RU2750865C1 - Polyvalent inactivated vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese, the method of its preparation - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: group of inventions relates to the field of veterinary medicine, it can be used to obtain a vaccine for the prevention of infectious pathologies in turkeys, ducks and geese caused by the bacteria Riemerella anatipestifer, Pasteurella multocida, Salmonella typhimurium, Salmonella infantis, Salmonella enteritidis. Polyvalent inactivated vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese contains the following components per 1 cm³ of the drug: inactivated protective antigen of the Riemerella anatipestifer strain No. 549-ARRIEVM (All-Russian Research Institute of Experimental Veterinary Medicine)

- 2 billion microbial cells, inactivated protective antigen of the R. anatipestifer strain No. 566-ARRIEVM

- 2 billion microbial cells, inactivated protective antigen of the R. anatipestifer strain No. 571-ARRIEVM - 2 billion microbial cells, inactivated protective antigen of the Pasteurella multocida strain No. 367-ARRIEVM - 2 billion microbial cells, inactivated protective antigen of the Salmonella enteritidis strain No. 162-ARRIEVM - 1 billion microbial cells, inactivated protective antigen of the Salmonella typhimurium strain No. 178-ARRIEVM - 1 billion microbial cells, inactivated protective antigen of the Salmonella infantis strain No. 1827-ARRIEVM - 1 billion microbial cells, as well as pharmaceutically acceptable target additives. The group of inventions also relates to the method for preparing this vaccine.

EFFECT: use of this group of inventions makes it possible to prepare a vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese, based on a balanced antigenic composition containing the most common serotypes of bacteria Riemerella anatipestifer, Pasteurella multocida, Salmonella spp. on the territory of the Russian Federation, ensuring the creation of long-term and intense immunity in vaccinated poultry.

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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 turkey breeding, duck breeding and goose breeding for specific prevention of infectious pathologies caused by bacteria of the species Riemerella anatipestifer, Pasteurella multocida, Salmonella typhimurium, Salmonella enteritis infantis. In addition to the veterinary field of application, the invention contributes to the improvement of the epidemiological situation in medical practice, as it helps to reduce the incidence of toxic infections in humans caused by bacteria of the Salmonella spp. Species associated with the consumption of food products from poultry processing enterprises.

Infectious diseases of birds of industrial species for agricultural producers and the state as a whole are the cause of significant economic losses resulting from the shortage of products and financial costs for preventive and therapeutic measures [Shome, R. An outbreak of Riemerella anatipestifer infection in ducks in Meghalaya. / R. Shome, BR. Shome, H. Rahman, HV. Murugkar, A. Kumar, BP. Bhatt, KM. Bujarbarauah. // Indian J. Comp. Microbiol. Immunol. Infect. Dis. - 2004. - 25. - P. 126-127.]. Intensive production technology leads to the need to simultaneously keep a large number of poultry in a limited area, which increases the likelihood of the occurrence and spread of infections [Sandhu, T. Riemerella anatipestifer infection. / T. Sandhu, R. Rimler. // Diseases of poultry. - 10th Ed London: Iowa State University Press, Ames Mosby-Wolfe. - 1997]. In addition, the risk of infectious pathologies at poultry farms should be associated with the import into the country of breeding hatching eggs that are not controlled for a number of diseases, as well as with the migrations of migratory birds that carry various pathogens, in particular the causative agents of riemerella, pasteurellosis and salmonellosis [Cha, S.Y. Surveillance and characterization of Riemerella anatipestifer from wild birds in South Korea. / S.Y. Cha, H.S. Seo, B. Wei, M. Kang, J.H. Roh, R.H. Yoon, J.H. Kim, H.K. Jang // J. Wild Dis. - 2015. - 2. P. 341-347; Pimenov, N.V. The identification of salmonella infection in hatching eggs and products of turkey-keeping. / N.V. Pimenov, A.I. Laishevtcev, V.V. Pimenova // Russian Journal of Agricultural and Socio-Economic Sciences. 2015. No. 10 (46). P. 9-17].

Riemerellosis (synonyms - "infectious serositis", "new disease of ducks", "septicemia of ducks", "influenza geese", "exudative septicemia of geese") is an infectious disease of turkeys, ducks, geese, characterized by sepsis, fibrinous pericarditis, aerosachepatitis, perichepatitis caseous salpingitis and meningitis [Laishevtcev, AI Clinico-morphological manifestation of waterfowls' riemerellosis and biological features of disease causative agent. // A.I. Laishevtcev, A.V. Kapustin, N.V. Pimenov, V.D. Iliesh // Russian Journal of Agricultural and Socio-Economic Sciences. 2017. No. 11 (71). P. 538-545]. The causative agent of the disease is the bacterium Riemerella anatipestifer. Outbreaks of the disease are recorded all over the world and lead to significant economic losses due to high mortality, reduced growth rates, poor feed conversion, costs of medical and recreational activities, etc. [Kardos, G. Development of a novel PCR assay specific for Riemerella anatipestifer. / G. Kardos, J. Nagy, M. Antal, A. Bistyak, M. Tenk, I. Kiss. // Lett Appl Microbiol. - 2007 .-- 44 (2). - P. 145-8]. The bird is most susceptible to the disease at the age of 3-4 weeks, in some cases up to 8 weeks. The recovered bird remains a carrier, without any clinical manifestations of the disease, but at the same time it is the main source of the spread of infection [Pierce, RL. Pasteurella anatipestifer infection in geese. / RL. Pierce, M.W. Vorhies. // Avian Dis. - 1973. - 17. - P. 868-870]. The mortality rate among young animals in dysfunctional enterprises can reach 75%, which indicates the high significance of this disease for industrial poultry farming [Sandhu, T. Riemerella anatipestifer infection. / T. Sandhu, R. Rimler. // Diseases of poultry. - 10th Ed London: Iowa State University Press, Ames Mosby-Wolfe. - 1997]. The migration of wild waterfowl, not only geese and ducks, but also swans, cranes, storks, etc. is essential in the spread of the disease around the world. In this case, the transmission of the pathogen can occur through direct contact between wild and poultry in grazing fields and water bodies [Cha, S.Y. Surveillance and characterization of Riemerella anatipestifer from wild birds in South Korea. / S.Y. Cha, H.S. Seo, B. Wei, M. Kang, J.H. Roh, R.H. Yoon, J.H. Kim, H.K. Jang // J. Wild Dis. - 2015. - 2. P. 341-347].

Pasteurellosis (cholera of birds) is a contagious disease of most species of animals and birds, caused by bacteria of the genus Pasteurella. The disease can be in a hyperacute, acute, subacute and chronic course, in addition, an asymptomatic form is quite common - bacterial carriage. The causative agent of the disease is the bacterium Pasteurella multocida. In the Russian Federation, three types of pasteurella are most common: A, B and D. P. multocida type A most often causes pasteurellosis infection of birds, rabbits and fur-bearing animals, and also contributes to the development of 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 on the territory of the Russian Federation are not widespread, while they are causative agents of hemorrhagic septicemia in cattle and wild ruminants in a number of foreign countries [Kapustin, A.V. and other Methodological instructions: Diagnostics of pasteurellosis of farm animals, birds and fur animals. 29 p. Moscow 2016]. In the acute course of the disease, septic processes, croupous pneumonia, pleurisy and edema of various localization are observed. 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 [Bisgaard, M. Ecology and significance of Pasteurellaceae in animals. / M. Bisgaard // Zentralbl. Bakteriol. 1993 279, 7-26]. Mortality in the acute course of pasteurellosis can reach 90%, in the absence of timely treatment [Christensen, J.P. Fowl cholera / J.P. Christensen, M. Bisgaard // Rev. sci. tech. Off. int. Epiz. 2000,19 (2) 626-637].

Salmonellosis is one of the leading problems in industrial and private poultry farming. This is an infectious disease of birds and animals, dangerous for humans by foodborne toxicoinfection. The causative agents of bird salmonellosis are various serovariants of bacteria of the species Salmonella enterica of the genus Salmonella of the family Enterobacteriaceae. Waterfowl and turkeys are most susceptible to Salmonella typhimurium (group "B"), Salmonella infantis (group "C"), Salmonella enteritidis (group "D"). Of course, there is still a large number of Salmonella serotypes adaptive to poultry, but their prevalence in the country is much lower than the three mentioned serotypes [Pimenov, N.V. The role of farming poultry's salmonella pathogens in infection and pathology of human disease. / N.V. Pimenov, A.I. Laishevtcev, V.V. Pimenova // Russian Journal of Agricultural and Socio-Economic Sciences. 2017. No. 2 (62). S. 282-289]. In acute and subacute course, the main clinical signs are lethargy, inactivity, lack of appetite, thirst, diarrhea. In the future, serous-mucous, mucopurulent develops, and in goslings and ducklings, sometimes fibrinous-purulent conjunctivitis. Mucopurulent discharge from the nasal openings is often observed. In addition, with a subacute course, difficulty breathing is noted, wheezing due to developing pneumonia, and arthritis is sometimes recorded in goslings [Lenev, S. Improvement of allocation and identification of Salmonella entericabacteria of Arizonae subspecies / S. Lenev, A. Laishevtsev, N. Pimenov , V. Semykin, I. Pigorev, V. Eremenko, O. Sein, A. Glinushkin, M. Ali Shariati // International Journal of Pharmaceutical Research and Allied Sciences. 2016. No. 5 (3). S. 342-348]. During the autopsy of the dead and forcibly killed birds, changes in character and severity are established. So, with an acute Salmonella infection in the corpse, changes in the intestines are found, especially in the thin section. In its lumen, accumulations of mucus and gases are visible. The mucous membrane is swollen, hyperemic, with petechial hemorrhages. In the large part of the intestine, the mucous membrane is covered with a pityriasis plaque, in places there are punctate hemorrhages and small erosions. In acute and subacute salmonellosis in the small intestine, pronounced changes are found to varying degrees, characteristic of acute serous or serous-mucous catarrh. In the thick section - catarrhal-fibrinous, less often - diphtheria inflammation with the development of necrosis of the integumentary epithelium and partially - the superficial layer of the mucous membrane with the saturation of necrotic masses with fibrin, exfoliation of the mucous membrane, as a result of which erosions and difficult-to-separate films are formed in such areas [Pimenov, N. IN. Modern aspects of the fight against salmonella infection in poultry / N.V. Pimenov, A.I. Laishevtsev // Monograph Moscow, 2017]. The incidence of salmonellosis can be 40-50%, while mortality ranges from 5-80% [Pimenov, N.V. Methodical recommendations for the prevention and elimination of salmonellosis of farm animals, including birds / N.V. Pimenov, E.A. Tinaeva, A.I. Laishevtsev, Yu.N. Kolesnikova // Moscow, 2016].

According to the non-profit organization Russian Poultry Union, the total number of turkeys, ducks and geese in Russia as of 2018 amounted to 38.84 million heads, of which 21.16 million ducks, 9.0 million geese and 8.67 million turkeys, which in the overall structure of the poultry population in the country is 3.9%, 1.7% and 1.6%, respectively. Despite the fact that the population of turkeys, ducks and geese in the Russian Federation is only 7.2% of the total population, this volume is significant for the country's economy. Detailed data on the poultry population for 2016-2018 are shown in Table 1.

State of the art

In view of the existing risks of the development and spread of rimerellosis, pasteurellosis and salmonellosis, which entail high mortality in poultry, ensuring the epizootic well-being of poultry enterprises is an important task for the veterinary service of the Russian Federation. In turn, this problem can be solved in two ways, the first of which is the use of antibacterial drugs when a disease occurs, and the second involves the use of specific prophylaxis. Against the background of widespread antibiotic resistance, the use of the first option is ineffective [Yakimova, E.A. Antibiotic resistance of the causative agent of riemerellosis in waterfowl / E.A. Yakimova E.A. // Veterinary and feeding. 2018. No. 7. S.25-28; Pimenova, V.V. The main directions of health-improving measures for salmonellosis of birds: principles and disadvantages of antibiotic treatments / V.V. Pimenova, A.I. Laishevtsev, N.V. Pimenov // Russian Journal of Agricultural and Socio-Economic Sciences. 2017. No. 11 (71). C. 496-510; Laishevtsev, A.I. Comparative analysis of antibiotic sensitivity of collection strains of Pasteurella multocida isolated in the period before 1990 with field isolates isolated during 2014-2016 from cattle and small ruminants on the territory of the Russian Federation / A.I. Laishevtsev, A.V. Kapustin, A.M. Gulyukin // Proceedings of the Kuban State Agrarian University. 2016. No. 63. S. 132-138]. Thus, the use of specific prophylaxis in the fight against riemerella, pasteurellosis and salmonellosis is more promising than antibiotic therapy.

For the specific prevention of pasteurellosis in turkeys, ducks and geese, the following are registered:

- inactivated sorbed vaccine against bird pasteurellosis produced by VNIVIP - branch of FSC VNITIP RAS;

- inactivated vaccine against salmonellosis, colibacillosis and pasteurellosis of birds "AVIVAC - SALMO - KOLI - PASTOVAK", produced by LLC NPP Avivak.

In addition, known vaccine against pasteurellosis of birds inactivated emulsified (Cholerin Triple), produced by ABIC Biological Laboratories, Ltd., which allows for specific prevention of pasteurellosis in turkeys and geese.

For the specific prevention of salmonellosis in ducks and geese, the following are registered:

- live dry vaccine against salmonellosis of waterfowl, produced by FKP "Stavropol Biofabrika". The disadvantage of this drug can be considered a limited specific activity, since the vaccine contains only the Salmonella typhimurium antigen, which does not allow the drug to be used to combat Salmonella infantis and Salmonella enteritidis.

For the specific prevention of salmonellosis in turkeys and ducks, the following are registered:

- live dry vaccine against salmonellosis of birds (AviPro SALMONELLA DUO), manufactured by Lohmann Animal Health GmbH. The product contains two attenuated strains of Salmonella enteritidis and Salmonella typhimurium, but not Salmonella infantis. The preparation does not provide for vaccination of geese.

There are no registered veterinary drugs intended for specific prophylaxis of ryemerellosis in waterfowl in the country. Nevertheless, earlier on the Russian market there were Biovac preparations intended for the specific prophylaxis of riemerellosis:

- Virsin 424 - the drug is made from inactivated antigens Pasteurella multocida strains Pm-1,3 and 4-7-12, and Riemerella anatipestifer strain RA.

- Virsin 424a - a preparation made from inactivated Pasteurella multocida antigens of Pm-1.3 and 4 strains, and Riemerella anatipestifer of RA strain.

- Vircine 570 - a preparation made from inactivated antigens of the Newcastle disease virus of birds strain "VH", Pasteurella multocida strains Pm-1,3 and 4-7-12, and Riemerella anatipestifer strain RA.

- Virsin 765 - the drug is made from inactivated antigens Pasteurella multocida strains Pm-1,3 and 4, Riemerella anatipestifer strain RA, and 3 strains of the bacterium Ornithobacterium rhinotracheale.

The above drugs were used in industrial duck breeding and goose breeding, but not in turkey breeding, and since April 2017 they have not been sold in the country and cannot be used to prevent the disease. In addition, a narrow specific activity against the causative agent of rimerellosis should be considered a disadvantage of the above funds, since they contain only one serotype.

In addition to the registered drugs against pasteurellosis, a live vaccine against pasteurellosis (cholera) of birds from a weakly virulent strain "K" for cutaneous vaccination is known [copyright certificate SU 119657 A1 publ. 1959.01.01]. The disadvantage of this product is that it is not intended for vaccination of ducks and geese, and also does not contain the antigens Riemerella anatipestifer and salmonella. In addition, the disadvantage of the proposed tool is the short duration of immunity in a twice vaccinated bird.

A known 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, the developers focused on the use of the proposed method exclusively for the cultivation of the Pasteurella multocida "K" strain used for the production of a live vaccine, which is indicated for the purpose of the claimed invention and the examples given.

The morphological characteristics and viability of pasteurella is not a parameter that affects the antigenic and immune activity of inactivated pasteurellosis vaccines, and therefore their quality. 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 may affect their harmlessness, since the substances that make up the nutrient media are proteins against which antibodies can form.

A known method of making a vaccine against pasteurellosis (cholera) birds [copyright certificate SU 135595 A1 publ. 1961.01.01]. The disadvantage of this method is that it is intended for the production of exclusively live biological products against pasteurellosis. The proposed method is not used for the production of inactivated vaccines. In addition, the method involves the use of semi-liquid mesopatamia agar for cultivation in the medium, while the proposed method is focused on the use of nutrient broths for submerged cultivation. This approach allows you to increase the concentration of bacterial cells per unit volume of the medium, thereby affecting the cost of the finished product.

A known 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 waste, 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-providing 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 known method of manufacturing a vaccine against pasteurellosis of birds [copyright certificate SU 1566533 A1 publ. 1994.11.15]. The disadvantage of this method is that it is oriented only for the production of live pasteurellosis vaccines, and is not intended for use for the production of inactivated agents.

A known method of manufacturing an emulsion anti-pasteurellosis vaccine [patent RU 2162339 C1 publ. 2001.01.27]. The method of production of the proposed biological product is limited only to Hottinger's broth for the cultivation of pasteurella strains, while to obtain a higher concentration of bacterial cells, it is more expedient to use brain heart broth, tryptone soy broth, etc., which have better growth-providing properties for pasteurella.

Known polyvalent immunizing and / or therapeutic composition for use in bacterial infections or food poisoning, in particular salmonellosis, a method of obtaining this composition, its use and a vaccine containing this composition [patent RU 2683027 C2 publ. 2019.03.26]. The method is distinguished by the laboriousness of obtaining the drug, since it initially provides for the production of not only a vaccine preparation, but also hyperimmune sera.

Known: vaccine against Salmonella [patent RU 2596208 C2 publ. 2016.08.27]; vaccine against Salmonella [patent RU 2455023 C2 publ. 2012.07.10]; inducing cross-immunity vaccines against Salmonella [patent RU 2679039 C2 publ. 2019.02.05]. The disadvantage of these inventions is the limited antigenic composition of drugs, which does not allow to simultaneously fight several infectious pathologies.

Known: a method of manufacturing a vaccine against salmonellosis of farm animals and birds [patent RU 2124366 C1 publ. 1999.01.20]. The disadvantage of the above invention is the narrow scope of production technology, limited to the production of only Salmonella drugs.

Known: Method for preparing bacteria liquid for Riemerella anatipestifer vaccine [patent - CN 101967458 A, publ. 2011.12.14, China]. The disadvantage of this invention is the lack of a complete technology for the production of the vaccine itself. The main focus is on the cultivation of Raymerell; issues of inactivation, concentration, layout were not touched upon.

Known: inactivated vaccine Riemerella anatipestifer, trivalent and method of its production (Riemerella anatipestifer tervalence inactivated vaccine and preparation method thereof) [patent - CN 102908616 B, publ. 2016.01.06, China]; oil emulsion vaccine against Riemerella anatipestifer 1, 2 and 4 serotypes (Riemerella anatipestifer serum 1 type, 2 type and 4 type trivalent oil emulsion inactivated vaccine) [patent CN 102309753 A, publ. 2012.01.11, China]. The disadvantage of these drugs is that the serotypes of Reimerellus included in them are most common in China, and there is no evidence that similar serotypes are circulating in Russia. Thus, one cannot expect high efficiency of the considered inventions on the territory of the Russian Federation.

Known: a method of obtaining a bivalent inactivated vaccine for infectious serositis of ducks (Production method for bivalent inactivated vaccine for infectious serositis of ducks) [patent - CN102125687B, publ. 2013.07.17]; method for preparing Riemerella anatipestifer capsular antigen and propolis vaccine [patent - CN 103656636 A, publ. 2015.08.12]. The proposed methods are limited by the technology for the production of monocomponent preparations, i.e. does not disclose the essence of the production of combined preparations intended for the simultaneous prevention of several poultry diseases. In addition, the proposed technologies for the production of drugs have confirmed data on harmlessness and effectiveness on ducks and geese, but not on turkeys.

According to the above characteristics for existing and / or previously used vaccines on the territory of the Russian Federation, the drug Virsin-424 [link on the Internet http://www.biovac.co.il/BioVac//userdata/SendFile.asp?DBID=l&LNGID = 3 & GID = 506] is the closest analogue (prototype) of the proposed vaccine. As the main active components, the vaccine contains formalin-inactivated antigens Pasteurella multocida of strains Pm-1,3 and 4-7-12, and Riemerella anatipestifer of RA strain. As auxiliary agents, the preparation includes: mineral oil 0.32 cm ³ , monosorbate T80 - 0.01 cm ³ , sorbinate mooleate A80 - 0.03 cm ³ and thiomersal 0.05 mg. Nevertheless, the given drug has significant drawbacks, in particular, it contains only one serotype Riemerella anatipestifer, while it was proved that at least three heterologous serotypes circulate in the country, and the drug is not intended for use on turkeys [Laishevtsev A.I. Experience in the development of the first domestic vaccine against rimerellosis / A.I. Laishevtsev, E.A. Yakimova, A.V. Kapustin // Veterinary Medicine. 2018. No. 8. P.24-29].

The closest analogue (prototype) of the proposed method for producing a vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese is a method for producing a bivalent inactivated vaccine against infectious serositis of ducks (Production method for bivalent inactivated vaccine for infectious serositis of ducks) [patent - CN102125687 ... 2013.07.17]. The essence of the prototype implies deep cultivation of Reimerell strains in tryptone soy broth at a temperature of 37-38 ° C and pH 7.2, for 9-10 hours. Subsequent inactivation of the culture fluid is carried out by adding 0.3% volumes of formalin (containing at least 37% formaldehyde). The inactivation exposure is 20 hours. The next stage of vaccine preparation involves preparing the water and oil phase for the emulsion vaccine. The aqueous phase is prepared using 4 parts of Tween-80 and 96 parts of antigen, which are mixed in a sterile container, until the Tween is completely dissolved. The oil phase is prepared by mixing 94 parts of white oil with 6 parts of aluminum stearate with constant mixing for 30 minutes while maintaining a temperature of 30 ° C. The next stage of obtaining a vaccine involved combining the water and oil phases, for this they were mixed in equal amounts in an emulsifier for 30 minutes at 4000 rpm.

The given method is limited by the technology of production of monocomponent preparations, i.e. does not disclose the essence of the production of combined preparations intended for the simultaneous prevention of several poultry diseases. In addition, the new proposed method for cultivating Reimerell provides not only a deep cultivation method, but also a surface cultivation method, which makes it possible to use it to reduce energy consumption and use it in low-tonnage bio-production.

Since birds of all three mentioned species are exposed to rimerellosis, pasteurellosis and salmonellosis, it is advisable to develop one associated means of specific prophylaxis against the listed diseases, which most fully covers the serotypic composition of pathogens circulating in the Russian Federation. Thus, the research carried out on the basis of the Federal State Budgetary Scientific Institution FSC VIEW RAS confirms the wide circulation at duck and goose-breeding, as well as turkey-breeding enterprises of the country, of at least three serotypes of Riemerella pathogens, as well as pasteurellosis pathogens belonging to type "A", and Salmonella. The data obtained are confirmed by the FGBI "ARRIAH" [Potekhin, A. New threats in poultry farming require new strategies / A. Potekhin // Veterinary Medicine and Life. - 2018. - 3 (10)].

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 (health care, 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 riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese, based on a balanced antigenic composition containing the most common serotypes of bacteria of the species Riemerella anatipestifer, Pasteurella multocida and Salmonella and the need to expand the arsenal of multivalent inactivated vaccines aimed at specific prevention of infectious diseases in turkeys, ducks and geese.

Disclosure of the essence of the invention The technical result of the proposed group of inventions is to increase the stability, antigenic and immunogenic activity of the vaccine and create long-term and intense immunity in vaccinated poultry (turkeys, ducks and geese) against riemerellosis, pasteurellosis and salmonellosis, expanding the arsenal of polyvalent inactivated vaccines aimed at specific prevention infectious diseases of poultry, the creation of a vaccine with increased stability, antigenic and immunogenic activity, the expansion of approaches and methods for the manufacture of polyvalent inactivated vaccines aimed at the specific prevention of infectious diseases of poultry.

Polyvalent inactivated vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese

The claimed polyvalent vaccine against riemerella, pasteurellosis and salmonellosis of turkeys, ducks and geese contains the following components per 1 cm ^{3 of the} drug: formalin-inactivated protective antigen of the Riemerella anatipestifer No. 549-VIEV strain - 2 billion micro cells; formalin-inactivated protective antigen of R. anatipestifer strain No. 566-VIEV - 2 billion micro cells; formalin-inactivated protective antigen of R. anatipestifer strain No. 571-VIEV - 2 billion micro cells; formalin-inactivated protective antigen of the Pasteurella multocida strain No. 367-VIEV - 2 billion micro cells; formalin-inactivated protective antigen of Salmonella enteritidis strains No. 162-VIEV - 1 billion micro cells, Salmonella typhimurium No. 178-VIEV - 1 billion micro cells, Salmonella infantis No. 1827-VIEV -1 billion micro cells cells, as well as pharmaceutically acceptable targeted additives ... The immunizing dose recommended for a single use is 0.5 cm ³ .

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

- An inactivant required for the disinfection of bacterial antigens. 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. As standard solvents can be used - sterile saline, sterile PBS - phosphate buffered saline, water for injection;

- An adjuvant required 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 PEG-6000 (polyethylene glycol-6000) in the amount of 10% of the composable batch of the drug; GOA (aluminum hydroxide) at a concentration of 3% in a volume of 10% of the composable batch of the drug; oil adjuvant in the amount of 50% of the composable batch of the drug, etc.;

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 PEG-6000. 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 PEG-6000 and GOA - 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 18 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 parent, replacement and commercial poultry livestock. The vaccine induces the formation of immunity to riemerella, pasteurellosis and salmonellosis of turkeys, ducks and geese 12-14 days after re-administration, which lasts at least 8 weeks for the riemerella component, and at least 6 months for the pasteurellosis and salmonella components. Despite the fact that the duration of immunity to Riemerella anatipestifer is rather low, this period is sufficient to ensure the safety of susceptible livestock, since birds are most susceptible to infection at the age of 1 to 4 weeks. Transovarian immunity in young animals obtained from immunized birds lasts up to 14 days.

Bacterial strains of Riemerella anatipestifer No. 549-VIEV, No. 566-VIEV and No. 571-VIEV included in the vaccine; Pasteurella multocida №367-VIEV and Salmonella enteritidis №162-VIEV, Salmonella typhimurium №178-VIEV, Salmonella infantis №1827-VIEV are new industrial crops and are used for the first time in the composition of 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 have been 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 Scientific Research Institute of Experimental Veterinary Medicine. 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 16S rRNA gene.

Characteristics of Riemerella anatipestifer strains

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 pathogenicity groups (danger) and causative agents of parasitic diseases", the bacteria Riemerella anatipestifer are not belong to none of the four pathogenicity groups.

All Riemerella cultures have morphological, tinctorial and cultural properties typical of the species: Riemerella anatipestifer is an immobile, non-spore-forming gram-negative bacillus with an average size of 0.3-0.5 * 1-2.5 microns. In smears, cells are located singly, in pairs or in the form of small chains. On solid culture media when cultured in a 5-10% strength CO ₂ atmosphere for 48 hours or more and a temperature of 37 ° C, the rise smooth, nonpigmented colonies mucoid consistency. When cultured on media containing defibrinated ram blood, hemolysis can be a variable property for the described species of bacteria. At the same time, in the course of the work, it was found that the growth of Riemerella anatipestifer on agar media can manifest itself in the form of large individual colonies with an average size of 2-5 mm on the 2nd day of cultivation or in the form of continuous growth.

Cultivation conditions of Reimerell strains: the microorganism grows on trypticase-soy broth and on agar media containing defibrinated blood.

1) The production strain Riemerella anatipestifer No. 549-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-1252

Date, source and place of allocation: Duck breeding enterprise of the Ryazan region. In 2017 from caterpillar fabrics.

Biochemical properties: Culture of oxidase-, catalase-, alpha-galactosidase-, a-maltosidase, a-chymotrypsin is positive. Beta-glucosidase and arginine decarboxylase are negative. The strain does not form pigment on blood agar. Liquefies gelatin, does not hydrolyze esculin, does not form indole. The culture ferments glucose, maltose, fructose, mannose, dextrin, and does not ferment trehalose. CAMP test negative, positive Voges-Proskauer test. Forms urease.

Virulence for laboratory animals: LD ₅₀ value - 2.20 * 10 ⁷ mcr class. for 10 day old ducklings.

2) The production strain Riemerella anatipestifer No. 566-VIEV is characterized by the following features and properties:

The number of the strain in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1251. The strain has a synonymous name - "Riemerella anatipestifer Ufa" [Laishevtsev, A.I. Experience in the development of the first domestic vaccine against rimerellosis / A.I. Laishevtsev, E.A. Yakimova, A.V. Kapustin // Veterinary Medicine. 2018. No. 8. P.24-29].

Date, source and place of separation: Duck breeding enterprise of the Republic of Bashkortostan. In 2017 from duck fabrics.

Biochemical properties: Culture of oxidase-, catalase-, alpha-galactosidase-, a-maltosidase, a-chymotrypsin is positive. Beta-glucosidase- and

arginine decarboxylase is negative. The strain does not form pigment on blood agar. Liquefies gelatin and does not hydrolyze esculin, but forms indole. The culture ferments glucose, maltose, fructose, mannose, dextrin, and does not ferment trehalose. CAMP test negative, positive Voges-Proskauer test. Does not form urease.

Virulence for laboratory animals: LD ₅₀ value - 5.31 * 10 ⁷ mcr class. for 10 day old ducklings.

3) The industrial strain Riemerella anatipestifer No. 571-VIEV is characterized by the following features and properties:

The number of the strain in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1253. The strain has a synonymous name - "Riemerella anatipestifer Kazan" [Laishevtsev, A.I. Experience in the development of the first domestic vaccine against rimerellosis / A.I. Laishevtsev, E.A. Yakimova, A.V. Kapustin // Veterinary Medicine. 2018. No. 8. P.24-29].

Date, source and place of allocation: Duck breeding enterprise of the Republic of Tatarstan. The strain was isolated in 2017 from the tissues of a duckling.

Biochemical properties: Culture of oxidase-, catalase-, alpha-galactosidase-, a-maltosidase, a-chymotrypsin is positive. Beta-glucosidase and arginine decarboxylase are negative. The strain does not form pigment on blood agar. Liquefies gelatin and does not hydrolyze esculin, but forms indole. The culture ferments glucose, maltose, fructose, mannose, dextrin, and does not ferment trehalose. CAMP test negative, negative Voges-Proskauer test. Forms urease.

Virulence for laboratory animals: LD ₅₀ value - 1.74 * 10 ⁸ μR class. for 10 day old ducklings.

Characteristics of Pasteurella multocida strains

1) Industrial strain Pasteurella multocida No. 367-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-1242

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 separation: Duck breeding enterprise of the Republic of Bashkortostan. The strain was isolated in 2017 from the tissues of a duckling.

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 must be 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 - "5" -shape. On PZhA - growth by injection.

Biochemical properties: Inherent in 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 ₅₀ value - 3.98 × 10 ⁸ μR class. for white mice. The death of animals occurs no later than 24 hours after 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. No more than 21 days for 0.25% RVA.

Characterization of Salmonella spp.

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 Salmonella enterica serovars Enteritidis, Typhimurium and Infantis belong to the IV pathogenicity group.

All cultures of production and control strains of Salmonella have morphological, tinctorial and cultural properties typical of the species, namely: in morphology, they are small gram-negative rods that do not form spores and capsules, and are mobile. The strains ferment glucose, xylose, sorbitol, arabinose, mannitol, rhamnose and dulcite with the formation of acid and gas; they do not ferment lactose and sucrose. They form hydrogen sulfide, do not form indole, do not curdle milk, do not liquefy gelatin. The oxidase activity is negative, the catalase activity is positive, the Voges-Proskauer reaction is negative.

Conditions for cultivating Salmonella strains: for cultivating Salmonella spp. use various nutrient media with the necessary growth-supporting properties: BCH, Hottinger's broth, tryptone-soy broth, Hottinger's agar, Eugonic, etc. They form a uniform opacity of the medium in BCH and Hottinger's broth; on MPA and Hottinger's agar in Petri dishes after 16-24 hours of cultivation - round, convex, translucent, with a smooth surface and smooth edges of the colony - "S" -shape.

1) The industrial strain of Salmonella enterica serovar Enteritidis No. 162-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-1257

Date, source and place of isolation: isolated from chicken feces in 2017 in the Moscow region

Serological properties: 1,9,12 g, m

Virulence in laboratory animals: Pathogenic in mice. LD ₅₀ -10 * 10 ⁵ md cl.

2) The industrial strain of Salmonella enterica serovar Typhimurium No. 178-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-1258

Date, source and place of isolation: isolated from the intestines of ducklings in 2018 in the Republic of Bashkortostan

Serological properties: 1.4, [5], 1.2 i 1.2

Virulence in laboratory animals: Pathogenic in mice. LD ₅₀ -10 * 10 ⁶ md cl.

3) The industrial strain of Salmonella enterica serovar Infantis No. 1827-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-1259

Date, source and place of isolation: isolated from chicken feces in 2016 in the Kaluga region

Serological properties: 6,7,14 r 1,5

Virulence in laboratory animals: Pathogenic in mice. LD ₅₀ -1.4 * 10 ⁸ md cl.

A method of obtaining a vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese.

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

1) Cultivation of industrial strains by deep and / or surface 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) Determination of the immunogenic activity of the vaccine.

For submerged cultivation of bacteria of the species Riemerella anatipestifer, trypticase-soy broth (with dextrose) and tryptone-soy broth can be used (on other media, the growth of Reimerell cultures is not observed). For surface cultivation, the recommended nutrient media are Brain Heart Agar, Trypticase Soy Agar, Beef Peptone Agar, Tryptone Soy Agar, Hottinger Agar, Mueller Hinton Agar supplemented with 10% defibrinated sheep blood.

For submerged cultivation of bacteria of the species Pasteurella multocida and Salmonella spp., In the production of a vaccine, trypticase-soy broth (with dextrose), tryptone-soy broth, eugonic broth, meat-peptone broth, Hottinger's broth, and other media with the necessary growth-providing properties can be used ... Recommended media for surface culture are Brainworm Agar, Trypticase Soy Agar, Beef Peptone Agar, Tryptone Soy Agar, Hottinger's Agar, Mueller Hinton Agar supplemented with 10% defibrinated sheep blood.

To obtain Riemerella anatipestifer antigens, one of two proposed cultivation methods can be used:

The first option involves the use of a surface cultivation method using brain heart agar, trypticase-soy agar, mesopatamia agar, tryptone-soy agar, Hottinger agar or Mueller-Hinton agar with the addition of 10% defibrinated sheep blood. The recommended procedure for surface cultivation of Riemerellas is as follows: on the first day, the first generation of strains is prepared by resuspension of a lyophilized culture of MSB (Master Seed Bacteria) in a test tube, followed by inoculation on blood agar; cultivation continues for 48 hours in a 5-10% CO ₂ atmosphere and a temperature of 37 ° C. On the third day of cultivation, if the growth of the cultures matches the morphological and tinctorial properties of Riemerella anatipestifer, as well as the absence of growth of foreign microflora, the culture must be washed off with sterile saline in a volume of 15 ml. On the same day, the resulting suspension of bacterial cells should be inoculated on previously prepared culture mattresses with blood agar with an area of 225 cm ² . The volume of the inoculum (uterine) bacterial suspension for each mattress should be 5 ml. Mattresses are cultured in a _CO2 - incubator for 48 hours, after which the bacterial mass is rinsed with saline in a volume of 50 ml. The resulting suspension is monitored for the absence of contamination by extraneous microflora, tinctorial properties and concentration. Thus, you can get 50 cm ³ suspension of bacterial cells with a concentration of 40-60 billion m.c. / cm ³ .

- the second option involves submerged cultivation using a commercial nutrient medium - trypticase-soy broth (with dextrose), or tryptone-soy broth. In the first variation of the submerged cultivation method, bottles of the corresponding volume can be used, stirred on a shaker in a CO ₂ incubator in a 5% carbon dioxide atmosphere with a temperature of 37 ° C. The preparation of the matte brood is carried out according to the above procedure. At the same time, the volume of the matt broth should be at least 10% of the total volume of the nutrient broth in the bottles. This method of cultivation on the 2nd day makes it possible to achieve an accumulation of up to 4.0 billion m.c. / cm ³ . The second variation of the method of submerged cultivation implies the use of a bioreactor of any model that has the ability to automatically control the enrichment of the medium with oxygen, regulate the level of carbon dioxide, maintain a given pH level of 7.4-7.8, and feed the culture with glucose. Cultivation in a fermenter makes it possible to achieve an accumulation of up to 6.6 billion μR cells / cm ³ after 24-26 hours of cultivation.

To obtain antigens Pasteurella multocida and Salmonella spp. the submerged cultivation method described earlier is applied. The difference is that not only trypticase soy broth can be used for cultivation, but also other nutrient media used in bio production. In addition, the cultivation is carried out without maintaining the level of carbon dioxide, while maintaining the pH level of 7.0-7.8 for Pasteurella and 7.4-7.6 for Salmonella. The concentration of pasteurella microbial cells as a result of cultivation in a cardiocerebral medium can reach 12 billion m.c. / cm ³ and more. The concentration of microbial cells of Salmonella as a result of cultivation on a cardio-cerebral medium can reach 30 billion m.c. / cm ³ and more.

Inactivation of the culture fluid containing Riemerella anatipestifer 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, within 3 days, and within 21 days for the culture fluid containing Salmonella spp. cells, at room temperature.

Concentration of cultures is carried out by centrifugation at 3-6 thousand rpm. min. within 1-3 hours or by separation at 10-15 thousand revolutions, depending on the equipment used.

The completeness of inactivation is assessed by the absence of viable cells in the concentrated antigen and the assembled preparation by sowing on growth-supporting nutrient media, as well as by the harmlessness of the inactivated bacterial mass in a biological sample on laboratory mice weighing 16-18 g. with subcutaneous administration of Pasteurella multocida and Salmonella spp. antigens, and in a bioassay on 10-day-old musk ducklings, with intramuscular administration for Riemerella anatipestifer antigens, in a single dose of 0.5 cm ³ . Three experimental mice are used to assess the safety of each batch of Pasteurella multocida and Salmonella spp. Antigen, and three musky ducklings are used to control the Riemerella anatipestifer antigen. Observation of the experimental animals continues for 10 days. The drug is considered inactivated if there is no growth of the culture of Riemerella anatipestifer, Pasteurella multocida, Salmonella spp., 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.

As mentioned earlier, various adjuvants can be used to manufacture 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 100 liters (200 thousand doses) are given.

Option number 1. - the layout of the vaccine using polyethylene glycol-6000 or aluminum hydroxide.

In a sterile reactor, inactivated bacterial antigens of seven industrial cultures (previously established concentration) are mixed with a diluent, adjuvant (10%) and sodium merthiolate, so as to obtain a concentration of bacterial antigens equal to 11 billion μR cells / cm ³ (2 billion μR cells ./cm ^{3 of} each strain of Riemerell and Pasteurella, and 1 billion μR cells / cm ^{3 of} each strain of Salmonella). After obtaining a homogeneous suspension with constant stirring, the recommended 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 70).

In a sterile reactor, inactivated bacterial antigens of seven 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 11 billion μR cells. cm ³ (2 billion μR cells / cm ³ antigen of each strain of Riemerell and Pasteurella, and 1 billion μR cells / cm ³ antigen of each strain of Salmonella). Obtaining a homogeneous emulsion is carried out using a dispersing machine at 6000-8000 rpm. The vaccine series 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".

Determination of the immunogenic activity of the vaccine is carried out using musky ducklings and white mice:

- Determination of the immunogenic activity of the Riemerellus component of the vaccine is carried out using 10-day-old musk ducklings. To achieve this goal, six groups of 10-day-old ducklings are formed, three of which are experienced, and three are control. The number of birds in each group should be at least 10. Ducklings of the experimental group are vaccinated twice with an interval of 14-16 days. 14-15 days after double vaccination, each group of immunized birds is infected with a control strain of Riemerell at a dose of 5LD ₅₀ . At the same time, birds of control groups are infected with all control strains of Riemerell. Observation of the bird is established for a period of 10 days, or until death. The vaccine is considered immunogenic if at least 80% of the birds survived after infection in the experimental groups, while at least 80% of the ducklings should die in the control group.

- Determination of the immunogenic activity of pasteurellosis and salmonella vaccine components in white mice. To achieve this goal, eight groups of 10 mice each, weighing 18-20 grams, are formed. Four groups are experimental (for each infecting strain) and four groups are control (for each infecting strain). Mice are pre-vaccinated twice with an interval of 14-16 days. On the 14-15th day after double vaccination, mice of the experimental and control groups are infected with the control strain Pasteurella multocida No. 1231, Salmonella enteritidis No. 1807-VIEV, Salmonella infantis No. 1841-VIEV, Salmonella typhimurium No. 371 at a dose of 5LD ₅₀ . Observation of animals is established for a period of 10 days, or until the moment of their death. The vaccine is considered immunogenic if, after infection, at least 80% of the mice survived in the experimental groups, while at least 80% of the mice should die in the control.

Control strains Riemerella anatipestifer No. 602-VIEV, No. 490-VIEV and No. 513-VIEV are well-studied cultures, with a known value of the LD ₅₀ index, which allows them to be used as “punch” strains when controlling the immunogenic activity of the proposed drug. In order to control the immunogenic activity of the Salmonella components of the vaccine, two new control strains of Salmonella enteritidis No. 1807-VIEW and Salmonella infantis No. 1841-VIEV with well-studied characteristics and properties have been proposed. To control the Salmonella component of the vaccine belonging to the group "B", it is proposed to use the well-known strain of Salmonella typhimurium No. 371 (deposited in the "All-Russian collection of microorganism strains used in veterinary medicine and animal husbandry" FGBU "VGNKI"), which is widely used in the manufacture and control of Salmonella vaccines and therapeutic sera [Method of manufacturing a vaccine against salmonellosis of pigs RU 2162340 C1 publ. 2001.01.27]. To control the immunogenic activity of the pasteurellosis component of the vaccine, the use of a well-studied and widespread strain Pasteurella multocida No. 1231 (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 medicinal sera [ ... RU 2414929 C1 publ. 2011.03.27].

Characteristics of control strains:

1) The control strain Riemerella anatipestifer No. 602-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-1254

Date, source and place of allocation: Duck-breeding and goose-breeding enterprise of the Kurgan region. The strain was isolated in 2017 from the tissues of a gosling.

Biochemical properties: The culture of oxidase, catalase, alpha-galactosidase, a-maltozidase, a-chymotrypsin is positive. Beta-glucosidase and arginine decarboxylase are negative. The strain does not form pigment on blood agar. Liquefies gelatin and does not hydrolyze esculin, but forms indole. The culture ferments glucose, maltose, fructose, mannose, dextrin, and trehalose. CAMP test negative, positive Voges-Proskauer test. Does not form urease.

Virulence for laboratory animals: LD ₅₀ value - 1.08 * 10 ⁹ mcr class. for 10 day old ducklings.

2) The control strain Riemerella anatipestifer No. 490-VIEV is characterized by the following features and properties:

The number of the strain in the "All-Russian state collection of pathogenic and vaccine strains of microorganisms-causative agents of infectious animal diseases": B-1255. The strain has a synonymous name - "Riemerella anatipestifer Kazan" [Laishevtsev, A.I. Experience in the development of the first domestic vaccine against rimerellosis / A.I. Laishevtsev, E.A. Yakimova, A.V. Kapustin // Veterinary Medicine. 2018. No. 8. P.24-29].

Date, source and place of allocation: Duck breeding enterprise of the Rostov region. The strain was isolated in 2018 from the tissues of a duckling.

Biochemical properties: The culture of oxidase, catalase, alpha-galactosidase, a-maltozidase is positive, and a-chymotrypsin is negative. Beta-glucosidase and arginine decarboxylase-negative. The strain does not form pigment on blood agar. Liquefies gelatin and does not hydrolyze esculin, but forms indole. The culture ferments glucose, maltose, fructose, dextrin, and does not ferment mannose and trehalose. CAMP test negative, positive Voges-Proskauer test. Forms urease.

Virulence for laboratory animals: LD ₅₀ value - 8.25 * 10 ⁷ mcr class. for 10 day old ducklings.

3) The control strain Riemerella anatipestifer 513-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-1256

Date, source and place of allocation: Duck breeding and goose breeding enterprise of Krasnodar Territory. The strain was isolated in 2017 from the tissues of a duckling.

Biochemical properties: The culture of oxidase, catalase, alpha-galactosidase, a-maltozidase, a-chymotrypsin is positive. Beta-glucosidase and arginine decarboxylase are negative. The strain does not form pigment on blood agar. Liquefies gelatin and does not hydrolyze esculin, but forms indole. The culture ferments glucose, fructose, mannose, dextrin, and does not ferment maltose and trehalose. CAMP test is positive, the Voges-Proskauer reaction is positive. Forms urease.

Virulence for laboratory animals: LD ₅₀ value - 4.95 * 10 ⁸ μR class. for 10 day old ducklings.

4) The control strain of Salmonella enterica serovar Enteritidis No. 1807-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-1260

Date, source and place of isolation: isolated from the blind processes of the intestine of a duck in 2016 in the Pskov region

Serological properties: 1,9,12 g, m

Virulence in laboratory animals: Pathogenic in mice. LD ₅₀ -3.5 * 10 ⁶ md cl.

5) The control strain of Salmonella enterica serovar Infantis No. 1841-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-1261

Date, source and place of isolation: isolated from turkey feces in 2016 in the Tver region

Serological properties: 6,7,14 r 1,5

Virulence in laboratory animals: Pathogenic in mice. LD ₅₀ -1.2 * 10 ⁸ md cl.

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 duck, goose-breeding and turkey breeding enterprises, unfavorable for rimerella, pasteurellosis and salmonellosis. Within the framework of approbation, the harmlessness of the drug and its protective efficacy were confirmed.

The proposed vaccine begins to be applied from the replacement poultry 42-45 days before the start of oviposition intramuscularly in the thigh area at a dose of 0.5 ml twice: the first time - 42-45 days before the start of oviposition, the second time 14-16 days after the first vaccination ... Subsequent parent stock is vaccinated once, every 6 months.

Young animals previously obtained from both vaccinated and unvaccinated birds are injected with the vaccine on the 7-10th day of life, twice with an interval of 14-16 days. If the young growth is planned to be used as a repair, it is subsequently vaccinated every 6 months, once.

Implementation of the invention

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

Example # 1. Surface cultivation of industrial strains of Riemerella anatipestifer

Receiving Riemerell antigens was carried out by the method of surface cultivation of industrial strains on heart-brain agar, which was additionally supplemented with defibrinated ram blood in a volume of 10%. On the first day of work, the first generation of the main seed of each production strain was prepared. For this purpose, a nutrient broth (Hottinger's broth) in a volume of 1 ml, necessary to restore the lyophilic mass to its original volume, was added to an ampoule with a lyophilized Riemerell strain. The resulting suspension in a volume of 0.5 cm ^{3 was} transferred with a Pasteur pipette into a test tube with Hottinger's broth (7 ml of medium). After that, from a test tube, the cell suspension was inoculated into Petri dishes containing 20 cm ³ of brain-heart agar according to the Drygalsky method. 5 Petri dishes were prepared from one tube (the number of dishes may vary depending on the required volume of matt brood production). The resulting inoculation dishes were cultured for 48 hours in a CO ₂ incubator in the constant presence of 5% carbon dioxide. The incubation temperature was 37 ° C. After confirmation of the cultural, morphological and tinctorial properties of the grown cultures, by the visual method and by the method of microscopy of Gram stained smears, the cultures were washed into physiological solution, at the rate of 15 cm ³ per dish. The total volume of the resulting suspension of each strain was 75 ml. The resulting suspension of bacterial cells was inoculated into mattresses with an area of 225 cm ² containing brain heart agar with 10% defibrinated ram blood, at the rate of 5 cm ^{3 of the} suspension per mattress. Thus, 15 mattresses were obtained with each strain. Cultivation of mattresses was carried out for 48 hours at a temperature of 37 ° C in the presence of 5% carbon dioxide. After cultivation, bacterial cultures were washed off the agar surface in mattresses with physiological saline at the rate of 50 cm ³ per mattress. As a result, 750 ml of bacterial suspension of each strain was obtained. Bacterial suspensions were monitored for typical tinctorial and morphological properties of cultures by microscopic examination of Gram stained smears, as well as for the absence of other morphological forms of bacteria.

All suspensions of Riemerell strains contained cells typical of the species and did not contain foreign inclusions.

Example # 2. Cultivation of industrial strains of Riemerella anatipestifer by the submerged method.

Cultivation of Riemerell strains was carried out using 3-liter bottles with trypticase-soy broth (2.5 liters each). The preparation of the main seed was carried out according to the method described in example No. 1, with some differences. So, on the first day of work, the first generation of the main inoculum was prepared, for which a nutritious broth (trypticase-soy broth) was added to the ampoule with a freeze-dried strain of Riemerell in a volume of 1 ml, necessary to restore the lyophilic mass to its original volume. Then the resulting suspension in a volume of 0.5 cm ^{3 was} transferred with a Pasteur pipette into a test tube with trypticase-soy broth, and in parallel, the cell suspension was plated on Petri dishes containing 20 cm ^{3 of} tryptone-soy agar with 10% defibrinated ram blood, according to the Drygalsky method ... The resulting tubes and dishes were cultured for 24 hours at 37 ° C in a CO ₂ incubator with 5% carbon dioxide. After cultivation of Petri dishes, the obtained Riemerella colonies were studied by cultural, morphological and tinctorial properties in order to determine the typical growth and the absence of growth of extraneous microflora. The bouillon culture in tubes was also subjected to microscopy after Gram staining. In case of confirmation of the properties of industrial strains and the absence of extraneous microflora on Petri dishes and in test tubes, the resulting cultures of Riemerell were subcultured into flasks with trypticase-soy broth (1 tube per 250 cm ^{3 of} nutrient medium) for subsequent reactor cultivation. For sowing, the volume of the matt brood is prepared at the rate of 10% to the volume of the nutrient medium in the bottle (2.5 l). The resulting flasks were further cultured for 24 hours at a temperature of 37 ° C, in a CO ₂ incubator with 5% carbon dioxide, to obtain the second generation of the strain. After culturing the flasks, before sowing in the flasks, the broth culture of Riemerell was controlled for cultural, morphological and tinctorial properties in order to determine the typical growth and the absence of growth of extraneous microflora. Culturing bottles with a third generation riemerell carried out for 48 hours at 37 ° C in a CO ₂ incubator with 5% carbon dioxide. During the indicated period, the pH level of the nutrient medium was maintained in the range of 7.4-7.8. Additionally, depending on the severity of the growth of the cultures, they were supplemented with glucose to a concentration of 0.5% by volume.

Example No. 3. Inactivation of the bacterial mass of Riemerella anatipestifer strains

Inactivation of the resulting bacterial suspension of Riemerell (example No. 1 and 2) was carried out by adding 0.3% formalin containing at least 37% formaldehyde to the volume of the culture liquid. Thus, to 750 ml of the cell suspension obtained by the surface cultivation method, 2.25 cm ^{3 of} formalin was added, and to 2750 ml of the broth culture obtained by the submerged cultivation method, 8.25 ml of formalin was added. Inactivation was carried out within 72 hours at a temperature of 21-22 ° C.

Example No. 4. Concentration of the bacterial mass of Riemerella anatipestifer obtained by cultivation by the surface method

Concentration of the resulting bacterial suspension of Riemerella after inactivation (example No. 3) 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 Production of Microorganisms of the Federal State Budgetary Institution "NCESMP" of the Ministry of Health of Russia. The results of surface cultivation and concentration of the bacterial mass of three industrial strains of Riemerell showed the following results: Riemerella anatipestifer No. 549-VIEV - 352 ml with a concentration of 76 billion μR cells / cm ³ ; Riemerella anatipestifer No. 566-VIEV - 390 ml with a concentration of 84 billion μR cells / cm ³ ; Riemerella anatipestifer No. 571-VIEV - 337 ml with a concentration of 84.5 billion μR cells / cm ³ .

The results of submerged cultivation and concentration of the bacterial mass of three industrial strains of Riemerell showed the following results: Riemerella anatipestifer No. 549-VIEV - 430 ml with a concentration of 21.3 billion μR cells / cm ³ ; Riemerella anatipestifer No. 566-VIEV - 520 ml with a concentration of 14.0 billion μR cells / cm ³ ; Riemerella anatipestifer No. 571-VIEV - 435 ml with a concentration of 25.9 billion μR cells / cm ³ .

Example No. 5. Determination of the completeness of inactivation of antigens of riemerella, pasteurella and salmonella

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 ". Additionally, while controlling the sterility of Riemerella anatipestifer antigens, inoculations were made on blood agar, followed by cultivation in a CO ₂ incubator for 7 days at 37 ° C.

Also, the completeness of inactivation of Pasteurella multocida and Salmonella spp. was evaluated by the harmlessness of the inactivated bacterial mass in a bioassay on laboratory mice weighing 16-18 g. when administered subcutaneously in a volume of 0.5 cm ³ . The completeness of inactivation of Riemerella anatipestifer antigens was carried out using 10-day-old ducklings, with intramuscular administration of antigens, in a volume of 0.5 cm ³ . Three experimental mice were used to assess the safety of each batch of Pasteurella multocida and Salmonella spp. Antigen, and three ducklings were used to control the Riemerella anatipestifer antigen.

Example No. 6. Cultivation of the Pasteurella multocida strain by the submerged method

Cultivation of the Pasteurella multocida strain was carried out using a Sartorius BIOSTAT-A reactor in tryptone-soy broth. The preparation of the main seed was carried out according to the method described in example No. 1, with some differences. So, on the first day of work, the first generation of the main inoculum was prepared; for this, a nutrient broth (tryptone-soy broth) was added to an ampoule with a freeze-dried pasteurella strain in a volume of 1 ml, necessary to restore the lyophilic mass to its original volume. Then the resulting suspension in a volume of 0.1 cm ^{3 was} transferred with a Pasteur pipette into three test tubes with tryptone-soy broth and in parallel, the cell suspensions were plated on Petri dishes containing 20 cm ³ of brain-heart agar, according to the Drygalsky method. The cultivation of the obtained tubes and cups was carried out for 18 hours at a temperature of 37 ° C. After the cultivation of Petri dishes, the obtained pasteurella colonies were studied for their cultural, morphological and tinctorial properties in order to determine the typical growth and the absence of growth of extraneous microflora. The broth culture from tubes was also subjected to microscopy after Gram staining. In case of confirmation of the properties of the industrial strain and the absence of extraneous microflora on Petri dishes and in test tubes, the resulting culture of pasteurella was inoculated into flasks with tryptone-soy broth (1 tube per 350 cm ^{3 of} nutrient medium) for subsequent reactor cultivation. In this case, the volume of the matt brood is prepared at the rate of 10% to the volume of the reactor nutrient medium (3.5 l). Reactor cultivation was carried out at a temperature of 37 ° C and pH 7.0-7.8 for 16 hours, upon reaching which the increase in the concentration of bacterial cells in the broth stopped. During cultivation, the level of hydrogen ions in the nutrient medium was regulated by alkali, and the presence of nutrients in the medium was maintained by glucose. The described cultivation scheme made it possible to achieve a concentration of the bacterial suspension of 7.3 billion μR cells / cm ³ .

Example No. 7. Inactivation of the bacterial mass of the Pasteurella multocida strain. Inactivation of the Pasteurella multocida culture was carried out according to the method described in example No. 3. ^{Thus, 11.55 cm 3 of} formalin was added to 3850 ml of the culture fluid.

Example No. 8. Concentration of Pasteurella multocida bacterial mass obtained by submerged cultivation

The concentration of the resulting bacterial suspension after inactivation was carried out according to the method described in example No. 4. Thus, 307 ml of Pasteurella multocida antigen No. 367-VIEV with a concentration of 86.2 billion μR cells / cm ³ were obtained.

Control of the completeness of inactivation of Pasteurella multocida antigens was carried out according to the method described in example No. 5.

Example No. 9. Cultivation of Salmonella spp. deep method

The cultivation of Salmonella strains was carried out similarly to the method described in example No. 7, but with some differences. Namely, while maintaining the pH of 7.4-7.6 for 10 hours, upon reaching which the increase in the concentration of bacterial cells in the broth stopped. During cultivation, the level of hydrogen ions was regulated by alkali, and the presence of nutrients in the medium was maintained by glucose. This cultivation scheme made it possible to obtain a bacterial cell concentration of 24.2 billion μR cells / cm ³ for the Salmonella enteritidis strain No. 162-VIEV; 29.6 billion microradiol cells / cm ³ for the Salmonella typhimurium strain No. 178-VIEV; 25.5 billion μR cells / cm ³ for the Salmonella infantis strain No. 1827-VIEV.

Example No. 10. Inactivation of Salmonella spp. Antigens.

Inactivation of the resulting bacterial suspension of Salmonella (example No. 9) was carried out by adding to its volume 0.3% formalin containing at least 37% formaldehyde. ^{Thus, 2.25 cm 3 of} formalin was added to 750 ml of the suspension, ^{11.55 cm 3 of} formalin was added to 3850 ml of the culture liquid. Inactivation was carried out for 21 days at a temperature of 21-22 ° C.

Example No. 11. Concentration of Salmonella spp. Antigens.

The concentration of the resulting bacterial suspension after inactivation was carried out according to the method described in example No. 3. Thus, 530 ml of Salmonella enteritidis antigen No. 162-VIEV with a concentration of 125.7 billion μR cells / cm ³ was obtained; 620 ml of Salmonella typhimurium antigen No. 178-VIEV with a concentration of 143.0 billion μR cells / cm ³ ; 480 ml of Salmonella infantis antigen No. 1827-VIEV with a concentration of 166.7 billion μR cells / cm ³ .

Determination of the completeness of inactivation of Salmonella antigens was carried out according to the method shown in example No. 5.

Example No. 12. Compilation of series No. 1 of a polyvalent inactivated vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese using saline as a diluent and polyethylene glycol (PEG-6000) as an adjuvant

When compiling a series of vaccines, inactivated bacterial antigens of seven industrial cultures were mixed in a sterile container with saline as a diluent, polyethylene glycol-6000 as an adjuvant, and sodium merthiolate as a preservative, setting the concentration of bacterial antigens to 11 billion μR cells / cm ³ ( 2 billion μR cells / cm ^{3 of} each strain of Riemerell and Pasteurella, and 1 billion μR cells / cm ^{3 of} each strain of Salmonella). 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 4. The composition is given on the basis of the volume of the produced batch of 5 thousand doses - 2.5 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. 13. Compilation of series No. 2 of a polyvalent inactivated vaccine against riemerellosis, pasteurellosis and salmonellosis of turkeys, ducks and geese using water for injection as a diluent and aluminum hydroxide (GOA) as an adjuvant

When compiling a series of vaccines, inactivated bacterial antigens of seven industrial cultures were mixed in a sterile container based on water for injection as a diluent and aluminum hydroxide as an adjuvant, and sodium merthiolate as a preservative, so as to obtain a concentration of bacterial antigens equal to 11 billion μR cells / cm ³ (2 billion μR cells / cm ^{3 of} each strain of Riemerell and Pasteurella, and 1 billion μR cells / cm ^{3 of} each strain of Salmonella). The components were mixed until a homogeneous suspension was obtained, after which the pH of the vaccine batch was adjusted to 7.3. The recipe for the composition of the vaccine when using this layout option is shown in table 5. The composition is given on the basis of the volume of the produced batch of 5 thousand doses - 2.5 liters of the drug.

The vaccine obtained in this way is in appearance

a suspension of light gray color with a gray-white precipitate, which easily breaks up when shaken into a homogeneous suspension.

Example No. 14. Compilation of series No. 3 of a polyvalent inactivated vaccine against riemerellosis, pasteurellosis and salmonellosis in turkeys, ducks and geese using phosphate buffered saline as a diluent and Montanide ISA 70 oil 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 seven industrial cultures were mixed on the basis of PBS - buffer as a diluent, Montanide ISA 70 as an adjuvant, sodium merthiolate as a preservative, in such a way as to obtain a concentration of bacterial antigens equal to 11 billion μR cells / cm ³ (2 billion μR cells / cm ^{3 of} each strain of Reimerell and Pasteurella, and 1 billion μR cells / cm ^{3 of} each strain of Salmonella). To obtain the final version of the vaccine, a Silverson L5M-A homogenizer is 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 6. The composition is given on the basis of the volume of the produced batch of 5 thousand doses - 2.5 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. 15. 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, from each vial (each batch) with the drug, inoculation was carried out 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 plating on Kitt-Tarozzi medium, plating was done on two test tubes and two flasks. Additionally, inoculation was carried out on blood agar, followed by cultivation in a CO ₂ incubator for 7 days at a temperature of 37 ° C. 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. 16. Determination of the value of the LD ₅₀ index of strains of rimerella, pasteurella and salmonella.

To determine the virulence of Riemerell strains, 10-day-old musk ducklings are used. Infection of ducklings is carried out by intramuscular injection of a tenfold titrated suspension of bacterial cultures in concentrations: 1.5 * 10 ⁹ m.c., 1.5 * 10 ⁸ m.c., 1.5 * 10 ⁷ m.c., 1.5 * 10 ⁶ m.c. in a volume of 0.5 cm ³ . For each contaminating concentration, 4-6 ducklings are used. Ducklings are monitored for a period of 10 days, or until they die. The results obtained are interpreted using probit analysis using BioStat 2009 software, or Kerber's formula modified by Ashmarin-Vorobiev.

The study of Pasteurella and Salmonella is carried out according to the indicated method, but white mice, not musky ducklings, are used as a model.

The studies carried out made it possible to establish the value of the virulence of the control strains of microorganisms used in assessing the immunogenic activity of the proposed vaccine. As a result of the research, the following results were obtained for the LD50 value for the control strains:

Riemerella anatipestifer No.602-VIEV LD ₅₀ - 1.08 * 10 ⁹ micro district;

Riemerella anatipestifer No. 490-VIEV LD ₅₀ - 8.25 * 10 ⁷ micro district;

Riemerella anatipestifer 513-VIEV LD ₅₀ - 4.95 * 10 ⁸ micro district;

Pasteurella multocida No. 1231 LD ₅₀ - 3.98 × 10 ⁸ mcr class;

Salmonella enterica serovar Enteritidis No. 1807-VIEV LD ₅₀ - 3.5 * 10 ⁶ mcr class;

Salmonella enterica serovar Infantis No. 1841-VIEV LD ₅₀ - 1.2 * 10 ⁸ mcr class;

Salmonella enterica serovar typhimurium No. 371 LD ₅₀ - 1.8 * 10 ⁴ mcr class.

Example No. 17. Determination of the immunogenic activity of the Riemerella component of the vaccine in 10-day-old ducklings.

Determination of the immunogenic activity of the Riemerella component of the proposed vaccine is carried out using musk ducklings. To achieve this goal, six groups of 10-day-old ducklings are formed, three of which are experienced, and three are control. The number of birds in each group must be at least 10 heads. The ducklings of the experimental group are vaccinated twice with an interval of 14 days. On the 15th day after double vaccination, the bird is infected with control strains of Riemerell, at a dose of 5LD ₅₀ . Observation of the bird is established within 10 days, or until death. The vaccine is considered immunogenic if, after infection, at least 80% of the birds survive in the experimental groups, while at least 80% of the ducklings should die in the control group.

The results of determining the immunogenic activity of the Riemerella component of the three series of the proposed vaccine are shown in table No. 7.

As can be seen from the results presented in Table 7, the proposed vaccine meets the requirements for immunogenic activity. So, in the control group, 100% of the birds died, and in the experimental group, 90% survived (the minimum obtained value is indicated).

Example No. 18. Determination of the immunogenic activity of pasteurellosis and salmonella vaccine components in white mice.

Determination of the immunogenic activity of pasteurellosis and salmonella components of the proposed vaccine is carried out using white mice. To achieve this goal, eight groups of 10 mice each, weighing 18-20 grams, are formed. Four groups are experimental (for each infecting strain) and four groups are control (for each infecting strain). Mice are pre-vaccinated twice with an interval of 14 days. On the 15th day after double vaccination, mice of the experimental and control groups were infected with the control strain Pasteurella multocida No. 1231, Salmonella enteritidis No. 1807-VIEV, Salmonella infantis No. 1841-VIEV, Salmonella typhimurium No. 371 at a dose of 5LD ₅₀ . Observation of animals is established for a period of 10 days, or until the moment of their death. A vaccine is considered immunogenic if, after infection, at least 80% of the mice survive in the experimental groups, while at least 80% of the animals in the control group must die.

The results of determining the immunogenic activity of the pasteurellosis and salmonella components of the three series of the proposed vaccine are shown in table No. 8.

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. 19. Assessment of the protective efficacy of the vaccine.

To assess the protective efficacy of the proposed vaccine, field trials were carried out in a duck enterprise in the Stavropol Territory, which is unfavorable for rimerellez and pasteurellosis. For the experiment, two groups of birds were formed. Evaluation of the effectiveness of the drug was carried out on ducklings at the age of 7 days, which were vaccinated twice with an interval of 14 days. The observation was carried out until the onset of 4 months of age (120 days). Evaluation of the effectiveness of the proposed drug was carried out according to the parameters of mortality and uniformity among poultry. The test results are shown in Table 9. In order to ensure the safety of the poultry population, in the event of an infectious pathology, antibacterial drugs were prescribed.

As can be seen from the data in Table 9, the number of ducklings that died and were discarded after the vaccine application in the experimental group is 7.17% less than in the control group. At the same time, there was an increase in the uniformity of the livestock in the experimental group by 3.8% in comparison with the control group. During the observed period of growing control ducklings, the cost per head was 24.89 rubles, and in the case of the estimated cost of two doses of the vaccine equal to 3.6 rubles. the savings amounted to 21.29 rubles. for 1 head.

In order to assess the effect of the proposed product on the parent stock and its performance indicators, two groups of ducks at the age of 150 days were formed. The bird was vaccinated twice with an interval of 14 days. The results of assessing the safety and productivity of the twice vaccinated population of ducks are shown in Table 10.

Analyzing the data in Table 10, we can conclude that the average value of the number of deaths in the experimental group is 2.33% lower than in the control group. The average value of the number of culled poultry in the experimental group is 1.69% less than in the control group. The average value of the indicator of total waste in the experimental group for the observed period is 4.02% lower than that in the control group. The average value of the productivity of birds of the vaccinated groups is 2.5% higher than that of the control group.

Claims (7)

1. Polyvalent inactivated vaccine against riemerella, pasteurellosis and salmonellosis of turkeys, ducks and geese, containing the following components per 1 cm ^{3 of the} preparation: inactivated protective antigen of the Riemerella anatipestifer No. 549-VIEV strain - 2 billion micro cells, inactivated protective antigen of the R. anatipestifer strain No. 566-VIEV - 2 billion μR cells, inactivated protective antigen of the R. anatipestifer strain No. 571-VIEV - 2 billion μR cells, inactivated protective antigen of the Pasteurella multocida strain No. 367-VIEV - 2 billion μR cells. , inactivated protective antigen of the Salmonella enteritidis strain No. 162-VIEV - 1 billion μR cells, inactivated protective antigen of the Salmonella typhimurium strain No. 178-VIEV - 1 billion μR cells, inactivated protective antigen of the Salmonella infantis strain No. 1827-VIEV - 1 billion μR cells ., as well as pharmaceutically acceptable target additives. 2. The vaccine according to claim 1, characterized in that a preservative is used as a pharmaceutically acceptable target additives in the form of an aqueous solution of sodium merthiolate 10%, introduced into the preparation at a rate of 1: 10000, 1 part of sodium merthiolate solution to 10000 parts of the preparation: per 100 liters of the finished product add 0.01 l of merthiolate solution. 3. The vaccine according to claim 1, characterized in that a diluent in the form of sterile saline, sterile PBS or water for injection is used as pharmaceutically acceptable target additives in the amount required to obtain 100% of the volume of the preparation. 4. The vaccine according to claim 1, characterized in that it contains one of the following adjuvants as pharmaceutically acceptable target additives: PEG-6000 in an amount of 10% of the volume of the preparation, aluminum hydroxide in a concentration of 3% in an amount of 10% of the volume of the preparation, or oil adjuvant in the amount of 50% of the volume of the drug. 5. The vaccine according to claim 4, characterized in that when PEG-6000 or aluminum hydroxide is used as an adjuvant, 10% sodium hydroxide is additionally used to establish an optimal pH level of 7.0-7.6. 6. A method of obtaining a vaccine according to claim 1, including the cultivation of industrial strains, inactivation of the culture fluid, concentration of antigens, determination of the completeness of inactivation of antigens, preparation of a series of vaccines, monitoring the sterility of the vaccine, determination of the immunogenic activity of the vaccine, characterized in that strains are used as industrial strains Riemerella anatipestifer No. 549-VIEV, R. anatipestifer No. 566-VIEV, R. anatipestifer No. 571-VIEV, Pasteurella multocida No. 367-VIEV, Salmonella enteritidis No. 162-VIEV, Salmonella typhimurium No. 178-VIEV No. 18, Salmonella-infantis infantis , and as control Riemerella anatipestifer No. 602-VIEV, Riemerella anatipestifer No. 490-VIEV, Riemerella anatipestifer 513-VIEV, Salmonella enterica Enteritidis serovar No. 1807-VIEV, Salmonella enterica serovar Infantis No. 1841-VIEV, two antigens are obtained options: using surface or submerged cultivation with maintaining the level of coal sludge gas; while to obtain antigens Pasteurella multocida and Salmonella spp. a submerged cultivation method is used without maintaining the level of carbon dioxide, but while maintaining a pH level of 7.0-7.6 for Pasteurella multocida and 7.4-7.6 for Salmonella spp. 7. The method according to claim 6, characterized in that the inactivant is used in the form of formalin containing at least 37% formaldehyde, at the rate of 0.3% by volume of the broth culture.