RU2763991C1 - Vaccine against infectious atrophic rhinitis and pasterellosis in pigs inactivated, method for its preparation - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: inventions group relates to the field of biotechnology and can be used by veterinary specialists in a pig farm for the specific prevention of infectious pathologies caused by bacteria of the species Bordetella bronchiseptica and Pasteurella multocida (infectious atrophic rhinitis and pig pasteurellosis). The vaccine against infectious atrophic rhinitis and pig pasteurellosis contains the following components per 2 cm3of the drug (immunizing dose): formalin-inactivated protective antigen of the Bordetella bronchiseptica B-1341 strain - 6 billion-micron cells; Pasteurella multocida B-1308 type D - 3 billion mcr cells; Pasteurella multocida B-1303 type A - 3 billion mcr cells, as well as pharmaceutically acceptable targeted additives. A method for obtaining a vaccine against infectious atrophic rhinitis and pasteurellosis of pigs is inactivated, according to the invention, the strains of Bordetella bronchiseptica B-1341 are used as industrial strains; Pasteurella multocida B-1303 type A; Pasteurella multocida B-1308 type D.EFFECT: inventions group increases the stability, safety of antigenic and immunogenic activity of the vaccine, providing long-term and intense immunity in vaccinated pigs against infectious atrophic rhinitis and pasteurellosis, expands the arsenal of multivalent inactivated vaccines aimed at specific prevention of infectious diseases of pigs, creating a vaccine with increased stability, antigenic and immunogenic activity, expansion of approaches and methods for the manufacture of multivalent inactivated vaccines aimed at specific prevention of infectious diseases of pigs.7 cl, 12 tbl

Description

SUBSTANCE: group of inventions relates to the field of veterinary medicine, veterinary microbiology and biotechnology, and can be used by veterinary specialists at pig complexes for the specific prevention of infectious pathologies caused by bacteria of the species Bordetella bronchiseptica and Pasteurella multocida (infectious atrophic rhinitis and pasteurellosis of pigs).

Respiratory diseases of pigs are one of the main causes of death and culling of animals in the pig industry around the world [Sorensen, V., Jorsal, S.E., Mousing, J., 2006. Diseases of the respiratory system. In: Straw, B.E., Zimmerman, J.J., d'Allaire, S., Taylor, D.J. (Eds.), Diseases of Swine, ninth ed. Blackwell Publishing, IA, USA, pp. 161-194]. Respiratory diseases in piglets can be caused by viral and bacterial infectious agents, and occur both in the form of monoinfections and their associations. The course of infections can be complicated by certain predisposing factors that provoke a decrease in the body's natural resistance to the pathogen. Such diseases include infectious atrophic rhinitis and pasteurellosis of pigs, the causative agents of which are Bordetella bronchiseptica and Pasteurella multocida. Designated microorganisms can be both primary etiological agents and secondary. It should be noted that both named types of bacteria may not have any specific etiological role, since some strains that do not have pathogenic properties (incapable of causing disease) [Brockmeier, SL, 2004. Prior infection with Bordetella bronchiseptica increases nasal colonization by Haemophilus parasuis in swine. Veterinary Microbiology 99, 75-78].

Porcine infectious atrophic rhinitis (IAR) is a widespread disease characterized by deformation, twisting or shortening of the nose as a result of atrophy of its bone structures. According to generally accepted data, toxigenic strains of Bordetella bronchiseptica and Pasteurella multocida are involved in the development of the disease [de Jong, M.F., 1999. Progressive and nonprogressive atrophic rhinitis. In: Straw, B.E., D'Allaire, S., Mengeling, W.L., Taylor, D.J. (Eds.), Diseases of Swine. 8th ed. Iowa State University Press, Ames, IA, pp. 355-384].

In an epizootic survey of pig enterprises from various regions of the Russian Federation, which are unfavorable for respiratory diseases, it was found that the incidence of Bordetella bronchiseptica from the lungs and upper respiratory tract of animals is in the range of 5-30%. Along with Bordetella bronchiseptica, Pasteurella multocida is found in 15-40% of cases (as of 2020), which entails the need for their combined specific prevention.

In accordance with the current taxonomy, the genus Bordetella has sixteen species: B. ansorpii, B. avium, B. bronchialis, B. bronchiseptica, B. flabilis, B. hinzii, B. holmesii, B. muralis, B. parapertussis, B. pertussis , B. petrii, B. pseudohinzii, B. sputigena, B. trematum, B. tumbae, B. tumulicola [https://lpsn.dsmz.de/genus/bordetella].

According to current taxonomy, the genus Pasteurella consists of thirteen species: P. aerogenes, P. bettyae, P. caballi, P. caecimuris, P. canis, P. dagmatis, P. langaaensis, P. mairii, P. multocida, P. oralis, P. skyensis, P. stomatis, P. testudinis [https://lpsn.dsmz.de/genus/pasteurella].

(2019). Bordetella bronchiseptica diguanylate cyclase BdcA regulates motility and is important for the establishment of respiratory infection in mice. Journal of Bacteriology.doi:10.1128/jb.00011-19; Kadlec, K., & Schwarz, S. (2018). Antimicrobial Resistance in Bordetella bronchiseptica. Microbiology Spectrum, 6(4). doi: 10.1128/microbiolspec.arba-0024-2017].Bordetella bronchiseptica is a non-spore-forming gram-negative coccus-like bacillus 0.2-0.5×1.0-2.0 µm in size, virulent strains form a microcapsule. In smears, cells are located singly, in pairs, rarely in short chains. Cultures of the pathogen are oxidase- and catalase-positive, have mobility. Cultivated at a temperature of 36-37°C. The optimum pH value is 7.2-7.6. It grows both on conventional nutrient media - MPB, MP A, etc., and on elective media - casein-coal agar (CUA), McConkey agar, Hartoch's medium, etc., forming small gray-white , shiny colonies with smooth edges with a diameter of 1-2 mm, when growing on blood agar, a zone of β-hemolysis is expressed [Belhart, K., Gutierrez, M. de la P., Zacca, F., Ambrosis, N., Cartelle Gestal, M., Taylor, D., …

(2019). Bordetella bronchiseptica diguanylate cyclase BdcA regulates motility and is important for the establishment of respiratory infection in mice. Journal of Bacteriology.doi:10.1128/jb.00011-19; Kadlec, K., & Schwarz, S. (2018). Antimicrobial Resistance in Bordetella bronchiseptica. Microbiology Spectrum, 6(4). doi: 10.1128/microbiolspec.arba-0024-2017].

Pasteurella multocida - are spherical, oval or rod-shaped cells 0.3-1.0×1.0-2.0 µm in size. When viewing smears from animal tissues, as well as primary cultures obtained from pathological material, P. multocida often exhibit bipolar staining and occur singly, in pairs, or in short chains. On subsequent subcultures, Pasteurella cultures lose their bipolarity and become similar to coccobacilli and capsuled rods. On MPA, Pasteurella can form three types of colonies: colonies with a smooth surface (S-colonies), colonies with a rough surface (R-colonies) and mucoid colonies (M-colonies) [Kapustin, A.V. Diagnosis of pasteurellosis in agricultural animals, birds and fur animals / A.V. Kapustin, A.I. Laishevtsev, I.Yu. Ezdakova, V.N. Skvortsov., T.V. Stepanova, M.I. Gulyukin // Moscow, 2016].

The antigenic structure of the Bordetella bronchiseptica cell wall O antigen is characterized by several types, which are listed in Table 1.

The generic antigen is factor 7, the species factor is antigen 12, typical factors are defined - 8, 9, 10, 11, 13. The agglutination reaction on glass is set with adsorbed species factor sera using 48-hour cultures. Using other factor sera, it is possible to determine the serotype [Litusov N.V. The causative agents of whooping cough and parapertussis. Illustrated teaching aid. - Ekaterinburg: Publishing house of UGMA, 2013. - 32 p.].

In the Russian Federation, three types of Pasteurella multocida are most common: A, B and D. Type A most often causes pasteurellosis infection in birds, rabbits and fur animals, and also contributes to the development of subacute or chronic enzootic pneumonia in calves and piglets; type B causes hemorrhagic septicemia in cattle and wild ruminants; type D - pig pasteurellosis. Isolates of P. multocida types E and F are not common in the Russian Federation, while they are the causative agents of hemorrhagic septicemia in cattle and wild ruminants in a number of foreign countries.

The detailed etiological significance of Pasteurella serotypes according to the Carter-Headleston classification is shown in Table 2.

Clinical signs of B. bronchiseptica bordetellosis range from mild with fever, cough, sneezing, conjunctival discharge, and swollen lymph nodes to severe pneumonia with dyspnea, cyanosis, and death. B. bronchiseptica also causes primary pneumonia in newborn piglets and secondary pneumonia in adult pigs [Ga Young Park, Hye Min Lee, Hyun Jin Yu, Jee Soo Son, Sang Joon Park, Kyoung Seob Song // Bordetella bronchiseptica bacteriophage suppresses B. bronchiseptica induced inflammation in swine nasal turbinate cells // 4 Received: 5 September 2018 / Accepted: 16 October 2018].

At autopsy of the corpses of pigs, typical and clear pathological changes in the internal organs are not observed, with the exception of the nasal cavity. With a longitudinal cut of the skull, an accumulation of mucopurulent exudate, partial or complete atrophy of the upper and lower turbinates, and thinning of the ethmoid bone are found. Necrotic changes in the nasal mucosa are rare [Temporary instruction on measures to combat infectious atrophic rhinitis of pigs, Approved by the State Veterinary Inspectorate of the Ministry of Agriculture of the USSR on September 6, 1960, as amended on January 20, 1961]. Microscopic examination fixes mucosal neutrophilic infiltration, loss of cilia, epithelial metaplasia and bone resorption with its replacement with fibrous tissue. Pulmonary lesions include neutrophilic airway infiltration, necrosis of the alveoli and blood vessels, hemorrhages and, ultimately, extensive fibrosis [Brockmeier S.L., Register K.B., Magyar T., Lax A.J., Pullinger G.D., Kunkle R.A. Role of the dermonecrotic toxin of Bordetella bronchiseptica in the pathogenesis of respiratory disease in swine. Infect. Immun. 2002; 70:481-490. doi: 10.1128/IAI.70.2.481-490.2002].

A significant representative in the etiological structure of infectious atrophic rhinitis is the species Pasteurella multocida, which has a vast area of distribution and high epizootic significance. As a rule, the disease reproduces only when B. bronchiseptica is associated with P. multocida.

Sporadic incidence of pasteurellosis is more often caused in pigs - P. multocida types A and D. Pigs are characterized by three forms of the course of the disease - hyperacute, acute and chronic. The hyperacute course of the disease is characterized by fever of 41°C and above, heart failure, bradycardia, decreased respiratory movements, pharyngitis, and edema in the intermaxillary region and neck. Redness and cyanosis appear behind the ears of the animal. Also, a change in color is observed on the surface of the ventral wall of the abdomen. Death with this form of flow occurs from asphyxia within 24-48 hours.

The acute course is characterized by a thoracic and pulmonary form of the disease. There is fibrous pneumonia, a strong cough with sputum and shortness of breath, with discharge of a mucopurulent nature, often with an admixture of blood streaks. Also, the disease is additionally manifested by damage to the digestive system, in the form of dyspepsia and cyanosis of visible mucous membranes, followed by a transition to diffuse clouding of the cornea of the eye. The final development of the disease is manifested by a weakening of cardiac function with the formation of hyperemic spots on the skin. Death also occurs from asphyxia 5-8 days after infection.

In the case of weak pathogenicity of the P. Multocida strain, the disease passes from an acute form into a chronic one. The clinical picture is smoothed compared to the acute course, but one can note the presence of a persistent cough, an increase in weakness and cachexia. Pigs can either die within 3-6 weeks or recover, but with the preservation of the carrier state of the disease [Kapustin, A.V. Diagnosis of pasteurellosis in agricultural animals, birds and fur animals / A.V. Kapustin, A.I. Laishevtsev, I.Yu. Ezdakova, V.N. Skvortsov., T.V. Stepanova, M.I. Gulyukin // Moscow, 2016].

The main pathoanatomical changes in pasteurellosis of pigs are: serous-gelatinous edema of the submandibular space passing to the neck, dewlap, pharynx and larynx, in addition, edema covers the limbs of the animal; hemorrhages are observed on the serous and mucous membranes, the parenchymal and intracellular tissue of the lungs is in a state of serous inflammation; croupous inflammation of the lungs; serous-fibrinous pleurisy; bronchial lymph nodes are hyperemic and enlarged; the lumen of the bronchi and bronchioles contains mucopurulent or fibrinous exudate; hemorrhages are found in parenchymal organs, as well as on the mucous membranes of the bladder, stomach, intestines; the liver contains scattered gray necrotic foci; under the capsule of the kidneys, anemic areas with petechial hemorrhages in the center are revealed; the abdominal cavity contains serous exudate with possible blood impurities [Kapustin, A.V. Diagnosis of pasteurellosis in agricultural animals, birds and fur animals / A.V. Kapustin, A.I. Laishevtsev, I.Yu. Ezdakova, V.N. Skvortsov., T.V. Stepanova, M.I. Gulyukin // Moscow, 2016.].

Histopathological features in pigs also include fibrous replacement of the bony lamellae of the ventral turbinates with varying degrees of inflammatory and reparative changes [Magyar T., Donko T., Repa I. & Kovacs M. (2013). Regeneration of toxigenic Pasteurella multocida induced severe turbinate atrophy in pigs detected by computed tomography. BMC Vet. Res., 9, 222].

State of the art

Specific prevention of infectious diseases of pigs is the most effective means of preventing cases of mass disease and death of animals.

Currently, the following veterinary drugs are registered in the Russian Federation for the prevention of infectious atrophic rhinitis and pasteurellosis in pigs:

Preparations for pigs:

- Polyvalent inactivated vaccine against porcine respiratory diseases PEC-VAK - produced by KOMIPHARM JSC, South Korea. The drug is intended for the prevention of infectious respiratory diseases of pigs (atrophic rhinitis, pasteurellosis, actinobacillus pleuropneumonia of pigs, hemophilic polyserositis and mycoplasmosis (pathogen Mycoplasma hyopneumoniae). One immunizing dose of the vaccine (2 cm ² ) contains: inactivated microbial cells of Bordetella bronchiseptica strains; Pasteurella multocida type D and type A - not less than 1.5×10 ¹⁰ CFU; Actinobacillus pleuropneumoniae serotypes 5 and 2 - not less than 1.0×10 ¹⁰ CFU; hemophilus Haemophilus parasuis, serotypes 4 and 5 - not less than 4×10 ⁹ CFU each and mycoplasma Mycoplasma hyopneumoniae - at least 2×10 ⁸ CFU, as well as dermonecrotic toxin (cDNT) of toxigenic strains of Bordetella bronchiseptica and Pasteurella multocida type D - at least 500 μg each; cell wall protein (OMR) of toxigenic strains of Actinobacillus pleuropneumoniae serotypes 5 and 2 - at least 10 mcg of each [https://galen.vetrf.ru/files/2b539f17-6d85-4de4-9a66-1b513d1 27c9c].

- Inactivated vaccine for the prevention of pneumonia in pigs Polypleurosin ARCH IM - produced by JSC BIOVETA, Czech Republic. The drug is intended for the prevention of pneumonia in pigs caused by Actinobacillus pleuropneumoniae serovars 2 and 9, Pasteurella multocida A and D, Bordetella bronchiseptica. One dose of the vaccine (1.0 cm ³ ) contains inactivated bacterial cells of Actinobacillus pleuropneumoniae serovars 2 and 9, Pasteurella multocida serovars A and D, Bordetella bronchiseptica, toxoids Arch I, ARC II, Arch III, as well as excipients - an oil adjuvant, formalin - 3.8 mg, thiomersal - 0.2 mg [https://galen.vetrf.ru/files/12efe1dc-f1a0-4b13-a7eb-6c99746fbf3e].

- Vaccine against infectious atrophic rhinitis of pigs inactivated RiniPig - produced by "Dae Sung Microbiological Labs.", Republic of Korea. The drug is intended for the prevention of infectious atrophic rhinitis in pigs in threatened and disadvantaged pig farms. One immunizing dose of the vaccine (2 cm ³ ) contains inactivated bacterial cells of Bordetella bronchiseptica (Bb1 DS strain) - not less than 6×10 ¹⁰ CFU and Pasteurella multocida (type D DS strain) - not less than 1.4×10 ¹⁰ CFU, as well as crude dermonecrotoxins Bordetella bronchiseptica (strain Bb1 DS) - not less than 0.5 mg and Pasteurella multocida (strain type D DS) - not less than 0.5 mg, formalin inactivated - not more than 0.25%, with the addition of a preservative thiomersal - not more than 0.01% and adjuvant of the fine oil-in-water emulsion "Montanid IMS 1313" - 10% [https://galen.vetrf.ru/files/8787ccc5-a881-49d7-91ba-cad05c6edae7].

- Vaccine against atrophic rhinitis of pigs inactivated RINISENG - produced by "Laboratories Hipra, SA", Spain. One inoculation dose of the vaccine (2 ml) contains inactivated Bordetella bronchiseptica bacteria (strain 833CER) - at least 2 × 10 ¹⁰ microbial cells, which corresponds to 9.8 BbCC Log and Pasteurella multocida recombinant type D toxin (PMTr) - at least 110 μg, inducing >1 MED63 with the addition of formaldehyde as a preservative (0.8 mg/dose and adjuvant: aluminum oxide hydrate (6.4 mg/dose) [https://galen.vetrf.ru/files/ae008d0e-7f59-4ce2- bae2-b2c378b6ba57].

- Polyvalent inactivated vaccine against respiratory diseases of pigs Donoban-10 - produced by "KBNP, INC.", Republic of Korea. The drug is intended for prophylactic immunization of pigs against atrophic rhinitis, pasteurellosis, actinobacillus pleuropneumonia, hemophilic polyserositis, streptococcosis and infection caused by Mycoplasma hyopneumoniae. The vaccine is made from inactivated bacterial cells of Bordetella bronchiseptica (ARDNT /SB-11 strain), Pasteurella multocida type A (NSPA /SB-40 strain) and Pasteurella multocida type D (NSA2 / SB-03 strain) and Actinobacillus pleuropneumoniae serotype 5 ( strain NSA5 /SB-04), mycoplasma Mycoplasma hyopneumoniae (strain NSM /SB-34), streptococcus Streptococcus suis type 2 (strain NSS2 /SB-50), haemophilus strains Haemophilus parasuis serotype 1 (strain NSH1 /SB-30), Haemophilus parasuis serotype 4 (strain NSH4 /SB-31) and Haemophilus parasuis serotype 5 (strain NSH5 /SB-32), as well as dermonecrotic toxin (cDNT) Bordetella bronchiseptica and Pasteurella multocida type D; cell wall protein (CMP) Pasteurella multocida type A; Actinobacillus pleuropneumonia 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 IMS 1313 (10%) up to 2 ml [https://galen.vetrf.ru/files/eee032b2-42b6-4e97-bdf6-201942a9cf33].

The disadvantage of these drugs is that they are all foreign-made, while the proposed invention is a domestic development, which fully complies with the principles of import substitution policy. In addition, the newly proposed drug uses new epizootic strains of Bordetella and Pasteurella isolated in the Russian Federation, which will have a positive effect on the effectiveness of the drug, since they are most homologous to strains of pathogens circulating in the country's pig breeding complexes.

- Vaccine associated against pasteurellosis and salmonellosis of pigs inactivated emulsion - produced by FGBU "ARRIAH", Vladimir. The drug is intended for the prevention of pasteurellosis and salmonellosis in pigs. The vaccine is made from cultures of strains of Pasteurella multocida No. 1231 (serogroup A) and No. 9 (serogroup D); Salmonella choleraesuis No. 370 and Salmonella typhimurium No. 371 inactivated with formaldehyde, with the addition of an adjuvant - Montanide ISA 70 (70%) [https://galen.vetrf.ru/files/52806702-4530-4839-b198-81cf2eb9a2b9].

- Vaccine against actinobacillary pleuropneumonia and pasteurellosis of pigs inactivated emulsified "Aptovak" - produced by "Biowet Pulawy", Poland. The vaccine is made from strains of Actinobacillus pleuropneumoniae serotypes 2 and 6, as well as Pasteurella multocida type D; with the addition of a BCI substrate (heart-brain broth, water for injection) - up to 2 ml, formaldehyde - 1 mg, aluminum oxide hydrate - 0.1 ml, Emulsigen mineral oil - 0.2 ml per dose [https://galen. vetrf.ru/files/aa9cccef-237c-465e-a2e2-ee9c7514c2d8].

- Vaccine against salmonellosis, pasteurellosis and enterococcal infection of piglets associated (inactivated) - produced by FKP "Armavir Biofactory", Krasnodar Territory. 1 cm ³ (0.2 commercial doses) of the vaccine contains at least 2 billion microbial cells of Salmonella choleraesuis No. 370, 6 billion, microbial cells of Pasteurella multocida No. 656 and 0.4 billion microbial cells of each strain of Streptococcus faecalis No. 356, No. 13, No. 345, Sokolovo and Konstantinovsky [https://galen.vetrf.ru/files/83ebe0d8-8f4b-45b4-8ec2-ef35dfcf3b0f].

- Vaccine associated against pasteurellosis, salmonellosis and hemophilic polyserositis of pigs inactivated emulsion - produced by FGBU "ARRIAH", Vladimir. The vaccine is made from cultures of strains of Pasteurella multocida No. 1231 (serogroup A) and No. 9 (serogroup D); Salmonella choleraesuis No. 370 and Salmonella typhimurium No. 371 inactivated with formaldehyde, with the addition of the adjuvant -Montanide ISA 70 (70%) [https://galen.vetrf.ru/files/6d74d410-5279-4a54-bb17-bf5eded44ff7].

- Vaccine against salmonellosis, pasteurellosis and enterococcal infection of piglets inactivated "Suigard" - produced by FKP "Stavropol biofactory", Stavropol. It is used for the prevention of salmonellosis, pasteurellosis and enterococcal infection of pigs in farms that are unfavorable according to these diseases. The vaccine consists of a culture of bacteria Salmonella choleraesuis No. 370, Pasteurella multocida No. 656, Streptococcus faecalis No. 13, 345, Sokolovo, Konstantinovsky, inactivated with formaldehyde with the addition of an adjuvant [https://galen.vetrf.ru/files/1a8724e5- 5fa6-4eeb-bf82-83dc81af60d3].

- Vaccine against pasteurellosis of sheep and pigs inactivated "Pastos" - production FKP "Stavropol biofactory", Stavropol. The vaccine contains a mixture of bacterial strains Pasteurella multocida (serogroup B) No. 796, No. 116, No. 656, No. 877, inactivated with formalin and precipitated with potassium alum [https://galen.vetrf.ru/files/181d850a-648c-4978-b4ee- 2be706d30cd7].

Vaccine associated against pasteurellosis, hemophilic polyserositis and actinobacillus pleuropneumonia of pigs "VERRES-PGA" - produced by LLC "Vetbiokhim", Moscow. The vaccine is made from a suspension of bacterial cells Pasteurella multocida serogroups A and D, Haemophilus parasuis, Actinobacillus pleuropneumonia © serotypes 2 and 5, inactivated with formalin (at a concentration of 0.3%) and adsorbed with a gel adjuvant in an amount of 15% of the vaccine volume [https:// galen.vetrf.ru/files/b80d939d-8015-4eca-8938-bb06b942274a].

- Vaccine against salmonellosis, pasteurellosis and streptococcosis of piglets "VERRES-SPS" - produced by LLC "Vetbiokhim", Moscow. The vaccine is made from industrial strains of Salmonella choleraesuis No. 370 and typhimurium No. 371, Pasteurella multocida type A No. 1231 and type D - "T-80", Streptococcus serogroup C strain P 2082 and Streptococcus suis serogroup R strain Kasli, inactivated by formalin (at a concentration of 0 .3%) and adsorbed on aluminum hydroxide gel (20%) [https://galen.vetrf.ru/files/ca86831a-8b5c-416e-be11-9baf9e1b623b].

- Vaccine against salmonellosis, pasteurellosis and streptococcosis in piglets - produced by FKP "Armavir Biofactory", Krasnodar Territory. The vaccine is made from cultures of strains of Salmonella choleraesuis No. 370, Salmonella typhimurium No. 371, Pasteurella multocida No. 1231, Pasteurella multocida No. 796, Pasteurella multocida No. T-80, serovars A, B, D, Streptococcus zooepidemicus No. P-2082 of serogroup C and Streptococcus suis type II No. "Kasli" serogroup R, formalin-inactivated (0.3% by volume), with the addition of alumina hydrate gel (30% by volume) as an adjuvant. 1 cm ³ (0.2 commercial doses) of the vaccine contains at least 2 billion salmonella microbial cells, 6 billion pasteurella and 2 billion streptococci [https://galen.vetrf.ru/files/c37b737d-29ef-4cec- a604-283c46b214ef].

The disadvantage of the indicated preparations is the absence of B. bronchiseptica antigens in their composition, despite the presence of P. multocida antigens in them, which indicates the possibility of using these agents for immunizing animals against pasteurellosis, but not against atrophic rhinitis.

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

- Vaccine associated inactivated aluminum hydroxide against infectious pneumonia and salmonellosis of pigs and method for its manufacture [patent RU 2246316 C1 publ. 2005.02.20]. According to the invention, the vaccine contains a bacterial mass of Pasteurella multocida serotypes A, B and D, Haemophilus pleuropneumoniae serogroups 1 and 2 and streptococci serogroups C and R, as well as Salmonella choleraesuis lysate antigens strain No. 370 and Salmonella typhimurium strain No. 415, mixed in a certain concentration . The vaccine has a high immunogenicity, provides reliable protection of pigs against infectious pneumonia of bacterial etiology and salmonellosis.

- Live attenuated bacterium Pasteurella multocida, a vaccine based on it, a method for producing a vaccine and using this bacterium to obtain a vaccine to protect animals or humans [patent RU 2415926 C2 publ. 2011.04.10]. Live attenuated bacterium Pasteurella multocida is modified by introducing a mutation in the Orf-15 gene. The mutation is an insertion and/or deletion in the Orf-15 gene. As a result of the modification, the bacterium is unable to express the functional Orf-15 protein, which leads to its attenuation. Also disclosed is the use of such a bacterium to create a vaccine based on it to protect humans or animals from infection with Pasteurella multocida bacteria or the pathogenic effects of infection.

- Vaccine against pasteurellosis pigs [application for invention RU 94023126 A1 publ. 1997.04.10]. The vaccine against pasteurellosis of pigs includes formalin-inactivated P. multocida antigens of serological variant A - strain DEP-8683, serological variant B - strain DEP-B681, serological variant D - strain DEP-T80 and an adjuvant consisting of mineral oil and lanolin. All strains are deposited in the collection of microorganisms of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Preparations. The concentration of each of the strains included in the vaccine is 3.0⋅10 ⁹ - 4.8⋅10 ⁹ microbial cells/cm ³ . The vaccine is administered intramuscularly to sows, regardless of age, once 4 to 5 weeks before the survey at a dose of 5.0 cm ³ , to piglets - twice up to 60 days of age, 2 cm ³ , over 60 days of age, 3 cm ³ . The use of the vaccine prevents hemosepticemia of pasteurellosis etiology, reduces the incidence of pneumonia by 50-80%, increases the average daily weight gain by 20.0-30.0 g. The proposed vaccine will be widely used in pig farms that are unfavorable for pasteurellosis.

- Animal pasteurellosis vaccine [patent RU 2311199 C1 publ. 2006.07.06]. The vaccine includes an inactivated backsuspension of Pasteurella multocida serological variants A, B, D and an adjuvant. At the same time, P. multocida serological variants A, B, D are used as P. multocida serological variants A, B, D with the activity of soluble surface antigens in the passive hemagglutination reaction as 1:256-4096, 1:512-8192, 1:256 -4096, respectively, in the following ratio, wt. %: Pasteurella multocida serological variants A, B, D, taken in mass ratios 1:(0.8-1.2):(0.8-1.2) - 45-55, adjuvant - the rest. The vaccine has a high activity and a long shelf life.

- Vaccine against pasteurellosis of animals [patent RU 2414929 C1 publ. 2011.03.27]. The vaccine includes an inactivated backsuspension of Pasteurella multocida serological variants A, B, D and an adjuvant. The vaccine additionally contains an inactivated backsuspension of Pasteurella haemolytica with the activity of soluble surface antigens in the passive hemagglutination reaction 1:32-64, and as an adjuvant, an ISA-70VG adjuvant in the following ratio, wt. %: inactivated backsuspension of Pasteurella multocida serological variants A, B, D and inactivated backsuspension of Pasteurella haemolytica, taken in mass ratios 1: (0.8-1.2): (0.8-1.2): (0.8- 1,2)-45-55, adjuvant ISA-70VG - the rest. The vaccine has a high immunological activity and a long shelf life.

A significant disadvantage of the above inventions is the limited antigenic composition in relation to pathogens of the etiology of atrophic rhinitis. The preparations do not contain Bordetella bronchiseptica antigens.

- Recombinant vaccine for preventing and treating porcine atrophic rhinitis (Recombinant vaccine for the prevention and treatment of atrophic rhinitis of pigs) [patent KR 100841207 B1 publ. 2018.06.24]. The peptide vaccine using the outer membrane protein H(OmpH) of Pasteurella multocida is designed to prepare a vaccine that shows excellent activity and is applicable in the body in various ways. The vaccine for the prevention and treatment of porcine atrophic rhinitis consists of a total or part of the recombinant outer membrane protein H of the Pasteurella multocida strain (D: 4), expressed from a recombinant expression vector, including a gene having at least one sequence encoding the total or part of the OmpH of the Pasteurella multocida strain (D:4) or a pharmaceutically acceptable carrier. The disadvantage of this invention, in relation to the newly proposed one, is the antigenic composition, which does not allow to fully prevent infectious atrophic rhinitis and pasteurellosis of pigs. So, the vaccine contains only Pasteurella multocida type D, but not type A, and there is also no Bordetella bronchiseptica.

- Killed vaccine for infectious porcine atrophic rhinitis and process for preparing the same 03/25/1975]. A killed vaccine against porcine infectious atrophic rhinitis, which is prepared from phase I Bordetella bronchiseptica, which is a gram-negative microbe with a K antigen and an antigen capable of providing mice with protective protection against intracerebral infection. This phase is characterized by a coccoid and coccobacillary morphological form, which has a capsule and/or membrane, but does not have flagella. The disadvantage of this invention, in relation to the newly proposed one, is an inferior antigenic composition for the specific prevention of IAR in pigs. The vaccine contains only Bordetella bronchiseptica., but not Pasteurella multocida type A and type D, which plays an important role in the pathogenesis of the disease.

In addition to vaccines, the following methods for their preparation are known:

- Method of preparation of anti-pasteurellosis vaccine [author's certificate SU 1839092 A1 publ. 1993.12.20]. The essence of this invention is to increase the immunogenicity of drugs, reduce their viscosity and reactogenicity. The problem is solved by using a certain ratio of the oil base - polyethylsiloxane liquid, and the emulsifier, during the assembly of the drug series. The disadvantage of the proposed method should be considered the sequence of its implementation, so according to the invention, the concentration of hydrogen ions is determined immediately after the cultivation of Pasteurella, and is not controlled during its implementation. In the case of maintaining a certain pH level during cultivation, it is possible to obtain a greater concentration of bacterial cells than under uncontrolled conditions. The next disadvantage of this method should be considered that the inactivation is carried out after the concentration of antigens, and not before. The concentration of non-inactivated culture fluid has its own risks, in particular, contamination of equipment, risks of human infection, risks of non-inactivated Pasteurella cells and its toxins getting into the sewer, risks of contamination of antigenic raw materials, etc.

- A method of manufacturing a vaccine against pasteurellosis of farm animals and birds [patent RU 2129015 C1 publ. 1999.04.20]. The disadvantage of this method is the high consumption of nutrient media during continuous-flow cultivation of Pasteurella. In addition, in this development there is no stage of concentrating pasteurellous antigens necessary to obtain inactivated vaccines, as a result of which the proteins of the nutrient medium enter the preparation with the necessary bacterial antigens. In the case of oral or aerosol use of live pasteurellosis vaccines, the presence of ballast residues of nutrient media in them most often does not contribute to any reactogenic reactions. In the case of the injection use of these preparations containing nutrient residues, this can affect both the safety and the level of the antigenic response, since the substances that make up the nutrient media are proteins that antibodies will be formed against.

- Method for the manufacture of 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 food 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 with better growth-producing properties, for example, heart-brain broth, tryptone-soy broth, eugonic broth, etc. In addition, culture media made from meat waste are more difficult to standardize than those made from standard raw materials.

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

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

- A method of obtaining a vaccine against pasteurellosis in animals [patent RU 2405567 C1 publ. 2010.12.10]; and also - A method for obtaining a vaccine against pasteurellosis in animals [patent RU 2310473 C1 publ. 2007.11.20]. The disadvantage of the proposed compositions and methods for their preparation is the excessive antigenic composition for different animal species. The use of the preparation made by the proposed method for pigs is not justified, since there is no evidence base for the susceptibility of pigs to the disease Mannheimia haemolytica.

- A method of manufacturing a vaccine preparation against infectious diseases of bacterial etiology of animals and birds [patent RU 2173560 C1 publ. 2001.09.20]. The disadvantage of this method is the cultivation of microorganisms in a medium based on expensive and scarce food protein - meat hydrolyzate according to Hottinger; preparations made using this hydrolyzate do not have sufficient stability, are non-standard, and the cost of their production is high.

- A method of obtaining an inactivated vaccine against infectious atrophic rhinitis of pigs [patent SU 403134 A1 publ. 1973.10.19]. The disadvantage of this invention is the limited antigenic composition in relation to pathogens of the etiology of atrophic rhinitis. The method for obtaining the vaccine does not contain the technology for obtaining Pasteurella multocida antigens.

According to the above characteristics, for vaccines existing and / or previously used in the territory of the Russian Federation, the drug Donoban-10 is a polyvalent inactivated vaccine against respiratory diseases of pigs [link on the network [http://www.kbnp.net/_mobile/sub02_1_l.html?number =721&xcode=&mcode=02&scode=01&page=2] is the closest analogue (prototype) of the proposed vaccine. The vaccine is made from inactivated bacterial cells of Bordetella bronchiseptica (ARDNT /SB-11 strain), Pasteurella multocida type A (NSPA /SB-40 strain) and Pasteurella multocida type D (NSA2 / SB-03 strain) and Actinobacillus pleuropneumoniae serotype 5 ( strain NSA5 /SB-04), mycoplasma Mycoplasma hyopneumoniae (strain NSM /SB-34), streptococcus Streptococcus suis type 2 (strain NSS2 /SB-50), haemophilus strains Haemophilus parasuis serotype 1 (strain NSH1 /SB-3O), Haemophilus parasuis serotype 4 (strain NSH4 /SB-31) and Haemophilus parasuis serotype 5 (strain NSH5 /SB-32), as well as dermonecrotic toxin (cDNT) Bordetella bronchiseptica and Pasteurella multocida type D; cell wall protein (CMP) Pasteurella multocida type A; Actinobacillus pleuropneumonia 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 IMS 1313 (10%) up to 2 ml .

One immunizing dose of the vaccine (2 ml) contains: inactivated microbial cells of Bordetella bronchiseptica strains - at least 4 * 10 ⁹ CFU, Pasteurella multocida type A and D - at least 2 * 10 ⁹ CFU each, actinobacillus Actinobacillus pleuropneumoniae serotype 2 and 5 - not less than 2*10 ⁹ CFU, mycoplasma Mycoplasma hyopneumoniae - not less than 2*10 ⁸ CFU, streptococcus Streptococcus suis, type 2 - not less than 42*10 ⁹ CFU, hemophilus Haemophilus parasuis, serotypes 1, 4, 5 - not less than 2* 10 ⁹ CFU each, as well as dermonecrotic toxin (cDNT) of toxigenic strains of Bordetella bronchiseptica and Pasteurella multocida type D - at least 400 μg, cell wall protein (OMR) of toxigenic strains of Pasteurella multocida type A - at least 60 μg, Actinobacillus pleuropneumoniae serotype 2 and 5 - at least 20 mcg of each. The vaccine causes the formation of an immune response to pathogens of respiratory diseases of pigs (atrophic rhinitis, pasteurellosis, actinobacillus pleuropneumonia, hemophilic polyserositis, streptococcosis and infection caused by Mycoplasma hyopneumoniae), 21 days after a double application, lasting 6 months.

The method of obtaining an inactivated vaccine against infectious atrophic rhinitis of pigs [patent SU 403134 A1 publ. 1973.10.19], is the closest prototype for the newly proposed invention. The disadvantage of the prototype should be considered the fact that the method is focused on obtaining the drug from the antigens of Bordetella bronchiseptica, and does not disclose the essence and features of obtaining associated drugs against IAR, taking into account all the antigens necessary for the full immunoprophylaxis of animals, in particular Pasteurella multocida.

The technical problem to be solved by this group of inventions is the need to create a safe polyvalent vaccine against infectious atrophic rhinitis and pasteurellosis of pigs, based on a balanced antigenic composition containing the most common bacterial serotypes of Bordetella bronchiseptica, Pasteurella multocida species in the Russian Federation and the need to expand the arsenal inactivated vaccines aimed at the specific prevention of infectious rhinitis and pasteurellosis of pigs.

Disclosure of the essence of the invention

The technical result of the proposed group of inventions is to increase the stability, safety, antigenic and immunogenic activity of the vaccine, providing long-term and intense immunity in vaccinated pigs against infectious atrophic rhinitis and pasteurellosis, expanding the arsenal of polyvalent inactivated vaccines aimed at the specific prevention of infectious diseases of pigs, creating a vaccine with increased stability, antigenic and immunogenic activity, expansion of approaches and methods for the manufacture of polyvalent inactivated vaccines aimed at the specific prevention of infectious diseases in pigs.

Vaccine against infectious atrophic rhinitis and pasteurellosis of pigs inactivated

The vaccine against infectious atrophic rhinitis and pasteurellosis of pigs contains the following components per 2 cm ^{3 of the} preparation (immunizing dose): formalin-inactivated strain Bordetella bronchiseptica B-1341 - 6 billion mcr. class; Pasteurella multocida B-1308 type D - 3 billion mkr. class; Pasteurella multocida B-1303 type A - 3 billion mkr. class, as well as pharmaceutically acceptable target additives.

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

- An inactivant needed to inactivate antigens and their toxins. For this, formalin was used at the rate of 0.3% by volume of the 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 10% is used, introduced into the preparation at the rate of 1:10,000 (1 part of sodium merthiolate and 10,000 parts of the finished product);

- The diluent required to dilute the concentrated bacterial antigen to the required concentration and 100% volume of the composable batch of the drug (according to 61-FZ "On the circulation of medicines", the batch of the drug is the amount of the drug produced as a result of one technological cycle by its manufacturer) . As standard solvents can be used - sterile saline, sterile PBS - phosphate buffered saline, water for injection;

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

- 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 7.2-7.6 for the vaccine variant made on GOA and Carbomer 971r. The pH level of the drug, made on an oil adjuvant, is not additionally regulated.

Depending on the adjuvant used, the appearance of the vaccine may have the following variations:

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

- When using oil adjuvants - in appearance, the vaccine is a white emulsion, sometimes with a yellowish tinge. During long-term storage, a slight stratification of the drug is possible, which, when shaken, goes into the initial state of a homogeneous emulsion.

Shelf life of the vaccine is 12 months from the date of issue, subject to appropriate storage conditions at a temperature of 2-8°C.

The vaccine is intended for the prevention of infectious atrophic rhinitis and pasteurellosis in pigs. The vaccine is administered to pregnant sows no later than 14 days before farrowing. The vaccine causes the formation of immunity to Bordetella bronchiseptica, Pasteurella multocida type A and D 14 days after repeated administration, which persists for at least 6 months. Colostral immunity in piglets obtained from immunized animals lasts up to 20 days.

The bacterial strains Bordetella bronchiseptica B-1341 and Pasteurella multocida B-1308 type D included in the vaccine are new production cultures and are used for the first time as part 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. The Pasteurella multocida B-1303 strain is known from the patent for invention No. RU 2744744 C1 publ. 03/15/2021.

All strains of bacteria used in the framework of the invention are deposited in the All-Russian Collection of Pathogenic and Vaccine Strains of Microorganisms Causing Infectious Animal Diseases of the Federal State Budget Scientific Institution Federal Scientific Center - All-Russian Research Institute of Experimental Veterinary Medicine named after V.I. K.I. Scriabin and Ya.R. Kovalenko of the Russian Academy of Sciences. All cultures were identified on the basis of the laboratory of microbiology with the museum of type cultures of the Federal State Budgetary Scientific Institution of the Federal Scientific Center of VIEV RAS, by studying the cultural, morphological, enzymatic properties and determining the nucleotide sequence of the 16S rRNA gene.

Characterization of industrial strains of Bordetella bronchiseptica, Pasteurella multocida

1) The production strain of Bordetella bronchiseptica No. 134-VIEV is characterized by the following features and properties:

Strain number in the All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms Causing Infectious Animal Diseases: B-1341

Pathogenicity: in accordance with the classification of microorganisms - pathogens of human infectious diseases, protozoa, helminths and poisons of biological origin according to pathogenicity groups of sanitary rules 1.3.2322-08 "Safety of working with microorganisms of III-IV groups of pathogenicity (danger) and pathogens of parasitic diseases" bacterial agents species Bordetella bronchiseptica belong to the fourth hazard group.

Date, source and place of isolation: pig lung. Pig-breeding enterprise of the Sverdlovsk region. 2018 year.

Where the culture is identified: Laboratory of Mycology and Antibiotics named after. OH. Sarkisov Federal State Budgetary Scientific Institution FNTs VIEV RAS.

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

Morphological features: small gram-negative rods with an average size of 0.2-0.5 * 1.0-2.0 μm, located singly in smears, sometimes in pairs, do not form spores, but form a microcapsule. Threads and other involutional forms are absent.

Cultural features: on Borde-Gangu medium, the microorganism forms smooth, convex, pearly, shiny colonies. May show slight hemolysis on blood media (Columbian agar base, MPA supplemented with 10% defibrinated sheep blood). The growth range is 25-42°C, but the optimum cultivation temperature is 37°C. Strict aerob. On MacConkey agar, the microorganism forms reddish colonies with a small discolored halo around them. Pigment does not form on any of the media. When grown on Bordetel agar medium, the colonies are round, convex, smooth, shiny with smooth edges, cream-colored, have a soft, oily consistency and are easily removed. The terms of the appearance of colonies - after 18-24 hours. The diameter of the colonies is 0.5-1 mm.

Biochemical properties: characteristic of Bordetella bronchiseptica: arginine -; aliphatic thiol -; triglyceride+; ρ-nitrophenyl-phosphate+; p-nitrophenyl-p-acetyl - b, d-glucosaminide+; ρ-nitrophenyl - a, d-glucoside+; ρ-nitrophenyl - b, d-glucoside+; ρ-nitrophenyl b, d-galactoside+; urease+; glucose+; xylose -; mannitol -; lactose -; sucrose -; maltose -; esculin -; malonate -; succinate -; glycerol -; inositol -; sorbitol -; trehalose -; proline-b-naphthylamide+; pyrrolidine-b-naphthylamide+; g-glutamyl-b-naphthylamide+; tryptophan-b-naphthylamide+; p-benzyl-arginine-b-naphthylamide+; tryptophan -; sodium nitrate+; catalase+; oxidase+; citrate+; indole -.

Virulence for laboratory animals: the value of LD ₅₀ - 4.6×10 ⁷ mcr. class The death of animals occurs no later than 24 hours after infection.

Cultivation conditions: the microorganism grows on most broth media used in bioproduction, in particular on Hottinger broth, heart-brain broth, trypton soy broth, etc., as well as on dense nutrient media: blood agar, MPA, Bordetel-agar , Borde-Jangu with the addition of 10% defibrinated sheep blood.

2) The production strain of Pasteurella multocida No. 1042-VIEV is characterized by the following features and properties:

Strain number in the All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms Causing Infectious Animal Diseases: B-1303

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

Date, source and place of isolation: from the lungs of a dead sheep. Sheep-breeding enterprise of the Kursk region. 2018

Where the culture was identified: the laboratory of microbiology with the museum of type cultures of the Federal State Budget Scientific Institution of the Federal Scientific Center of the VIEV RAS and on the basis of the FBSI MNIIEM. G.N. Gabrichevsky.

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

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

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

Biochemical properties: characteristic of Pasteurella multocida. The strain ferments glucose, sucrose, galactose, mannitol, sorbitol with the formation of acid without gas. Does not ferment dulcite, arabinose, maltose, raffinose, lactose. Oxidazo- and catalase-positive culture. Do not coagulate milk and do not liquefy gelatin. They form indole and hydrogen sulfide.

Serological properties: type A.

Virulence for laboratory animals: the value of LD ₅₀ - 20±4 mcr. class for white mice, with intraperitoneal infection.

Culture Conditions: The microorganism grows on most broth media used in biomanufacturing, such as Hottinger Broth, Brain Heart Broth, Tryptone Soy Broth, etc. MPB, MPA, broth and Hottinger agar with the addition of 5%-10% sterile defibrinated sheep blood, M-144 (HiMedia) at pH-7.2 with a temperature of 37°C for 18-24 hours.

3) The production strain of Pasteurella multocida No. 820-VIEV is characterized by the following features and properties:

Strain number in the "All-Russian State Collection of Pathogenic and Vaccine Strains of Microorganisms Causing Infectious Animal Diseases": B -1308

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

Date, source and place of isolation: from the lungs of a dead piglet. Belgorod region, 2018.

Where the culture was identified: laboratory of microbiology with a museum of type cultures of the Federal State Budgetary Scientific Institution of the Federal Scientific Center of VIEV RAS.

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

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

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

Biochemical properties: characteristic of Pasteurella multocida. The strain ferments glucose, sucrose, galactose, mannitol, sorbitol with the formation of acid without gas. Does not ferment dulcite, arabinose, maltose, raffinose, lactose. Oxidazo- and catalase-positive culture. Do not coagulate milk and do not liquefy gelatin. They form indole and hydrogen sulfide.

Serological properties: type D.

Virulence for laboratory animals: the value of LD ₅₀ - 5.8*10 ⁷ mcr. class for white mice, with intraperitoneal infection.

Culture Conditions: The microorganism grows on most broth media used in biomanufacturing, such as Hottinger Broth, Brain Heart Broth, Tryptone Soy Broth, etc. MPB, MPA, broth and Hottinger agar with the addition of 5%-10% sterile defibrinated sheep blood, M-144 (HiMedia) at pH-7.2 with a temperature of 37°C for 18-24 hours.

A method for obtaining a vaccine against infectious atrophic rhinitis and pasteurellosis of pigs.

Obtaining the claimed drug involves the implementation of the following successive steps:

1) Cultivation of industrial strains by the deep method;

2) Inactivation of the culture fluid;

3) Concentration of antigens;

4) Determination of the completeness of inactivation of antigens;

5) Drawing up a series of vaccines;

6) Vaccine sterility control;

7) Assessment of the safety of the vaccine.

For deep cultivation of Bordetella bronchiseptica and Pasteurella multocida species in the production of a vaccine, trypticase-soy broth, tryptone-soy broth, eugonic broth, heart-brain broth, Hottinger broth, and other media with the necessary growth-promoting properties can be used.

The claimed method uses the following production strains of microorganisms: Bordetella bronchiseptica B-1341; Pasteurella multocida B-1303 type A; Pasteurella multocida B-1308 type D.

The sequence of preparation of uterine brood strains of Bordetella bronchiseptica and Pasteurella multocida from MSB (Master Seed Bacteria - the main inoculum):

- on the first day, the first generation of production strains is prepared by resuspension of the lyophilized MSB culture in a test tube, followed by inoculation on nutrient agar. Each strain is prepared separately. Cultivation of the first generation continues for 24-36 hours under aerobic conditions at a temperature of 37°C;

- after 24-36 hours, the first generation is checked for the typicality of the culture in terms of morphological, tinctorial and growth properties, as well as for the absence of foreign microflora, both on a Petri dish and in a test tube. If the first generation corresponds to all the indicated properties, the second generation is obtained. To do this, individual colonies from a Petri dish are inoculated into test tubes with fresh nutrient medium. The resulting crops are cultivated for 24-36 hours under aerobic conditions at a temperature of 37°C. To obtain the maximum accumulation of bacterial cells in the broth medium, the test tubes are placed on a shaker with constant stirring;

- on the third - fourth day, the second generation is monitored for the typicality of the culture in terms of morphological, tinctorial and growth properties, as well as for the absence of foreign microflora, both on a Petri dish and in a test tube. If the second generation corresponds to all the indicated properties, the third generation is obtained. To do this, the broth culture from test tubes is inoculated into vials with fresh nutrient medium (at least 100 ml). The resulting crops are cultivated for 24-36 hours under aerobic conditions at a temperature of 37°C. To obtain the maximum accumulation of bacterial cells in the broth medium, the vials are placed on a shaker, at 50-150 rpm, for constant stirring;

- on the fourth or fifth day, after preliminary control of the brood, the entire volume of the vial is reseeded into bottles with a nutrient medium, which will subsequently be used for seeding in reactors. The mother culture is prepared at the rate of 10% of the volume of the nutrient medium in the reactor. Cultivation is carried out for 24-36 hours under aerobic conditions at a temperature of 37°C;

- on the fifth or sixth day, the production strains are directly cultivated in the reactor. The cultivation of strains of Bordetella bronchiseptica and Pasteurella multocida is carried out at a pH of 7.2-7.8, at a temperature of 37°C.

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

Each strain of microorganisms is cultivated separately.

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

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

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

Compilation of a series of vaccines.

For the production of the proposed vaccine can be used in various adjuvants, in view of this, the stages of drawing up a series of vaccines may vary. Series options are listed below. Calculations are given for the production of a series of vaccines with a volume of 300 liters (150 thousand doses of the vaccine).

Option number 1. - layout of the vaccine using aluminum hydroxide or carbomer 971r.

In a sterile reactor, inactivated bacterial antigens of three production cultures (at a previously established concentration) are mixed with a diluent, adjuvant (10%) and sodium merthiolate, so that the concentration of Bordetella bronchiseptica bacterial antigens is equal to 3 billion mcr. cells/cm ³ , and the concentration of Pasteurella multocida type A and D antigens is 1.5 billion mc. cells / cm ³ . With constant stirring, a homogeneous suspension is obtained, the required level of hydrogen ions is set (7.2-7.6). The formulation of the composition of the vaccine when using this layout option is shown in table No. 3.

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

In a sterile reactor, the inactivated bacterial antigens of three production cultures are mixed at a previously established concentration with a diluent, an adjuvant (50%), and sodium merthiolate, so that the concentration of Bordetella bronchiseptica bacterial antigens is equal to 3 billion mcr. cells/cm ³ , and the concentration of Pasteurella multocida type A and D antigens is 1.5 billion mc. cells / cm ³ . Obtaining a homogeneous emulsion is carried out using a dispersing machine at 6000-8000 rpm. The resulting vaccine batch has an average pH of 6.0. The recipe for the composition of the vaccine when using this layout option is shown in table No. 4.

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

The assessment of the safety of the vaccine is carried out in a bioassay on laboratory white mice weighing 16-18 g. with a single subcutaneous or intramuscular injection of the drug at a dose of 0.5 cm ³ . Five experimental animals are used to evaluate the safety of each vaccine lot. Mice were observed for 14 days. The vaccine is considered harmless if all animals remain alive during the observation period.

Implementation of the invention

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

Example #1. Cultivation of strains of Bordetella bronchiseptica and Pasteurella multocida by the deep method.

Cultivation of strains of Bordetella bronchiseptica and Pasteurella multocida was carried out using a BIOSTAT-A reactor (Sartorius AG, Germany) on trypton-soy broth, heart-brain and eugonic broths (each strain was sequentially cultivated on all the given media). The preparation of the main inoculum was carried out according to the following procedure: on the first day, the first generation of production strains was obtained by resuspension of the lyophilized MSB culture in a test tube, followed by inoculation on nutrient agar. Each strain was prepared separately. Cultivation of the first generation of cultures continued for 20 hours under aerobic conditions at a temperature of 37°C; on the second day, after confirming the properties of production cultures, individual colonies from Petri dishes were transferred to test tubes with nutrient broth (10 ml each), for subsequent cultivation for 20 hours under aerobic conditions at a temperature of 37°C, with constant stirring on a shaker (100 rpm); on the third day, after controlling the properties of the grown cultures, the broth culture from the test tubes was subcultured into vials with heart-brain and/or tryptone-soy broth (1 test tube per 250 ml of medium). The obtained crops were cultivated for 18 hours under aerobic conditions at a temperature of 37°C, with constant stirring on a shaker (100 rpm); on the fourth day, after a preliminary control of the brood, the entire volume of the vial was reseeded into the BIOSTAT-A reactor containing 5 liters of nutrient medium. At the same time, the amount of mat brood for reactor cultivation was 10% of 5 liters, i.e. 500 ml. The cultivation of strains of Bordetella bronchiseptica and Pasteurella multocida was carried out at a pH of 7.2-7.8, at a temperature of 37°C. To maintain the required level of nutrients in the medium, a 40% glucose solution was added at the rate of 10 ml of glucose per 1 liter of nutrient medium 1 time per hour, depending on the intensity of culture growth. The pH level during cultivation is maintained with 10% alkali solution (NaOH).

As a result of cultivation on various nutrient media, the results were obtained, reflected in table No. 5.

The results reflected in table No. 5 indicate the interchangeability of nutrient media for deep cultivation of strains of Bordetella bronchiseptica and Pasteurella multocida.

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

Example #2. Culture fluid inactivation

Inactivation of broth cultures of Bordetella bronchiseptica and Pasteurella multocida strains (example No. 1) was carried out by adding formalin (containing at least 37% formaldehyde) to them, in an amount of 0.3% of the volume of the culture liquid. So, the introduced volume of formalin in each batch of culture liquid is reflected in table No. 6.

Example #3. Concentration of culture liquid

The concentration of the obtained broth culture after inactivation (example No. 5) was carried out by centrifugation on the MPW-380R equipment for 1 hour at 3 thousand revolutions, RCF - 1861. After centrifugation and separation of the supernatant (fugate), the bacterial mass was collected in a separate sterile container ( vial) for further use. At this stage, the concentration of the bacterial mass and its volume were determined. The determination of the suspension concentration was carried out using turbidity standards - a set of RSD for turbidity of bacterial suspensions of the GKPM FGBU "NTsESMP" of the Ministry of Health of Russia. The results of concentration are shown in table No. 7.

According to the data reflected in the table J№/, the culture liquid of the oil is concentrated on average 10 times.

Example number 4. Determination of the completeness of antigen inactivation

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

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

Example number 5. Compilation of series No. 1 of the vaccine against infectious atrophic rhinitis and pasteurellosis of pigs using saline as a diluent and GOA as an adjuvant.

When compiling a vaccine series, inactivated bacterial antigens of three production cultures were mixed in a sterile container with a diluent (saline solution), an adjuvant (3% aluminum hydroxide solution) and a preservative (sodium merthiolate), setting the concentration of Bordetella bronchiseptica bacterial antigens - 3 billion. md. cells / cm ³ , Pasteurella multocida type A and D, 1.5 billion mkr. cells / cm ³ . The components were mixed until a homogeneous suspension was obtained, the pH of the vaccine series was adjusted to 7.6. The formulation of the composition of the vaccine with this arrangement is shown in table No. 8. The composition is given on the basis of the volume of the produced series of 1.5 thousand doses (3 liters of the drug).

The vaccine thus obtained in appearance is a light yellow suspension with a gray-white precipitate, which breaks up into a homogeneous suspension when shaken.

Example number 6. Compilation of vaccine series #2 against porcine infectious atrophic rhinitis and pasteurellosis using water for injection as a diluent and carbomer 971P as an adjuvant

When compiling a vaccine series, inactivated bacterial antigens of three production cultures were mixed in a sterile container with water for injection as a diluent, a 2% solution of carbomer 971P as an adjuvant, and sodium merthiolate as a preservative, setting the concentration of bacterial antigens of Bordetella bronchiseptica - 3 billion md. cells / cm ³ , Pasteurella multocida type A and D, 1.5 billion mkr. cells / cm ³ . The components were mixed until a homogeneous suspension was obtained, after which the pH of the vaccine batch was adjusted to 7.6. The formulation of the composition of the vaccine when using this layout option is shown in table No. 9. The composition is given on the basis of the volume of the produced series of 1.5 thousand doses (3 liters of the drug).

The vaccine thus obtained in appearance is a light yellow suspension with a gray-white precipitate, which breaks up into a homogeneous suspension when shaken.

Example number 7. Compilation of batch #3 of porcine infectious atrophic rhinitis and pasteurellosis vaccine using phosphate buffered saline as diluent and Montanide ISA 70 oil adjuvant as adjuvant

A pre-emulsion is prepared in a sterile container. To do this, in a mixer with constant stirring (250 rpm for 1.5 hours), inactivated bacterial antigens of three production cultures were mixed based on 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 of Bordetella bronchiseptica - 3 billion microns. cells / cm ³ , Pasteurella multocida type A and D, 1.5 billion mkr. cells / cm ³ . To obtain the final version of the vaccine, a homogenizer of the Silverson L5M-A type was used. Emulsification is carried out for 3 hours at 6 thousand rpm. The formulation of the composition of the vaccine when using this layout option is shown in table No. 10. The composition is given based on the volume of the produced series of 1.5 thousand doses (3 liters of the drug).

The vaccine thus obtained in appearance is a white emulsion, sometimes with a yellowish tint. During long-term storage, a slight separation of the drug was observed, which, when shaken, goes into the initial state of a homogeneous emulsion.

Example number 8. Determining Vaccine Sterility

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

Example #9. Assessment of the safety of the vaccine

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

Example number 10. Determination of the safety of an experimental vaccine against atrophic rhinitis and pasteurellosis of pigs inactivated for pregnant sows.

For the experiment, two groups of pregnant sows were formed (6 weeks before farrowing), 10 heads each. Animals of the first group were injected with the drug at the recommended dose of 2 cm ³ , animals of the second group were immunized by administering a double dose of 4 cm ³ . All animals were clinically healthy and previously marked with special ear tags with individual numbers. The vaccine was administered according to the temporary instructions for use once, intramuscularly, in compliance with the rules of asepsis.

The vaccinated animals were observed for 10 days, conducting a daily clinical examination of the animals, thermometry, and palpation of the injection site.

During the clinical examination, the absence of any systemic and local undesirable consequences of the administration of the drug was noted: the animals did not show signs of depression, loss of appetite, adynamia, abortions. On palpation of the injection site, the formation of slight edema and hyperemia of the skin was noted, which persisted in animals for 12-24 hours, and then disappeared on their own. In some animals (~20%), the administration of the drug in the recommended and doubled doses caused a short-term increase in temperature, on average by 0.5-0.7°C, and in sows it was 38.3°C before vaccination, 6 hours after vaccination 38.9°C, which did not go beyond the physiological norm for animals of this age group. 24 hours after the injection, the body temperature of the animals returned to the original parameters.

The experience has shown that the proposed vaccine is safe for pregnant sows with a single intramuscular injection in the recommended and doubled doses.

Example number 11. Determination of the effectiveness of an experimental vaccine against atrophic rhinitis and pasteurellosis of pigs inactivated (a series of vaccines made on the basis of aluminum hydroxide).

To evaluate the effectiveness of the developed vaccine against porcine atrophic rhinitis and pasteurellosis, two groups of pregnant sows were formed, one of which, in the amount of 246 heads, was immunized with the drug previously used at the enterprise - Inactivated Porcilis AR-T DF vaccine against porcine atrophic rhinitis DF, MSD Animal Health. The second group of sows in the amount of 114 heads was immunized with the inactivated vaccine against atrophic rhinitis of pigs, developed by the Federal State Budget Scientific Institution of the Federal Scientific Center of the VIEV RAS.

The evaluation criterion was the absence of cases of disease in piglets obtained from immunized sows, atrophic rhinitis, as well as the identification of cases of undesirable local and / or systemic reactions in sows to the administration of drugs. The conditions of keeping and feeding experimental sows were similar. The control (non-vaccinated) group was not used to avoid the risk of disease outbreaks in pigs.

Immunization of sows was carried out in accordance with the instructions for the use of drugs. The vaccines were administered to sows twice intramuscularly behind the ear in compliance with the rules of asepsis at a dose of 2 ml. The results of assessing the safety of vaccines are presented in Table 11.

As can be seen from the data presented in Table 11, in the experimental groups there were no cases of death of animals, no cases of illness, abortions and birth of piglets with congenital deformities, both in the experimental group immunized with the developed vaccine, and in the group of sows immunized with the reference drug, which indicates the safety of the experimental vaccine.

The evaluation of the effectiveness of the vaccination was assessed by the safety of piglets obtained from sows vaccinated with the experimental vaccine and the reference drug, the average daily gain and the average live weight of the piglets when they were transferred to fattening at 102 days of age.

The results of the experiment are presented in table No. 12.

The data reflected in table No. 12 indicate that the use of an experimental vaccine against atrophic rhinitis and pasteurellosis of pigs inactivated ensures 97.16% safety of piglets, which is commensurate with the same indicator in groups of animals immunized with the reference drug - 96.96%.

To confirm the specific efficacy of the vaccine, all dead animals were opened to determine the causes of death on the basis of the available pathological signs, as well as from animals that had any signs of an infectious disease, material was taken for bacteriological studies.

It was established that the cause of death of animals during the experiment was traumatism, streptococcosis, actinobacillus pleuropneumonia, as well as a number of other causes that occurred in isolated cases.

As a result of bacteriological studies, pathogens of atrophic rhinitis (Pasteurella and Bordetella) were not identified. There were no cases of clinically pronounced disease in piglets with damage to the bones of the facial part of the skull in the experimental groups.

Reliable data were also obtained on the impact of the vaccination on economic indicators, in particular, the average daily weight gain in piglets. So in the group of animals immunized with the test drug, the average daily weight gain was 0.887, while in the comparison group it was 0.854 kg, which is 33 g less when recalculated for each animal.

Thus, it can be argued that the developed experimental vaccine against porcine atrophic rhinitis, inactivated, provides a reliable level of protection in the vaccinated livestock, which has a positive effect on veterinary and economic indicators, and also reduces the level of carriage of the pathogen.

Claims (7)

1. Vaccine against infectious atrophic rhinitis and pasteurellosis of pigs, containing in its composition the following components per 2 cm ^{3 of the} drug (immunizing dose): formalin-inactivated protective antigen of the strain Bordetella bronchiseptica B-1341 - 6 billion micro cells; Pasteurella multocida B-1308 type D - 3 billion micro cells; Pasteurella multocida B-1303 type A - 3 billion micro cells, as well as pharmaceutically acceptable targeted additives. 2. The vaccine according to claim 1, characterized in that an inactivator in the form of formalin is used as pharmaceutically acceptable target additives at the rate of 0.3% by volume of the broth culture, containing at least 37% formaldehyde. 3. The vaccine according to claim 1, characterized in that as pharmaceutically acceptable target additives, a preservative is used in the form of an aqueous solution of sodium merthiolate 10%, introduced into the preparation at the rate of 1:10,000 (1 part of sodium merthiolate and 10,000 parts of the finished product). 4. The vaccine according to claim 1, characterized in that as pharmaceutically acceptable target additives, a diluent is used in the form of sterile saline, sterile PBS - phosphate-buffered saline, water for injection in the amount necessary to obtain 100% of the volume of the drug. 5. The vaccine according to claim 1, characterized in that it contains an oil adjuvant in an amount of 50% of the total volume as pharmaceutically acceptable target additives. 6. The vaccine according to claim 1, characterized in that it contains one of the following adjuvants as pharmaceutically acceptable target additives with the addition of 0.2% of the total volume of 10% sodium hydroxide solution: 3% aluminum hydroxide solution in an amount of 10% of the total volume , Carbomer 971r 2% solution in PBS in the amount of 10% of the total volume. 7. A method for obtaining a vaccine according to claim 1, including cultivating production strains, inactivating cultures, concentrating antigens, determining the completeness of antigen inactivation, drawing up a series of vaccines, controlling vaccine sterility, assessing the safety of the vaccine, characterized in that strains of Bordetella bronchiseptica are used as production strains B-1341; Pasteurella multocida B-1303 type A; Pasteurella multocida B-1308 type D.