RU2761379C1 - Polyvalent inactivated vaccine against swine streptococcosis, method for its production and use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: inventions group relates to the field of biotechnology. The proposed vaccine against swine streptococcosis, polyvalent inactivated, contains the following components per one immunizing dose of the drug (2 cm³): formalin-inactivated protective antigens of Streptococcus suis strains No. UR-4-VBH - 3.5 billion mcr. cl.; Streptococcus porcinus UR-3-VBH - 3.5 billion mcr. cl.; Streptococcus pyogenes OB-4-VBH - 3.5 billion mcr. cl.; Streptococcus dysgalactiae UR-16-VBKh - 3.5 billion mcr. cl., as well as pharmaceutically acceptable target additives. The method for obtaining a vaccine includes the cultivation of industrial strains; inactivation of the culture fluid; concentration of antigens; determination of the completeness of antigen inactivation; preparation of a vaccine batch; control of the sterility of the vaccine and the safety of the vaccine; determination of the immunogenic and antigenic activity of the vaccine. The vaccine is used as a prophylactic agent when administered to pregnant sows intramuscularly in the upper third of the neck (behind the ear) in a dose of 2 ml twice: the first time - 50-55 days before farrowing, the second time - 25-30 days before farrowing. Subsequently, the vaccine is administered 25-30 days before each subsequent farrowing once in the same dose. It is not allowed to vaccinate sows later than 25-30 days before farrowing, as well as lactating animals. Piglets are vaccinated starting at 14 days of age. The vaccine is injected intramuscularly into the femoral muscle group twice with an interval of 21-24 days at a dose of 2 ml, additional revaccination is carried out at the age of 4 months. once.

EFFECT: vaccine has enhanced immunogenic and antigenic activity, induces intense and long-term immunity in pigs against streptococcosis caused by S. suis, S. dysgalactiae, S. porcinus and S. pyogenes species, which significantly distinguishes it from the existing vaccines of this direction.

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Description

The group of inventions relates to veterinary medicine, namely to veterinary microbiology and biotechnology, and can be used in pig breeding to ensure epizootic welfare for streptococcosis caused by Streptococcus suis, Streptococcus pyogenes, Streptococcus porcinus and Streptococcus dysgalactiae.

Streptococcosis is a whole group of infectious diseases, widespread in pig breeding, characteristic of various age and physiological groups of animals [Spiridonov A.G. et al. Etiology of infectious diarrhea in newborn piglets and calves / Veterinary medicine. 2011. V. 95. S. 264-265; Efanova L.I. et al. Etiological structure of factorial infections of pigs and cattle in the farms of the Central ChZ of Russia / Bulletin of the Kursk State Agricultural Academy, 2012. No. 12. S. 71-72; Malik E.V. Etiological structure of streptococcosis of pigs / the dissertation Author's abstract on scientific degree competition kvn / 2000, p. 28; Panin A.N. Swine streptococcosis / Ph.D. thesis / 1992, p. 45]. The main economic losses are formed due to the death and culling of piglets, a decrease in the productivity of sows due to the emerging mastitis and endometritis, a decrease in weight gain, as well as an increase in the cost of carrying out therapeutic and prophylactic measures [Manzhurina OA, Skogoreva AM, Parkhomenko Yu.S. , Chernyshova I.S., Semenova E.V. Study of resistance to antibacterial drugs Streptococcus suis in the pig-breeding complex of the Belgorod region // In the collection: Veterinary and sanitary aspects of the quality and safety of agricultural products Materials of the II International Conference on Veterinary and Sanitary Expertise. 2017. S. 175-179].

Streptococcosis in pig breeding enterprises often causes the development of arthritis, omphalitis, meningitis, mastitis, septicemia, lymphadenitis, pneumonia and other lesions. In this case, the group of infections itself refers to factorial and, as a rule, manifests itself against the background of primary viral or bacterial infections, mycoplasmosis, stress and violation of zooveterinarian standards [Kapustin A. V., Laishevtsev A. And. The modern etiological structure of swine streptococcosis // In the collection: One world - one health 2018. S. 218-220].

In turn, ensuring the epizootic welfare of pig farms is an important task for the veterinary service. In this case, the problem can be solved in two ways, the first of which is the use of antibacterial drugs for treatment, and the second is the use of specific prophylaxis. Against the background of widespread antibiotic resistance, including among microorganisms of the genus Streptococcus, the first option is ineffective [Lebel G., Piche F., Frenette M., Grenier D., Gottschalk M. Antimicrobial activity of Nisin against the swine pathogen Streptococcus suis and its synergistic interaction with antibiotics Peptides. 2013. T. 50. C. 19-23]. The use of specific prophylaxis (vaccines) is more promising than antibiotic therapy. An important condition for achieving a high protective activity of the vaccine is its antigenic composition, which should be focused on modern epizootic data and the etiological structure of streptococcosis.

Currently, a number of immunobiological agents have already been developed and are actively used against the pathology under consideration, but they all have a limited antigenic composition and are not fully focused on the etiological structure of the infection. In addition, the antigenic composition of vaccines for the streptococcal component has not been updated for the last 30-35 years, and this has led to the fact that the preparations include streptococcal species that are not of primary etiological importance today, for example, Streptococcus zooepidemicus, but there is no many actual species [Terekhov P. Yu., Matyash EA, Yakimova EA, Kapustin AV, Belyaeva AS, Laishevtsev AI A new look at the etiological structure of pig streptococcosis // In the collection: YR Conference Series: Earth and Environmental Science conference proceedings. Krasnoyarsk Science and Technology City Hall of the Russian Union of Scientific and Engineering Associations. 2020. C. 32052]. Thus, registered drugs contain Streptococcus suis and Streptococcus zooepidemicus antigens, despite the fact that in recent years, studies have shown a significant change in the etiological structure of the infection. Most often, sick and dead piglets with a typical clinical and morphological manifestation of streptococcal infection are distinguished: Streptococcus dysgalactiae and Streptococcus massiliensis in 50% of enterprises; Streptococcus porcinus, Streptococcus mutans and Streptococcus pyogenes in 37.5% of cases; Streptococcus agalactiae, Streptococcus gallolyticus, Streptococcus rubneri and Streptococcus uberis in 25% of cases; Streptococcus iniae, Streptococcus lutetiensis, Streptococcus pneumoniae and Streptococcus suis in 12.5% of cases, that is, the number of cases of Streptococcus suis isolation is insignificant, and Streptococcus zooepidemicus is not detected in swine streptococcosis in general [Laishevtcev A.I. Etiological structure of streptococcosis of pigs in various regions of the Russian Federation // Russian Journal of Agricultural and Socio-Economic Sciences. 2018. No. 2 (74). S. 257-260]. Of all the isolated streptococci, it was Streptococcus dysgalactiae, Streptococcus porcinus, Streptococcus pyogenes, and Streptococcus suis that possessed pathogenicity, while other species did not possess this property.

These results are confirmed by foreign scientists, who in their works also demonstrate the important etiological role of bacteria of the species Streptococcus dysgalactiae, Streptococcus porcinus, Streptococcus pyogenes and Streptococcus suis [Kasuya, K., Yoshida, E., Harada, R., Hasegawa, M., Osaka, H., Kato, M., Shibahara, T. Systemic Streptococcus dysgalactiae Subspecies equisimilis Infection in a Yorkshire Pig with Severe Disseminated Suppurative Meningoencephalomyelitis. Journal of Veterinary Medical Science. 2014.76 (5), 715-718 .; Oh, S.-I., Kim, J. W., Jung, J.-Y., Chae, M., Lee, Y.-R., Kim, J. H., ByungJae So, Kim, H.-Y. Pathologic and molecular characterization of Streptococcus dysgalactiae subsp. Equisimilis infection in neonatal piglets. Journal of Veterinary Science. 2018.19 (2), 313 .; Katsumi, M., Kataoka, Y., Takahashi, T., Kikuchi, N., Hiramune, T. Biochemical and Serological Examination of. BETA, -hemolytic Streptococci Isolated from Slaughtered Pigs. Journal of Veterinary Medical Science. 1998. 60 (1), 129-131 .; Arai, S., Kim, H., Watanabe, T., Tohya, M., Suzuki, E., Ishida-Kuroki, K., Sekizaki, T. Assessment of pig saliva as a Streptococcus suis reservoir and potential source of infection on farms by use of a novel quantitative polymerase chain reaction assay. 2018. American Journal of Veterinary Research, 79 (9), 941-948 .; Van Samkar, A., Brouwer, M. C, Schultsz, C, van der Ende, A., & van de Beek, D. Streptococcus suis Meningitis: A Systematic Review and Meta-analysis. PLOS Neglected Tropical Diseases. 2015.9 (10), e0004191 .; Tang, J., Wang, C, Feng, Y., Yang, W., Song, H., Chen, Z., Gao, G. F. Streptococcal Toxic Shock Syndrome Caused by Streptococcus suis Serotype 2. PLoS Medicine. 2006.3 (5), e151.]. Due to the fact that various types of streptococci do not have the ability to form a cross-immune response, existing drugs are not able to fully protect animals from streptococcosis caused by other species.

The solution to the problem is the development of an effective modern vaccine with an expanded and updated antigenic composition from streptococcus species etiologically significant for pig breeding, a method for its production and use.

The proposed invention is implemented within the framework of the following provisions: "Strategies 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 of 25.09.17, No. 2045-r, the National Security Strategy of the Russian Federation, approved. By the Decree of the President of the Russian Federation of December 31, 15, 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 until 2025 and Further Prospect, approved. President of the Russian Federation of 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).

State of the art

In the Russian Federation, at the time of the implementation of this development, the following drugs were registered for the specific prevention of swine streptococcosis:

1) Vaccine against salmonellosis, pasteurellosis and streptococcosis of piglets "VERRES-SPS" - LLC "Vetbiochim", Russia. The product is made from industrial strains Salmonella choleraesuis and Salmonella typhimurium, Pasteurella multocida types A and D, Streptococcus suis serogroups C and R, inactivated with formalin and adsorbed on an aluminum hydroxide gel;

2) Vaccine against salmonellosis, pasteurellosis and streptococcosis of piglets - FKP "Armavir Biofabrika", Russia. The vaccine is made from cultures of bacteria Salmonella choleraesuis 370, Salmonella typhimurium 371, Pasteurella multocida serovars A, B, D, Streptococcus serogroups C and R, inactivated with formaldehyde with the addition of an adjuvant gel of aluminum oxide hydrate;

3) Polyvalent inactivated vaccine against swine respiratory diseases "Donoban-10" - "KBNP, INC.", Republic of Korea. The product is made from a suspension of inactivated bacterial cells of Bordetella bronchiseptica cDNT (SB-11), Pasteurella multocida type A NSPA (SB-40) and Pasteurella multocida type D cDNT (SB-54), Actinobacillus pleuropneumoniae serotype 2 NAS2 (SB-54) ) and Actinobacillus pleuropneumoniae serotype 5 NAS5 (SB-04), mycoplasma Mycoplasma hyopneumoniae NSM (SB-34), streptococci Streptococcus suis type 2 NSS2 (SB-50), hemophilia strains Haemophilus parasuis serotype 1 NSH1 (SB-30), Haemophilus serotype 4 NSH4 (SB-31) and Haemophilus parasuis serotype a 5 NSH5 (SB-32), as well as dermonecrotic toxin (cDNT) Bordetella bronchiseptica and Pasteurella multocida type D; cell wall protein (OMP) Pasteurella multocida type A; Actinobacillus pleuropneumoniae serotype 2 and 5, inactivated with formalin (at a concentration of 0.2%), with the addition of thiomersal (0.01%) as a preservative, adjuvants: aluminum oxide hydrate (20%) and ISA 25 (3%) and isotonic solution sodium chloride up to 2 ml;

4) Inactivated emulsified vaccine against streptococcosis "StreptVac-P", FGBI "ARRIAH", Russia. The vaccine is made from formaldehyde-inactivated bacterial cells of Streptococcus suis serogroup D type 2, strain 4232 with the addition of an oil adjuvant - Montanide ISA 206 (50%) and a phosphate-buffered solution (up to 50%).

In addition to the means of specific prevention of streptococcosis of pigs registered in the territory of the Russian Federation, there are known (discontinued or registered in the countries of the Customs Union):

1) Inactivated emulsified vaccine against pigs streptococcosis "Streptovac-S", JSC "BelVitunifarm", Republic of Belarus. The vaccine contains Streptococcus suis antigen with M-protein, inactivated with formalin at a concentration of 0.4% and emulsified in an oil adjuvant;

2) Vaccine against streptococcosis of farm animals, carnivores and rodents "Streptoevac", OOO "Trading house" BiAgro ", Russia. The vaccine contains cultures of serological group C streptococci, inactivated with formalin and sorbed on aluminum oxide hydrate.

In addition to these agents, biological products are known, which include streptococci classified in the genus Enterococcus: in particular, Enterococcus faecalis (formerly Streptococcus faecalis), which are not considered in the present invention.

All the above funds aimed at the specific prevention of swine streptococcosis are limited in antigenic composition, which in turn reduces their protective activity against all streptococcus species important for the industry that are not included in the drug, namely Streptococcus dysgalactiae, Streptococcus porcinus, Streptococcus pyogenes ...

In addition to the well-known commercial drugs, an associated inactivated vaccine against parvovirus disease, streptococcosis and salmonellosis of pigs is known [dissertation Nurgaliev FM, Kazan, 2006]. The proposed combination of antigens is due to the fact that these bacterial and viral diseases in 40% of cases are the cause of dysfunctions of the organs of the reproductive system of pigs [Gaffarov, Kh.Z. Infectious diseases of pigs and modern means of combating them / Kh.Z. Gaffarov, E.A. Romanov. Moscow: "Aquarium", 2004. -200 p.]. The vaccine is made from the U-13 strain of parvovirus, the C-05 strain of Streptococcus suis and the K-03 strain of Salmonella choleraesuis isolated from aborted sow fetuses. The strains have been deposited and used as production strains. Inactivation of the bacterial mass of all strains is carried out with formalin. Adjuvant - an alumina hydrate gel (GOA) and / or an oil adjuvant (MA), consisting of mineral oil with lanolin. The viral suspension of the PVA strain, having a hemagglutinating activity of 1: 512 before inactivation, was mixed with bacterial suspensions of Salmonella and streptococci containing 10 billion mc / ml in equal proportions, the vaccine pH 7.2-7.4. The disadvantages of the drug include: a narrow species spectrum of streptococci introduced into the vaccine; being immunogenic against other topical infections of piglets, it protects animals from only one species of Streptococcus suis; contains very high doses of bacterial antigens (10 billion mc / ml, while similar vaccines contain 3-10 times less antigen), which makes production unprofitable.

From sources of patent information known "Associated aluminum hydroxide vaccine against infectious pneumonia of pigs of bacterial etiology and a method for its manufacture" [patent RU 2191598 C2 publ. 2002.10.27]. The vaccine contains the bacterial mass Pasteurella multocida serovariants A, B and D, Haemophilus pleuropneumonia serogroups 1 and 2 and streptococci serogroups C and R, mixed in equal volumes. In the manufacture of the vaccine, sparing modes of inactivation and a controlled cultivation process are used. The vaccine provides protection against infectious swine pneumonia caused by a wide range of bacteria. The disadvantages of this vaccine are a narrow spectrum of antigenic activity against streptococci, and the species S. zooepidemicus (serogroup C), as already mentioned, does not present clinical significance in porcine streptococcosis at the present time.

Known "A method of obtaining a vaccine associated against colibacillosis, streptococcosis and enterococcal infection of calves and pigs" [patent RU 2429012 C1 publ. 2011.09.20; patent RU 2650268 C1 publ. 2018.04.17]. The specified vaccine is made from epizootic strains of Escherichia coli, Streptococcus bovis, Enterococcus faecalis, isolated from the affected organs of calves and pigs. The disadvantage of this method is that field isolates of microorganisms are used in the manufacture of the drug, which contradicts the requirements of F3-61 "On the Circulation of Medicines". In addition, the disadvantage of the method and the vaccine itself is the overloading of the bacterial mass of Escherichia coli cells carrying an excess antigenic load, which negatively affects the formation of immune defense in animals against streptococci and enterococci. The disadvantage of the preparation method is the unbalanced antigenic composition, more focused on the prevention of calf diseases, since it contains Streptococcus bovis antigens that are not relevant for pigs, and Enterococcus faecalis, which is an opportunistic microorganism and has a weak etiological significance for coccal infections in piglets.

Known "Vaccine composition against infection caused by Streptococcus suis" [patent RU 27355101 A publ. 2018.05.29]. This development is focused on the use of a polypeptide and a vector with a polynucleotide as an active immunogenic ingredient in the production of a vaccine against Streptococcus suis. Known "Composition of a vaccine against Streptococcus suis, a method for its preparation and use" (Streptococcus suis vaccine composition, and preparation method and application thereof) [patent CN104248754A publ. 2014.2.31]. The proposed preparation contains two types of immunoprotective antigens of Streptococcus suis - 34KDA and SaoA proteantigen and an adjuvant. Obtaining an antigen is possible by using genetic engineering methods, in particular, by means of a polynucleotide expression vector. Additionally, the present invention may contain a complete bacterial antigen of Streptococcus suis strain SC type 2. In addition, the proposed composition may contain immunostimulants such as interferon alpha, interferon beta, interferon gamma, and interleukin-22, an antioxidant, surfactant, coloring agent, essential oil, buffering agent, dispersant, propellant and antiseptic. Known "Attenuated Streptococcus suis vaccine and a method for its production" (It is attenuated Streptococcus suis vaccine and production and preparation method thereof) [patent CN 104780935 B publ. 2015.7.15]. This invention is based on the use of an attenuated strain of Streptococcus suis as the main component of a vaccine against swine streptococcosis. Known invention "Attenuated Streptococcus suis vaccines and methods for their production and use" (Attenuated Streptococcus suis vaccines and methods of making and use thereof) [patent AU2016222520B2 publ. 2018.03.22]. The invention is focused on the use of attenuated strains of Streptococcus suis in the production of live vaccines against streptococcosis. Known invention "A combined inactivated vaccine against contagious pleuropneumonia and streptococcosis of pigs caused by Streptococcus suis and a method for its production" (Porcine contagious pleuropneumonia and Streptococcus suis disease combined inactivated vaccine and preparation method thereof) [patent CN 103566364 A publ. 2014.2.12]. The invention is directed to the creation and use of an associated inactivated vaccine made in a combination of strains of Actinobacillus pleuropneumonia serotype 1, 5, 7 and two highly virulent strains of Streptococcus suis. The disadvantage of these inventions should be considered a narrow antigenic spectrum, which does not allow to provide the necessary protective activity against infections caused by other types of streptococci, typical for pig breeding.

The closest analogue (prototype) of the claimed vaccine is "Vaccine against salmonellosis, pasteurellosis and streptococcosis of pigs" - "VERRES-SPS" - LLC "Vetbiohim", Russia. The tool is a suspension for injection (inactivated vaccine). The vaccine is made from industrial strains of Salmonella choleraesuis No. 370 and Salmonella typhimurium No. 371, Pasteurella multocida type A No. 1231 and type D - "T-80", Streptococcus serogroup C, strain P-2082 and Streptococcus suis of serogroup R strain, inactivated with formalin (at a concentration of 0.3%) and adsorbed on the gel of aluminum hydroxide (20%). In appearance, the vaccine is a light yellow liquid with a gray-white precipitate that easily breaks down when shaken. The shelf life of the vaccine is 18 months from the date of issue, subject to the storage and transportation conditions. In animals, the vaccine induces the formation of immunity to salmonellosis, pasteurellosis and streptococcosis 10-12 days after the administration of the second dose of the drug, which lasts up to 5 months. One immunizing dose of the vaccine (3 ml) contains: inactivated microbial cells of Salmonella choleraesuis and Salmonella typhimurium strains - at least 4 * 10 ⁹ CFU each; Pasteurella Pasteurella multocida type A and D - not less than 5 * 10 ⁹ CFU each; Streptococci Streptococcus serogroup C and Streptococcus suis serogroup R - at least 3 * 10 ⁹ CFU each. The vaccine is harmless and areactogenic, does not possess medicinal properties. The disadvantage of the prototype is its limited antigenic composition. In particular, the drug contains Streptococcus zooepidemicus and Streptococcus suis antigens, but not Streptococcus porcinus, Streptococcus pyogenes, Streptococcus dysgalactiae.

The closest analogue (prototype) of the method of manufacturing the proposed vaccine is "Associated aluminum hydroxide vaccine against infectious pneumonia of pigs of bacterial etiology and a method for its manufacture" [patent RU 2191598 C2 publ. 2002.10.27]. A method of manufacturing an associated aluminum hydroxide vaccine against infectious pneumonia of pigs of bacterial etiology includes inoculation of vaccine strains of pasteurella, hemophilic bacteria and streptococci, their separate cultivation in a liquid nutrient medium based on Hottinger's digestion in fermenters, followed by inactivation of cultures with formaldehyde, adsorption and inactivation of cultures differs in that during the cultivation of streptococcal strains immediately after inoculation, the redox potential of the culture liquid is reduced to (-150) - (-200) mV, after which, until the end of the cultivation process, the oxygen level pO ₂ in the culture liquid is maintained at a level of 15-25% from saturation with air oxygen, the pH of the culture liquid is adjusted at the level of 6.8-7.2 units. pH, and fractional glucose supply is carried out in doses up to a concentration of 0.05-0.2% while limiting the growth of streptococci by glucose. In addition, this method involves the inactivation of streptococcal cultures with formaldehyde at a temperature of 43-48 ° C for 12-36 hours, and the final concentration of formaldehyde in the culture is 0.02-0.08%. The disadvantage of this method is the emphasis of the invention only on the method of cultivating streptococcal strains, and not on the complete sequence of production of the finished product and its control. In addition, in the cultivation of streptococci, it is proposed to use only Hottinger's broth, which limits the technology itself for the production of vaccines against streptococcosis, since other growth providing nutrient media are also known. Unlike the prototype, the claimed invention implies the use of carbopol-971 instead of aluminum hydroxide, which makes it possible to enhance the antigenic activity of the drug and ensure its high specific efficacy. In addition, in the newly proposed preparation and method of its production, methods for controlling the antigenic and immunogenic activity of vaccines against swine streptococcosis are presented and characterized.

The closest analogue (prototype) of the method of application is the invention "Attenuated Streptococcus suis vaccines and methods of making and use thereof" [patent AU 2016222520 B2, publ. 2018.03.22]. The invention is focused on the use of attenuated strains of Streptococcus suis in the production of live vaccines against streptococcosis. Compared to epizootic (field) streptococcal cultures, the proposed vaccine strains may have one or more polymorphic variations (more than 25) in the nucleotide sequence, which makes it possible to reduce the virulence of these cultures. In particular, such amino acid substitutions are found in the virulence genes rpsL-S12 (ribosomal protein of the 30S subunit), the ATC-binding membrane protein of the ABC transporter (ABC-ATPBMP), and the transcriptional regulator marR. The method of obtaining the considered invention includes several sequential stages, namely: 1) growing the parental strain of Streptococcus suis in the presence of a mutagenic agent; 2) sowing the surviving cells into an appropriate medium; 3) testing of individual colonies by infection of pigs to determine virulence. A strain is considered weakened if, after infection of pigs, it does not show any clinical signs of infection. When composing a batch of a preparation according to the invention, an adjuvant can be used, which is any substance that enhances the immune response, compared to a batch of vaccines that does not have an adjuvant in its composition. The developers emphasize that chitosan-based adjuvants are the most suitable for the preparation of a batch of the drug. The method of application of the present invention to pigs has several variations. The first option implies “simple” (prime) immunization of animals, when the proposed drug is administered to the animals once or repeatedly. The second vaccination scheme is focused on a prime-boost system, which implies one primary immunization and one booster immunization. Thus, the primary vaccine lot must be different from the boosted vaccine lot. Animals are vaccinated at intervals of 1-6 weeks, but an interval of 3-5 weeks is preferred. Evaluation of the effectiveness of the proposed drug is carried out due to infection of animals (pigs) 2-4 weeks after their last immunization. The infection procedure itself can be performed intramuscularly, subcutaneously, by spray method, intranasally, intratracheally, orally, intraocularly. The disadvantage of this invention is the limited antigenic composition of the proposed drug, which is not able to provide the necessary protective protection of animals against streptococci, except for Streptococcus suis. The method of production of the product does not fully cover all the technological manipulations necessary for the release of a series of vaccines, but is focused exclusively on the stage of cultivation of industrial strains. The method of using the considered prototype, according to the patent, focuses on the immunization of sows, while the proposed invention emphasizes the importance of immunizing not only sows, but also their offspring. This approach helps to increase the duration of immunity in piglets, which favorably affects the provision of epizootic well-being in the pig farm.

The technical problem to be solved by this invention is the need to create a safe vaccine against swine streptococcosis based on a balanced antigenic composition containing the most common Streptococcus spp. Species in the territory of the Russian Federation, a method for its production and use.

The technical result of the proposed invention is the creation of a vaccine with enhanced immunogenic and antigenic activity, inducing intense and long-term immunity in pigs against streptococcosis caused by S. suis, S. dysgalactiae, S. porcinus and S. pyogenes species, which leads to an expansion of the arsenal of polyvalent inactivated vaccines , aimed at the specific prevention of infectious diseases of pigs.

Disclosure of the essence of the invention

Vaccine against pigs streptococosis, polyvalent inactivated

The declared inactivated vaccine against swine streptococcosis contains the following components per one immunizing dose of the drug (2 cm ³ ): formalin-inactivated protective antigens of Streptococcus suis strains No. УР-4-ВБХ - 3.5 billion mcr. cl .; Streptococcus porcinus UR-3-VBH - 3.5 billion microns. cl .; Streptococcus pyogenes OB-4-VBH - 3.5 billion microns. cl .; Streptococcus dysgalactiae UR-16-VBKh - 3.5 billion mcr. class, as well as pharmaceutically acceptable target additives.

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

- an inactivant necessary for the disinfection of bacterial antigens. As an inactivant, formalin is used at the rate of 0.3% to the volume of broth culture, containing at least 37% formaldehyde;

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

- a diluent required to dilute the concentrated bacterial antigen to the required concentration. The solvent is sterile phosphate buffered saline (PBS);

- an adjuvant necessary for the nonspecific enhancement of the immune response to antigens. As an adjuvant in the proposed vaccine, a 2% solution of Carbopol-971 is used, which is applied in an amount of 10% of the volume of the drug;

- to establish the optimal level of hydrogen ions in the preparation, a 20% sodium hydroxide solution is used, the pH level of the preparation is 6.2-7.8.

In the finished form, the inactivated vaccine against swine streptococcosis is a suspension for injection, the color of which can vary from light gray to yellow-gray, with a gray-white precipitate that easily breaks down when shaken into a homogeneous suspension.

The shelf life of the drug is 18 months from the date of release.

The bacterial strains of Streptococcus suis No. УР-4-ВБХ, Streptococcus porcinus УР-3-ВБХ, Streptococcus pyogenes ОВ-4-ВБХ, Streptococcus dysgalactiae УР-16-ВБХ are new industrial crops and are used for the first time in vaccine preparations for widespread use. All newly proposed bacterial strains are deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" Federal Budgetary Institution of Science "State Scientific Center for Applied Microbiology and Biotechnology" (FBUN SSC PMB). 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 FSC VIEW RAS by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Characterization of strains

Production control strains Streptococcus suis UR-4-VBH, Streptococcus porcinus UR-3-VBH, Streptococcus pyogenes OB-4-VBH, Streptococcus dysgalactiae UR-16-VBH are used for the production of immunobiological drugs for veterinary use for the first time. Cultures are resistant to dissociation, are technologically advanced, forming growth on most liquid nutrient media, and are also virulent for laboratory animals. Establishing the value of LD ₅₀ for each strain of the pathogen allows them to be used as strains of "piercers" when monitoring the immunogenic activity of the proposed drug.

Characteristics of production control strains:

1) Strain of Streptococcus suis No. УР-4-ВБХ has the following characteristics and properties:

The strain was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under registration number B-8345.

Date, source and place of isolation: 08.2016 from dead piglets belonging to OOO SPK Mashkino, Kolomensky district, Moscow region.

Morphology: spherical or slightly oval cells, 0.5-2.0 µm in diameter, arranged in pairs or chains, motionless, non-spore-forming. Gram positive. Catalase negative. Optional anaerobes. Grows best on rich nutrient media.

Cultural properties: when growing in liquid nutrient media, forms a uniform turbidity of the medium, followed by precipitation of the culture; on blood agar in Petri dishes after 18-24 hours of cultivation - small, about 1 mm in diameter, round, translucent colonies. The colonies are surrounded by a zone of β-hemolysis.

Biochemical properties: Culture S. suis No.UR-4-VBH: VP -, hydrolysis of esculin +, beta-glucuronidase +, ferments to form acid: glucose +, inulin +, lactose +, maltose +, salicin +, trehalose +, sucrose +, arabinose -, glycerol -, mannitol -, melicytosis -, ribose -, sorbitol -.

Virulence: Industrial strain S. suis No. UR-4-VBH causes the death of white mice weighing 16-18 g. with intraperitoneal injection of culture. The value of LD ₅₀ is 1.08 * 10 ⁹ md. cl. The death of white mice occurs within 4-10 days after infection.

2) Strain of Streptococcus pyogenes No. OB-6-VBH has the following characteristics and properties:

The strain was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under registration number B-8344.

Date, source and place of isolation: 07.2015 from dead piglets PskovAgroInvest LLC, Pskov district, Pskov region.

Morphology: Cells are spherical or slightly oval, 0.5-2.0 µm in diameter, arranged in pairs or chains, motionless, non-spore-forming. Gram positive. Optional anaerobes. Catalase negative. Grows best on rich nutrient media.

Cultural properties: when growing in broth, they form a uniform turbidity of the medium, followed by precipitation of the culture; on blood agar in Petri dishes after 18-24 hours of cultivation - small, about 1 mm in diameter, round, translucent colonies. The colonies are surrounded by a zone of β-hemolysis.

Biochemical properties: S. pyogenes No. OB-6-VBH: ONPG -, VP -, ferments to form acid: fructose +, glucose +, galactose +, lactose +, salicin +, sucrose +, adonitol -, arabinose -, dulcitol - , sorbitol -.

Virulence: Industrial strain S. pyogenes No. OB-6-VBH causes the death of white mice weighing 16-18 g. with intraperitoneal injection of culture. The value of LD ₅₀ is 1.08 * 10 ⁹ md. cl. The death of white mice occurs within 4-10 days after infection.

3) Strain of Streptococcus porcinus No. УР-3-ВБХ has the following characteristics and properties:

The strain was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under registration number B-8343.

Date, source and place of isolation: 12.2015 from dead piglets SPA "Kolkhoz named after Voroshilov", Novoaleksandrovsky district, Stavropol Territory.

Morphology: spherical or slightly oval cells, 0.5-2.0 µm in diameter, arranged in pairs or chains, motionless, non-spore-forming. Gram positive. Optional anaerobes. Catalase negative. Grows best on rich nutrient media.

Cultural properties: when growing in broth, they form a uniform turbidity of the medium, followed by precipitation of the culture; on blood agar in Petri dishes after 18-24 hours of cultivation - small, about 1 mm in diameter, round, translucent colonies. The colonies are surrounded by a zone of β-hemolysis.

Biochemical properties: culture of S. porcinus No. UR-3-VBH: VP +, esculin +, arabinose -, dulcitol -, glucose -, inositol -, lactose -, maltose +, mannitol +, raffinose +, rhamnose -, salicin +, sorbitol +, trehalose +.

Virulence: Industrial porcinus strain No. UR-3-VBH: causes the death of white mice weighing 16-18 grams. with intraperitoneal injection of culture. The value of LD ₅₀ is 1.08 * 10 ⁹ md. cl. The death of white mice occurs within 4-10 days after infection.

4) Strain of Streptococcus dysgalactiae No. UR-16-VBH has the following characteristics and properties:

The strain was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under registration number B-8342.

Date, source and place of isolation: 11.2016 from a mastitis sow of JSC "Agrofirma Dmitrova Gora", Konakovsky district, Tver region.

Morphology: spherical or slightly oval cells, 0.5-2.0 µm in diameter, arranged in pairs or chains, motionless, non-spore-forming. Gram positive. Optional anaerobes. Catalase negative. Grows best on rich nutrient media.

Cultural properties: when growing in broth, it forms a uniform turbidity of the medium, followed by precipitation of the culture; on blood agar in Petri dishes after 18-24 hours of cultivation - small, about 1 mm in diameter, round, translucent colonies. The colonies are surrounded by a zone of β-hemolysis.

Biochemical properties: Culture of S. dysgalactiae strain No. UR-16-VBH: VP-, esculin-, arabinose +, dulcitol-, fructose +, galactose +, glucose +, inositol-, lactose +, maltose +, mannitol-, raffinose- , salicin-, sorbitol-, sucrose +, trehalose +, xylose +.

Virulence: Industrial strain S. dysgalactiae No. УР-16-ВБХ causes the death of white mice weighing 16-18 grams. with intraperitoneal injection of culture. The Ld ₅₀ value is 1.08 * 10 ⁹ mcr. cl. The death of white mice occurs within 4-10 days after infection.

Vaccine production method:

The vaccine is made from four strains of the genus Streptococcus: S. suis No. UR-4-VBH, S. pyogenes No. OB-6-VBH, S. porcinus No. UR-3-VBH, S. dysgalactiae No. UR-6-VBH. All strains were isolated during bacteriological studies of pathological material from animals that died or were forcedly killed, with signs of an infectious disease clinically corresponding to streptococcal infection.

All strains are stored as live lyophilized cultures.

For the manufacture of experimental and pilot-industrial series of vaccines, semi-finished products are used - formalin-inactivated (0.3% by volume) antigens of Streptococcus suis, S. porcinus, S. pyogenes, S. dysgalactiae, obtained by submerged cultivation and concentrated up to 100 ± 5 billion. m.c. / cm ³ .

A method of obtaining a vaccine against pigs streptococcosis inactivated.

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

1) Cultivation of industrial strains by the deep method;

2) Inactivation of the obtained culture liquid;

3) Concentration of antigens;

4) Determination of the completeness of antigen inactivation;

5) Compilation of a series of vaccines;

6) Control of vaccine sterility;

7) Determination of the harmlessness of the vaccine;

8) Determination of the immunogenic and antigenic activity of the vaccine.

For deep cultivation of streptococci in the production of vaccines, Hottinger's broth, tryptone-soy broth, mesopatamia buffered broth and other media with the necessary growth-supporting properties can be used.

The sequence of preparation of the uterine brood of streptococcal strains:

- on the first day, the lyophilized cultures are resuspended in saline, transferred to tubes for primary accumulation and plated on agar growth medium to assess colony morphology. Each strain is prepared separately. The cultivation of the first generation continues for 18-24 hours under aerobic conditions at a temperature of 37 ° C;

- on the second day, the primary brood is monitored according to the typical growth, the similarity of the morphological and tinctorial properties of the culture, as well as the absence of foreign microflora in liquid and agarized broods. If the first generation of the strain meets the stated requirements, reseeding is carried out to obtain the second generation. The obtained crops are cultivated for 18-24 hours under aerobic conditions at a temperature of 37 ° C. To obtain the maximum accumulation of bacterial cells in the broth medium, the tubes are placed on a shaker for constant stirring at 120-150 rpm;

- on the third day, after the quality control of the brood according to the previously indicated parameters, the broth culture of each strain is subcultured from test tubes into vials with fresh nutrient medium (at least 100 ml). The obtained crops are cultivated for 18-24 hours under aerobic conditions at a temperature of 37 ° C. To obtain the maximum accumulation of bacterial cells in the broth medium, the vials are placed on a shaker for constant stirring at 120-150 rpm;

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

- on the fifth day, the production strains are cultivated in a fermenter at a temperature of 37 ° C and a pH value of 7.2-7.8. To achieve the maximum accumulation of bacterial cells, 1-3% of blood serum of cattle or horses is additionally added to the nutrient medium. With a weakly expressed growth of streptococci, 2-3% of freshly prepared yeast extract is additionally introduced, which ensures a consistently high accumulation of bacterial mass.

To maintain the required level of nutrients, a 40% glucose solution is introduced into the medium at the rate of 0.5-1 liter of glucose per 100 liters of the nutrient medium 1 time per hour, depending on the intensity of culture growth. The pH level during cultivation is maintained with a 10% alkali solution. This method of cultivation makes it possible to achieve biomass accumulation up to 8.0 billion MC / cm after 12-14 hours of cultivation.

To inactivate the cultured culture liquid of streptococci, formalin is added to the fermenter containing at least 37% formaldehyde, 0.3% by volume of the culture liquid. Inactivation is carried out for 3 days at room temperature (22 ± 2 ° C) and periodic stirring every 12 hours.

Concentration of cultures is carried out by centrifugation at 5-6 thousand rpm. / min. within 2 hours or by separation at 12-15 thousand revolutions, depending on the equipment used.

The completeness of inactivation of the bacterial mass of streptococci is checked by inoculating a concentrated antigen on growth-supporting nutrient media in accordance with GOST 25085, as well as by the harmlessness of the inactivated bacterial mass in a bioassay on laboratory mice weighing 16-18 grams, which are once subcutaneously injected with antigens previously diluted 1:10 with water for injection, in a volume of 0.5 cm ³ . Three experimental mice are used to assess the safety of each series of antigen for each strain of streptococci. Observation of the experimental animals continues for 10 days. The drug is considered inactivated if there is no growth of cultures of streptococci 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.

The proposed inactivated vaccine against swine streptococcosis is produced by mixing certain volumes of inactivated S. suis, S. pyogenes, S. porcinus and S. Dysgalactiae antigens with an adjuvant - 2% carbopol-971 solution (10% by volume), preservative - 10% merthiolate solution sodium (to a final concentration of 1: 10000) and a diluent - phosphate buffered saline (PBS) to the required volume.

When assembling the preparation, the required amount of PBS is introduced into a sterile reactor, after which antigens of four industrial cultures with a specified concentration are added in stages. After uniform mixing of inactivated bacterial antigens, an adjuvant is fed into the reactor in an amount of 10% of the volume of the preparation, and the preservative is a solution of sodium merthiolate in a volume of 1 part of the preservative per 10,000 parts of the preparation, after which the diluent is added to the required volume. The concentration of bacterial antigens in the preparation is 14 billion mc / cm ³ , which corresponds to 3.5 billion mcr. cl. / cm of each strain. After receiving, with constant stirring of a homogeneous suspension, the recommended level of hydrogen ions (6.2-7.8) is established. The recipe for the composition of the vaccine with a volume of 100 liters (50 thousand doses) is shown in Table 1.

Formalin, used as an inactivant at the stage of obtaining inactivated antigens, also has a second purpose - the preservation of the finished product, after the preparation of a batch of the drug. This approach is especially necessary for drugs produced in multi-dose vials, since repeated insertion of a needle into a vial can lead to contamination of the vaccine, in view of this, formalin should also be considered as a targeted additive.

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 white mice and cultures of streptococci S. suis UR-4-VBH, S. porcinus UR-3-VBH, S. pyogenes OB-4-VBH, S. dysgalactiae UR-16-VBH.

For testing, eight groups of white mice weighing 16-20 g are formed, 10 heads each (a total of 4 experimental groups and 4 control groups: 1 experimental and control group for each strain). The vaccine is administered to white mice of the experimental groups twice with an interval of 21-24 days subcutaneously in the area of the withers in a dose of 0.5 ml. Control mice are not immunized. In 14-16 days after repeated administration, vaccinated and control analogous mice are infected with each strain included in the preparation (for example: 1 experimental and control group - Streptococcus suis UR-4-VBH; 2 experimental and control group - Streptococcus porcinus UR- 3-VBH; 3 experimental and control group - Streptococcus pyogenes OB-4-VBH; 4 experimental and control group - Streptococcus dysgalactiae UR-16-VBH). For infection, daily agar cultures of streptococci are used, which are administered to white mice intraperitoneally at a dose of 5 LD ₅₀ in a volume of 0.5 ml. The observation period for all animals is 10 days. The LD ₅₀ value of infecting streptococcal cultures is determined by titrating on white mice of the same batch, each time before conducting an experiment to assess the immunogenicity of a vaccine batch in connection with the rapid loss of virulence by strains during passages. Simultaneously with the infection of the mice, the method of administration is monitored, for which 10 mice are additionally used, which are injected intraperitoneally with a sterile saline solution at a dose of 0.5 ml. The observation period for all animals is 10 days. A series of vaccines is considered active if at the end of the observation period at least 80% of immunized white mice in each experimental group remain alive, with the death of at least 80% of white mice in each control group. At the same time, at the time of the end of the registration of the results, at least 90% of the mice injected with saline should survive; otherwise the experiment is repeated.

Released by the described method, three series of vaccines were tested in vivarium conditions, as well as at pig breeding enterprises, unfavorable for streptococcosis. Within the framework of approbation, the harmlessness of the drug and its specific efficacy were confirmed.

Control of the antigenic activity of the proposed drug is carried out in a test tube agglutination reaction.

For the study, the vaccine is preliminarily administered to 40 white mice weighing 16-18 g twice subcutaneously with an interval of 21-24 days at the withers in a volume of 0.5 ml. 14-16 days after re-administration, the vaccinated and control analog mice are bled to obtain sera (fatal). All the obtained blood serum from mice is combined into a common pool and used for staging an agglutination reaction in order to determine the titer of antibodies to S. suis, S. dysgalactiae, S. porcinus, S. pyogenes - a positive test. In order to control the background level of antibodies in experimental animals, blood is additionally taken from 10 unvaccinated mice. Before use, the sera are heated in a water bath at 56 ° C for 30 min.

The resulting serum is subjected to a two-fold dilution: 1: 2, 1: 4, 1: 8, 1:16, 1:32 with sterile saline. The antigen for the experiment is obtained by culturing control-production strains on MPA agar. Subsequently, the culture is washed off with sterile saline. The mixture is heated at 56 ° C for 30 minutes and brought to a concentration of 2 billion microns. cl. cm ³ . Subsequently, the serum and antigen of the corresponding strain are mixed in equal volumes (0.5 cm ³ each). A test culture mixed with saline (pH 7.2-7.4) and with normal rabbit serum diluted 1:10 (to exclude self-agglutination of the culture) serves as a control. In addition, sera obtained from unvaccinated mice are also examined in the control. The mixed sera with antigen are incubated at 37 ° C for 18-24 hours.

The research results are interpreted as follows:

"++++" - complete clarification of the liquid, the antigen has settled at the bottom of the test tube in the form of an “umbrella”, when the test tube is shaken, the precipitate breaks up into flakes with a clear liquid (100% agglutination);

"+++" - incomplete clarification of the liquid, pronounced "umbrella" (75% agglutination);

"++" - there are liquid clarifications, the "umbrella" is moderately pronounced (50% agglutination);

"+" - barely noticeable enlightenment, the "umbrella" is barely noticeable, when shaking, a small amount of agglutinate flakes is found (25% agglutination);

“-” - there is no clarification of the liquid, there is no “umbrella”, the sediment is easily agitated in the absence of agglutinate flakes.

The vaccine is considered immunogenic if in the blood serum of vaccinated mice in the agglutination reaction the titer of specific antibodies to the antigens of S. suis, S. dysgalactiae, S. porcinus and S. pyogenes is not less than 2 (log ₂ ) higher than the corresponding value in serum of mice of the control group. In this case, the agglutination of antigens and serum should take place at least 3 crosses. There should also be no self-agglutination of the antigen with saline and normal rabbit serum.

Method of application of polyvalent inactivated vaccine against swine streptococcosis

The vaccine is intended for the prevention of streptococcosis in pigs of all age groups in farms that are unfavorable for these diseases. The vaccine induces the formation of immunity to streptococcosis caused by S. suis, S. pyogenes, S. porcinus and S. dysgalactiae 12-14 days after repeated administration, which lasts for at least 6 months. Colostral immunity in piglets obtained from immunized sows lasts up to 21 days. The vaccine is harmless, has no medicinal properties.

The vaccine is administered to pregnant sows intramuscularly in the upper third of the neck (behind the ear) in a dose of 2 ml twice: the first time - 50-55 days before farrowing, the second time - 25-30 days before farrowing. Subsequently, the vaccine is administered 25-30 days before each subsequent farrowing once in the same dose. It is not allowed to vaccinate sows later than 25-30 days before farrowing, as well as lactating animals.

Piglets are vaccinated starting at 14 days of age. The vaccine is injected intramuscularly into the femoral muscle group twice with an interval of 21-24 days at a dose of 2 ml, additional revaccination is carried out at the age of 4 months. once.

It is forbidden to immunize clinically sick and / or weakened animals.

Implementation of the invention

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

Example # 1. Cultivation of industrial strains S. suis UR-4-VBH, S. porcinus UR-3-VBH, S. pyogenes OB-4-VBH, S. dysgalactiae UR-16-VBH was carried out by the submerged method, each strain was grown separately.

The preparation of inoculum was carried out as follows: on the first day of work, the first generation of inoculum was prepared, for which a nutrient broth in a volume of 1 ml was added to the ampoule with the freeze-dried strain to restore the lyophilisate. The resulting suspension in a volume of 0.5 cm ^{3 was} transferred into tubes with nutrient broth and in parallel, the suspension was plated on Petri dishes with Hottinger's agar with 10% defibrinated ram blood. The inoculations were cultivated for 24 ± 2 h at a temperature of 37 ± 1 ° C, after which the obtained streptococcal colonies were studied by cultural, morphological and tinctorial properties in order to determine the typical growth and the absence of growth of extraneous microflora. Cultures were also microscoped after Gram staining. If the production strains comply with the stated requirements and the absence of extraneous microflora, the resulting cultures were subcultured into flasks with nutrient broth to accumulate biomass (1 test tube per 250 cm ³ nutrient medium).

The resulting flasks were cultured for 24 ± 2 hours at a temperature of 37 ± 1 ° C to obtain the second generation of the strain. Cultures in flasks were monitored for cultural, morphological and tinctorial properties in order to determine the typical growth and the absence of growth of extraneous microflora.

To obtain the required amount of bacterial mass of streptococcal strains, the Sartorius "BIOSTAT-A" fermenter was inoculated. The cultivation of the strains was carried out in Hottinger's broth, while the volume of brood brood introduced into the fermenter was 10% by volume of the nutrient medium. Reactor cultivation was carried out at a temperature of 37 ° C and a pH of 7.0-7.8 for 14-16 hours, until the end of a stable increase in the concentration of bacterial cells. During the cultivation, the level of hydrogen ions in the nutrient medium was maintained at the level of 7.6-7.8 by adding a 20% sodium hydroxide solution. Additionally, the medium was enriched by introducing at regular intervals a 40% glucose solution to a concentration of 0.5% of the medium volume.

The described cultivation scheme made it possible to achieve a concentration of a bacterial suspension of streptococci of 8.3 ± 1.8 billion microns. cl. / cm ³ .

Example No. 2. Inactivation of the bacterial mass of streptococcal strains.

The resulting bacterial mass of streptococci (example No. 1) was inactivated by introducing formalin containing at least 37% formaldehyde, at the rate of 0.3% by volume of the culture fluid. Thus, 8.25 ml of formalin was added to 2750 ml of broth culture obtained by submerged cultivation. Inactivation was carried out within 72 hours at a temperature of 21-22 ° C.

Example No. 3. Concentration of the bacterial mass of streptococci.

Concentration of the resulting inactivated bacterial suspension (example No. 2) 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, it was determined volume and concentration and stored at 2-8 ° C until further use. Determination of the concentration of the suspension was carried out using turbidity standards - a set of TOC of turbidity of bacterial suspensions of the State Committee for the Provision of Mineral Resources of the Federal State Budgetary Institution "NTsESMP" of the Ministry of Health of Russia. As a result of this manipulation, it is possible to concentrate bacterial cells 10-15 times compared to the initial volume.

Example No. 4. Determination of the completeness of inactivation of streptococcal antigens.

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 ". 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.

Also, the completeness of inactivation was assessed by the safety of the obtained antigens in a bioassay on laboratory mice weighing 16-18 grams. The bacterial suspension of each strain was diluted 1:10 with water for injection, mixed thoroughly and injected subcutaneously in mice in a volume of 0.5 cm, using four experimental mice for each antigen. All antigens used for the composition of the three series of vaccines were sterile and did not cause death of laboratory animals within 14 days of observation.

Example No. 5. Compilation of a series of inactivated vaccine against swine streptococcosis was carried out according to the following method.

When composing a series of vaccines in a sterile container, four inactivated bacterial antigens of industrial cultures were mixed with phosphate buffered saline as a diluent, carbopol 971 as an adjuvant, and sodium merthiolate as a preservative, setting the concentration of bacterial antigens to 14 billion μR. cells / cm ³ (3.5 billion microns cells / cm ^{3 of} each strain). The components were mixed until a homogeneous suspension was obtained, after which the pH of the vaccine batch was set at 6.2-7.8. The recipe for the composition of the vaccine when using this layout option is shown in Table 2. The composition is given on the basis of the volume of the produced batch of 10 thousand doses - 20 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. 6. 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 series) with the drug, inoculation was carried out on liquid nutrient media - MPB, MPPB (Kitt-Tarozzi medium), Saburo, and on solid nutrient media - MP A and Saburo. Inoculation from each vial was carried out on three tubes and two dishes with a nutrient medium. When sowing on Wednesday Kitt-Tarozzi, sowing was done in two test tubes and two bottles. 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. Tubes and flasks with inoculations on all media except Sabouraud's medium were cultured 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.

Example No. 7. Determination of the Ld value of ₅₀ strains of streptococci.

To determine the virulence of streptococcal strains, white mice weighing 16-18 g were used. Infection of mice was carried out by intraperitoneal injection of two-fold dilutions of a suspension of bacterial cultures at concentrations: 3.0 * 10 ⁹ m.c., 3.0 * 10 ⁸ m.c., 3.0 * 10 ⁷ m.c., 3.0 * 10 ⁶ m.c. in a volume of 0.5 cm ³ . For each challenge concentration, 10 mice were used. The observation period for the animals was 10 days from the moment of the introduction of the cultures. The results are interpreted using probit analysis using BioStat 2009 software.

The studies carried out made it possible to establish the value of the virulence of the control-production strains of streptococci used to assess the immunogenic activity of the proposed vaccine. As a result of the research, the following results were obtained _{for the Ld 50} value for the control strains: LD ₅₀ Streptococcus suis UR-4-VBH - 1.08 * 10 ⁹ μR. cl .; LD ₅₀ Streptococcus porcinus UR-3-VBH - 3.25 * 10 ⁹ md. cl .; LD ₅₀ Streptococcus pyogenes OB-4-VBH - 2.95 * 10 ⁸ md. cl .; LD ₅₀ Streptococcus dysgalactiae UR-16-VBH - 3.98 × 10 ⁸ μR. cl.

Example No. 8. Determination of the immunogenic activity of the vaccine against S. suis, S. pyogenes, S. porcinus and S. dysgalactiae.

For testing, eight groups of white mice weighing 16-20 g were formed, 10 heads each (a total of 4 experimental groups and 4 control groups, 1 experimental and control group for each strain). The vaccine was administered to white mice of the experimental groups twice with an interval of 21-24 days subcutaneously in the area of the withers in a dose of 0.5 ml. Control mice were not immunized. 14 days after re-vaccination, 10 vaccinated and 10 control mice-analogs were infected with each strain included in the preparation. Namely: 1 experimental and control group were infected with the strain of Streptococcus suis UR-4-VBH; 2 experimental and control groups were infected with the strain of Streptococcus porcinus UR-3-VBH; 3 experimental and control groups were infected with the Streptococcus pyogenes OB-4-VBH strain; 4 experimental and control groups were infected with the Streptococcus dysgalactiae UR-16-VBH strain. The suspension was injected intraperitoneally to white mice at a dose of 5 Ld ₅₀ in a volume of 0.5 ml. Simultaneously with the infection of the mice, the method of administration was monitored, for which 10 mice were additionally used, which were injected with a sterile saline solution intraperitoneally at a dose of 0.5 ml. The observation period for all animals was 10 days.

The results of determining the immunogenic activity of three series of the proposed vaccine are shown in table No. 3.

The results obtained indicate that the survival rate of the animals of the experimental groups, regardless of the batch of the drug used, was 90-100%, while at least 90% of the animals of the control groups died within 4-9 days after infection (the minimum value is indicated for three series preparation), which indicates a high level of immunogenic activity of the vaccine.

Example No. 9. Determination of the antigenic activity of the vaccine against strains of Streptococcus suis, Streptococcus dysgalactiae, Streptococcus porcinus, Streptococcus pyogenes in the agglutination reaction.

Testing

Determination of antigenic activity of the proposed vaccine was carried out using white mice. The vaccine was administered to 40 white mice weighing 16-18 g twice subcutaneously with an interval of 21 days in the area of the withers in a volume of 0.5 ml. 14 days after re-administration, the vaccinated and control analog mice were bled to obtain sera. All the obtained blood serum from mice of each group was combined into a common pool and used for staging an agglutination reaction in order to determine the titer of antibodies to S. suis, S. dysgalactiae, S. porcinus, S. pyogenes. The obtained sera were heated in a water bath at 56 ° C for 30 min before use. The resulting serum was subjected to a two-fold dilution: 1: 2, 1: 4, 1: 8, 1:16, 1:32 with sterile saline. The antigen for the experiment was obtained by culturing control-production strains on MPA agar. Subsequently, the culture was washed off with sterile saline. The mixture was heated at 56 ° C for 30 min and brought to a concentration of 2 billion μR. cl. cm ³ . Subsequently, the serum and antigen of the corresponding strain were mixed in equal volumes (0.5 cm ³ ). A test culture mixed with saline (pH 7.2) and with normal rabbit serum diluted 1:10 (to exclude self-agglutination of the culture) served as a control. In addition, serum obtained from unvaccinated mice was also studied in the control. The mixed sera with antigen were incubated at 37 ° C for 18 hours.

When testing the proposed way of three batches of vaccine, the results were obtained indicating that 14 days after the second vaccination of laboratory animals, the level of antibodies was in the range of 1: 8-1: 16 expressed by at least 3 crosses. At the same time, antibody titers were absent in unvaccinated animals. Self-agglutination with saline and normal rabbit sera was absent in all cases. Thus, it was proved that the vaccine has the required level of antigenic activity.

Example No. 10. Evaluation of the protective efficacy of the vaccine in the vaccination of pregnant sows.

To assess the protective efficacy of the proposed vaccine, field trials were carried out in a pig-breeding complex in the Smolensk region, which is unfavorable for streptococcosis caused by S. suis, S. dysgalactiae, S. porcinus and S. pyogenes. The pigs for the experiment were selected according to the principle of analogs, that is, all animals in the experimental and control groups had identical parameters of body weight, age, and state of health. The animals were kept in the premises of pig farms separately in groups. Feeding was carried out with special concentrated feed intended for these age groups of animals.

Evaluation of the effectiveness of the drug was carried out on pregnant sows, which were vaccinated twice with an interval of 24 days. Observation of vaccinated animals, as well as piglets obtained from vaccinated and intact (control) sows, was carried out before the onset of weaning of piglets and their transfer to growing at the age of 24 days. Evaluation of the effectiveness of the proposed drug was carried out according to the following parameters: the safety of the obtained piglets, the average weight of the pigs at weaning, the average daily weight gain. The test results are shown in table No. 4.

The results show that the use of the vaccine provides 94.14% of the safety of piglets obtained from vaccinated sows, while in the control group (piglets from non-immune sows) the safety was 89.22%. At the same time, the sick and dead piglets showed signs of streptococcal infection with joint damage and a meningitis form of infection. In the group of piglets obtained from vaccinated sows, no diseases with signs characteristic of streptococcal infection were recorded.

Also, piglets obtained from vaccinated sows had a greater weight when transferred to growing up on average by 0.48 kg. A clearly pronounced positive effect of vaccination was also established on the average daily weight gain of piglets: in the experimental group, they were 239 g / day, and in piglets in the control group, 218 g / day, which is 8.78% less.

Example No. 11. Evaluation of the protective efficacy of the vaccine in the vaccination of piglets.

To assess the protective efficacy of the proposed vaccine, tests were carried out on a pig-breeding complex in the Tyumen region, which is unfavorable for streptococcosis caused by S. suis, S. dysgalactiae, S. porcinus and S. pyogenes. When conducting clinical studies, pigs of different age groups were selected according to the principle of analogues, that is, all animals in the experimental and control groups had identical parameters of body weight, age, and health status. The animals were kept in the premises of pig farms separately in groups. Feeding was carried out with special concentrated feed intended for these age groups of animals.

Evaluation of the effectiveness of the drug was carried out on weaned piglets, which were vaccinated twice with an interval of 21 days at the age of 14 and 25 days. Observation of vaccinated and control piglets was carried out during the period of growing up to transfer to fattening from 27 to 85 days of life (growing period - 58 days). Evaluation of the effectiveness of the proposed drug was carried out according to the parameters of the safety of the obtained pigs, the average weight of the pigs when transferring to fattening, the average daily weight gain. The test results are shown in table 5.

The use of the vaccine provides 96.50% of the safety of vaccinated pigs, while in the control group the safety was 87%, while the sick and dead pigs showed signs of streptococcal infection: septicemia, joint damage, meningitis. In the group of vaccinated pigs, no disease of piglets with signs characteristic of streptococcal infection was recorded.

In vaccinated piglets, the average weight when transferred to fattening was on average 1.49 kg higher than in animals of the control group. A clearly pronounced positive effect of vaccination was also established on the average daily weight gain of piglets: in the experimental group, they were 363 g / day, in piglets in the control group, 335 g / day, which is 7.71% less.

As can be seen from the presented data, the polyvalent inactivated vaccine against swine streptococcosis is a safe and effective immunobiological preparation and can be recommended for use in pig farms for the prevention of swine streptococcosis.

Claims (8)

1. Polyvalent inactivated vaccine against swine streptococosis, containing in its composition the following components per one immunizing dose of the drug in 2 cm ³ : inactivated protective antigen of the Streptococcus suis strain No. B-8345 "GPM-Obolensk" - 3.5 billion microns. cl .; inactivated protective antigen of the Streptococcus porcinus strain No. B-8343 "GKPM-Obolensk" - 3.5 bln. md. cl .; inactivated protective antigen of the Streptococcus pyogenes strain No. B-8344 "GKPM-Obolensk" - 3.5 billion microdistrict. cl .; inactivated protective antigen of the Streptococcus dysgalactiae strain No. B-8342 "GKPM-Obolensk" - 3.5 bln. md. class, as well as pharmaceutically acceptable target additives. 2. The vaccine according to claim 1, characterized in that a preservative in the form of an aqueous solution of sodium merthiolate 10%, introduced into the preparation at a rate of 1: 10000, namely 1 part of sodium merthiolate solution per 10,000 parts of the preparation, is used as pharmaceutically acceptable target additives. 3. The vaccine according to claim 1, characterized in that a diluent in the form of sterile PBS - phosphate-buffered saline solution is used as pharmaceutically acceptable target additives in the amount necessary to obtain 100% of the volume of the drug. 4. The vaccine according to claim 1, characterized in that an inactivant in the form of formalin containing at least 37% formaldehyde, at the rate of 0.3% by volume of the culture liquid, is used as pharmaceutically acceptable target additives. 5. The vaccine according to claim 1, characterized in that, as pharmaceutically acceptable target additives, it contains an adjuvant in the form of a 2% solution of carbopol-971 in an amount of 10% of the volume of the preparation. 6. The vaccine according to claim 5, characterized in that when carbopol-971 is used as an adjuvant, it additionally contains a 20% sodium hydroxide solution in the amount necessary to establish an optimal pH level of 6.2-7.8. 7. 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 antigen inactivation; preparation of a vaccine batch; control of the sterility and safety of the vaccine; determination of the immunogenic and antigenic activity of the vaccine, characterized in that the strains of Streptococcus suis No. В-8345 "ГКПМ-Obolensk", Streptococcus porcinus No. В-8343 "ГКПМ-Obolensk", Streptococcus pyogenes No. В-8344 "ГКПМ- Obolensk ", Streptococcus dysgalactiae No. В-8342" GKPM-Obolensk "; antigens are obtained using submerged cultivation while maintaining a pH of 6.2-7.8. 8. The method of application of the vaccine according to claim 1, characterized in that the vaccine is administered intramuscularly to pregnant sows in the upper third of the neck, behind the ear, in a dose of 2 ml twice: the first time - 50-55 days before farrowing, the second time - 25 -30 days before farrowing, then the vaccine is administered 25-30 days before each subsequent farrowing once in the same dose; piglets are vaccinated starting from 14 days of age using intramuscular double injection into the femoral muscle group with an interval of 21-24 days at a dose of 2 ml, and additional revaccination is carried out at the age of 4 months. once.