material was mixed with lyophilization medium to contain a minimum of 10%. The virus strain was obtained from a flood-pulsed visus isolated from infectious materials via serial passages on the FE-labeled feline. Virus population homogeneity and stability of immunoblological properties were achieved by isolating the line by the 6-fold repeat dilution method. Distemper strains CPV-F-BN 10/83 and CAPM V-289 collection were boladapted to primary or secondary cultures prepared from germs and cultures of stable cell vein ladvin VOR. As the growth medium, the least esendal medium of bovine-enriched Eagle was used in which CS 267937 B1 3 virus was propagated at 36-38 ° C for 24-48 h, i.e. until a monolayer of the culture surface was formed. Thereafter, the growth medium was exchanged for maintenance minimally essential medium of Eagle's, in which the vaccine virus was produced at 36-38 ° C for 5-9 days. After the formation of distinct cytopathic changes, the viral mate-4-rial was mixed with lyophilized medium to contain at least 10 µm .ml

The distemper virus strain was vaccinated from the psinky virus of the Onderstepoort strain, attenuated by serial passages on chick embryo membranes, by its adaptation to cell cultures prepared from chicken embryos and the cell cultures of the stable tadvin VEROZ line, followed by selection of a virus line characterized by high irradiation efficiency with very good the growth properties on said species of tissue with the development of pronounced cytopathic changes accompanying captive virus multiplies.

The infectious laryngotracheitis virus strain with the designation CADV-2 H-5/85-HDCK40 by CAPH V-320 was adapted to cultures of stable kidney kidney MDCK cells. In the propagation of the infectious laryngotracheitis virus, growth medium-depleted cell lines of the MEKK line were infected with the vaccine strain with a primultiplicity of infection of 0 to 0.01 and, after adsorption of the virus on the cells, was coated with minimal oral medium according to Eagles. Infected cultures were infected at 36-38 ° C for 3-5 days. Upon formation of distinct cytopathic changes, the viral material was mixed with the lyophilization medium so as to contain at least 10%. ml

Infectious laryngotracheitis virus is a virulent strain of foreign tissue obtained in the form of lyophilisate of infectious tissue fluids in the 5th passage of cell spheres. Virus attenuation, homogeneity of the virus population, and stability of immunobiological properties were achieved in passage 35 at b.1. HDCK. To prepare the vaccine, a virus was used in passage 40 on the aforementioned tissue type attenuated and defined at our site.

The paranfluenza virus strain 2 dogs labeled CPIV-2 BN4 / 85/32 and CAPH V-321 collection was adapted to cultures of stable HDCKa malignant cell line cultures on stable FE line. As a growth medium, at least essential bovine-enriched medium according to Eagle was used, in which the virus was propagated at 36-38 ° C for 4-7 days. Virus growth at 1. The FE is accompanied by distinct cytopathic changes in the cellular raonolayer. The infectious tissue fluids obtained contain 16 to 128 hemaglucination units in 0.05 ml and the infectious titer is 10 to 107.5 TKIDcn. ml-1. Upon completion of the incubation, the viral material was mixed with the thyophilizer 3U 45-1 medium to contain a minimum of 10 µm. Direct isolate propagated in the annular cavities of 10-day-old chicken embryos from SPF eggs, was adapted to embryonic cell cultures with stably FE and HDCK cell lines. The homogeneity of the viral population and the stability of the immunoblogistic properties of the attenuated strain was achieved by the cross-border dilution method in the 32nd tissue passage.

Parvovirus virus strains of dogs CPVA-BN 80/82 CAPH V-290, distemper CDV-F-BN 10 / 83CAPH V-289, infectious laryngotracheitis CADV-2 H-5/85 HDCK40 CAPM V-320 and parainflu-enzyme 2 CPIV- 2 BN4 / 85/32 CAPM V-321 ss deposited in the Czech hunting collection of microorganisms at the Veterinary Research Institute in Brno, Hudcova 60.

Vaccine Production Procedure:

The method of producing a vaccine is based on the fact that said vaccine strains, non-harmful to all age categories of dogs and carnivorous fur animals in any application, are propagated on defined cell substrates grown on glass, plastic mass, cultivated in a stationary manner or in rotated fats. / rolleroch /. CS 267937 B1 falls on microcarriers.

Method of Cell Cell Infection with Vaccine Parvovlrosis Virus:

A cellulosic suspension of 5 to 10.10 cells. ml in growth medium is mixed with the vaccine virus at a multiplicity of infection from 0.0001 to 0.001. The virus-cell mixture is incubated at 36-38 ° C for 30-60 minutes. Thereafter, the mixture of virus and cells is diluted in growth medium, which is at least essential medium according to Eagles with a sH of 6.8 to 7.3 and a preparation of five to nine percent by weight of bovine serum with the indicated quality.

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

The cell suspension thus prepared for the preparation of the vaccine is cultured on a cello-cellular mass, by cloning method or in rotary flasks, optionally on a microcarrier, at 36-38 ° C for 4-8 days.

Infection of attached cells on the culture surface is also possible - after four to six hours of incubation of the FE cell line at 36-38 ° C. A vaccine virus vaccine pr1 multiplicity of infection of 0.0001 to 0.001 is added to the growth medium. Incubation was 36-38 ° C for 4-8 days. The propagation of the virus, as in point A and B, is accompanied by significant cytopathic changes.

Method of infection of KEB chicken embryo fibroblast cells and distemper cell lines:

Cell cultures of KEB or VĚRO are infused with canine distemper virus strains primultiplicity of infection of at least 0.005. Infecting 24 to 8 hours of cell growth with the culturing surface by replacing the growth medium with a maintenance to which an infectious inoculum is added. The maintenance medium for the individual cell type is the same as the growth medium, but does not contain serum and has a adjusted sodium bicarbonate content of 0.1 to 0.2 weight percent.

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

Also, infectious inoculum can be added to the cell suspension in the growth medium and poured into the culture vessel without prior incubation. In 24-48 hours, the growth medium is changed, without rinsing the monolayer cells under maintenance and incubation, the same conditions continue. After detection of the first cytopathic lesions (usually 72-96 h), the medium is removed at 24 hour intervals, added to the lyophilization medium and stored frozen until preparation of the combined vaccine prior to lyophilization, or the sacultivation is allowed to proceed for as long as 50 percent of the monolayer cells are affected by virus specific changes and the virus suspension is collected only once. The method of infection of MDCK cells with infectious laryngotracheitis virus:

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

Also, infectious inoculum can be added to the cell suspension in growth medium and poured into a culture vessel without prior incubation. 2a 24 to 48 hours, the growth medium is changed, without rinsing the monolayer cells with maintenance and incubation is continued under the same conditions for 3-5 days. Cultivation in both cases does not last as long as more than 80 percent of the monolayer is affected by virus-specific changes.

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

A suspension of FE or MDCK cells at a density of 5 to 10 < 10 > [mu] l < -1 > in growth medium is mixed with the vaccine virus at a multiplicity of infection of 0.001 to 0.01. The viral / cell mixture is incubated at 36-38 ° C for 30-60 minutes, then diluted in raspberry medium which, at least, represents a medium of pH 6.8-7.3 with the addition of five up to ten weight percent of bovine serum.

It is also possible to add a vaccine virus inoculum with a multiplicity of infection of 0.001 to 0.01 to the cell suspension in the growth medium and pour it immediately into the culture vessel. Incubation is continued at 36-38 ° C for 4-7 days. Virus growth on the FE line is accompanied by the development of distinct cytopathic changes in cellular monolayer. Upon completion of the culture, the infectious fluids of the individual viruses are mixed with the lyophilization medium in a ratio of 50 to 75% by weight of virus to 50 to 25% by weight of lyophilization medium and stored at -15 to -20 ° C until lyophilized. The lyophilizing medium can be added just prior to co-amplification and lyophilization of the vaccine while maintaining the same weight ratios. Infectious fluids with a content of at least 10 µC are used for the preparation of lyophilized vaccine. ml of CPVA-BN80 / 82 CAMP V-290, 104 virus particles CDV-F-BN 10/83 CAPM V-289, 103 Virus CADV-2 H-5/85 MDCK40 CAMP V-320 and 105 ΤΚΙΟ ^ θ particles of CPIV-2 virus BN4 / 85 / 32CAMP V-321, bacteriologically sterile. The virus suspensions thus prepared mixed in 0.5 parts of parvovirus + 1 part of distemper virus + 1 part of infectious la ryn-gotracheit1dy virus + 1 dl of parainfluenza 2 virus with lyophilized starting medium containing sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin but or another source of protein, dextran or gelatin / BOVARNICK, MR - MILLER, J.C. - SNYDER, J.C., The influence of certain salts, amonoacids, sugaes, and proteins on the stability of rickettsiae. J. Bacteriol., 59, 1950, 509-522) are dispensed into ampoules or vials in the form of a homogenate, lyophilized, sealed and stored at refrigerator temperature. The vaccine prepared according to the above procedure contains at least 103 µL of canine distemper virus, 102'3 ΤΚΙΰ, -θ infectious laryngotra-eheitis virus and 104 ΤΚΙΟ 3 θ ain parainfluenza 2 virus per administration dose, representing one immunization dose. 1

Sposob. cultivation:

Suspensions of FE, MDCK, VERO cells prepared by proteolytic fermentation of chicken embryonic cell (KEB) cells prepared by proteolytic ferment from 11 day chicken germs in growth media containing a defined amount of amino acids and minerals with 5 weight percent addition sera of cultures KEB and VERO and 10% by weight of sera intended for tissue cultures of the FE and MDCK cultures are cultured on glass in a sterile and suspension manner. Example 2

Method of Cell Culture Infection with Parvovirus Vaccine Virus:

Cell cultures prepared and cultured according to step 1 are infected with the vaccine virus CPVA-BN 80/82 at a multiplicity of infection of at least 0.001 non-follow-up: A 10.10 6 cell line suspension of FE line cells. ml ”1 in the growth medium is mixed with the vaccine virus at a multiplicity of infection of 0.001. The virus / cell mixture is incubated at 37 ° C for 30 minutes. Thereafter, the mixture of virus and cells is diluted in a growth medium that is at least essential to the medium of Eagle's substrate at pH 7.1 and the addition of 10 weight percent bovine serum. 5-1 bZ Inoculum of the vaccine virus with a multiplicity of infection of 0.001 is added to the cell suspension of the FE line at a density of 5.10 ml in the growth medium, and the infected suspension is immediately poured into the culture vessel. The thus prepared cell suspension for virus preparation into culture is cultured in Roux flasks at 37 ° C for 6 days. cZ Infection of adherent cells on the culture surface after six hours of culture of FE cells at 37 ° C is added to the growth medium with a vaccine virus inoculum of primitiplicity of infection of 0.001. Incubation is continued at 37 ° C for 6 days. Propagation of the virus, as well as at a / a and bZ, is accompanied by marked cytopathic changes.

The vaccine virus is produced in growth medium in FE cell cultures grown according to step 1 and infected according to 2. After large-scale changes have been made microscopically, the culture is terminated and lyophilization medium is added directly to the culture medium with the propagated virus. 50% by weight of the virus suspension and 50% by weight of the lyophilization medium. The mixture is kept frozen in culture vessels. Example 3

Cell culture infection method of 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 cultured according to step 1 are infected with at least 0.005 following methods: a / Infection of growth of single cell KEB: Growth medium is removed from cell monolayer after growth for 24 hours and cell culture is infected with inoculum added to maintenance medium to replace rastoizé médium. 7-1 bZ Cell suspension in growth medium with a density of 10.10 cells is mixed with the vaccine virus inoculum and the mixture is incubated at 37 ° C for 30 minutes, after the infection, 5 µl of the infected cell suspension is diluted in growth medium to contain 4.10 . ml cells in 1 ml and cultured at 37 ° C for 48 hours, i.e. until a single-layer cell is formed on the culture surface. Thereafter, the growth medium is exchanged for maintenance. 5-1 cZ To KEB cell suspension in growth medium and density of 4.10. ml of the vaccine virus is added. The suspension is immediately poured into a culture flask and incubated at 37 ° C for 24 hours, i.e. until a single layer of cells is formed. This is followed by a replacement of the growth medium with the maintenance medium.

The vaccine virus is produced in a maintenance medium that is as basic as its growth medium at least essential to Eagle's, except that it does not contain serum and the concentration of soda bicarbonate is up to 0.30 weight percent. The virus from the cell cultures grown according to point 1 and infected according to point 3 is obtained by the following methods: The maintenance medium is left unchanged on the cultures until extensive cytopathic changes are established determined microscopically. The lyophilization medium is added directly to the virus culture medium into the virus maintenance medium in a proportion of 2 parts of the virus suspension and one part of the lyophilization medium. The mixture is further stored in culture vessels in a crushed state. b) After the appearance of the first signs of specific cytopathic changes, the maintenance medium is discharged and the same amount of maintenance medium is poured into the culture vessels. The procedure is repeated at 24 hour intervals until a large-scale cytopathic effect is produced, whereby the final treatment is performed with a / a. The lyophilization medium is added to the viral suspension obtained by replacing the holding medium repeatedly and then frozen. Example 4

Method of Cell Culture Infection with Infectious Laryngotracheitis Virus:

The cell cultures prepared and cultured at point 1 are infected with the vaccine virus CADV-2 H-5/85 MDCK40 at a multiplicity of infection of at least 0.01 in the following ways: a) Infection of an increased single cell MDCK: by growth at 37 ° C for one hour, the growth medium which represents at least the essential medium of the Eagle is removed and the cell culture is infected by adsorption of the virus to the cells for up to 60 minutes at 37 ° C. Thereafter, a maintenance medium is added which is the same as the growth medium but does not contain serum. 5-1 b / To cell suspension in growth medium with density of 4.10. ml of vaccine virus is added. The suspension is immediately poured into a culture flask and incubated at 37 ° C for 48 hours, i.e. until single cell formation. Thereafter, the growth medium is changed to maintenance and incubation is continued at 37 ° C for 2 days. Virus multiplication in both cases is accompanied by significant cytopathic changes in the cell monolayer. The vaccine virus is produced in growth medium on MDCK cell cultures grown at point 1. and infected with step 4. After extensive cytopathic changes have been made, the culture is terminated and lyophilization is added directly to the culture medium with the enriched virus. medium in a ratio of 2 parts of the suspension and one part of the lyophilization medium. The mixture is further stored in culture vessels in the frozen state. Example 5

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

Cell cultures prepared and cultured at 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: 6 "* 1 a / 10.10 ml FE cell line suspension is mixed The virus / cell mixture is incubated at 37 ° C for 30 minutes, after which the virus / cell mixture is diluted in growth medium, which is at least essential to Eagle's medium and 5% by weight of serum serum. 5-1 b / Inoculum of a vaccine virus of a multiplicity of infection of 0.01 is added to a suspension of 5.10 ml MDCK cells in growth medium, and the infected suspension is immediately poured into a culture vessel. is cultured at 37 ° C for 5 days, and the proliferation of virus on FE cells is accompanied by distinct cytopathic changes in cellular monolayer. e 10 TKID θ.f.nu m / »t 1 n3

The shedding virus is produced in growth medium in cell cultures of FE and MDCK-treated podes of point 1. and infected according to point 5. After the culture is completed, lyophilization medium is added directly to the culture medium with the multiplication virus in the ratio of 2 diets of virus suspension and one diet of lyophilization medium. The garlic is further stored in frozen culture vessels. Example 6

Preparing the combined vaccine:

For the preparation of the thyophilized vaccine, infectious fluids containing mini- are used. ml-1 particles of CPVA-BN80 / 82, 10 ^ TKID virus. of CDV-3 virus particles in 5 -1 TKIDjq of the CADV-2 H-5/85 MDCK4O virus and 10 TKID50 particles. ml of CPIV-2 BN4 / 85/32 virus particles; bacteriologically sterile. The virus suspensions thus prepared are mixed in a ratio of 0.5 parvovirus diet and 1 part distemper virus and 1 part virus infection taryngotracheitis and 1 part para influenza virus 2. The homogenized virus material with the desired content of infectious units is adjusted, lyophilized, air-sealed and stored at refrigerator temperature.

The vaccine prepared according to the above procedure contains at least 10 3 TKI / µg parvovirus dogs, 10 5/10 5 psi virus, 10 3 TKID infectious laryngotracheitis virus and 10 5/5 5 parainfluenza 2 virus per dose.

Prove the effectiveness of the combination vaccine:

I compare ! Figures 1 and 2. The figures shown graphically represent the mean haemagglutination-inhibitory / HX / antibodies against parvovirus, infectious taryngotracheitis virus and parainfluenza 2 virus, and virus-neutralizing antibodies / VN7 against distemper virus. From the present results it is clear that after vaccination with the polyvalent vaccine, the specific antibody level was found to be at the same level or higher than after the mono challenge. resp. bivalent vaccines. It has not been anticipated and yet been found to induce an immune response. From the results of Table 1, neither vaccinated nor non-reactive non-reactive infection of the dog's parvovirus field strain with a noticeable impairment of health can be seen. These findings are consistent with those of McAdaragh, J.P. - Eustis, S.T. - Nelson, D. Ti - et al., Experimente infection of conventlonal doge with vanine parvovirs. A. J. Vet, Res., Vol 43 No.4, 1982 p.693-696; Pollock, R.V.H. - Carmichael, L.E., Dog respon-se inactivated eanlne parvovirus and feline panleukopenia virus vaccines. Corne (Vol.1982, 72, 16-35 and i. That infection of susceptible animals with field parvirus virus strain strains will remain undetectable). All vaccinated animals remained healthy after infection with virulent canine virus, & The result of the vaccinated animals being vaccinated with the parainfluenza 2 virus is characteristic of the Infection-Infant Animals with the virus whose pathogenicity is determined by the involvement of other infectious agents in the disease.

In clamping protection of vaccinated animals for infection with infectious laryngotra-cheitis virus, both controls responded to anterior but apparent airway disease.

Likewise, all vaccinated animals infected with infectious hepatitis virus have remained clinically healthy, showing close antigenic relationships of both viruses and the cross-protection of infectious laryngotracheitis virus-infected animals against vi -

Claims (5)

P R E D M E T OF THE INVENTION 1. Live attenuated parvovirus vaccine containing strain CPVA-BN 80/82 V-290 in an amount of 10 ³ ΤΚΙΟ ^ θ, canine distemper containing strain CDV-F-BN 10/83 CAPM V-289 in an amount of 10 ³ TKIDjq, infectious laryngotracheitis, infectious hepatitis and parainfluenza 2, characterized in that it contains in 2 ml a strain of canine parvovirus CPVA-BN 80/82 CAPM V-290 in an amount of at least 10 ³ ΤΚΙ05θ, a strain of canine distemper CDV-F-BN 10/83 CAPM v- 289 in an amount of at least 10 ³ TKID-n, infectious laryngotracheitis virus strain CADV-2 H-5/85 2 5 MDCK 40 CAPM V-320 in an amount of at least 10 'ΤΚΙΟ ^ θ and a parainfluenza strain 2 CPIV-2 BN4 / 85/32 CAPM V-321 in an amount of at least ΙΟ ⁴ ' ^ TKID ₅₀ virus particles and lyophilization medium. 2. The live attenuated vaccine according to item 1, characterized in that it contains a lyophilization medium in an amount of 25 to 50% by weight, based on the weight of the whole vaccine. · 3. The live attenuated vaccine according to items 1 and 2, characterized in that the lyophilization medium contains sucrose, phosphate buffer, sodium or potassium glutamate, bovine albumin or another source of protein and gelatin or dextran. 4. A process for the production of a vaccine according to item 1 by culturing parvovirus CPVA-BN 80/82 CAPM V-290 and canine distemper virus CDV-F BN 10/83 CAPM V-289 in a known manner, characterized in that the cultivars of parvovirus and canine distemper virus are mixed. with CADV-2 H 5/85 MDCK40 CAPM V-320 infectious laryngotrachitis virus culture obtained by propagation on a stable MDCK cell line after 30 to 60 minutes of infection with a 24 to 48 hour monolayer in minimal essential medium according to Eagle at a temperature of 36 to 38 ° C for 3 to 6 days, until the formation of cytopathic changes, mixed with lyophilization medium to a content of at least 10 ΤΚΙΒ ^ θ. ml and a CPIV-2 BN4 / 85/32 CApM V-321 2 dog parainfluenza virus culture obtained by multiplication on stable FE and MDCK lines in Eagle's minimal essential medium and pH 6.8 to 7.3 enriched with bovine serum at a temperature of 36 to 38 ° C for 4 to 8 days supplemented with lyophilisate medium for at least 10 ^ τκίΡ ^ θ. . ml ~ ^ and this virus mixture is lyophilized 5. The method for producing a vaccine according to item 4, characterized in that the lyophilization medium is added to the virus suspensions immediately after their acquisition and the mixture is stored in a frozen state until lyophilization or is lyophilized immediately.