RU2517733C1 - Inactivated emulsion vaccine associated against adenoviral, herpes virus infection, parainfluenza-3 and viral diarrhea - mucosal disease of cattle - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: proposed vaccine contains concentrated antigenic materials from the strain "Adeno III WBR-1-DEP" of the third serotype of subgroup I, reproduced in a passaged culture of cow embryo kidney cells, from strain "Adeno IV Weybridge CT2-DEP" of 4th serotype of subgroup II, reproduced in a passaged culture of calf kidney cells Taurus-1, from the strain "TKA-VIEV-B2-DEP" of herpesvirus of type I, reproduced in a passaged culture of cow embryo kidney cells, from the strain "SF-4-DEP" of virus of parainfluenza-3, reproduced in a passaged culture of cow embryon lung cells from the strain "VK-1-DEP" of viral diarrhoea virus - mucosal disease of cattle, reproduced in a passaged culture of cow embryo kidney cells. The concentrated and avirulent antigenic materials, formalin inactivated, are combined with an adjuvant in an effective ratio.

EFFECT: vaccine induces the high level of antibodies against the said infections and provides reliable protection of newborn calves, young stock of rearing and fattening period.

3 cl, 7 tbl, 9 ex

Description

The vaccine associated against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - a disease of the mucous membranes of cattle inactivated emulsion.

The invention relates to the field of veterinary virology and biotechnology, in particular against viral respiratory and intestinal infections of calves and the resulting violations of the reproductive function of adult livestock of cattle (cattle).

Respiratory and gastrointestinal diseases of young cattle remain one of the most difficult problems of infectious diseases of animals in many countries of the world, including Russia. In etiopathogenesis, parainfluenza-3 virus (PG-3), herpesvirus type I, viral diarrhea virus - mucosal diseases (VD-BS), respiratory syncytial virus, parvo- and reoviruses, as well as rota-, coronaviruses, are detected with greater consistency pathogenic bacteria, chlamydia and mycoplasmas. These diseases, occurring mainly in the form of a mixed infection, are widespread in modern large dairy complexes and the loss of young animals in them reaches 35-50% (Gaffarov Kh.Z. Infectious diseases of cattle of chlamydial, mycoplasma and viral etiology, the development of diagnostic and therapeutic prophylactic Preparations // Abstract of thesis ... Doc. Vet. Sciences. - Kazan. - 1986. - 39 p.).

Reliably established participation in this pathology of adenoviruses of cattle I and II subgroups, which, according to some scientists, is one of the four most likely causative agents of respiratory diseases of cattle and in terms of prevalence are in second place after parainfluenza-3. In addition, they are recognized as the etiological agent of neoplastic processes and asymptomatic infections in humans and animals and therefore have much greater epizootic and socio-economic significance than previously thought (Gunenkov V.V., Syurin V.N., Vertinskaya K.K. Adenovirus cattle infection / Little-known viral infections of cattle. - Moscow, 1972.- 47 pp .; Mocsari E. et al., 1974. - // Acta Vet. Acad. Sci. Hung. - 1974. -24. - No. 1-2. - P.39-43; Mohanty SB Comparative studies of Bovine Adenovims // Amer. J. Vet. Res. - 1971. - V.32. - No. 12. - P.1899-1905).

Regarding the pathogenicity spectrum of cattle adenoviruses in vivo, it should be noted that adenovirus infection in calves from 2 weeks to 2-4 months of age is characterized by an acute course, bronchopneumonia, enteritis and conjunctivitis develop due to inflammatory processes of the mucous membranes of the respiratory tract and digestive tract. In adult cattle, the infection proceeds latently, accompanied by prolonged virus carrier. Isolation of adenovirus from the fetus indicates the possibility of transplantation of the pathogen.

Adenoviruses of cattle are represented by six serotypes that show differences in antigenic structure: adenoviruses of the 1st, 2nd and 3rd serotypes react with each other, but do not interact with serums of the 4th, 5th and 6th serotypes, which caused them to be divided into two antigenic subgroups ( Bartha A. Proposal for sub grouping of bovine adenoviruses // Acta vet. Acad. Sci. Hung. - 1969. - 19. - No. 3. - P.313-321; Rondhuis PR Bovine Adenoviruses. A Comparative Serological and Experimental Study / / Drukkerij-Uitgeverij G. van Dijk NV Breukelen. - 1970.- P.218).

Adenovirus infection of cattle was first established in the USA in 1959 (Klein H., Maitzman W., Levine AJ Structure function relationships of the adenovirus DNA-binding protein // J. Biol. Chem. - 1979. - V.254. - P .11051-11060) then in England (Darbyshire JH Bovine Adenoviruses // J. Amer. Vet. Med. Assoc. - 1968. - V.152. - P.786-792), Hungary (Aldasy P., Csontos L. , Bartha A. Pneumoenteritis in Caives Caused by Adenoviruses // Acta Vet. Acad. Sci. Hung. - 1965. - 15. - P.175-176), Holland (Rondhuis PR Anew Bovine Adenovirus // Arch. Ges. Virusforsch. - 1968. - 25. - P.235-236), Japan (Inaba Y., Tanaka Y., Sato Y., Ito K., Ito H., Matumoto M. Bovine adenovirus. II A. Serotype. Fukuroi, Recoverend from Japanene Cattle // Jap.J. MicrobioL - 1968. - 12. - P.219-229). A little later, this disease was reported in Italy, Poland, Germany, Canada, Australia, Bulgaria and other countries.

The widespread prevalence of adenovirus infection is evidenced by frequent cases of isolation in infectious respiratory diseases of calves, as well as a high percentage of seropositivity in adult livestock. So, in the neutralization reaction (PH) adenoviral antibodies were detected in 66.8% (Sawhney DS Vadehre DV, Baker RC // Poultry Sci. - 1973. - 52. - No. 2. - P.465-473), 60% ( Adamesteanu L., Adamesteanu C. // Rev. zootehn.si med. Vet. - 1973. - V.23. - No. 9.- P.51-56) from the entire surveyed population and in 73.41% of animals in those groups in which the clinical symptoms of respiratory and intestinal diseases were observed during the study period.

In most cases, the etiology of respiratory diseases of calves was able to establish the participation of not only adenoviruses, but also others, such as PG-3 viruses, infectious rhinotracheitis and viral diarrhea and chlamydia (Sawhney D.S. et al., 1973). However, Mocsari, E. et al. believes that adenoviruses play a more important role in the etiology of respiratory and intestinal diseases of calves than the virus of infectious rhinotracheitis and chlamydia.

Experimental infection of calves at the age of 20-25 days (Augustinavichus B.V. / in the book. Scientific and technical information. - Lithuanian scientific. - researched by the Veterinary Institute. - 1966. - P.3-5), 8 weeks (Pons WA, Cucullu AF, Franz AO, Lee Louise S., Goldblatt Leo A. // J. Assoc. Offic. Anal. Chem. - 1973. - 56. - No. 4. - P.803-807), 6-12 weeks and even 8-month-old animals with cattle adenovirus caused a clinically expressed disease. Moreover, viruses of the 3rd and 4th serotypes cause a more severe disease in calves than viruses of the 1st and 2nd serotypes, which cause a mild reaction or do not cause it at all in calves lacking colostrum or fed on a normal diet ( Gillespie J. // J. Amer. Vet. Med. Assoc. - 1973. - 163. -No. 7 - P.901).

The first reports of the troubles of farms regarding adenovirus infection in the USSR appeared in the late 60s (Alieva N.A., Ganiev M.K., Dreizin R.S., Alieva R.A., Saryev G.A. Adenovirus infection calves // Transactions of AzNIVI. - 1967. - T. XXI; Gunenkov VV, Khalenev GA, Syurin VN Viral and chlamydial respiratory and intestinal infections of cattle // Animal husbandry and veterinary medicine. Problems of veterinary medicine, a series of “Results of science and technology.” - Moscow, 1975. - T. 8. - P.87-95).

In the period 1998-2003. the highest incidence of calves with adenovirus infection was recorded in the farms of the Moscow region. Of the 108 households examined, in all cases, the circulation of PG-3 viruses (100%), RTI (63.8%), VD-BS virus (38.8%), adenovirus (36.2%), and respiratory syncytial virus ( 4.6%). The data obtained indicate a significant role of adenovirus infection in the etiological structure of respiratory and intestinal pathology of cattle in the farms of the Russian Federation (Lobova T.P. Improvement of laboratory diagnosis of adenovirus infection in cattle // Abstract of thesis ... Candidate of Biological Sciences. - FGOU VPO MGAVMiB. - Moscow. - 2006. - 19 p.).

In order to determine the etiology of mixed respiratory diseases in various regions of the Middle Volga and Urals, in 2001-2004, 1665 blood serum samples of various sex and age groups of cattle from 76 dysfunctional households were studied for the presence of antibodies to the viruses PG-3, IRT, VD-BS and adenovirus infection. The research results showed that the largest percentage of positive samples was detected for PG-3 virus - 76.6% and for IRT virus - 35%, and for adenovirus - 18.4%, VD-BS - 17.9% (Akhmetsafin R.G. Improvement of diagnostic tools and specific prophylaxis of mixed respiratory infections of calves // Abstract of thesis ... Candidate of Veterinary Sciences. - Kazan. - 2005. - 19 p.).

The measures for the prevention and control of adenovirus infection in the Russian Federation (RF), which are traditionally used at present, do not solve the problem due to insufficient disclosure of the etiology of this symptom complex in each individual case, the unexplored fundamental features of its manifestation in modern large complexes, and the absence of a systemic seroimmunological monitoring in relation to the main causative agents of respiratory and intestinal pathology, which creates difficulties in determining the leading role of a particular inf ktsionnogo agent in mixed infections, and, finally, the veterinary service of the country at the present time is not really a highly effective methods and means of its diagnostics and any specific biologics for its prevention.

Noteworthy is the statement of a number of scientists that the achievements in the immunoprophylaxis of adenovirus infection are more modest than with other viral diseases of the calves of the respiratory intestinal complex (Edwards GS, Wogan GN // Biochim. Biophys. Acta. - 1970. - V.224 .- 597 p .; Darbyshire JH Bovine Adenoviruses // J. Amer.Vet. Med. Assoc. - 1968. - V.152. - P.786-792). This is due to a number of difficulties, obviously, primarily due to the many types of pathogen and latent course in adult animals (Syurin N.V., Samuilenko A.Ya. Adenovirus infection in cattle // Viral animal diseases. - Moscow. - VNITIBP , 1998. - S.795-801).

In our country, in the 70-80s, the search for effective methods of immunoprophylaxis of this infection was carried out in two directions: by creating passive protection with the help of multivalent hyperimmune serums and by forming active immunity with the help of vaccines.

For specific prophylaxis of viral respiratory and intestinal infections of young animals and uterine livestock of cattle, live and inactivated vaccines are used in the Russian Federation.

Known virus vaccine "TKA-VIEV" against infectious cattle rhinotracheitis, which is used in cases of this infection, immunizes all animals located in the epizootic focus, as well as in meat-producing farms with inpatient distress - all animals suspected of illness and suspected of infection.

The well-known "Vaccine inactivated against adenovirus infection in cattle", which was developed on the basis of the field strains "CLB" and "Russia 18/81", belonging to the I and II subgroups, respectively (Syurin V.N., Belousova R.V., Kolenkova L.M., Litvinov T.I., Lobova T.P. Vaccine against adenovirus infection in cattle / Author's certificate No. 44486434 / 30-13 / 138050 of 08.29.89). The vaccine was manufactured by the Pokrovsky plant of biological preparations in the amount of 125 liters. The biological product was used in the farms of the Krasnodar Territory, the Kyrgyz SSR and the Belarusian SSR. The tested vaccine was recognized as harmless and immunogenic for deep-shelled cows and calves aged 10-15 days.

However, the mixed nature of the manifestation of viral respiratory and intestinal infections and the peculiarities of the circulation of these viruses suggests greater efficacy of associated vaccines and the use of monovaccines does not provide positive dynamics of diseases (Vazir Ya., Belousova R.V., Ustinova GI. Tests of a bivalent vaccine against adenovirus infection and cattle viral diarrhea in rabbits using immunomodulators // Veterinary Pathology. - 2007. - No. 3 (22). - P.199-202; Matveeva I. N. Industrial technologies for the manufacture of of components of mono- and complex diagnostics of infectious diseases of animals // Abstract of thesis ... doc. Biol. Sciences. - Schelkovo. - 2008. - 53 p.).

A known vaccine against parainfluenza-3 and infectious rhinotracheitis of cattle is dry, culture associated, containing the lyophilized vaccine virus PG-3 (strain "PTK-45/86") and the vaccine virus of infectious rhinotracheitis (strain "TKA-VIEV-B2"), calf kidney grown in cell culture; and a protective drying environment. The vaccine is administered subcutaneously to calves aged 1-6 months twice, 7 months and older - once.

Known vaccine associated against parainfluenza-3, infectious rhinotracheitis and cattle chlamydia inactivated emulsion. The vaccine contains inactivated PG-3 antigens (strain "PTK-45/86"), infectious rhinotracheitis (strain "TKA-VIEV-B2") and the causative agent of chlamydial abortion of cows (strain "250"), emulsified in an oil adjuvant. Calves are immunized from 10-20 days of age twice, at a dose of 1 cm ³ , the vaccine is administered intramuscularly; calves older than 6 months, cows and bulls in a dose of 2 cm ³ .

A well-known inactivated combined vaccine against viral diarrhea, rota-, coronavirus diseases and calf Escherichiosis (Kombovak-K), which is a comprehensive drug for the prevention of 3 viral and bacterial diseases of cattle, characterized by gastrointestinal syndrome. The vaccine is made from inactivated strains of three viruses: rota-, corona- and viral diarrhea, Escherichia protective antigens: somatic 09, 078, 0115; capsular polysaccharide K80, K30; adhesive antigens K99, F-41; thermostable and thermolabile inactivated enterotoxins.

Known vaccine inactivated combined against infectious rhinotracheitis, parainfluenza-3, viral diarrhea and calf pasteurellosis (Combovac-R). The vaccine is made from inactivated strains of the viruses of infectious rhinotracheitis, parainfluenza-3, viral diarrhea and two types of pasteurella: Pasteurella multocida (serovars A, B and D) Pasteurella haemolitica.

A known vaccine is combined against infectious rhinotracheitis, parainfluenza-3, viral diarrhea, respiratory syncytial, rota- and coronavirus diseases of calves (Combovac), which is a composition of 6 virus strains inactivated with formalin and adsorbed on a 6% aluminum hydroxide gel (GOA ) and saponin, intended for the immunization of calves, heifers and cows, in farms that are unsuccessful for gastrointestinal and respiratory diseases.

Known vaccine "Trivak" (VIEV) for the prevention of infectious rhinotracheitis, parainfluenza-3 and viral diarrhea - diseases of the mucous membranes of cattle (Yurov K.P., Vecherkin A.S., Shulyak A.F., Zhidkov S.A. / Patent application 96123539/13 of 12/11/1996). The vaccine contains the antigen from the attenuated virus of infectious rhinotracheitis (strain "TKA-VIEV-B2"), the antigen from the attenuated virus VD-BS (strain "VK-1), the attenuated parainfluenza virus-3 (strain" PTK-45/86 ") and a protective environment based on gelatin and sucrose. The vaccine is administered intradermally, twice, to calves at a dose of 0.2 cm ³ , for adult animals 0.4 cm ³ .

A common disadvantage of the above known vaccines is the lack of antigen of cattle adenoviruses of the 1st and 11th subgroups in their composition, which significantly reduces their effectiveness.

From the above information it follows that at present, domestic analogues of vaccines intended for specific prophylaxis of viral respiratory infections of calves and the pathologies of uterine reproductive organs caused by them, made on the basis of antigens of cattle adenoviruses I and II subgroups, herpesvirus type I, parainfluenza 3 and viral diarrhea - diseases of the mucous membranes of cattle does not exist.

Literature data indicate that in many countries mono-, bi- and polyvalent vaccines are produced against adenovirus infection in cattle, which differ both in the set of adenoviral components, their combination with other viruses, as well as in the methods of their manufacture and their use ( Gole J. // J. Amer. Vet. Med. Assoc. - 1973. - No. 7. - P. 836-839; Phillipp JIH, Sands JH The Isolation of Bovine Adenovirus Serotypes 4 and 7 in Britain // Res. Vet ScL - 1972. - 13. - P.386-387; Tribe GW, Kerry JB Combined bovine parainfluenza and adenovirus vaccine // Vet. Rec. - 1969. - V.84. - P.299-303). A number of authors have shown the promise and feasibility of creating associated vaccines with the mandatory inclusion of two subgroups of adenoviruses in them: both the 1st, 3rd, and 4th and 5th serotypes. Moreover, it was found to be more immunogenic with an adjuvant from an emulsion of tween arlazel-bayol than with an adjuvant of 10-20% aluminum hydroxide (Bürki F., 1973; Schipper E., Nicolet J., König H., Steck F. Virus bedingte Respirations-Krankheiten in Kälber und Ridermastbetreiben // Schweiz. Arch. Tierheilkunde. - 1972. - H.114. - S.334-361). Active immunization of calves was carried out according to various programs depending on the epizootic situation. Moreover, both a milk herd and young animals of different ages are vaccinated.

1. In Hungary, for the first time, a live adenoviral vaccine was used, prepared from type 4 TNT / 62 strain, which passed 72 passages in a bull testicle cell culture (Bartha A. Immunization experiments on calves with type 4 bovine adenovirus // Acta vet. Acad. Sci Hung. - 1967. - V.-17. - P.209-216).

2. In Bulgaria, an inactivated vaccine with adjuvant was designed based on the antigens of the infectious rhinotracheitis virus, PG-3 virus, type 1 adenovirus (Bovine-10 strain) and type 3 adenovirus (WBR-1 strain). 16 series of vaccines were made and tested on 170 calves aged 45-180 days. Animals were vaccinated subcutaneously, twice with a 23-day interval at a dose of 7 ml per calf, as well as 54 pregnant cows. In calves, specific antibodies were first detected on the 14th day after vaccination, and maximum antibody titers were reached 28 days after revaccination (Haralambiev H., Draganov, M., Zwetkov P. Hyperimmunserum Against Myxovirus Parainfluenza-3 and Bovine Adenovirus in Prophylaxis of Respiratory Disease in Calves // Arch. Exptl. Vet. Med. - 1972.- V.26. - P.273-276).

3. In the UK, the Pneumovac Plus (C-Vet) vaccine has been used to specifically prevent respiratory viral infections. The vaccine includes parainfluenza-3 antigens (strain SF-4), serotype 3 adenovirus (strain "WBR-1"), type 1 reovirus (strain "Lang strain"), and viral diarrhea virus - mucous membrane disease (strain "Nadi" ), bovine infectious rhinotracheitis virus and aluminum hydroxide gel. The drug is administered intramuscularly to young cattle, starting at 14 days old, re-vaccinated at 5 and 10 weeks of age (Morzaria SP, Richards MS, Harkness JW, Maund BA A field trial with a multicomponent inactivated respiratory viral vaccine // The Veterinary Record. - 1979. - V.11. - No. 3. - P.410-414).

The well-known "Vaccine against enzootic bronchopneumonia for cattle inactivated" containing inactivated antigens of parainfluenza virus-3 (strain "SF-4"), cattle adenovirus 1st (strain "Bovine 10"), 3rd (strain " WBR-1 ") serotypes of the first subgroup and the 5th (strain" Munich 293/3 ") serotype of the second subgroup, reovirus of the 1st (strain" Lang ") and 3rd (strain" Dearing ") serotypes reproduced in the corresponding sensitive biological systems and subjected to inactivation with formalin and GOA adjuvant in an effective ratio (Animal He alth Products / Vaccine against enzootic bronchopneumonia, inactivated, for cattle ("Bovigrip") Aqueous suspension for subcutaneous injection. / Hoechst Veterinar GmbH. - 1992. - P.37-39)

Therefore, the vaccine contains antigens of adenoviruses of the 1st and 2nd subgroups, serogroup reoviruses I and III, as well as parainfluenza-3 virus. The vaccine is administered subcutaneously at a dose of 5 cm ³ , calves are immunized twice with an interval from 4 weeks to 6 weeks of age.

Known "Vaccine against enzootic bronchopneumonia and infectious rhinotracheitis in cattle, inactivated for cattle" containing inactivated antigens of parainfluenza-3 (strain "SF-4"), cattle adenovirus 1st (strain "Bovine 10") and 3- go (strain "WBR-1") serotypes, infectious rhinotracheitis virus (strain "Calw"), reovirus 1st (strain "Lang") and 3rd (strain "Dearing") serotypes reproduced in sensitive biological systems and subjected to inactivation with formalin and GOA adjuvant in an effective ratio. The vaccine called Bovigrip plus is produced by the world's largest German company, Hoecht Veterinar GmbH, successfully used in many countries of Western Europe (Animal Health Products / Vaccine against enzootic bronchopneumonia and infectious bovine rhinotracheitis (IBR), inactivated, for cattle. Aqueous suspension for subcutaneous injection. / Hoechst Veterinar GmbH, 1992. - P.41-43).

1. Closest to the proposed invention in terms of essential features is "Inactivated concentrated multivalent vaccine against infectious rhinotracheitis, parainfluenza-3, adenovirus infection and viral diarrhea of cattle" (Belousova RV, Tretyakova IV, Lobova T. P., Tiganova N.V., Kish L.K., Kuznetsov D.P. / Application for invention No.201529545 / 10 dated 12.24.2010), including separate cultivation of infectious rhinotracheitis viruses, parainfluenza-3, viral diarrhea, collection of culture liquids and getting an tigens, characterized in that the strain Bovine-10 of the adenovirus of the 1st subgroup is additionally introduced, which is cultured in a PT-80 cell culture, a virus with an infectious titer of 6-7 lg TCD ₅₀ / ml is obtained, then the resulting viruses are inactivated with formalin up to 0.2-0.3% of the final concentration at a temperature of 37-38 ° C for 72 hours at a pH of 7.2-7.4 and concentrate 5 times PEG-6000 in a final concentration of 9-11% in solution 0, 5-0.6 M sodium chloride, then the resulting antigens are mixed in equal volumes 1: 1: 1, add the official adjuvant - aluminum hydroxide to 2-3% of the final concentration and get the vaccine.

An analysis of the above information shows that the problem of specific prophylaxis of adenovirus infection in cattle has not yet been resolved. The creation of associated vaccines against cattle adenovirus infection, taking into account the prevailing etiological viral agents detected by seroimmunological monitoring, could be promising and economically feasible.

The prototype - the initial sample when creating a new vaccine, the proposed invention was "Inactivated bivalent vaccine against viral diarrhea and adenovirus infection in cattle and a method for the prevention of viral diarrhea and adenovirus infection in cattle" (Belousova R.V., Lobova T.P., Tretyakova I.V., Wazir J. / Application for invention No. 2008103716/13 of 06.02.2008). From the description of the invention to the patent it follows that the vaccine includes antigens of the first serotype adenovirus (Bovine-10 strain), Oregon strain of viral diarrhea virus, formalin inactivated, 5 times concentrated with polyethylene glycol-6000 (PEG-6000) and deposited on aluminum hydroxide in an effective ratio. The proposed vaccine is administered to animals intramuscularly with an interval of 13-16 days at a dose of 1 cm ³ .

The vaccine prototype has significant disadvantages:

- it does not protect animals from herpesvirus infection type I and parainfluenza-3;

- it does not protect animals from adenovirus infection caused by adenoviruses of the second subgroup, which are antigenically different from each other and can cause various pathological processes in the animal organism, despite the use of vaccines containing adenovirus antigens of other subgroups;

- a five-fold concentration of the antigenic viral components of the prototype vaccine may not be sufficient to create intense immunity in vaccinated animals;

- aluminum hydroxide is a relatively weak adjuvant, unable to provide long-term release of antigen from the vaccine injection site, the oil adjuvant used by us gives a water-oil emulsion that releases antigens for a longer time than aluminum hydroxide and similar adsorbents, it explains the more powerful immune stimulation after the first immunization of animals with oil vaccines.

In this regard, the problem of creating a vaccine associated, inactivated, concentrated and emulsion against adenovirus infection, including antigens of I and II subgroups, herpesvirus infection type I, parainfluenza-3 and viral diarrhea-disease of the mucous membranes of cattle with high antigenic and immunogenic activity and harmlessness, continues to remain relevant and is the main direction of research to improve it.

The need for the development and production of this associated vaccine is due to the variety of clinical manifestations of adenovirus infection in calves of various age groups, its widespread prevalence in the livestock complexes of the Russian Federation, and the course of the disease, mainly in the form of mixed infections, most often associated with herpesvirus type I, parainfluenza-3 and viral diarrhea - diseases of the mucous membranes.

The results of our studies showed that 65.0% of the tested samples were found to be seropositive for PG-3 virus, 48.3% for adenovirus, 41.0% for type I herpesvirus, and 39.4% for VD-BS. The results obtained indicate the fact of the circulation of these pathogens among various age groups of cattle (Table 1).

It is known that under natural conditions, the simultaneous or sequential effect on the organism of animals of several pathogens, interdependent action of each other, exacerbates and worsens the course of the infectious process. This is confirmed by the research results of both foreign and domestic scientists.

The objective of the present invention is to obtain a highly immunogenic associated inactivated emulsion vaccine against adenovirus, herpes virus type I, parainfluenza-3 and viral diarrhea - a disease of the mucous membranes of cattle based on production strains with high biological, antigenic and immunogenic activity in their native form and after inactivation, suitable for the manufacture of an associated emulsion preparation that creates effective protection for the fetus, newborn calves, young animals the period of rearing and fattening, as well as pregnant cows and heifers from the above respiratory-intestinal viral infections and the pathologies of the reproductive organs of the uterine population caused by them, which are widespread and constantly circulating in cattle-breeding enterprises of the Russian Federation.

The technical result from the use of the invention is to expand the arsenal of effective, harmless and areactogenic associated vaccines against viral infections of cattle.

The indicated technical result was achieved by the creation of a vaccine with high immunogenic activity, significantly differing in the complex of viral antigenic components from the existing means of prophylaxis currently being produced in the Russian Federation, and with a wide range of specific protection for the fetus, newborn calves, young animals during the growing and fattening period, pregnant cows and heifers from infection with epizootic (field) strains of adenoviruses I and II subgroups, herpesvirus type I, parainfluenza-3 and VD-BS virus circulating in the territory Russian Federation.

The proposed vaccine contains an active biological substance in the form of avirulent purified and concentrated antigens of the Adeno III WBR-1 strains of the 3rd serotype of the first subgroup and the Adeno IV Wey bridge CT2 of the 4th serotype of the second subgroup, the TKA-VIEV-B2 strain herpesvirus type I, strain SF-4 of parainfluenza virus 3, strain VK-1 of viral diarrhea virus of mucous membrane of cattle obtained in mammalian cell cultures and target additives in the form of an oil adjuvant in an effective ratio. The first two adenovirus strains were obtained from the Central Veterinary Laboratory of the UK Ministry of Agriculture, and their immunobiological properties were studied. The strains of adenoviruses Adeno III WBR-1, Adeno IV Weybridge CT2 and parainfluenza virus 3 SF-4 were deposited in the laboratory for the storage and study of strains of especially dangerous animal diseases (Museum of strains) used in veterinary medicine and animal husbandry of the Federal State of the budgetary institution “Federal Center for Toxicological, Radiation and Biological Safety of Animals (FSBI“ FTsTRB-VNIVI ”), the strain“ TKA-VIEV-B2 ”of type I herpesvirus and“ VK-1 ”of the virus of diarrhea - cattle mucous membrane diseases were deposited in the collection of FSBI "VGNKI".

The essence of the invention is as follows: "Associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - inactivated emulsion mucous membrane diseases of cattle" contains concentrated antigens of cattle adenoviruses I and II subgroups, herpesvirus type I, VD-B virus cattle emulsified in an oil adjuvant, with the following ratio of components, vol.% 1: 1: 1: 1: 1 (inactivated by formalin to a final concentration of 0.2-0.3%).

Avirulent, purified and concentrated culture suspension of cattle adenovirus subgroup I strain Adeno III WBR1-DEPT) obtained in a transplanted culture of cow embryonic kidney cells (MDBK line) with an infectious titer of at least 6.25 lg TCD ₅₀ / ml 10.0-11.0 Avirulent, purified and concentrated culture suspension of cattle adenovirus II subgroup strain Adeno IV Weybridge CT2-DEP obtained in a transplanted culture of Taurus-1 (T-1) calf kidney cells with an infectious titer of at least 6.5 lg TCD ₅₀ / ml 10.0-11.0 Avirulent, purified and concentrated culture suspension of bovine herpesvirus type I strain TKA-VIEV-B2-DEP obtained in a transplantable MDBK cell culture with an infectious titer of at least 7.5 lg TCD ₅₀ / ml 10.0-11.0 Avirulent, purified and concentrated culture suspension of bovine parainfluenza virus-3 obtained in the transplantable culture of lung cells of a cow embryo (LEC) strain SF-4-DEPT with an infectious titer of at least 6.5 lg TCD ₅₀ / ml and activity in RGA not less than 6 log ₂ (1:64) 10.0-11.0 Avirulent, purified and concentrated culture suspension of VD-BS virus in cattle strain "VK-1-DEPT" obtained in transplantable cell culture "MDBK" with an infectious titer of at least 6.5 lg TCD ₅₀ / ml 10.0-11.0 Targeted Adjuvant up to 100.0

The strains of viruses used as production viruses are characterized by the following features and properties:

- Adeno III WBRl-DEPT strain of bovine adenovirus subgroup I and Adeno IV Weybridge CT2-DEP strain of bovine adenovirus subgroup II are representatives of the genus Mastadenovirus of the Adenoviridae family. Viral particles of regular polyhedral (icosahedron) shape. The viral genome is represented by a double-stranded, unfragmented linear DNA molecule. Virions have a diameter of 70-80 nm, viruses are resistant to ether, chloroform, saponin, sodium deoxycholate, trypsin. Treatment with chloroform increases the infectious titer of viruses by 0.5 lg. Adenoviruses are resistant to changes in pH from 3.0 to 9.0 for 3 hours at room temperature. At 4 ° C, they are stored for more than 6 months, at room temperature for 1-4 months, for a long time they maintain infectious activity at -30 ° C. Viruses are resistant to repeated freezing and thawing, persist at pH 6.0–9.0, and tolerate freeze drying. Strains do not have hemagglutinating properties.

The Adeno III WBR-1-DEP adenovirus strain in the MDVK cell line culture and the Adeno IV Weybridge CT2-DEP strain in the Taurus-1 calf kidney cell culture (T-1) cause CPD 48-72 hours after infection. Visible cytopathic changes induced by the virus are characterized by enlargement and rounding of the cells. Subsequently, the cells are gradually grouped into conglomerates in the form of lumps, clusters, forming voids in a monolayer, resembling honeycombs.

- The strain “TKA-VIEV-B2-DEP" of herpesvirus type I cattle of herpesvirus type I of the genus Herpesvirus of the family Herpesviridae is a homogeneous population of DNA-containing virus obtained by exposure to ultraviolet rays on the initial virulent virus IRT in a dose close to the threshold. The hexogonal capsid, diameter 100-110 nm, consists of 162 capsomeres. The vaccine strain "TKA-VIEV-B2-DEPT" has high immunogenic properties and we have identified as a manufacturing one for the manufacture of the associated vaccine.

Herpesvirus type I is reproduced in a monolayer of cells of the kidney of a cow embryo and its cytopathogenic effect occurs after 48-96 hours. The virus grown in a monolayer of transplantable MDVC lines has stable activity and reaches 6.75-7.25 lg TCD ₅₀ / cm ³ . Reproduction of the virus is accompanied by a suppression of the mitotic activity of cells and the formation of intranuclear inclusions. The virus is easily concentrated and purified by centrifugation in a gradient of colloidal silicon oxide - polyethylene glycol. It can be precipitated with methanol, acetone, magnesium sulfate and ammonium sulfate. Acetone, ether, chloroform and ethyl alcohol inactivate the virus immediately. At a temperature of -60-70 ° C, the virus survives for 7-9 months, a temperature of 56 ° inactivates it after 20 minutes, 37 ° - after 4-10 days, 22 ° - after 50 days. At 4 ° C, the activity of the virus decreases after 30-40 days by only 1 lg. Lyophilization almost does not reduce its activity.

- strain "SF-4-DEPT" of the parainfluenza virus-3 of cattle is a representative of the genus Paramyxovirus of the family Paramyxoviridae. Virions are round-oval in shape, consisting of a shell and an internal component. The diameter of the virions is 150-250 nm, the genome of the virus is represented by non-segmented single-stranded linear RNA. At 4 ° without a preservative, after 3-4 months it completely loses its infectious activity, at -60 ° the virus remains infectious for several months. With repeated freezing and thawing of the viral suspension, the infectious activity is rapidly lost. The strain "SF-4-DEPT" does not agglutinate erythrocytes of chickens, horses, agglutinates erythrocytes of guinea pigs and, in a lower titer, erythrocytes of rabbit, pig, cow, monkey, mouse, human (group 0). The strain is sensitive to ether and deoxychylate, to acidic pH (3.0).

The strain "SF-4-DEPT" of the PG-3 virus in the cell culture LEC causes CPD 72 hours after infection. Visible cytopathic changes induced by the virus were characterized by the formation of granular and vacuolated round cells or multinucleated symplasts resulting from the fusion of several cells. Cells infected with PG-3 virus cause diffuse adsorption of guinea pig erythrocytes.

- strain "VK-1-DEPT" of the virus VD-BS of cattle is a representative of the genus Pestivirus of the family Flaviviridae, the virus genome is represented by single-stranded RNA. The size of the virions is 40-100 nm, the floating density in the gradient of sucrose density is 1.5-1.19 g / cm ³ .

Sensitive to ether, chloroform and trypsin. Warming at 50 ° C for 10 min leads to inactivation of the attenuated virus. Freezing at -20 ° C and thawing reduces the titer of infectious activity in cell culture. In contrast to the initial epizootic strain of the virus, the attenuated strain acquired greater resistance to pH 3.0.

The red blood cells of guinea pig, ram, cattle and birds do not agglutinate. It multiplies with the development of a cytopathic effect in cell cultures of PEC, calf kidney (PT), and pig embryo kidney (PES). As the CPD develops, the cells gradually tear away from the surface of the glass, forming islands. The virus reaches an infectious titer of 10 ^6.0-6.5 TCD ₅₀ / ml.

In the body of guinea pigs and cattle causes the formation of virus-neutralizing antibodies. In vaccinated cattle, it stimulates the formation of local and humoral immunity. It causes a weak reaction in some vaccinated calves with subcutaneous, intramuscular and intranasal administration of a five-fold immunizing dose: a 1-2-day increase in the body temperature reaction by 0.3-0.5 ° C, a slight decrease in the number of leukocytes, without other deviations from the clinical and physiological norms (Zhidkov S.A., Kryukov N.N. The strain of the virus Bovine viral Diarrhea-mucosal disease virus for the manufacture of a vaccine against viral diarrhea in cattle // Authors certificate SU 1322673 from 09.09.85). In a lyophilized state at 4-10 ° C it is stored for a year, practically without reducing infectious activity.

Virus strains accepted by the applicant as industrial have high biological, antigenic and immunogenic activity in their native form and after inactivation, are highly productive in sensitive biological cultivation systems. They are free from contamination by bacteria, fungal microflora, mycoplasmas and related viruses. The possibility of their use for the manufacture of an associated vaccine, creating intense and prolonged immunity in vaccinated animals, was confirmed experimentally in laboratory conditions and production experiments.

2. The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the invention, allowed us to establish that the applicant did not find sources characterized by features that are identical (identical) to all the essential features of the invention .

Determination from the list of identified analogues of the prototype (associated vaccines "Bovigrip", "Bovigrip plus", which includes inactivated antigens of parainfluenza-3 (strain "SF-4"), cattle adenovirus 1st (strain "Bovine 10") and 3 (strain “WBR-1”) serotypes, infectious rhinotracheitis virus (strain “Calw”), manufactured by “Hoecht Veterinar GmbH”) as the closest in combination of features to the applicant's perceived technical result of the distinctive features of the proposed vaccine, set forth in an independent claim.

Therefore, the claimed solution meets the condition of patentability "Novelty."

To verify the conformity of the proposed solution to the patentability condition "Inventive step", an additional search of known solutions was carried out to identify features included in the distinctive part of the independent claim.

The search results showed that the proposed solution does not follow for the specialist explicitly from the prior art set forth in the corresponding section of the description, and the effect of the transformations provided for by the essential features of the invention to achieve a technical result is not revealed.

Therefore, the proposed associated vaccine meets the condition of patentability "Inventive step".

The essence of the invention is illustrated by examples of its execution, which do not limit the scope of the invention.

Example 1. The production strain "Adeno III WBR1-DEPT" of cattle adenovirus subgroup I for the manufacture of the proposed vaccine is reproduced in a transplantable culture of kidney cells of a cow embryo (line MDBK) grown in three-liter roller bottles on a combined growth medium consisting of medium 199 - 10%, GLA - 40%, MEM Needle - 40%, cattle serum - 10% and antibiotics. The culture is grown for 3-4 days at a sowing concentration of 140-150 thousand cells in 1 cm ³ environment.

To obtain 10 l of the viral suspension take 28-35 bottles with cell culture with a capacity of 3.0 DM ³ each.

The growth medium is drained, a supporting medium is introduced (GLA - 60%, MEM Needle - 40% and antibiotics: streptomycin - 100 μg / cm ³ , penicillin - 100 PIECES / cm ³ ) in an amount of 300-350 cm ³ , into which they are preliminarily added bovine adenovirus strain "Adeno III WBR1-DEPT" at the rate of 0.2-1 TCD ₅₀ / cage. The bottles are incubated in a roller unit at 37-38 ° C for 48-72 hours. The collection of culture fluid is carried out during a period of pronounced cytopathic effect - the maximum accumulation of the virus. The infectious titer of the virus should be at least 6.25 lg TCD ₅₀ / ml.

The virus is released from cells by freezing and thawing three times. The resulting virus-containing material is purified from cell detritus by centrifugation at 3000 rpm for 20 minutes, subject to sterile conditions.

The purified virus-containing suspension is subjected to inactivation with formalin (active formaldehyde content of 37-38%) at a final concentration of 0.2-0.3% at a temperature of 37 ° C for 72 hours with periodic stirring.

The concentration of the antigen of the adenovirus subgroup I is carried out using PEG-6000 to a final concentration of 7-8% (wt. Volume) and sodium chloride to a final concentration of 3% for 24 hours at 4 ° C. The viral protein is precipitated by centrifugation at 5000 rpm for 60 minutes and the suspension is resuspended using a 0.85% sodium chloride solution. The resulting concentrated antigen is standardized by the quantification of a specific protein on a spectrophotometer. The protein concentration should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is checked for sterility, avirulence according to the generally accepted method.

Example 2. The production strain "Adeno IV Weybridge CT2-DEP" of cattle adenovirus II subgroups for the manufacture of the proposed vaccine is reproduced in a cell culture of an inoculable cell culture of Taurus-1 (T-1) calf kidney cells grown in three-liter roller bottles on growth medium Igla MEM -90%, serum of cattle - 10% and antibiotics (streptomycin - 100 μg / cm ³ , penicillin - 100 PIECES / cm ³ ). The culture is grown for 3-4 days at a sowing concentration of 140-150 thousand cells in 1 cm ³ environment.

To obtain 10 l of the viral suspension take 35-37 bottles with cell culture with a capacity of 3.0 DM ³ each.

The growth medium is drained, a support medium of 300 cm ³ , consisting of Needle MEM medium and antibiotics, is introduced into which cattle adenovirus Adeno IV Weybridge CT2-DEPT is preliminarily added at a rate of 0.2-1 TCD ₅₀ / cell. The bottles with the infected monolayer are incubated in a roller unit at 37-38 ° C for 48-72 hours. The infectious titer of the virus in the culture suspension should be at least 6.5 lg TCD ₅₀ / ml.

The resulting virus-containing material was frozen three times at -20 ° C and thawed at 37 ° C, then centrifuged at 3000 rpm for 20 minutes in order to release cell detritus.

The virus-containing suspension is inactivated with formalin (active formaldehyde content of 37-38%) at a final concentration of 0.2-0.3% at 37 ° C for 72 hours with periodic stirring.

The concentration of the antigen of the adenovirus II subgroup is carried out using PEG-6000 to a final concentration of 7-8% (wt. Volume) and sodium chloride to a final concentration of 3% for 24 hours at 4 ° C. The viral protein is precipitated by centrifugation at 5000 rpm for 60 minutes, the resulting precipitate is resuspended in a 0.85% sodium chloride solution. Standardization of antigen is carried out on a spectrophotometer. The concentration of a specific protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is tested for sterility and avirulence.

Example 3. The production strain "TKA-VIEV-B2-DEPT" of herpesvirus type I cattle for the manufacture of the proposed vaccine is reproduced in a transplantable culture of kidney cells of a cow embryo (MDBK line) grown on a combined growth medium consisting of 199 - 10% medium , GLA - 40%, MEM Needle - 40%, cattle serum - 10% and antibiotics. At a sowing concentration of 140-150 thousand cells in 1 cm ^{3 of the} medium, the formation of a continuous monolayer in three-liter roller bottles occurs within 3-4 days at a temperature of 37 ± 0.5 ° C in a thermal room.

To obtain 10 l of the viral suspension take 28-35 bottles (capacity 3.0 DM ³ ) with a monolayer of cell culture line MDBK.

After the growth medium is drained, the supporting medium is added (GLA-60%, MEM-40% Needle and antibiotics: streptomycin - 100 μg / cm ³ , penicillin - 100 PIECES / cm ³ ) in an amount of 300-350 cm ³ , into which they are preliminarily added herpesvirus type I strain "TKA-VIEV-B2-DEPT" at the rate of 0.1-1 TCD ₅₀ / cell. The bottles are incubated in a roller unit at 37-38 ° C for 48-72 hours. The collection of culture fluid is carried out during a period of pronounced cytopathic effect - the maximum accumulation of the virus. The infectious titer of the virus should be at least 7.5 lg TCD ₅₀ / ml.

The virus-containing material is frozen three times at -20 ° C and thawed at 37 ° C, then centrifuged at 3000 rpm for 20 minutes in order to release cell detritus.

The purified virus-containing suspension is subjected to inactivation with formalin (active formaldehyde content of 37-38%) at a final concentration of 0.2-0.3% at a temperature of 37 ° C for 72 hours with periodic stirring.

The concentration of the herpesvirus type I antigen is carried out by PEG-6000, which is added to the purified virus-containing suspension to a final concentration of 7-8% (weight. Volume), sodium chloride is added to a final concentration of 3%. Concentration is carried out for 24 hours at a temperature of 4 ° C. Viral protein is precipitated by centrifugation at 5000 rpm for 60 minutes. The resulting concentrated antigen is standardized by the quantification of a specific protein on a spectrophotometer. The concentration of viral protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is checked for sterility, avirulence.

Example 4. Production strain "SF-4-DEPT" virus parainfluenza-3 cattle for the manufacture of the proposed vaccine is reproduced in a transplantable culture of lung cells of a cow embryo (LEC).

The cell culture "LEK" at a sowing concentration of 140-150 thousand cells in 1 cm ^{3 is} grown in mattresses with a capacity of 1.5 dm ³ for 3-4 days on a combination medium consisting of GLA medium - 60%, MEM Needle - 30% and 10% cattle serum.

To obtain 10 l of the viral suspension, 65-70 mattresses with a cell culture of 1.5 dm ³ each are taken. After the growth medium is drained, a parainfluenza-3 suspension is added to the mattresses at a rate of 0.2-1 TCD ₅₀ / cell. Virus adsorption is carried out at 37 ° C for 1 hour. After that, 150 cm ^{3 of the} support medium, consisting of MEM Needle 40% and GLA 60%, antibiotics, is introduced into the mattresses. Cultivation is carried out at 37 ° C for 72-96 hours. As a control, 3 mattresses are left uninfected, in which there should not be any destructive changes during the cultivation period. Infected mattresses with 80-90% damage to the cell monolayer are selected, frozen three times at -20 ° C and thawed.

The resulting virus-containing material is purified from cell detritus by centrifugation at 3000 rpm for 20 minutes, subject to sterile conditions. The hemagglutinating activity of the SF-4-DEPT strain of bovine PG-3 virus is determined in an RGA with a 1% suspension of guinea pig erythrocytes at 20-22 ° C. The hemagglutinating activity of the cattle PG-3 virus should be at least 6 log ₂ (1:64).

The purified virus-containing suspension is subjected to inactivation with formalin (active formaldehyde content of 37-38%) at a final concentration of 0.2-0.3% at a temperature of 37 ° C for 72 hours with periodic stirring.

Example 5. The production strain "VK-1-DEP" VD-BS of cattle for the manufacture of the proposed vaccine is reproduced in a culture of kidney cells of a cow embryo (MDBK line) grown on a combined growth medium consisting of 199-10%, GLA- 40%, MEM-40% needle, cattle serum - 10% and antibiotics. The inoculum concentration of 140-150 thousand cells in 1 cm ^{3 of} medium, a monolayer in three-liter roller bottles is formed within 3-4 days at a temperature of 37 ± 0.5 ° C in a thermal room.

To obtain 10 l of the viral suspension take 28-35 bottles (capacity 3.0 DM ³ ) with a monolayer of cell culture line MDBK.

Before infection, the growth medium is drained, a support medium is introduced (GLA - 60%, MEM Needle - 40% and antibiotics: streptomycin - 100 μg / cm ³ , penicillin - 100 PIECES / cm ³ ) in an amount of 300-350 cm ³ , in which pre-introduce the virus VD-BS strain "VK-1-DEPT" at the rate of 0.2-1 TCD ₅₀ / cell. Incubation is carried out in a roller unit at 37-38 ° C for 48-72 hours. The collection of culture fluid is carried out during a period of pronounced cytopathic effect - the maximum accumulation of the virus. The infectious titer of the virus should be at least 6.5 lg TCD ₅₀ / ml.

The virus-containing material is frozen three times at -20 ° C and thawed at 37 ° C, then centrifuged at 3000 rpm for 20 minutes in order to release cell detritus.

The purified virus-containing suspension is subjected to inactivation with formalin (active formaldehyde content of 37-38%) at a final concentration of 0.2-0.3% at a temperature of 37 ° C for 72 hours.

The concentration of the VD-BS virus antigen is carried out by PEG-6000, which is added to the purified virus-containing suspension to a final concentration of 7-8% (weight volume), sodium chloride is added to a final concentration of 3%. Concentration is carried out for 24 hours at a temperature of 4 ° C. Viral protein is precipitated by centrifugation at 5000 rpm for 60 minutes. The resulting concentrated antigen is standardized by the quantification of a specific protein on a spectrophotometer. The concentration of viral protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is checked for sterility, avirulence.

Example 6. An oil adjuvant for the manufacture of the proposed vaccine is prepared from synthetic oil PES-3M (organosilicon fluid GOST 13004-77) and anhydrous lanolin, taken in a ratio of 84:16 volume parts. A mixture of oil and lanolin is mixed, autoclaved at 120 ° C (1.0 atm.) For 40 minutes. Ready adjuvant is cooled, checked for sterility according to the generally accepted method.

Example 7. The vaccine associated against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - diseases of the mucous membranes of cattle inactivated emulsion is prepared by dispersing a mixture of concentrated antigenic materials from strains of "Adeno III WBR1-DEPT" cattle adenovirus subgroup I subgroup I Adeno IV Weybridge ST2-DEP "of cattle adenovirus subgroup II," TKA-VIEV-B2-DEP "herpesvirus type I," SF-4-DEPT "parainfluenza-3 virus," VK-1-DEPT "virus of diarrhea - mucosal diseases cattle nuts.

For this, concentrated antigenic materials obtained as described in examples 1, 2, 3, 4 and 5 are mixed together in a ratio of 1: 1: 1: 1: 1, i.e. 1 part of cattle adenovirus antigenic material of subgroup I, 1 part of cattle adenovirus subgroup II, 1 part of herpesvirus type I, 1 part of parainfluenza-3 and 1 part of VD-BS virus are taken.

Concentrated antigenic material from the Adeno III WBRl-DEP strain of cattle adenovirus 10.0-11.0 Antigenic material concentrated from Adeno IV Weybridge CT2-DEP strain of cattle adenovirus 10.0-11.0 Concentrated antigenic material from strain TKA-VIEV-B2-DEP of herpesvirus type I 10.0-11.0 Antigenic material concentrated from strain "SF-4-DEPT" parainfluenza-3 10.0-11.0 Antigenic material concentrated from strain "VK-1-DEPT" of virus VD-BS 10.0-11.0

The resulting mixture of concentrated antigens is thoroughly emulsified with an oil adjuvant in a ratio of 1: 1 (vol.%: 50:50).

The associated vaccine is dispensed into sterile glass vials and controlled according to specifications.

In appearance, the vaccine is a stable pinkish-white emulsion. During prolonged storage in the upper part of the vial, insignificant (up to 10%) delamination of the emulsion, which is restored by shaking, is allowed.

Sterility of the vaccine is determined in accordance with GOST 28085-89, by plating on MPA, MPB, MPPB and Saburo agar. The vaccine should not produce growth of bacteria and fungi in any of the seeded tubes, i.e. must be sterile.

To determine the residual virulence, the associated vaccine is diluted in a ratio of 1:10 and in a volume of 0.1 cm ^{3 a} pipette is pipetted into 5 vials with transplantable cultures of T-1, LEK, MDVC cells and kept for 1.5-2 hours at 37 ° C , and 5 vials with the specified cell culture are left as a control. After adsorption, the inoculum is poured out, into the vials with T-1, LEK, MDVK cell cultures, a support medium with antibiotics is introduced and incubated in a thermostat at 37 ° C for 5-7 days. All bottles with cell cultures are periodically examined under a microscope in order to detect the CPD of adenovirus, herpesvirus type I, PG-3 and diarrhea virus - a disease of the mucous membranes of cattle.

The resulting culture suspensions are used for the next two “blind” passages. At the third passage in the transplanted cell cultures of T-1, LEK, MDVK, the characteristic CPD of adenovirus, herpesvirus, PG-3 and VD-BS was not detected, which indicates the avirulence of the associated vaccine.

To check the safety, the vaccine is administered intraperitoneally to 10 white mice at a dose of 0.25 cm ³ . The vaccine is considered harmless if white mice remain clinically healthy for 10 days without any pathological changes.

The viscosity of the vaccine is determined on a VPZh-2 viscometer, and it is expressed in mm ² / s. The proposed vaccine containing, as the target additive, an oil adjuvant (synthetic oil PES-3M and anhydrous lanolin), forming a type of water-oil emulsion, has a viscosity of 38 ± 0.5 mm ² / s.

To determine the stability of the drug, vaccine samples are frozen at -40 ° C for 1 hour. After the specified time, the test tubes with the vaccine are placed for 1 hour in a thermostat at 37 ° C. In test tubes, after freezing and incubation in a thermostat, detachment of 0.5-1% transparent oil can be observed in the upper part, and the rest is represented by an emulsion. In this case, the vaccine is considered stable.

To test the reactogenicity of the drug, the vaccine is administered intramuscularly in the thigh area of 3 rabbits at a dose of 2 cm ³ . The clinical condition of the animals is monitored for 14 days. In the process of monitoring the clinical condition of animals to which the vaccine was administered, there were no significant deviations of a general or local nature. In experimental animals, there was a slight increase in local temperature at the injection site, but the general condition remained satisfactory. There were no signs of inflammation in the tissue section. Therefore, the associated vaccine is a type of areactogenic drugs.

Antigenic activity is monitored on three rabbits with a live weight of 2.5-3.0 kg, to which the vaccine is administered intramuscularly, twice with an interval of 21 days in a dose of 2 cm ³ . Blood from rabbits is taken from the ear vein 21-30 days after the second vaccination. Serum is examined for the presence of specific antibodies to antigens of adenovirus, herpesvirus type I and VD-BS in ELISA, parainfluenza-3 in rtga. At the same time, blood is drawn from two unvaccinated rabbits and serum is used for control.

The vaccine series is considered to have passed the control for antigenic activity if the titer of virus-specific antibodies 21-30 days after vaccination II amounts to adenovirus, herpesvirus type I, VD-BS virus, at least 1: 1600 in ELISA, to PG-3 1: 160 and more in RTGA (table 2). The vaccine made has the optimal composition of the components, vol.%:

Antigenic material concentrated from Adeno III WBR1-DEP strain of cattle adenovirus reproduced in a transplanted culture of cow embryonic kidney cells (MDBK line) with biological activity of at least 6.25 lg TCD ₅₀ / ml and inactivated with formalin (final concentration 0.2 -0.3%) 10.0-11.0 Concentrated antigenic material from Adeno IV Weybridge CT2-DEP strain of cattle adenovirus reproduced in an inoculated culture of Taurus-1 (T-1) calf kidney cells with biological activity of at least 6.5 lg TCD ₅₀ / ml and inactivated with formalin (final concentration 0.2-0.3%) 10.0-11.0 Concentrated antigenic material from the strain TKA-VIEV-B2-DEP of herpesvirus type I reproduced in a culture of cow embryonic kidney cells (MDBK line) with biological activity of at least 7.5 lg TCD ₅₀ / ml and inactivated with formalin (final concentration 0, 2-0.3%) 10.0-11.0 Concentrated antigenic material from the SF-4-DEP strain of parainfluenza-3 cattle reproduced in a transplantable culture of cow embryonic lung cells (LEC) with biological activity of at least 6.5 lg TCD ₅₀ / ml and hemagglutinating activity of at least 6 logs (1 : 64), inactivated by formalin (final concentration 0.2-0.3%) 10.0-11.0 Concentrated antigenic material from the VK-1-DEP strain of VD-BS cattle virus reproduced in a culture of kidney embryo of a cow (line MDBK) with biological activity of at least 6.5 lg TCD ₅₀ / ml inactivated with formalin (final concentration 0.2 -0.3%) 10.0-11.0 Targeted Adjuvant up to 100

The content of antigenic materials for the vaccine within the above ranges is their effective amount in the preparation, ensuring the achievement of a technical result from the use of the invention.

The vaccine is intended for specific prophylaxis of respiratory and gastrointestinal diseases of young animals.

The vaccine is used to prevent adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea, a disease of the mucous membranes of cattle in threatened and permanently dysfunctional farms.

The vaccine is administered to calves and adult livestock intramuscularly in the middle third of the neck, observing the rules of aseptic and antiseptic, according to the following scheme:

- deep-shelled cows and heifers are vaccinated twice: the first time 40-50 days before calving, the second time 15-20 days before calving at a dose of 2 cm ³ in order to prevent respiratory and gastrointestinal diseases in newborn calves, thereby creating colostral immunity.

- calves are immunized from 25-30 days of age in a dose of 1 cm ³ twice, with an interval of 21 days, in order to prevent viral respiratory and gastrointestinal infections. Revaccination is carried out after 6 months.

- calves older than 6 months and bulls are vaccinated twice, with an interval of 21 days, at a dose of 2 cm ³ . After 6 months, they are revaccinated.

The vaccine causes the formation of an immune response in cattle to adenovirus, herpesvirus type I, parainfluenza-3 and viral diarrhea-disease of the mucous membranes. Immunity in vaccinated animals occurs 21-30 days after a double use and lasts for 6 months. Specific antibodies from vaccinated cows are transmitted with colostrum to newborn calves. The vaccine is harmless, does not possess medicinal properties.

Example 8. Tests were conducted to determine the vaccine dose of the “Associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - inactivated emulsion cattle mucous membrane diseases” made from concentrated antigenic materials of the Adeno III WBR1-DEPT strains of large adenovirus cattle of I subgroup, “Adeno IV Weybridge ST2-DEP” of cattle adenovirus II subgroup, “TKA-VIEV-B2-DEP” of herpesvirus type I, “SF-4-DEP” of parainfluenza-3 virus, “VK-1-DEP” diarrhea virus - mucus disease ies skins of cattle as described in Example 7.

The graft dose of the experimental series of the associated vaccine was determined on cattle under the production conditions of Zolotaya Niva LLC, Kaybitsky district of the Republic of Tatarstan.

For this purpose, a preliminary study of blood serum from animals formed three groups of animals of different age groups according to the principle of an analogue in the amount of 15 animals, seronegative or having a low level of antibodies against adenovirus, herpesvirus type I, PG-3 and VD-BS of cattle . Each dose of the drug was administered to 5 animals. The vaccine was administered intramuscularly in the middle third of the neck twice with an interval of 21 days according to the scheme: deep-growing cows and heifers - 1 cm ³ , 2 cm ³ and 3 cm ³ (the first time - for 40-50 days, the second time - for 15-20 days before calving); calves 25-30 days of age - 0.5 cm, 1 cm ³ , 2 cm ³ ; young animals older than 6 months - 1 cm ³ , 2 cm ³ and 3 cm ³ .

In order to determine the dynamics of the increase in titers of specific antibodies, blood was taken from animals of all groups before vaccination, 21 days after the first and second immunization. Serum samples were examined for the presence of antibodies to PG-3 in rtga, to adenoviruses, herpesvirus type I and VD-BS virus in ELISA. Statistically processed research results are presented in tables 3, 4, 5.

Double administration of the associated vaccine on average ensured the production of antibodies in ELISA within the limits of deep-growing cows and heifers to adenovirus I subgroup 2400.00 ± 565.69, adenovirus II subgroup 2880.00 ± 357.77, to herpes virus 2560.00 ± 438.18 to VD-BS 2240.00 ± 438.18 and to PG-3 in RTGA 512.00 ± 87.64; in calves of 25-30 days of age, to adenovirus I subgroup 2080.00 ± 536.66, adenovirus II subgroup 2240.00 ± 438.18, to herpes virus 1600.00 ± 0.00, to VD-BS 1440.00 ± 178 89 and to PG-3 164.00 ± 53.10; in young animals older than 6 months of age, the adenovirus I subgroup 2560.00 ± 438.18 adenovirus II subgroup 2560.00 ± 438.18, the herpes virus 2560.00 ± 438.18, VD-BS 2880.00 ± 357, 77 and to PG-3 384.00 ± 71.55.

Thus, the optimal two-time vaccine doses for cattle of different age groups were determined, which were: for deep-skinned cows and heifers 2 cm ³ , calves 25-30 days of age 1 cm ³ , young animals over 6 months of age 2 cm ³ .

Example 9. Tests of antigenic activity and efficacy of the “Associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - inactivated emulsion cattle mucous membranes diseases” were carried out on cattle under production conditions.

According to the veterinary service of the economy, gastrointestinal and respiratory diseases of young animals are observed annually and occur mainly in the autumn-winter and spring. In 2011 (from January to October) 281 calves were obtained, 75 calves fell ill, which is 26.6%, 20 calves fell - 7.1% of the total number of young animals received.

The calves were vaccinated from 25-30 days of age, deep-shelled cows and heifers, young animals up to 12 months old. The vaccine was administered intramuscularly in the middle third of the neck: deep-growing cows and heifers for 40-50 days and 15-20 days before calving, 2 cm ³ ; young animals up to 6 months of 1 cm ³ and young animals older than 6 months of 2 cm ³ twice with an interval of 21 days. 800 cattle were vaccinated. In the post-vaccination period in vaccinated animals, deviations from the physiological norm were not observed.

The dynamics of the increase in titers of specific antibodies in the blood serum of cattle was studied before vaccination and 21 days after the second immunization (n = 20). In order to determine the intensity of colostral immunity, we examined the blood serum of young animals up to 14 days of age (n = 20). Serum samples were examined for the presence of antibodies to PG-3 in rtga, to adenovirus, herpesvirus type I and VD-BS virus in ELISA and neutralization (pH) reactions (Table 6).

The level of specific antibodies in colostrum serum of calving vaccinated cows was determined for PG-3 in RTGA, for adenovirus, herpesvirus type I and VD-BS virus in ELISA, unvaccinated animals served as a control group (n = 10). Statistically processed research results are presented in table 7.

Twice administration of the associated vaccine on average ensured the production of antibodies in ELISA (PH) within the limits of deep-growing cows and heifers to adenovirus I subgroup 2500.00 ± 234.20 (56.00 ± 7.85), adenovirus II subgroup 2740.00 ± 195 86 (60.00 ± 7.71), herpes virus 2340.00 ± 234.95 (54.40 ± 8.20), VD-BS 2220.00 ± 219.75 (54.40 ± 9.63 ) and to PG-3 in RTGA 552.00 ± 55.19; in calves 25-30 days of age, adenovirus I subgroup 2240.00 ± 286.19 (57.26 ± 8.46), adenovirus II subgroup 2120.00 ± 266.46 (56.00 ± 9.11), herpesvirus 1040.00 ± 124.19 (49.60 ± 7.23), VD-BS 1360.00 ± 246.23 (51.20 ± 5.53) and PG-3 158.00 ± 58.36 ; in young animals up to 14 days of age, to adenovirus I subgroup 1120.00 ± 92.25 (38.40 ± 3.01), adenovirus II subgroup 1100.00 ± 98.18 (39.20 ± 3.47), to herpes virus 1080.00 ± 136.76 (36.00 ± 3.55) to VD-BS 1200.00 ± 94.15 (44.00 ± 3.93) and PG-3 168.00 ± 19.66. In 2012, 268 calves were obtained from vaccinated cows and heifers, of which 32 calves fell ill, which is 11.9%, 12 calves fell - 4.4% of the total number of young animals received.

A production test of an associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea, an inactivated emulsion of mucous membranes of cattle, showed that it is harmless, reactogenic and causes the formation of a tense immune response in animals after double vaccination. A study of the effectiveness of the associated vaccine in the economy found that it provides protection for newborn calves, young growth period from viral respiratory and gastrointestinal infections, as well as pregnant cows and heifers from pathologies of reproductive organs.

Table 1 Serological monitoring results for adenovirus, herpesvirus type I, parainfluenza-3 and VD-BS of cattle No. p / p Farm Name Number of samples PG-3 IRT VD-BS Adenovirus RTGA IFA IFA IFA Republic of Tatarstan one LLC Navruz, Agryz district 47 39 9 9 36 2 Agro-Invest CJSC, Aksubaevsky District, RT 55 thirty 7 6 17 3 Ak Bars Agro LLC, Arsky district 36 16 21 13 17 four LLC "Golden Niva" Kaybitsky district 105 42 28 45 27 5 Zolotaya Niva LLC (branch Ulyankovo), Kaybitsky district fifty fifty 2 9 41 6 LLC "Bahetle-Agro" Nizhnekamsk district 33 26 33 32 eighteen 7 LLC A / F "South" of the Nurlat region 33 17 10 10 5 8 Peasant farm "Suleymanova" Nurlatsky district 19 10 6 9 3 Nizhny Novgorod region of the Russian Federation 9 SEC "Poshatovsky" Krasnooktyabrsky district eleven eleven 9 7 5 10 LLC "Trekhozerskoye" Krasnooktyabrsky district 10 2 10 8 8 eleven Development LLC Krasnooktyabrsky district 25 25 twenty 23 23 The Republic of Mordovia 12 LLC AF Ryazanovka, Staro-Shaigovsky district 28 10 8 10 fifteen 13 CJSC "VKM-Agro" of Ruzayevsky district thirty 29th 22 7 21 Mari El Republic fourteen CJSC Mariyskoye, Medvedevsky District 25 23 23 12 9 The number of samples studied, total: 507 of which are positive: qty 330 208 200 245 at % 65.0 41.0 39,4 48.3

Table 3 The level of antibodies in the blood serum of cattle before vaccination with the associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - inactivated, emulsion diseases of the mucous membranes of cattle (M ± m, n = 5, p <0.05) No. p / p Age group Dose, cm ³ Antibody titer AB-III (IFA) AB-IV (IFA) IRT (IFA) PG-3 (RTGA) VD-BS (IFA) one deep cows and heifers one 120.00 ± 54.77 40.00 ± 44.72 160.00 ± 44.72 52.00 ± 13.42 160 ± 44.72 2 280.00 ± 54.77 80.00 ± 54.77 200.00 ± 70.71 36.00 ± 4.47 160 ± 44.72 3 160.00 ± 44.72 80.00 ± 54.77 160.00 ± 44.72 40.00 ± 12.25 120 ± 36.51 2 calves 25-30 days of age 0.5 160.00 ± 83.67 0.00 ± 0.00 40.00 ± 44.72 16.00 ± 8.37 0.00 ± 0.00 one 120.00 ± 9.44 0.00 ± 0.00 40.00 ± 44.72 20.00 ± 17.32 80 ± 54.77 2 40.00 ± 44.72 40.00 ± 44.72 0.00 ± 0.00 12.00 ± 8.94 0.00 ± 0.00 3 young growth over 6 months one 160.00 ± 44.72 120.00 ± 89.44 40.00 ± 44.72 4.00 ± 4.47 80 ± 54.77 2 160.00 ± 44.72 80.00 ± 54.77 160.00 ± 109.54 4.00 ± 4.47 80 ± 54.77 3 80.00 ± 54.77 120.00 ± 54.7 120.00 ± 54.77 12.00 ± 8.94 120 ± 54.77

Table 4 The level of antibodies in the blood serum of cattle after I vaccination with the associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - inactivated, emulsion disease of the mucous membranes of cattle (M ± m, n = 5, p <0.05) No. p / p Age group Dose, cm ³ Antibody titer AB-III (IFA) AB-IV (IFA) IRT (IFA) PG-3 (RTGA) VD-BS (IFA) one deep cows and heifers one 1200.00 ± 282.84 880.00 ± 328.63 1,280.00 ± 219.09 192.00 ± 35.78 560.00 ± 109.54 2 1840.00 ± 657.27 1200.00 ± 282.84 1760.00 ± 438.18 208.00 ± 53.67 800.00 ± 0.00 3 2240.00 ± 438.18 2080.00 ± 536.66 2400.00 ± 565.69 320.00 ± 97.98 880.00 ± 328.63 2 calves 25-30 days of age 0.5 1,280.00 ± 219.09 880.00 ± 219.09 480.00 ± 89.44 24.00 ± 4.47 400.00 ± 0.00 one 1360.00 ± 558.57 960.00 ± 303.32 560.00 ± 109.54 32.00 ± 5.48 480.00 ± 89.44 2 1520.00 ± 536.66 1,280.00 ± 219.09 640.00 ± 109.54 52.00 ± 30.50 640.00 ± 268.33 3 young growth over 6 months one 1920,00 ± 357.77 1280,00 ± 536.66 880.00 ± 219.09 160.00 ± 0.00 960.00 ± 303.32 2 2080.00 ± 536.66 1,280.00 ± 219.09 1120.00 ± 219.09 256.00 ± 43.82 1120.00 ± 219.09 3 2880.00 ± 357.77 1440.00 ± 178.89 1920.00 ± 606.63 320.00 ± 97.98 1600,00 ± 0,00

Table 5 The level of antibodies in the blood serum of cattle after II vaccination with the associated vaccine against adenovirus, herpes virus infection, parainfluenza-3 and viral diarrhea-disease of the mucous membranes of cattle inactivated, emulsion (M ± m, n = 5, p <0.05) No. p / p Age group Dose, cm ³ Antibody titer AB-III (IFA) AB-IV (IFA) IRT (IFA) PG-3 (RTGA) VD-BS (IFA) one deep cows and heifers one 2240.00 ± 657.27 2080.00 ± 536.66 2400.00 ± 565.69 320.00 ± 97.98 1920.00 ± 606.63 2 2400.00 ± 565.69 2880.00 ± 357.77 2560,00 ± 438.18 512.00 ± 87.64 2240.00 ± 438.18 3 2560,00 ± 438.18 3200.00 ± 0.00 2560,00 ± 438.18 768.00 ± 141.11 2400.00 ± 565.69 2 25-30 day old calves 0.5 1920,00 ± 357.77 1920,00 ± 357.77 1200.00 ± 282.84 68.00 ± 27.93 960.00 ± 178.89 one 2080.00 ± 536.66 2240.00 ± 438.18 1600,00 ± 0,00 164.00 ± 53.10 1440.00 ± 178.89 2 2560,00 ± 438.18 2240.00 ± 438.18 1920,00 ± 357.77 192.00 ± 60.66 1840.00 ± 657.27 3 young growth over 6 months one 2240.00 ± 438.18 1440.00 ± 178.89 1360.00 ± 558.57 320 ± 0,00 1760.00 ± 438.18 2 2560,00 ± 438.18 2560,00 ± 438.18 2560,00 ± 438.18 384.00 ± 71.55 2880.00 ± 357.77 3 2880.00 ± 357.77 2880.00 ± 357.77 2560,00 ± 438.18 448.00 ± 87.64 2880.00 ± 357.77

Table 7 The level of antibodies in colostrum of cattle after immunization with the associated vaccine against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - a disease of the mucous membranes of cattle inactivated emulsion (M ± m, n = 10, p <0.05) Group of animals Antibody titer AB-III (IFA) AB-IV (IFA) IRT (IFA) PG-3 (RTGA) VD-BS (IFA) vaccinated cows and heifers 2560,00 ± 275.41 2880.00 ± 490.10 2560,00 ± 674.62 256.00 ± 51.52 2560,00 ± 674.62 control 160.00 ± 52.59 120.00 ± 56.22 240.00 ± 87.77 28.00 ± 10.52 200.00 ± 62.85

Information sources

1. Aliyev, N.A. Adenovirus infection of calves / N.A. Aliyeva, M.K. Ganiev, Dreizin R.S., Alieva R.A., G.A. Saryev // Transactions of AzNIVI. - 1967. - T.XXI.

2. Augustinavichus, B.V. / in the book. Scientific information. - Litas. scientific research vet. institute. - 1966. - P.3-5.

3. Akhmetsafin, R.G. Improvement of diagnostic tools and specific prophylaxis of mixed respiratory infections of calves / R.G. Akhmetsafin // Abstract. dis. ... cand. vet. sciences. - Kazan. - 2005. - 19 p.

4. Belousova, R.V. Inactivated bivalent vaccine against viral diarrhea and adenovirus infection in cattle and a method for the prevention of viral diarrhea and adenovirus infection in cattle / R.V.Belousova, T.P. Lobova, I.V. Tretyakova, I.Vazir / Application for invention No. 2008103716/13 dated 02/06/2008.

5. Belousova, R.V. A method of manufacturing an inactivated concentrated multivalent vaccine against infectious rhinotracheitis, parainfluenza-3, adenovirus infection and viral diarrhea of cattle / R.V. Belousova, I.V. Tretyakova, T.P. Lobova, N.V. Tiganova, L.K. .Kish, D.P. Kuznetsov / Application for invention No. 201052945/10 of 12.24.2010.

6. Vazir, Ya. Tests of a bivalent vaccine against adenovirus infection and viral diarrhea of cattle in rabbits using immunomodulators / Ya. Vazir, RV Belousova, GI Ustinova // Veterinary pathology. - 2007. - No. 3 (22). - S.199-202.

7. Gaffarov, Kh.Z. Infectious diseases of cattle of chlamydial, mycoplasma and viral etiology, the development of diagnostic and therapeutic drugs / Kh.Z. - Gaffarov // Abstract. dis. ... doc. vet. Sciences - Kazan. - 1986. - 39 p.

8. Gunenkov, V.V. Adenovirus infection of cattle / V.V. Gunenkov, V.N.Syurin, K.K. Vertinsky / Little-known viral infections of cattle. - Moscow, 1972.- 47 s.

9. Gunenkov, V.V. Viral and chlamydial respiratory and intestinal infections of cattle. / V.V. Gunenkov, G.A. Khalenev, V.N. Syurin // Livestock and veterinary medicine. Problems of Veterinary Medicine, a series of "Results of science and technology." - Moscow, 1975 .-- T.8. - S.87-95.

10. Zhidkov, S.A. The strain of the virus Bovine viral Diarrhea-mucosal disease virus for the manufacture of a vaccine against viral diarrhea in cattle / S.A. Zhidkov, N.N. Kryukov // Auth. Certificate SU 1322673 A1 dated 09.09.85.

11. Lobova, T.P. Improvement of laboratory diagnosis of adenovirus infection in cattle / T.P. Lobova // Abstract. dis. ... can. biol. sciences. - FGOU VPO MGAVMiB. - Moscow. - 2006. - 19 p.

12. Matveeva, I.N. Industrial technologies for the manufacture of components of mono - and complex diagnostic kits of infectious diseases of animals / I.N. dis. ... doc. biol. sciences. - Schelkovo. - 2008 .-- 53 p.

13. Syurin, V.N. Vaccine against adenovirus infection in cattle / V.N.Syurin, R.V. Belousova, L.M. Kolenkova, T.I. Litvinov, T.P. Lobova / Auth. certificate No. 44486434 / 30-13 / 138050 of 08.29.89.

14. Syurin, N.V. Adenovirus infection of cattle / V.N.Syurin, A.Ya. Samuilenko // Viral diseases of animals. - Moscow. - VNITIBP. - 1998. - S.795-801.

15. Yurov, K.P. The vaccine "Trivak (VIEV) for the prevention of infectious rhinotracheitis, parainfluenza-3 and viral diarrhea - diseases of the mucous membranes of cattle" / K.P. Yurov, A.S. Vecherkin, A.F. Shulyak, S.A. Zhidkov / Patent Application No. 96123539/13 of 12/11/1996.

16. Adamesteanu, L., Adamesteanu C. // Rev. zootehn. si med. vet. - 1973. - V.23. - No. 9. - P.51-56.

17. Aldasy, P. Pneumoenteritis in Caives Caused by Adenoviruses / P. Aldasy, L. Csontos, A. Bartha // Acta Vet. Acad. Sci. Hung - 1965. - 15. - P.175-176.

18. Animal Health Products / Vaccine against enzootic bronchopneumonia and infectious bovine rhinotracheitis (IBR) ("Bovigrip plus"), inactivated, for cattle. Aqueous suspension for subcutaneous injection. / Hoechst Veterinar GmbH, 1992. - P.41-43.

19. Animal Health Products / Vaccine against enzootic bronchopneumonia, inactivated, for cattle ("Bovigrip") Aqueous suspension for subcutaneous injection. / Hoechst Veterinar GmbH. - 1992. - P.37-39.

20. Bartha, A. Immunization experiments on calves with type 4 bovine adenovirus / A. Bartha // Acta vet. Acad. Sci. Hung - 1967. - V.-17. - P.209-216.

21. Bartha, A. Proposal for sub grouping of bovine adenoviruses / A. Bartha // Acta vet. Acad. Sci. Hung - 1969. - V.19. - Number 3. - P.313-321.

22. Burki, F. Experimental Adenovirus Vaccines in Cattle / F. Burki // J. Amer. Vet. Med. Assoc. - 1973. - V.163. - No. 7. - P.897-900.

23. Darbyshire, J.H. Bovine Adenoviruses / J.H. Darby shire // J. Amer. Vet. Med. Assoc. - 1968. - V.152. - P.786-792.

24. Edwards, G.S. Wogan G.N. // Biochim. biophys. acta. - 1970. - V.224. - 597 p.

25. Gillespie, J. // J. Amer. Vet. Med. Assoc. - 1973. - 163. - No. 7. - P.901.

26. Gole, J. // J. Amer. Vet. Med. Assoc. - 1973. - No. 7. - R. 836-839.

27. Haralambiev, H. Hyperimmunserum Against Myxovims Parainfluenza-3 and Bovine Adenovirus in Prophylaxis of Respiratory Disease in Calves / H. Haralambiev, M. Draganov, P. Zwetkov // Arch. Exptl. Vet. Med. - 1972. - V.26. - P.273-276.

28. Inaba, Y. Bovine adenovirus. II A.Serotype. Fukuroi, Recoverend from Japanene Cattle / Y. Inaba, Y. Tanaka, Y.Sato, K.Ito, H. Ito, M. Matumoto // Jap. J. Microbiol. - 1968. - 12. - P.219-229.

29. Klein, H. Structure function relationships of the adenovirus DNA-binding protein / H. Klein, W. Maitzman, A. J. Levine // J. Biol. Chem. - 1979. - V.254. - P.11051-11060.

30. Mocsari, E., Saghy E., Kiss J., Fulessy E., Ovary. // Acta Vet. Acad. Sci. Hung - 1974. - 24. - No. 1-2. - P.39-43.

31. Mohanty, S.V. Comparative studies of Bovine Adenovirus // Amer. J. Vet. Res. - 1971. - V.32. - No. 12. - P.1899-1905.

32. Morzaria, S.P. A field trial with a multicomponent inactivated respiratory viral vaccine / S.P. Morzaria, M.S. Richards, J.W. Harkness, B.A. Maund // The Veterinary Record. - 1979. - V.11. - No. 3 - P.410-414.

33. Phillipp, J.I.H. The Isolation of Bovine Adenovirus Serotypes 4 and 7 in Britain / J.I. H. Phillipp, J. H. Sands // Res. Vet. Sci. - 1972. - 13. - P.386-387.

34. Pons, W. A., Cucullu A. F., Franz A. O., Lee Louise S., Goldblatt Leo A. // J. Assoc. Offic. Anal. Chem. - 1973. - 56. - No. 4. - P.803-807.

35. Rondhuis, P.R. Anew Bovine Adenovirus / P.R. Rondhuis // Arch. ges. Virusforsch. - 1968. - 25. - P.235-236.

36. Rondhuis, P.R. Bovine Adenoviruses. A Comparative Serological and Experimental Study / P.R. Rondhuis // Drukkerij-Uitgeverij G. van Dijk N.V. Breukelen. - 1970. - P.218.

37. Sawhney, D.S. Vadehre D.V., Baker R.C. // Poultry Sci. - 1973. - 52. - No. 2. - P. 465-473.

38. Schipper, E. Virus bedingte Respirations-Krankheiten in Kalber und Ridermastbetreiben / E. Schipper, J. Nicolet, H. Konig, F. Steck // Schweiz. Arch. Tierheilkunde. - 1972. - H. 114. - S.334-361.

39. Tribe, G.W. Combined bovine parainfluenza and adenovirus vaccine / G.W. Tribe, J. B. Kerry. // Vet. Rec. - 1969. - V.84. - P.299-303.

Claims (3)

1. The vaccine associated against adenovirus, herpes virus infections, parainfluenza-3 and viral diarrhea - diseases of the mucous membranes of cattle inactivated emulsion, including the cattle adenovirus antigen I of the subgroup "Adeno III WBR1-DEPT" reproduced in a transplanted culture of kidney cells line MDBK) with the biological activity of at least 6,25 lg TCD ₅₀ / ml and inactivated with formalin to a final concentration of 0.2-0.3%, viral diarrhea virus - mucosal disease "VK-DEP-1" in a reproduced pereviva my culture cow embryo kidney cells (MDBK line) with the biological activity of at least 6,5 lg TCD ₅₀ / ml, officinal formalin, a concentrated PEG 6000 at a final concentration of 7-8%, an adjuvant, wherein the vaccine further comprises an antigen adenovirus Cattle of the II subgroup “Adeno IV Weybridge ST2-DEP” reproduced in a transplanted culture of Taurus-1 (T-1) calf kidney cells with a biological activity of at least 6.5 lg TCD ₅₀ / ml and formalin inactivated to a final concentration of 0.2-0 , 3%, herpesvirus type I "TKA-VIEV-B2-DEPT" reproduced in a transplanted culture of kidney embryonic cells of a cow (MDBK line) with biological activity of at least 7.5 lg TCD ₅₀ / ml and formalin inactivated to a final concentration of 0.2-0.3%, parainfluenza virus-3 cattle strain "SF-4- DEPT ”reproduced in a transplanted culture of lung cells of a cow embryo (LEC) with biological activity of at least 6.5 lg TCD ₅₀ / ml and hemagglutinating activity of at least 6 log ₂ (1:64), inactivated with formalin to a final concentration of 0.2- 0.3%, in a ratio of 1: 1: 1: 1: 1. 2. The vaccine according to claim 1, characterized in that it contains concentrated antigenic material from the Adeno III WBR1-DEP strain of cattle adenovirus subgroup I, concentrated antigenic material from the Adeno IV Weybridge CT2-DEP strain of cattle adenovirus Subgroup II, concentrated antigenic material from the TKA-VIEV-B2-DEP strain of herpesvirus type I, concentrated antigenic material from the SF-4-DEP strain of cattle parainfluenza-3 virus, concentrated antigenic material from the VK-1 strain -DEP "virus VD-BS to cattle, while the components are part of the vaccine in the following ratio, vol.%:
Concentrated antigenic material from the Adeno III WBRl-DEP strain of cattle adenovirus reproduced in a transplanted culture of cow embryonic kidney cells (MDBK line) 10.0-11.0 Concentrated antigenic material from Adeno IV Weybridge ST2-DEP strain of cattle adenovirus reproduced in an inoculated Taurus-1 (T-1) calf kidney cell culture 10.0-11.0 Concentrated antigenic material from the TKA-VIEV-B2-DEP strain of herpesvirus type I reproduced in a transplanted culture of cow embryonic kidney cells (MDBK line) 10.0-11.0 Concentrated antigenic material from the SF-4-DEP strain of parainfluenza 3 reproduced by the transplanted culture of lung cells of a cow embryo (LEK) 10.0-11.0 Concentrated antigenic material from the strain “VK-1-DEPT” of the VD-BS virus of cattle reproduced in a transplanted culture of kidney cells of a cow embryo (MDBK line) 10.0-11.0 Adjuvant up to 100 3. The vaccine according to claim 1, characterized in that as adjuvant use synthetic oil PES-3M and anhydrous lanolin in a ratio of 5: 1.