Vaccine against plague, infectious hepatitis and parvovirus enteritis carnivorous

 

(57) Abstract:

The invention relates to the specific prophylaxis of plague, infectious hepatitis and parvovirus enteritis in dogs. The vaccine contains a new attenuated strain "Vniivvim-88 virus canine distemper infectious activity of 3.5-4.5 lg 50 TPD/cm3. The strain is grown in compared to CV-1 transplantable kidney cells of the African green monkey. The vaccine also contains vaccinal strain Cornell-2 virus of infectious hepatitis in dogs with infectious activity of 5,5-6,5 lg 50 TPD/cm3. The strain grown in human cell cultures of pig testicles (PTP). In addition, it includes a vaccinal strain of virus, panleukopenia in cats with infectious activity of 3.5-4.5 lg 50 TPD/cm3and hemagglutinine activity in RSA 1:32 to 1:512. The strain grown in human cell cultures kidney kitten (subline Fk). The vaccine also includes a environment drying on the basis of lactose, sucrose, sorbitol and gelatin. The vaccine is characterized by safety, reactogenicity, high antigenic and immunogenic activity, storage stability, standardization and high technological obtain. 3 table.

The invention relates to veterinarnogo enteritis in dogs.

For prevention of these diseases using live and inactivated mono - and associated vaccines, obtained on the basis of primary and transplantable cell cultures.

For immunization of carnivores, including dogs against plague apply cultural vaccines from strains of PEM, Vniivvim-88, 668-KF, Rockburn modified Snyder-hill and others (Author, SV-VA USSR class. C 12 N 7/08 N 527072, 1977; class A 61 K 39/12 N 1287592, 1984. Patents U.S. CL 424-89 N 4224412, 1980; N 4351827, 1982; N 5000951, 1991).

For protection against infectious hepatitis is used mainly preparations from strain Cornell, and adenoviruses dogs type 1 and 2 (Patents U.S. CL 195-1.3 N 3432391, 1969; CL 195-1.8 N 3616203, 1971; CL 424/89 N 3950512, 1976. Patent of Russian Federation CL A 61 K 39/295, N 2030917, 1995).

Vaccines against parvovirus enteritis contain live attenuated and inactivated virulent strains of parvovirus in dogs, feline panleukopenia in cats and mink enteritis (Patents U.S. CL 424-89, N 4193990, 1980; N 4303645, 1981; N 4810494, 1989. The author. SV-VA. Czechoslovakia class. A 61 K 39/23 N 262736, 1988; N 252746, 1988; N 252747, 1988. Patent of Russian Federation CL A 61 K 39/23 N 871371, 1994. THE 46-21-705-80 on the culture vaccine against viral enteritis of mink).

To generate immunity against the listed infections in the shortest possible time vaccination against every disease, successfully apply the associated vaccine (Patent England class. A 61 K 3/62 N 1016586, 1966. Patent U.S. CL 424-89 N 5000951, 1991. The author. SV-VA. Czechoslovakia class. A 61 K 39/295 N 267937, 1989. Patent of Russian Federation CL A 61 K 39/295, N 2030917, 1995. THE 08064-019-004-94 the liquid vaccine inactivated against parvovirus enteritis and hepatitis carnivorous. THE 10-09-98-91 to the vaccine dry culture against plague, hepatitis and parvovirus carnivorous).

Most are similar in composition (a prototype of the proposed drug is a vaccine dry culture against plague, hepatitis and parvovirus, distemper, containing strains: "668-KF" distemper, "Cornell-2 virus of infectious hepatitis and dogs "And" virus, panleukopenia in cats, developed by the staff of the Pokrovsky plant biologics (TU 10-09-98-91). Its main drawback is that to get vaccinated materials are expensive, non-standard, Molodejnaya and not available from the primary contaminants trypsinization cell culture: from the tissues of the chick embryo ("668-KF"), kidney pig ("Cornell-2) and kidney kitten ("And"). In addition, the strain 668 - KF" has a relatively low immunogenicity. The need to Refine the vaccine was indicated in the order on the Main uprawia against plague, infectious hepatitis and parvovirus enteritis carnivores, possessing a high immunogenic activity, safety, storage stability and high technological production.

This objective is achieved in that the vaccine contains a new attenuated strain "Vniivvim-88 virus canine distemper (HFI), infectious activity of 3.5-4.5 lg TCD 50/cm3produce as compared to CV-1 transplantable kidney cells of the African green monkey, strain Cornell-2 virus of infectious hepatitis in dogs (VIGS) infectious activity of 5,5-6,5 lg TCD 50/cm3grown in human cell cultures of pig testicles (PTP), and the strain of virus, panleukopenia in cats (MIC) infectious activity of 3.5-4.5 lg EID 50/cm3and hemagglutinine activity in RSA 1:32-1:512 grown in human cell cultures kidney kitten (subline Fk), as well as the environment drying, including lactose, sucrose, sorbitol and gelatin, in the following ratio of components,%:

The viral suspension of strain Vniivvim-88" with a titer of infectious activity of 3.5-4.5 lg TCD 50/cm3- 32-33

The viral suspension of strain Cornell-2 with a titer of infectious activity of 5,5-6,5 lg TCD 50/cm3- 9-11

Viral suspense is 1:512 - 32-33

20% solution of lactose - 4,8-5,2

60% sucrose solution - 4,8-5,2

40% solution of sorbitol - 4,8-5,2

a 20% solution of gelatin - Rest

The cultivation of these three strains is high-tech roller method using high yield, standard, not tumorogenic transplantable cell lines that are used in the Institute for cultivation of various viruses.

For growing distemper use subline CV-1 monolayer culture of cells obtained in Vniivvim from the original culture transplantable cells CV-1 (USA, ATCC, CCL 70) kidney of the African green monkey (room 43.2 directory cell collection Vniivvim). The Bank, working cell manufacturer (BRCI) as compared to CV-1 contains cells 17 of the passage in the amount of 165 million cells. Monoclonal culture cells as compared to CV-1, the received serial passirovannym factor reseeding 1:3-1:5, used within 50 serial passages.

For the cultivation of the virus of infectious hepatitis in dogs use monoclonal culture transplantable cells PTP received by the Institute of Ukrainian scientific-research veterinary Institute in 1989. (10.5 culture in cat the cell culture PTP is used for the cultivation of the virus within 50 consecutive subcultures.

For the cultivation of the virus of feline panleukopenia in cats use monoclonal culture cells as compared to FK obtained in Vniivvim source of transplantable line kidney cats CRFK (USA, ATCC, CCL 94). Subline registered under number 34.1 directory cell collection Vniivvim. BRKI compared to the FK contains cells 22-47 passages in the amount of 100 million cells. Monoclonal culture cells as compared to FK, the received serial passirovannym factor reseeding 1:2-1:4, used for 50 consecutive subcultures.

Vaccinal strain "Vniivvim-88" (registration number "Vniivvim-88 Dept" in the collection VGNKI) obtained from a strain of PEM by adapting it to the as compared to CV-1 through a series of intermittent passages and subsequent cloning method of limiting dilutions. The new strain is characterized by the following properties:

- has morphological properties characteristic of canine distemper virus of the genus Morbillivirus of the family Paramyxoviridae;

- breeds in transplantable cell culture as compared to the CV-1 with the development of a pronounced JRS as simpleton and subsequent destruction of cells, the titer of virus in a given culture reaches 3.5 to 4.5 lg TCD 50/cm3. Without adaptation, the virus multiplies in the culture of cells from Dane ;

- cultivated in monolayer transplantable cells as compared to CV-1 in the stationary and rolling conditions, under optimal conditions, the maximum biological activity is achieved through 44-52 hours;

- harmless to laboratory and susceptible animals, contagiosum, previsible, non-pathogenic for humans;

- induces the formation of intense and long-lasting immunity against plague and vaccinated fur-bearing animals (mink, tortoreto, foxes, foxes, raccoons and dogs;

- causes the formation of neutralizing antibodies in titers of 1:8-1:32; hemagglutination properties had not;

- genetically stable within 10 serial passages in cell culture compared to the CV-1.

The vaccine against plague, infectious hepatitis and parvovirus enteritis carnivorous impose adult dogs once in a volume of 2 cm3and the puppies twice with an interval of 14-30 days in the amount of 1 cm3.

Example 1.

Vaccinated materials of all three strains grown in monolayer transplantable cell cultures using a roller installed capacity of 50 DM3for one cycle.

Viral suspension vaccine strain Vniivvim-88 virua-MEM with 10% vol. the blood serum of cattle. As supporting the use environment of the Needle-MEM with Ob.% the blood serum of cattle.

The production strain Vniivvim-88" HFI with a title not below the 3.5 lg TCD 50/cm3make 20 cm3in the roller receptacles formed with 1-2 days of a monolayer of cells, pre-draining of these growth environment. The contact of the virus with cells is conducted for 30 to 40 minutes at a temperature of 36.5 - 37,5oC, then in each vessel add 480 cm3environment support, pH 7,2-7,4.

The virus cultivation is carried out at a temperature of 36.5-37,5oC and the speed of rotation of the vessel about 10-12. in the hour. After 40-48 hours at the development of specific JRS 50% of the cells viral suspension is poured, after taking samples from each vessel to control for contamination by bacteria, fungi, mycoplasmas and determine the titer of the virus by the JRC in cell culture compared to the CV-1. After freezing at a temperature of minus 60oC and thawing at 25oC the resulting virus is poured into one container.

Viral suspension vaccine strain Cornell-2" WIGS get in transplantable cell culture PTP. As the growth medium used Wednesday Needle-MEM with 10% vol. the blood serum of cattle. As growth and supportive environments use environment is the GS with a titer of at least 5.5 lg TCD 50/cm3make 6 cm3in the roller receptacles formed with 1-2 days of a monolayer of cells, pre-draining of these growth environment. The contact of the virus with cells is conducted within 60 - 90 minutes at a temperature of 36.5 - 37,5oC, then in each vessel add 494 cm3environment support, pH 7,2-7,4.

The virus cultivation is carried out at a temperature of 36.5-37,5oC and the speed of rotation of the vessel about 10-12. in the hour. After 36-48 hours when the specific manifestation of the JRS at 50-70% of the cells viral suspension is poured, after taking samples from each vessel to control for contamination by bacteria, fungi, mycoplasmas and determine the titer of the virus by the JRC in the cell culture PTP. After freezing at a temperature of minus 60oC and thawing at 25oC the resulting virus is poured into one container.

Viral suspension vaccine strain "And" MIC get in transplantable cell culture Fk. As growth and supportive environments used eagle medium-MEM with 10 and 5 vol.% the blood serum of cattle, respectively.

Production strain "And" MIC with a title not below the 3.5 lg EID 50/cm3make 20 cm3in the roller receptacles formed with 1-2 days of a monolayer of cells, pre-draining of these growth with the d add 480 cm3environment support, pH of 7.2 to 7.4. The virus cultivation is carried out at a temperature of 36.5-37,5oC and the speed of rotation of the vessel about 10-12. in the hour. After 6 days of viral suspension is poured, after taking samples from each vessel to control for contamination by bacteria, fungi, mycoplasmas, determination of biological activity in cell culture Fk and hemagglutinine activity in the DSA. After freezing at a temperature of minus 60oC and thawing at 25oC the resulting virus is poured into one container.

In table. 1 shows the results of three experiments get vaccinated materials used for the production of vaccines.

Contamination by bacteria, fungi and mycoplasmas were absent.

As can be seen from the table. 1, all series vaccinated material derived from transplantable cell cultures were stable performance and possessed high activity.

Example 2

To 3,2 DM3the distemper add 3,2 DM3virus, panleukopenia in cats and 0.9 DM3the virus of infectious hepatitis in dogs. To the resulting mixture of viruses with thorough stirring in shuttel machine type environment drying, containing 0,48 DM33, monomitsina 200 IU/cm3and polymyxin b 100 U/cm3. Set the pH to 6.6 with succinic acid at a final concentration of 6%.

Similarly cook another 2 series vaccinated mixture with the following quantities of components (DM3) (see table. 2).

The mixture is Packed in 2 cm3in sterile ampoules (series 1 and 2), glass bottles (series 3) and lyophilizers. Just made 15000 immunizing doses for dogs (1 dose vial and the vial).

Example 3

By a Commission (ACL Institute) 3 series of the vaccine was studied by the following indicators: biological activity, safety, reactogenicity and immunogenic. Requirements for vaccine set forth in the technical specifications 9384-051-00008064-96 "Vaccine against plague, infectious hepatitis and parvovirus enteritis carnivorous culture dry", approved in 1996 by the veterinary Department of the Ministry of agriculture and PRF.

The biological activity of the vaccine is determined by titration vaccine strains in cell cultures: HFI in cell culture compared to the CV-1, VIGS in cell culture PTP, MIC in cell culture compared to the Fk. Activity of the strains in the dry vaccine should cm3.

The safety of the vaccine is determined on the dogs 2-4 months of age weighing at least 3 kg, clinically healthy, not vaccinated against diseases studied, past deworming. Four dogs injected into the muscles of the thigh 5 cm3the vaccines. Animals provide clinical supervision for 21 days. All vaccinated animals must remain alive and healthy during the observation period. Pets within 1-3 days. refusal or incomplete eat the feed and the dilution of feces.

Reactogenicity of the vaccine is determined on the dogs 2-4 months. age weighing at least 3 kg, clinically healthy, not vaccinated against diseases studied, past deworming. Four dogs administered the vaccine in the thigh in volume 1 vaccination dose (2 cm3). For vaccinated animals provide clinical supervision for 21 days. The vaccine is considered reactogenic, if all vaccinated animals not observed deviations from the physiological norm during the period of observation. Pets within 1-2 days. refusing feeds not complete his eating, as well as dilution of feces.

Immunogenic activity of a vaccine against canine distemper determine tortoreto 2-2,5 months of age, polirovannyj against plague and not in a rut. The vaccine is administered 4 dorsomedial intramuscularly on 1 cm3containing 500-1000 TCD 50 HFI. After 21 days after vaccination, vaccinated and 4 control animals infect virulent strain "Snyder hill" in a dose of 1000 LD 50 intramuscularly. The observation period of 25 days. The vaccine is immunogenic, if she was protected from infection and destruction of all once grafted tortoreto at the end of 4 animals in the control group.

Immunogenic activity of a vaccine against parvovirus enteritis is determined on the mink 2-6 months. age derived from successful in infectious and parasitic diseases farms, clinically healthy, not vaccinated against plague and not in a rut. The vaccine is administered 4 Minks intramuscularly on 1 cm3containing 1000 ID 50 PC virus. After 21 days after vaccination, vaccinated and 4 control animals infect virulent strain of "Springs" in a dose of 1000 ID 50 intramuscularly. The observation period of 14 days. The vaccine is immunogenic, if she was protected from infection and destruction of all once vaccinated mink at the end of 4 animals in the control group. Pets survival of the two control mink when parabolani their PE with the characteristic symptoms of the disease.

Immune is usedsuch from experience, by definition, reactogenicity. After 21 days after vaccination, vaccinated and 4 control animals infect virulent strain of "Karabash" in a dose of 1000 ID 50 IPR (5 cm3and in conjunctive eyes (0.2 cm3). The observation period of 25 days. The vaccine is immunogenic, if it protects from the disease once all vaccinated puppies. While the control animals show rise in temperature to 40.3-41oC, depression, loss of appetite, keratitis of the cornea. Possible loss of puppies for 18-25 days.

The results of Commission trial 3 series vaccines are summarized in tab. 3.

It is established that in the dry vaccine contagious vaccine strain "Vniivvim-88" was 3.0-3.75 to lg TCD 50/cm3, strain Cornell-2" of 3.75 to 4.5 lg TCD 50/cm3, strain "And" - 3,0-4,0 lg EID 50/cm and RSA 1:8-1:256. The vaccine was harmless and reactogenic for dogs, immunogenic for tortoreto, dogs and mink. After double vaccination of puppies (the second vaccination in 14-30 days. ) in animals were identified neutralizing and inhibiting the hemagglutination antibodies, which reached a maximum titer after 10-14 days. after secondary immunization against HFI - 1:64 - 1:128 against WIGS - 1: 16 - 1:64 against VPK - 1:64 - 1:512. Antibodies against three infections is ASS="ptx2">

All immunobiological characteristics of the 3 vaccine series comply with the requirements of TU-9384-051-00008064-96 and stored for 12 months. storage at a temperature of 4-6oC within specified standards.

The use of a new vaccine against plague, infectious hepatitis and proviruses enteritis carnivores in veterinary practice will reduce the incidence of dogs these diseases and improve the effectiveness of epidemic events.

Vaccine against plague, infectious hepatitis and parvovirus enteritis carnivores, including lyophilized mixture of three culture vaccine strains and the environment-drying, characterized in that it contains at effective concentrations,% vol.:

The suspension of the new vaccine strain "Viewm-88" distemper, grown as compared to the SU-1 transplantable kidney green monkey with a titer of infectious activity of 3.5 - 4.5 lg 50 TPD/cm3- 32 - 33

Suspension vaccine strain Cornell-2" infectious hepatitis dog, grown in human culture cells of the testes Piglet gnotobiotic with the titer of infectious activity of 5,5 - 6,5 lg 50 TPD/cm3- 9/11

Suspension vaccine strain "And" panleukopenia in cats, the EID 50/cm3and hemagglutinine activity 1 : 32 - 1 : 512 - 32 - 33

20% solution of lactose - 4,8 - 5,2

60% sucrose solution - 4,8 - 5,2

40% solution of sorbitol - 4,8 - 5,2

a 20% solution of gelatin - Rest

 

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