SE421748B - Vaccine against distemper and a process for its preparation - Google Patents

Abstract

Live-virus vaccine against distemper in carnivores and process for its preparation. The vaccine in question preferably consists of a freeze- dried mixture of a virus and a stabilizer. The virus in the vaccine is one that is registered in the Pasteur Institute under number I-129 and is an attenuated strain of a virus causing distemper in carnivorous animals. This strain is prepared from a wild virus isolated from a mink with distemper by repeated passages in cell cultures of different origins. One of these cultures comes from canine kidneys, another from the kidneys of a human Rh embryo, and a third is a mixed cell culture consisting of cells from canine kidneys and cells from a Japanese quail embryo, this strain having been adapted to the cell culture of the embryo of a Japanese quail embryo and cultured on this medium. In the process for the preparation of the vaccine, an attenuated strain of the distemper virus causing this disease in carnivores is cultured in a cell culture on a nutrient medium, the virus-containing liquid is collected, and the said liquid is then freeze-dried (lyophilized) in the presence of a stabilizer. The process is characterized in that the attenuated virus strain is a virus that is registered in the Pasteur Institute under number I-129 and is one causing distemper in carnivores. It is obtained from a wild-type virus isolated from the blood of a mink with distemper. This procedure involves the following steps: a) a 20-40-fold passage in a cell culture from canine kidneys at a temperature of 37 +/- 1 degrees C, b) a 5-15 fold passage in a cell culture from Rh human embryo kidneys at a temperature of 32 +/- 1 degrees C, c) a 2-7 fold passage in a mixed cell culture consisting of cells from canine kidneys and cells from a Japanese quail embryo at a temperature of 35 +/- 1 degrees C, and finally d) adaptation by a 4-10 fold passage in a cell culture from a Japanese quail embryo. The advantages of the vaccine thus obtained are e.g. that it is not harmful and that it has a high immunogenic and antigenic activity. The vaccine does not contain any alien viruses and can be used for the prophylactic treatment of distemper in carnivores and for eliminating epizootic infections. The vaccine can also be used in aerosol form.

Description

a 8008185-4 2 time be contaminated with foreign virus. To prevent the dogs from becoming infected, you must have a special animal breeding institute, where the animals are bred under strictly controlled conditions, which, however, entails large investment costs. Another disadvantage of these known vaccines is that their biological activity is difficult to determine, since during viral reproduction one obtains a vaguely marked cytopathogenic effect, which is manifested on the 20th day after infection, when non-specific de- generative cell changes may occur, which make the estimation of the results more difficult.

A further disadvantage of these known vaccines prepared for cell culture from dog kidneys is a slow growth of said cells (a so-called monolayer of cells is not formed until the fifth-seventh day, which increases the processing time for the production of the vaccine).

Viral vaccines, which are intended to control puppy disease in carnivorous animals and are prepared by culturing a virus strain on a cell culture from chicken embryos aged 9 days or on coronary allantoic membranes from chicken embryos, can be dangerous due to the fact that these chicken embryos are highly contaminated with foreign viruses, especially viruses belonging to the leukosis and sarcomatosis group (compare, for example, U.S. Pat. Nos. 2,912,361 and 2,965,51414).

Said disadvantage is also typical of a vaccine, prepared in a cell culture from chicken embryos, of a strain known as KF-668 (USSR).

In the American vaccine known under the name ASL is non-harmful and is free from the mentioned disadvantage, because it is prepared in a cell culture from leukosis-free chicken embryos. In order to be able to breed leukosis-free hens, one must have access to special breeding facilities, which are difficult and expensive to achieve and maintain. The vaccine strain of the virus also does not give a uniform cytopathic effect in the cell culture from chicken embryos, which makes the process technique for producing the vaccine complicated.

Furthermore, a viral vaccine against puppy disease virus is known, which was prepared by culturing a puppy disease in carnivorous animals causing virus on a cell culture from green markets (compare, for example, U.S. Pat. No. 4 OOÄ 97Ä). This vaccine is fairly immunogenic, non-reactogenic, highly standardized and non-harmful. 8008185-4 3 Licensing for the capture of monkeys (or market monkeys) has been restricted in many countries due to the steadily declining number of monkeys and market monkeys. In addition, the aforementioned known vaccines are based on the utilization of waste products from the production of a polio vaccine, which is intended for humans, so that the production of the known puppy disease vaccine is directly linked to the production of the polio vaccine. A separate department required for the production of the puppy disease vaccine, which is based on the cell culture from monkey or market kidneys, is expensive and not profitable.

The choice of cell substrate for the production of the viral vaccine is thus complicated. The necessary condition is that the cell substrate must not be contaminated with foreign viruses and that it must be possible to produce the vaccine virus in sufficient quantities.

An object of the present invention is to provide a vaccine against distemper in carnivorous animals which is highly immunogenic, antigenic, non-harmful and free from foreign viruses, while being inexpensive to manufacture.

Another object of the invention is to provide a method for producing the proposed vaccine using a new strain of puppy disease virus, which strain can be grown on a harmless and easily accessible substrate, which method makes it possible to prepare the vaccine which is highly immunogenic, antigenic, non-harmful and free of foreign viruses.

This is achieved according to the invention by means of a live viral vaccine against distemper, which vaccine contains an attenuated strain EPM of the infectious virus. This strain (quail embryo Moscow) is deposited partly with the Soviet institutes listed below under number 10-76, and partly with the Institut Pasteur, France (27 February 1980), under registration number 1-129; and this virus strain has been produced from a wild-type virus, isolated from the blood of a mink infected with puppy disease, through multiple repeated passages successively on the following cell cultures: 1) on a cell culture from dog kidneys, 2) on an ungrafted cell culture Rh from kidneys of human embryos , 3) on a mixed cell culture, consisting of cells from dog kidneys and cells from embryos of Japanese quail, which strain is adapted to the cell culture from embryos of Japanese quails and grown on this culture.

The said object is further achieved by means of a process for the production of the live virus vaccine against distemper in carnivores, which process comprises on the one hand culturing - on a cell culture from embryos of Japanese quail in a suitable nutrient medium - the attenuated the strain EPM of the carnivorous disease of carnivorous animals causing the virus, which strain as mentioned above is deposited under the number 10-76 in the Soviet Union and under the number 1-129 in France (Inst. Pasteur) and is produced from a wild-type virus, which is isolated from the blood of a mink infected with puppy disease, through multiple repeated passages successively on the following cell cultures: 1) on a cell culture from dog kidneys, 2) on an ungrafted cell culture from kidneys from human embryos Rh, 3) on a mixed cell culture consisting of cells from dog kidneys and cells of embryos from Japanese quail, with subsequent adaptation to the cell culture from embryos of Japanese quails, partly collection of a virus-containing fluid and and on the other hand freeze-drying the virus-containing liquid in the presence of a suitable stabilizer.

Said attenuated virus strain, which was used for the preparation of the puppy disease vaccine, has been termed EPM by viruses which cause puppy disease in carnivorous animals. EPM is an abbreviation for "quail embryo, Moscow" in Russian. This strain is a newly produced vaccine strain, is stored in "Vseso- juznom Gosudarstvennom nautchno-kontroljnom institute veterinarnykh preparator Ministerstva Seljskogo Khozjajstva SSR" and is, as mentioned above, deposited under number 10-76 in the Soviet Union and under number 1-129 in France (Inst. Pasteur).

The one under the number 10-76 resp. 1-129 dcponoradu utammun EPM of viruses that cause puppy disease in carnivorous animals is produced by a wild-type virus isolated from the blood of a mink infected with puppy disease. The preparation took place through genomic H0 passages on a cell culture from dog kidneys at a temperature of 37,100, then 5-15 passages on a cell culture from kidneys of human embryo Rh at a temperature of 32 É 1oC and 2-7 passages on a mixed cell culture consisting of cells from dog kidneys and cells of embryos of Japanese quail at a temperature of 35 É 1 ° C, with subsequent adaptation during H-10 passages on a cell culture of embryos of Japanese quails (Coturnix coturnix japonica) .

For the preparation of the vaccine, said strain is grown on the cell culture of embryos of Japanese quail in a suitable nutrient medium, for example a medium 199 or an Earle medium, with the addition of 10% blood serum from cattle. The first harvest of the viral fluid is taken after the emergence of the virus' cytopathic 8008185-4 f; effect i20 ~ HO% of the cells. The collected virus-containing liquid is lyophilized in the presence of a suitable stabilizer, for example 5.5% sorbitol and 1H-1-6% gelatose. By "gelatoo" is meant the product obtained by hydrolysis of gelatin (product of gelation hydrolysis).

The vaccine strain EPM deposited under no. 10-76 in the Soviet Union and under the number 1-129 in France (Dept. Pasteur) of the porcine disease in carnivorous animals is highly attenuated and multiplies very well in the cell culture of embryos of Japanese quail, with clearly pronounced effect, which makes the preparation of the vaccine considerably easier. Said strain is storage-stable.

The vaccine proposed according to the invention has the following advantages: a) the vaccine is not harmful, has no toxic effect and is produced in cell culture from embryos of Japanese quail, which is a cheap and easily accessible raw material which can be reliably controlled for the absence. of foreign viruses; Japanese quails are inherently not susceptible to viruses belonging to the leukosis and sarcomatosis group, so the cell culture from embryos of Japanese quails is an optimal substrate for the production of the virus vaccine; b) the cell culture from Japanese quail embryos exhibits high proliferative activity, so one can infect the cell suspension without waiting for the formation of a monolayer of cells; c) the vaccine exhibits high antigenic activity and induces vaccinated animals a strong and long-lasting immunity, the use of the vaccine in the puppy-nuknepinnntin area for carnivorous animals rapidly eliminating the infectious spot; d) the vaccine can be used not only parenterally but also in the form of an aerosol, which makes the vaccine prophylaxis considerably easier and cheaper; e) because the vaccine is highly active, preparations can be marketed with very varying numbers of doses (1-500) in ampoules or bottles, which is very convenient for individual use or mass use of the vaccine.

These and other advantages will be apparent from the following detailed description of the vaccine, the method of its preparation and its use.

The procedure for the administration of the live virus is based on a n.k. vaccine virus, in which a large amount of vaccine virus is prepared, which meets all the requirements imposed on the weakened strain, the vaccine virus is stored in the frozen state and the same is used, when necessary, for production. status of the vaccine.

To prepare the vaccine for the control of distemper in carnivores, the attenuated strain EPM 10-76 and the 1-11 in France (Inst. Pasteur) are used as indicated above; it is hereinafter referred to as "strain 1-129".

The strain 1-129 has been produced from a virulent wild-type virus, which causes puppy disease in carnivorous animals and which has been isolated from the blood of a mink that has been ill with puppy disease. The wild-type virus is attenuated by multiple repeated passages on different cell cultures in the following order: I 1) on a cell culture from dog kidneys; 20-H0 passages at a temperature of 37 É 1 ° C, 2) on an ungrafted cell culture from kidneys of human embryos Rh; 5-15 passages at a temperature of 32 É 100 and 3) on a mixed cell culture, consisting of cells from canine kidneys and cells from embryos of Japanese quails; 2-7 passages at a temperature of 35 É 1 ° C.

After the attenuation process is completed, the virus is adapted to the cell culture from embryos of Japanese quails for 4-10 passages. The resulting virus strain is cloned according to a method for so-called border dilution in cell culture from embryos of Japanese quail. Through this treatment, the virus strain acquires a clearly marked cytopathic activity, which makes it possible to control the vaccine preparation, collect the virus harvest for optimal periods of time, greatly simplify the determination of the virus 'infectious activity, determine the virus' specificity in the oil culture and detect antibodies in the blood serum. from vaccinated animals. The resulting virus strain exhibits the following properties: 1) specificity in neutralization reaction in cell culture from embryos of Japanese quail; 2) innocuousness for laboratory animals, ie. mice, guinea pigs, rabbits, chicken embryos and minks and blue foxes; 3) infectious activity in cell culture from embryos of Japanese quails; _ U) antigenic activity; 5) immunogenic activity (in neutralizing reaction in animals and in cell culture from embryos of Japanese quail); 6) absence of contaminating virus (viral contaminants); 8008185-4 7 7) bacterial sterility, absence of mycoplasma PPIO; 8) storage stability; 9) is not contagious.

From this virus strain with all these properties, a vaccine virus is prepared on cell culture from the space of Japanese quails. All the quality characteristics of the vaccine virus must be identical to the quality characteristics of the said strain. The inoculum virus is stored in a frozen state at a temperature of not more than -EOOC and is used when necessary.

To produce the vaccine, a trypsinized tissue cell culture was used from embryos of Japanese quail, for which purpose the tissue from 9-10 day old embryos of Japanese quail is pulverized and trypsinized according to the generally known method. The resulting cell suspension is infected with the inoculum virus and supplemented with a suitable nutrient medium. This may be any of the known media used for culturing cells and viruses, for example a medium 199 containing 10% of bovine serum. pH is T- The infected cell culture is incubated in a (slowly) rotating device at 35 É 100. The rotational speed can e.g. be 2ü-25 rpm. After a monolayer of cells has formed and the cytopathic effect of the drug has given rise to nosuria -10% of the cells, the precursor fluid is collected (for the first time) for the first time. In order to increase the amount of nppunmlnt vivur and tüttrr utilization of the cell culture, the virulence-containing liquid is recovered from a similar sample of the culture many times at the same time as fresh nutrient medium is added. These operations are repeated until the culture cells have completely degenerated.

In order to be able to extract the intracellular virus, the cell culture must be frozen and thawed. The total virus-containing liquid is poured - after the cone has been checked for sterility and infectious activity - into a single container, added with a stabilizer, poured into bottles or ampoules and lyophilized. The dried preparation has a residual moisture content of not more than 5% by weight.

The shelf life of the freeze-dried vaccine is at least one year.

The lyophilized vaccine is stored at a temperature of not more than H00.

If the vacrinrt is stored at a lower temperature, its life will be .filmn u fi lqrn lllän-'uh-r lfinp fi rv. I ~ l.'í | - man vill: | nv.'ín-J: | v: 1f'f-Ir | --t, ul.- ngaäflui-: null «i: d, L-, adt: n1«: v: 1r <-irl - I nu - 1 --ri [15 lh- | »k ;; - lF3n11i | 1r3 ~: | t-: 1u |: rv1d saline. 8008185-4 8 The vaccine preparation prepared is checked for sterility, innocence, specificity, absence of contaminating virus, infectious activity, immunogenic and antigenic activity and residual moisture content, after which the vaccine can be used for prophylactic immunization of fur animals and dogs and for therapeutic use during the first days. after contact with a carnivorous animal, which is sick with puppy disease. A single mean dose for vaccination of animals must contain at least 10 cytopathic tissue doses (CVDSO). An increase in the virus content to 100,000 CVD50 does not cause any undesirable side reactions in the animals.

The animals are immunized by intramuscular injection of 1 ml of vaccine.

In order to be able to vaccinate the animals reliably, serological examinations of the immunized animals can be carried out after 10 days and the titer of antibodies in the neutralization reaction on cell culture from embryos of Japanese quail can be determined by using the said vaccine strain, which gives a pronounced cytopathy. technical effect. The antibodies appear in all the immunized animals in a titer of 1:16 to 1:32. The largest possible amount of antibodies (in a titer of 1: 256) accumulates already on the 50th day. The antibodies remain in the blood for an observation period of 18 months. In the subsequent treatment of the immunized animals with a virulent strain Styder Hill after 1, 3, 6, 12 and 18 months after the immunization with said vaccine, no cases of disease were detected. The vaccine proposed according to the invention thus shows 100% immunogenic and antigenic activity.

The vaccine according to the invention has been used in fur animal breeding at a time when puppy disease has broken out among silver foxes. After one month after the vaccination, the puppy disease ceased, and it was not detected during the following time period. Near the fox farm was a mink farm. Because puppy disease in carnivorous animals is highly contagious, in some cases the disease was also detected in minks. Immediate injection of the vaccine according to the invention prevented a regular outbreak of puppy disease among the minks.

The results obtained thus testify that the vaccine, which l'r-: urx: zi '..'. Il'l 1. :: av: ztznu l-1; '_ O: lv dx-l. valpujuku hm: kïittåitarvlc 'animal' inducing virus, can within 5-N weeks eliminate the valpujukan on a fur farm, where this disease has broken out. The vaccine according to the invention can also be used for immunizing animals by a so-called aerosol injection procedure.

For this purpose, a vaccine intended for injection was used, i.e. conventional series of vaccines. The aerosel method makes it possible to immunize a large number of animals in a short time.

By testing the vaccine according to the invention on animals, it has been discovered during the prophylaxis period that in practically 100% of the cases antibodies are formed in a high titer, which completely prevent the animals from becoming infected with puppy disease. The vaccine was also effective as a pesticide for eliminating the focus of infection.

The vaccine according to the invention is relatively inexpensive to produce by using as a substrate the cell culture from embryos of Japanese quail. In terms of vaccine production, this cell culture has the following advantages over the other known cultures: An important advantage of the substrate is that Japanese quails are by nature not susceptible to viruses belonging to the avian leukosis group and are resistant when infected.

Another important advantage of the cell culture of Japanese quail is that it has a high proliferative activity and that the monolayer of cells is formed during the first or second day after cell inoculation.

A further advantage of said cell culture is that one can infect a cell suspension, which results in an extra reduction of the percentage gene for the production of the vaccine. Another advantage of the invention is that it is possible to carry out a multiple repeated collection of the virus-containing liquid, for example up to 5 collections from a single culture sample.

Considering that Japanese quails are easily accessible and inexpensive, while the maintenance of the quail farms is not complicated and does not require significant investment costs, it will be readily understood that the vaccine of the invention is commercially valuable.

The invention is illustrated below by means of the following exemplary embodiments, in which Example 1 illustrates the preparation of the strain 1-129 of the puppy disease in carnivorous animals causing the virus, for example, 'llf- lf /: zf ~ 1' F¿31 * I ': | 1 ~ :: r | <| -! I'iï | - I'z ~ '| n .. I FSI 1 rlï Int ": -v -I: -1. * L -' 1: 1r1-if- vf1- ~ '- ïrif-I. Uvh wxvmprl 3-U Jifnr Jnvïndninpfn dv vn ~~ inrï on animals. n 8008185-4 10 Example 1. Preparation of the attenuated strain 1-129 of the puppy disease in carnivorous animals causing the virus, starting from virulent wild-type virus.

In each of 10 test tubes filled with a primary kidney culture from canine kidneys, 0.2 ml of defibrinized blood from a mink sick in puppy disease is introduced. After a contact time of 3 hours at room temperature, the cell culture is thoroughly washed with nutrient medium 199.

In each test tube, 1.5 ml of fresh nutrient medium 199 with 10% blood serum from cattle is introduced, after which the cell cultures are incubated at a temperature of 5700. The nutrient medium is changed 1-3 times per bag. After a period of 62 days after infection, a virus-containing fluid is collected from the test tubes, which is used to infect a new portion of cell culture from dog kidneys, for which purpose in each of ten test tubes, filled with a new primary cell culture from dog kidneys, before 0 , 3 ml of the resulting virus-containing fluid and 1 ml of the nutrient medium 199 with an addition of 10% blood serum from cattle, after which the cell cultures are incubated at 3700.

During the first ten passages, the infectious material consists of a virus-containing fluid, which is collected 35-H0 days after the infection of the previous cultures. For subsequent =; virus passages, a virus-containing fluid collected on the 15-20th day after infection is used, and a virus extracted from the cells after freezing and thawing the infected cells is used.

12 virus passages are then performed - according to a similar infection method - in an inoculated cell culture from kidneys of human embryo Rh at} PÜC. The material for each subsequent infection consists of the virus-containing fluid, which was collected on the 15-20th day after the previous infection.

The virus is then attenuated by at 5,500 passages performed on a mixed culture, consisting of 2 million cells from dog kidneys and 2 million cells from embryos of Japanese quail. In such a mixed culture, 5 virus passages are performed, after which the virus is allowed to pass 5 times in cell culture from embryos of Japanese quails at BSOC in a rotating device at a speed of U rpm.

Starting from the fifth passage, the virus regularly caused a cytopathic cell destruction, which was observed 5-7 days after infection. Such a virus was cloned by a so-called border dilution method in cell culture from embryos of Japanese quails and used as the vaccine strain. The resulting attenuated strain of the carnivorous disease of carnivorous animal-producing virus, which strain has been adapted to the cell culture from Japanese quail embryos, is checked for: 1) specificity (identification) in the neuralization reaction of the Japanese culture embryonic cell culture; 2) innocence (in laboratory animals such as mice, embryos, guinea pigs, rabbits, as well as minks and blue foxes); 3) the infectious activity in the cell culture from embryos of Japanese quails; U) antigenic activity; 5) immunogenic activity; 5) absence of viral contaminants; 7) bacterial sterility, absence of mycoplasma (PPiO); 8) the storage stability of the preparation; 9) non-infectious. §§Gm§¿l_§. Preparation of a vaccine virus and the vaccine: a) Preparation of the vaccine virus. The inoculum virus is prepared from the strain 1-JD9 prepared according to Example 1, for which purpose embryos of Japanese quails with an age of 9-11 days are used, which were delivered from a specially controlled quail farm. 100 embryos are trypsinized according to the approved method, thereby obtaining 3,300,000,000 cells, which are suspended in 9,120 liters of the nutrient medium 199 and added: with 10% serum from cattle and 100 mg monomycin per ml. 300 ml of the cell suspension is poured into two 1 liter. bottles (150 ml in each bottle); these samples are saved for control purposes. Into the second portion of the cell suspension is introduced 10 ml of a culture broth containing the vase strain 1-129, after which 150 ml is poured into every 1 liter: bottle. All bottles are incubated at a normal temperature by BSOC in a rotating device. On the third day after inoculation and infection of the cells, the nutrient medium is removed, after which fresh nutrient medium 199 and 100 mg of monomycin per ml are introduced into the vials. On the sixth day after infection of the cells, when the cytopathic activity foci of the virus appear, the virus-containing liquid is collected from the vials, after which samples are taken to carry out the nv-checked and introduced fresh medium 199 into the vials. The viral fluid is then collected repeatedly until 50% of the cells are maintained. During the final step, 70 ml of fresh medium 199 are introduced, after which the cell culture is frozen in dry ice and thawed in order to be able to extract the intrauellular virus. 8008185-4 12 During all preparation steps for the inoculum virus, samples are taken for control purposes. The vaccine virus must be checked for: specificity, innocuousness, infectious activity, antigenicity, immunogenicity, absence of contaminating virus, sterility and non-infectivity. The vaccine virus, which meets all control requirements, is stored in a frozen state at a temperature of not more than -2050. b) Preparation of the vaccine. For this purpose, embryos were used by Japanese quails with an age of 9-11 days, which were delivered from a controlled special quail farm. 200 embrgc: 'according to the approved method, whereby 6,600 cells are obtained, which are suspended in 8.25 liters of the nutrient medium 199 and supplemented with 10% serum from cattle and 100 mg monomycin per ml. 750 ml of the cell suspension is poured into five 1 liter. bottles (150 ml in each bottle); design is saved for control purposes. In the second portion of the cell suspension, 10 ml of a culture broth containing the inoculum prepared according to a) of strain 1-129 are introduced. bottle is poured 150 ml. All bottles are incubated at 3500 in a rotating device. On the third day after inoculation and infection of the cells, the nutrient medium is removed, after which fresh medium 199 and 100 mg of monomycin per ml are introduced into the vials. On the sixth day after infection of the cell suspension - when the cytopathic activity foci of the virus appear in 50% of the cells - the virus-containing liquid is collected from the vials, after which samples are taken for control purposes and a fresh portion of medium 199 is introduced into the vials. The virus-containing fluid is then collected repeatedly until 50% of cells remain, after which 70 ml of medium 199 are introduced. The cell culture is frozen in dry ice and thawed in order to extract the intracellular virus.

All collected portions of virus-containing liquid are poured into a single container and added with a stabilizer consisting of 5% sorbitol and 1.5% gelatose, after which the vaccine is poured into ampoules or bottles and lyophilized. Such a "pool" constitutes a single series of the vaccine. The prepared vaccine can be poured into bottles or ampoules with a volume of 1-35 ml, whereby a single bottle or ampoule may contain 1-500 doses depending on the consumer's requirements.

During all vaccine preparation steps, samples are taken for control.

The vaccine must be controlled for: specificity, innocuousness, infectious activity, antigen and immunogenic activity, absence of virus contaminants, sterility and lack of infectivity.

The vaccine cry, which meets all control requirements, can be used for immunization of animals. In this embodiment, a single series contains 360,000 vaccine doses.

Example 3. Testing of the vaccine for innocence.

The vaccine is used in a dose of 1 ml containing 100 CVD50 of the virus after it is pre-diluted with Henks' solution.

The vaccine was injected intramuscularly. 2U mink puppies and 12 blue fox puppies were vaccinated. For control purposes, an additional 16 mink puppies and 8 blue fox puppies were included in the experiment. The vaccinated animals were observed daily for a period of 28 days. All animals remained clinically healthy. No pathological reactions were detected, ie. refusal to eat the feed, depression, indigestion. The animals that had been in contact with the vaccinated did not become ill with puppy disease.

Example H. Testing of the vaccine for antigenicity (antigenicity test).

From the animals vaccinated with a dose of the preparation as set forth in Example 3, blood samples were taken after 10 days and the titers of the antibodies were determined in the neutralization reaction on cell culture from Japanese quail embryos using the vaccine strain 1-129 as antigen. of the puppy disease in carnivorous animals causing the virus. The antibodies were determined for all animals; the titer was 1:16 to 1:32. The largest possible amount of antibodies (in a titer of 1: 256) was collected on the 30th day. The antibodies were maintained for an observation period of 18 months.

Example 5. Testing of the vaccine for immunogenicity and duration of immunity. a) 1 ml of a diluted vaccine (1 ml in each puppy) containing 100 CVDSO of the virus was injected intramuscularly into bra mink puppies and 12 blue fox puppies. An additional 16 mink puppies and 8 blue fox puppies were used as control animals. After 30 days, the vaccinated and control animals were infected with a virulent virus (Snyder Hill For minks and Gaujasskij for blue foxes).

The animals were observed daily. The control animals (ie the unvaccinated) became ill with (died of) puppy disease, while the vaccinated animals did not show any evidence of the injection of the virulent puppy disease virus. Throughout the observation period (21 days after infection), the vaccinated animals remained clinically healthy. ; sons1ss-4 lä b) After one year and after one year and six months after vaccination, the animals were re-infected with the virulent virus Snyder Hill resp. Gaujasskij. No vaccinated animals became ill with puppy disease and all of these animals remained alive.

Example 6. Testing of the vaccine under field conditions.

During the summer vaccination period, 1 H00 000 fox, H5 500 blue fox, 2500 sables, 300,000 minks and 255 dogs were vaccinated on 15 fur animal farms - for prophylactic treatment. No animals became ill with puppy disease, regardless of the fact that outbreaks of puppy disease took place in the areas where the said Farmer was located.

Example 7. Testing of the vaccine in a puppy disease center.

On a fur farm, where many cases of puppy disease in carnivorous animals, especially young silver foxes, were discovered, up to 120 puppies died daily. (Disease cases were examined and confirmed by laboratory tests.) All animals were vaccinated. After two months, the number of nntulut deaths due to puppy poop had halved. After another month, the puppy week ended completely with the foxes.

On a mink farm, which was in the immediate vicinity of the fox farm, isolated cases of puppy disease were discovered. After rapid vaccination of all those minks, a regular outbreak of puppy disease on the farm was prevented.

Example 8. Test of the preparation according to the invention in comparison with the known vaccines.

On a mink farm, where numerous cases of puppy disease in carnivorous young animals were found, all the animals were immunized with the known vaccine KF-668 (Soviet Union). After the vaccine was injected, the number of deaths among the animals decreased, with this number amounting to 180 mink pups per day. For this reason, a repeated immunization of 612 animals was rapidly performed with the vaccine of the invention and 1580 animals with the known American ASL vaccine. Following immunization with these vaccines, the number of deaths suddenly decreased. After another two months, the disease on this farm was eliminated. In terms of efficacy, the vaccine of the invention is not inferior to the known ASL vaccine.

Example 9. Testing of the vaccine in the so-called the aerosol injection method.

For the so-called The aerosol injection used a vaccine prepared in the same way as the vaccine intended for intramuscular injection. The aerosol immunized 300 mink puppies aged 45-60 days. The vaccine was sprayed so that it

Claims (4)

One group of minks received a single conventional dose, while the other group of minks received three conventional doses. The conventional dose is a dose that is approved for intramuscular injection. The antibodies to the puppy disease virus in blood serum from the immunized animals were determined in the neutralization reaction to cell culture from Japanese quail embryos by using the vaccine strain 1-129 as antigen. For the group of minks vaccinated with a single dose, the antibodies were determined in a dilution ratio of 1: 8. In the second group of minks, treated with three doses, the antibodies were determined in a dilution ratio of 1:32. No significant difference was detected in the titer of virus-neutralizing antibodies in the aerosol-immunized animals and those immunized intramuscularly (according to Example 3). To control the so-called the immunity strength of the aerosol vaccinated animals used a method of infection with the virulent virus Snyder Hill. After infection with this virus, no puppy disease was detected in the aerosol vaccinated animals. As shown in Example 9, the vaccine proposed according to the invention, prepared from the puppy disease virus strain 1-129, is effective not only if used intramuscularly but also if used according to the aerosol immunization method. P a t e n t k r a v Live virus vaccine against distemper in carnivorous animals, consisting of a preferably lyophilized mixture of virus and stabilizer, wherein the virus is an attenuated strain of a puppy disease in carnivorous animal-producing virus, characterized in that as said attenuated virus strain a strain of the puppy disease virus, which has been deposited with the Institut Pasteur under No. I-129 and has been produced from a wild-type virus isolated from the blood of a mink sick in puppy disease, through multiple repeated passages successively on the following cell culture 1) on a cell culture from dog kidneys, 2) on a cell culture from kidneys of human embryos Rh and 3) on a mixed cell culture, consisting of cells from dog kidneys and cells from embryos of Japanese quail, which strain has been adapted to cell culture from embryos of Japanese quails and grown thereon culture. 8008185-4 16 A method for producing a vaccine according to claim 1, comprising culturing an attenuated strain of the puppy disease in carnivorous animals causing the virus on a cell culture in a nutrient medium, collecting a virus-containing liquid and lyophilizing the collected virus-containing medium. the liquid in the presence of a stabilizing agent, characterized in that, as said attenuated strain, the strain deposited with Institut Pasteur under No. I-129 is used in the puppy disease of carnivorous animals causing the virus, which strain is produced by a wild-type virus isolated from mink, which is sick in puppy disease, by a) 20-NO passages on a cell culture from dog kidneys at a temperature of 37,3100, then b) 5-15 passages on a cell culture from kidneys of human embryos Rh at a temperature of 32 1 1 ° C, then c) 2-7 passages on a mixed cell culture consisting of cells from canine kidneys and cells from embryos of Japanese quails at a temperature of 1 1 ° C , and thereafter d) adaptation during 4-10 passages on cell culture from embryos of Japanese quails. 3. A method according to claim 2, characterized in that the virus-containing fluid is collected for the first time when the virus, when incubated with cells from embryos of Japanese quail, has a cytopathic effect in 20-40% of the cells. Process according to Claim 2 or 3, characterized in that the virus-containing liquid is lyophilized in the presence of 4.5-5.5% by weight of sorbitol and 1.4-1.6% by weight of gelatose. 8008185-4 Gathering Live virus vaccine to control puppy disease in carnivorous animals and method of producing the same. Said vaccine consists of a preferably lyophilized mixture of virus and stabilizer. The vaccine contains, as mentioned, a virus attenuated by Institut Pasteur under No. I-129, an attenuated strain of a puppy disease in carnivorous animals, which strain is produced by a wild-type virus isolated from a mink which is sick with puppy disease. by multiple passages in cell cultures of varying origin: on a cell culture from dog kidneys, on a cell culture from kidneys of human embryos Rh and on a mixed cell culture consisting of cells from dog kidneys AND cells from embryos of Japanese quail, to which strain is adapted to cell culture from embryos of Japanese quails and grown_on this culture. The process for preparing the vaccine comprises cd attenuating a weakened strain of the carnivorous disease of carnivorous animals causing the virus on a cell culture in a nutrient medium, collecting a virus-containing liquid and lyophilizing the collected viral fluid in the presence of a stabilizer. The feature of the attenuated strain is that, as said attenuated strain, the strain of the puppy disease of carnivorous animals induced by Institut Pasteur under No I-129 is used, which strain is produced from a wild-type virus isolated from the blood of a mink. , which is sick in puppy disease, by a) 20-H0 passages on a cell culture from dog kidneys at a temperature of 37 I 10C, then b) 5-15 passages on a cell culture from kidneys of human embryos Rh at a temperature of 32 I 10C, then c) 2-7 passages on a mixed cell culture consisting of cells from dog kidneys and cells from embryos of Japanese quails at a temperature of 35 É 1 ° C, and thereafter d) adaptation during H-10 passages on cell culture from Japanese quail embryos. The advantage of the vaccine according to the invention is i.a. that this vaccine is not harmful and that it has high immunogenic and antigenic activity. The vaccine is free of foreign viruses and can be used for the prophylactic treatment of puppy-nosed carnivores and for the elimination of epizootics. The vaccine can also be used in the form of an aerosol.