RU2145236C1 - Mixed vaccine against rota-, corona-, herpesviral and escherichia diarrhea in newborn calves - Google Patents

Abstract

FIELD: veterinary science, vaccines. SUBSTANCE: invention relates to preparing vaccine used for specific prophylaxis of mixed infectious diarrheas in newborn calves. Industrial strains of rota-, corona-, herpes viruses of cattle and E. coli synthesizing adhesive antigens K99, A20 are used as source of specific antigens. Strains are cultured separately. Rotaviruses are cultured in pig kidney transplanted culture cells (strain SPEV). Coronaviruses are cultured in cow embryo kidney transplanted culture cells (strain MDVK). Herpes viruses are cultured in cow embryo trachea transplanted culture cells (TR) in roller units of industrial type. E. coli cells synthesizing adhesive antigens K99, A20 are cultured on Mink's medium and beef-extract agar. Rota- and herpes viruses are collected in 24-48 h, coronaviruses - in 72-96 h and E. coli cells - in 24-36 h. Viruses are inactivated with 0.5-% formalin solution at 25-27 C for 24-36 h. E. coli culture is inactivated with 0.5-% formalin at 36-37 C for 72 h. Inactivated cultures are mixed. To obtained mixture 6-% solution of aluminium hydroxide gel is added at amount 10-%. For prophylaxis aim vaccine is administrated in deep-pregnant cows and heifers (30-35 days before calving) twice at break for 14-15 days at the dose 10 cm³ by intramuscular route. Vaccine is able to elevate the level of accumulation of specific protective antibodies in vaccinated cows serum blood and colostrum and prevent calves death due to colostrum immunity. EFFECT: enhanced effectiveness of vaccine. 3 tbl, 7 ex

Description

 The invention relates to the field of veterinary virology, microbiology and biotechnology, in particular, to the production of a vaccine for the specific prophylaxis of mixed infectious diarrhea of newborn calves, the main etiological agents of which are rota, corona, herpes viruses and enteropathogenic strains of E. coli., Synthesizing adhesive antigens K99. and A20.

 The information available in the literature of recent years and the knowledge accumulated by us indicate that mass enteritis and enterocolitis of newborn calves with clinical signs of diarrhea are caused by rota-, corona-, herpes viruses and E. coli, which synthesize adhesive antigens K99 and A20, which can independently cause symptoms diarrhea accompanied by general intoxication and dehydration.

 In addition, a certain etiological significance was found in this form of pathology in calves of parvovirus, cattle diarrhea virus, chlamydia, some bacteria, cryptosporidia and coccidia (Mebus C. 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; Koromyslov G.F., Sidorov M, A., Kryukov N. H. "Infectious Diseases in Industrial Cattle Breeding", Moscow, 1980, pp. 1-169; Hall GA et. Al. - Res. In Veter. Se., 1988, 45, 2, 240-250; Pospisil Z. et. al., Zbl. Veter. Med. Reihe, 1979, 26, 4, 325-335; Sokolova N.L., Mnikova L.A., Sattorov I.T. - Actual problems of virology and bacterio ogii. VIEV Transactions, v. 64, Moscow, 1988, pp. 16-17).

 The infectious process is compounded by the fact that these pathogens act on a defenseless organism in combination with each other, causing a mixed infection (Wellemans G., Van Opdenbosch E., - Elevages belges, 1981, 35, 10, 10-11; Sizov I. et al. ., - Veter. - medical science, 1984, 21, 10, 89-94; Bondar V.M. et al., - Moldavian Research Institute of Livestock and Veterinary Medicine, N 681-M, Chisinau, 1986; Morris JA et. Al ., - Veter. Rec., 1987, 121, 9, 189-191; L. Belyanko, Viral diseases of agricultural animals (Abstracts of the All-Russian Scientific and Practical Conference), Vladimir, 1995, p. 218).

 The study of the features of the epizootic process with a mixed form of diarrhea showed that, as a rule, 90-100% of newborn calves become infected, mortality of sick calves is reached 27-55%. The occurrence and spread of infectious diarrhea is due to a high concentration of livestock, latent persistence of pathogens in the body of cows, but causing acute infections in newborn calves, untimely feeding of full colostrum, and non-observance of the veterinary-sanitary regime in livestock buildings.

 Based on the foregoing, great importance is attached to the consumption of colostrum calves with a high content of specific antibodies for at least two weeks, since only antibodies administered to the calves orally protect them from the disease. This provision determines the strategy for the development of specific prophylaxis. Therefore, the specific protection of newborn calves in the first days of life is carried out by active immunization of mothers in order to stimulate their formation of antibodies and ensure their transmission in colostrum. To this end, vaccine cows in the second half of pregnancy.

 In 1982, the USDA announced the receipt and use of a new combined trivalent vaccine against rota-, coronaviruses, and E. coli bacteria, designed to vaccinate pregnant cows and heifers. Extensive testing of this vaccine (Scour-Gnord-3) stimulated the formation of antibodies in mothers and facilitated their transmission with colostrum to newborn calves, thereby providing a 100% effect of immunity in calves to mixed etiology enteritis (Anon, Scour, vaccine now availadle "Colorado Rancher Farmer "1982, 10, 37, 28).

 A combination vaccine from formaldehyde-inactivated calf rotavirus and E. coli K99 antigen with oil adjuvant was created at the Moredun Research Institute (Edinburgh, UK) and was used intramuscularly to immunize pregnant cows. The vaccine stimulated the secretion of specific antibodies in colostrum and milk. Calves born from vaccinated cows were completely protected against colibacteriosis during experimental infection. The effectiveness of the rotavirus component of the combination vaccine was studied in field experiments. In groups of calves from vaccinated cows, the number of calves with diarrhea decreases by about half, its duration decreases, weight gain increases (Snodgrass DR, - Ann. Rec. Veter., 1983, 14,4, 519-521; Snodgrass DR, - 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, Preobrazhensky D.S., Sokolova N.L., Gogolev M.M., Mnikova L.A. studied the possibility of immunization of deep-skinned cows with the associated preparations of rota-, coronaviruses in order to increase antibody titers in colostrum of cows against both viruses simultaneously. Based on the results of the studies, the authors came to the conclusion that an increase in antibody titers in the colosse of cows is ensured, and it is advisable to combine this vaccine preparation with other microbial vaccines from E. coli and Cl. perfringens (VIEV Bulletin, issue 66, M., 1988, pp. 21-26).

 It is known and produced by the biological industry "Formolthiomersal vaccine against colibacteriosis and paratyphoid of fur animals, birds, calves and piglets" (Schuster B.Yu., Malakhov Yu.A., Soloviev V.S., Tugarinov O.A., - Veterinary preparations, Handbook, Moscow, Kolos, 1981, pp. 223-224). However, the results of the use of this vaccine were found to be unsatisfactory (Dvornin G.L., Lenkova V.A. in the book "Problems of Veterinary Immunology", Scientific Works, Moscow, Agropromizdat, 1985, p. 108).

 Also known polyvalent aluminum hydroxide formolthiomersal vaccine VGNKI against calves escherichiosis, which can cause significant immunological changes in the body of vaccinated cows.

 The antimicrobial immunogenicity of these vaccines is low, and the amount of protective K antigens is insufficient in it. The vaccine is recommended for use not only for pregnant cows, but also for calves from 3-10 to 17-20 days of age. The effectiveness of such vaccinations seems doubtful because of the possible neutralization of colostral antibodies with specific vaccine antigens.

 The closest analogue is the "Associated vaccine against calf diarrhea", which is a composition of 10 strains of enterobacteria inactivated with ethanol and deposited on aluminum hydroxide. The strains in the vaccine are selected from representatives of enterobacteria: 3 Escherichia strains, two Klebsiels, and two proteins (Lavrovskaya V.M., Voronin E.S., Devrishchev D.A., Brusnigina N.F., RF Patent N 2035916, 1993).

 A disadvantage of the known associated vaccine is not high enough efficiency due to the inferiority (incompleteness) of the antigenic composition of its components. The vaccine does not contain antigens of rota-, corona, and herpes viruses in cattle, as the etiology of outbreaks of diarrheal diseases of calves in the country's farms is dominated by the aforementioned viruses.

 Therefore, the problem of creating a single associated vaccine against the main causative agents 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 K99 and A20 for the specific prevention of diarrhea of newborn calves caused by mixed infection.

 The technical result of the invention is to obtain an associated vaccine with high immunogenic activity and increased costly effect.

The essence of the invention is as follows: the associated vaccine against rota-, corona, herpesvirus diarrhea of newborn calves contains antigens of rota-, corona, herpes viruses of cattle, E. coli, synthesizing adhesive antigens K99 and A20, deposited on aluminum hydroxide in the following ratio components, vol.%:
Inactivated culture suspensions:
Bovine rotavirus strain "III-I" with a titer of 10 ^7.0 - 10 ^7.8 TCD ₅₀ / ml - 27.0 - 30.0
strain Bovine coronavirus "SM-1" with a titer of 10 ^5.0 - 10 ^5.8 TCD ₅₀ / ml - 27.0 - 30.0
strain Bovine Herpesvirus "TKA-VIEV-B2" with a titer of 10 ^7.6 - 10 ^8.0 TCD ₅₀ / cell - 27.0 - 30.0
Inactivated cell suspension of the strain Escherichia coli containing adhesive antigen K99 with a concentration of 100-120 billion mk in 1 ml of physical solution - 3.0 - 4.0
Inactivated cell suspension of the strain Escherichia coli containing adhesive antigen A20 with a concentration of 100-120 billion, mk in 1 ml of physical solution - 3.0 - 4.0
Gel aluminum hydroxide, 6% solution - The rest.

 The strains of viruses included in the associated vaccine are sophisticated and are industrial in the manufacture of monovalent vaccines. Of the antigens Escherichia vaccine contains any of the production or local strains of E. coli, actively producing adhesins K99 and A20.

 Example 1. The preparation of native suspensions of rotavirus, coronavirus and herpes virus of cattle.

Cattle rotavirus (strain III-I) is propagated in three-liter roller bottles with a grown single-layer transplanted culture of pig embryonic kidney cells (SPEV line) on a combined growth medium consisting of lactoalbumin hydrolyzate (GLA) - 45%, from medium 199 - 45%, cattle serum - 10% and antibiotics. The culture is grown for 3-4 days at a sowing concentration of 100-120 thousand cells per 1 ml of medium. Before introducing into the culture, the virus is activated with trypsin (0.6 ml of 0.25% trypsin per 100 ml of medium for 30-35 minutes at 37-38 ^o C) in a ratio of 1: 10 - 1:12 and add it to the support serum-free medium at the rate of 5 ml per 500 ml of medium, as well as a 3% solution of glutamine (5 ml per 500 ml of medium) and antibiotics.

The growth medium is discarded, 350–370 ml of support medium is introduced into the culture bottles and incubated at 37–38 ^° C. for 24–48 hours. After a pronounced destruction of the cell culture, the virus titer should be 10 ^7.0 - 10 ^7.8 TCD ₅₀ / ml. To free the virus, the culture was frozen three times at minus 18-20 ^° C and then thawed quickly at 36-37 ^° C. The resulting viral culture suspension was clarified by centrifugation for 20 minutes at 2000 rpm.

 Cattle coronavirus (strain "SM-1") is propagated in a transplanted culture of kidney cells of a cow embryo (MDVC line) grown in three-liter roller bottles on a combination medium consisting of 199-60% medium, 30% GLA and large fetal serum cattle - 10%. A monolayer is formed on the 3-4th day at a seed concentration of 120-150 thousand cells per 1 ml of medium.

For breeding of cattle coronavirus, the growth medium is drained, a support medium (medium 199 - 50% and GLA - 50%) is added in an amount of 350-370 ml, into which the virus is preliminarily added at the rate of 15-20 ml per 500 ml of medium, 0, 25% trypsin 3 ml, 3% glutamine solution - 5 ml and antibiotics. The bottles are incubated in a roller unit at 37-38 ^o C for 3-4 days. After 72-96 hours of growth at 37 ^° C., a 50% CPD is manifested. The cytopathogenic effect is characterized by granulation of the cytoplasm, decay of isolated cells, the formation of symplasts with subsequent degeneration. The collection of culture fluid is carried out during a period of pronounced cytopathic effect. The titer of the virus should be at least 10 ^5.0 - 10 _5.8 TCD ₅₀ / ml.

 The virus is released from the cells by freezing and thawing, the resulting viral suspension is clarified by centrifugation.

 Cattle herpesvirus (strain "TKA-VIEV-B2") is propagated in a transplanted culture of cells of the tracheal cells of a cow embryo (TP) grown in three-liter roller bottles on a combined medium consisting of 199 - 10% medium, GLA - 40%, Igla medium MEM - 40% and cattle serum - 10%. The culture is grown for 3-4 days at a sowing concentration of 140-150 thousand cells.

The growth medium is drained, a support medium is introduced (medium 199 - 20%, GLA - 40% and MEM Needle - 40%) in an amount of 350-370 ml, into which the virus is preliminarily added at the rate of 10 ml per 500 ml of medium, 3% glutamine solution - 5 ml and antibiotics. The bottles are incubated in a roller unit at 37-38 ^o C for 3-4 days.

 Example 2. Combining viral suspensions in a series and its inactivation.

 The resulting viral suspensions are combined and formalin with an active formaldehyde content of 37-38% (based on 0.5 ml per 100 ml of the viral suspension) is added in order to inactivate the viruses, mixed and left for 24 hours at room temperature, then checked for bacterial and fungal contamination . To prepare the vaccine take viral fees without contamination. The completeness of inactivation is controlled by administering 5 ml of the vaccine preparation to two newborn calves that have not received colostrum, and the other two calves are left as a control. A vaccine preparation is considered inactivated if the calves remain clinically healthy for 7 days after administration.

 Example 3. Obtaining seed brood of each strain of E. coli.

As production strains in the manufacture of the associated vaccine against rota-, corona-, herpesvirus and Escherichia diarrhea of newborn calves using:
"KV-1" - synthesizing adhesive antigen K99;
"PZ-3" - synthesizing adhesive antigen A20;
which cause the death of white mice weighing 14-16 g within two days after intraperitoneal infection in a dose of 0.5 cm ^{3 with a} suspension of daily culture containing 1 billion m. in cm ³ according to the bacterial or optical standard of turbidity.

To obtain E. coli cultures, nutrient media are used - meat-peptone agar (for the strain synthesizing the adhesive antigen A20) and Mink's medium (for the strain synthesizing the adhesive antigen K99). The strain of these cultures of E. coli is grown separately on appropriate nutrient media at a temperature of 37 ^o C. After 24 hours, the grown colonies of E. coli are examined in RA on glass with monovalent agglutinating anti-adhesive coli serums with A20 and K99, respectively, to clarify the purity of the cultures.

The maters of E. coli strains are stored in a lyophilized form or in semi-liquid MPA under liquid paraffin at a temperature of 4-6 ^o C. To maintain the active state, the strains are passaged annually through white mice.

 Example 4. The cultivation of the bacterial mass separately for each strain and its inactivation.

To obtain biomass of E. coli cultures, MPA (for a strain synthesizing adhesive antigen A20) and Mink's medium (for a strain synthesizing adhesive antigen K99) are used. To this end, these media are poured into mats of 200 cm ³ and sterilized at 0.5 atmospheric (110 ^o C) for 15 minutes. After solidification of the media do the sowing of these cultures at 1.0 cm ³ 1 billion m Crops are kept in an incubator at 37 ^o C. After 24 hours, the grown colonies of E. coli cultures are washed off with a 0.85% sodium chloride solution, and a suspension is prepared at a concentration of 100 billion m. in 1 cm ³ according to the bacterial or optical standard turbidity GISK them. Tarasevich. Cultures are checked for purity, morphological and serological typicality.

Inactivation of a bacterial suspension having 100 billion cubic meters in 1 cm ³ is carried out by formalin. For this, formalin with a content of 37-38% active formaldehyde (0.5 cm ³ per 100 cm ^{3 of} suspension) is added to the microbial suspension. Stirred and put in a thermostat for 3 days. Then take a sample to control the completeness of inactivation, which is carried out by seeding inactivated microbial suspensions on the appropriate nutrient medium.

 Example 5. Preparation of an associated vaccine.

 The vaccine is prepared from antigens of rotavirus, corona, and herpes viruses of calves containing a specific protein of at least 30-35 mg / mm and strains of E. coli, "P3-3" and "KV-1" producing adhesive antigens A20 and K99, respectively.

The rota, corona, and herpes virus suspensions are taken in equal proportions and a 100 billion suspension of E. coli is added to them, based on the calculation of 3 cm ^{3 of} each strain per 100 cm ^{3 of the} vaccine preparation, as well as 6% aluminum hydroxide gel from calculating 10 cm ³ per 100 cm ^{3 of} vaccine. The pH of the vaccine is adjusted to 7.2-7.4 and packaged with periodic thorough stirring into bottles, which are then closed with rubber stoppers, wrapped in metal caps and labeled.

 Table No. 1 shows the composition of the vaccine.

 Example 6. The control of the vaccine for sterility, safety and antigenic activity.

To test for sterility, from 6 bottles of vaccine of each series, 0.5 ml inoculations are made on MPB, MPA, MPPB under liquid paraffin and Saburo agar, 2 tubes per bottle. Crops with all media are kept in a thermostat at 37 ^o C, and with Saburo agar - at a temperature of 20-22 ^o C for 15 days. Culture media with crops should remain sterile.

To test for harmlessness, 10 white mice with a live weight of 17-18 g are selected and a vaccine is administered at a dose of 0.5 cm ³ subcutaneously. The vaccine is considered harmless if the mice remain alive and clinically healthy for 10 days after administration of the vaccine.

The antigenic activity of the vaccine is monitored in 3 rabbits with a live weight of 2.0-2.5 kg, to which the drug is administered subcutaneously at a dose of 2 cm ³ twice with an interval of 14-15 days. 14 days after the last vaccination, blood is taken from the ear vein in rabbits and the serum is examined in order to establish the level of specific antibodies to the company-, corona-, herpesvirus, and also escherichia antigens. Antibody titers to rota, herpes viruses should be in the range of 1: 800 - 1: 1600 - in ELISA, to coronavirus 1: 128 - 1: 256 in RTGA and Escherichia coli 1: 320 - 1: 640 in RA.

 The results of the control of the experimental series of the associated vaccine showed that all series of the biological product were sterile, harmless and antigenically active.

A production test of the experimental series of the associated vaccine was carried out on deep-shelled cows of two farms of the Republic of Tatarstan, dysfunctional in mixed forms of infectious diarrhea of newborn calves. In this case, the animals were divided into 2 groups: experimental and control. The animals of the experimental group, the vaccine was administered subcutaneously twice for 30-35 and 15-20 days before calving at a dose of 10 cm ³ . No vaccine was administered to animals of the control group. The test results are presented in table No. 2, from which it follows that the associated vaccine has a pronounced immunogenic activity: in the groups of calves obtained from vaccinated cows, the incidence of calves ranged from 20.2 to 35.8%, safety - 96.7 - 97.2 %, while in calves of the control group, these indicators were 90.6 - 100% and 68.5 - 79.1%, respectively.

 Example 7. The purpose, general information and procedure for the use of the associated vaccine against rotavirus, corona, herpes virus and sherichiosis diarrhea of calves.

 The vaccine is intended for the prevention of diarrhea of newborn calves caused by a mixed infection caused by company-, corona-, herpesvirus, and enterotoxigenic Escherichia strains producing adhesion factors K99 and A20.

 The vaccine is an equal mixture of a suspension of antigens of company-, corona-, herpesviruses, as well as E. coli cultures containing K99 and A20 antigens in optimal proportions.

 In appearance, the vaccine deposited with aluminum hydroxide is an opaque liquid with a white precipitate, which, when the bottle is shaken, easily breaks up, forming a homogeneous suspension of a pale pink color.

The vaccine is produced in glass bottles of 50, 100 and 200 cm ³ with tightly closed rubber stoppers, rolled in metal caps, and labeled in accordance with TU.

The vaccine is suitable for use within 12 months from the date of its manufacture, subject to storage and transportation at a temperature of 2-10 ^o C.

The vaccine is used for prophylactic purposes in farms that are permanently unfavorable for infectious diarrhea of newborn calves. The drug is injected into the middle third of the neck subcutaneously to deep cows and heifers twice up to 30-35 and 15-20 days before calving at a dose of 10 cm ³ .

 The vaccine provides the creation in newborn calves of colostral immunity to rota-, corona-, herpes viruses and colibacteriosis pathogens that produce the corresponding adhesion factors - K99 and A20 antigens.

 For the period 1994 - 1998 79,200 doses of the associated vaccine were manufactured and used on farms in 12 regions of the Republic of Tatarstan. 3 - of the Chuvash Republic, 4 - of the Republic of Bashkortostan, 3 - of the Republic of Mari-El, dysfunctional infectious diarrhea of newborn calves.

Claims (1)

Associated vaccine against rota-, corona-, herpesvirus and Escherichia diarrhea of newborn calves, characterized in that it contains inactivated culture suspensions of Bovine rotavirus "III-1", Bovine coronavirus "CM-1" strains as antigens , Bovine Herpesvirus "TKA-VIEV-B2" with titers of 10 ^7.0 - 10 ^7.8 TCD _{50 / ml} , respectively, 10 ^5.0 - 10 ^5.8 TCD _{50 / ml} , 10 ^7.6 - 10 ^8.0 of Escherichia antigens - inactivated cell suspensions of Ecsherichia coli strains containing adhesins K99 and A20 with a concentration of 100 - 120 billion m.k. in 1 ml of saline, and a gel of aluminum hydroxide in the following ratio of components, vol.%:
Inactivated culture suspensions: Bovine rotavirus strain "III-1", with a titer of 10 ^7.0 - 10 ^7.8 TCD _{50 / ml} - 27.0 - 30.0
Strain Bovine coronavirus "CM-1", with a titer of 10 ^5.0 - 10 ^5.8 TCD _{50 / ml} - 27.0 - 30.0
The strain Bovine Herpesvirus "TKA-VIEV-B2" with a titer of 10 ^7.6 - 10 ^8.0 TCD _{50 / ml} - 27.0 - 30.0
Inactivated cell suspension of the strain Ecsherichia coli containing adhesive antigen K99 with a concentration of 100 - 120 billion m. in 1 ml of physiological solution - 3.0 - 4.0
Inactivated cell suspension of the strain Ecsherichia coli containing adhesive antigen A20 with a concentration of 100 - 120 billion m. in 1 ml of physiological solution - 3.0 - 4.0
Gel of aluminum hydroxide, 6% solution - The rest

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