RU2708891C1 - Vaccine for rota-, coronaviral infection and cattle colibacillosis - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention refers to veterinary science, namely to virology, and can be used to produce a vaccine for rota-, coronaviral infection and cattle colibacillosis. Vaccine as an active substance contains a mixture of inactivated suspension of rotavirus strain RM, family Reoviridae, genus Rotavirus, inactivated suspension of the coronavirus KL-2 strain of the Coronaviridae family, of the genus Coronavirus, with activity of each of viral strains 10^5.5 - 10^6.5 TCDD₅₀ in cm³ a vaccine grown in a transplantable cell culture of MDVK or LEC, and adhesive antigens K99, F41, Att25 of Escherichia coli bacteria; purification of adhesive antigens K99, F41, Att25 of Escherichia coli bacteria taken in equal ratio (1:1:1), with concentration 50–100 ml of microbial cells in 1 cm³ vaccines are carried out with concentration of the solution of phosphate-urea buffer increased to 1.5–3.0 M.

EFFECT: using the given invention enables to obtain an associated trivalent vaccine exhibiting high immunogenicity in association with adhesive Escherichia coli strains.

1 cl, 4 ex, 4 tbl

Description

The present invention relates to veterinary microbiology and virology.

The vaccine contains the active substance and targeted additives.

As an active substance, the vaccine contains a mixture of an inactivated suspension of a strain of rotavirus PM, family Reoviridae, genus Rotavirus (certificate of deposit No. 193/19 of 02/04/2004), an inactivated suspension of a strain of coronavirus KL-2 of the family Coronaviridae, genus Coronavirus (certificate of deposit No. 3064/37 dated 06/02/1989 g), with the activity of each of the viral strains 10 ^5.5 - 10 ^6.5 TCD ₅₀ in cm ^{3 of} vaccine and adhesive antigens of Escherichia coli bacteria taken in equal proportion with a concentration of 50-100 mld. microbial cells in 1 cm ³ vaccine.

The vaccine is recommended for use in farms that are unsuccessful for intestinal diseases of viral and bacterial etiology, such as rota-, coronavirus and colibacteriosis of calves.

Colibacteriosis (Escherichiosis) is a worldwide widespread infectious disease of newborn farm animals, manifested in the form of diarrhea, sepsis and accompanied by high morbidity, mortality and a decrease in weight gain in sick animals.

On the other hand, mass enteritis and enterocolitis of newborn calves caused by rota-, coronaviruses have a high incidence with E. coli. In this regard, great importance is attached to the active immunization of mother cows with the aim of obtaining from them colostrum and milk with a high level of antibodies against rota-, coronavirus and colibacteriosis for

feeding newborn calves and creating passive immunity to these infections.

Microbial adhesion is one of the main pathogenicity factors. 60% of E. coli strains circulating on farms, the causative agents of colibacteriosis, have various adhesive antigens and their combinations. Adhesins K99, F41, Att25 are most often found in calves. Active protection against adhesion factors of microorganisms with the help of specific drugs can be especially effective for intestinal infections of animals, especially those caused by E. coli.

In our country, a series of commercial adsorbed inactivated antiviral vaccines were proposed - against rotavirus enteritis of calves "ROTA-WAC VIEV", vaccine inactivated aluminum hydroxide against coronavirus enteritis in cattle "KOR-WAK VIEV", vaccine associated inactivated against rotavirus and coronavirus diarrhea of cattle, “RODICOR-VAK VIEV” (Gulukin MI, Mnikova LA, Ishkova TA, Sokolova NL, Zhidkov SA Development and implementation in practice of effective su- diagnostics and prophylaxis of viral gastro-intestinal diseases in cattle // Proceedings of the All-Russian Research Institute of Experimental Veterinary Medicine. YR Kovalenko. 2015. T. 78. S. 179-186).

A number of vaccines based on adhesive antigens have been developed. In particular, the VIEV proposed a vaccine against colibacteriosis of young farm animals based on adhesive antigens K99, K88av, K88as, K88ad, F41, Att25, 987P, as well as thermostable (ST) and thermolabile (LT) enterotoxin esherichia conjugate (Sokolova N.A. , Golovko A.N., Kurashvili T.K. et al. A method for producing a vaccine against colibacteriosis of farm animals, patent RU 2053792, C1, 02/10/1996).

Vaccines had high immunogenic properties and wide

spectrum of action on individual pathogens of colibacillosis of young farm animals - calves, piglets and lambs.

A multivalent vaccine with adhesive antigens against colibacteriosis (Escherichiosis) of calves is known, which includes 50% of the antigenic part of E.coli A20 (Att25), E.coli F41, E.coli K88 (F4) E.coli K99 (F5) and 50% adjuvant Montanide ISA-206 (Amosova L.A., Lomako Yu.V., Novikova O.N., Zubovskaya I.V., Karpovich V.K., Pukslis A.I. Experimental studies on the development of technology for the manufacture and use of the multivalent vaccine with adhesive antigens against colibacteriosis (Escherichiosis) of calves / Epizootology, immunobiology, pharmacology, sanitation. - 2015. - No. 1. - S. 3-7.).

An associated vaccine was developed against coronavirus and rotavirus infection, colibacteriosis and calostridiosis of calves - which contains inactivated bovine rotaviruses and coronaviruses, inactivated adhesion-producing E. coli strains with K99 and F41 antigens, Clostridium perfringens type C beta beta toxoid (C).

All available commercial vaccines include an incomplete spectrum of causative agents of diarrhea of newborn calves, or not all specific adhesive antigens characteristic of causative agents of Escherichiosis. In these vaccines, an inactivated cell suspension is used as a bacterial component, which is a mixture of cell wall antigens that are not responsible for the colonizing ability of bacteria.

The associated inactivated aluminum hydroxide vaccine against rota-, corona, herpesvirus and Escherichia diarrhea of newborn calves is also known (Gaffarov Kh.Z., Spiridonov Ch.N., Ivanov A.V. et al. Patent RU 2137499, C1, 10.02.2000)

The associated vaccine has low antigenic and immunogenic activity of vaccine strains, and as a result, the lack of immunity in

animals to Escherichiosis, proceeding in the form of colidia and colienterotoxemia.

The closest in technical essence, selected as a prototype, is a vaccine inactivated against viral diarrhea, rota-, coronovirus disease, and bovine Escherichiosis (Patent RU No. 2403063 C1; A61K 39/295, A61P 31/00; 08/28/2009) ( prototype).

The combined vaccine has a relatively low titer of immunogenic activity of the viral suspensions included in its composition, the presence of Escherichia cells and congestion with antigens of its composition.

The objective of the research was to obtain a vaccine with high immunogenic properties, an expanded prophylactic spectrum of action on intestinal infections of calves of viral and bacterial etiology.

We have proposed a vaccine associated inactivated against rotavirus, coronavirus enteritis and bovine Escherichiosis.

A comparative analysis of the proposed method for producing the vaccine and the prototype shows that the proposed vaccine is characterized in that it combines the purified adhesive antigens K99, F41, Att25 with viral material, and the adhesins themselves are obtained using increased to 1.5-3.0 M concentration of a solution of phosphate-urea buffer.

The invention consists in the following.

The composition of the vaccine includes cattle rotavirus strain PM, grown in a transplanted cell culture of MDVC or LEK; cattle coronavirus, strain KL-2 grown in the same cultures and purified adhesins of E. coli strains - K99, F41, Att25. The vaccine is inactivated by formalin and adsorbed onto aluminum hydroxide.

The vaccine against rota-, coronavirus infection, and bovine Escherichiosis is a light yellow liquid; during storage, a loose white precipitate forms, which easily breaks up into a homogeneous suspension with shaking.

The vaccine is stable and remains active at a temperature of 2-8 ° C for at least 12 months.

The vaccine is administered to pregnant animals twice for 40 and 20 days before calving in a volume of 5 ml subcutaneously in the middle third of the neck or in the inguinal fold.

Example 1

The technological process consists of several stages: the stage of obtaining the viral and Escherichia components, the addition of an adjuvant, the mixing of all components of the vaccine with the subsequent packaging of the finished product.

A. Stage of obtaining the viral component of the vaccine.

The cultivation of the company and coronaviruses is carried out in LEK or MDVK cell cultures simultaneously or in several cycles with the gradual accumulation of viral material, stored until the vaccine is manufactured at -10-20 ° C. Each batch of rota-, coronaviruses is checked for sterility by plating on nutrient media, and activity is studied in ELISA (not less than 1:16) for rota- and in RGA (not less than 1: 512) for coronavirus. Suspensions containing a separate company and coronavirus inactivate formalin at a final concentration of 0.05%.

After inactivation, the antigenic activity of a suspension of a strain of rotavirus PM should be 10 ^5.5 - ^{6.5 6.5} TCD ₅₀ in cm ³ vaccines, suspensions of a strain of coronavirus KL-2 10 ^5.5 - 10 ^6.5 TCD ₅₀ in cm ^{3 of} vaccine.

B. The stage of obtaining the bacterial component of the vaccine.

Selecting strains producing adhesive antigens K99, F41, Att25 (reference or field cultures of E. coli) that react in the agglutination reaction with standard monospecific anti-adhesive serums in titers of 1: 320 - 1: 1280 and higher, possessing immunogenic properties. Inoculated into Hottinger broth or any other medium that promotes the expression of adhesive antigens. Cultivation is carried out at 37-38 ° C for 6-8 hours when shuttling. Received

the broth culture is centrifuged at 3000-5000 rpm for 30-40 minutes at 4-10 ° C. The supernatant is removed and the precipitate resuspended in a 1.5-3.0 M solution of phosphate-urea buffer to a concentration of 50 × 10 ⁹ -100 × 10 ⁹ mic. cells / ml The resulting suspension is heated in a water bath at 60-64 ° C for 20-25 minutes with periodic shaking (60 U / min), after which it is cooled and centrifuged at 10,000-15,000 rpm for 10-20 minutes.

The supernatant is poured into a sterile dish, inactivated by adding 0.3-0.4% formalin and kept at 37-38 ° C for 24 hours, after which it is checked for sterility. Aluminum hydroxide is added to a final concentration of 25%.

B. Stage of combining the viral and bacterial components of the vaccine.

To obtain a complex preparation, the adhesin and virus components prepared in this way are taken in equal proportions (1: 1: 1) and mixed in a sterile container, i.e. 1 cm ^{3 of the} vaccine contains 1 part of a suspension of a strain of rotavirus PM, 1 part of a strain of coronavirus KL-2 and 1 part of adhesive bacteria antigens of Escherichia coli. In turn, the adhesive antigens of bacteria Escherichia coli K99, F41, Att25 are also taken in equal proportions.

The prepared vaccine is packaged in sterile glass bottles and sealed. Store 12 months. at a temperature of + 2-8 ° C.

Methods for producing adhesive antigens for a vaccine tested in laboratory mice.

Option 1. The method of obtaining the vaccine is carried out as described above, but to separate the adhesive antigens from bacteria, use a 1.5 M concentration of a solution of phosphate-urea buffer.

The resulting preparation, when administered once to white mice, stimulates the production of antibodies to adhesins in titers of 1: 160-1: 640 (in the agglutination reaction).

Option 2. The method of obtaining the vaccine is carried out as described above, but to obtain individual adhesive antigens using a 2.0 M concentration of a solution of phosphate-urea buffer.

Such a preparation, when administered once to white mice, stimulates the production of antibodies to adhesins in titers of 1: 640-1: 1280 (in RA).

Option 3. The method of obtaining the vaccine is carried out as described above, but to obtain a purified adhesive antigens using a 3.0 M concentration of a solution of phosphate-urea buffer.

Example 2. Evaluation of the antigenic activity of the vaccine in laboratory animals.

The resulting preparation during a single immunization of white mice stimulates the production of antibodies to adhesins in titers of 1: 320-1: 1280 (in RA).

In the rat experiment (Table 1), the antigens included in the vaccine were administered without adjuvants in three variants of rotavirus (PB) and adhesive antigens of E. coli, coronavirus (KB) and adhesive antigens of E. coli, as well as all antigens subcutaneously together in the back 3 ml twice with an interval of 20 days.

Blood was taken 14, 21 and 28 days after the second immunization and examined for the presence of specific antibodies by ELISA for viruses and RA for adhesive antigens. At all stages of the study, an increase in titers of antibodies to antigenic components of the vaccine was noted. To viral

components of the vaccine together with adhesive antigens of E. coli, titers reached a value of 1: 6400 to RV and 1: 400 to KR with a decrease by the 28th day. Titles to adhesive antigens when used together with viruses were to K99 1: 320, to F41 1:80 and Att25 1: 1280, with a gradual decrease by the 28th day.

Guinea pigs and rabbits were given the trivalent vaccine with adjuvant intramuscularly twice with an interval of 7 days. 14 days after the first and second immunization, blood was taken to determine antibodies to the components of the vaccine.

The results of a study of the antigenic properties of the trivalent vaccine in guinea pigs and rabbits showed that administration of the vaccine to animals stimulates the formation of specific antibodies against all components of the vaccine (Table 2). It has been established that aluminum hydroxide is an effective adjuvant in the immunization of laboratory animals.

So, when examining the blood serum of guinea pigs and rabbits immunized with the vaccine by enzyme-linked immunosorbent assay, the following was established: titers of antibodies to the company, and to the coronaviruses after two vaccinations increased to a value of 1: 1600 - 1: 3200 with an initial reading of an average of 1: 200. Antibody titers to E. coli adhesive antigens in RA on day 14 after the second immunization in guinea pigs and rabbits reached for K99 1:80 and 1: 960, for F41 1: 448 and 1: 960, Att25 1: 1296 and 1: 800 respectively. The results obtained indicated that the fabricated

the laboratory vaccine series was characterized by pronounced antigenic properties.

Example 3. Production test of experimental series of vaccines in experiments on cows.

The immunogenic properties of the associated vaccine were studied on pregnant cows and calves in the farms of the Vologda Oblast in 2015-2017. During the trial, 76 pregnant cows were immunized with the associated vaccine; the control group consisted of 23 animals.

The vaccine was administered to animals 40 and 20 days before calving in a volume of 5-6 ml in the neck. In animals before vaccine administration, 40 and 20 days before the expected calving, blood serum samples were taken, and after calving, colostrum samples were taken on days 1, 2, and 3. Blood serum and feces of calves were selected on days 3 and 7 for testing for the presence of antibodies to rotavirus, and cattle coronaviruses and bacterial components of the vaccine. The results are shown in (Table 3).

It was established that immunization of pregnant cows significantly affects the accumulation of specific antibodies in the blood and colostrum of animals and does not adversely affect the physiological parameters of the body. So, before vaccination, the titer of antibodies in serum samples

cows was 1: 400-1: 800 for viruses and 1: 10-1: 80 for adhesins, 20 days before calving, the titer increased to 1: 1600-1: 3200 for viruses and up to 1: 278-1: 1105 for adhesins.

In colostrum of the first day, the highest titer of specific antibodies was noted. So, to the rota and coronaviruses, antibody titers reached a value of 1: 25600, to the adhesive antigen K99-1: 3595, to F41-1: 2324, to Att25-1: 7498.

Compared to colostrum from control animals, the titers of antibodies to adhesins increased by 17-268 times.

Colostrum on the 2nd and 3rd day of antibodies contained significantly less antibodies, but their titers still exceeded the control values by 8-100 times.

Example 4. Production test of experimental series of vaccines in experiments on calves.

The immunogenic properties of the vaccine were also determined by the degree of incidence of newborn calves obtained from vaccinated and control cows. Colostrum was fed to calves 2-4 hours after birth. The calves were monitored and clinically examined daily. In this case, the number of specific antibodies obtained by the animal with colostrum of the mother, the frequency, duration, severity of calf disease, was taken into account. As a result of the studies, a direct correlative relationship was established between the number of antibodies in the colostrum of cows and their presence in the blood serum and feces of calves fed with immune colostrum. So, in the serum of 3 day old calves from immunized cows, antibody titers against adhesins K99, F41, Att25 were 13, 10, and 32 times higher than in control animals, respectively (Table 4). The feces of calves fed colostrum from vaccinated cows contained antibodies in 65% of cases, and only one calf showed antibodies in the feces of calves in the control group. The titers of coproantibodies to adhesive antigens in calves from immunized cows were 6-28 times higher than the titers in control animals.

About 20% of calves from vaccinated cows during the first week

fell ill with the clinical manifestation of diarrhea, however, the disease proceeded in a mild form, lasted no more than 3-4 days and ended in recovery.

In control calves obtained from non-immunized cows, the disease proceeded for a long time, in acute form, accompanied by severe diarrhea with a lot of mucus, dehydration, fever, and exhaustion. Of all the cases, 4 calves fell (17.3%).

As a result of the studies, the immunogenic properties of the trivalent vaccine against rotavirus-coronavirus infections and calf colitis were studied and the protective properties of colostrum from cows vaccinated with the trivalent vaccine and their effect on calf resistance were given.

Based on the results, normative and technical documentation was developed and approved for the trivalent associated vaccine against rotavirus, coronavirus infections, and calf colienteritis, including instructions for the manufacture of the vaccine, instructions for its use, and STO (standard organization of the Federal State Budgetary Institution of Higher VET).

Thus, the developed vaccine will make it possible to properly organize measures for the prevention and control of gastrointestinal diseases of calves. The use of the vaccine will significantly reduce the incidence and departure of calves, facilitate the course of the disease, and reduce the possibility of infection in healthy calves. The developed vaccine corresponds to the level presented to the drugs of this

destinations, since it contains the rota-, coronavirus components of viruses that exhibit high incidence in association with adhesive strains of E. coli, and purified adhesins provide a significantly higher level of antibodies compared to using a whole bacterial cell or its other components.

The vaccine was tested with a positive result on cattle in dysfunctional farms. This confirms the harmlessness of the drug, the increased immunogenic activity of the components of the vaccine in comparison with other drugs, expressed in the induction of the immune response in mother cows to different variants of the antigens of pathogens circulating on the farm.

Testing results indicate that the vaccine against rota-, coronavirus infection, and Escherichiosis in cattle has high immunogenic properties, provides the creation of intense colostral immunity, and has protective properties.

Given the widespread spread of gastrointestinal diseases in Russian farms and the need to vaccinate a large number of animals, the economic effect of using the vaccine being developed can be significant.

List of references

1. Gulyukin M.I., Mnikova L.A., Ishkova T.A., Sokolova N.L., Zhidkov S.A. Development and implementation of an effective system for the diagnosis and prevention of viral gastrointestinal diseases in cattle // Proceedings of the All-Russian Research Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko. 2015.V. 78.P. 179-186.

2. Sergeev V.A., Nepoklonov E.A., Alipper T.I. et al. Inactivated vaccine against viral diarrhea, rota-, coronovirus disease, and bovine Escherichiosis / RU patent No. 2403063 C1; A61K 39/295, A61P 31/00, 08/28/2009. Prototype.

3. Sokolova N.A., Golovko A.N., Kurashvili T.K. and others. A method of obtaining a vaccine against colibacteriosis of farm animals / patent RU 2053792, C1, 02/10/1996.

4. Amosova L.A., Lomako Yu.V., Novikova O.N., Zubovskaya I.V., Karpovich V.K., Pukslis A.I. Experimental studies on the development of technology for the manufacture and use of a multivalent vaccine with adhesive antigens against colibacteriosis (Escherichiosis) of calves / Epizootology, immunobiology, pharmacology, sanitation. - 2015. - No. 1. - C. 3-7.

5. Gaffarov Kh.Z., Spiridonov Ch.N., Ivanov A.V. and other Associated vaccine against rota-, corona-, herpes virus and Escherichia diarrhea of newborn calves / patent RU 2137499, C1, 02/10/2000.

Claims (2)

1. A vaccine against rota-, coronavirus infection, and bovine Escherichiosis, comprising an active substance and target additives, characterized in that as an active substance it contains a mixture of an inactivated suspension of a strain of rotavirus PM, family Reoviridae, genus Rotavirus, an inactivated suspension of a strain of coronavirus CL -2 of the Coronaviridae family, of the genus Coronavirus, with the activity of each of the viral strains 10 ^5.5 - 10 ^6.5 TCD ₅₀ in cm ^{3 of the} vaccine grown in the transplanted culture of MDVC or LEC cells and adhesive antigens K99, F41, Att25 of Escherichia c bacteria oli; purification of adhesive antigens K99, F41, Att25 of Escherichia coli bacteria taken in equal proportions (1: 1: 1), with a concentration of 50-100 mld microbial cells in 1 cm ^{3 of the} vaccine, is carried out with an increase to 1.5-3.0 M concentration of a solution of phosphate-urea buffer. 2. The vaccine according to claim 1, characterized in that it contains an aluminum hydroxide adjuvant as a target additive.