RU2452512C2 - Inactivated emulsion associated vaccine for parvoviral, rheoviral, type i herpes-viral infections and viral diarrhoeia - cattle mucosal disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology. What is described is an emulsion inactivated associated vaccine for parvoviral, rheoviral, type I herpes-viral infections and a viral diarrhoeia - cattle mucosal disease (CMD). The vaccine contains a mixture of the concentrated antigen materials of Parvo 32459-DEP parvovirus strains of type I CMD, Reo I - Lang-DEP rheovirus strain of type I CMD, TK-A- (VIEV)-V2-DEP herpes-virus strain of type I CMD and NADL-DEP virus strain of viral diarrhoeia - cattle mucosal disease. The antigen material of the strains is inactivated by 1.2.-aminoethylaziridine. The concentrated and avirulent antigen materials are bound with an adjuvant in the effective relation.

EFFECT: vaccine induces a high level of antibodies to parvoviral, rheoviral, type I herpes-viral infections and viral diarrhoeia - cattle mucosal disease, provides reliable protection of a foetus, newborn calves, young growth of completion of growing and sagination, and also pregnant cows.

4 cl, 5 tbl, 6 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular to a technology for the manufacture of a vaccine against viral respiratory and intestinal infections of calves and the resulting reproductive dysfunctions of adult cattle.

It is known that in the structure of the incidence of agricultural animals in the Russian Federation, respiratory and gastrointestinal infections of calves and the pathology of the reproductive organs of the uterine livestock of cattle are associated with early embryonic mortality, abortions, the prevailing pathogens of which are viruses belonging to various taxonomic groups. Among them, according to the severity of the manifestation of the clinical picture, herpesvirus infection type I (IRT), viral diarrhea - mucous membrane disease (VD-BS), respiratory syncytial (PC), parvovirus, adenovirus, reovirus, and coronary virus infections of the cattle come to the forefront. cattle, various combinations of which cause not only damage to the respiratory and digestive organs of calves, but also impaired reproductive organs of the adult population. It has also been proven that semen from bulls infected with these viruses is a source of infection for cows and heifers. In this case, the incidence of calves reaches 80-100%, is accompanied by frequent relapses and death from 20-30 to 56% of sick animals, causing significant economic damage to livestock.

The information available in the literature of recent years and the knowledge accumulated by us indicate that there is an increase in the level of infection of cows and heifers with pathogens that can affect the embryo and fetus. These are, along with herpes virus and VD-BS virus, and parvovirus and cattle reovirus, which are poorly studied in the Russian Federation, which can penetrate the placenta through the placenta, causing abortion and stillbirth. In this case, the emergence and spread is not limited by external factors, and the introduction of these viruses into the economy naturally leads to outbreaks of the above forms of pathologies.

It is noteworthy that foreign authors attach great importance in the etiopathogenesis of these pathologies to parvovirus type I of cattle, which is antigenically different from parvoviruses of other animal and human species, causing not only damage to the digestive and respiratory organs in calves, but also impaired reproductive function in cows and heifers, manifesting themselves clinically as embryonic mortality, abortion, stillbirth, endometritis, and ovarian hypofunction (Elschner, M. Untersuchungen zur Diagnostik von bovinen Parvoviren in Kotproben von Kalbern / Berl. mun. ch, tierarztl. Wschr. - 1994. - Jg. 108. - H.7. - S.256-260; David Sandals WC, Charles Povey R. and Alan H. Meek. Prevalence of Bovine Parvovirus Infection in Ontario Dairy Cattle / Can J. Vet. Res. 1995. 59: 81-86).

Parvovirus infection of cattle is also known to be widespread among naturally susceptible animals in the United States, Europe, Japan, Canada and Algeria. At the same time, the level of antibodies in the blood serum of cows in the USA was 65-85% of the examined cattle, in Algeria - 70%, in the UK - 46% (Storz, J., Bates RC Parvovirus infection in calves / J. Am. Vet. Med. Assoc. - 1973. - V.163. - P.884-886).

Clinically, parvovirus infection in calves is manifested mainly in the form of damage to the digestive system: diarrhea develops 24-48 hours after infection (Spahn, GJ, et al., Characteristics of hemadsorbing enteric (HADEN) virus / Canad. J. Microbiol. - 1966. - V.12. - P.653-660 .; Durham P. JK et al., Epidemiological studies of parvovirus infection in calves on endemically infected properties / J. Res. Vet. Sci. - 1985. - V.38 . - No. 2. - P.234-240; Sandals, WCD Prevalence and seroepidemiology of bovine parvovirus / MSc Thesis, University of Guelph, 1989. - P.432-437).

In addition, it turned out that in experimentally infected calves, the virus is regularly detected in the lymph nodes, spleen, thymus; it accumulates in high titers in the adrenal gland cells. Moreover, the most common specific viral antigen is found in the epithelial cells of the jejunum, ileum, and cecum, as well as in blood leukocytes (Storz J. et al., Parvovirus infection in calves / J. Am. Vet. Med. Assoc. - 1973. - V .163. - P.884-886).

During experimental infection of cows at the 3-4th month of pregnancy intravenously or at the 5-6th month in the amnion, they aborted, respectively, after 3-5 weeks. The virus was found in the thymus, lungs and spleen of mummified fruits. Moreover, parvovirus persisted for a long time in the body of a cow (Bachman, PA Properties of a bovine parvovirus // Zbl. Vet., Med., 1871. - P.80-85; Bodine, AB, et al., Possible "immuno-protection" of the bovine parvovirus in the uterus: Preliminary communication / J. Possible Theriogenology. - 1981. - V.16. - P.201-206).

In our country, the first reports of the isolation of the B-2 strain of bovine parvovirus, assigned to the third serotype of parvoviruses and antigenically different from the reference Haden strain, were published in 1976 (Zudilina ZF et al. Isolation of large parvovirus cattle / Actual issues of veterinary virology. - Vladimir. - 1976; Kryukov, NN et al. Parvovirus infection of cattle / Veterinary medicine. - 1988. - No. 7. - P.27-29; Kurov P. et al. Parvovirus Infection of Cattle / Veterinary Medicine - 1994. - No. 5. - C.2 6-27; V. Mishchenko et al. Features of parvovirus infection in cattle / Veterinary medicine. - 2000. - No. 5. - S.19-21).

At the same time, the authors emphasized the widespread prevalence of parvovirus infection, the pathogenicity of this virus for cattle, its ability to penetrate the tissues of various parts of the intestine and adjacent lymph nodes, as well as to overcome the placental barrier, causing embryonic mortality.

Since 1959, it has been known that in calves, reovirus infection is manifested by pneumoenteritis in the first 3 months of life. In adult animals, the disease is latent. It was first registered in the USA, Belgium, Germany and other countries. In Germany, the participation of reoviruses in the defeat of the respiratory tract of calves, in the pathology of the fetus and newborn is proved. In a study of 283 animals 2-6 years old at 47 Swedish farms with clinical signs of acute respiratory and (or) intestinal diseases, it was found that 71-81% of the studied sera reacted with type I reovirus (Moreno-Lopez I. A Serosurvey of viruses during outbreaks of acute respiratory and / or enteric disease in Swedish cattle. / Zbl. Veter. - Med. Reihe B., 1979, 26, 8: 634-640).

Also noteworthy are reports that strains of human and animal reoviruses cause the same changes: they multiply in the same tissue cultures and cause similar reactions in natural hosts. Possible diseases of people with infection with bovine reovirus type I and calves with infection with any of the serotypes of the human virus. Therefore, reovirus infection can be attributed to zoonoses, which simultaneously affect cattle, horses and humans, like the flu. Reoviruses due to their high contagiousness and stability in the environment are often the cause of epidemic outbreaks. In the circulation of the virus, asymptomatic virus carriage plays an important role. The virus is pathogenic for sucker mice when infected in the brain, subcutaneously, intraperitoneally or intranasally, their death occurs in 5-12 days with damage to the liver, myocardium, nerve cells (Ritova V.V. et al. Reoviruses and their role in human infectious pathology and animals / Questions of virology. - 1977. - No. 2. - S.134-141).

It is known that the efficiency of dairy cattle breeding is determined by the high productivity of cows and their safety. The accelerated development of livestock breeding, provided for by the priority national project “Development of the agro-industrial complex”, is carried out by increasing the number of highly productive cows not only by raising the local population, but also by importing livestock from abroad. So, for example, since 2004 about 30 thousand cattle from Germany, Holland, Hungary, Denmark, Canada, Austria, Estonia, and Australia were brought to the Republic of Tatarstan (RT).

The widespread spread of steam and reovirus infections in Western Europe, given the likelihood of these viruses being imported with imported livestock, determined the need to study 838 cattle blood serum samples obtained from 21 households in 6 regions of the Republic of Tatarstan. The results of our studies showed that seropositivity in ELISA for parvovirus antigen is 72.2% (605 samples) and reovirus antigen - 33.7% (194 samples). A higher percentage of positively responding animals was observed on farms where imported livestock was housed.

The results obtained indicate the fact of the circulation of these pathogens among various age groups of cattle.

Their distribution is facilitated by the large concentration of animals coming from various epizootic farms, the ever-increasing industrialization of keeping and exploitation, the widespread exchange of animals within the country and the importation of valuable highly productive animals from abroad, the difficulties of organizing the continuous production of full-fledged feeding and creating an optimal microclimate. Therefore, favorable conditions are created for the circulation and increased virulence of the above pathogens, which are most often in association, against the background of stress factors, can cause an epizootic process.

For many years, the role of parvovirus and reovirus infections in cattle pathology has been underestimated in the Russian Federation, which hampered the development of laboratory diagnostics and specific prophylaxis.

Noteworthy are the statements of a number of researchers that the vaccines so far created have not solved the problem, although with their use the incidence decreases, but a decisive fracture is not achieved. For example, in the recommendations of the Russian Academy of Agricultural Sciences (Siberian Branch), GNU IEVS and DV “Planning of vaccination programs for viral respiratory diseases of cattle”, prepared by a group of scientists with the participation of heads of the veterinary department of the Tyumen and Novosibirsk regions (A.G. Glotov, T. I. Glotova, A.V. Nefedchenko, V.A. Kachanov, Yu.N. Zaitsev / RAAS, Sib. Department, GNU IEVS and DV. - Novosibirsk, 2006. - 36 pp.), It is also emphasized that unfortunately, the protection of the fetus induced by vaccines produced in the Russian Federation is not always complete and there are no such vaccines yet, showing full protection. This suggests that the etiological structure of respiratory and intestinal infections of young animals and pathologies of the reproductive organs of adult cattle are not fully disclosed in the country.

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 rhinotracheitis of cattle, which is used in the Russian Federation in cases of this infection, and immunize all animals located in the epizootic focus, as well as in farms of the meat direction with stationary dysfunction vaccinate all animals suspected of disease and suspected of infection.

Known vaccine against parainfluenza-3 and infectious rhinotracheitis of cattle dry, cultured, associated, containing lyophilized parainfluenza vaccine virus-3 from strain PTK-45/86 and vaccine virus of infectious rhinotracheitis from strain TKA (VIEV) grown in a calf kidney cell culture , and a protective drying environment. The vaccine is administered subcutaneously to calves aged 1-6 months twice, 7 months and older - once (O.I.Sukharev, N.G. Ozidze, N.R. Gladchenko and others. Associated vaccine "Bivak" against infectious rhinotracheitis and parainfluenza 3 cattle, a method for its manufacture and a method for the prevention of infectious rhinotracheitis paragippa-3 / A.S. No. 1092778. - Declared December 17, 1982, No. 3523289).

Known vaccine associated against parainfluenza-3, infectious rhinotracheitis and chlamydia in cattle inactivated, emulsion. The vaccine contains inactivated concentrated antigens of parainfluenza virus 3 strain PTK-45/86, infectious rhinotracheitis strain TKA (VIEV) and the causative agent of chlamydial abortion of cows strain "250", emulsified in an oil adjuvant (Certificate of state registration No. PVR-1- 4.8 / 02246 dated August 27, 2008, valid for August 27, 2013).

A well-known inactivated combined vaccine against viral diarrhea, rota-, coronavirus disease and calf Escherichiosis (Kombovak K), which is a comprehensive drug for the prevention of four 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, F41; 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) and Pasteurella haemolitica.

Known inactivated combined vaccine against infectious rhinotracheitis, parainfluenza-3, viral diarrhea, respiratory syncytial, rota-, coronavirus disease of calves (Combovac), which is a composition of six strains of viruses inactivated with formalin containing not less than 37% formaldehyde and adsorbed on 6% gel of aluminum hydroxide (GOA) and saponin, intended for immunization of calves, heifers and cows in farms that are not healthy for gastrointestinal respiratory diseases. A common drawback of the above known vaccines is the absence of pathogens such as parvovirus and reovirus in their composition, which significantly reduces its effectiveness. A similar picture is observed when analyzing the composition of the components of the Bovie Shield Gold FP5 L5 vaccine (Pfhizer Animal Health Products. - 1992. - P.37, 41, 117.USA), which contains live attenuated pathogens of ИРТ, ВД, ПГ-3, RSV, as well as inactivated leptospira 5 serotypes. This vaccine imported into the Russian Federation also does not contain the antigens of parvo and reoviruses that are so widespread according to the results of seromonitoring for parvo and reovirus infections among livestock, especially in animals imported from Western Europe.

From the above information, it follows that at present, there are no domestic analogues of vaccines intended for specific prophylaxis of viral respiratory and intestinal infections of calves and the pathologies of reproductive organs of the uterine livestock made on the basis of parvovirus and cattle reovirus antigens.

Closest to the proposed invention in terms of essential features is the “Combined vaccine against infectious rhinotracheitis, pragrippa-3, viral diarrhea, respiratory syncytial, rota-, coronavirus calf disease” (Combovac) manufactured by NPO Narvak, which is a composition of six strains viruses inactivated by formalin and sorbed on a 6% gel of aluminum hydroxide (GOA) and saponin (Sergeev V.A., Nepoklonov A.E., Alipper T.I., RF patent for the invention No. 2261111 of 04/08/2004). A common drawback of these known vaccines against viral respiratory and intestinal infections of calves and pathologies of the reproductive organs of adult livestock is their low antiepizootic efficacy due to the fact that the strains of respiratory and intestinal viruses of cattle used for their manufacture have a narrow antigenic spectrum against pathogens causing pathology of the reproductive organs of the uterine livestock. The “Inactivated combined vaccine against infectious rhinotracheitis, pragrippa-3, viral diarrhea, respiratory syncytial, rota-, coronavirus calf disease” vaccines (“Combovac”) contains no antigens of bovine paro- and reoviruses. Foreign manufacturers of biological products attach great importance to these viruses in the etiology of respiratory and intestinal infections of calves and in the pathology of the reproductive organs of the uterine livestock. They experimentally confirmed the feasibility of using them for the manufacture of vaccine preparations. In this regard, attention was drawn to insufficiently studied viruses - potential pathogens of cattle diseases, suitable for inclusion in the composition of the claimed object.

Literature data indicate that in many countries of the world mono-, bi- and polyvalent vaccines are produced against IRT, PG-3, VD-BS and RS infection, parvovirus and reovirus infections and carry out active immunization of calves according to various programs, depending on epizootic situation. Moreover, both the uterine herd and young animals of different ages are vaccinated.

1. Vaccines associated under the names "Lactovac" containing the inactivated antigen of the strain "Haden" of parvovirus type I cattle, "Bovigrip" and "Bovigrip plus", which include inactivated reovirus antigens (strains "Lang" and "Dearing") , produced by the world's largest German company Hoecht Veterinär GmbH, successfully used in many countries of Western Europe: France, Germany, England, Yugoslavia, Sweden, Belgium, Denmark and others (Morzaria SP, Richards MS, Harkness JW, Maund BA A field trial with a multicomponent inactivated respiratory viral vaccine / Veterinary Record. - 1979. - 105. - P.410-414; Soulebot JP Prophylaxie medicate des affections respiratoires des bovines d'origine virale (IBR, PL ₃ , reovirus, maladie des muqueuses) / Rec. Med. veter. 1985. - 161, 12: 1271-1275; Thomson JR, Nettleton, PP, Greig, A. , Barr, J. A bovine respiratory virus vaccination trial / Veter. Rec. - 1986. - 119. - 18: 450-453; Animal Health Products. - 1992. - P.37, 41, 117).

The prototype - the initial sample when creating a new vaccine, the proposed invention was a “Vaccine against enzootic bronchopneumonia and infectious bovine rhinotracheitis inactivated for cattle" containing inactivated parainfluenza-3 antigens (strain "SF-4"), bovine adenovirus 1st (strain "No. 10") and 3rd (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 prototype has significant disadvantages:

- it does not provide protection of animals against parvovirus infection, viral diarrhea - diseases of the mucous membranes of cattle;

- as an inactivant, it contains formalin, which causes destructive changes in viral proteins and reduces the immunogenic activity of the vaccine;

- it has insufficient antigenic and immunogenic activity due to the use of non-concentrated cultural viral suspensions in its manufacture. In this regard, the problem of creating an associated emulsion inactivated vaccine against parvovirus, reovirus, herpes virus type I infections and viral diarrhea, a disease of cattle mucous membranes with high antigenic and immunogenic activity and harmlessness, remains relevant and is the main direction of research to improve it .

The need to develop and manufacture this vaccine is due to the polyetiological structure of the observed forms of pathologies in calves of various age groups and reproductive organs in cows and their widespread occurrence in various regions of the Russian Federation. This is confirmed by the research results of both foreign and domestic scientists (Table 1).

The objective of the present invention is to obtain a highly immunogenic vaccine associated inactivated emulsion against parvovirus, reovirus, herpes virus type I infections and viral diarrhea - mucosal diseases based on production strains that have high biological, antigenic and immunogenic activity in their native form and after inactivation and suitable for the manufacture of associated emulsion inactivated drug, creating effective protection of the fetus, newborn calves, young during the rearing and fattening period, 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 available prophylactics currently 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 parvovirus type I, reovirus type I, herpesvirus type I and VD-BS virus circulating in Russia Federation.

The proposed vaccine contains an active biological substance in the form of avirulent purified and concentrated antigens of Parvo 32459 strains of parvovirus type I cattle identical to the Haden strain in PBH and RTGA, Reo I - Lang strain of Reovirus type I, Nadl strain virus VD-BS and strain "TK-A-VIEV-B2" of herpesvirus type I obtained in cultures of mammalian cells and target additives in the form of an oil adjuvant in an effective ratio. The first three strains of viruses were obtained from the Central Veterinary Laboratory of the UK Ministry of Agriculture. Their immunobiological properties were studied, and the strains 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 Institution “Federal Center for Toxicological and Radiation Safety of Animals” of the Ministry of Agriculture of the Russian Federation (Federal State Institution FTsTRB-VNIVI) ) The strain “TK-A-VIEV-B2" of herpesvirus type I was deposited in the collection of FGU "VGNKI".

The essence of the invention is as follows: "Associated vaccine against parvovirus, reovirus, herpesvirus type I infections and viral diarrhea - inactivated emulsion cattle mucous membrane diseases" contains concentrated antigens of parbo-, re-, herpesviruses and VD-BS virus in cattle, emulsified in an oil adjuvant, in the following ratio of components, wt.%: 1: 1: 1: 1 (inactivated by 1.2-aminoethylaziridine).

Avirulent, purified and concentrated culture suspension of parvovirus type I cattle pcs. "Parvo 32459-DEPT" obtained in the primary cell culture of kidney cells of a cow embryo "PEC" with an infectious titer of at least 6.0-6.5 log ₂ TCD ₅₀ / cm ³ and activity in the RGA of at least 11 log ₂ (1: 2048 ) 12.5-14.5 wt.%.

Avirulent, purified and concentrated culture suspension of Reovirus type I strain "Reo I - Lang-DEPT" obtained in transplantable cell cultures of "Vero" and (or) PT-80, MDVC with an infectious titer of at least 6.0-6.5 log ₂ TCD ₅₀ / cm ³ and activity in the RGA not less than 10 log ₂ (1: 1024) 12.5-14.5 wt.%.

Avirulent, purified and concentrated culture suspension of herpesvirus type I (RTI) of cattle strain "TK-A-VIEV-B2-DEPT" obtained in transplantable cell culture "MDBK" with an infectious titer of at least 6.5-7.1 log ₂ TCD ₅₀ / cm ³ 12.5-14.5 wt.%.

Avirulent, purified and concentrated culture suspension of VD-BS virus in cattle strain "Nadl-DEPT" obtained in transplantable cell culture "MDBK" with an infectious titer of at least 6.0-6.5 log ₂ TCD ₅₀ / cm ³ 12, 5-14.5.

The target additive adjuvant is up to 100.0.

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

- strain “Parvo 32459-DEPT” of parvovirus type I cattle isolated from feces of a dead 3-month-old calf with signs of enteritis, identical to the reference strain of “Haden” virus in the neutralization reaction and in rtga. The most characteristic property of this strain of parvovirus in cattle is the ability to stably agglutinate erythrocytes of guinea pigs, humans of the 0-group. It is pathogenic for calves, it is able to penetrate the tissues of various parts of the intestine and adjacent lymph nodes, as well as to overcome the placental barrier, causing death of the embryo, causing abortion in cows, and to persist for a long time in the body of a cow. The strain "Parvo 32459-DEPT" parvovirus type I cattle is stable in the external environment. Reproduction of the virus occurs most actively in dividing cells.

The strain Parvo 32459-DEPT belongs to parovovirus type I cattle, belongs to the family Parvoviridae, the genus Parvovirus, DNA-containing, was isolated in the UK (1975) from the feces of a fallen 3-month-old calf with signs of enteritis, identical to the reference strain Haden "Virus in the neutralization reaction and in the rtga (cited by N. Kryukov et al., 1988). The most characteristic property of this strain of parvovirus in cattle is the ability to stably agglutinate erythrocytes of guinea pig and human 0-group. It is pathogenic for calves, it is able to penetrate the tissues of various parts of the intestine and adjacent lymph nodes, as well as to overcome the placental barrier, causing death of the embryo, causing abortion in cows, and to persist for a long time in the body of a cow. Reproduction of the virus occurs most actively in dividing cells. The “Parvo 32459-DEP” strain of bovine type I parvovirus is resistant to changes in pH from 3 to 8, ether, chloroform and 1% trypsin, and heating at 70 ° C for 2 hours reduces the infectious activity by 100 times , thermostable at 37 and 56 ° C.

- The strain “Reo I - Lang-DEPT” of the first type belongs to the genus Reovirus, the family Reoviridae, the genome of which consists of several fragments of double-stranded RNA. The name “reoviruses” (reo-respipatory enteric orphan viruses) reflects the relationship between the respiratory tract and the intestinal tract. Mammalian reoviruses are classified into three serological types based on RVN and RTGA. All three serotypes of reoviruses have a group-wide complement-binding antigen. Reoviruses isolated from humans and animals do not differ from each other and are able to infect the epithelial cells of the mucous membrane of the respiratory tract and digestive apparatus.

Reovirus virions are spherical particles with a diameter of 75-80 nm, have a clear two-capsid structure. In reovirus preparations, along with full particles, empty covers are detected, which is the result of the absence of viral nucleic acid.

Reoviruses have a pronounced resistance to the influence of the temperature factor. For example, type I reovirus at 36.7 ° C reduced the infectious titer by 2 lg for 3 weeks and at 56 ° C for 30 minutes. In the cold, samples can be stored at -20 ° C from 8-10 months to 2 years. At 4 ° C, the virus will retain its infectious properties for 2 months. They tolerate a wide range of pH (from 2.2 to 8), but are completely inactivated by 70% ethanol or 3% formalin at 56 ° C for 30 minutes.

In cattle, disease can be caused by intranasal infection with each of the three serotypes of human reovirus. Experimentally infected calves transmit the infection to healthy animals upon contact.

- The strain "TK-A- (VIEV) -B2-DEPT" of herpesvirus type I of the family Herpesviridae is a homogeneous population of DNA-containing virus, obtained by exposure to ultraviolet rays on the initial virulent virus of IRT at a dose close to the threshold. The vaccine strain "TK-A- (VIEV) -V2-DEPT" has high immunogenic properties and we have identified as a manufacturing one for the manufacture of the associated vaccine.

Herpesvirus type I (IRT) is reproduced in the monolayer of the cells of the kidney of the cow embryo, and its cytopathogenic effect occurs after 48-96 hours. The virus grown in a monolayer of transplantable cow embryonic kidney cell lines (MDVC) has stable activity and reaches 6.75-7.25 log ₂ 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 strain "Nadl-DEP" of the virus of viral diarrhea - diseases of the mucous membranes is a representative of the genus Pestivirus of the family Flaviviridae, isolated from outbreaks of respiratory infections in cattle along with other reference strains "Oregon", "Singer", "Osloss", refers to 1- subgroup of cytopathogenic strains. It was adapted by transplanted calf kidney cell culture lines (MDVC), and the infectious titer was 6.5 lg TCD ₅₀ / cm ³ . The most technologically advanced and highly productive are considered transplantable cultures of saiga kidney and Taurus-2 kidney cells. For 96-120 hours, the virus accumulates in these cells at the level of 6.5-6.7 lg TCD ₅₀ / cm ³ and 6.8-7.0 lg TCD ₅₀ / cm ³ , respectively (S. Levchenko. Study of immunobiological properties the causative agent of viral diarrhea - diseases of the mucous membranes of cattle used for the manufacture of inactivated vaccines: Abstract of thesis, Candidate of Biological Sciences. - Vladimir, 2008. - 25 p.).

When cows are infected with the Nadl-DEPT strain of the VD-BS virus, early mortality of embryos, abortion, stillbirth, underdevelopment of fetuses, a small mass of born calves, the presence of viral persistence and immunological tolerance are observed.

The virus is sensitive to ether, chloroform, trypsin; when the pH of the medium changes from 5.7 and 9.3, its infectivity decreases, it is most stable at pH 7.4. It is well preserved in its native form without the addition of stabilizing solutions at 4, -20, -40 ° C and in a lyophilized state, restoring the specified characteristics.

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 viruses. The possibility of their use for the manufacture of an associated vaccine, which creates intense and prolonged immunity in vaccinated animals, has been 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 . The definition from the list of identified analogues of the prototype (associated vaccines "Lactovac" containing inactivated antigen of the strain "Haden" parvovirus cattle, "Bovigrip", "Bovigrip plus", which includes inactivated antigens of reovirus strains "Lang-I" and "Dearing -2 ", manufactured by" Hoecht Veterinär GmbH "(Animal Health Products. - 1992. - P.37, 41, 117)) as the closest in combination of features with respect to the technical result perceived by the applicant as distinctive features of the proposed vaccine set forth in the independent clause formulas from grip.

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 antigenic material from the Parvo 32459-DEP strain of parvovirus type I cattle for the manufacture of the proposed vaccine is prepared from the mother virus grown on a monolayer of the primary culture of cow embryonic kidney cells (PEC), on a combination medium, and exhibiting infectious activity in the cell culture of PEC not less than 6.0 lg TCD ₅₀ / cm ³ and GA activity of not less than 1: 2048 GAE. To obtain antigenic material, 3-day-old PEC cell culture grown in mattresses is used, with a sowing cell concentration of 140-150 thousand cells per 1 ml of medium. To obtain 10 liters of parvovirus suspension, 55-60 mattresses are taken with a cell culture of 1.5 dm ³ each.

The growth medium, consisting of GLA medium - 80%, medium 199 - 10%, embryonic serum of cattle - 10% and antibiotics, is drained and 3-5 cm ^{3 of} parvovirus of mother seed are drained. After 40-60 minutes of incubation at a temperature of (37 ± 2) ° C, 150-200 cm ^{3 of} supporting medium are introduced into the mattresses (GLA - 90%, medium 199 - 10%, antibiotics). Infected mattresses are placed in a cultivation thermostat at a temperature of (37 ± 2) ° С. The cultivation period is from 72 to 96 hours. As a control, 3 uninfected mattresses are left, in which the growth medium is changed to 200 cm ^{3 of} maintenance medium without serum. Infected and control mattresses are microscopied daily. Mattresses in which cytopathic changes with a lesion are observed, more than 80-90% of the cells, are selected, frozen three times at a temperature of 20 ° C and thawed. The virus-containing material is then cleaned of cellular detritus by centrifugation at 2000 rpm for 20 minutes, subject to sterile conditions.

The purified virus-containing suspension is inactivated with 1.2-aminoethylaziridine. For this, aminoethyl aziridine is diluted with buffer to 5% concentration, adjusted to pH 8.2–8.4 with glacial acetic acid (working solution) and introduced into the viral suspension with constant stirring to a final concentration of 0.1%. Inactivation is carried out in a thermostat at a temperature of (37 ± 2) ° C for 24 hours with periodic stirring.

The concentration of the parvovirus suspension is carried out using PEG-600 to a final concentration of 7% (weight / volume) and NaCl to 0.3 M for 16-18 hours at 4 ° C. The precipitate was collected by centrifugation at 4000 rpm for 60 minutes and resuspended. The parvovirus antigen is standardized by the quantification of a specific protein on a spectrophotometer. The concentration of protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is tested for sterility, avirulence and HA activity.

Example 2

Antigenic material from the Reo I Lang-DEP strain of type I reovirus for the manufacture of the proposed vaccine is prepared from the mother virus grown in transplantable cultures of Vero and (or) PT-80 and MDVC cells of 3 days old concentration cells 120-140 thousand cells / cm ³ . A master virus is considered suitable for producing viral raw materials if it meets the following requirements: the infectivity titer after reproduction in a monolayer of Vero and (or) PT-80 and MDVC cells 6.0 lg TCD ₅₀ / cm ³ , GA- activity not less than 1:64, pH 7.2-7.4 and in the absence of contamination.

For the manufacture of 10 liters of culture production reovirus take 55-60 mattresses with a cell culture with a capacity of 1.5 DM ³ each. After the growth medium is drained, 5 cm ^{3 of the} virus-containing material of the mother seed are introduced into the mattresses. Virus adsorption is carried out at 37 ° C for an hour. After that, a supportive medium (MEM needle 40%, GLA medium 60%, trypsin 20 μg / cm ³ , antibiotics) in a volume of 150-200 cm ^{3 is} introduced into the mattresses.

Cultivation is carried out at 37 ° C for 4-5 days. For 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.

Cellular detritus is precipitated by centrifugation at 2000 rpm for 20 minutes. The supernatant virus-containing suspension is inactivated. Inactivation is carried out using a 5% working solution of 1.2-aminoethylaziridine in a final concentration of 0.08-0.1% in a thermostat at a temperature of (37 ± 2) ° C for 24 hours with periodic stirring.

The concentration of the reovirus suspension is carried out using PEG-600 to a final concentration of 7% (weight / volume) and NaCl to 0.3 M for 16-18 hours at 4 ° C. The precipitate was collected by centrifugation at 4000 rpm for 60 minutes and resuspended. Reovirus antigen is standardized by the quantification of a specific protein on a spectrophotometer. The concentration of protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is tested for sterility, avirulence and HA activity.

Example 3

The antigenic material from the strain "TKA-VIEV-B2-DEP" of herpesvirus type I for the manufacture of the proposed vaccine is prepared from the mother virus reproduced in the transplanted cell culture "LEK".

The LEK cell culture is grown for 3-4 days at an inoculum concentration of 140-150 thousand cells in a combination medium consisting of GLA medium - 60%, MEM Needle - 30% and cattle serum - 10%.

To prepare 10 liters of culture production herpesvirus type I take 55-65 mattresses with a cell culture of 1.5 dm ³ each.

The growth medium is drained, 3 cm ^{3 of} herpes virus with an infectious titer of 6.5 lg TCD ₅₀ / cm ³ , free of bacterial and fungal microflora, are introduced into the mattresses. Virus adsorption is carried out at 37 ° C for an hour. After that, the following is introduced into the mattresses: a supportive environment (GLA - 60%, MEM Needle - 40% and antibiotics) in a volume of 150-200 cm ³ .

Cultivation is carried out at 37 ° C for 48-72 hours until the appearance of cytopathic changes in the cells. As a control, 3 mattresses are left uninfected.

Mattresses in which cytopathic changes are observed with damage to more than 80-90% of the cell monolayer are selected, frozen three times, thawed and the contents are poured into one container, subject to sterile conditions. The virus-containing suspension is freed from cell detritus by centrifugation at 2000 rpm for 20 minutes.

The purified virus-containing suspension is inactivated with 1.2-aminoethylaziridine, which is previously diluted with buffer to 5%, and the pH is adjusted to 7.4-7.6 with glacial acetic acid. Aminoethylaziridine working solution is introduced with constant stirring into a virus-containing suspension to a final concentration of 0.2%. Inactivation is carried out for 24 hours at a temperature of (37 ± 2) ° C with periodic stirring.

The concentration of herpes virus antigen is carried out using PEG-6000 to a final concentration of 7-8% (weight / volume) and NaCl to a final concentration of 3% for 18 hours at 4 ° C. The precipitate was collected by centrifugation at 4000 rpm for 60 minutes and resuspended. Herpes virus antigen is standardized by quantifying a specific protein in it on a spectrophotometer. The concentration of protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is checked for sterility, avirulence.

Example 4

Antigenic material from the NADL-DEP strain of the viral diarrhea virus - mucous membrane disease (VD-BS) of cattle for the manufacture of the proposed vaccine is prepared from the mother virus grown in a transplanted cell culture LEC 3-4 days old with a cell concentration of 140-150 thousand cells.

VD-BS master virus with an infectivity titer of at least 6.0 lg TCD ₅₀ / cm ³ , pH 7.2-7.4 and in the absence of contamination is considered suitable for producing viral raw materials.

To prepare 10 liters of VD-BS culture production virus, 50-65 mattresses with a cell culture of 1.5 dm ³ each are taken.

The growth medium is drained, 3 cm ^{3 of the} virus-containing material of the mother seed are introduced into the mattresses and left to contact at 37 ° C for an hour. After that, a supportive medium of 150-200 cm ^{3 is} introduced into the mattresses. As a control, 3 mattresses are left uninfected.

Cultivation is carried out at 37 ° C for 4-5 days until the appearance of cytopathic changes in the cells. Infected and control mattresses are microscopied daily. The culture fluid is collected at the time of the highest degeneration of monolayer cells (75-80%), while the virus titer should be 6.0-6.5 lg TCD ₅₀ / cm ³ .

The virus is released from cells by freezing and thawing three times. It is clarified by centrifugation at 2000 rpm for 20 minutes.

Inactivation of the purified viral suspension is carried out using a 5% working solution of 1.2-aminoethylaziridine in a final concentration of 0.15% at a temperature of 37 ° C for 24 hours.

Antigen concentration is carried out by adding 50% PEG-6000 to a virus-containing suspension to a final concentration of 7% (w / v) and NaCl to 0.3 M. The mixture is kept for 16-18 hours at 4 ° C until a precipitate forms. The precipitate was collected by centrifugation at 4000 rpm for 60 minutes and resuspended. VD-BS antigen is standardized by the quantification of a specific protein. The concentration of protein should be in the range of 1.8-2 mg / cm ³ . Concentrated antigenic material is tested for sterility and avirulence.

Example 5

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 in a ratio of 84:16 volume parts. A mixture of oil and lanolin is sterilized at 120 ° C (1.0 atm) for 40 minutes. The adjuvant is cooled, checked for sterility.

Example 6

Inactivated emulsion inactivated vaccine against parbo-, re-, herpesvirus infection and VD-BS of cattle is prepared by dispersing a mixture of concentrated antigenic materials from Parvo 32459-DEPT strains of parvovirus type I, “Reo I - Lang-DEPT”, reovirus type I, “ TK-A-VIEV-B2-DEP "herpesvirus type I," NADL-DEPT "virus VD-BS.

For this, concentrated antigenic materials obtained as described in examples 1, 2, 3 and 4 are mixed with vigorous stirring in a ratio of 1: 1: 1: 1, i.e. 1 part of parvovirus antigenic material, 1 part of reovirus, 1 part of herpes virus, 1 part of VD-BS virus, which corresponds to their content in the proposed vaccine, wt.%:

Concentrated antigenic material from the Parvo 32459-DEP strain of parvovirus type I 12.5-14.5 Concentrated antigenic material from strain "Reo I-Lang-DEPT" reovirus type I 12.5-14.5 Concentrated antigenic material from the strain "TK-A-VIEV-B2-DEPT" of herpesvirus type I 12.5-14.5 Concentrated antigenic material from strain "NADL-DEPT" virus VD-BS 12.5-14.5

The mixture of concentrated antigens is thoroughly emulsified with an oil adjuvant in the ratio, wt.%: 50:50.

If necessary, as a target additive, the proposed vaccine may contain a preservative sodium mertiolate in an amount of 0.001 wt.%, Which corresponds to 0.1 g of the drug per 10000 doses of the vaccine.

The finished vaccine is poured into sterile glass bottles and controlled in accordance with the technical conditions.

The vaccine is an emulsion of a white with a pink tint color, a slightly viscous consistency.

With long-term storage (over 12 months), a slight peeling of mineral oil over a non-stratified homogeneous emulsion is possible. When shaken, the vaccine gains a homogeneous uniform structure.

Sterility of the vaccine is determined in accordance with GOST 28085-89.

The virulence of the drug is determined by the method of three consecutive passages of inactivated antigens in the primary cell culture "PEC" and in transplantable cell cultures "Vero" and (or) "PT-80", "MDVK", "LEK" by the absence of CPD.

To test the safety, the vaccine is administered subcutaneously 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. At the injection site, a local tissue reaction is allowed.

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 38.3 ± 0.5 mm ² / s.

The vaccine is monitored for emulsion stability by centrifugation at 3000 rpm for 30 minutes, as well as during storage at (+ 2 ° C) - (+ 8 ° C) and 37 ° C, followed by a visual assessment of the uniformity of the emulsion. The proposed vaccine with oil adjuvant has a high stability of the emulsion during storage at various temperature conditions and during centrifugation.

To test the reactogenicity of the drug, the vaccine is administered intramuscularly in the thigh area to 3 rabbits and 5 guinea pigs at a dose of 2 cm ³ and 1 cm ^3, respectively. The clinical condition of the animals is monitored for 14 days.

After immunization in rabbits and guinea pigs, a slight local increase in body temperature and the formation of swelling of about 1 cm ³ at the injection site were noted. These signs completely disappeared by the end of the observation period.

Thus, the proposed vaccine is a type of weakly reactive drugs.

The antigenic activity of the vaccine is tested in rabbits. For this, 5 rabbits weighing 2.0-2.5 kg are taken. The vaccine is administered to three rabbits subcutaneously in the back region of 2 cm ³ . 14 days after administering the vaccine twice with an interval of 20-25 days, 2-3 cm ^{3 of} blood is taken from the ear vein in rabbits, kept at 37 ° C, serum is separated and examined for the presence of antibodies to parvovirus and reovirus in rtga, to herpes virus and VD-BS in IFA. At the same time, blood is taken from unvaccinated animals (2 heads) and used for control.

The vaccine series is considered to have passed antigenicity control if the titer of virus-specific antibodies to parvovirus is 1: 640, to reovirus 1: 320 in rtga, to herpes virus 1: 1600, to virus VD-BS 1: 1600 in ELISA.

The vaccine made has the optimal composition of the components, wt.%:

Concentrated antigenic material from the Parvo 32459-DEPT strain of parvovirus type I reproduced in the primary cell culture “PEC” with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and GA activity of at least 1: 2048 12.5-14.5 Concentrated antigenic material from the strain "Reo I - Lang-DEPT" of type I reovirus reproduced in the transplanted cell culture of "Vero" and (or) "PT-80" and "MDVC" with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and GA activity of at least 1:64 12.5-14.5 Concentrated antigenic material from the strain “TK-A-VIEV-B2-DEPT” of herpesvirus type I reproduced in the transplanted cell culture “LEK” with biological activity of at least 6.5 lg TCD ₅₀ / cm ³ 12.5-14.5 Concentrated antigenic material from strain "NADL-DEPT" virus VD-BS, reproduced in transplantable culture cells "LEK" with biological activity not less than 6.0 lg TCD ₅₀ / cm ³ 12.5-14.5 Adjuvant up to 100.0

The content of antigenic materials in 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 intestinal infections of young animals and pathologies of the reproductive organs of adult livestock.

The vaccine is used as a preventive measure in threatened and permanently dysfunctional farms for parvovirus, reovirus, herpesvirus type I infections and viral diarrhea, a disease of cattle mucous membranes.

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

- calves are immunized from 10-20 days of age in a dose of 1 cm ³ twice, with an interval of 21-30 days in order to reliably protect against viral respiratory infections caused by parbo-, reo-, herpesviruses and VD-BS virus. Revaccination is carried out after 6 months;

- heifers of a random period are vaccinated subcutaneously in a dose of 2 cm ³ twice, with an interval of 21-30 days for 1.0-1.5 months before the first mating in order to reliably protect the fetus and prevent impaired reproductive organs (ovarian hypofunction, infertility). Revaccination is carried out after 6 months;

- deep-shelled cows and heifers are vaccinated in a dose of 2 cm ³ twice for 50-45 days and 25-20 days before calving in order to reliably protect newborn calves from pneumonia and diarrhea, creating colostral immunity. Revaccination is carried out after 6 months;

- bulls are vaccinated every 6 months at a dose of 2 cm ³ .

Immunity in vaccinated animals occurs on 21-30 days after two immunizations lasting at least 6 months. The vaccine causes the formation of a humoral immune response in cattle to causative agents of parvovirus, reovirus, herpesvirus type I infections, viral diarrhea - diseases of the mucous membranes and provides reliable passive maternal immunity in young animals up to 10-20 days of age, which allows it to be protected from field virulent causative agents of parbo-, re-, herpes viruses and VD-BS of cattle in early life.

Example 7

Tests were conducted to study antigenic activity and determine the vaccine dose of an experimental series of inactivated emulsion inactivated vaccines against parvovirus, reovirus, herpes virus type I infections and VD-BS in cattle based on the Parvo 32459-DEPT strain of parvovirus type I, Reo I - Lang -DEP "type I reovirus," TK-A- (VIEV) -V2-DEP "herpesvirus type I," NADL-DEPT "virus VD-BS, made as described in example 6, and containing, wt.%:

Concentrated antigenic material from strain "Parvo 32459-DEPT" reproduced type I parvovirus in the primary cell culture "PEC" with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and HA activity not less than 1: 2048 and inactivated 1,2-aminethylaziridine 12.5-14.5 Concentrated antigenic material from the strain "Reo I-Lang-DEPT" reovirus type I, reproduced in a transplanted cell culture "Vero" and (or) "PT-80" and "MDVK" with biological activity not less than 6.0 lg TCD ₅₀ / cm ³ and HA activity not less than 1: 1024 and inactivated 1,2-aminethylaziridine 12.5-14.5 Concentrated antigenic material from the strain "TK-A-VIEV-B2-DEPT" herpesvirus type I reproduced in transplantable cell culture "LEK" with biological activity not less than 6.5 lg TCD ₅₀ / cm ³ and inactivated by 1,2-aminethylaziridine 12.5-14.5 Concentrated antigenic material from strain "NADL-DEPT" virus VD-BS, reproduced in transplantable culture cells "LEK" with biological activity not less than 6.0 lg TCD ₅₀ / cm ³ and inactivated by 1,2-aminethylaziridine 12.5-14.5 Adjuvant up to 100.0

To conduct a test to study antigenic activity and determine the vaccine dose of an experimental series of associated vaccines in the conditions of the Zolotaya Niva LLC farm in the Kaybitsky district of the Republic of Tajikistan, three groups of animals of different age groups were formed according to the principle of an analogue in the amount of 45 animals, seronegative or having a low level of antibodies against parvovirus , reovirus, herpesvirus type I and VD-BS of cattle.

Three age groups of animals were vaccinated. Three corresponding vaccine doses were tested for each age group. Each dose of the drug was administered to 5 animals. The vaccine was administered subcutaneously in the middle third of the neck twice with an interval of 21-30 days: calves 10-20 days of age in doses of 0.5 cm ³ , 1 cm ³ and 2 cm ³ ; heifers of a random age - 1 cm ³ , 2 cm ³ , 3 cm ³ ; deep-growing cows and heifers - 1 cm ³ , 2 cm ³ , 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,

after the first and second vaccinations. Serum and colostrum samples were examined for the presence of antibodies to parvovirus, reovirus in rtga and ELISA, herpesvirus type I and viral diarrhea virus in ELISA. Statistically processed research results are presented in tables 1, 2, 3, 4, 5.

Table 1 Percentage of serological monitoring results for parvovirus, reovirus, herpesvirus type I and VD-BS in cattle on farms in the Republic of Tajikistan. No. p / p Farm Name Number of samples Parvovirus cattle VD-BS Cattle reovirus IRT RTGA IFA IFA RTGA IFA IFA Apastovsky district of the Republic of Tatarstan one LLC SHP them. Rakhimova thirty 33.3 fifty 63.3 46.7 80 80 2 LLC SHP "Tran" twenty fifty 70 70 0 0 35 3 LLC SHP "Ibragimov and K" 12 0 fifteen fifty 0 13 58.3 four LLC SHP "Alga" thirty 16.7 83.3 83.3 10 26.6 86.7 5 LLC SHP "Ibragimov and K" 29th 44.8 62 96.5 6.9 20.7 65.5 6 MTF "Utyamyshevo" 27 18.5 29.6 96.3 3,7 22.2 85,2 7 LLC SHP "Enali" d. Yumrals thirty 40 63.3 fifty 36.6 43.3 63.3 8 MTF "Azbaba" thirty 43.3 60 fifty 15.3 34 83.3 9 LLC SHP "Enali" d. Baltai 29th 13.8 34.5 62.1 3.4 17,2 58.6 10 LLC SHP "Enali" d. Devlekeevo 31 45,2 61.3 32.3 19,4 29th 64.5 eleven LLC SHP "Tabar" d. Tabar. Teal 31 19,4 41.9 55 3.2 6.5 58.1 12 MTF "B.Kokuzi" thirty 10 33.3 60 10 23.3 fifty 13 LLC SHP "Alga" d. Shutay 19 15.8 31.6 63,2 10.5 26.8 31.6 Kaybitsky district of the Republic of Tatarstan one LLC Golden Niva fifty 28 fifty eighteen fifty 58 four Kamsko-Ustyinsky district of the Republic of Tatarstan one LLC "Syukeyevo" 55 29.1 38,2 n.i. 23.6 34.5 40 Baltasinsky district of the Republic of Tatarstan one LLC "Pine" 48 18.7 41.7 n.i. 27.1 33.3 n.i. 2 LLC Mayak fifty 44 56 n.i. thirty 46 n.i. 3 LLC Igenche 24 20.8 29.3 n.i. 12.5 20.8 n.i. Novosheshminsky district of the Republic of Tatarstan one AF Tatarstan LLC 28 eleven n.i. 60.7 21,4 n.i. 70.3 Yutazinsky district of the Republic of Tatarstan one LLC Nur-Agro 19 15.8 n.i. n.i. 31.6 n.i. 26.3 Note: “n.i.” - not investigated

It follows from the table that a study of the blood sera of various sex and age groups of animals established the seropositivity of a significant number of animals with respect to herpesvirus type I, parvo- and reovirus in both RTHA and ELISA. The results obtained indicate the fact of the circulation of these pathogens among the examined livestock of cattle.

table 2 The level of humoral antibodies before immunization with the associated vaccine against parvovirus, reovirus, herpesvirus type I infections and VD-BS of cattle inactivated emulsion (M ± m, n = 5, p <0.05) Age group Dose, cm ³ Antibody titer, M ± m to pavovirus to reovirus to ИРТ to VD-BS RTGA IFA RTGA IFA IFA IFA Deep-shelled cows and heifers one 28.0 ± 5.4 50.0 ± 0.0 32.0 ± 5.4 30.0 ± 13.6 50.0 ± 17.6 50.0 ± 17.6 2 28.0 ± 8.9 70.0 ± 22.3 40.0 ± 12.2 30.0 ± 13.6 40.0 ± 11.1 50.0 ± 17.6 3 12.0 ± 5.4 30.0 ± 13.6 36.0 ± 13.0 60.0 ± 11.1 30.0 ± 13.6 40.0 ± 11.1 Chicks of a random period one 16.0 ± 8.3 50.0 ± 25.0 24.0 ± 8.3 40.0 ± 11.1 50.0 ± 0.0 0,0 ± 0,0 2 28.0 ± 8.9 40.0 ± 11.1 28.0 ± 5.4 50.0 ± 0.0 50.0 ± 0.0 60.0 ± 20.9 3 160 ± 8.3 30.0 ± 13.6 16.0 ± 4.4 40.0 ± 11.1 50.0 ± 0.0 30.0 ± 13.6 Calves 10-20 days. age 0.5 12.0 ± 5.4 30.0 ± 13.6 12.0 ± 5.4 30.0 ± 13.6 30.0 ± 13.6 0,0 ± 0,0 one 24.0 ± 8.3 60.0 ± 20.9 20.0 ± 10.0 30.0 ± 13.6 0,0 ± 0,0 0,0 ± 0,0 2 44.0 ± 10.9 60.0 ± 11.1 16.0 ± 8.3 40.0 ± 20.9 0,0 ± 0,0 0,0 ± 0,0

Table 3 The level of humoral antibodies after the first immunization with the associated vaccine against parvovirus, reovirus, herpesvirus type I infections and VD-BS of cattle inactivated emulsion (M ± m, n = 5, p <0.05) Age group Dose, cm ³ Antibody titer, M ± m to parvovirus to reovirus to ИРТ to VD-BS RTGA IFA RTGA IFA IFA IFA Deep-shelled cows and heifers one 200.0 ± 60.0 1040.0 ± 268.3 192.0 ± 60.6 960.0 ± 178.8 640.0 ± 109.5 560.0 ± 109.5 2 352.0 ± 87.6 1280.0 ± 219.0 320.0 ± 0.0 1120.0 ± 219.0 1,080.0 ± 357.7 1040.0 ± 268.3 3 384.0 ± 71.5 1360.0 ± 268.3 336.0 ± 99.6 1160.0 ± 319.3 1120.0 ± 219.0 1,080.0 ± 357.7 Chicks of a random period one 216.0 ± 71.5 960.0 ± 178.8 192.0 ± 35.7 840.0 ± 249.0 680.0 ± 134.1 600.0 ± 141.4 2 352.0 ± 87.6 1200.0 ± 282.8 328.0 ± 106.2 1120.0 ± 219.0 1040.0 ± 268.3 960.0 ± 178.8 3 368.0 ± 131.4 1280.0 ± 219.0 336.0 ± 99.6 1280.0 ± 219.0 1040.0 ± 268.3 1000.0 ± 300.0 Calves 10-20d. age 0.5 168.0 ± 49.8 640.0 ± 109.5 144.0 ± 17.8 640.0 ± 109.5 520.0 ± 134.1 400.0 ± 0.0 one 208.0 ± 53.6 960.0 ± 303.3 192.0 ± 35.7 920.0 ± 328.6 800,0 ± 0,0 720.0 ± 89.4 2 224.0 ± 65.7 980.0 ± 317.0 216.0 ± 71.5 960.0 ± 303.3 880.0 ± 219.0 760.0 ± 268.3

Table 4 The level of humoral antibodies after the second immunization with the associated vaccine against parvovirus, reovirus, herpesvirus type I infections and VD-BS of cattle inactivated emulsion (M ± m, n = 5, p <0.05) Age group Dose, cm ³ Antibody titer, M ± m to parvovirus to reovirus to ИРТ to VD-BS RTGA IFA RTGA IFA IFA IFA Deep-shelled cows and heifers one 672.0 ± 199.2 1280.0 ± 536.6 576.0 ± 71.5 1280.0 ± 219.0 960.0 ± 178.8 720.0 ± 89.4 2 736.0 ± 262.9 2560.0 ± 438.1 704.0 ± 175.2 2400.0 ± 565.6 2160.0 ± 715.5 1920.0 ± 357.7 3 832.0 ± 214.6 2880.0 ± 357.7 768.0 ± 143.1 2560.0 ± 438.1 2240.0 ± 657.2 2080.0 ± 536.6 Chicks of a random period one 528.0 ± 125.2 1120.0 ± 219.0 480.0 ± 113.1 1440.0 ± 178.8 1200.0 ± 565.6 1,080.0 ± 357.7 2 704.0 ± 280.5 2400.0 ± 565.6 672.0 ± 199.2 2240.0 ± 657.2 2080.0 ± 536.6 1840.0 ± 657.2 3 736.0 ± 262.9 2560.0 ± 438.1 704.0 ± 175.2 2320.0 ± 638.7 2240.0 ± 438.1 1920.0 ± 357.7 Calves 10-20 days old 0.5 320.0 ± 0.0 1040.0 ± 268.3 304.0 ± 107.3 960.0 ± 178.8 880.0 ± 219.0 800,0 ± 0,0 one 576.0 ± 71.5 1320.0 ± 313.0 512.0 ± 87.6 1200.0 ± 282.8 1040.0 ± 268.3 960.0 ± 178.8 2 640.0 ± 0.0 1360.0 ± 268.3 512.0 ± 87.6 1280.0 ± 219.0 1160.0 ± 319.3 1040.0 ± 268.3

Table 5 The level of specific antibodies in colostrum serum of calving cows 30-35 days after double immunization with the associated vaccine against parvovirus, reovirus, herpes virus type I infections and VD-BS of cattle inactivated emulsion (M ± m, n = 5, p <0.05 ) Age group Dose, cm ³ Antibody titer, M ± m to parvovirus to reovirus to ИРТ to VD-BS RTGA IFA RTGA IFA IFA IFA Deep-shelled cows and heifers one 384.0 ± 71.5 800,0 ± 0,0 352.0 ± 87.6 720.0 ± 89.4 640.0 ± 109.5 520.0 ± 134.1 2 480.0 ± 113.1 1120.0 ± 219.0 448.0 ± 87.6 1040.0 ± 268.3 1000.0 ± 300.0 920.0 ± 328.6 3 512.0 ± 87.6 1200.0 ± 282.8 480.0 ± 113.1 1120.0 ± 219.0 1040.0 ± 268.3 960.0 ± 178.8 The control 8.0 ± 5.48 20.00 ± 13.69 8.0 ± 5.4 20.0 ± 13.6 40.0 ± 11.1 10.0 ± 11.1

As a result of the studies, it was found that the associated emulsion inactivated vaccine against parvovirus, reovirus, herpes virus type I infections and VD-BS of cattle is sterile, harmless and causes a sufficiently high level of specific antibodies in the blood serum of vaccinated animals.

The optimal vaccination dose of the associated emulsion vaccine was: for deep-growing cows, heifers and heifers of a random period - 2 cm ³ , for calves 10-20 days of age - 1 cm ³ . Two-time administration of an emulsion vaccine on average ensured the production of antibodies in ELISA within the limits of: in deep-growing cows, heifers and heifers of a random period to parvovirus 1: 2400.0 ± 565.6-1: 2560.0 ± 438.1, to reovirus 1: 2240 , 0 ± 657.2-1: 2400.0 ± 565.6, to herpes virus 1: 2080.0 ± 536.6-2160.0 ± 715.5, to VD-BS 1840.0 ± 657.2-1 : 1920.0 ± 357.7; in calves 10-20 days of age, to parvovirus 1: 1320.0 ± 313.0, to reovirus 1: 1200.0 ± 282.8, to herpesvirus 1: 1040.0 ± 268.3, to VD-BS 1: 960.0 ± 178.8; in colostrum - to parvovirus 1: 1280.0 ± 219.0, to reovirus 1: 1040.0 ± 268.3, to herpes virus 1: 1000.0 ± 300.0, CVD-BS 1: 920.0 ± 328, 6 (P <0.05).

Information sources

1. Glotov, A.G. Isolation and characterization of viral diarrhea virus isolates - cattle mucosa diseases / A.G. Glotov, T.I. Glotova, E.I. Ryabchikova and others // Issues of Virology. M. - 2006. - No. 1. - S. 41-45.

2. Zudilina, Z.F. Isolation of bovine parvovirus / Z.F. Zudilina, P.N. Mikhailov, S.A. Zhidkov // Actual issues of veterinary virology. - Vladimir. - 1976.

3. Zhidkov, S.A. The role of viral diarrhea in the etiology of respiratory and gastrointestinal diseases of calves / S.A. Zhidkov, A.I. Lebedev, M.M. Gogolev and others // News. Russian Acad. agricultural sciences. 1995. - No. 3. - S.50-53.

4. Koromyslov, G.F. Infectious abortion of cattle / G.F. Koromyslov. - The results of science and technology. Livestock and veterinary medicine, t.13, Moscow, 1980. - S.163-167.

5. Kryukov, N.N. Parvovirus infection of cattle / Kryukov N.N., Zudilina Z.F. and others // Veterinary medicine. - 1988. - No. 7. - S.27-29.

6. Levchenko S.V. Study of the immunobiological properties of the causative agent of viral diarrhea - a disease of the mucous membranes of cattle used for the manufacture of inactivated vaccines / S. Levchenko // Abstract. dis. Cand. biol. sciences. - Vladimir, 2008 .-- 25 p.).

7. Mishchenko V.A. et al. Features of parvovirus infection in cattle / V.A. Mishchenko et al. // Veterinary medicine. - 2000. - No. 5. - S.19-21).

8. Orlyankin, B.G. Parvovirus infections and their effect on animal productivity / B.G. Orlyankin, V.A.Sergeev, S.P. Kachanova // VNIITEEISKH, M., 1985. - P.33-35.

9. Sukharev O.I., Osidze N.G., Gladchenko N.R. et al. Associated vaccine "Bivak" against infectious rhinotracheitis and parainfluenza-3 in cattle, method for its manufacture and method for the prevention of infectious rhinotracheitis parainfluenza-3 / Sukharev OI, Osidze NG, Gladchenko N.R. et al. // A.S. No. 1092778. - Declared. 12/17/1982, No. 3523289.

10. Ritova V.V., Trofimov N.M. Reoviruses and their role in the infectious pathology of humans and animals / V.V. Ritova, N. M. Trofimov / Questions of virology - 1977. - No. 2. - S.134-141.

11. Syurin, V.N. Private Veterinary Virology / V.N.Syurin, N.V. Fomina. - M., Kolos, 1979.- 164 p.

12. Yurov, K.P. Enzyme-linked immunosorbent assay for the detection of parvovirus of cattle of the 3rd type (strain B-2) / K.P. Yurov, TB Shcherbakova // All-Russian. Research institute experiment. Veterinary Medicine Ya.R. Kovalenko. - M., 1993 .-- 7 p.

13. Animal Health Products. - 1992. - P.37, 41, 117.

14. Bachman, P.A. Properties of a bovine parvovirus // Zbl. Vet., Med., 1871. - P.80-85.

15. Bodine, A.B. Possible "immuno-protection" of the bovine parvovirus in the uterus: Preliminary communication / A.B.Bodine, C.F. Alberty, C.S. Buck, M.E. Richardson, R.E. Wright. - J. Possible Theriogenology. - 1981. - V.16. - P.201-206.

16. David Sandals W.C., Charles Povey R. and Alan H. Meek. Prevalence of Bovine Parvovirus Infection in Ontario Dairy Cattle / W. C. David Sandals., R. Charles Povey and H. Alan Meek // Can J. Vet. Res. - 1995 .-- 59: 81-86.

17. Durham, P.J.K. Epidemiological studies of parvovirus infection in calves on endemically infected properties / P.J.K. Durham, R. H. Johnson, H. Isles, R. J. Parker, R. G. Holroyd, I. Goodchild // J. Res. Vet. Sci. - 1985. - V.38. - No. 2. - P.234-240.

18. Elschner, M. Untersuchungen zur Diagnostik von bovinen Parvoviren in Kotproben von Kalbem / M.Elschner // Berl.u.munch.tierarztl.Wschr. - 1994 .-- Jg. 108. - H.7. - S.256-260.

19. Moreno-Lopez I. A Serosurvey of viruses during outbreaks of acute respiratory and / or enteric disease in Swedish cattle./ I. Moreno-Lopez // Zbl. Veter.-Med. Reihe B., 1979, 26, 8: 634-640.

20. Morzaria S.P., Richards M.S., Harkness J.W., Maund B.A. A field trial with a multicomponent inactivated respiratory viral vaccine / S.P. Morzaria, M.S. Richard, J.W. Harkness, B.A. Maund // Veterinary Record. - 1979. - 105. - P.410-414.

21. Sandals, W.C.D. Prevalence and seroepidemiology of bovine parvovirus / W.C.D. Sandals // MSc Thesis, University of Guelph, 1989 .-- P.432-437.

22. Storz, J. Parvovirus infection in calves / J. Storz, R. C. Bates // J. Am. Vet. Med. Assoc. - 1973. - V.163. - P.884-886.

23. Spahn, G.J. Characteristics of hemadsorbing enteric (HADEN) virus / G.J.Spahn, S.B. Mohanty, F.M. Hetrick // Canad. J. Microbiol. - 1966. - V.12. - P.653-660.

24. Soulebot JP Prophylaxie medicate des affections respiratoires des bovines d'origine virale (IBR, PL ₃ , reovirus, maladie des muqueuses / JPSoulebot // Rec. Med. Veter. 1985. - 161, 12: 1271-1275.

25. Thomson J.R., Nettleton, P.P., Greig, A., Barr, J. A bovine respiratory virus vaccination trial / J.R. Thomson, P.P. Nettleton, A. Greig, J. Barr // Veter. Rec. - 1986. - 119. - 18: 450-453.

Claims (4)

1. Vaccine associated against parvovirus, reovirus, herpesvirus type I infections and viral diarrhea - a disease of the mucous membranes of cattle inactivated emulsion containing antigenic material of the causative agent of parvovirus infection of cattle reproduced in the primary cell culture of kidney cells of the embryo of cows ("PEC") subjected to inactivation with 1.2.-aminoethylaziridine and an adjuvant, characterized in that it contains concentrated antigenic material as an antigenic material and «Parvo 32459-DEP" parvovirus type I bovine reproduced in primary culture embryonic kidney cells cows ( "PEC") with biological activity, at least, 6,0 lg TCD ₅₀ / ml and ³ gemagglyutiniruyuuschey activity at at least 1: 2048 GAE inactivated with 1.2.-aminoethylaziridine, concentrated antigenic material of the Reo I - Lang-DEP strain of type I reovirus reproduced in transplantable cell cultures of Vero and (or) PT-80 and MDVC with biological activity of at least 6.0 lg TCD ₅₀ / ml ³ and hemagglutinating activity at least 1: 1024 GAE, inactivated by 1.2, α-aminoethylaziridine, concentrated antigenic material of the strain “TK-A (VIEV) -V2-DEP” of herpesvirus type I cattle reproduced in the transplantable cell culture “LEK” with biological activity of at least 6.5 lg TCD ₅₀ / ml ³ inactivated with 1.2-aminoethylaziridine, concentrated antigenic material of the NADL-DEPT strain of the VD-BS virus of cattle reproduced in the transplantable cell culture “LEC” with biological activity at least 6.0 lg CD ^_50/3 mL, 1.2.-aminoetilaziridinom inactivated and adjuvant in a ratio of at least 1: 1: 1: 1. 2. The vaccine according to claim 1, characterized in that it contains concentrated antigenic material of the strain "Parvo 322459-DEPT" parvovirus type I cattle, concentrated antigenic material of the strain "Reo I - Lang-DEPT" reovirus type I, concentrated antigenic material strain TK-A- (VIEV) -V2-DEPT of herpesvirus of type I cattle, concentrated antigenic material of strain NADL-DEPT of virus BD-BS of cattle, in the ratio, wt.%:
antigenic material, concentrated Parvo 32459-DEPT strain of parvovirus type I cattle reproduced in the primary culture of cow embryonic kidney cells (PEC) 12.5-14.5 antigenic material, concentrated Reo I - Lang-DEPT strain "Type I reovirus reproduced in transplantable cell cultures of Vero and (or) PT-80 and MDVK 12.5-14.5 antigenic material, concentrated strain TK-A- (VIEV) -V2-DEPT "Herpesvirus type I cattle reproduced in transplantable cell cultures" LEK "12.5-14.5 antigenic material, concentrated strain "NADL-DEPT" virus VD-BS of cattle reproduced in transplantable cell cultures "LEK" 12.5-14.5 adjuvant to 100.0. 3. The vaccine according to claim 1, characterized in that the synthetic oil PES-3M and anhydrous lanolin in the ratio 84:16 are used as adjuvant. 4. The vaccine according to claim 3, characterized in that it contains an adjuvant in an amount of at least 50.0 wt.%.