CS218120B1 - Nutrient vaccine of the virus culture against the distemper of dogs and fur animals and method of making the same - Google Patents

Abstract

The nature of the vaccine according to the invention resides in that it contains 102 to 104 TCID50 in 1 ml viruses. distemper dogs, strain EPM 10-76, which it is deposited in the Scientific General Union Institute for Veterinary Testing ASSCJVP. The way in which it is produced consists in being strain distemper virus EPM 10-76 deposited in ASSCJVP cultures on cell culture Japanese quail embryo fibroplasts Parker broth of bovine serum, containing 10% carbon source, amino acid a mineral salts, at a pH of 7.0 to 7.5 and at a cultivation temperature of 34-36 ° C, wherein with the first collection of culture liquid which it contains a virus; effect in 20 to 40% of the infected Cell Culture Embryo Japanese Quail and the following subscriptions are made for so long if less than 50% live cells.

Description

The essence of the vaccine according to the invention is that it contains 10 ² to 10 ⁴ TKID ₅₀ viruses per ml. canine distemper, strain EPM 10—76, which is deposited with the Scientific Union General Institute for Veterinary Preparations ASSCJVP.

A method for producing the same is to cultivate the canine distemper virus strain EPM 10-76 deposited in ASSCJVP on a cell culture culture of Japanese quail embryos on Parker's bovine serum broth containing 10% carbon, amino acids and mineral salts at pH 7, 0 to 7.5 and at a culture temperature of 34 to 36 ° C, wherein the first harvest of the culture liquid containing the virus is performed at 20 to 40% of the infected cells of the Japanese quail embryo culture to achieve a cytopathic effect, unless less than 50% of the living cells remain.

The present invention relates to a live vaccine of a virus culture against canine distemper and fur animals used in veterinary medicine and to a process for its production.

Canine distemper is an acute contact disease of fur animals and dogs. Infectious chicks of mink, silver-black foxes and blue foxes are particularly susceptible to this disease. Due to the high mortality and lack of effective drugs, this disease causes considerable economic damage in animal husbandry.

At present, a number of live virus culture vaccines are used for the prophylaxis of canine fur animals and dogs, which contain attenuated strains of canine virus adapted to kidney cell cultures of canine or monkey or hen embryos.

The vaccine, which contains an attenuated strain of canine distemper virus "Rochborn", obtained from a virulent strain of virus isolated from the blood of a sick dog, is prepared for a dog's kidney culture. For the production of such a vaccine, said virus strain adapts to a dog kidney cell culture by multiple passages (> 50-fold) and is cultured on said cell culture in Henks ' solution containing 0.5% lactalbumin ohydrolysate and 10% horse blood serum or 20 % of calf serum or in Erl's medium containing 0.5% lactalbumin hydrolyzate and 2% equine or calf blood serum The adaptation temperature is kept constant between 30 and 37 ° C, preferably 35 to 37 ° C. the vaccine is stabilized and lyophilized (U.S. Pat. No. 3,098,011).

Also known is a vaccine against canine fur animals and dogs obtained from a virus which has been subjected to 10 passages on a dog's kidney cell culture in Erl's medium containing 5% lactalbumin hydrolyzate and 5% horse serum at 38.5 Deň: 32 ° C. (U.S. Patent No. 3,354,038).

These vaccines have high immunogenic efficacy, but have a number of drawbacks. One of these drawbacks lies in the low reproducibility of the virus in the dog's kidney cell culture.

Another drawback of these vaccines lies in the use of dog kidney cultures as a substrate for virus attenuation and vaccine production. Dog kidneys can also be contaminated with foreign viruses. In order to rule out the infection of dogs, it is necessary to create a special animal husbandry farm in which the animals are kept under strictly controllable conditions. This, however, requires considerable costs.

A further drawback of vaccines lies in the difficulty of determining their biological efficacy, since virus reproduction is accompanied by a faint cytopathogenic effect, which manifests itself up to the 20th day after infection, when non-specific degenerative cell changes may occur, making it difficult to evaluate the results.

Furthermore, the deficiency of the vaccines produced on the dog's kidney cell culture is due to the slow growth of these cells (monolayer cells are only formed on days 5-7), which prolongs the technological production process of the vaccine.

Virus vaccines for combating distemper of fur animals and dogs produced by culturing the virus strain on a 9-day-old hen embryo cell culture or chick embryo chorioallantoin membranes may be dangerous due to high contamination of hen embryos with foreign viruses, especially leucosis and sarcoma group viruses. U.S. Patent Nos. 2,912,361 and 2,965,544).

This deficiency also has a vaccine prepared from a strain in hen embryo cell culture and is known as KF-668 (USSR).

The American vaccine, known as the ASL vaccine, is harmless and does not have the stated drawback, since it is prepared in a culture of cells of non-leucous hen embryos. However, special breeding poultry farms are needed for the breeding of non-leukosis-free hens, the establishment and maintenance of which is complicated and costly. In addition, the virus strain vaccine does not provide any regular cytopathic effect in the hen embryo cell culture, making the vaccine production technology difficult.

There is also a known vaccine against fur canine and canine distemper, which is prepared by culturing fur canine and canine distemper virus on kidney cell culture of young monkeys (U.S. Pat. No. 4,004,974). This vaccine is characterized by good immunogenicity, is areagenic and meets the highest standards.

However, at present, the capture of monkeys has been limited in many countries with a view to reducing animal status. Taking into account the fact that the vaccine is aware of and is directly linked to the waste arising from the production of a vaccine against poliomyelitis intended for humans, it is easy to understand that the creation of a special cell-based vaccine production for fur dogs and dogs monkey kidney culture is costly and inappropriate.

In this way, the selection of the cell substrate for the preparation of the virus vaccine is complicated. A prerequisite is that the cell extract does not contact foreign viruses as well as the possibility of obtaining a virus vaccine in sufficient quantities.

It is an object of the present invention to provide a vaccine against distemper of fur animals and dogs that has high immunogenicity, areactogenicity, harmlessness, no foreign viruses and can be obtained at low cost.

In accordance with this objective, the task was to develop a method of producing a vaccine using a new strain of fur canine and canine viruses that can be cultivated on a harmless and accessible substrate and allows to obtain a vaccine with high immunogenicity, antigenicity and harmlessness due to the absence of viruses.

The essence of the live vaccine culture of canine distemper virus and fur animals, in the lyophilized state, according to the invention is that it contains in 1 ml 10 ² to 10 ⁴ TKID _{5 ()} canine distemper virus strain EKM 10-76, which is deposited in the Scientific Union General Institute for Veterinary Preparations ASSGJVP. This strain was obtained from a virulent virus isolated from mink blood, distempered by multiple passage passages on the following cell cultures: 1) in dog kidney cell culture; 2) a booster culture of human Rh embryo kidney cells; 3) on a mixed cell culture consisting of dog kidney cells and Japanese quail embryo cells, adapted to and cultured on Japanese quail embryo cell culture.

The principle of the vaccine production method according to the invention is characterized in that the strain of distemper virus EKM 10-76 deposited in ASSCJVP is cultivated on a cell culture of Japanese quail embryo fibroblasts on Parker's bovine serum medium containing 10% carbon, amino acid and mineral sources salts, at a pH of 7.0-7.5 and at a culture temperature of 34-36 ° C, wherein the first harvest of the culture liquid containing the virus is performed in 20-40% of the infected cells of the Japanese quail embryo culture to achieve a cytopathic effect; sampling is performed as long as less than 50% of the living cells remain.

Lyophilization of the virus-containing liquid is carried out in the presence of 4.5 to 5.5% by weight. % sorbine and 1.4 to 1.6 wt. gelatin.

The attenuated virus strain that is used to produce a vaccine against canine distemper and fur animals has been called the canine distemper and fur animal EPM strain. The term EPM means the initial letters of the words: embryo quail, Moscow.

The strain is a recently obtained vaccine strain and is deposited in the collection of cultures of the State Union Scientific and Control Institute for Veterinary Preparations of the Ministry of Agriculture of the USSR, Moscow, D-22, Svenigorodskoye Sossé, 5 under number EPM 10-76.

The canine and fur animal EPM strain deposited under EPM No. 10-76 was obtained from a virulent virus isolated from the blood of canine distemper mink 20 to 50 by passage! on a dog's kidney cell culture at 37 (+1) ° C, then 5 to 15 passages on a human Rh embryo cell culture at 32 (+ 1) ° C, and 2 to 7 passages on a mixed cell culture consisting of canine kidney cells and from Japanese quail embryo cells at a temperature of 35 (+1) ° C and subsequent adaptation during 4 to passage on the Japanese quail (Coturnix japonica) embryo cell culture.

For vaccine production, said strain is cultured on Japanese quail embryo cell culture in a suitable nutrient medium such as medium 199, which is a mixture consisting of amino acids, vitamins, nucleic acid components, etc. in Henk's saline solution or Erl medium with 10% blood beef cattle sera.

First, a dose of virus-containing fluid is collected according to evidence of a cytopathic effect in 20-40% of the cells. The collected virus-containing fluid is dried lyophilically in the presence of 4.5-5.5 sorbitol and 1.4-1.6 why. gelatin.

The EPM vaccine strain of canine and fur animals, deposited under the number EPM 10-76, is highly attenuated, multiplies well in Japanese quail embryo cell culture with a distinctly pronounced cytopathic effect, which greatly simplifies the vaccine manufacturing process. Said strain has post-storage stability.

The offered vaccine has the following advantages:

the vaccine is harmless, has no toxic effect, is produced in a cell culture of Japanese quail embryos, which is a cheap and accessible raw material that can be reliably checked for foreign viruses;

Japanese quails are not naturally susceptible to leukosis and sarcoma viruses and therefore cell culture from their embryos is an optimal substrate for vaccine production.

bj cell culture of Japanese quail embryos has a high proliferative efficiency, which allows to infect the cell suspension without having to expect the formation of a cell monolayer.

The cj vaccine has high antigenic efficacy, induces desired and long-term immunity in animals vaccinated with the vaccine, the use of the vaccine in herds of canine distemper and fur animals will kill the herd rapidly.

d) the vaccine can be administered not only parenterally, but also in the form of an aerosol, which greatly simplifies and cheaper prophylaxis with the vaccine.

(e) since the vaccine is highly effective, it is possible to produce a commercial product which contains a wide dosage range of from 1 to 500 doses per ampoule or bottle, which is very convenient for individual and mass use.

These and other advantages may be apparent from the following detailed description of the vaccine, the method of manufacture and use thereof.

The method of producing a live virus culture vaccine is based on a virus vaccination system.

The vaccine virus system takes into account the production of the virus vaccine in large volume, which meets all the requirements of the attenuated strain, its storage in the frozen state and its use as needed for the production of the vaccine.

. As mentioned above, an attenuated EPM strain, which has been deposited under the number EPM 10-76, is used to produce a vaccine against canine distemper and fur animals.

The strain was obtained from a "wild" virulent canine distemper virus and fur animals, which was isolated from the blood of distemper sick mink. This virulent virus is attenuated by multiple passages on different cell cultures in the following order: 1) on canine kidney cell culture during 20 to 40 passages at 37 (+1) ° C, 2) on a booster culture of human Rh embryo kidney cells during 5 to 15 passages at 32 (+

1) ° C and 3] on a mixed cell culture consisting of canine kidney cells and Japanese quail embryo cells for 2 to 7 passages at 35 (+1) ° C.

Upon completion of the attenuation process, the virus adapts during 4 to 10 passages to the Japanese quail embryo cell culture. The virus strain obtained is processed by the method of maximum dilution in Japanese quail embryo cell culture. As a result of these processes, the virus strain clearly obtains cytopathic efficacy that allows to control the vaccine production process, collect virus doses at optimal times, greatly simplify the determination of infectious virus efficacy, and further determine virus specifications in cell culture and prove in the blood serum of vaccinated animals. antitélíska. The virus strain obtained is characterized by the following features:

1) specificity in neutralization reaction in ambergris cell culture! Japanese quail;

(2) harmless to laboratory animals: mice, guinea pigs, rabbits, hen embryos, mink and fox;

3) infectious efficacy in cell culture of Japanese quail embryos;

4) antigenic activity;

5) immunogenic efficacy in a neutralizing response to animals and cell cultures of Japanese quail embryos;

6) absence of contaminating viruses;

7) bacterial sterility, absence of mycoplasma PPI O;

(8) storage stability;

9) is not contagious.

A vaccine virus is prepared from the above-mentioned strain of virus on a cell culture of Japanese quail embryos. The vaccine virus must have, by analogy with the above strain, all qualitative characteristics. The vaccine virus is stored frozen at a temperature of -20 ° C or less and used as necessary.

Trypsin-treated Japanese quail embryo tissue culture cells are used for vaccine production. To this end, the tissue of 9-10 days old Japanese quail embryos is ground and processed in a conventional manner with trypsin. The cell suspension obtained is infected with the vaccine virus and an appropriate culture medium is added. Known media used for culture of cells and viruses, for example media 199 containing 10% bovine serum at pH 7.0-7.5, can be used as the culture medium. The infected cell culture is incubated under rotation conditions at 35 (+1) ° C. After formation of the cell monolayer and evidence of cytopathic efficacy in 20-40% of the cells, a first virus-containing fluid collection is performed.

To increase virus collection and to better utilize cell culture, multiple collection of virus-containing fluid is performed in one; and the same culture sample by adding fresh culture medium. These processes are repeated until complete degeneration of the cell culture.

To withdraw intracellular virus, the cell culture is frozen and thawed. The collected virus-containing fluids, after sterility and infectious activity control, are combined into a single tank, a stabilizer is added, filled into bottles or ampoules and lyophilized. The dried preparation has a residual moisture of up to 3%.

The lifetime of the lyophilized vaccine preparations is at least one year. The freeze-dried vaccines should be stored at a maximum of 4 ° C. If the vaccines are stored at a lower temperature, the period during which the vaccines are usable will be extended by several months. Prior to use, saline solution is used for the diluted vaccine, which is prepared based on Henks' solution.

The vaccine preparation obtained is subjected to control for stability, harmlessness, specificity, absence of contaminating viruses, infectious activity, immunogenic and antigenic activity, and residual moisture.

Thereafter, the vaccine can be used both for the prophylactic immunization of furry animals and dogs, and for therapeutic use during the first days after contact with distempered animals. The average dose for the animal vaccine must contain a minimum titer of 10 TKID ₅₀ (cytopathic tissue dose). Increasing the virus content up to a titer of 1,000,000 TKID ₅₀ does not cause any adverse reactions in animals.

Immunization of the animal is performed by intramuscular injection of 1.0 ml vaccine.

To assess the reliability of vaccine vaccination, after 10 to 14 days, serological detection in immunized animals and determination of antithelial titers by neutralizing response to Japanese quail embryo cell culture may be performed by using said vaccine strain, which exhibits a distinctly significant cytopathic effect.

Antibodies were demonstrated in all immunized animals at a titer of 1: 16 to 32. Maximum antifungal enrichment (at titer 1:

: 256) appears on day 30. The antithelia are maintained in the blood for 18 months of observation. In the following solution, no cases of disease were detected in immunized animals vaccinated with the virulent Snyder Hill strain 1, 3, 6, 12 and 18 months after immunization with the vaccine. In this way, it has been found that the vaccine of the invention has 100% immunogenic and antigenic activity.

The offered vaccine was used in animal husbandry at the time of the distemper outgrowth among silver-black foxes. One month after vaccination, the disease disappeared and no disease was observed in the following period. Next to the fox farm there was a mink farm. Due to the high contagiosity of canine distemper virus and fur animals, individual cases of mink disease have been registered. The immediate introduction of the vaccine of the invention prevented canine distemper outgrowth.

This indicates the ability of a vaccine prepared from the EPM strain of canine distemper and fur animals, which is deposited under the number EPM 10-76, to dispose in animal husbandry starting an canine outbreak within 3 to 6 weeks.

The vaccine of the invention may also be used to immunize animals with an aerosol delivery method.

For this purpose, a ready-to-use vaccine, i.e. a conventional marketed vaccine series, may be used. The aerosol method allows to immunize large numbers of animals in a short time.

As a result of animal testing of the offered vaccine, it has been found during prophylaxis that in almost 100% of cases, high titer antithelia are formed which perfectly protect the animals from canine distemper disease. In addition, the vaccine was also effective as a means of controlling herd diseases.

The vaccine of the invention is cheap due to the use of a Japanese quail embryo cell culture as a substrate. Compared to other known cultures, said culture has the following advantages for vaccine production. The main advantage of the substrate is that Japanese quails are naturally insensitive to the viruses of the group of poultry leucosis and remain resistant when infected with it.

Another important advantage of cell cultures of Japanese quail embryos is that it has high ioroliferative activity and that the cell monolayer is formed on the first to second days after cell inoculation.

A further advantage of said cell culture is that the cell suspension can be infectious. This leads to an additional reduction in the duration of the technological process of vaccine production. Another advantage of the invention is the multiple collection of fluid containing virus up to 5 collections per culture sample.

Given the fact that Japanese quails are accessible and inexpensive, and keeping quail farms does not cause any inconvenience at no great cost, it is easy to understand the commercial value of the vaccine offered.

In order to illustrate the invention, examples are given, of which Example 1 relates to the production of the EPM strain of dog and fur animals deposited under EPM 10-76, Example 2 of the method of producing a live virus culture vaccine and Examples 3 to 9 using the vaccine on animals.

Example 1

Production of an attenuated strain of the canine distemper virus and fur animals deposited under the number EPM 10-76 from a wild virulent strain.

Ten tubes containing canine kidney primary cell culture received 0.2 ml of canine distemper mink blood. After three hours of contact at room temperature, the cell culture is washed carefully with nutrient medium 199, 1.5 ml of nutrient medium 199 with 10% bovine serum is introduced into each tube and the cell cultures are incubated at 37 ° C. changes the nutrient medium twice a week. 62 days after infection, the virus-containing fluid is collected from the tubes and a new dose of canine kidney cell culture is used for infection. To this end, 0.3 ml of the recovered virus-containing fluid and 1 ml of 199 medium are added to each of the 10 tubes containing the new primary canine kidney cell culture and 10% bovine serum serum is added and the cell cultures are incubated for temperature 37 ° C.

For the first 10 passages, the virus-containing fluid collected as the infectious agent is collected 35-40 days after infection of the previous culture. For the next 23 passages, the virus-containing fluid collected 15 to 20 days after infection is used, and the virus recovered from the infected cells after freezing and thawing is also used.

Then, according to a similar infection method, 12 passages of the virus are performed in a booster culture of human Rh embryo kidney cells at 32 ° C. Substance for subsequent infection is a virus-containing fluid collected 15 to 20 days after the previous infection.

Furthermore, the virus is attenuated by passaging on a mixed culture consisting of 2 ml canine kidney cells and 2 ml Japanese quail embryo cells at 35 ° C. In such a mixed culture, three passages of the virus were performed, after which the virus was passaged five times with a Japanese quail embryo cell culture at 35 ° C under rotation conditions of 4 rpm. Beginning with passage 5, the virus periodically induces cytopathic cell destruction, which was demonstrated 5 to 7 days after infection. Such a virus was processed as a vegetatively pure line by the method of maximum dilution in the Japanese quail embryo cell culture and used as a vaccine strain. The attenuated strain of canine distemper virus and fur animals that has been adapted to the Japanese quail cell culture is checked for the following characteristics:

lj to the specificity (identity) in the neutralization response to the Japanese quail embryo cell culture;

(2) harmlessness (for laboratory animals: mice, embryos, guinea pigs, rabbits, mink and fox);

3) infectious activity in Japanese quail embryo cell culture;

4) for antigenic activity;

5) for immunogenic efficacy;

(6) the absence of contaminating viruses;

7) for bacterial sterility, absence of mycoplasma (PPIO);

8) storage stability of the preparations;

9) that it is not contagious.

Example 2

Preparation of vaccine virus vaccine

(a) Preparation of the vaccine virus. The vaccine virus is produced from the EPM strain deposited under the number EPM 10-76 obtained according to Example 1. For this purpose, the use of 9 to 11 day old Japanese quail embryos that were brought from a controllable special quail farm was used. 100 embryos were treated in the usual way with trypsin and 3,300,000,000 cells were obtained, which were suspended in 4,120 liters of broth 199 with 10% bovine serum and 100 mg / ml monomysin. 300 ml of cell suspension is dispensed in 150 ml in two 1 liter bottles and left for inspection.

The second part of the cell suspension is charged with 10 ml of culture fluid containing the EPM vaccine strain, swirled under the number EPM 10-76, and filled into 150 ml bottles of 1 liter. All bottles were incubated at 35 ° C with rotation. Three days after cell inoculation and infection, the culture medium is removed and fresh broth 199 and 100 mg / ml monomycin are added to the bottles. 6 days after infection of the cells, evidence of the cytopathic activity of the virus is collected by the fluid containing the virus from the bottles, sampled for appropriate controls, and fresh culture medium is added to the bottles 199. Subsequent harvesting of the fluid containing the virus is performed several times until preserves 50% of the cells. In the final step, 70 ml of fresh broth 199 is introduced, the cell culture is frozen in dry ice and thawed to withdraw intracellular virus. Control samples are taken at all stages of vaccine virus preparation. The vaccine virus must be checked for the following characteristics: specificity, harmlessness, infectious activity, antigenicity, immunogenicity, absence of contaminating viruses, sterility, contagiousness.

The vaccine virus complying with the above control requirements shall be kept frozen at not more than - 20 ° C.

(b) Production of the vaccine. For this, 9 to 11 days old Japanese quail embryos are used, which were brought from a controllable special quail farm. 200 embryos were treated in the usual way with trypsin to obtain 6,600,000,000 cells which were suspended in 8,250 liters of broth 199 with the addition of 10% bovine serum and 100 mg / ml monomycin. 750 ml of cell suspension is filled in · 150 ml into 5 1 liter bottles and left for inspection. The second part of the cell suspension is introduced with 10 ml of culture fluid containing the vaccine virus strain EPM 10-76 obtained by the method described above, and is now filled in 150 ml bottles into 1 liter bottles. All flasks were incubated at 35 ° C. Three days after cell inoculation and infection, the culture medium is removed and bottles of fresh culture medium 199 and 100 mg / ml monomycin are added to the bottles. 6 days after infection of the cell suspension, in 30% of the cells showing the cytopathic activity of the virus, the virus-containing fluid in the flask is collected, sampled for appropriate controls, and fresh culture medium 199 is added to the flasks. several times until 50% of the cells are retained; 70 ml of broth 199 are added. The cell culture is frozen in dry ice and thawed to withdraw intracellular virus.

All sterile virus-containing collections are pooled in a single tank, a stabilizer consisting of 5% sorbitol and 1.5% gelatin is added, the vaccine is filled into ampoules or bottles and lyophilized.

This dose is a series of vaccines. The obtained vaccine may be filled into bottles or ampoules of varying contents ranging from 1 ml up to 33 ml, and from 1 to 500 doses may be contained in one bottle or ampoule as desired by the consumer.

Control samples are taken at all stages of vaccine production. The vaccine must be inspected for the following characteristics: specificity, harmlessness, infectious activity, antigenic and immunogenic activity, absence of contaminating viruses, sterility, and whether it is contagious.

A series of vaccines corresponding to the con218120 trolling requirements can be used to immunize animals. In the example, 360,000 doses of vaccine are contained in one series.

Example 1 a d 3

Testing the vaccine for harmlessness

The vaccine was used at a dose of 1 ml containing 100 TKIP ₅₀ virus after dilution with Henks' solution. The vaccine was administered intramuscularly. 24 mink and 12 fox cubs were vaccinated with the vaccine. 16 mink and 8 fox cubs were used as controls. Animals vaccinated with the vaccine were observed daily for 28 days. All animals remained clinically healthy. There were no pathological reactions - food rejection, discouragement, indigestion. Animals in contact with the animals vaccinated with the vaccine did not become sick with distemper.

Example 4

Testing the vaccine for antigenicity

Animals that were vaccinated with the dose of the preparation of Example 3 were sampled after 10-14 days and the anti-body titer was determined by neutralizing the cell culture of Japanese quail embryos by using the EPM vaccine strain of canine and fur animals as antigen, deposited under EPM number 10—76. Antithelia in all animals were found in titers ranging from 1: 16 to 32.

The maximum titer enrichment (at 1: 256 titer) was observed as early as day 30. The antithelia were maintained for 18 months of observation.

Vaccine testing for immunogenicity and duration of immunity as well as 24 mink and 12 fox cubs were vaccinated intramuscularly with 1 ml of a diluted vaccine containing 100 TKID _{5 (} ) virus each. 16 mink and 8 fox cubs were taken for inspection. Thirty days later, infection of vaccinated animals and control animals infected with virulent virus (Snyder Hill for mink and Gaujasski for fox j.

Animals were observed daily. Control animals (i.e., non-vaccinated animals) were treated with distemper and no responses to the introduction of virulent distemper virus were demonstrated in the vaccinated animals. Throughout the observation period - (21 days after infection) the observed animals remained clinically healthy.

bj 1 year and 1.5 years after vaccination, the animals were again infected with the virulent virus Snyder Hill and Gaujasski. No animal vaccinated with the vaccine became sick with distemper and all remained alive.

Example 6

Field vaccine testing.

During the summer prophylaxis vaccine, 1,400,000 foxes, 45,500 blue foxes, 25,000 sable, 300,000 mink, and 255 dogs were vaccinated in 15 livestock farms. No animal has been affected by distemper, ignoring the fact that distemper disturbances have been observed in countries where animal husbandry farms were located.

Example 7

Vaccine testing in canine distemper and fur animals

In animal husbandry, where numerous cases of canine distemper and fur animals among silver-black chicks of foxes were registered (confirmed by laboratory analyzes), the mortality of animals (chicks) reached 120 per day. All animals were vaccinated. After two months, the mortality rate of fox chicks was doubled and ceased at all after another month.

Individual cases of canine distemper were registered at mink farms in the direct vicinity of the fox farm. After the special vaccination of all mink with vaccine, the outbreak of distemper on the farm was wound up.

Example 1 a d 8

Testing the offered preparation in comparison with known vaccines

In animal breeding on a mink farm with numerous cases of canine fur animals among the offspring, a whole herd of animals was known to be known by KF vaccines (USSR). After vaccination, animal mortality did not decrease and reached 180 pups per day. Accordingly, immunization of 14,612 animals with the vaccine offered and 15,580 animals with the known ASL vaccine (USA) was performed without delay. After immunization with these vaccines, the mortality of the offspring was greatly reduced and the outbreak of canine distemper was eliminated after two months. In this way, it has been shown that the vaccine according to the invention does not lag behind the known ASL vaccine in any way.

Example 9

Vaccine testing with aerosol delivery method

For aerosol delivery, a vaccine was used which was made similar to the bag218120 tin intended for intramuscular delivery. 300 mink pups aged 45 to 60 days were immunized by the aerosol method. Vaccine spray exposure was performed such that one group of mink received the usual dose and the other group three conventional doses; "usual dose" means the dose used for intramuscular administration. The antithelia of canine distemper and fur animals in the blood serum of immunized animals were determined by a neutralizing response to the Japanese quail embryo cell culture by using the vaccine strain EPM 10-76 as the antigen. In the mink group receiving a single dose of vaccine, anti-bodies at a 1: 8 dilution were detected, in another group of minks who received three doses, anti-bodies at a 1: 32 dilution were detected.

There was no significant difference in titer size of virus neutralizing anti-bodies in animals immunized by the aerosol method and intramuscularly (according to Example 4).

To test the immunity of animals treated with the vaccine by the aerosol method, the method of infection with the virulent strain Snyder Hill was used. No animal disease was observed following infection with the virulent virus in animals which were used for vaccination by the aerosol method.

As can be seen from Example 9, the vaccine offered, which was made from an EPM strain of canine distemper and fur animals, is deposited under number 10-76, made for injection, also effective in aerosol immunization.

Further data on the vaccine of the invention can be seen from Tables 1 and 2 below.

Stability of several genetic traits

Degree of passage of distemper virus vaccine:

amount of virus introduced (TKID ₅₀ / 1.0 ml):

number of animals vaccinated with the vaccine: mink:

polar foxes: animal response to vaccine: body temperature:

mink:

polar foxes:

catarrhal diseases of mucous membranes (eyes, nose, stomach-intestinal tract) antigen activity:

mink:

polar foxes:

Table 1 strain of canine distemper virus

1 10 10 000 10 000 25 25 25 25 39,5—40,0 39,5—40,0 39,0—39,5 39,0—39,5 OTC * OTC * 1: 63 1: 43 1: 74 1: 71

Note:

* OTC - absence of clinical signs within 30 days after vaccination

Conclusion:

The results of the experiments suggest that the reactogenic and antigenic properties of distemper virus do not vary depending on the degree of passage (1 to 10 passages).

Table 2

Effect of the amount of distemper virus strain introduced into animals on the appearance of the reactogenic and antigenic properties of the vaccine The amount of virus in TKID _so / 1.0 ml

introduced by 1 animal: 10 50 100 ALIGN! Number of animals vaccinated: mink: 15 Dec 15 Dec 15 Dec polar foxes: 15 Dec 15 Dec 15 Dec animal response to vaccination vaccine: body temperature: mink: 39,5—40,0 39,5—40,0 39,5—40,0 polar foxes: 39,0—39,5 39,0—39,5 39,0—39,5 catarrhal diseases of the mucous membranes (eyes, nose, stomach and intestinal tract J OTC * OTC * OTC * antigen activity: mink: 1: 16 1: 45 1: 54 polar foxes: 1: 27 1: 60 1: 77 number of diseased animals after introduction virulent virus **: mink: 0 0 0 polar foxes: 0 0 0

Note:

OTC * = absence of clinical signs within 30 days after vaccination of animals with the vaccine ** = virulent distemper virus introduced 21 to 22 days after vaccination (Gaujaskij for Arctic foxes, Spyder Hill for mink).

Conclusion:

The data presented in Table 2 show that the vaccine response in vaccinated mink and polar foxes will not occur within 30 days after vaccination, regardless of the amount of virus strain introduced. All animals vaccinated with the vaccine were infected 21 to 22 days after vaccination with a virulent strain of distemper virus (mink strain Snyder Hill, polar foxes strain Gaujaskij). Animals vaccinated with the 10-100 TKD ₅₀ vaccine strain did not become ill and remained alive. Control animals became ill and died due to the appearance of canine distemper.

Claims (3)

Live vaccine of a culture of canine distemper virus and fur animals, in a lyophilized state, characterized in that it contains in 1 ml 10 ² to 10 ⁴ TKID ₅₀ canine distemper viruses, strain EKM 10-76, which is deposited in the scientific Union of the European Union Institute for Veterinary Preparations ASSCJVP. 2. The method of claim 1, wherein the strain of canine distemper EKM 10-76 deposited in ASSCJVP is cultured on a cell culture of Japanese quail embryo fibroblasts on bovine serum Parker broth containing 10% carbon, amino acid recovery. and mineral salts, at a pH of 7.0-7.5 and at a culture temperature of 34-36 ° C, wherein the first harvest of the virus-containing culture fluid is performed in 20-40% of the infected cells of the Japanese quail embryo culture to achieve a cytopathic effect; subsequent harvesting is performed as long as less than 50% of living cells remain. 3. A process according to claim 2, wherein the lyophilization of the virus-containing liquid is carried out in the presence of 4.5 to 5.5% by weight. % sorbitol and 1.4 to 1.6 wt. gelatin.