The associated vaccine against rota-, corona-, herpes and exericises diarrhea of newborn calves

 

(57) Abstract:

The invention is intended for the manufacture of a vaccine for prophylaxis of mixed infectious diarrhea of newborn calves. As a source of specific antigens using production strains Rota-, corona-, herpes viruses of cattle and E. coli synthesizing adhesive antigens C, A20. The strains are grown separately. Rotavirus - in transplantable cell cultures kidney pig (line SPM). Coronaviruses in transplantable culture of kidney cells of the embryo cows (line MDVC). The herpes viruses in human cell cultures of tracheal embryo cows (TR) in roller units industrial type. E. coli synthesizing adhesive antigen C, A20 - on environment minka and mesopatamia agar. Collection Rota-, herpes viruses carried out 24-48 hours of coronaviruses - through 72 - 96 h, E. coli after 24 - 36 hours Inactivation of viruses carried out with 0.5% formalin at 25-27oC for 24-36 h Culture of E. coli inactivate with 0.5% formalin for 72 h at 36-37oC. Mixed inactivated culture. To the resulting mixture add 6% solution gel of aluminium hydroxide in the amount of 10%. Prophylactic vaccines injected deep intramuscularly is and can increase the level of accumulation of specific protective antibodies in the serum and colostrum from vaccinated cows and save newborn calves due celastrales immunity. 3 table.

The invention relates to the field of veterinary Virology, Microbiology and biotechnology, in particular, to the production of vaccines for specific prophylaxis of mixed infectious diarrhea of newborn calves, the main etiological agents of which is recognized as the Rota-, corona-, herpes viruses and enteropathogenic strains of E. coli., synthesizing adhesive antigens C and A20.

Literature information in recent years and our accumulated knowledge suggests that the mass enteritis and enterocolitis of newborn calves with clinical signs of diarrhea caused Rota-, corona-, herpes viruses, and E. coli synthesizing adhesive antigens C and A20, which is able to cause symptoms of diarrhea, followed by General intoxication and dehydration.

In addition, it appeared certain etiological significance in this form of the disease in calves of parvovirus, a virus diarrhea, bovine, chlamydia, some bacteria, Cryptosporidium and coccidia (C. Mebus et. al., Amer. Vet. Med. Assoc., 1973, 163, N 7, 2, 880-883; Moussa A. et. al. - Rec. Med. Veter., 1983, 159, 3, 185-190; Raisers Using, F., Jones M,A., Kryukov N. N. "Infectious diseases in industrial farming", Moscow is. Sattorov I. T. - Actual problems of Virology and bacteriology. Proceedings of VIEW, T. 64, M, 1988, pp. 16-17).

The infectious process is compounded by the fact that these agents act on the naked body in combination with each other, causing a mixed infection (Wellemans, G. , Van Opdenbosh E., - Elevages belges, 1981, 35, 10, 10-11; Sizov, I. et al., - Wind. - honey. science, 1984, 21, 10, 89-94; Bondar, C. M. et al. - Moldovan research Institute of animal husbandry and veterinary, N 681-M, Kishinev, 1986; Morris J. A. et. al., - Veter. Rec., 1987, 121, 9, 189-191; Belenko L. C. , Virus diseases of farm animals (Abstracts of all-Russian scientific-practical conference), Vladimir, 1995, page 218).

Study of the characteristics of epizootic process when mixed form of diarrhoea showed that infected, usually 90-100% of newborn calves, mortality sick calves is achieved 27-55%. The emergence and spread of infectious diarrhea due to the high concentration of livestock, latent persistence of pathogens in the body of a cow, but cause acute infection in newborn calves, delayed breastfeeding full of colostrum and non-veterinary-sanitary regime in livestock pens.

Considering the above, the importance of prenatal, so as soon as antibodies, entered calves oral, protect them from disease. This provision defines the strategy and development of specific prevention. Consequently, the specific protection of newborn calves during the first days of life is carried out by active immunization of mothers in order to encourage their formation of antibodies, and transmit their colostrum. To this end immunized cows in the second half of pregnancy.

In 1982, the USDA announced the receipt and application of a new combination of trivalent vaccine against Rota-coronavirus and E. coli bacteria intended for the vaccination of pregnant cows and heifers. Extensive testing of this vaccine (Scour - Gnord -3) stimulate the formation of antibodies in mothers and facilitated transfer them with colostrum for newborn calves, thereby providing 100% effect of immunity in calves to enteritis mixed etiology (Anon, Scour, vaccine now availadle "Colorado Rancher Farmer" 1982, 10, 37, 28).

In Macedonski research Institute (Edinburgh, UK) created a combined vaccine of inactivated by formaldehyde rotavirus in calves and antigen C E. coli with oil adjuvant and applied intramuscularly d is at, born from vaccinated cows, were completely protected against colibacillosis in experimental infection. Effectiveness of rotavirus component of the combination vaccine was studied in field experiments. In groups of calves from vaccinated cows approximately twice reduced the number of calves suffering from diarrhoea, reducing the duration, increase the gain (Snodgrass D. R., Ann. Rec. Veter., 1983, 14.4V, 519-521; Snodgrass, D. R., - Veter. Rec. 1986, 119, 2, 39-42).

Such biological products have been developed and found to be effective in Germany, Austria, France, Czechoslovakia and other European countries).

In our country Transfiguration D. S., Sokolova N. L., Gogolev M. M., Mnichov L. A. studied the possibility of immunization globorotalia cows associated drugs Rota-coronavirus, with the goal of increasing titers of antibodies in the colostrum of cows to both viruses at the same time. Based on the research results, the authors concluded that there is increase in antibody titers colosio cows, and it is advisable to combine this vaccine formulation with other microbial vaccines from E. coli and Cl. perfringens (Bulletin VIEW, issue 66, M, 1988, pp. 21-26).

Known and available biological industry "Formation.A., Soloviev, C. S., Tugarinov, O. A., Veterinary drugs, Handbook, Moscow, Kolos, 1981, page p.223-224). However, the results of the use of this vaccine was found to be unsatisfactory (Dworkin, L., Lenkova Century A. in the book "problems of veterinary immunology", proceedings, Moscow, Agropromizdat, 1985, page 108).

Also known polyvalent gidrookisaljuminievuju formationally vaccine VGNKI against escherichiosis calves, which can cause significant immunological changes in body vaccinated cows.

Antimicrobial immunogenicity of these vaccines is low enough in it and the number To protective antigens. The vaccine is recommended for use not only pregnant cows, and calves, but with 3-10 to 17 to 20 days of age. The effectiveness of such vaccines is questionable due to the potential for neutralizing colostral antibodies to specific antigens of the vaccine.

The closest analogue is Associated vaccine against diarrhea of calves, which is a composition of 10 strains of enterobacteria, inactivated by ethanol and deposited on aluminium hydroxide. The strains in the vaccine composition selected from representatives of antirobe is N. F., RF patent N 2035916, 1993).

The disadvantage associated vaccine is not sufficiently high efficiency due to inferiority (incomplete) antigenic composition, its components. In the composition of vaccines antigens Rota-, corona-, herpes viruses of cattle, because in the etiology of outbreaks of diarrhoeal diseases calves in farms of the country is dominated by the mentioned viruses.

Therefore, the problem of creating a single associated vaccines against major pathogens of infectious diarrhea of newborn calves continues to be relevant.

The objective of the invention is to develop a harmless vaccine based on the synergistic Association of Rota-, corona-, herpes viruses, and E. coli synthesizing adhesive antigens C and A20 with the purpose of prophylaxis of diarrhea of newborn calves caused by mixed infection.

The technical result of the invention consists in obtaining the associated vaccine with high immunogenic activity and increased cost effect.

The invention consists in the following: the associated vaccine against Rota-, corona-, herpes virus diarrhea newborn bodies shall iheni C and A20, deposited on aluminium hydroxide in the following ratio of components,%:

Inactivated culture suspension:

strain of Bovine rotavirus "III-I" with a titer of 107,0- 107,8TCD50/ml - 27,0 - 30,0

strain Bovine coronavirus "CM-1" with title 105,0- 105,8TCD50/ml - 27,0 - 30,0

strain of Bovine Herpesvirus "TKA-VIEW-B2" with a titer of 107,6- 108,0TCD50/CL - 27,0 - 30,0

Inactivated suspension of cells of Escherichia coli containing adhesive antigen C with a concentration of 100-120 billion m K. in 1 ml of physiologic saline - 3,0 - 4,0

Inactivated suspension of cells of Escherichia coli containing adhesive A20 antigen concentration 100-120 billion, M. K. in 1 ml of physiologic saline - 3,0 - 4,0

Gel aluminum hydroxide, 6% solution - Rest.

Included in associated vaccine strains of viruses found and are productive in the manufacture of monovalent vaccines. From antigens of Escherichia vaccine contains any of the production or local strains of E. coli, actively producing adhesin C and A20.

Example 1. Preparation of native suspensions rotavirus, coronavirus and herpesvirus cattle.

Rotavirus bovine strain "III-I) multiply the year (line SPM) on the combined growth environment, consisting of hydrolyzed milk albumin (GLA) - 45%, from 199 - 45%, bovine serum at 10% and antibiotics. The culture is grown for 3-4 days at a seeding concentration of 100-120 thousand cells per 1 ml of medium. Before entering to culture the virus activates trypsin (0.6 ml of 0.25% trypsin in 100 ml of medium for 30-35 minutes at 37-38oC) in a ratio of 1: 10 to 1:12 and add it to the support medium without serum at a rate of 5 ml per 500 ml of medium, and 3% glutamine solution (5 ml per 500 ml of medium) and antibiotics.

Growth medium is drained, bring in bottles with culture maintenance medium in the amount of 350-370 ml and incubated at 37-38oC for 24-48 hours. After a pronounced destruction of the cell culture titers of virus should be 107,0- 107,8TCD50/ml. For the liberation of virus culture three freeze at minus 18-20oC and later quickly thawed at 36-37oC. the Obtained culture viral suspension clarify by centrifugation for 20 minutes at 2000 rpm

Coronavirus bovine (strain CM-1) was propagated in human culture of kidney cells of the embryo cows (line MDVC) grown in three-liter roller bottles at is. onosai is formed on 3-4 days when seeding concentration 120-150 thousand cells per 1 ml of medium.

For the seed of coronavirus bovine growth environment is drained, make a support medium (medium 199 (50%) and CHAP - 50%) in the number of 350-370 ml, which previously contributed virus based 15-20 ml per 500 ml of medium, of 0.25% trypsin 3 ml of a 3% solution of glutamine 5 ml and antibiotics. Bottles incubated in roller installed at 37-38oC for 3-4 days. After 72-96 hour cultivation at 37oC appears JRS 50%. Cytopathogenic action is characterized by granulation of the cytoplasm, the collapse of the isolated cells, the formation of simpleton with subsequent degeneration. Collecting the culture fluid is produced in a period of pronounced cytopathic effect. The titer of the virus should not be below 105,0- 105,8TCD50/ml.

The release of virus from the cells by freezing and thawing, received the virus suspension clarify by centrifugation.

Herpesvirus bovine strain "TKA-VIEW-B2") propagated in human cell cultures of tracheal embryo cows (TR) grown in three-liter roller bottles at combo is grown for 3-4 days at a seeding concentration of 140-150 thousand cells.

Growth medium is drained, make a support medium (medium 199 - 20%, CHAP 40% and the Needle MEM - 40%) in the number of 350-370 ml, in which pre-make the virus at the rate of 10 ml per 500 ml of medium, 3% solution of glutamine 5 ml and antibiotics. Bottles incubated in roller installed at 37-38oC for 3-4 days.

Example 2. The Association of viral suspensions in the series and its inactivation.

Received the virus suspension unite and to inactivate viruses add formalin to a concentration of active formaldehyde 37-38% (from a rate of 0.5 ml per 100 ml of viral suspension), mix and leave for days at room temperature, then tested for bacterial and fungal contamination. For preparation of the vaccine take viral fees without any contamination. Control of completeness of inactivation is carried out by giving reg OS 5 ml of vaccine preparation two newborn calves not receiving colostrum, the other two calves leave as a control. Vaccine formulation consider inaktivirovannye, if within 7 days after the administration of calves remain clinically healthy.

Example 3. Getting motroway the seed of each strain of E. coli.

As made in the heat of diarrhea of newborn calves used:

"KV-1" - synthesizing adhesive antigen C;

"PZ-3" - synthesizing adhesive A20 antigen;

who kill white mice weighing 14-16 g within two days after intraperitoneal infection at a dose of 0.5 cm3the suspension of the daily culture containing 1 billion m K. in cm3for bacterial or optical standard turbidity.

To obtain cultures of E. coli using nutrient - mastopathy agar (for strain synthesizing adhesive antigen A20) and the environment minka (strain synthesizing adhesive antigen C). The strain of these cultures of E. coli grown separately in appropriate nutrient media at a temperature of 37oC. After 24 hours, the grown colonies of E. coli examined in RA on the glass with monovalent agglutinating anti-adhesive coli-sera respectively with A20 and K to Refine the purity of the cultures.

Metrovia seed strains of E. coli are stored in lyophilized form or in semi-MPA under paraffin oil at a temperature of 4-6oC. To maintain active strains annually passedout through white mice.

Example 4. Cultivation of bacterial mass separately for each strain and its inactivation.

Minka (strain synthesizing adhesive antigen C). To this end the said medium is poured into Matri 200 cm3and sterilized with 0.5 atmospheric (110oC) for 15 minutes. After solidification of the media doing the sowing of these crops by 1.0 cm31 billion, M. K. Crops incubated at 37oC. After 24 hours the grown colony cultures of E. coli wash of 0.85% sodium chloride solution, preparing a suspension at a concentration of 100 billion, M. K. 1 cm3for bacterial or optical turbidity standard of gisk named after. Tarasevich. The culture is checked for purity, morphological and serological typicality.

Inactivation of bacterial suspension with 100 billion, M. K. 1 cm3is formalin. To do this in the microbial suspension add formaldehyde content 37-38% active formaldehyde (0.5 cm3100 cm3suspension). Mixed and placed in the incubator for 3 days. Then take the sample to control for completeness of inactivation, which is carried out by inoculation of inactivated microbial suspensions in appropriate nutrient media.

Example 5. Preparation of associated vaccine.

The vaccine is prepared from antigens Rota-, corona-, herpes viruses calves, containing a specific protein n>/P>Rota-, corona-, herpes virus suspension take in equal proportions and add 100 billion bacterial suspension of E. coli, based on 3 cm3each strain 100 cm3vaccine preparation, as well as 6% gel of aluminium hydroxide at the rate of 10 cm3100 cm3the vaccines. Bring the pH of the vaccine to 7.2-7.4 and Packed with periodic thorough mixing vials, which are then closed with rubber stoppers, running-metal caps and etecetera.

In table No. 1 shows the composition of the vaccine.

Example 6. Control vaccines for sterility, safety and antigenic activity.

To test for sterility of the 6 vials of vaccine each series make the crops in 0.5 ml BCH, MPA, MPB under vaseline oil and agar Saburo 2 tubes on each bottle. Crops with all the media incubated at 37oC, and with agar Saburo is at a temperature of 20-22oC for 15 days. Nutrient crops must remain sterile.

To test for harmlessness select the 10 white mice live weight of 17-18 g and introduce the vaccine in a dose of 0.5 cm3subcutaneously. The vaccine is considered harmless if the mouse within 10 days after the injection WACC the orders of the live weight of 2.0 to 2.5 kg, which the drug is administered subcutaneously at a dose of 2 cm3twice with an interval of 14-15 days. 14 days after the last vaccination in rabbits from the ear veins take blood and test serum with a view to establishing the level of specific antibodies against Rota-, corona-, herpesvirus, and esherichiosis antigens. The titers of antibodies to the company, the herpes viruses should be in the range of 1:800 and 1:1600 in ELISA, cov 1:128 to 1:256 in rtga and Escherichia coli 1:320 to 1:640 in RA.

The inspection results of experimental series of associated vaccine showed that all series of the biological product have been sterile, harmless and antigenically-active.

Production test pilot series of associated vaccine was conducted on globorotalia cows on two farms of the Republic of Tatarstan, troubled by mixed forms of infectious diarrhea of newborn calves. While the animals were divided into 2 groups: experimental and control. Animals of the experimental group, the vaccine was administered subcutaneously twice for 30-35 and 15-20 days to calving in the dose of 10 cm3. Animals of the control group, the vaccine was not introduced. The results of the tests are presented in table No. 2, from which it follows that the associated vaccine has expressed immune is a 20.2 to 35.8%, safety - 96,7 - 97,2%, whereas the calves of the control group, these values were of 90.6 - 100% and 68.5 - 79,1%, respectively.

Example 7. The purpose of General information and the application procedure associated vaccine against Rota-, corona-, herpes and escherichiosis diarrhea of calves.

The vaccine is intended to prevent diarrhea of newborn calves caused by a mixed infection due to Rota-, corona-, herpesvirus and enterotoxigenic strains of Escherichia producing factors of adhesion K and A20.

The vaccine represents an equal mixture of the suspension of antigens Rota-, corona-, herpes viruses, and cultures of E. coli containing antigens C and A20 in optimal proportions.

In appearance the vaccine is deposited by hydrooxysaluminium, opaque liquid with a white precipitate, which upon shaking the container can be easily broken, forming a homogeneous suspension pale pink color.

The vaccine is produced in glass bottles of 50, 100 and 200 cm3with tightly closed with rubber corks, rolled metal caps, and etecetera in accordance with the specifications.

The vaccine is suitable for use within 12 months from the date of manufacture, provided storage is farms, permanently affected with infectious diarrhea of newborn calves. The drug is injected in the middle third of the neck subcutaneously globorotalia cows and heifers twice before and 15-20 30-35 days before calving in the dose of 10 cm3.

The vaccine provides creation of newborn calves celastrales immunity to Rota-, corona-, herpes viruses and pathogens colibacillosis, producing relevant factors adhesion antigens C and A20.

For the period 1994 - 1998, manufactured 79200 doses associated vaccine and used in the farms of the 12 districts of the Republic of Tatarstan. 3 - the Chuvash Republic, 4 - Republic of Bashkortostan, 3 - Republic of Mari-El, affected with infectious diarrhea of newborn calves.

The associated vaccine against Rota-, corona-, herpes and exericises diarrhea of newborn calves, characterized in that as antigens Rota, corona and herpes viruses it contains inactivated culture suspensions of strains of Bovine rotavirus "III-1", Bovine coronavirus "CM-1", Bovine Herpesvirus "TKA-VIEW-B2" with titers of 107,0- 107,8TCD50/ml, 105,0- 105,8TCD50/ml, 107,6- 108,0from antigens of Escherichia - inactivated cell suspension ø the gel of aluminium hydroxide in the following ratio of components, vol.%:

Inactivated culture suspension: strain of Bovine rotavirus "III-1", with a titer of 107,0- 107,8TCD50/ml- 27,0 - 30,0

Strain Bovine coronavirus "CM-1", with a titer of 105,0- 105,8TCD50/ml- 27,0 - 30,0

Strain of Bovine Herpesvirus "TKA-VIEW-B2" with a titer of 107,6- 108,0TCD50/ml- 27,0 - 30,0

Inactivated suspension of cells of strain Ecsherichia coli containing adhesive antigen C with a concentration of 100 to 120 billion m K. in 1 ml of physiological solution - 3,0 - 4,0

Inactivated suspension of cells of strain Ecsherichia coli containing adhesive A20 antigen with a concentration of 100 to 120 billion m K. in 1 ml of physiological solution - 3,0 - 4,0

Gel aluminium hydroxide, 6% solution - Rest

 

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