RU2744744C1 - Vaccine against manchemiosis, bibershteiniosis and pasterelosis of large and small cattle associated inactivated, method for its preparation - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: group of inventions relates to the field of veterinary medicine, veterinary microbiology and biotechnology and can be used for the specific prevention of infectious pathologies caused by bacteria of the species Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida. Associated inactivated vaccine against mancheimiosis, biebersteinosis and pasteurellosis of cattle and small ruminants contains the following components per 1 cm³ of the preparation: inactivated protective antigen of Mannheimia haemolytica strain No. 822-VIEV serotype A1 - 2 billion microbial cells; inactivated protective antigen of Mannheimia haemolytica strain No. 1050-VIEV serotype A2 - 2 billion microbial cells; inactivated protective antigen of the Bibersteinia trehalosi strain No. 984-VIEW - 2 billion microbial cells; inactivated protective antigen of the Pasteurella multocida strain No. 1042-VIEV type A - 2 billion microbial cells; inactivated protective antigen of the Pasteurella multocida strain No. 163-VIEV type B - 2 billion microbial cells as well as pharmaceutically acceptable target additives. The group of inventions also relates to a method for producing this vaccine.

EFFECT: use of this group of inventions makes it possible to obtain multivalent inactivated vaccines based on a balanced antigenic composition containing the most common serotypes of bacteria of the species Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida in the territory of the Russian Federation and providing the creation of long-term and intense immunity in vaccinated animals.

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Description

The group of inventions relates to the field of veterinary medicine, veterinary microbiology and biotechnology, and can be used by veterinary specialists in cattle breeding, sheep breeding and goat breeding for the specific prevention of infectious pathologies caused by bacteria of the species Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida.

The etiological significance of bacteria of the Pasteurellaceae family in the development of respiratory infections in cattle and small ruminants is recognized by veterinarians around the world as the most acute problem that entails huge economic losses. So, in accordance with the current epizootic situation on the territory of the Russian Federation, of the respiratory infections of pasteurellosis and pasteurellosis-like etiology, the greatest role is played by Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida, respectively causing mancheimiosis, bibersteiniasis and pasteurellosis. These infections are common not only in the Russian Federation, but also abroad [Shegidevich, E.A. State and prospects of studying pasteurellosis of agricultural animals / E.A. Shegidevich // Actual problems of veterinary medicine in industrial animal husbandry. Collection of scientific papers. VIEV. - M .: - 1984 .-- T.60. - S. 58-63; Kann, O. Frequency and antibiotic susceptibility of Pasteurella multocida and Mannheimia haemolytica isolates from nasal cavities of cattle. / O. Kann, S. Kahya, K. Tayfun // Turk. J. Vet. Anim. Sci. - 2010 .-- 34 (1). - P. 91-94; Biberstein, E.L. Serological types of Pasteurella haemolytica. / E.L. Biberstein, M. Gills, H. Knight // Cornell Vet. - 1960. - P. 283-300].

// Vet. Мех. - 2009. - 40. - P. 293-314]. Манхеймиоз проявляется как оппортунистическая инфекция с неспецифичной клинико-морфологической картиной и сложной лабораторной диагностикой.Mancheimiosis is a pasteurellosis-like infectious disease, mainly of animals of ruminant species, caused by the causative agent Mannheimia haemolytica, accompanied by sepsis, extensive damage to the respiratory system in cattle and small ruminants [Jaramillo, A.S. Mannheimiosis bovina: etiologia, prevencion in control. / AC. Jaramillo, TF Trigo, F.

// Vet. Fur. - 2009. - 40. - P. 293-314]. Mancheimiosis manifests itself as an opportunistic infection with a nonspecific clinical and morphological picture and complex laboratory diagnostics.

When studying the epizootic state of dysfunctional agricultural enterprises for respiratory diseases in various regions of the Russian Federation, an important etiological role of Mannheimia haemolytica was established, which in the structure of pasteurellosis pathology is 9.5% (as of 2018). It has been established that calves up to 6 months of age and lambs up to 3 months of age are most susceptible to mancheimiosis. Adult animals are also susceptible to disease, but the disease usually does not lead to their death, but goes into a chronic form, in which the animals remain carriers of bacteria. The main clinical manifestations of the disease are: fever, dry cough, gradually turning into moist, abundant mucous and serous-mucous discharge from the nasal passages, sometimes signs of catarrhal enteritis, keratoconjunctivitis in lambs. When conducting a postmortem examination, the prevailing signs were established in young animals: lobular fibrinous bronchopneumonia or lobar pneumonia; interstitial pulmonary edema; serous-fibrinous pleurisy, serous-fibrinous pericarditis, bronchial lymphadenitis, congestive hyperemia of the tracheal vessels with multiple hemorrhages. In adult animals, inflammation of the supra-lymph nodes and bronchial lymph nodes is noted. For lactating animals, a characteristic feature is a decrease in milk production [Laishevtsev, A.I. Clinical and epizootic substantiation of vaccine prevention and development of a vaccine against mancheimiosis in cattle and small ruminants, Dissertation for the degree of candidate of biological sciences / Federal Scientific Center - All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko RAS. Moscow, 2018, 155 pages]. In sheep and goats, the mastitis form of mancheimiosis is found. In this case, the pathological process most often covers one lobe of the mammary gland, during which necrotic processes occur, which causes a number of common systemic lesions - fever, anorexia, depression, loss of appetite, weakness. The form of the disease, in which the mammary gland is affected, has a synonymous name - "blue udder" [Laishevtsev, A.I. Mancheimiosis of cattle ("blue udder") / A.I. Laishevtsev // Veterinary medicine and feeding. 2019. No. 6. S. 32-34].

In accordance with current taxonomy, the genus Mannheimia has five species: M. haemolytica, M. glucosida, M. granulomatis, M. raminalis and M. varigena. The division of M. haemolytica into biotypes is based on the fermentation of sugars: biotype A is able to ferment arabinose, and T - trehalose [Smith, G.R., The characteristics of two types of Pasteurella haemolytica associated with different pathological conditions of sheep / G.R. Smith // J. Pathol. Bacteriol. 81 (1961) 431 440]. Subsequently, biovar T was classified as Pasteurella trehalosi, and even later was named Bibersteinia trehalosi. Currently, the Mannheimia haemolytica species includes 12 serotypes A (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16 and A17), the Mannheimia haemolytica A11 serotype is considered a species of Mannheimia glucosida. Serotypes A1 and A2 have the greatest prevalence and epidemiological significance [Binham, D.P .; Moore, R. and Richards, A.B. Comparison of DNA: DNA homology and enzymatic activity between Pasteurella haemolytica and related species / D.P. Binham, R. Moore, A.B. Richards // Am. J. Vet. Res. 51: 1161-1166,1990; Kapustin, A.V. Pasteurellosis of cattle caused by Mannheimia haemolytica / A.V. Kapustin, A.I. Laishevtcev // Russian Journal of Agricultural and Socio-Economic Sciences. 2016. No. 4 (52). C. 3-12].

Biebersteiniasis is a pasteurellosis-like disease of cattle and small ruminants, manifested by the defeat of the respiratory system and sepsis [Bowersock, TL. Efficacy of a multivalent modified-live virus vaccine containing a Mannheimia haemolytica toxoid in calves challenge exposed with Bibersteinia trehalosi / T.L. Bowersock, B.E. Sobecki, S.J. Terrill, N.C. Martinon, T.R. Meinert, R.D. Leyh // Am J Vet Res 2014 No. 75 P. 770-776].

It is known that the causative agent of the disease belongs to the representatives of the microflora of the conjunctiva, nasopharynx and tonsils of clinically healthy animals, but at the same time it is able to be involved in the pathological process as a secondary agent. There are two main forms of manifestation of biebersteinosis, namely septic and respiratory. The septic form of infection is accompanied by a high mortality rate among young animals, reaching from 3 to 70%, depending on the effectiveness of the treatment and prophylactic measures used. The main clinical signs are fever, decreased activity, frothy discharge from the mouth and nasal passages, and shortness of breath. Death occurs within 6-8 hours. At autopsy of dead animals, hemorrhages are noted on the pleura, epicardium, endocardium, heart valves, visceral peritoneum, mesenteric lymph nodes, spleen. In the pleural and abdominal cavity, there is a large amount of transparent exudate with a blood tint. There are known cases of development of fibrinous pericarditis, focal hemorrhagic necrosis of the liver [Szeredi, L. Immunohistochemical detection of Bibersteinia (Pasteurella) trehalosi in a lamb died of acute systemic infection Bibersteinia (Pasteurella) trehalosi kimutatasa immunhisztoztoztoketemi mods , T. Csabai, L. Makrai, S. Janosi // Magyar Allatorvosok Lapja 2008, 130 (11) P. 658-662]. The respiratory form of bibersteinosis is manifested by proliferative and exudative pneumonia in animals of all age groups. In cattle, purulent emphysematous pneumonia with fibrinous pleurisy, enlarged bronchial lymph nodes, and necrotizing laryngitis may occur. Clinical signs of this form include cough and shortness of breath, which are aggravated by exertion. Mucopurulent discharge from the nasal passages. In adult animals, the disease is manifested by the sudden development of hyperthermia, toxemia and tachypnea, which indicates the development of endotoxemia [Gilmour, N.J.L. Clinical and pathological findings in an outbreak of pneumonia in sheep / N.J.L. Gilmour, J.G.F. Brotherston // Journal of Comparative Pathology and Therapeutics 1963, 73 (3) P. 329-333; Stamp, J.T. Pneumonia of sheep / J.T. Stamp, D.I. Nisbet // Journal of Comparative Pathology and Therapeutics 1963, 73 (3) P.319; Collins, R.L. Bibersteinia trehalosi in cattle - another component of the bovine respiratory disease complex? / R.L. Collins // Cattle Practice 2011, 19 (1). P.9-12]. The serotypic composition of Bibersteinia trehalosi is represented by 4 serotypes - T3, T4, T10, T15.

Pasteurellosis is a contagious disease of most species of animals and birds caused by bacteria of the genus Pasteurella. In the acute course of the disease, septic processes, croupous pneumonia, pleurisy and edema of various localization develop. The subacute and chronic course of the disease is characterized by purulent-necrotizing pneumonia, lesions of the joints, mammary glands, organs of vision and hemorrhagic enteritis. All age and physiological groups of animals are susceptible to pasteurellosis, but the greatest mortality is observed among young animals [Kapustin, A.V. Pasteurellosis of cattle caused by Mannheimia haemolytica / A.V. Kapustin, A.I. Laishevtcev // Russian Journal of Agricultural and Socio-Economic Sciences. 2016. No. 4 (52). C. 3-12].

A typical representative of the genus is Pasteurella multocida, which has a wide distribution area and high epizootic significance. The species is subdivided into three subspecies: P. multocida subspecies gallicida, P. multocida subspecies multocida, P. multocida subspecies septica [Laishevtcev, A.I. Specific identification of Pasteurella bacteria on the basis of biochemical properties in accordance with modern classification // A.I. Laishevtcev, A.V. Kapustin, N.V. Pimenov // Russian Journal of Agricultural and Socio-Economic Sciences. 2017. No. 1 (61). C. 320-328]. According to the OIE “Guidelines for Diagnostic Tests and Vaccines for Terrestrial Animals”, there are three distinct entities caused by Pasteurella multocida: hemorrhagic septicemia in cattle, porcine atrophic rhinitis and avian cholera. Other species of the genus Pasteurella spp. Have less epizootic significance [Kapustin, A.V. Diagnostics of pasteurellosis of farm animals, birds and fur animals / A.V. Kapustin, A.I. Laishevtsev, I. Yu. Ezdakova, V.N. Skvortsov., T.V. Stepanova, M.I. Gulyukin // Moscow, 2016.].

For pasteurellosis of cattle, 4 types of the course of the disease are typical - hyperacute, acute, subacute and chronic. The forms of pasteurellosis include: septic, edematous, chest and intestinal. The main clinical manifestations of the disease are: an increase in body temperature up to 40-42 ° C; violation of cardiac activity; development of pulmonary edema and acute gastroenteritis; refusal to feed and lethargy; swelling in various tissues of the body; fibrinous, purulent-fibrinous, serous-catarrhal or lobar pneumonia or pleuropneumonia; copious foamy or mucopurulent discharge from the nasal passages; keratitis, rhinitis, conjunctivitis; diarrhea and wasting of the body; joint damage.

The main pathological changes in cattle pasteurellosis are: hemorrhages on the serous and mucous membranes of the parenchymal organs and chest cavity; the mucous membranes of the nasal cavity have highly blood-filled vessels; in the subcutaneous tissue and intermuscular tissues, inflammatory edema is found together with gelatinous infiltration; the lungs are swollen or with fibrinous inflammation; necrotic encapsulated lesions in the lungs; the trachea and bronchi contain a foamy liquid; the epiglottis, trachea and catarrhal pleura are hyperemic with punctate hemorrhages; in the lymph nodes of the head, neck and chest cavity, there are signs of serous, hemorrhagic or serous-purulent inflammation, characterized by a decrease in the number of lymphocytes; the spleen has a flabby consistency; signs of granular degeneration in the liver, kidneys and myocardium; parenchymal tissue of the liver, lungs, spleen and kidneys contains hemorrhages and necrotic foci; catarrhal or hemorrhagic inflammation of the abomasum and small intestine; inflammation of the spleen, in which the content of polynuclear cells and eosinophils in the red pulp increases; the marginal and central sinuses of the lymph nodes are split and contain serous effusion with leukocytes [Kapustin, A.V. Diagnostics of pasteurellosis of farm animals, birds and fur animals / A.V. Kapustin, A.I. Laishevtsev, I. Yu. Ezdakova, V.N. Skvortsov., T.V. Stepanova, M.I. Gulyukin // Moscow, 2016.].

In the Russian Federation, three types of pasteurella are most common: A, B and D.

Type A most often causes pasteurellosis infection of birds, rabbits and fur-bearing animals, and also contributes to the development of a subacute or chronic course of enzootic pneumonia in calves and piglets; type B causes hemorrhagic septicemia in cattle and wild ruminants; type D - pig pasteurellosis.

Isolates of P. multocida types E and F are not widespread on the territory of the Russian Federation, while they are the causative agents of hemorrhagic septicemia in cattle and wild ruminants in a number of foreign countries.

The detailed etiological significance of Pasteurella serotypes according to the Carter-Hedlestone classification is shown in Table 1.

State of the art

Specific prophylaxis of infectious diseases of farm animals is the most effective means of preventing mass diseases and death of animals.

Currently, the following veterinary drugs are registered in the Russian Federation for the prevention of pasteurellosis and mancheimiosis in cattle and small ruminants (the Bibersteinia trehalosi component is absent in registered and commercially available vaccines):

Preparations for cattle:

- Vaccine against pasteurellosis of cattle inactivated emulsified (Pasteurvacarm) produced by FKP "Armavir Biofabrika". The drug is intended for the specific prophylaxis of pasteurellosis in cattle in dysfunctional cattle farms that are threatened by respiratory pathology. The vaccine includes the strains Pasteurella multocida serotype A and Mannheimia (Pasteurella) haemolytica serogroup A1, inactivated with formaldehyde to a concentration of formaldehyde (0.3%) and enclosed in a water-oil emulsion.

- Inactivated combined vaccine against infectious rhinotracheitis, parainfluenza-3, respiratory syncytial disease, viral diarrhea and pasteurellosis of cattle (KOMBOVAK-R) - produced by Vetbiochim LLC. The drug is intended for the immunization of pregnant cows and calves. The vaccine includes formalin-inactivated antigens of 4 industrial strains of viruses: infectious rhinotracheitis, parainfluenza-3, diarrhea virus, respiratory syncytial and viral diarrhea in cattle, as well as antigens of Pasteurella strains Pasteurella multocida serogroups A, B, D, and Mannaticheim A1 with aluminum hydroxide as an adjuvant.

- Vaccine against pasteurellosis of cattle inactivated emulsion produced by FGBU "ARRIAH". The drug is intended for the prevention of pasteurellosis in cattle in dysfunctional and threatened farms for this disease. The vaccine includes formaldehyde-inactivated bacterial cells Mannheimia haemolytica serotype A: 1, strain No. 1412, Pasteurella multocida serogroup A, strain No. 1414 and Mannheimia haemolytica toxoid, strain No. 1412 with the addition of an oil adjuvant - 70% and ISA buffer solution (up to 30%).

- Inactivated vaccine against pasteurellosis of cattle and buffaloes (Boviret) - produced by FKP "Stavropol Biofabrika". The drug is intended for the prevention of pasteurellosis in cattle and buffaloes. The vaccine includes antigens of Pasteurella multocida serogroup B strains inactivated with formalin, adsorbed on a gel of alumina hydrate based on phosphate buffer.

- Inactivated vaccine for the prevention of clostridiosis and pasteurellosis in cattle (VanShotUltra 8) - produced by "Zoetis Inc." The drug is intended for the prevention of clostridiosis and pasteurellosis in cattle. The vaccine includes a culture of bacteria Mannheimia (Pasteurella) haemolytica serotype A1, inactivated with formalin; cultures of bacteria Clostridium chauvoei, Cl. septicum, Cl. haemolyticum, Cl. novyi, Cl. sordellii, Cl. perfringens type C and D, inactivated with formalin with the addition of saponin, potassium sulfate, aluminum and water for injection.

- Vaccine against parainfluenza-3, respiratory syncytial infection and bovine pasteurellosis, inactivated (Bovilis® Bovipast RSP) - produced by "Intervet International B.V." The drug is intended for the prevention of parainfluenza-3, respiratory syncytial infection and pasteurellosis of cattle in threatened and permanently dysfunctional farms. The vaccine includes the culture fluid of a continuous MDBK cell line infected with parainfluenza-3 and respiratory syncytial viruses, and antigens of Pasteurella haemolytica serotypes A1 and A6, inactivated with formaldehyde, with the addition of adjuvants (aluminum hydroxide 37.5 mg, saponin - 0.625 mg) , stabilizer (sodium thimerfonate - 0.05 mg), antifoam (baysilon EBZ - 0.0625 mg), phosphate buffer solution up to 5 ml.

Preparations for cattle and sheep:

- Inactivated emulsified vaccine against pasteurellosis of ruminants (Pulmovac) - produced by FKP "Stavropol Biofabrika". The drug is intended for the specific prophylaxis of pasteurellosis in sheep and cattle in threatened and unfavorable farms for pasteurellosis. The vaccine includes inactivated microbial cells Pasteurella multocida serogroups A, B, D and Mannheimia (Pasteurella) haemolytica serotype A1, with the addition of formaldehyde (0.3%) as a preservative and an oil adjuvant Montanid ISA 70 (SEPPIC) in a ratio of 51:49 ...

- Vaccine emulsified against pasteurellosis of cattle, buffalo and sheep produced by LLC "AGROVET". The drug is intended for immunization of cattle, buffaloes and sheep. The vaccine includes formalin-inactivated concentrated culture of Pasteurella multocida and water-oil emulsion, emulsifier.

Sheep preparations:

- Inactivated vaccine against pasteurellosis of sheep and pigs (Pastos) - produced by FKP "Stavropol Biofabrika". The drug is intended for the prevention of pigs and sheep against pasteurellosis. The vaccine includes antigens of serogroup B Pasteurella multocida strains, inactivated with formalin and precipitated with potassium alum.

Preparations for cattle, sheep and goats:

- Formolvaccine against pasteurellosis of ruminants, hydroxide aluminum - produced by FKP "Armavir Biofabrika". The drug is intended for the specific prevention of pasteurellosis in cattle and small ruminants, as well as reindeer. The vaccine includes antigens of the Pasteurella multocida strains of serogroups A, B, D, inactivated with formalin with the addition of an adjuvant of alumina hydrate.

The above drugs are widely used in the cattle-breeding and sheep-breeding industry, but nevertheless they have a number of disadvantages, in particular:

- not all drugs are simultaneously intended for vaccination of cattle, sheep and goats, i.e. has application restrictions;

- not all preparations contain a combination of Pasteurella multocida and Mannheimia haemolytica;

- none of the preparations contains the component Mannheimia haemolytica serotype A2;

- none of the preparations contains Bibersteinia trehalosi antigens.

In addition to commercial drugs registered in the Russian Federation, the following developments are known:

Inactivated combined vaccine against infectious rhinotracheitis, parainfluenza-3, respiratory syncytial disease, viral diarrhea and pasteurellosis in cattle [patent RU 2403061 C1 publ. 2010.11.10]. The disadvantage of the above invention is the limited antigenic composition against the causative agents of pasteurellosis etiology and the narrow specific purpose of the drug. So, given in the invention, the antigenic composition is focused exclusively on cattle, but not on sheep and goats. In addition, the preparation does not contain the antigens Mannheimia haemolytica serotype A2 and Bibersteinia trehalosi. In view of the fact that the etiological role of pathogens of infectious pathologies of animals is changing, the newly proposed vaccine is more consistent with the epizootic situation in the Russian Federation.

- Vaccine against animal pasteurellosis [patent RU 2311199 C1 publ. 2006.07.06]; and also - Vaccine against pasteurellosis of animals [patent RU 2414929 C1 publ. 2011.03.27]. The disadvantage of these inventions, in relation to the newly proposed one, is the unreasonable antigenic composition of the preparation for cattle. There is no information about the wide distribution of Pasteurella multocida types A and D in cattle breeding, sheep breeding and goat breeding. Types A and D are more common in poultry and pig production. At the same time, the vaccine under consideration does not contain the antigens of Mannheimia haemolytica and Bibersteinia trehalosi, which can negatively affect the epizootic situation.

- Cross-protection of bovines against b. Trehalosi infection by a multi-valent vaccine [patent CA 2907098 A1 publ. 09/25/2014]. The disadvantage of the drug is the possibility of a narrow specific application of the drug, since the tool is not intended for vaccination of small ruminants. In addition, the invention under consideration is focused on the development of an immunobiological agent against mancheimiosis and bibersteinosis, but not pasteurellosis caused by Pasteurella multocida, which indicates a limited antigenic composition of the drug. Since Bibersteinia trehalosi, Mannheimia haemolytica and Pasteurella multocida are simultaneously circulating on the territory of the Russian Federation, the drug in question is limited in antigenic composition and does not correspond to modern epizootic data.

- Bivalent inactivated vaccine against diseases caused by Pasteurella multocida and Mannheimia haemolytica, and a method for its preparation (Ox pasteurella multocida disease, Mannheimia haemolytica disease bivalent inactivated vaccine and preparation method thereof) [patent CN 109847060 A publ. 06/07/2019]. The difference between the invention under consideration is the fact that in China the prevailing serotype of Mannheimia haemolytica is A6, while in the territory of the Russian Federation, serotypes A1 and A2 are predominantly circulating. Thus, the proposed antigenic composition of the vaccine is not able to fully provide protective protection against pasteurellosis in our country. The second drawback of the drug in question is its limited specific purpose, so the tool is not focused on small ruminants.

In addition to vaccines, the following methods of obtaining them are known:

- Method of preparation of antipasterella vaccine [copyright certificate SU 1839092 A1 publ. 1993.12.20]. The essence of this invention is to increase the immunogenicity of drugs, reduce their viscosity and reactogenicity. The problem is solved by using a certain ratio of the oil base - polyethylsiloxane liquid, and the emulsifier, during the composition of the series of the drug. The disadvantage of the proposed method is the sequence of its implementation, so according to the invention, the concentration of hydrogen ions is determined immediately after the end of the cultivation of pasteurella, and not controlled during its implementation. In the case of maintaining a certain pH value during cultivation, it is possible to obtain a higher concentration of bacterial cells than in uncontrolled conditions. The next disadvantage of the above method is that inactivation is carried out after the concentration of antigens, and not before. Concentration of non-inactivated culture liquid has its own risks, in particular - contamination of equipment, risks of human infection, risks of non-inactivated pasteurella cells and its toxins entering the sewer system, risks of contamination of antigenic raw materials, etc.

- A method of manufacturing a vaccine against pasteurellosis of farm animals and birds [patent RU 2129015 C1 publ. 1999.04.20]. The disadvantage of this method is the high consumption of nutrient media during continuous flow cultivation of pasteurella. In addition, this development lacks the stage of concentration of pasteurellosis antigens, which is necessary to obtain inactivated vaccines, as a result of which the proteins of the nutrient medium itself enter the preparation with the necessary bacterial antigens. In the case of oral or aerosol use of live pasteurellosis vaccines, the presence of ballast residues of nutrient media in them most often does not contribute to any reactogenic reactions. In the case of the injection use of these preparations containing nutrient residues, this can affect both the harmlessness and the level of the antigenic response, since the substances that make up the nutrient media are proteins against which antibodies will be formed.

- A method of manufacturing vaccines against pasteurellosis of animals and birds [patent RU 2129440 C1 publ. 1999.04.27]. This method is focused on the use of dry protein hydrolyzate from meat production wastes in order to replace high-grade edible meat raw materials. The disadvantage of this method is that a specific medium is recommended for the cultivation of Pasteurella strains, while there are a number of nutrient media that have the best growth-supporting properties, for example, brain heart broth, trypton soy broth, eugonic broth, etc. In addition, a culture medium made from meat waste is more difficult to standardize than one made from standard raw materials.

- A method of making a formol-alum vaccine against pasteurellosis in cattle, sheep and pigs [copyright certificate SU 94044 A1 publ. 1952.01.01]. The disadvantage of this method is that it cannot be implemented in modern conditions of industrial production of biological products, in accordance with the requirements of good manufacturing practices, GMP (English Goodmanufacturing practice). In accordance with GMP requirements, the composition of the drug cannot change from batch to batch, which is why the use of 7-8 pasteurella cultures for the production of formol-alum vaccine is unacceptable, since the formulation must be accurate. In addition, the disadvantage of the above drug is the limited duration of immunity - 5-6 months.

- A deep method of making a vaccine against pasteurellosis in cattle, sheep and pigs in a tanker [copyright certificate SU 127365 A1 publ. 1960.10.10]. The disadvantage of this invention is its non-compliance with GMP requirements. Thus, it is impossible to standardize the production of biological products, the antigens of which were obtained by co-cultivation.

- A method of obtaining a vaccine against pasteurellosis of animals [patent RU 2405567 C1 publ. 2010.12.10]; and also a Method for producing a vaccine against pasteurellosis of animals [patent RU 2310473 C1 publ. 2007.11.20]. The disadvantage of the proposed compositions and methods for their preparation is the excessive antigenic composition for different types of animals. As already indicated earlier, cattle are susceptible to infection with type B pasteurella, to a lesser extent type A, which makes the use of antigens of other types of pasteurella unreasonable and increases the cost of the vaccine. In turn, the use of the preparation made by the proposed method for pigs is also not justified, since there is no evidence base for the susceptibility of pigs to Mannheimia haemolytica. In addition, the presence of an excessive amount of antigens in the preparation can adversely affect the level of immunity.

A method of manufacturing an associated concentrate vaccine against emphysematous carbuncle, malignant edema and pasteurellosis in cattle [copyright certificate SU 122251 A1 publ. 1959.01.01]. The disadvantage of this method is the narrow specificity of the vaccine, since the product is intended exclusively for cattle. In addition, the lack of technology aspects related to the production of Mannheimia haemolytica and Bibersteinia trehalosi antigens can be attributed to the disadvantage.

According to the above characteristics, for existing and / or previously used vaccines on the territory of the Russian Federation: Formol vaccine against ruminant pasteurellosis, aluminum hydroxide [link on the Internet http://www.armbio.info/catalog/item36.html], is the closest analogue ( prototype), the proposed vaccine. As the main active ingredients, the vaccine contains antigens of the strains Pasteurella multocida 796 (serogroup B), Pasteurella multocida 1231 (serogroup A) and Pasteurella multocida T-80 (serogroup D) inactivated with formalin (at a concentration of up to 0.3%), with the addition of an adjuvant - 6% aluminum oxide hydrate in the amount of 25% to the volume of the vaccine. 1 cm ^{3 of the} vaccine contains at least 5 billion, pasteurella microbial cells of each strain. The vaccine induces the formation of an immune response in vaccinated animals to pasteurellosis pathogens 14 days after the double injection of the vaccine, which lasts 12 months, with subsequent revaccination 6 months after the second immunization. The vaccine is intended for the specific prevention of pasteurellosis in cattle and small ruminants, as well as reindeer in farms that are threatened and unfavorable for pasteurellosis. Nevertheless, the given drug has significant drawbacks, in particular, it does not contain antigens necessary for the prevention of mancheimiosis and bibersteinosis. The limited antigenic composition of the drug reduces the protective efficacy of the agent, since pasteurellosis caused by Pasteurella multocida of various types can occur in an associated form with Mannheimia haemolytica and Bibersteinia trehalosi [Laishevtsev, A.I. Clinical and epizootic substantiation of vaccine prevention and development of a vaccine against mancheimiosis in cattle and small ruminants / A.I. Laishevtsev // Dissertation for the degree of candidate of biological sciences / Federal Scientific Center - All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko RAS. Moscow, 2018].

The closest analogue (prototype) of the proposed method for producing a vaccine against mancheimiosis, bibersteinosis and pasteurellosis in cattle and small ruminants associated inactivated is a method for producing a vaccine against pasteurellosis in animals [patent RU 2405567 C1 publ. 2010.12.10]. The essence of the prototype implies individual submerged cultivation of Pasteurella multocida type A, B, D and Mannheimia haemolytica strains. The cultivation is carried out by introducing 10% matt bacterial culture into the reactor, followed by cultivation at a temperature of 37 ° C, until the maximum accumulation of soluble surface antigens is achieved. The next stage of obtaining the vaccine is the inactivation of the culture liquid by adding 0.3% formalin (containing at least 37% formaldehyde) within 24 hours at a temperature of 37 ± 1 ° C. After inactivation, the bacterial mass of each strain is mixed in equal proportions, centrifuged at 6-8 thousand rpm. The sediment obtained after centrifugation is resuspended in 0.1-0.2 M phosphate buffer with pH 7.2-7.4, bringing the concentration to 40-100 billion microns. cl. cm ³ . Subsequently, the resulting bacterial mass is heated for 30-60 minutes at 56-60 ° C, and then centrifuged at 10-15 thousand rpm to separate the supernatant. The resulting supernatant is subsequently concentrated 3-5 times, after which the activity of surface soluble antigens in RPHA is determined. When the activity of the surface soluble antigens of Pasteurella multocida serological variants A, B, D and Pasteurella haemolytica reaches 1: 2048-4096, 1: 4096-5120, 1: 2048-8192 and 1: 16-64, respectively, a vaccine is obtained.

The method for preparing the vaccine, which is the prototype of the drug, does not reveal the scheme for the production of Bibersteinia trehalosi antigens. In addition, the principle of antigen concentration after mixing the culture liquid complicates the process of control and standardization of the finished vaccine. So, it is more expedient to concentrate the culture liquid of each strain separately, i.e. do not mix them, since during cultivation, different strains, the activity of surface soluble antigen (PRA) in practice is significantly different. In addition, the indicated range of possible concentration of ballast does not meet the requirements of good manufacturing practices GMP, which are aimed at the production of standardized products, i.e. containing in its composition antigens and additives in certain concentrations and ratios. The next significant difference between the prototype and the present invention is that a whole-cell protective antigen is used as an antigen, rather than a surface soluble antigen.

Thus, the proposed invention is fully implemented within the framework of the following provisions of the "Strategy for preventing the spread of antimicrobial resistance in the Russian Federation for the period up to 2030", approved by the order of the Government of the Russian Federation dated 09.25.17, No. 2045-r, the National Security Strategy of the Russian Federation, approved By Decree of the President of the Russian Federation of December 31, 2015 No. 683 "On the National Security Strategy of the Russian Federation", and the Fundamentals of State Policy in the Field of Chemical and Biological Safety of the Russian Federation for the Period up to 2025 and Beyond, approved. President of the Russian Federation dated 01.11.13, No. Pr-2573, as well as the provisions of the Political Declaration of the High-level Meeting of the General Assembly on the problem of antimicrobial resistance, adopted at the 71st session of the UN General Assembly (resolution A / RES / 71/3 of 05.10 .16), and the 68th World Health Assembly's Global Plan of Action to Combat Antimicrobial Resistance (resolution WHA68.7 of May 26, 2015), defining an integrated and cross-sectoral approach for industries that use antimicrobials (healthcare, agriculture, including crops, livestock, aquaculture, and food and water treatment).

The technical problem to be solved by this group of inventions is the need to create a safe vaccine against Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella, based on a balanced antigenic composition, containing the most common serotypes of bacteria of the species Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella. multocida and the need to expand the arsenal of multivalent inactivated vaccines aimed at specific prevention of infectious diseases in cattle, sheep and goats.

Disclosure of the essence of the invention

The technical result of the proposed group of inventions is to increase the stability, safety, antigenic and immunogenic activity of the vaccine, providing long-term and intense immunity in vaccinated animals (cattle, sheep and goats) against mancheimiosis, bibersteinosis and pasteurellosis, expanding the arsenal of polyvalent inactivated vaccines aimed at specific prevention of infectious diseases of farm animals, creation of a vaccine with increased stability, antigenic and immunogenic activity, expansion of approaches and methods for the manufacture of polyvalent inactivated vaccines aimed at specific prevention of infectious diseases of farm animals.

Vaccine against mancheimiosis, bibersteinosis and pasteurellosis in cattle and small ruminants associated inactivated

The declared polyvalent vaccine against mancheimiosis, biebersteinosis and pasteurellosis of cattle and small ruminants contains the following components per 1 cm ^{3 of the} preparation: formalin-inactivated protective antigen of Mannheimia haemolytica strain No. 822-VIEV serotype A1 - 2 billion mcr. cl .; Mannheimia haemolytica No. 1050-VIEV serotype A2 2 billion md. cl .; Bibersteinia trehalosi No. 984-VIEV - 2 billion microdistrict. cl .; Pasteurella multocida No. 1042-VIEV type A - 2 billion microns. cl .; Pasteurella multocida No. 163-VIEV type B - 2 billion microns. class, as well as pharmaceutically acceptable target additives. The immunizing dose recommended for a single use is 3 cm ³ for adult cattle and 2 cm ³ for calves and small ruminants.

As pharmaceutically acceptable target additives, the vaccine, in addition to antigens, contains:

- An inactivant required for the disinfection of bacterial antigens and their toxins. As this agent, formalin is used at the rate of 0.3% to the volume of broth culture, containing at least 37% formaldehyde;

- A preservative required for the production of multi-dose packaging of the drug. As a standard preservative, an aqueous solution of sodium merthiolate 10% is used, introduced into the preparation at a rate of 1: 10000;

- The diluent required to dilute the concentrated bacterial antigen to the required concentration. Standard solvents can be sterile saline, sterile PBS — phosphate buffered saline, water for injection;

- An adjuvant necessary for the deposition of bacterial components in the vaccine and for the nonspecific enhancement of the immune response to antigens. As a target additive, the proposed vaccine composition may contain GOA (aluminum hydroxide 3% solution) in a volume of 10% of the composable batch of the drug; oil adjuvant (for example, Montanide ISA 50, Montanide ISA 61, Montanide ISA 70) in the amount of 50% of the composited batch of the drug, etc .; Carbomer 971p 2% solution in PBS in a volume of 10% of the composited batch of the drug;

Additionally, in order to normalize the level of hydrogen ions in the preparation, it is necessary to use 10% sodium hydroxide (caustic soda). The optimal pH level of the drug should be considered 7.0-7.6 for the variant of the vaccine performed on GOA and Carbomer 971r. The pH level of the preparation made on the oil adjuvant is not additionally regulated.

Depending on the adjuvant used, the appearance of the vaccine can vary as follows:

- When using GOA and carbomer 971p - in appearance, the vaccine is a suspension of light gray color with a gray-white precipitate that easily breaks down when shaken into a homogeneous suspension.

- When using oil adjuvants - in appearance, the vaccine is a white emulsion, sometimes with a yellowish tinge. With prolonged storage, slight stratification of the drug is possible, which, upon shaking, turns into the initial state of a homogeneous emulsion.

The shelf life of the vaccine is 12 months from the date of issue, regardless of the adjuvant used, provided that the appropriate storage conditions are observed at a temperature of 2-8 ° C.

The vaccine is intended for the prevention of mancheimiosis, bibersteinosis and pasteurellosis of cattle and small ruminants of all age groups in farms that are unfavorable for these diseases. The vaccine induces the formation of immunity to Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida 14 days after repeated administration, which lasts for at least 12 months. Colostral immunity in young animals obtained from immunized animals lasts for 21 days.

Bacterial strains of Mannheimia haemolytica No. 822-VIEV, Mannheimia haemolytica No. 1050-VIEV, Bibersteinia trehalosi No. 984-VIEV, Pasteurella multocida No. 1042-VIEV, Pasteurella multocida No. 163-VIEV, and are for the first time used in vaccines in vaccines, are included in the vaccine preparations for widespread use. Earlier, at the stage of preclinical and clinical trials, the suitability of these cultures for use as industrial strains of microorganisms was confirmed.

All newly proposed bacterial strains are deposited in the All-Russian collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases of the Federal State Budgetary Scientific Institution Federal Scientific Center - All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences. All cultures were identified on the basis of the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky, by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16 S rRNA gene.

Characteristics of industrial strains Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida

1) The production strain Mannheimia haemolytica No. 822-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1300

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria Mannheimia haemolytica does not belong to any of the four pathogenicity groups.

Date, source and place of excretion: from the lungs of a dead calf. Cattle breeding enterprise of the Republic of Mari-El. 2018 year.

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, ovoid rods, located in isolation, in pairs and / or chains, motionless, do not form spores. In smears from blood and organs, they are stained bipolar, sometimes with a pronounced capsule. Threads and other involutional forms are absent.

Cultural features: on blood agar, colonies are smooth, translucent, shiny 1-2 ml in diameter with a manifestation of a β-hemolysis zone in the size of 1-1.5 mm around the entire colony. Growth in small translucent colonies on MacConkey agar. On mesopatamia agar, bacteria show poor growth of small convex, translucent colonies 0.5-1 mm in size, easily removable with a bacterial loop or a Pasteur pipette. On medium for the cultivation of hemophilus and actinobacillus Haemophilus test medium HTM base manufactured by Oxoid, Mannheimia haemolytica shows abundant growth of smooth, transparent, shiny colonies 1-1.5 mm in size. In broth media, i.e. mesopatamia broth, tryptone-soy broth, Hottinger's broth, Eugonic broth, as well as in brain-heart broth, there is a uniform turbidity of the medium after 12 hours of cultivation. Growth in semi-liquid agar was manifested only during the injection with a loop.

Biochemical properties: strain Mannheimia haemolytica No. 822-VIEV ferments glucose, arabinose, dextrin, fructose, galactose, glycerol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, xylose to form acid; does not ferment adonitol, arbutin, cellobiose, dulcitol, inulin, mannose, melicytosis, melibiose, rhamnose, salicin, sorbose, trehalose. The strain restores nitrate. Lysine decarboxylase, ornithine decarboxylase and arginine decarboxylase show negative results.

Serological properties: type A1.

Virulence for laboratory animals: LD ₅₀ value - 4.3 × 10 ⁸ μR. cl. for white mice, previously immunosuppressed with dexamethasone, with intraperitoneal infection.

Cultivation conditions: grows on most broth nutrient media used in bio-production, in particular: brain heart, tryptone soy broth, Hottinger broth, etc., as well as agar media (MPA, Hottinger, M-144 (HiMedia) with the addition of 10% sterile defibrinated ram blood, at a pH of 7.2 and a temperature of 37 ° C for 18-24 hours.

2) The production strain Mannheimia haemolytica No. 1050-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1301

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria Mannheimia haemolytica do not belong to any of the four pathogenicity groups.

Date, source and place of excretion: from the lungs of a fallen sheep. Sheep breeding enterprise of the Kursk region. Oct 2018

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G. N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, ovoid rods, located in isolation, in pairs and / or chains, motionless, do not form spores. In smears from blood and organs, they are stained bipolar, sometimes with a pronounced capsule. Threads and other involutional forms are absent.

Cultural features: on blood agar, colonies are smooth, translucent, shiny 1-2 mm in diameter with a manifestation of a β-hemolysis zone in the size of 1-1.5 mm around the entire colony. Growth in small translucent colonies on MacConkey agar. On mesopatamia agar, bacteria show poor growth of small convex, translucent colonies 0.5-1 mm in size, easily removable with a bacterial loop or a Pasteur pipette. On the medium for the cultivation of hemophilus and actinobacillus Haemophilus test medium HTM base produced by Oxoid, bacteria form abundant growth of smooth, transparent, shiny colonies 1-1.5 mm in size. In broth media, i.e. mesopatamia broth, tryptone-soy broth, Hottinger's broth, Eugonic broth, as well as in brain-heart broth, there is a uniform turbidity of the medium after 12 hours of cultivation. Growth in semi-liquid agar was manifested only during the injection with a loop.

Biochemical properties: strain Mannheimia haemolytica No. 1050-VIEV ferments glucose, arabinose, dextrin, fructose, galactose, glycerol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, xylose to form acid; does not ferment adonitol, arbutin, cellobiose, dulcitol, inulin, mannose, melicytosis, melibiose, rhamnose, salicin, sorbose, trehalose. The microorganism recovers nitrate. Lysine decarboxylase, ornithine decarboxylase and arginine decarboxylase show negative results.

Serological properties: type A2.

Virulence for laboratory animals: the LD ₅₀ value is 1.28 × 10 ⁹ μR. cl. for white mice, previously immunosuppressed with dexamethasone, with intraperitoneal infection.

Cultivation conditions: grows on most broth nutrient media used in bio-production, in particular: brain heart, tryptone soy broth, Hottinger broth, etc., as well as agar media (MPA, Hottinger, M-144 (HiMedia) with the addition of 10% sterile defibrinated ram blood, at a pH of 7.2 and a temperature of 37 ° C for 18-24 hours.

3) Industrial strain Bibersteinia trehalosi No. 984-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1302

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria Bibersteinia trehalosi do not belong to any of the four pathogenicity groups.

Date, source and place of excretion: from lungs of a dead kid. Goat breeding enterprise of the Tver region. 2018 year.

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G. N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, ovoid rods, located in isolation, in pairs and / or chains, motionless, do not form spores. Threads and other involutional forms are absent.

Cultural features: on blood agar, colonies are smooth, translucent, shiny 1-2 mm in diameter with a manifestation of a β-hemolysis zone in the size of 1-1.5 mm around the entire colony. Growth in small translucent colonies on MacConkey agar. On mesopatamia agar, bacteria show poor growth of small convex, translucent colonies 0.5-1 mm in size, easily removable with a bacterial loop or a Pasteur pipette. On the medium for the cultivation of hemophilus and actinobacillus Haemophilus test medium HTM base manufactured by Oxoid, bacteria form abundant growth of smooth, transparent, shiny colonies 1-1.5 mm in size. In broth media, i.e. mesopatamia broth, tryptone-soy broth, Hottinger's broth, Eugonic broth, as well as in brain-heart broth, uniform turbidity of the medium is observed after 12 hours of cultivation. Growth in semi-liquid agar was manifested only during the injection with a loop.

Biochemical properties: Bibersteinia trehalosi strain No. 984-VIEV ferments glucose, mannitol, sorbitol, mannose, maltose, sucrose, trehalose, esculin and salicin to form acid. Does not ferment arabinose, xylose, dulcitol, galactose. The microorganism recovers nitrate.

Serological properties: not determined.

Virulence for laboratory animals: LD ₅₀ value - 1.42 × 10 ⁸ μR. cl. for white mice, with intraperitoneal infection.

Cultivation conditions: grows on most broth nutrient media used in bio-production, in particular: brain heart, tryptone soy broth, Hottinger broth, etc., as well as agar media (MPA, Hottinger, M-144 (HiMedia) with the addition of 10% sterile defibrinated ram blood, at a pH of 7.2 and a temperature of 37 ° C for 18-24 hours.

4) Industrial strain Pasteurella multocida No. 1042-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1303

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria of the species Pasteurella multocida belongs to the third hazard group.

Date, source and place of excretion: from the lungs of a fallen sheep. Sheep breeding enterprise of the Kursk region. Oct 2018

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, often ovoid rods, located in isolation, sometimes in pairs and less often in chains, motionless, do not form spores. In smears from blood and organs, they are stained bipolar, sometimes with a pronounced capsule. Threads and other involutional forms are absent.

Cultural features: form uniform turbidity of the medium in Hottinger's broth; on Hottinger's agar in Petri dishes after 18-24 hours of cultivation small, round, convex, translucent, with a smooth surface and smooth edges of the colony - "S" -shape. On PZhA - growth by injection.

Biochemical properties: Inherent to Pasteurella multocida. The strain ferments with the formation of acid without gas, glucose, sucrose, galactose, lures, sorbitol. Does not ferment dulcite, arabinose, maltose, raffinose, lactose. Oxidazo and catalase-positive culture. They do not curdle milk and do not thin gelatin. Form indole and hydrogen sulfide.

Serological properties: type A.

Virulence for laboratory animals: LD value ₅₀ - 20 μR. cl. for white mice, with intraperitoneal infection.

Cultivation conditions: The microorganism grows on most broth media used in bio-production, in particular Hottinger broth, brain heart broth, tryptone soy broth, etc. MPB, MPA, Hottinger's broth and agar with the addition of 5% -10% sterile defibrinated ram blood, M-144 (HiMedia) at pH-7.2 with a temperature of 37 ° C for 18-24 hours.

5) Industrial strain Pasteurella multocida No. 163-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1304

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria of the species Pasteurella multocida belongs to the third hazard group.

Date, source and place of excretion: from the lungs of a dead calf. Moscow region, 2017.

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, often ovoid rods, located in isolation, sometimes in pairs and less often in chains, motionless, do not form spores. In smears from blood and organs, they are stained bipolar, sometimes with a pronounced capsule. Threads and other involutional forms are absent.

The cultured features: form a uniform cloudiness of the medium in Hottinger's broth; on Hottinger's agar in Petri dishes after 18-24 hours of cultivation - small, round, convex, translucent, with a smooth surface and smooth edges of the colony - "S" -shape. On PZhA - growth by injection.

Biochemical properties: Inherent to Pasteurella multocida. The strain ferments with the formation of acid without gas, glucose, sucrose, galactose, lures, sorbitol. Does not ferment dulcite, arabinose, maltose, raffinose, lactose. Oxidazo and catalase-positive culture. They do not curdle milk and do not thin gelatin. Form indole and hydrogen sulfide.

Serological properties: type B.

Virulence for laboratory animals: LD ₅₀ - 10 μR. cl. for white mice, with intraperitoneal infection.

Cultivation conditions: The microorganism grows on most broth media used in bio-production, in particular Hottinger broth, brain heart broth, tryptone soy broth, etc. MPB, MPA, Hottinger's broth and agar with the addition of 5% -10% sterile defibrinated ram blood, M-144 (HiMedia) at pH-7.2 with a temperature of 37 ° C for 18-24 hours.

A method of obtaining a vaccine against mancheimiosis, biebersteiniasis and pasteurellosis in cattle and small ruminants.

Obtaining the proposed drug implies the implementation of the following sequential steps:

1) Cultivation of industrial strains by the deep method;

2) Inactivation of the culture fluid;

3) Concentration of antigens;

4) Determination of the completeness of antigen inactivation;

5) Compilation of a series of vaccines;

6) Control of the sterility of the vaccine;

7) Assessment of the safety of the vaccine;

8) Determination of the immunogenic activity of the vaccine.

For submerged cultivation of bacteria of the species Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida in the production of vaccines, trypticase soy broth, tryptone soy broth, eugonic broth, brain heart broth, Hottinger's broth, and other media can be used that have the necessary growth-supporting properties.

The sequence of preparation of the brood brood of Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida strains from MSB (Master Seed Bacteria - the main seed):

- on the first day, the first generation of industrial strains is prepared by resuspending the lyophilized MSB culture in a test tube, followed by inoculation on nutrient agar. Each strain is prepared separately. The cultivation of the first generation continues for 18-24 hours under aerobic conditions at a temperature of 37 ° C;

- on the second day, the first generation is monitored for the typicality of the culture in terms of morphological, tinctorial and growth properties, as well as for the absence of extraneous microflora, both on a Petri dish and in a test tube. If the first generation matches all the specified properties, the second generation is obtained. For this, from a Petri dish, individual colonies are inoculated into tubes with fresh culture medium. The obtained crops are cultivated for 18-24 hours under aerobic conditions at a temperature of 37 ° C. To obtain the maximum accumulation of bacterial cells in the broth medium, the tubes are placed on a shaker for constant stirring.

At this stage of the work, from the obtained agar culture of the industrial strain of bacteria, it is possible to prepare WSB (Working Seed bacteria). To do this, individual colonies of the production strain are selected from Petri dishes and inoculated into PZhA (semi-liquid agar), followed by cultivation for 18-24 hours under aerobic conditions at 37 ° C. Then the resulting culture is sterilely packaged in pipettes and sealed for later use. When sealed, production crops of Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida retain their properties for 2-3 weeks, after which the preparation of WSB from MSB is repeated. The use of WSB significantly reduces the time spent on the preparatory stage of the production of serial drugs;

- on the third day, the second generation is monitored for the typicality of the culture in terms of morphological, tinctorial and growth properties, as well as for the absence of extraneous microflora, both on a Petri dish and in a test tube. If the second generation matches all the specified properties, the third generation is obtained. For this, the broth culture from test tubes is subcultured into vials with a fresh nutrient medium (at least 100 ml). The obtained crops are cultivated for 18-24 hours under aerobic conditions at a temperature of 37 ° C. To obtain the maximum accumulation of bacterial cells in the broth medium, the vials are placed on a shaker at 50-150 rpm for constant stirring;

- on the fourth day, after preliminary control of the brood, the entire volume of the bottle is reseeded into bottles with a nutrient medium, which will subsequently be used for inoculation into reactors. The mother culture is prepared at the rate of 10% to the volume of the nutrient medium in the reactor. The cultivation is carried out for 18-24 hours under aerobic conditions at a temperature of 37 ° C.

- on the fifth day, the production strains are directly cultivated in the reactor. The cultivation of strains Mannheimia haemolytica and Bibersteinia trehalosi is carried out at a pH of 7.2-7.8, and Pasteurella multocida at a pH of 7.0-7.8, at a temperature of 37 ° C. To achieve the maximum accumulation of bacterial cells, 5% yeast extract, 3-5% of cattle blood serum are additionally introduced into the nutrient medium. When cultivating strains of Mannheimia haemolytica, hemin and nicotinamide-adenine-dinucleotide are additionally included in the medium at the rate of 5-10 mg per liter of nutrient medium.

To maintain the required level of nutrients in the medium, a 40% glucose solution is added at the rate of 0.5-1 liter of glucose, per 100 liters of the nutrient medium 1 time per hour, depending on the intensity of culture growth. The pH level during cultivation is maintained with a 10% alkali solution (NaOH).

The sequence of preparation of brood brood of Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida strains from WSB (Working Seed bacteria - working inoculum containing bacteriological material):

- on the first day, the preparation of the first generation of industrial strains is carried out by introducing the WSB culture into flasks with broth medium and subsequent sowing on nutrient agar. Each strain is prepared separately. The cultivation of the first generation continues for 18-24 hours under aerobic conditions at a temperature of 37 ° C. To obtain the maximum accumulation of bacterial cells in the broth medium, the vials are placed on a shaker at 50-150 rpm for constant stirring;

- on the second day after the preliminary control of the brood, the entire volume of the bottle is reseeded into bottles with a nutrient medium, which will subsequently be used for inoculation into reactors. The mother culture is prepared at the rate of 10% to the volume of the nutrient medium in the reactor. The cultivation is carried out for 18-24 hours in aerobic conditions at a temperature of 37 ° C;

- on the third day, the production strains are directly cultivated in the reactor. The cultivation of strains Mannheimia haemolytica and Bibersteinia trehalosi is carried out at a pH of 7.2-7.8, and Pasteurella multocida at a pH of 7.0-7.8, at a temperature of 37 ° C. To achieve the maximum accumulation of bacterial cells, 5% yeast extract, 5% of bovine blood serum are additionally introduced into the nutrient medium. To maintain the required level of nutrients in the medium, a 40% glucose solution is added at the rate of 0.5-1 liter of glucose, per 100 liters of the nutrient medium 1 time per hour, depending on the intensity of culture growth. The pH level during cultivation is maintained with a 10% alkali solution (NaOH).

Inactivation of the culture fluid containing Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida cells is carried out by adding formalin (containing at least 37% formaldehyde) in a volume of 0.3% to the volume of the inactivated fluid, for 3 days, at room temperature and constant stirring.

Concentration of cultures is carried out by centrifugation at 2-10 thousand vol. min. within 0.5-3 hours or by separation at 5-15 thousand revolutions, depending on the type of equipment used.

The completeness of inactivation is assessed by the absence of viable cells in the concentrated antigen and the assembled preparation by plating on growth-supporting nutrient media. In addition, the assessment of the completeness of inactivation is carried out according to the harmlessness of the inactivated bacterial mass for white mice weighing 16-18 g. For this, the experimental animals are injected intraperitoneally, previously diluted up to 6 billion microns. cl. cm ³ , antigen in a volume of 0.5 cm ³ . To assess the safety of each series of antigen, five experimental mice are used. Observation of the experimental animals continues for 14 days. The drug is considered inactivated in the absence of growth of cultures of Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida and other microorganisms on nutrient media, as well as the absence of any local and systemic reactions, including death, in experimental animals.

Compilation of a vaccine batch.

Various adjuvants can be used for the production of the vaccine according to the invention, therefore, the steps involved in formulating a vaccine batch may vary. Series options are shown below. Calculations of the production of a series of vaccines with a volume of 300 liters (100 thousand doses of vaccine) are given.

Option number 1. - the layout of the vaccine using aluminum hydroxide and carbomer 971p.

In a sterile reactor, inactivated bacterial antigens of five industrial cultures (at a previously established concentration) are mixed with a diluent, adjuvant (10%) and sodium merthiolate, so that the concentration of bacterial antigens is equal to 10 billion microns. cells / cm ³ (2 billion microns. cells / cm ^{3 of} each production strain). With constant stirring, a homogeneous suspension is obtained, the required level of hydrogen ions is set (7.0-7.6). The recipe for the composition of the vaccine when using this layout option is shown in table 2.

Option number 2. packaging the vaccine using an oil adjuvant (eg Montanide ISA 50, Montanide ISA 61, Montanide ISA 70).

In a sterile reactor, inactivated bacterial antigens of five industrial cultures are mixed in a previously established concentration with a diluent, an adjuvant (50%), and sodium merthiolate, so as to obtain a concentration of bacterial antigens equal to 10 billion microns. cl. cm ³ (2 billion microns. cells / cm ³ antigen of each production strain). Obtaining a homogeneous emulsion is carried out using a dispersing machine at 6000-8000 rpm. The vaccine batch obtained in this way has an average pH value of 6.0. The recipe for the composition of the vaccine when using this layout option is shown in Table 3.

The control of the sterility of the vaccine is carried out in accordance with GOST 28085-2013 "Biological medicinal products for veterinary use".

The assessment of the safety of the vaccine is carried out in a bioassay on laboratory white mice weighing 16-18 grams. with a single intramuscular injection of the drug, at a dose of 0.5 cm ³ . Five experimental animals are used to assess the safety of each vaccine batch. The observation of the mice continues for 14 days. The vaccine is considered harmless if all animals remain alive during the observation period.

Determination of the immunogenic activity of all components of the vaccine is carried out on white mice. To control the immunogenic activity of the Mannheimia component of the vaccine, control strains of Mannheimia haemolytica No. 1540-VIEV serotype A1 and No. 1494-VIEV serotype A2 were proposed, respectively. To control the immunogenic activity of the Bibersteinia trehalosi component in the vaccine, a control strain of Bibersteinia trehalosi No. 1586-VIEV was proposed. In order to control the immunogenic activity of the type A pasteurellosis component of the vaccine, it is envisaged to use the well-studied and widespread strain of Pasteurella multocida No. 1231 type A (deposited in the All-Russian collection of microorganism strains used in veterinary medicine and animal husbandry of the Federal State Budgetary Institution "VGNKI"), used in the manufacture of pasteurellosis vaccines and medicinal sera [Vaccine against pasteurellosis of animals. RU 2414929 C1 publ. 2011.03.27]. To control the pasteurellosis component of the vaccine according to type B, it is proposed to use the well-known strain Pasteurella multocida No. 656 type B (deposited in the All-Russian collection of strains of microorganisms used in veterinary medicine and animal husbandry FGBU "VGNKI"), used in the manufacture of pasteurellosis vaccines and therapeutic sera [Inactivated vaccine against combined infectious rhinotracheitis, parainfluenza-3, respiratory syncytial disease, viral diarrhea and pasteurellosis in cattle. RU 2403061 C1 publ. 2010.11.10].

To achieve this goal, ten groups of 10 mice each, weighing 18-20 grams, are formed. Five groups are experimental (for each infecting strain), and five control groups (for each infecting strain). For example: experimental group No. 1 and control group No. 1 are designed to control the mannheimia component according to serotype A1 and are infected with the Mannheimia haemolytica strain No. 1540-VIEV serotype A1; experimental group No. 2 and control group No. 2 are designed to control the mannheimia component according to serotype A2 and are infected with the Mannheimia haemolytica strain No. 1494-VIEV serotype A2; experimental group No. 3 and control group No. 3 are designed to control the activity of the Bibersteinia trehalosi component and are infected with the Bibersteinia trehalosi strain No. 1586-VIEV; experimental group No. 4 and control group No. 4 are designed to control the activity of the pasteurellosis component according to type A and are infected with the Pasteurella multocida strain No. 1231; experimental group No. 5 and control group No. 5 are designed to control the activity of the pasteurellosis component according to type B and are infected with the Pasteurella multocida strain No. 656 (the principle of forming groups may differ from the above).

Evaluation of the immunogenic activity of the mancheimial component is carried out by the method of preliminary immunosuppression with dexamethasone, the rest of the components are controlled by the method of direct infection. So, when assessing the immunogenic activity of the mancheimia component of the drug, mice of experimental and control groups No. 1 and No. 2 are pre-vaccinated, twice with an interval of 14-16 days, at a dose of 1 cm ³ . 10 days after the last vaccination, these animals are injected intramuscularly with 0.01 ml of a 0.4% solution of dexamethasone at the rate of 0.5 ml of active ingredient per 1 injection. This manipulation is repeated 4 times, i.e. 10, 11, 12, 13 days after the second vaccination. On the 14th day after the second vaccination, the designated experimental and control groups of animals are subjected to intraperitoneal infection with the corresponding strain of Mannheimia haemolytica at a dose of 5LD ₅₀ . Evaluation of the immunogenic activity of the remaining components of the vaccine is carried out by the method of direct intraperitoneal infection on the 14th day after the second vaccination at a dose of 5LD ₅₀ .

The proposed drug has a high immunogenic activity, since it provides 80-100% safety among the animals of the experimental group (after infection), while the mortality in the control group is at least 80-100%.

Characteristics of the control strains Mannheimia haemolytica, Bibersteinia trehalosi

1) The control strain Mannheimia haemolytica No. 1540-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1306

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria Mannheimia haemolytica do not belong to any of the four pathogenicity groups.

Date, source and place of excretion: from milk of a cow with clinical manifestation of mastitis. Moscow region - 2018.

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, ovoid rods, located in isolation, in pairs and / or chains, motionless, do not form spores. In smears from blood and organs, they are stained bipolar, sometimes with a pronounced capsule. Threads and other involutional forms are absent.

Cultured peculiarities: on blood agar, colonies are smooth, translucent, shiny in diameter 1-2 mm with the manifestation of a zone of β-hemolysis in the size of 1-1.5 mm around the entire colony. Growth in small translucent colonies on MacConkey agar. On mesopatamia agar, bacteria show poor growth of small convex, translucent colonies 0.5-1 mm in size, easily removable with a bacterial loop or a Pasteur pipette. On the medium for the cultivation of hemophilus and actinobacillus Haemophilus test medium HTM base produced by Oxoid, bacteria form abundant growth of smooth, transparent, shiny colonies 1-1.5 mm in size. In broth media, i.e. mesopatamia broth, tryptone-soy broth, Hottinger's broth, Eugonic broth, as well as in brain-heart broth, uniform turbidity of the medium is observed after 12 hours of cultivation. Growth in semi-liquid agar was manifested only during the injection with a loop.

Biochemical properties: strain Mannheimia haemolytica No. 1540-VIEV ferments glucose, arabinose, dextrin, fructose, galactose, glycerol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, xylose to form acid; does not ferment adonitol, arbutin, cellobiose, dulcitol, inulin, mannose, melicytosis, melibiose, rhamnose, salicin, sorbose, trehalose. The microorganism recovers nitrate. Lysine decarboxylase, ornithine decarboxylase and arginine decarboxylase show negative results.

Serological properties: type A1.

Virulence for laboratory animals: LD ₅₀ value - 2.42 × 10 μR. cl. for white mice previously immunosuppressed with dexamethasone during intraperitoneal infection.

Cultivation conditions: grows on most broth nutrient media used in bio-production, in particular: brain heart, tryptone soy broth, Hottinger broth, etc., as well as agar media (MPA, Hottinger, M-144 (HiMedia) with the addition of 5% sterile defibrinated ram blood, at a pH of 7.2 and a temperature of 37 ° C for 18-24 hours.

2) The control strain Mannheimia haemolytica No. 1494-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1305

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria Mannheimia haemolytica do not belong to any of the four pathogenicity groups.

Date, source and place of excretion: from the lungs of a dead lamb. Sheep breeding enterprise of the Smolensk region. 2018 year.

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, ovoid rods, located in isolation, in pairs and / or chains, motionless, do not form spores. In smears from blood and organs, they are stained bipolar, sometimes with a pronounced capsule. Threads and other involutional forms are absent.

Cultured peculiarities: on blood agar, colonies are smooth, translucent, shiny in diameter 1-2 mm with the manifestation of a zone of β-hemolysis in the size of 1-1.5 mm around the entire colony. Growth in small translucent colonies on MacConkey agar. On mesopatamia agar, bacteria show poor growth of small convex, translucent colonies 0.5-1 mm in size, easily removable with a bacterial loop or a Pasteur pipette. On the medium for the cultivation of hemophilus and actinobacillus Haemophilus test medium HTM base produced by Oxoid, bacteria form abundant growth of smooth, transparent, shiny colonies 1-1.5 mm in size. In broth media, i.e. mesopatamia broth, tryptone-soy broth, Hottinger's broth, Eugonic broth, as well as in brain-heart broth, uniform turbidity of the medium is observed after 12 hours of cultivation. Growth in semi-liquid agar was manifested only during the injection with a loop.

Biochemical properties: strain Mannheimia haemolytica No. 1494-VIEV ferments glucose, arabinose, dextrin, fructose, galactose, glycerol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, xylose; does not ferment adonitol, arbutin, cellobiose, dulcitol, inulin, mannose, melicytosis, melibiose, rhamnose, salicin, sorbose, trehalose. The microorganism recovers nitrate. Lysine decarboxylase, ornithine decarboxylase and arginine decarboxylase show negative results.

Serological properties: type A2.

Virulence for laboratory animals: LD ₅₀ value - 1.61 × 10 ⁸ μR. cl. for white mice, previously immunosuppressed with dexamethasone, with intraperitoneal infection.

Cultivation conditions: grows on most broth nutrient media used in bio-production, in particular: brain heart, tryptone soy broth, Hottinger broth, etc., as well as agar media (MPA, Hottinger, M-144 (HiMedia) with the addition of 10% sterile defibrinated ram blood, at a pH of 7.2 and a temperature of 37 ° C for 18-24 hours.

3) The control strain Bibersteinia trehalosi No. 1586-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1307

Pathogenicity: in accordance with the classification of microorganisms - causative agents of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of work with microorganisms of III-IV groups of pathogenicity (danger) and causative agents of parasitic diseases" bacteria Bibersteinia trehalosi do not belong to any of the four pathogenicity groups.

Date, source and place of excretion: from the lungs of a dead lamb. Goat breeding enterprise of the Republic of Mari-El. 2018 year.

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution FNTs VIEV RAS and on the basis of the FBUN MNIIEM named after V.I. G.N. Gabrichevsky.

Identification methods: by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Morphological signs: small gram-negative, ovoid rods, located in isolation, in pairs and / or chains, motionless, do not form spores. Threads and other involutional forms are absent.

Cultured features: on blood agar, colonies are smooth, translucent, shiny 1-2 mm in diameter with a manifestation of a β-hemolysis zone in the size of 1-1.5 mm around the entire colony. Growth in small translucent colonies on MacConkey agar. On mesopatamia agar, bacteria show poor growth of small convex, translucent colonies 0.5-1 mm in size, easily removable with a bacterial loop or a Pasteur pipette. On the medium for the cultivation of hemophilus and actinobacillus Haemophilus test medium HTM base produced by Oxoid, bacteria form abundant growth of smooth, transparent, shiny colonies 1-1.5 mm in size. In broth media, i.e. mesopatamia broth, tryptone-soy broth, Hottinger's broth, Eugonic broth, as well as in brain-heart broth, uniform turbidity of the medium is observed after 12 hours of cultivation. Growth in semi-liquid agar was manifested only during the injection with a loop.

Biochemical properties: Bibersteinia trehalosi strain No. 1586-VIEV ferments with the formation of acid lures, sorbitol, glucose, mannose, maltose, sucrose, trehalose, esculin and salicin. Does not ferment arabinose, xylose, dulcitol, galactose. The microorganism recovers nitrate.

Serological properties: not determined.

Virulence for laboratory animals: LD ₅₀ value - 3.66 × 10 ⁸ μR. cl. for white mice, with intraperitoneal infection.

Cultivation conditions: grows on most broth nutrient media used in bio-production, in particular: brain heart, tryptone soy broth, Hottinger broth, etc., as well as agar media (MPA, Hottinger, M-144 (HiMedia) with the addition of 10% sterile defibrinated ram blood, at a pH of 7.2 and a temperature of 37 ° C for 18-24 hours.

The series of vaccines produced in this way were tested in the vivarium of the Federal State Budgetary Scientific Institution FSC VIEV RAS, as well as at cattle-breeding and goat-breeding enterprises, unfavorable for mancheimiosis, bibersteinosis and pasteurellosis. Within the framework of approbation, the harmlessness of the drug and its protective efficacy were confirmed.

When using the vaccine, it is not allowed to vaccinate animals later than two weeks before calving or lambing.

The vaccine is injected subcutaneously into the middle third of the neck or intramuscularly into the croup, heifers and cows at a dose of 3 cm ³ , calves 2 cm ³ each. Sheep and goats of all return groups, the drug is injected subcutaneously or intramuscularly into the area of the inner thigh at a dose of 2 cm ³ .

The vaccination schedule for animals can be as follows:

# 1. Cows and heifers of breeding age are vaccinated twice with an interval of 21-24 days 4 and 1 week before insemination, and then revaccinated before calving twice according to scheme No. 2.

# 2. Pregnant cows and heifers are vaccinated twice: the first time no later than 45 days before calving, the second vaccination is carried out 21-24 days after the first vaccination, but no later than 14 days before calving.

Number 3. Calves obtained from previously vaccinated animals, and from unvaccinated ones, are vaccinated on the 7-10th day of life twice with an interval of 21-24 days. Revaccination of calves is carried out once at the age of 6 months.

No. 4. Sheep and goats are vaccinated twice: the first time no later than 45 days before lambing, the second vaccination is carried out 21-24 days after the first, but no later than 14 days before lambing. An offspring obtained from previously vaccinated animals and from an unvaccinated livestock is vaccinated on the 7-10th day of life twice with an interval of 21-24 days. Revaccination of young animals is carried out once at the age of 6 months with the prescribed dose.

No. 5. All livestock of small ruminants older than one year are vaccinated every 6 months twice with an interval of 21 days.

Implementation of the invention

The examples below are illustrative and thus do not limit the scope of the present invention.

Example # 1. Differentiation of P. multocida by sensitivity to hyaluronidase, to confirm the properties of control-production strains.

A daily broth culture of P. multocida with a bacteriological loop is plated on Petri dishes with Hottinger's agar. Sowing is carried out, without tearing off the loop from the surface of the agar, in parallel strokes at a distance of 7-10 mm from one another. Then a daily broth culture of Staphylococcus aureus is inoculated perpendicularly to the inoculation of the pasteurella culture with a bacteriological loop in one stroke along the diameter of the dish.

The inoculation dishes are incubated at 37 ° C, the test results are taken into account after 18-24 hours.The strains forming near (up to 5 mm) from the staphylococcus growth line, smaller colonies than at a distance from this line, are referred to serotype A.

Example # 2. Differentiation of P. multocida serotypes using acriflavin test to confirm the properties of control-production strains.

A daily broth culture of P. multocida is centrifuged at 3000 rpm for 15 minutes. The supernatant is decanted, the bacterial cells are resuspended in the same test tube with the remnants of the supernatant until a homogeneous suspension is obtained. To the resulting suspension add 0.5 cm ^{3 of an} aqueous freshly prepared solution of acriflavine 1: 1000 and mix thoroughly.

The strains belonging to serotype D, within 10-15 minutes after the addition of the acriflavine solution, form a large-cotton flocculate that does not break when shaken. Strains that fail the hyaluronidase and acriflavine test are type B.

Example No. 3. Determination of the serotype of Mannheimia haemolytica strains.

Since on the territory of the Russian Federation two serotypes of manchemia are predominantly circulating, namely A1 and A2, these two serotypes are included in the antigenic composition of the proposed vaccine [Laishevtsev, A.I. Clinical and epizootic substantiation of vaccine prevention and development of a vaccine against mancheimiosis in cattle and small ruminants Dissertation for the degree of candidate of biological sciences / Federal Scientific Center - All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko RAS. Moscow, 2018, 155 pages]. When studying the epizootic situation of mancheimiosis in cattle and small ruminants, a huge problem is the lack of commercial diagnostic sera on the Russian market. In this regard, the serotyping of field isolates is carried out by comparing their homology with each other and with strains of known serotypes. So, the known strain collection ATCC No. 33396, related to serotype A2, and production control strains No. 169 and KMIEV-B158, related to serotype A1.

To obtain antisera to known serotypes of manchemia, four guinea pigs weighing 350-450 g are used. for each strain. Additionally, two guinea pigs are used to control the survival of hyperimmunization.

The capsular type of manchemia antigens was determined by the following method:

1) hyperimmunization of animals using complete microbial antigens of Mannheimia haemolytica. To implement this stage of the study, blood serum was obtained from the animals used in the experiment as a negative control. Then the animals were injected subcutaneously once every 7 days with an inactivated suspension of bacterial cells of the corresponding strain at a concentration of 6 billion μR. cl. in 1 cm ³ . The volume of a single dose administered was 2 ml, i.e. the final concentration of antigen was 12 billion microns. cl. For each group of animals, the procedure was performed 7 times over 42 days. Blood sampling to obtain serum was performed 14 days after the last injection. Sera of the respective groups of gilts were pooled;

2) obtaining the capsular antigen of Mannheimia haemolytica for staging the reaction of indirect hemagglutination was performed using an overnight culture grown in tryptone-soy broth. The broth culture was subjected to thermal inactivation at 60 ° C for 30 minutes. In order to obtain the supernatant, the bacterial mass inactivated by heating was centrifuged at 3000 rpm for 30 minutes, after which the supernatant was taken into a sterile Falkon tube and subsequently used as a capsular antigen when staging an indirect hemagglutination reaction;

3) obtaining erythrocytes from the blood of cattle was performed according to the following scheme. The presence of erythrocytes of susceptible animal species was a prerequisite for staging the reaction of indirect hemagglutination. For this, defibrinated blood in a volume of 250 ml was obtained from a young bull. Then, the defibrinated blood was centrifuged at 2500 rpm, for a total duration of 5 minutes, to precipitate heavy cells, after which the erythrocytes were washed twice with phosphate buffered saline, followed by repeated centrifugation under the same mode until a clear supernatant was obtained;

4) adsorption of capsular antigens on the surface of erythrocytes. Erythrocytes of cattle in a volume of 0.2 ml were mixed with 20 cm ^{3 of the} capsular antigen of each strain. In order to firmly fix the adsorbed antigens on erythrocytes, 200 μl of glutaraldehyde was added to the mixture. Incubated at 37 ° C for 1 hour with gentle stirring. As a result of this procedure, antigens adsorbed by erythrocytes were obtained at a low concentration; for their concentration, the mixture was subjected to double centrifugation at 2500 rpm. / min. within 5 minutes with rinsing with PBS. After the end of the second centrifugation and removal of the supernatant, the remaining volume of red blood cells was suspended in 20 cm ³ PBS to achieve a final concentration of red blood cells equal to 1%;

5) setting the reaction of indirect hemagglutination (RIGA). For this step, disposable, sterile 96-well U-bottom plates are used, in which serum dilutions from 1: 2 to 1: 1024 are obtained. As a result of the study, the strains of Mannheimia haemolytica No. 822-VIEV and No. 1540-VIEV belong to the serotype A1 serotype, and demonstrate agglutination at a dilution of 1: 64-1: 128. Mannheimia haemolytica strains No. 1050-VIEV and No. 1494-VIEV belong to the A2 serotype, and show agglutination at a dilution of 1: 64-1: 128.

Example No. 4. Cultivation of Mannheimia haemolytica, Bibersteinia trehalosi, and Pasteurella multocida strains by submerged method.

The cultivation of the strains Mannheimia haemolytica, Bibersteinia trehalosi, and Pasteurella multocida was carried out using a Sartorius BIOSTAT-A reactor on tryptone soybean, brain heart infusion, and eugonic broths (each strain was sequentially cultivated on all the above media). The preparation of the main inoculum was carried out according to the following method: on the first day, the first generation of industrial strains was obtained by resuspension of a lyophilized MSB culture in a test tube, followed by inoculation on nutrient agar. Each strain was prepared separately. The cultivation of the first generation of cultures continued for 18 hours under aerobic conditions at a temperature of 37 ° C; on the second day, after confirming the properties of industrial cultures, individual colonies from Petri dishes were subcultured into tubes with nutrient broth (10 ml each), for subsequent cultivation for 18 hours under aerobic conditions at 37 ° C, with constant stirring on a shaker (100 rpm); on the third day, after controlling the properties of the grown cultures, the broth culture from test tubes was subcultured into vials with cerebral heart and / or tryptone-soy broth (1 tube per 250 ml of medium). The obtained crops were cultivated for 18 hours under aerobic conditions at a temperature of 37 ° C, with constant stirring on a shaker (100 rpm); on the fourth day, after preliminary control of the brood, the entire volume of the vial was subcultured into the Sartorius BIOSTAT-A reactor containing 5 liters of culture medium. In this case, the amount of matt brood for reactor cultivation was 10% of 5 liters, i.e. 500 ml The cultivation of strains Mannheimia haemolytica and Bibersteinia trehalosi was carried out at a pH of 7.2-7.8, and Pasteurella multocida at a pH of 7.0-7.8, at a temperature of 37 ° C. To achieve the maximum accumulation of bacterial cells of industrial strains Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida, 5% yeast extract and 5% bovine blood serum were additionally added to the nutrient medium. To maintain the required level of nutrients in the medium, a 40% glucose solution was added at the rate of 10 ml of glucose, per 1 liter of the nutrient medium 1 time per hour, depending on the growth rate of the culture. When cultivating strains of Mannheimia haemolytica, hemin and nicotinamide-adenine-dinucleotide were additionally included in the medium at the rate of 10 mg per liter of nutrient medium. The pH level during cultivation is maintained with a 10% alkali solution (NaOH).

As a result of the cultivation carried out on various nutrient media, the results shown in table 4 were obtained.

The results shown in Table 4 indicate the interchangeability of nutrient media for submerged cultivation of Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida strains.

In all cases, broth cultures of industrial strains were cells typical of the species in terms of morphology, and did not contain extraneous microflora.

Example No. 5. Inactivation of the culture fluid

Inactivation of broth cultures of Mannheimia haemolytica, Bibersteinia trehalosi and Pasteurella multocida strains (example No. 4) was carried out by adding formalin (containing at least 37% formaldehyde) to them, in an amount of 0.3%, based on the volume of the culture fluid. So, to the volume of 5800 ml of culture fluid, 17.4 ml of formalin was added. Inactivation was carried out within 72 hours at a temperature of 21-22 ° C.

Example No. 6. Concentration of the culture liquid

The concentration of the resulting broth culture after inactivation (example No. 5) was carried out by centrifugation on MPW-380R equipment for 1 hour at 3 thousand revolutions, RCF - 1861. After centrifugation and separation of the supernatant (centrifuge), the bacterial mass was collected in a separate sterile container ( bottle) for future use. At this stage, the concentration of the bacterial mass and its volume were determined. Determination of the concentration of the suspension was carried out using turbidity standards - a set of TOC of turbidity of bacterial suspensions of the State Committee for the Provision of Mineral Resources of the Federal State Budgetary Institution "NTsESMP" of the Ministry of Health of Russia. Concentration results are shown in Table 5.

According to the data shown in Table 5, the culture fluid was concentrated on average 10 times.

Example No. 7. Determination of the completeness of antigen inactivation

The completeness of antigen inactivation was assessed by the absence of viable cells in the bacterial mass by inoculating samples on growth-supporting nutrient media in accordance with GOST 28085-2013 “Biological medicinal products for veterinary use. Method of bacteriological control of sterility ".

Also, the completeness of antigen inactivation was assessed by the harmlessness of the inactivated bacterial mass in laboratory mice weighing 16-18 g. with intraperitoneal injection of 0.5 cm ³ . Before the introduction of the antigen to animals, it is preliminarily diluted to a concentration of 6 billion microns. cl. cm ³ . Five experimental mice were used to assess the safety of each series of antigen. All antigens used to assemble the three series of vaccines were sterile and did not cause death of laboratory animals within 14 days of observation.

Example No. 8. Compilation of series No. 1 of the vaccine against mancheimiosis, bibersteinosis and pasteurellosis in cattle and small ruminants using saline as a diluent and GOA as an adjuvant

When compiling a series of vaccines, inactivated bacterial antigens of five industrial cultures were mixed in a sterile container with a diluent (saline solution), an adjuvant (3% aluminum hydroxide solution) and a preservative (sodium merthiolate), setting the concentration of bacterial antigens to 10 billion μR. cells / cm ³ (2 billion microns cells / cm ³ each). The components were mixed until a homogeneous suspension was obtained, the pH of the vaccine batch was adjusted to 7.4. The recipe for the composition of the vaccine with this arrangement is shown in table 6. The composition is given on the basis of the volume of the produced batch of 1 thousand doses (3 liters of the drug).

The vaccine thus obtained in appearance is a suspension of light gray color with a gray-white precipitate, which easily breaks down when shaken into a homogeneous suspension.

Example No. 9. Compilation of series No. 2 of vaccines against mancheimiosis, bibersteinosis and pasteurellosis in cattle and small ruminants using water for injection as a diluent and carbomer 971P as an adjuvant

When composing a series of vaccines, inactivated bacterial antigens of five industrial cultures were mixed in a sterile container with water for injection as a diluent, a 2% solution of carbomer 971P as an adjuvant, and sodium merthiolate as a preservative, setting the concentration of bacterial antigens to 10 billion μR ... cells / cm ³ (2 billion microns cells / cm ³ each). The components were mixed until a homogeneous suspension was obtained, after which the pH of the vaccine batch was adjusted to 7.4. The recipe for the composition of the vaccine when using this layout option is shown in Table 7. The composition is given on the basis of the volume of the produced batch of 1 thousand doses (3 liters of the drug).

The vaccine thus obtained in appearance is a suspension of light gray color with a gray-white precipitate, which easily breaks down when shaken into a homogeneous suspension.

Example No. 10. Compilation of series No. 3 of the vaccine against mancheimiosis, bibersteinosis and pasteurellosis in cattle and small ruminants using phosphate buffered saline as a diluent and an oil adjuvant Montanide IS A 70 as an adjuvant

A pre-emulsion is prepared in a sterile container. For this, in a mixer with constant stirring (250 rpm for 1.5 hours), the inactivated bacterial antigens of five industrial cultures were mixed on the basis of PBS - buffer as a diluent, Montanide ISA 70 as an adjuvant, sodium merthiolate as a preservative, so as to obtain a concentration of bacterial antigens equal to 10 billion microns. cells / cm ³ (2 billion microns cells / cm ^{3 of} each strain). To obtain the final version of the vaccine, a Silverson L5M-A homogenizer was used. Emulsification is carried out within 3 hours at 6 thousand rpm. The recipe for the composition of the vaccine when using this layout option is shown in Table 8. The composition is given on the basis of the volume of a batch of 1 thousand doses (3 liters of the drug).

The vaccine thus obtained is in appearance a white emulsion, sometimes with a yellowish tinge. During long-term storage, a slight stratification of the preparation was observed, which, upon shaking, turns into the initial state of a homogeneous emulsion.

Example No. 11. Determination of vaccine sterility

The control of the sterility of the vaccine was carried out in accordance with GOST 28085-2013 “Biological medicinal products for veterinary use. Method of bacteriological control of sterility ". So, three vials of each batch of the drug prepare a combined sample, after which it is inoculated on liquid nutrient media Sabouraud, MPB and MPPB (Kitt-Tarozzi medium) and on solid culture media Sabouraud and MPA. Three test tubes and two plates with a nutrient medium were used for inoculation from each vial. When sowing on Kitt-Tarozzi medium, sowing was done in two test tubes and two vials. To identify aerobic microorganisms and facultative anaerobic microorganisms, 0.5 cm ^{3 of} inoculum was sown in one tube and 1-2 cm ³ in one bottle, and to identify anaerobic microorganisms - 1 and 5 cm ³ , respectively. Test tubes and vials with inoculations on all media, except for Sabouraud's medium, were kept in a thermostat at a temperature of (37 ± 1) ° С, in Sabouraud's medium - at a temperature of (22.5 ± 2.5) ° С, for 7 days (for anaerobes - 14 days). After the expiration of the specified period, reseeding was done, with the exception of crops on MPA. Samples were subcultured on the same nutrient media and in the same volumes as at inoculation. Secondary crops were also kept for 7 days (for anaerobes - 14 days). As a result of the control carried out on three batches of the preparation, their sterility was established. No extraneous microflora was detected on any of the used growth media.

Example No. 12. Vaccine safety assessment

The study of three series of the drug was carried out on white mice weighing 16-18 g. To test each series of vaccines, 5 mice were used, which were injected intramuscularly with 0.5 ml of the vaccine. The observation of the experimental animals continued for 14 days. All three series of the drug turned out to be harmless, since they did not provoke any local and systemic reactions in animals, including not leading to their death.

Example No. 13. Determination of the immunogenic activity of the vaccine in laboratory animals (white mice weighing 18-20 g.)

To achieve this goal, ten groups of 10 mice each, weighing 18-20 grams, were formed. Five groups were experimental (for each infecting strain), and five control groups (for each infecting strain). Experimental group No. 1 and control group No. 1 were designed to control the mannheimia component in serotype A1 and were infected with Mannheimia haemolytica strain No. 1540-VIEV serotype A1; experimental group No. 2 and control group No. 2 were designed to control the mannheimia component according to serotype A2 and were infected with the Mannheimia haemolytica strain No. 1494-VIEV serotype A2; experimental group No. 3 and control group No. 3 were designed to control the activity of the Bibersteinia trehalosi component and were infected with the Bibersteinia trehalosi strain No. 1586-VIEV; experimental group No. 4 and control group No. 4 were designed to control the activity of the pasteurellosis component according to type A and were infected with the Pasteurella multocida strain No. 1231; experimental group No. 5 and control group No. 5 were designed to control the activity of the pasteurellosis component by type B and were infected with the Pasteurella multocida strain No. 656.

Evaluation of the immunogenic activity of the mancheimia component was carried out by the method of preliminary immunosuppression with dexamethasone, the rest of the components were controlled by the method of direct infection. So, when evaluating the immunogenic activity of the mancheimiosis component of the proposed agent, mice of experimental and control groups No. 1 and No. 2 were previously vaccinated, twice with an interval of 14-16 days, at a dose of 1 cm ³ . 10 days after the last vaccination, these animals were injected intramuscularly with 0.01 ml of a 0.4% solution of dexamethasone at the rate of 0.5 ml of active ingredient per 1 injection. This manipulation was repeated 4 times, i.e. 10, 11, 12, 13 days after the second vaccination. On the 14th day after the second vaccination, the designated experimental and control groups of animals were subjected to intraperitoneal infection with the corresponding strain of Mannheimia haemolytica at a dose of 5LD ₅₀ .

Evaluation of the immunogenic activity of the remaining components of the vaccine is carried out by the method of direct intraperitoneal infection on the 14th day after the second vaccination at a dose of 5LD ₅₀ .

The 5LD ₅₀ value for the control strains was as follows:

Mannheimia haemolytica No. 1540-VIEV - 1.21 × 10 ⁹ md. cl .;

Mannheimia haemolytica No. 1494-VIEV - 8.05 × 10 ⁸ md. cl .;

Bibersteinia trehalosi No. 1586-VIEV - 1.83 × 10 ⁹ md. cl .;

Pasteurella multocida No. 1231 - 1.99 × 10 ⁹ md. cl .;

Pasteurella multocida No. 656 - 2.41 × 10 ⁹ md. cl.

The results of determining the immunogenic activity of three series of the proposed vaccine are shown in table No. 9.

When carrying out this study, the results were obtained indicating that the survival rate of animals in the experimental groups, regardless of the batch of the drug used, was 90%, while 90% (the minimum value for the three groups is indicated) of the animals of the control groups died within 1-2 days. after infection, which indicates a high level of immunogenic activity of the vaccine.

Example No. 14. Determination of the safety and efficacy of the vaccine series No. 1 in sheep

Tests of the vaccine series No. 1 were carried out in a sheep-breeding enterprise in the Tver region, which is unfavorable for mancheimiosis, bibersteinosis and pasteurellosis, which was previously confirmed by laboratory diagnostic studies. The enterprise formed two groups of ewes, 40 heads each, the experimental group of animals was immunized with the proposed drugs, while the control group was not vaccinated. The animals were vaccinated according to the scheme No. 4, i.e. pregnant sheep were vaccinated twice: the first time 45 days before lambing, the second time 21 days later, i.e. 24 days before lambing. The vaccine dose for a single use was 2 cm ³ . The injection was made intramuscularly into the area of the inner thigh. The offspring obtained from the vaccinated animals was also subjected to two vaccinations at 7 and 28 days of age, with the same dose and method of administration.

As a result of the studies, it was found that the drug is harmless when used once or twice both on young animals and young animals. Intramuscular use of the drug did not provoke any systemic or local reactions. Mobility, appetite, body temperature in animals of the experimental group did not differ from that in the control group.

The results of assessing the safety of young animals in the experimental and control groups after vaccination were carried out for the period when they reached 5 months of age. Thus, 64 lambs were obtained from 40 heads of the experimental group, while 71 lambs were received from the control group. Upon reaching 5 months of age, the safety of lambs obtained from the experimental group of ewes was 90.6%, while this value in the control group was 77.46%. In addition, in order to ensure the safety of animals in the control group, young animals were prescribed a course of antibacterial therapy. Antibiotics were not prescribed to the experimental group due to lack of need.

Thus, the results obtained indicate the harmlessness of the proposed tool and its effectiveness in the conditions of a sheep breeding enterprise.

Example No. 15. Determination of the safety and efficacy of the vaccine series No. 3 in cattle

Tests of the vaccine series No. 3 were carried out in a cattle-breeding enterprise in the Moscow region, which was unfavorable for mancheimiosis and pasteurellosis, which was previously confirmed by laboratory diagnostic tests. At the enterprise, two groups of cows were formed, 55 heads each, the experimental group of animals was immunized with the proposed drugs, while the control group was not vaccinated. The animals were vaccinated according to the scheme No. 2, i.e. pregnant cows were vaccinated twice: the first time 45 days before calving, the second time 21 days later, i.e. 24 days before calving. The vaccine dose for a single use was 3 cm ³ . The injection was made subcutaneously into the region of the middle third of the neck. The offspring obtained from the vaccinated animals was vaccinated according to the scheme No. 2. So, vaccination was carried out on 10 and 21 days of life, subcutaneously, at a dose of 2 cm ³ .

As a result of the studies, it was found that the drug is harmless when used once or twice both on pregnant cows and on young animals obtained from them. Subcutaneous use of the product did not provoke any systemic or local reactions. Mobility, appetite, body temperature in animals of the experimental group did not differ from that in the control group.

The results of assessing the safety of young animals in the experimental and control groups after vaccination were carried out for the period they reached 6 months of age. So, from 55 heads of the experimental group, 57 calves were received, while from the control group, 59 calves, respectively. Upon reaching 6 months of age, the safety of calves obtained from the experimental group of cows was 94.7%, while this value in the control group was 81.35%. In addition, in order to ensure the safety of animals in the control group, young animals were prescribed a course of antibacterial therapy. Antibiotics were not prescribed to the experimental group due to lack of need.

Thus, the results obtained indicate the harmlessness of the proposed tool and its effectiveness in the conditions of a cattle-breeding enterprise.

Claims (7)

1. Vaccine against mancheimiosis, biebersteiniasis and pasteurellosis of cattle and small ruminants associated inactivated, containing the following components per 1 cm ^{3 of the} preparation: inactivated protective antigen of the Mannheimia haemolytica strain No. 822-VIEV serotype A1 - 2 billion mcr. cl .; inactivated protective antigen of the Mannheimia haemolytica strain No. 1050-VIEV serotype A2 - 2 billion microns. cl .; inactivated protective antigen of the Bibersteinia trehalosi strain No. 984-VIEV - 2 billion microns. cl .; inactivated protective antigen of the Pasteurella multocida strain No. 1042-VIEV type A - 2 billion microns. cl .; inactivated protective antigen of the Pasteurella multocida strain No. 163-VIEV type B - 2 billion microns. class, as well as pharmaceutically acceptable target additives. 2. The vaccine according to claim 1, characterized in that a preservative is used as pharmaceutically acceptable target additives in the form of a 10% aqueous solution of sodium merthiolate, introduced into the preparation at a rate of 1: 10000, 1 part of sodium merthiolate solution to 10000 parts of the drug: 300 l of the finished solution of the drug add 0.03 l of merthiolate solution. 3. The vaccine according to claim 1, characterized in that a diluent in the form of sterile saline solution, sterile PBS - phosphate buffered saline solution, water for injection in the amount required to obtain 100% of the volume of the composited batch of the drug is used as pharmaceutically acceptable target additives. 4. The vaccine according to claim 1, characterized in that, as pharmaceutically acceptable target additives, it contains 3% oil adjuvant in a volume of 50% by volume of the preparation. 5. The vaccine according to claim 1, characterized in that, as pharmaceutically acceptable target additives, it contains one of the following adjuvants with the addition of 0.2% of the total volume of the composited batch of the drug 10% sodium hydroxide solution: 3% aluminum hydroxide solution in a volume of 10% on the volume of the preparation, Carbomer 971p 2% solution in PBS in the volume of 10% of the volume of the preparation. 6. A method of obtaining a vaccine according to claim 1 against mancheimiosis, bibersteinosis and pasteurellosis of cattle and small cattle associated inactivated, including the cultivation of industrial strains, inactivation of the culture fluid, concentration of antigens; determination of the completeness of antigen inactivation; preparation of a vaccine batch; control of vaccine sterility; assessment of the safety of the vaccine; determination of the immunogenic activity of the vaccine, characterized in that strains of Mannheimia haemolytica No. 822-VIEV serotype Al are used as industrial strains; Mannheimia haemolytica No. 1050-VIEV serotype A2; Bibersteinia trehalosi No. 984-VIEW; Pasteurella multocida No. 1042-VIEV type A; Pasteurella multocida No. 163-VIEV, and as control strains Mannheimia haemolytica No. 1540-VIEV; Mannheimia haemolytica No. 1494-VIEW; Bibersteinia trehalosi No. 1586-VIEW; Pasteurella multocida No. 1231 - VGNKI; Pasteurella multocida No. 656 - VGNKI, and the assessment of the immunogenic activity of the mancheimia component is carried out by the method of preliminary immunosuppression with dexamethasone. 7. The method according to claim 6, characterized in that an inactivant in the form of formalin containing at least 37% formaldehyde, at the rate of 0.3% to the volume of the broth culture, is used as pharmaceutically acceptable target additives.