RU2741643C1 - Associated vaccine against myxomatosis, pasteurellosis and viral haemorrhagic disease 1 and type 2 of rabbits - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a vaccine associated with myxomatosis, pasteurellosis and rabbit viral haemorrhagic disease containing two components as an active substance: a liquid component - a mixture of inactivated antigenic material from strain No. 1231 Pasteurella multocida, deposited under registration number: No.307-dep/20-95 POIM FGBU “VNIIZZh”; of inactivated and purified antigenic materials of strain “VGBK1/VNIIZZh” type 1, deposited under registration number: No.305-dep/20-93-GKShM FGBU “VNIIZZh”, and from “VGBK 2/VNIIZZh” strain, deposited under registration number: No.306-dep/20-94-GKShM FGBU “VNIIZZh”; and a dry component - lyophilised attenuated purified antigenic material from “Mixo/VNIIZZh-18” strain, deposited under registration number: No.120 -dep/19-10-KShM FGBU “VNIIZZh”; and target additives: 3% aluminum hydroxide gel and stabilizing medium, taken in volume ratio of 1.0:0.5:0.5:0.9:2.0:0.1, respectively, and in amounts providing protective immunogenic activity of each antigen in rabbits body.

EFFECT: invention widens the range of high-immunogenic, harmless and areactogenic associated vaccines against infectious agents of rabbits circulating in the territory of the Russian Federation.

8 cl, 4 tbl, 6 ex, 3 dwg

Description

The invention relates to the field of biotechnology, relates to an associated vaccine that can be used for the specific prophylaxis of rabbit diseases induced by viruses of viral hemorrhagic disease types 1 and 2, myxomatosis and pasteurellosis bacteria.

Rabbit farming is a livestock industry that produces valuable products such as dietary meat, skin and fur. Among the viral diseases of rabbits, which are currently relevant and cause large economic losses, myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits should be distinguished.

Myxomatosis is an acute highly contagious viral disease of rabbits, characterized by inflammation of the mucous membranes, serous or serous-purulent conjunctivitis, the formation of gelatinous edema (mix) in the head and other parts of the animal's body. The disease is caused by a DNA-containing virus belonging to the Poxviridae family, genus Leporipoxvirus [1, 2].

Pasteurellosis is an acute highly contagious infectious disease of rabbits, characterized by various clinical signs: rhinitis, conjunctivitis, otitis media, abscesses, bronchopneumonia. The acute course of the disease is most often caused by various serovars of P. multocida belonging to serogroup A, the chronic form of the disease is caused by serogroups A and D in association. P. multocida is divided into 5 capsular serogroups (A, B, D, E, and F) and 16 somatic antigen serovariants. In rabbits, the disease is caused by Pasteurella serogroups A, B, and D for the capsular antigen [3, 4].

Rabbit viral hemorrhagic disease (VGBD) is an acute highly contagious viral disease of rabbits, characterized by the phenomena of hemorrhagic diathesis in various organs, mainly in the liver, lungs and kidneys. The disease is caused by an RNA virus belonging to the Caliciviridae family, genus Lagovirus. The disease proceeds mainly without clinical signs; depression of animals, decreased appetite can be observed. With HBV, hemorrhagic inflammation of almost all organs occurs and there is a high mortality rate in animals [5].

Myxomatosis, pasteurellosis and viral haemorrhagic disease of rabbits cause significant economic damage to rabbit breeding in the form of deaths, the introduction of quarantine measures, a sharp drop in productivity, a decrease in the quality of products obtained, and a decrease in the increase in livestock.

In the acute form, myxomatosis in rabbits proceeds rapidly and ends with a 95-100% lethal outcome [6, 7]. In this regard, when myxomatosis occurs, rabbit farms incur huge economic damage in the form of mass death of animals, the introduction of quarantine measures and a sharp drop in productivity. According to the OIE, over the past three years, an outbreak of myxomatosis was recorded in the Kirov region (2018).

Also recently, viral hemorrhagic disease of rabbits has become a serious problem for rabbit breeding - a highly contagious, acute infection, accompanied by symptoms of hemorrhagic diathesis in various organs, especially in the liver and lungs [8, 9, 10, 11].

Both diseases owe their spread across continents to humans and are historically interrelated, since their pathogens were consistently cultivated by Australian farmers in order to reduce the rabbit population, which is an ecological problem of local agriculture [9, 12, 13, 14, 15].

The prevalence of HBV morbidity is very high. According to the OIE, outbreaks of HBV have been observed over the past three years in Sweden (2017), Finland (2017-2019), Kazakhstan (2017), Israel (2017-2018), USA (2018-2019), New Zealand (2018), Canada ( 2018-2019), Norway (2019), Ireland (2019). In 2019, countries such as New Zealand, Denmark, Canada, Norway, the United States and Finland are unfavorable in terms of VGBK. In Russia, over the past three years, VGBV outbreaks have been recorded in the Trans-Baikal Territory (2017), the Moscow Region (2018), the Tver Region (2018), and the Krasnoyarsk Territory (2019) [2, 8].

Myxomatosis and viral hemorrhagic disease of rabbits cause great economic damage to rabbit breeding - the mortality rate is on average 70-90% of the total livestock [6, 7, 16, 17]. The main routes of infection are alimentary and respiratory. Sources of infection are sick, recovered and dead rabbits. The factors of transmission are fodder contaminated with the pathogen, objects of the environment, including soil, water, surfaces of premises, equipment, transport and technical means, people [6, 8, 18, 19].

The emergence of a new HBV serotype is of great concern. This serotype differs from the previous one in that young animals less than 2 weeks of age are also susceptible to it. Another feature of the pathogen is that it overcomes the species barrier and affects not only rabbits, but also some species of hares.

Vaccines containing RHDV and RHDVa do not protect rabbits from RHDV2. It was first registered in France in 2010, and later in Australia (2015), Canada (2016) [2]. In 2018, this serotype was detected in the Tver and Moscow regions of the Russian Federation, and there are currently no registered vaccines against this serotype in our country. Therefore, the development of a vaccine against HBV 2 is an urgent task.

Pasteurellosis is a dangerous infectious disease of rabbits, which, along with viral hemorrhagic disease of rabbits and myxomatosis, causes enormous economic damage to rabbit farms. Although respiratory distress (from rhinitis to pneumonia) is a common manifestation of pasteurellosis in rabbits, it can also present with various clinical signs such as genital infections, abscesses, otitis media and sepsis, or it can occur latently without showing any signs. The mortality rate ranges from 15 to 75%. Economic damage during pasteurellosis consists of the death of animals, deterioration in the quality of meat and skins, poor development of young animals [3, 4].

In Russia, according to the OIE, pasteurellosis was registered in the following regions: Republic of Mari El (2015), Krasnoyarsk Territory (2018), Oryol Region (2019).

Monovaccines and associated vaccines are available to prevent outbreaks of myxomatosis and HBV. In the Russian Federation, vaccines against myxomatosis and VGBV are registered both by domestic manufacturers (FGBNU FITSViM, Pokrovsky Biopreparation Plant OJSC, BiAgro Trading House LLC) and foreign (Merial, France; Laboratorios Hipra, SA, Spain; Intervet International BV ", Netherlands). There are no officially registered vaccines against rabbit pasteurellosis in the Russian Federation. Rabbit breeders are forced to use vaccines against pasteurellosis of cattle, ruminants, pigs.

An associated vaccine against myxomatosis, pasteurellosis and HBV of types 1 and 2 has not been registered in the Russian Federation, despite the fact that these diseases are especially dangerous for rabbits, highly contagious infections, causing great economic damage, leading to the death of a large number of livestock.

At the moment, the development of new drugs for the eradication of myxomatosis, pasteurellosis and viral hemorrhagic disease in rabbits is one of the most important tasks of vaccine prevention [6, 8, 11, 14, 18].

The effectiveness of the prevention of these diseases all over the world is associated mainly with the use of inactivated mono- and bivalent vaccines. At the same time, vaccine prophylaxis until recently remains the only fundamentally worked out methodological method for effective resistance to infectious diseases when raising rabbits.

However, each type of vaccine used for the specific prevention of the above diseases in rabbits has advantages and disadvantages.

The use of inactivated monovalent vaccines is limited by the need for simultaneous vaccination against several infections in a short period of time. In addition, the repeated repetition of the individual method of vaccination affects labor costs, prolongs the period of immunity formation in rabbits, and contributes to the occurrence of stressful events and complications.

These disadvantages are partially eliminated by the development and use of associated inactivated vaccines in rabbit breeding, which contain a varied quantitative and qualitative ratio of antigens.

A known method of preparation and inactivation of a vaccine against rabbit pasteurellosis [UA 27906 (U) from 04.16.2007,]. The method of obtaining a vaccine includes the cultivation of epizootic strains of the genus Pasteurella for the accumulation of bacterial mass, preparation of corpuscular inactivated antigen, addition of an adjuvant and determination of the level of protection of the drug. Inactivation of a strain of the genus Pasteurella is carried out with a 0.5% aqueous solution of hydrogen peroxide for 12 hours [20, 26].

Known propolis bivalent vaccine against hemorrhagic disease of rabbits and pasteurellosis inactivated and a method for its preparation [patent CN 105169380, from 15.09.2015]. This vaccine contains a tissue suspension of the rabbit haemorrhagic disease virus and a liquid antigen of the Pasteurella multocida strain, inactivated with propolis. The vaccine has a 100% rate of immune protection of rabbits against hemorrhagic disease and 90% rate of immune protection against pasteurellosis, long shelf life [21].

Known vaccine associated with streptococcosis and viral hemorrhagic disease of rabbits [patent RU 2429879 from 12.04.2010]. This vaccine is intended for the prevention of infectious diseases in rabbits, in particular streptococcosis and viral hemorrhagic disease, contains a cell suspension of a pure culture of the causative agent of streptococcosis Str. pneumoniae and a suspension of rabbit liver with rabbit viral hemorrhagic disease virus isolated from a local epizootic focus were inactivated with formalin and aluminum hydroxide [22].

Known vaccine associated against colibacillosis, streptococcosis and viral hemorrhagic disease of rabbits [patent RU 2443775 from 29.06.2010]. The vaccine contains as antigens a suspension of cells of a pure culture of the causative agent of colibacillosis E. coli, streptococcosis Str. pneumoniae and rabbit haemorrhagic disease virus obtained by selection of affected organs from dead rabbits from a local epizootic focus [23].

Known trivalent vaccine for rabbits [patent CN 101708332 from 30.11.2009]. The vaccine is made, mainly from the viral suspension of the rabbit haemorrhagic disease virus, from the bacterial mass of Pasturella multocida type A and Clostridium perfringens strains, is inactivated and has a volume ratio of 1: 1: 2 [24].

According to the data of the patent and scientific and technical literature, a similar set of features, in terms of components, was not found, which makes it possible to judge the inventive level of the claimed technical solution.

As for the criterion "industrial applicability", the components that make up the vaccines are known and widely used in veterinary practice in the manufacture of vaccines: an associated vaccine against myxomatosis and viral haemorrhagic disease of rabbits (STO 00495549-0044-2007); associated vaccine against salmonellosis, pasteurellosis and enterococcal infection of piglets TU 10.09-62-90, approved on 01.08.90, inactivated tissue-based aluminum hydroxide vaccine against hemorrhagic disease of rabbits STO 00495549-0005-2006; provides all the necessary information about microorganisms. However, for rabbits, some vaccines are not used, or contain components that differ from the proposed invention.

A common significant disadvantage of the known associated vaccines is their insufficiently high efficiency due to the inconsistency of the antigenic profile of the specific antigens used in the vaccine for the epizootic setting.

Thus, with all the variety and rather wide range of vaccines offered against rabbit diseases, there is not a single drug that would include viral antigens (HBV, myxomatosis) and bacterial antigen (pasteurellosis), while HBV and pasteurellosis antigens are inactivated, and the antigen myxomatosis is an attenuated viral material. For the first time, the sorbed inactivated component of the vaccine is used as a diluent for the dry component of the live virus, thereby reducing the level of dilution of the viral components.

It is also known that in the manufacture of associated vaccines with a valence higher than three, a significant mutual dilution of the antigenic components contained in them occurs. As a result, their concentration in the inoculated volume of the drug decreases. At the same time, the possibility of increasing the concentration of viruses by cultivation is limited. In this regard, one of the most important technological indicators is their optimal ratio in the inoculated volume of the vaccine, which would ensure the formation of intense immunity in rabbits. Important in the production of inactivated vaccines is the choice of an adjuvant that would ensure the stability of the vaccine, the induction and persistence of high antibody titers in vaccinated rabbits in the absence of side effects at the sites of its administration, as well as the convenience of using the vaccine in industrial conditions.

In the associated vaccine, the components against rabbit haemorrhagic disease virus and pasteurellosis are inactivated. To reduce the reactogenicity of the vaccine and complete inactivation of the virus, it is necessary to purify it from cellular debris. There are physical, physicochemical, and chemical methods to purify viruses from ballast proteins [25]. The present invention presents a purification method in which the least loss of antigen occurs and which maximally cleans it from cellular debris; chloroform was used in the manufacture of this vaccine.

In monovaccines against viral hemorrhagic disease of rabbits, formaldehyde is used to inactivate the virus. The disadvantage of using formaldehyde to inactivate viruses is the often observed "tails" of an infectious virus, the formation of cross-links in viral proteins, and a significant decrease in antigenic activity. In the present invention, aminoethylethyleneimine (AEEI) was used to inactivate viral material. The advantage of its use is minimal damage to the antigen structure during inactivation, the reliability and duration of the reaction, as well as the effect only on the genetic material of the virus. Virus inactivation occurs due to depurenization and subsequent depolymerization of nucleic acids [25].

However, formaldehyde (produced as an aqueous 36-40% solution) is used to inactivate bacterial antigens. For bacterial strains, this inactivant has a relatively low toxicity and cost, is easy to use, and ensures reliable preservation of immunobiological preparations.

In the manufacture of the proposed vaccine for inactivation of the virus-containing suspension, AEEI was used at a final concentration for VGBV1 - 0.3%, for VGBK2 - 1.0%, for inactivation of bacteria of the Pasteurella multocida strain, formaldehyde was used at a final concentration of 0.1%.

Therefore, the problem of obtaining a highly effective associated vaccine against the main pathogens of rabbit diseases remains relevant and is the main direction of research to create the desired drug.

The problem to be solved by the present invention is to obtain an associated vaccine containing inactivated viral and bacterial agents and a live attenuated viral agent, which creates effective protection of rabbits from infection with pathogens of myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2. The main indicator of the effectiveness of the drug is the protection of rabbits from myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2 - modern epizootic strains, as well as the creation of protection against cross-infection of rabbits with the virus of hemorrhagic disease of types 1 and 2.

The technical result from the use of the proposed invention is to expand the arsenal of highly immunogenic, harmless and areactogenic associated vaccines against pathogens of infectious diseases of rabbits circulating in the Russian Federation.

The specified technical result was achieved by creating an associated vaccine against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2, characterized by the following set of features.

According to the invention, the proposed vaccine contains, as an active substance, a mixture of attenuated antigenic material from the Mixo / ARRIAH-18 strain of the rabbit myxomatosis virus; from inactivated purified antigenic material from the VGBK1 / ARRIAH strain of the rabbit hemorrhagic disease virus type 1; from inactivated purified antigenic material from the VGBK2 / ARRIAH strain of the rabbit hemorrhagic disease virus type 2; from inactivated purified antigenic material from the strain No. 1231 Pasteurella multocida, taken in effective amounts that provide protective immune activity in the body of the animal after the introduction of the target drug. As a targeted additive, the proposed vaccine contains aluminum hydroxide (manufactured by ARRIAH) and a stabilizing medium as an adjuvant.

In the proposed vaccine, the active substance and the target additive are combined in the ratio: for the liquid component, vol. %: 50.0 ÷ 50.0; for the dry component - the active substance and the target additive are combined in the ratio, vol. %: 91.8 ÷ 8.8: the liquid component and the dry component are mixed in a 1: 1 ratio.

The proposed invention includes the following set of essential features that provide a technical result in all cases for which legal protection is sought:

1. Vaccine associated against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2.

2. The active substance in the form of a mixture of freeze-dried live attenuated antigenic material from the Mixo / ARRIAH-18 strain of the rabbit myxomatosis virus, fam. Poxviridae, subfamily Chordopoxvirinae, genus Leporipoxvirus, collection of FGBI "ARRIAH" under registration number: №120 - dep / 19-10 - KShM FGBI "ARRIAH"; from inactivated purified antigenic material from the VGBK1 / ARRIAH strain of the rabbit hemorrhagic disease virus, family Caliciviridae, genus Lagovirus, Rabbit hemorrhagic disease virus type 1, collection of FGBI "ARRIAH" under registration number: №305 - dep / 20-93 - SCSM FGBU "ARRIAH"; from inactivated purified antigenic material from the VGBK2 / ARRIAH strain of the rabbit hemorrhagic disease virus, fam. Caliciviridae, genus Lagovirus, Rabbit hemorrhagic disease virus type 2, collection of FGBI "ARRIAH" under registration number: №306 - dep / 20-94 - SCSM FGBU "ARRIAH"; from inactivated purified antigenic material from strain No. 1231 Pasteurella multocida, family. Pasteurellaceae, genus Pasteurella, type A, collection of FGBI "ARRIAH" under registration number: №307 - dep / 20-95 - GKShM FSBI "ARRIAH"

3. Targeted supplements

Significant distinctive features of the proposed vaccine are that, as an active substance, it contains a mixture of inactivated purified antigenic materials from the strains "VGBK1 / ARRIAH" and "VGBK2 / ARRIAH" HBV virus and strain No. 1231 Pasteurella multocida, and attenuated lyophilized viral material from strain "Mixo / ARRIAH-18" of rabbit myxomatosis virus, in an effective amount.

The proposed invention is also characterized by other distinctive features expressing specific forms of implementation or special conditions for its use:

1. Antigenic material from the Myxo / ARRIAH-18 strain of the rabbit myxomatosis virus, preferably obtained in a continuous culture of RK-13/91 cells, in an effective amount.

2. Antigenic material from the Myxo / ARRIAH-18 strain of the rabbit myxomatosis virus, obtained preferably in a continuous culture of RK-13/91 cells, which is an attenuated lyophilized viral suspension with an infectious activity of at least 6.5 ± 0.25 lg TCID ₅₀ / cm ³ and infectious activity on rabbits - not less than 3.0 ± 0.25 lg ID ₅₀ / cm ^{3 in the} amount of 91.8 vol. %.

3. Inactivated and purified antigenic material from the VGBK1 / ARRIAHI strain of type 1 haemorrhagic disease virus of rabbits, preferably obtained from a 10% liver suspension of a rabbit infected with the VGBK1 / ARRIAHI strain of type 1 haemorrhagic disease virus type 1, which is a viral suspension in an effective amount.

4. Inactivated and purified antigenic material from the VGBK1 / ARRIAH strain of the type 1 haemorrhagic disease virus of rabbits, preferably obtained from a 10% hepatic suspension of a rabbit infected with the VGBK1 / ARRIAHI strain of the type 1 haemorrhagic disease virus of rabbits, which is a viral suspension with infectious activity on rabbits not less than 4.0 ± 0.25 lg LD ₅₀ / cm ³ , an amount of 12.5 vol. %.

5. Inactivated and purified antigenic material from the strain "VGBK2 / ARRIAH" of the virus of rabbit hemorrhagic disease type 2, preferably obtained from a 10% liver suspension of a rabbit infected with the strain "VGBK2 / ARRIAH" of the virus of hemorrhagic disease of rabbits type 2, which is a suspension in effective amount.

6. Inactivated and purified antigenic material from the VGBK2 / ARRIAH strain of rabbit hemorrhagic disease virus type 2, preferably obtained from a 10% liver suspension of a rabbit infected with the VGBK2 / ARRIAHI strain of rabbit hemorrhagic disease virus type 2, which is a viral suspension with infectious activity on rabbits not less than 5.25 ± 0.25 lg LD ₅₀ / cm ³ , in an amount of 12.5 vol. %.

7. Inactivated and purified antigenic material from strain No. 1231 Pasteurella multocida type A, obtained preferably by submerged cultivation in a laboratory bioreactor AK-210, in a liquid nutrient medium prepared on the basis of Hottinger tryptic hydrolyzate of meat with a concentration of amine nitrogen of 250-300 mg%, with the addition of 2% yeast extract as a source of V-growth factor, in an effective amount.

8. Inactivated and purified antigenic material from the strain No. 1231 Pasteurella multocida type A, obtained by submerged cultivation in a laboratory bioreactor AK-210, in a liquid nutrient medium prepared on the basis of a tryptic hydrolyzate of meat according to Hottinger with an amine nitrogen concentration of 250-300 mg%, with adding as a source of V-growth factor 2% yeast extract, which is a bacterial suspension with infectious activity in rabbits 3.0 ± 0.25 LD ₅₀ / cm ³ , in an amount of 25.0 vol. %.

9. Of targeted additives, the vaccine contains aluminum hydroxide.

10. 3% aluminum hydroxide gel.

11. The vaccine contains 3% aluminum hydroxide gel in an amount of at least 50.0 vol. %.

12. In addition to targeted additives, the vaccine contains a stabilizing medium.

13. The stabilizing medium consists of: lactalbumin hydrolyzate (GLA) - 4%, sucrose - 4%, gelatose - 0.8%.

14. The vaccine contains a stabilizing medium: lactalbumin hydrolyzate (GLA) - 4%, sucrose - 4%, gelatose - 0.8%, in an amount of 8.8 vol. %.

15. A mixture of inactivated and purified antigenic materials from the strain "VGBK1 / ARRIAH" of the virus of rabbit hemorrhagic disease of type 1, from the strain of "VGBK2 / ARRIAH" of the virus of hemorrhagic disease of rabbits type 2, from the strain No. 1231 Pasteurella multocida type A; lyophilized attenuated antigenic material from the Myxo / ARRIAH-18 strain of the rabbit myxomatosis virus and targeted additives: stabilizing medium and adjuvant aluminum hydroxide in the amount, vol. %:

Dry component:

- Lyophilized antigenic material from attenuated strain "Mixo / ARRIAH-18" 91.2 rabbit myxomatosis virus - Stabilizing environment 8.8

Liquid component:

Antigenic material from the strain "VGBK1 / ARRIAH" hemorrhagic virus rabbit disease type 1 12.5 Antigenic material from the strain "VGBK2 / ARRIAH" hemorrhagic virus rabbit disease type 2 12.5 Antigenic material from strain No. 1231 Pasteurella multocida type A 25.0 Adjuvant 3% aluminum hydroxide gel 50.0.

As a result of the studies carried out, the authors identified the most important technological indicators that made it possible to create a drug in which there is no competition between the antigens that make up the proposed vaccine, and its immunogenic properties were comparable to those after the use of monopreparations. A high immunological effect in relation to specific antigens that make up the vaccine was achieved by obtaining viral and bacterial components in the vaccine that have harmlessness, immunogenicity and antigenic activity, as well as the option of their optimal ratio in the vaccine, ensuring the production of a protective level of antibodies, those. creation of intense immunity to diseases in rabbits.

The proposed vaccine has high immunogenic activity and provides reliable protection against myxomatosis virus and viral hemorrhagic disease of rabbits of types 1 and 2; against pasteurellosis isolates circulating in the territory of the Russian Federation.

An additional technical result from the use of the proposed invention is achieved due to the fact that aminoethylethyleneimine (AEEI) is used to inactivate vaccinated materials, which can significantly reduce labor and energy costs for vaccine production and improve the quality of antigenic materials.

An additional technical result from the use of the proposed invention is achieved due to the fact that the presented active substances (liquid and dry components), before using the finished product, are mixed in a ratio of 1: 1, providing the antigenic and immunogenic activity of the target product.

The essence of the invention is reflected in the graphic materials:

FIG. 1 - Dendrogram, reflects the phylogenetic relationship of Myxo / ARRIAH-18 myxomatosis virus strains with epizootic and vaccine myxomatosis virus strains. The dendrogram is based on a comparison of the complete nucleotide sequences of the m071L gene.

FIG. 2 - Dendrogram, reflects the phylogenetic relationship of VGBV1 / ARRIAH rabbit haemorrhagic disease virus strains with epizootic and vaccine HBVV strains. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP60 gene.

FIG. 3 - Dendrogram, reflects the phylogenetic relationship of VGBV2 / ARRIAH rabbit haemorrhagic disease virus strains with epizootic and vaccine HBVV strains. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP60 gene.

The original virus for obtaining a strain of rabbit myxomatosis virus was obtained from the Institute of Experimental Zooprophylaxis (Italy), as a classic variant of rabbit myxomatosis, which was isolated in Northern Italy "Mukho No. 278". By successive passages in the cell culture of rabbit kidney RK-13/91 from the Mukho No. 278 strain, the culture virus “Mixo / ARRIAH-18” of rabbit myxomatosis was obtained.

The strain "Mixo / ARRIAH-18" of the rabbit myxomatosis virus was deposited in the Collection of microorganism strains of the FGBU "ARRIAH" under the registration number: No. 120 - dep / 19-10 - KShM of the FGBU "ARRIAH".

The strain "Mixo / ARRIAH-18" of the rabbit myxomatosis virus is characterized by the following features and properties.

Morphological signs

The strain "Mixo / ARRIAH-18" belongs to the Poxviridae family, the Chordopoxvirinae subfamily, the Leporipoxvirus genus, the Myxoma virus species: the virion is brick-shaped, ~ 280 nm in size. The virion consists of a nucleoid shaped like a biconcave disc and containing DNA. On both sides of the nucleoid there are oval structures - lateral bodies. The lipoprotein envelope of the virus is covered with randomly arranged villi with a tubular structure. The genome is represented by double-stranded, filamentous, non-fragmented DNA, approximately 147.6 kb in length. up to 161.8 kb

Antigenic properties

According to its antigenic properties, the strain "Mixo / ARRIAH-18" of the rabbit myxomatosis virus has 3 antigens: L and S (often in the LS complex) and the nucleoprotein NP. Antigens L and S are non-infectious, located on the surface of the virion, have precipitating and complement-binding activity, L is thermolabile. NP antigen is a structural nucleocapsid protein characteristic of all members of the Poxviridae family. The virus is stably neutralized by a homologous antiserum, in recovered rabbits, antibodies are formed in the blood serum, which are detected in an enzyme-linked immunosorbent assay (ELISA). When rabbits are immunized with a vaccine, it induces the formation of specific antibodies detected in an ELISA.

By the method of nucleotide sequencing, the primary structure of the gene of the Myxomatosis virus strain "Mixo / ARRIAH-18" was determined and the primary structure of the protein was derived. Comparative analysis of nucleotide sequences showed that the Myxo / ARRIAH-18 strain of the myxomatosis virus does not differ from isolates and strains isolated in Europe, with a bootstrap value in the range of 70 to 100% (Fig. 1).

On the territory of the Russian Federation, strains and isolates of the causative agent of myxomatosis circulate, having almost 100% homology with European strains in the m071L gene region encoding the main envelope protein of the virus.

Thus, phylogenetic analysis showed that the strain "Mixo / ARRIAH-18" belongs to the genetic line of representatives of IUD, in accordance with the GenBank data.

Geno- and chemotaxonomic characteristics

The strain "Mixo / ARRIAH-18" is a double-stranded DNA-containing virus, coated with an envelope. The lipoprotein membrane is a tubular villi. In its composition, the virus contains proteins (88% of the virion mass), DNA (5% of the virion mass), lipids (4% of the virion mass), carbohydrates (3% of the virion mass).

The strain "Mixo / ARRIAH-18" exhibits genetic stability.

Biotechnological characteristics

The strain "Mixo / ARRIAH-18" of rabbit myxomatosis virus is cultivated in continuous cell lines: rabbit kidney (RK-13/91), causing a cytopathic effect (CPE), and within 48-96 hours of incubation accumulates in titers from 6.5 to 7.0 lg CPP ₅₀ / cm ³ . The results of adaptation of the VMC virus strain "Mixo / ARRIAH-18" to the cell culture RK-13/91 are presented in table 1.

Cytomorphological changes during the cultivation of the strain "Mixo / ARRIAH-18" IUD have specific parameters that allow to fairly objectively establish its CPP.

The strain "Mixo / ARRIAH-18" is stable and retains its properties for 10 passages (observation period).

Resistance to external factors

The myxomatosis virus is sensitive to the action of trypsin, is completely inactivated by ether, and is resistant to drying. Heating the virus for an hour at a temperature of 56 ° C causes its inactivation. At subzero temperatures, the virus remains infectious for up to 6 months.

In the corpses of animals that died from myxomatosis, it lasts up to 7 days, in dried skins - up to 10 months, in the soil - up to 2 years. The myxomatosis virus in fresh-dry preservation rabbit skins is inactivated at a temperature of 19-23 ° C after 159 days; at a temperature of 4-6 ° С - after 570 days, and at 0-3 ° С after 720 days.

Reliable disinfectants against myxomatosis virus are solutions of formaldehyde and caustic alkali.

Additional signs and properties

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for rabbits of any breed and age.

Virulence - virulent for naturally-susceptible animals through contact infection, in a transmissible way and mechanically through feed, equipment, animals, birds and people contaminated with myxomatosis virus.

Stability - retains the original biological properties when passaged for 10 passages (observation period) in continuous cultures of rabbit kidney cells (RK-13/91).

Free from contamination by bacteria, mycoplasmas and foreign viruses.

To obtain antigenic material of their strain "Mixo / ARRIAH-18" of the myxomatosis virus of rabbits, a monolayer continuous culture of rabbit kidney cells RK-13/91 (KK PK RK-13/91) grown in 1.5 dm 3 is used as a sensitive biological system ³ mattresses.

To preserve the infectious activity during the drying process, a stabilizer or stabilizing medium is added to the vaccinated material, which ensures the maximum preservation of the biological activity of the Myxo / ARRIAH-18 strain of the rabbit myxomatosis virus. A medium of the following composition is used as a stabilizer: lactalbumin hydrolyzate (GLA) - 4.0%, gelatose - 0.8%, sucrose - 4.0%. Lyophilization is carried out in a lyophilic installation, the live attenuated antigenic material and the stabilizing medium are premixed in a quantitative ratio of 0.9: 0.1; the finished product is packaged in vials, cooled to minus 45 ° C in a freezer. Then the frozen material in cassettes is quickly transferred (no more than 2-3 minutes) from the freezer to the sublimation chamber, which is hermetically closed and a vacuum pump is turned on. The amount of vacuum in the apparatus is from 4 Pa to 7 Pa. The initial temperature of the shelves with the material to be dried is from minus 50 ° С to minus 55 ° С. The final temperature of the shelves during the drying process is from 30 ° C to 40 ° C. The gradient of the shelf temperature rise from the initial to the final value varies from 10 to 15% per second. The final temperature of the dried material is from 20 ° C to 23 ° C. Drying of the material at the specified temperature is carried out from 4 to 6 hours.

For the manufacture of the proposed vaccine, freeze-dried live material from the strain "Mixo / ARRIAH-18" of the rabbit myxomatosis virus is used, obtained in a cell culture RK-13/91 with an infectious activity of at least 6.5 ± 0.25 lg TCD ₅₀ / cm ³ , in an amount of at least 91.8 vol. %.

The viral strain, which served as a source for obtaining the production strain "VGBK1 / ARRIAH" of rabbit hemorrhagic disease virus (type 1), was isolated by refreshing and scaling the strain "RHDVaPv97" VGBK type 1, obtained from the Institute of Experimental Zooprophylaxis (Italy), in the body of susceptible animals ( rabbits).

The strain "VGBK1 / ARRIAH" of the type 1 HBVV virus was deposited in the collection of microorganism strains of the FGBI "ARRIAH" under the registration number: №305 - dep / 20-93 GKShM FGBU "ARRIAH".

The VGBK1 / ARRIAH strain has genetic and phenogenetic characteristics. It is cultivated on naturally susceptible rabbit animals in vivo, while it has infectious, antigenic and immunogenic activity in its native form and retains high antigenic and immunogenic activity after inactivation.

Morphological signs

The "VGBK1 / ARRIAH" strain of rabbit hemorrhagic disease virus belongs to the order Picornavirales, class Pisoniviricetes, family Caliciviridae, genus Lagovirus, species Rabbit hemorrhagic disease virus. The virus is cubic symmetry, has the shape of an icosahedron, ~ 30 nm in diameter, has cup-shaped depressions. The genome is represented by a single-stranded positive linear non-segmented RNA, approximately 7.5-8.5 kb in length.

Antigenic properties

In terms of antigenic properties, the VGBK1 / ARRIAH strain belongs to type 1.

The virus is stably neutralized by a homologous antiserum.

The virus reacts with antibodies of recovered or immunized animals in an ELISA reaction. When vaccinating naturally susceptible animals, the inactivated virus induces the formation of virus-specific antibodies at a protective level, which contributes to the formation of immunity against this disease (the values of the protective level of antibodies depend on the diagnostic kit used).

Genotaxonomic characteristics

With the genetic identification of a sample of the strain "VGBK1 / ARRIAH" by PCR and nucleotide sequencing, it was established that it belongs to VGBK1 type (Fig. 2), i.e. the first type of genetic group G6, and full correspondence of the nucleotide sequences of the VP60 gene to the declared strain in accordance with the GenBank database.

Biotechnological characteristics

The strain "VGBK1 / ARRIAH" of the rabbit hemorrhagic disease virus is cultivated in the organism of susceptible animals (rabbits). Viral material is obtained by infecting animals with a 10% hepatic suspension obtained from the liver of a rabbit that has died from "VGBK1 / ARRIAH". The VGBK1 / ARRIAH strain, refreshed during 2 consecutive passages on rabbits, causes lethality in the period from 24 to 72 hours with direct and contact infection with the manifestation of clinical and pathological signs characteristic of VGBK.

The strain "VGBK1 / ARRIAH" is stable and retains its original characteristics when passaged on the indicated susceptible animals for 5 consecutive passages (observation period).

Physical properties The virus is resistant to treatment with ether, chloroform, pH 3.0 and a temperature of 50 ° C for 60 minutes. It remains in a suspension of an infected liver at minus 40 ° С for 1 year, minus 20 ° С - 3 years, minus 50 ° С - 5 years without decreasing virulence; inactivated by 0.1% formaldehyde at 40 ° C during the day. Additional signs and properties

Antigenic activity - possesses antigenicity in the composition of an inactivated vaccine and a preparation for hyperimmunization of diagnostic sera donors.

Reactogenicity - does not possess reactogenic properties.

Stability - retains the original biological (antigenic) properties when passaged on rabbits for 10 serial passages (observation period).

Pathogenicity - pathogenic for rabbits of any breed. Pathogenic for rabbits over 1.5 months of age.

Virulence is expressed.

Contagiousness is expressed.

The infectious activity of the strain when titrated on rabbits with a live weight of 2.5-3.0 kg was not less than 4.0 ± 0.25 lg LD ₅₀ / cm ³ .

Contamination by bacteria, fungi and mycoplasmas is absent. Sowing a suspension of a sample of the VGBK1 / ARRIAH virus strain on the appropriate nutrient media without seedlings during the entire observation period.

To obtain antigenic material from the VGBK1 / ARRIAHV strain of type 1 haemorrhagic disease virus in rabbits, a biological system is predominantly used as a 10% hepatic suspension obtained from the liver of a rabbit infected with the VGBK1 / ARRIAHI strain of type 1 haemorrhagic disease virus in rabbits. The resulting vaccinated material is purified from ballast impurities by centrifugation or by another known method.

To inactivate the strain "VGBK1 / ARRIAH" type 1 hemorrhagic disease virus in rabbits, aminoethylethyleneimine (AEEI) is used, which is added to the purified vaccinated suspension to a concentration of 0.3%. Virus inactivation is carried out at a temperature of 37 ° C for 72 hours at a pH value of 7.6-7.8 with periodic stirring of the suspension (every 5-6 hours for 3-5 minutes). At the end of the inactivation, the AEE is neutralized by introducing sodium thiosulfate into the antigenic material. For this, a 2 M sodium thiosulfate solution is added to the cooled antigen suspension at the rate of 10% to the volume of the AEE used.

For the manufacture of the sorbed - liquid part of the associated vaccine, inactivated purified antigenic material is used from the VGBK1 / ARRIAH strain of the rabbit hemorrhagic disease virus type 1, obtained from a 10% hepatic suspension of a sick rabbit infected with the VGBK1 / ARRIAHI strain of the rabbit hemorrhagic disease virus 1 type, with infectious activity on rabbits not less than 4.0 ± 0.25 lg LD ₅₀ / cm ³ , in an amount of 12.5 vol. %

The viral strain that served as a source for the production of the production strain "VGBK2 / ARRIAH" of the virus of rabbit hemorrhagic disease (type 2) was isolated by refreshing and scaling the strain "RHDV2Ch14" of the virus of rabbit hemorrhagic disease type 2, obtained from the Institute of Experimental Zooprophylaxis (Italy), in the body susceptible animals (rabbits).

VGBK2 / ARRIAHV strain of type 2 HBVV was deposited in the collection of microorganism strains at ARRIAH under registration number: No. 306 - dep / 20-94 of the SCHM FGBU ARRIAH.

The strain "VGBK2 / ARRIAH" is cultivated only on naturally susceptible animals - rabbits in vivo, while it has infectious, antigenic and immunogenic activity in its native form and retains high antigenic and immunogenic activity after inactivation.

Morphological signs

The "VGBK2 / ARRIAH" strain of the rabbit hemorrhagic disease virus belongs to the order Picornavirales, class Pisoniviricetes, family Caliciviridae, genus Lagovirus, species Rabbit hemorrhagic disease virus. It possesses morphological features characteristic of the rabbit haemorrhagic disease virus: the virus of cubic symmetry, has the shape of an icosahedron, ~ 30 nm in diameter, has cup-shaped depressions. The genome is represented by a single-stranded positive linear non-segmented RNA, approximately 7.5-8.5 kb in length. Antigenic properties

The main protective antigen of the virus is Vp 60, upon injection of which immunity is formed in the rabbit.

In terms of its antigenic properties, the VGBK2 / ARRIAH strain of the rabbit hemorrhagic disease virus belongs to the RHDV2 genogroup (type 2).

The virus is stably neutralized by a homologous antiserum.

When vaccinating naturally susceptible animals, the viral antigen induces the formation of specific antibodies that are detected by ELISA.

Thus, the presented strain "VGBK2 / ARRIAH" of rabbit haemorrhagic disease virus provides protection against infection with isolates of the RHDV2 serotype.

Geno- and chemotaxonomic characteristics

The VGBK2 / ARRIAH strain belongs to the RHDV2 serotype.

The causative agent is an RNA-containing virus with an average molecular weight of 2.1 kDa. The genome is represented by a single-stranded positive linear non-segmented RNA, approximately 7.5-8.5 kb in length. The virion is a spherical nucleocapsid of icosahedral symmetry devoid of a supercapsid envelope, ~ 30 nm in diameter. There are 32 cup-shaped depressions on the surface of the virions.

There are 4 virion proteins in the capsids of the virus - VP1, VP2, VP3, VP4. VP1 is the main capsid protein, which accounts for 54.7% of the mass of capsid proteins, VP2 is a minor structural protein. Protein VP60, a unique component of the VGBK capsid, has been synthesized.

The phylogenetic relationship of the VGBK2 / ARRIAH strain was studied in the GSC by comparison with the strains and isolates presented in the GenBank NCBI international database using the MEGA 6.0 program. The strain "VGBK2 / ARRIAH" belongs to the serotype RHDV2 and is closely related to isolates 13-71 France, E13-37 France, CBVal16, Zar2014-8 and Ssl1 Italy (Fig. 3).

Biotechnological characteristics

The strain "VGBK2 / ARRIAH" of the virus of haemorrhagic disease of rabbits type 2 is cultivated only on naturally susceptible animals - rabbits in vivo. Rabbits were infected with 10% tissue suspension prepared from the liver of infected rabbits at a dose of 4.0-5.0 lg LD ₅₀ / cm ³ .

Biological activity during titration of the virus of each passage on naturally susceptible animals - rabbits, within 48-144 hours, the virus titer reaches high values from 4.0 ± 0.25 to 5.0 ± 0.25 lg LD ₅₀ / cm ³ . The VGBK2 / ARRIAH strain is stable and retains its original characteristics when passaged on the indicated susceptible animals for 5 consecutive passages (observation period).

Resistance to external factors

The "VGBK2 / ARRIAH" strain of the rabbit haemorrhagic disease virus is resistant to treatment with ether, chloroform, pH 3.0 and a temperature of 50 ° C for 60 minutes. In a suspension of an infected liver, it is stored at 4 ° C for 1 year, minus 20 ° C for 3 years, minus 50 ° C for 5 years without decreasing virulence.

It is sensitive to formalin and glutaraldehyde. It is inactivated by 0.1% formaldehyde or theotropin during the day. Additional signs and properties Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for rabbits of any breed. Pathogenic for rabbits 1 month of age.

Virulence - highly virulent for naturally susceptible animals - rabbits, upon contact infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 10 passages (observation period) on rabbits.

To obtain antigenic material from the VGBK2 / ARRIAHV strain of type 2 hemorrhagic disease virus, a 10% hepatic suspension obtained from the liver of a rabbit infected with the VGBK2 / ARRIAHI strain of type 2 hemorrhagic disease virus is used. The resulting vaccinated material is freed from cell detritus and other ballast impurities by low-speed centrifugation or any other known method.

To inactivate the VGBK2 / ARRIAH strain of the rabbit haemorrhagic disease virus type 2, aminoethylethyleneimine (AEEI) is used, which is added to the purified vaccinated suspension to a concentration of 1.0%. Virus inactivation is carried out at a temperature of 37 ° C for 24 hours at a pH value of 7.6-7.8 with periodic stirring of the suspension (every 5-6 hours for 3-5 minutes). At the end of the inactivation, the AEE is neutralized by introducing sodium thiosulfate into the antigenic material. For this, a 2 M sodium thiosulfate solution is added to the cooled antigen suspension at the rate of 10% to the volume of the AEE used.

For the manufacture of the sorbed - liquid part of the associated vaccine, avirulent purified antigenic material from the VGBK2 / ARRIAH strain of the rabbit hemorrhagic disease virus type 2 obtained from a 10% hepatic suspension of a dead rabbit infected with the VGBK2 / ARRIAHI strain of rabbit hemorrhagic disease virus 2 is used type, with infectious activity on rabbits not less than 5.25 ± lg LD ₅₀ / cm ³ , in an amount of 12.5 vol. %.

Based on the data obtained from the analysis of morphological, biotechnological, antigenic and additional characteristics, it can be argued that the strains "VGBK1 / ARRIAH" and "VGBK2 / ARRIAH" of rabbit hemorrhagic disease virus are highly stable during in vivo reproduction in rabbits, are characterized by high titer of infectious activity , strain "VGBK1 / ARRIAH" of rabbit haemorrhagic disease virus provides protection against infection with isolates and strains of the RHDVa / G6 subtype, and strain "VGBK2 / ARRIAH" of rabbit haemorrhagic disease virus allows protection against infection with isolates of serotype RHDV2.

The viral isolate, which served as a source for obtaining the industrial bacterial strain No. 1231 Pasteurella multocida type A, was isolated from a piglet and obtained from the All-Russian State Collection of Microorganism Strains Used in Veterinary Medicine and Animal Husbandry, VGNKI Federal State Budgetary Institution.

Strain No. 1231 Pasteurella multocida type A was deposited in the FGBI "ARRIAH" under the registration number: No. 307 - dep / 20-95 GKShM FGBU "ARRIAH".

Strain No. 1231 P. multocida pasteurellosis of rabbits is characterized by the following characteristics and properties.

Morphological signs

P. multocida strain No. 1231 of rabbit pasteurellosis belongs to the family Pasteurellaceae, genus Pasteurella, species Pasteurella multocida. It has the shape of short, oval rods 0.3-1.0 x 1-2 microns in size; in culture, along with the typical shape, there are small coccoid cells, located singly, in pairs and in chains. In freshly isolated cultures, a capsule is found that is not a continuation of the cell culture and its loss does not cause significant changes in the vital activity of bacteria. With prolonged cultivation, the capsule disappears. Gram stained - gram-negative. Non-spore-forming facultative anaerobe.

Antigenic structure

In strains of P. multocida in RIGA with sheep erythrocytes, 5 types of capsular antigens A, B, D, E and F, which are polysaccharides, are identified. According to the P. multocida capsular antigen, strain No. 1231 belongs to serogroup A.

Cultural properties

The optimum temperatures for the growth of pasteurella are 36-38 ° C and a slightly alkaline environment.

During the day on MPA, small (1-3 mm), similar to dew drops, translucent, grayish, slightly convex, smooth colonies with smooth edges, have a bluish tint, grow on the MPA during the day. With an increase, the colonies acquire a whitish color, in shape they resemble a button with a raised center and a peripheral ridge. Over time, the colonies merge with each other, forming a continuous, thin and delicate film on the surface of the medium.

P. multocida grows on blood agar in the same way as on MPA, but much more luxuriant. P. multocida does not grow on McConkey agar. On semi-liquid meat-peptone agar, growth occurs along the line of the prick with a loop, at first in the form of separate colonies, and then a whitish rod without processes is formed. On the surface of gelatinous media, the bacterium grows in the same way as on agar media, gelatin does not liquefy.

Growth on BCH is accompanied by a uniform cloudiness of the medium on the first day after sowing. Shaking the tube produces "moiré waves". On the broth, according to Hottinger, it grows in the form of a uniform turbidity with a small sediment, which, when shaken, rises in the form of a "pigtail".

On 1.5% agar-based hydrolyzate according to Hottinger, it forms translucent colonies of regular rounded shape with smooth edges, with a smooth, convex, shiny surface, slimy consistency, with a diameter of 3.0 to 5.0 mm. In subsequent passages, dissociation and the appearance, along with the S-form colonies, of the rough R-form colonies can be observed.

Biochemical properties

The P. multocida strain No. 1231 does not possess gelatinase, lysine and arginine decarboxylase. Reduces nitrates to nitrites. Gives a positive reaction to catalase, alkaline phosphatase, and oxidase. Does not synthesize urease. Forms indole. Has ornithidine decarboxylase. Ferments glucose, sucrose, mannitol to form acid without gas. Does not ferment lactose, maltose.

Biotechnological characteristics

The most suitable method for obtaining bacterial mass is submerged cultivation. Using this method, the P. multocida strain is cultivated on AK-210 apparatus in a liquid nutrient medium prepared on the basis of Hottinger's tryptic hydrolyzate of meat. The temperature of the nutrient medium in the bioreactor during the cultivation of bacteria was maintained at a constant level of 37.0 ± 0.2 ° C. The initial environment before the introduction of brood material is characterized by the following physicochemical parameters: pH - 7.5 units, eH - + 90 mV, pO ₂ - 100%.

Biological activity during titration of the causative agent of each passage on naturally susceptible animals - rabbits, within 24-48 hours, the titer of the virus reaches 3 LD ₅₀ (60 CFU). Pasteurella retains its original characteristics when passaged on the indicated susceptible animals for 7 consecutive passages (observation period).

Geno- and chemotaxonomic characteristics

According to the capsular antigen, P. multocida "1231" belongs to serogroup A.

The pasteurella genome is concentrated on a circular chromosome of 2.257 million base pairs. It includes over 2000 coding zones, 6 rRNA operons and 57 tRNAs with an average size of one gene of 997 base pairs. Over 100 genes (5% of the genome) are associated with pasteurella virulence.

Identified 50 genes (2.5% of the genome), whose products are associated with iron metabolism. Genes responsible for capsule formation, drug resistance of pasteurella, and bacterial chemotaxis were found.

Resistance to external factors

In frozen corpses, the P. multocida strain persists for up to 1 year. High temperature, on the other hand, causes rapid death of the pathogen: 45-55 ° C in 45 minutes, 58-62 ° C in 15-20 minutes, 70-90 ° C in 5-10 minutes.

The P. multocida strain remains viable under direct sunlight at a temperature of 15-20 ° C for 2 months. In dry substrates, especially with limited air access, it dies in 2-3 days. In rotting corpses, the pathogen persists for 1-3 months, and in manure for up to 14 days. In the surface layers of the soil and on the grass, Pasteurella, depending on humidity, shade and temperature, remain viable from 5 days to 4 months, in water - from 4 months to 1 year, in ice - up to 3 weeks. P. multocida survives in liquid culture media under liquid paraffin oil or in sealed pipettes for 5-12 months. When storing frozen liquid cultures, 15% glycerol is a good cryoprotectant.

Reliable disinfectants at room temperature are 3% NaOH solution (3 h), 0.34% formaldehyde solution (1 h), 3% water emulsion of creolin (2 h), solutions with chlorine concentration 1: 30,000-1: 40,000 (1 -2 h), mercuric chloride solution 1: 5000 and 5% phenol solution (1 min), 3% cresol and 1% milk of lime solution (10 min).

P. multocida is sensitive to ampicillin, baytril, neomycin, penicillin, tetracycline, chloramphenicol, cephalosporins.

Additional signs and properties

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for rabbits and white mice. The titer LD ₅₀ / cm ³ with subcutaneous infection of white mice (16-18 g) is 25 CFU / cm ³ .

Virulence - virulent to rabbits and white mice; the transmission of the pathogen occurs by aerogenic and alimentary, vertical, through the damaged cover or mucous membranes.

Stability - retains the original biological properties when passaged in nutrient media for 7 passages (observation period).

Free from contamination with mycoplasmas and foreign viruses.

Based on the data obtained from the analysis of morphological, biotechnological, antigenic and additional characteristics, it can be argued that P. multocida strain No. 1231 is highly stable during reproduction in Hottinger broth, is characterized by high titer of infectious activity and provides protection against type A rabbit pasteurellosis.

To obtain antigenic material from P. multocida type A strain No. 1231, predominantly liquid nutrient medium prepared on the basis of Hottinger tryptic hydrolyzate of meat with an amine nitrogen concentration of 250-300 mg% is used as a sensitive biological system, with the addition of V-growth factor as a source 2% yeast extract. The medium is freed using flow centrifugation at 4000 rpm. The resulting bacterial suspension is resuspended with phosphate-buffered saline to the original volume, inactivated with 0.1% formaldehyde (in the form of an aqueous 36-40%) solution) at 37 ° C and constant stirring (60 rpm).

For the manufacture of the sorbed form of the proposed vaccine use inactivated purified antigenic material from strain No. 1231 P. multocida type A, obtained by submerged cultivation in a bioreactor AK-210, with infectious activity on rabbits 3.0 ± 0.25 LD ₅₀ / cm ³ , in an amount not less than 25.0 vol. %.

An associated vaccine for prophylactic immunization of rabbits against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2 has been obtained.

The essence of the invention is illustrated by examples of its use, which do not limit the scope of the invention.

Example 1. Preparation of the proposed vaccine

To prepare an associated vaccine against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of type 1 and 2, we used strains of viruses and bacteria: strain "Mixo / ARRIAH-18", strain "VGBK1 / ARRIAH", strain "VGBK2 / ARRIAH" and strain No. 1231 P multocida type A.

The technological process consists of the following technological stages:

obtaining viral suspensions from strains "Mixo / ARRIAH-18", strain "VGBK1 / ARRIAH", strain "VGBK2 / ARRIAH" and bacterial suspension of strain No. 1231 P. multocida;

determination of the activity of each suspension;

lyophilization of viral material from the attenuated strain "Mixo / ARRIAH-18" separately;

inactivation of virus and bacteriological liquids of the strains "VGBK1 / ARRIAH", "VGBK2 / ARRIAH" and No. 1231 P. multocida separately;

combining the obtained inactivated suspensions of viruses and bacteria;

adding an adjuvant to the mixture of inactivated viruses and bacteria;

packaging of the finished product - lyophilized attenuated antigen of the Mixo / ARRIAH-18 strain separately from the mixed combined inactivated sorbed components.

The inoculum used for the manufacture of the vaccine was obtained in continuous cell culture, nutrient medium and naturally susceptible animals (rabbits).

For the manufacture of a vaccine associated against myxomatosis, pasteurellosis, viral hemorrhagic disease of rabbits of types 1 and 2, we used a cultured attenuated virus of the Mixo / ARRIAH-18 strain grown in 1.5 dm mattresses; 10% hepatic suspension containing the virus "VGBV1 / ARRIAH" VGBV type 1; 10% hepatic suspension containing the virus "VGBK2 / ARRIAH" VGBV type 2, bacterial suspension of strain No. 1231 P. multocida, grown in a bioreactor AK-210.

The proposed vaccine has an optimal component composition, vol. %:

- Dry component:

1. Antigenic material from the Mixo / ARRIAH-18 strain of the rabbit myxomatosis virus, reproduced in a continuous culture of RK-13/91 cells, with an infectious activity of at least 6.5 ± 0.25 lg TCD ₅₀ / cm ³ in an amount of 91, 8 vol. %.

2. The stabilizing medium, in the amount of 8.8 vol. %.

- Liquid component:

3. Antigenic material from the VGBK 1 / ARRIAHV strain of type 1 hemorrhagic disease virus, obtained from a 10% hepatic suspension of a rabbit infected with the type 1 hemorrhagic disease virus type 1, with no infectious activity on rabbits. less than 4.0 ± 0.25 lg LD ₅₀ / cm ³ , in an amount of 12.5 vol. %.

4. Antigenic material from the strain "VGBK2 / ARRIAH" of the virus of rabbit hemorrhagic disease of type 2, obtained from a 10% hepatic suspension of a rabbit infected with the strain "VGBK2 / ARRIAH" of the virus of hemorrhagic disease of rabbits type 2, with infectious activity on rabbits of at least 5 , 25 ± 0.25 lg LD ₅₀ / cm ³ in an amount of 12.5 vol. %.

5. Antigenic material from strain No. 1231 Pasteurella multocida type A, obtained by submerged cultivation in a laboratory bioreactor AK-210, in a liquid nutrient medium prepared on the basis of Hottinger tryptic hydrolyzate of meat with a concentration of amine nitrogen 250-300 mg%, with the addition as source of V-growth factor 2% yeast extract, with infectious activity in rabbits 3.0 ± 0.25 LD ₅₀ / cm ³ , in the amount of 25.0 vol. %.

6. Adjuvant 3% aluminum hydroxide gel in an amount of at least 50.0 vol. %.

The content of antigenic materials in the proposed vaccine in the above concentrations is their effective amount in the preparation, ensuring the achievement of the technical result from the use of the invention.

The resulting vaccine is:

- lyophilized dry homogeneous mass of light or dark beige color;

- beige-brown liquid with sediment. When shaken, it easily breaks up into a homogeneous suspension.

Vessels in which CPV of rabbit myxomatosis virus was noted with lesions of 70-80% of the cell monolayer were tested for sterility. Selected vessels (V = 1.5 dm ³ ) with cell culture RK-13/91 containing the virus strain "Mixo / ARRIAH-18", passed control for the absence of contamination, were once frozen and thawed at room temperature, while the cells of the vaccinated suspension were disaggregated , and then the suspension of the melted monolayer was shaken. The mattresses were poked horizontally three times over a period of two hours. The virus-containing culture suspension from all vessels was combined, the total sample was re-plated on bacterial media to exclude contamination with microbes, fungi and mycoplasmas, after which samples were taken to determine the infectious activity.

The contents of the vessels, with a 10% vaccinated suspension of rabbit liver homogenate, containing the strains "VGBK 1 / ARRIAH" and "VGBK2 / ARRIAH" were poured into separate containers and cooled at a temperature of 2-8 ° C, freeing from cellular debris in a known manner, used chloroform and low speed centrifugation. The suspension, freed from detritus, should have the form of a translucent liquid from light brown to dark brown with a reddish tint.

The contents of the AK-210 reactor, with submerged cultivation of strain No. 1231 Pasteurella multocida type A, during the entire cultivation time was checked for sterility and growth of the bacterial mass. When the concentration of bacterial cells is not less than 10 billion m.c / cm in “5, 6 hours, the contents of the reactor were sterilely poured into sterile butyls for cooling to 2-8 ° C. By the method of flow centrifugation, the nutrient medium was removed. The resulting bacterial cell pellet was resuspended in phosphate-buffered saline with pH 7.4-7.6. to the original volume and pumped into a vessel for inactivation. The concentration of bacteria in the suspension was determined visually using a turbidity standard.

Then, samples were taken from the containers to control viral raw materials.

A series of virus strain "Mixo / ARRIAH-18" myxomatosis of rabbits must have an infectious activity of at least 5.0 lg TCD ₅₀ / cm ³ .

A series of virus strain "VGBV1 / ARRIAH" VGBV type 1 must have antigenic activity not lower than the minimum positive value - 0.300 in optical density;

A series of virus strain "VGBK2 / ARRIAH" VGBV type 2 must have antigenic activity not lower than the minimum positive value of 0.300 in optical density;

The production batch of the strain No. 1231 Pasteurella multocida type A should have antigenic activity ≥1: 200 in ELISA.

Lyophilization of the viral suspension of the Mixo / ARRIAH-18 strain of rabbit myxomatosis with the addition of a stabilizing medium 8.8 vol. %, packaged in vials, was carried out using a lyophilizer. The virus suspension with a stabilizer was cooled in a freezer to minus 45 ° C. Then the frozen material in cassettes was placed in a sublimation chamber, which was hermetically closed and a vacuum pump was turned on. The amount of vacuum in the apparatus was from 4 Pa to 7 Pa. The initial temperature of the shelves with the material to be dried was in the range from minus 50 ° C to minus 55 ° C. The final temperature of the shelves during the drying process is from 30 ° C to 40 ° C. The gradient of the shelf temperature increase from the initial to the final value varied from 10 to 15% per second. The final temperature of the dried material ranged from 20 ° C to 23 ° C. Drying of the material at the specified temperature was carried out from 4 to 6 hours.

Inactivation of viral raw materials was carried out by adding aminoethylethyleneimine (AEEI): added to the viral suspension of the VGBK1 / ARRIAH strain, VGBK1 type 1 0.3% concentration at an exposure of 72 h and an inactivation temperature of 37 ° C; and for the strain "VGBK2 / ARRIAH" VGBK type 2 was injected with 1% concentration at an exposure of 24 h and a temperature of 37 ° C. Inactivation was carried out with periodic stirring (every 5-6 hours for 3-5 minutes)

The bacterial suspension of P. multocida strain No. 1231 was inactivated by adding 0.1% formaldehyde at 37 ° C and constant stirring (60 rpm).

At the end of the inactivation, the antigenic material of each strain was cooled to a temperature of 2-8 ° C and samples were taken to determine pH, sterility, and completeness of inactivation.

To neutralize AEE, sodium thiosulfate was added to the viral suspension of the antigenic material at the rate of 10% to the volume of the used AEE.

The obtained antigenic material of the VGBK1 / ARRIAH strain, VGBK2 / ARRIAH strain, P. multocida strain No. 1231 was used to prepare a sorbed form of a vaccine against myxomatosis, pasteurellosis, viral hemorrhagic disease of rabbits of types 1 and 2.

For this, the antigenic material is thoroughly mixed with a 3% aluminum hydroxide gel in a quantitative ratio of 12.5: 12.5: 25.0: 50.0, respectively.

The vaccine was monitored for stability, sterility and antigenic activity.

The sterility of the vaccine is determined in accordance with GOST 28085-89 “Biological preparations. Methods for biological control of sterility ". The essence of the method is to determine the absence of growth of bacterial and fungal microflora in crops from vaccine samples on nutrient media.

The control of the lyophilized part was carried out for residual moisture after lyophilization, control of biological activity in the cell culture, absence of contamination of the strain with other viruses by conventional methods. The contents of the vials in the form of a reconstituted suspension were plated on the following media: MPA (meat-peptone agar), MPB (meat-peptone broth), MPPB (meat-peptone liver broth), Sabouraud agar (liquid), and thioglycol medium. To exclude contamination with mycoplasmas, inoculations were made on liquid Kagan medium with 0.3% agar and Kagan semi-solid medium with 1.3% agar; 3 passages were carried out with an interval of 4-7 days. In the crops, no growth of bacterial, fungal microflora and mycoplasmas was observed during the established observation periods.

To determine the residual moisture content was carried out in accordance with GOST 24061-89 "Dry biological preparations. Moisture determination method ". For testing, 3 vials with the strain "Mixo / ARRIAZZH-18" VMC were used. A sample of a biological product, crushed to a powdery state, was introduced into a pre-weighed weighing bottle in an even layer. Samples and lids were weighed and placed in an open oven in an oven. The start of the final drying was considered the time to reach a temperature of 105 ° C, which lasts 1 hour. After finishing the drying, the weighing bottles were covered with lids and transferred to a desiccator to reach room temperature, after which they were weighed to the fourth decimal place. The moisture content of each of the samples was calculated with an accuracy of two decimal places. The arithmetic mean of three parallel determinations was taken as the final result. The mass fraction of moisture in the sample of the strain "Mixo / ARRIAH-18" VMK was 3.9%, which meets the requirements of ND.

Control of biological activity was carried out in a continuous culture of RK-13/91 cells by determining the titer of biological activity of the strain "Mixo / ARRIAH-18". The essence of titration is based on the determination of the cytopathogenic action (CPE) of the virus, which manifested itself in the formation of numerous symplasts containing 20 or more nuclei without fusion into a single syncytium. The nuclei in the cells were tightly packed, and the nucleoli were well contrasted, which indicated their functional activity. The titre of the virus was considered its highest dilution, causing CPP in 50% of the cell culture.

Tests for the biological activity of the strain "Mixo / ARRIAH-18" IUD were carried out according to the generally accepted titration technique using successive 10-fold dilutions of the test sample. For this purpose, it was diluted from 10 ^-1 to 10 ^-8 on a mixture of PSS + Igla (2: 1) media with 2% cattle blood serum. For titration of the strain "Mixo / ARRIAH-18" IUD used 1-2 day monolayer of cell culture RK-13/91, grown in penicillin flasks. The titer of the virus was determined by the method of Kerber in the modification of Ashmarin. The titer of biological / infectious activity of the strain "Mixo / ARRIAH-18" IUD, after lyophilization was 6.5 ± 0.25 lg TCD ₅₀ / cm ³ .

The freeze-dried part of the vaccine (dry component) containing the attenuated virus strain "Mixo / ARRIAH-18" IUD was monitored for harmlessness and avirulence in naturally susceptible animals (rabbits) according to GOST 31926. A sample of the strain "Mixo / ARRIAH-18" dose of rabbits not immune to myxomatosis. The contents of three vials were dissolved in saline to the original volume before lyophilization and combined. A dilution of vaccinated material containing 3.0 lg ID ₅₀ / cm ³ IUD was prepared from the mixture. The drug was injected into three rabbits (C, 10 ^-1 and 10 ^-2 ) intramuscularly in the thigh area by 1.0 cm and intradermally at 0.25 cm at four points (two on the left and two on the right) into the hairless area of the skin of the back and sides, for which cut the wool on the sides of the animals with the left and right stripes perpendicular to the spine. The assessment of the general condition of the animals and the manifestations of clinical signs of myxomatosis was carried out within 28 days. Lyophilized drug from the strain "Mixo / ARRIAH-18" IUD is harmless, because during the observation period, all rabbits remained alive and clinically healthy.

The liquid part of the vaccine was monitored for completeness of inactivation (avirulence), harmlessness and packed in sterile vials.

The completeness of inactivation of the drug is determined by a generally known method, similar to the definition of harmlessness.

The harmlessness of the vaccines was determined on rabbits of two months of age, weighing at least 2.5 kg, in the amount of 4 heads of rabbits at the rate of two heads per vaccine sample and two control animals (not vaccinated). The liquid part of the vaccine of the invention was injected intramuscularly into the thigh area in a volume of 3 cm ³ , which is 3 times the recommended inoculation volume. Clinical observation of the animals was carried out for 10 days. According to the results of the safety check, the animals remained alive during the entire observation period. It is known that local and general reactions, depending on the toxic effect of the vaccine, are most pronounced after the first injection of the drug, while allergic properties appear during repeated vaccination.

The reactogenicity of the sorbed liquid part of the vaccine was studied on rabbits of two months of age, weighing at least 2.5 kg, using three heads per test preparation. Observation of the animals for 10 days after the introduction of the second dose of the vaccine did not reveal any deviations from the general state of the animals' organism, which indicates the harmlessness and areactogenicity of the liquid part of the vaccine.

The liquid component of the vaccine must be sterile, avirulent and harmless.

The harmlessness and areactogenicity of the associated vaccine against IUD, pasteurellosis, VGBV1 and VGBK2 were tested on rabbits over one month of age weighing over 1 kg, not vaccinated against IUD, pasteurellosis, VGBK1 and VGBK2. The contents of five vials with a dry component were combined with the contents of five vials with a liquid component of the vaccine, mixed, received an average sample and injected into three rabbits intramuscularly at 3.0 cm ³ (3-fold inoculation dose). The animals were observed for 10 days. During the entire observation period, all the animals remained alive, no deviations from the physiological norm were observed in the condition of the rabbits. No necrotic changes were recorded at the injection site. The absence of death of rabbits after vaccination additionally indicated the absence of residual virulence of the vaccine components.

Example 2. The use of the proposed associated vaccine for immunization of rabbits

The proposed vaccine consists of 2 components:

- dry component - lyophilisate for preparation of suspension for injection (made from a continuous culture of rabbit kidney cells RK-13/91 infected with an attenuated rabbit myxomatosis virus strain "Mixo / ARRIAH-18", with the addition of stabilizing components (% on dry matter): hydrolyzate lactalbumin (4%), sucrose (4%) and gelatose (0.8%));

- a liquid component - a mixture of antigens obtained from a suspension of the liver of rabbits infected with the virus of rabbit hemorrhagic disease type 1 strain "VGBK1 / ARRIAH" and type 2 strain "VGBK2 / ARRIAH" inactivated by aminoethylethyleneimine (AEEI) and cells of bacteria Pasteurella multocida type A strain 1231, inactivated with formaldehyde, with the addition of 3% aluminum hydroxide gel as an adjuvant.

In appearance, the dry component is a homogeneous porous mass of light or dark beige color, completely dissolving in the liquid component of the vaccine within 2-3 minutes without the formation of flakes. The liquid component is a beige-brown liquid with a precipitate that easily breaks down when shaken into a homogeneous suspension.

In a vial with a dry component of the vaccine, containing 5 vaccine doses of the vaccine, observing the rules of asepsis, 5.0 cm ^{3 of the} liquid component was introduced using a syringe and thoroughly shaken.

The vaccine was administered to rabbits intramuscularly at a dose of 1.0 cm ³ in the area of the middle third of the thigh in compliance with the rules of asepsis.

The vaccine induces the formation of an immune response to myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits 10-14 days after a single use, lasting about 12 months against myxomatosis and viral hemorrhagic disease of rabbits of types 1 and 2 and at least 6 months against pasteurellosis.

Example 3. Efficiency of vaccination with the proposed vaccine The efficacy of the vaccine associated against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits type 1 and 2, made as described in example 2, is shown in table 2.

The test of the immunogenic activity of the obtained preparation was carried out on 73 rabbits from 4-5 weeks of age to 2 months of age not vaccinated against myxomatosis, HBV and pasteurellosis, and 8 rabbits were left unvaccinated as a control. Sampling for obtaining blood sera was carried out before vaccination, 14 and 21 days after it. Blood sera were examined by ELISA for the presence of antibodies to IUD viruses, VGBV and pasteurellosis. The results of studies of antibody titers for each vaccine component are shown in Table 2.

The data in Table 2 indicate that the titers of antibodies against IUD, VGBV and rabbit pasteurellosis after 14 days significantly increased compared with the initial values, exceeded the required minimum titers to all antigenic components of the vaccine, and remained at a high level by 21 days of observation. Antibody titers to all antigenic components in the control group remained approximately at the same level throughout the observation period.

The data indicate that the resulting vaccine has a high antigenic activity and causes the formation of high titers of antibodies to the Myxomatosis virus of rabbits "Mixo / ARRIAH-18" in rabbits, and the increase in antibody titers was constant throughout the observation period and reached a maximum value of 1: 295 21 days after vaccination.

The data indicate that the associated vaccine induces in vaccinated rabbits the formation of high levels of antibodies to viruses "HBV1 / ARRIAH" and "HBV2 / ARRIAH". The maximum level of antibody titers was observed in rabbits 21 days after vaccination and amounted to 0.429; 0.478 (optical density), respectively.

The data indicate that the obtained preparation has high antigenic activity for the antigenic component of P. multocida strain No. 1231 type A and causes the formation of an antibody titer in rabbits at a level of 1: 325 21 days after vaccination.

21 days after vaccination in all groups after diagnostic slaughter, the site of administration of the proposed vaccine was examined, the result of which was the absence or insignificant amount of vaccine residues, inflammatory changes or areas of necrosis in them.

Thus, the vaccine associated against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2 has high immunogenic and antigenic activity against all 4 antigenic components and is harmless to rabbits.

Example 3. Determination of the antigenic activity of the myxomatosis virus

An average sample of the vaccine was prepared, the contents of five vials with a dry component were combined with the contents of five vials with a liquid component of the vaccine, mixed, an average sample was obtained and injected into five rabbits, 1.0 cm each (one inoculated dose) intramuscularly in the thigh area. A control group of two rabbits were left unvaccinated and housed separately. The animals were clinically observed for 21 days, after which individual blood samples were taken to determine serum antibody titers by ELISA. Serum samples from vaccinated and non-immune (control) groups of rabbits were examined by ELISA.

A vaccine is considered antigenically active if, 21 days after vaccination, at least 80% of vaccinated rabbits have an antibody titer of at least 1: 190 in ELISA.

Example 4. Determination of antigenic activity of strain No. 1231 Pasterurella multocida type A

The combined sample of the vaccine prepared as in example 3 was injected intramuscularly into the area of the inner side of the right thigh to 5 rabbits once, 1.0 cm ³ (one inoculation dose). Before vaccination and 21 days after vaccination, 2-3 cm ^{3 of} blood was taken from the ear vein from the animals, kept at a temperature of (37.0 ± 0.5) ° С, blood serum was separated and examined for the presence of antibodies.

The vaccine is antigenic, the titer of specific antibodies in ELISA to P. multocida in rabbits 21 days after vaccination was at least 1: 200.

Example 5. Determination of the immunogenic activity of the virus of the strains "VGBK1 / ARRIAH" and "VGBK2 / ARRIAH"

Breakdown of the vaccine obtained according to example 3, eight rabbits were vaccinated intramuscularly in the area of the inner side of the thigh, 1.0 cm ³ (one inoculated dose). A control group of six rabbits were left unvaccinated and housed separately. 7 days after immunization, all vaccinated and control rabbits were infected intramuscularly in the thigh area with 1.0 cm ³ at a dose of 3 lg LD ₅₀ / cm ^{3 with} control viruses "VGBK1 / ARRIAH" and "VGBK2 / ARRIAH" (4 vaccinated and 3 control rabbit with each virus). The animals were observed for 10 days.

All vaccinated rabbits survived (100% protection). In the control group, two out of three animals fell ill and died.

Example 6. Control of the immunogenicity of the proposed vaccine for the component VGBV1 and VGBV2

Conducted on rabbits from 4-5 weeks of age to 2 months of age, not vaccinated against myxomatosis and VGBV, using various associated vaccines: VGBK1 + VGBK2 + IUD; VGBK1 + VGBK2 + P.m. and VGBK1 + VGBK2 + VMC + P.m. (proposed invention).

Used 38 rabbits. The animals were kept in isolators with autonomous watering and feeding. Mutual contamination with strains "VGBK1 / ARRIAH" and "VGBK2 / ARRIAH" was excluded. When infected, the animals were injected intramuscularly with 1.0 cm ^{3 of} vaccinated suspension, and their physiological state was monitored daily for 7 days. Vaccine samples were prepared as follows:

- associated vaccine against HBV1 + HBV2 + IUD.

The lyophilized part of the vaccine against IUD (2 cm ³ ) was dissolved in 5 cm ^{3 of the} inactivated vaccine against VGBK1 + VGBK2, containing 2.5 cm ^{3 of the} strains “VGBK1 / ARRIAH” and “VGBK2 / ARRIAH”. The vaccine was injected intramuscularly at 1.0 cm ³ ;

- associated vaccine against HBV1 + HBV2 + P.m. An inactivated vaccine was prepared containing 1.25 cm ^{3 of} the VGBK1 / ARRIAH strain; 1.25 cm ^{3 of} the VGBK2 / ARRIAH strain; 2.5 cm ³ strain No. 1231 P. multocida type A. The vaccine was injected intramuscularly at 1.0 cm ³ ; VGBK1 + VGBK2 + P.m. + IUD (present invention).

Lyophilized dry vaccine against IUD (2 cm ³ ) was dissolved in 5 cm ^{3 of} inactivated liquid vaccine containing strains “VGBK1 / ARRIAH” and “VGBK2 / ARRIAH” and strain No. 1231 P. multocida type A. The vaccine was administered intramuscularly at 1.0 cm ³ .

To test the immunogenicity of the associated vaccine for the HBV1 and HBV2 components, 5 groups were formed. Associated vaccines with different component composition were administered to three groups of animals (10 heads each), two groups (4 heads each) were used as controls. On the 14th day after vaccination, animals of all groups, including the control group, were injected with the strains "VGBK 1 / ARRIAH" and "VGBK2 / ARRIAH" at a dose of 3.0 lg LD ₅₀ / cm ³ per head, intramuscularly, 1.0 cm ³ ... The research results are presented in table 3.

From the data in Table 3 it can be seen that the deaths of animals were recorded only in the control groups. In the experimental groups, all the animals survived. There were no abnormalities in the physiological state.

Thus, it was concluded that samples of associated vaccines containing the component - HBV1 and HBV2, protect immunized rabbits with direct control infection by 100% from homologous strains of HBV, i.e. the vaccine (invention) for this component is immunogenic.

Example 6. Control of the immunogenicity of the proposed vaccine by component P. multocida

To test the immunogenicity of the associated vaccine (the invention) for the P. multocida component, four groups were formed. Associated vaccines with different component composition were administered to three groups of animals (10 animals each), one group (10 animals) was used as a control. The scheme for making vaccine samples was similar to the scheme presented in the previous experiment. On the 14th day after vaccination, the animals of all groups, including the control, were injected with strain No. 1231 P. multocida type A with an infection dose of 3.0 LD ₅₀ / cm ³ per 1 head. The results of the studies are presented in Table 4. It can be seen from the data in the table that in the group of rabbits vaccinated with the associated vaccine, which did not include the P. multocida component, 100% deaths of animals were observed, while in the group immunized with the vaccine with the bacterial component ( proposed invention), the safety of animals was 100%.

It is revealed that the samples associated vaccines comprising in its composition components - P. multocida, protect rabbits immunized with direct control the infection at a dose of 3,0 lg LD ₅₀ / cm ³ to 100% of the homologous strain of P. multocida, i.e. the vaccine for this component is immunogenic and provides a protective level of antibodies.

Sources of information taken into account in the preparation of the description of the invention to the application for the grant of a patent of the Russian Federation for the invention "Associated vaccine against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits of types 1 and 2":

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Claims (14)

1. Vaccine associated against myxomatosis, pasteurellosis and viral hemorrhagic disease of rabbits, containing an active substance and a targeted additive, characterized in that it contains two components as an active substance: a liquid component - a mixture of inactivated antigenic material from strain No. 1231 Pasteurella multocida, seed ... Pasteurellaceae, genus Pasteurella, deposited in the collection of strains of microorganisms of the FGBI "ARRIAH" under registration number No. 307 - dep / 20-95 POIM FGBU "ARRIAH"; from inactivated and purified antigenic material from the strain "VGBK1 / ARRIAH" viral hemorrhagic disease of rabbits, this. Caliciviridae, genus Lagovirus, type 1, deposited in the collection of strains of microorganisms of the FGBI "ARRIAH" under registration number №305 - dep / 20-93 - GKShM FGBU "ARRIAH"; from inactivated and purified antigenic material from the strain "VGBK 2 / ARRIAH" viral hemorrhagic disease of rabbits, family. Caliciviridae, genus Lagovirus, type 2, deposited in the collection of microorganism strains of the FGBI "ARRIAH" under the registration number №306 - dep / 20-94 - GKShM FGBU "ARRIAH"; and a dry component - lyophilized attenuated purified antigenic material from the Mixo / ARRIAH-18 strain of the rabbit myxomatosis virus, the Poxviridae family, the Leporipoxvirus genus, deposited in the collection of microorganism strains of the ARRIAH under registration number 120 - dep / 19-10 - KSHM FGBI "ARRIAH"; and targeted additives: 3% aluminum hydroxide gel and a stabilizing medium, taken in a volume ratio of 1.0: 0.5: 0.5: 0.9: 2.0: 0.1, respectively, and in amounts that provide a protective immunogenic activity of each antigen in the body of rabbits after administration of the target drug. 2. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the strain "VGBK1 / ARRIAH" viral hemorrhagic disease of rabbits, family. Caliciviridae, genus Lagovirus, type 1, obtained preferably from a 10% liver suspension of a rabbit infected with the "VGBK1 / ARRIAH" strain of rabbit haemorrhagic disease virus type 1, which is a viral suspension in an amount of 12.5 vol.%. 3. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the strain "VGBK2 / ARRIAH" viral hemorrhagic disease of rabbits, family. Caliciviridae, genus Lagovirus, type 2, obtained preferably from a 10% liver suspension of a rabbit infected with the "VGBK2 / ARRIAH" strain of rabbit hemorrhagic disease virus type 2, which is a viral suspension in an amount of 12.5 vol.%. 4. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the strain No. 1231 Pasteurella multocida type A, obtained by submerged cultivation in a laboratory bioreactor AK-210, in a liquid nutrient medium prepared on the basis of a tryptic hydrolyzate of meat according to Hottinger with an amine nitrogen concentration of 250-300 mg%, with the addition of 2% yeast extract as a source of V-growth factor, which is a bacterial suspension with infectious activity on rabbits 3.0 ± 0.25 LD ₅₀ / cm ³ , in the amount of 25.0 vol.%. 5. The vaccine according to claim 1, characterized in that as an adjuvant it contains 3% aluminum hydroxide gel in an amount of at least 50 vol.%. 6. The vaccine according to claim 1, characterized in that it contains dry attenuated antigenic material from the Myxo / ARRIAH-18 strain of the rabbit myxomatosis virus, preferably obtained in a transplantable cell culture RK-13/91, which is an attenuated lyophilized viral suspension with infectious activity not less than 6.5 ± 0.25 lg TCD ₅₀ / cm ³ and infectious activity on rabbits not less than 3.0 ± 0.25 lg ID ₅₀ / cm ³ , in an amount of 91.8 vol.%. 7. The vaccine according to claim 1, characterized in that it contains a stabilizing medium of: lactalbumin hydrolyzate (GLA) - 4%, sucrose - 4%, gelatose - 0.8%, in an amount of 8.8 vol.%. 8. The vaccine according to any one of paragraphs. 1-7, characterized in that it contains a mixture of inactivated and purified antigenic materials from the VGBK1 / ARRIAH strain of the rabbit haemorrhagic disease virus type 1, preferably obtained from a 10% liver suspension of a rabbit infected with the VGBK1 / ARRIAH virus strain hemorrhagic disease of rabbits of type 1, with an infectious activity of at least 4.0 ± 0.25 LD ₅₀ / cm ³ ; from the strain "VGBK2 / ARRIAH" of the virus of hemorrhagic disease of rabbits type 2, with infectious activity of at least 5.25 ± 0.25 LD ₅₀ / cm ³ ; from strain No. 1231 Pasteurella multocida type A, with infectious activity of at least 3.0 ± 0.25 LD ₅₀ / cm ³ ; lyophilized antigenic material from the attenuated strain "Mixo / ARRIAH-18" of the rabbit myxomatosis virus, with an infectious activity of at least 6.5 ± 0.25 lg TCD ₅₀ / cm ³ and targeted additives: a stabilizing medium and an oil adjuvant in an amount, vol.% : Dry component: - Lyophilized antigenic material from strain "Mixo / ARRIAH-18" virus 91.2 rabbit myxomatosis - Stabilizing environment 8.8 Liquid component: Antigenic material from the strain "VGBK1 / ARRIAH" hemorrhagic virus rabbit disease type 1 12.5 Antigenic material from the strain "VGBK2 / ARRIAH" hemorrhagic virus rabbit disease type 2 12.5 Antigenic material from strain No. 1231 Pasteurella multocida type A 25.0 Adjuvant 3% Aluminum Hydroxide Gel 50.0

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