CA1054053A - Live virus culture vaccine against carnivore distemper and method of producing same - Google Patents
Live virus culture vaccine against carnivore distemper and method of producing sameInfo
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- CA1054053A CA1054053A CA225,696A CA225696A CA1054053A CA 1054053 A CA1054053 A CA 1054053A CA 225696 A CA225696 A CA 225696A CA 1054053 A CA1054053 A CA 1054053A
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- virus
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- vaccine
- kidney tissue
- carnivore
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
- A61K39/175—Canine distemper virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Pulmonology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A lime virus culture vaccine against carnivore dis-temper.
Said vaccine is an attenuated stain of the, carnivore distemper virus adapted to a cell culture of the kidney tissue of green monkeys and cultivated in said culture.
The method of producing said vaccine comprises infect-ing a cell culture of the kidney tissue of green monkeys with a carnivore distemper virus strain adapted to said culture and cultivating it in said culture.
The advantages offered by the proposed vaccine are stan dard properties, safety, adequate immunological activity and epizootological effectiveness as well as low produc-tion cost.
A lime virus culture vaccine against carnivore dis-temper.
Said vaccine is an attenuated stain of the, carnivore distemper virus adapted to a cell culture of the kidney tissue of green monkeys and cultivated in said culture.
The method of producing said vaccine comprises infect-ing a cell culture of the kidney tissue of green monkeys with a carnivore distemper virus strain adapted to said culture and cultivating it in said culture.
The advantages offered by the proposed vaccine are stan dard properties, safety, adequate immunological activity and epizootological effectiveness as well as low produc-tion cost.
Description
~o~
Fhe present invention relates to industrial biology;
more specifically, it is directed to a method of producing a live virus culture vaccine for immunization against carni-vore distemper~
The prior art vaccines widely employed for immunization a~aLnst carnivore distemper are likewise live virus culture vnccines prepared from chick-embryo or puppy~kidney tissue cultures.
Thus~ the German vaccines "Candur ~" and "Candur C' used for mink distemper prevention are prepared from a pup-py-kidney tissue culture; the U.S. vaccine ASL and the Canna-ught" are prepared from a cluck-embryo tissue culture.
Said vaccines, though inducing an adequately high level ` of protection, all have the following disadvantages: the va~
ccine prepared from the puppy-kidney tissue culture is liab-le to be contaminated with viruses pathogenic for fur bearers ` ~ e.g. hepatitis or rabies viruses; the vaccines prepared from the chick-embryo tissue culture are not standard enough~ For both types of vaccines, each series usually includes virus ~`` yields from numerous stock cultures of said tissues, so that i it is impossible to obtain high yields of an entirely homoge-neous vaccine from a single culture (i.e. one produced from one embryo) with minimal spending on biological control of the -:. :
~', .,~.
Fhe present invention relates to industrial biology;
more specifically, it is directed to a method of producing a live virus culture vaccine for immunization against carni-vore distemper~
The prior art vaccines widely employed for immunization a~aLnst carnivore distemper are likewise live virus culture vnccines prepared from chick-embryo or puppy~kidney tissue cultures.
Thus~ the German vaccines "Candur ~" and "Candur C' used for mink distemper prevention are prepared from a pup-py-kidney tissue culture; the U.S. vaccine ASL and the Canna-ught" are prepared from a cluck-embryo tissue culture.
Said vaccines, though inducing an adequately high level ` of protection, all have the following disadvantages: the va~
ccine prepared from the puppy-kidney tissue culture is liab-le to be contaminated with viruses pathogenic for fur bearers ` ~ e.g. hepatitis or rabies viruses; the vaccines prepared from the chick-embryo tissue culture are not standard enough~ For both types of vaccines, each series usually includes virus ~`` yields from numerous stock cultures of said tissues, so that i it is impossible to obtain high yields of an entirely homoge-neous vaccine from a single culture (i.e. one produced from one embryo) with minimal spending on biological control of the -:. :
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suitability o each tissue culture source.
It is an obJect of the present invention to obviate the foregoing disadvantages.
Accordin~ly, the invention seeks to provide a novel method o produclng an antidistemper vaccine whereby the tlssue cult~lres of a slngle animal could yield large series of a highly standard, sufficiently immunogenic, safe and cheap vaccine.
It is an object of the present invention to provide an antidistemper vaccine which would be free from viruses pathogenic for the animals to be vaccinated.
It is another object of the present invention to pro~
vide a vaccine for carnivore distemper prevention which would be highly standard, sufficiently immunogenic, safe and cheap.
It is a further object of the invention to provide a method of producing an antidistemper vaccine exhibiting the above-described properties.
It is yet another object of the present invention to provide a method of producing said vaccine which would en-sure a high yield of wirus from the tissue cultures of a single animal.
It is still another object of the present invention to -:. ~
~,provide a method of producing said vaccine which would uti- ` `
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lize ~idely available Material.
These and other ob~ects are attained in an antidistem~
per vaccine ~hich, in accordance ~it~ the invention, is constitutecl by an attenuated strain of the carnivore dis-temper virus aclapted to the culture of green monkey kiclney cells ancl gown ln saicd culture.
Said vaccine will be referred to hereinafter as "Va-kchuml' (a Russian abbreviation for antidistemper vacc-ine ).
"Vakchum" offers the following advantages: it is ade-quately immunogenic, safe ior the vaccinated animal re-gardless of their age, highly standard and free from con-taminant-viruses pathogenic for carnivorous animals.
The proposed method of producing "Vakchum", in accorda_ nce with the invention, includes the steps of preadapting an attenuated strain of the carnivore distemper virus to the tissue culture of green monkey kidney cells~ growing the this adapted vaccine strain in the cell culture of gr-een monkey kidney tissue in the presence of a suitable nu~
trient medium~ removing from the resultant virus-contain-ing fluid the degenerated cell detritus of said culture~
and lyophlizing the liquid vaccine in the presence of a suitable virus thermostabilizer.
The te m "cell culture ot = nkey-kidney tissue" impli-1~540~;;3 es the primary cell culture of momkey-kidney tissue~ the subculture and the continuous cell line, however, it mi-ght turn out more efficient to produce the proposed vacci~
ne from the continuoua cell lines of green monkey embryonal kidney.
The term "continuous cell lines" ls to be construed as Lmplyin~ the cell cultures of green monkey embryonal kid-ney capable of unlimited growth, which may be cultivated in the course of an unlimited number of passages after being stored in liquid nitrogen.
Thanks to the above-described features of the continuo-us cell lines~ ~'Vakchum~ may be produced in any desired quantity no matter how many~ if any, green monkeys are ;
available.
However~ the fore~oing subculture of the green mon-key kidney cells is also an effective feedstock for the production of the proposed vaccine~ as subcultivation of the primary cells over 2 or 3 passages enables the yield of the cell mass, and consequintly the?yield of the vaccine, ~;
to be increased accordingly.
.:
~` In accordance with the invention, use is made of an attenuated strain of the carnivore destemper virus which is preadapted to the cell culture of green monkey kidney tissue~ The adaptation is carried out by several passages~ ~;
e.g. 8 to 10 passages~ at a temperature of fro~ 33.8 to ~
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:,.. . : .: ..... , . . , :: .. . . : ;:. ~ ,, . : i , -Lo5~L~S3 39.2C until the incllhation period of v;ral ~3rowth ;6 cut back -to 4 or 5 days.
Since ~he a-ttenuated strain of the carnivore distemper virus is subjected to 8 to ]0 passa~es in the yreen monkey kidney tissue cul-ture, said strain becomes fully adapted to the cell culture (as confirmed by the shortening of -the incubation period oE vlrus development and by the rise in its -I-itre), and ~he primary level of strain attenlla-tion can be preserved. It is ]ikewise posslble to ernploy an attenuated strain adap ed less stringently (4 or 5 passages), but in such a case the incuba-tion period of virus development is lengthened by S to 10 days and its titres decrease by a substantial margin. The level of virus attenuation may be affected by varying the temperature of the procedure beyond the above-cited limits.
The virus adapted as shown hereabove is used to pre-pare an inoculum from which the vaccine is procuded.
The production of the vaccine is based on the inocula-tion principle.
The inoculum system provides for the preparation of a large amount of vaccine virus meeting all the requirements to the attenuated strain, i.e. it must withstand storage in liquid nitrogen and furnish material for the production of the vaccine as such a need arises.
-~ The proposed vaccine may be produced both from freshly trypsinized cell culture of green monkey kidney tissue and from one withdrawn from storage in liquid nitrogen.
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:: - . - . . , :: - . , . . ,. . -~s~0~;3 The vacclne virus shall be grown in said cultures un-der rigirous temperature conditions (34.0 + 0.2 C.).
In order to increase the virus yield and fully utilize the cell culture of said lcidney tissue, it is recommended to grow the strain in such a way that the virus-contalning 1uld is dralned off and replaced by a fresh nutrient medi-um several times, the operation of virus-containing fluid replacement bcing effected or the first time when 30 to 40 percent of cells show a cytopathigc effect (CPE); and this operation is repeated s i.e. the virus-containing fluid is drained off and a fresh nuerient medium placed instead, to the point of total cell degradation~ usually 3 to 5 ti-mes.
The virus-containing fluid batches collected from the cell culture are pooled , freed from cell defritus by any known method, e.g. by settling followed by decantation~
or using a separator, or else by low-speed centrifugation.
To stabilize the virus, a suitable stabilizer is added to the product virus-contain$ng fluid, e.g. 4~5 to 5.5 per_ cent sorbite by weight and 1.4 to 1.6 percent gelatose by weight, after which the liquid is lyophilized.
At all the stages of "Vakchum" preparation sterile condi-tions are required.
; The lyophlized preparation is subjected to biological ., , ~
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~ests on laboratory ani~als and in a culture tissue.
The ai~ of these tests consists in determining the level of the vaccine ViFUs in the preparation, sterility including control for the level of mycoplasm, sa~ety and the level of contaminan~-viruses pathoginic for carnivorous animals~
In case the test results are satisfactory~ the prepara~
tion ls regarded as a ready-for-use vaccine.
The invention offers severàl advantages over the prior art.
The proposed vaccine against carnivore distemper conta-ins no viruses pathogenic for said animals as it is prepared in a green monkey kidney tissue culture heterogenous for car-nivorous animals. Should said cultures happen to be contami-nated with zoonotic viruses, the latter are readily identi-fied by common biologlcal control methods, and the affected cultures are discorded, whereas the prior art vaccines are susceptible to contamination with viruses which are patho-genic for carnivorous animals.
The high immunogenicity and low reactogenicity of the ;
proposed vaccine are guaranteed by the adaptation conditions under which the strain retains all its attenuated properties, high immunological effectiveness and low reactogenicity.
The "Vakchum" preparation is highly standardized, which ~ -is achieved by the very high yield of the preparation in one series ~from 100~000 to 2,000,000`vaccination doses)~
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whereas the prior art vaccines are not standard enollgh9 what with the small volume of the series.
The proposed vaccine con~ains a minimum of foreing proteins, ensuring the anlmals against allergic reac~ions; and the lack of contaminant-viruses pathogenic for carnivorous animals ~uarantees vaccine safety.
~ further advantage of the present invention consists in the possibility of utilizing the cell cultures of green monkey kidney tissue which are discarded, for this or that reason, in the ~ ;
production of antipolio vaccines but quite fit for "Vakchum"
production. This feature favourably affects the economics of the process and permits cutting back heavily on the cost of the preparation, a substantial commercial advantage of "Vakchum" over the prior art antidistemper vaccines. ~ ~ `
The method of producing "Vakchum" in accordance with the present invention conduces to a far cheaper vaccine than all the prior art ones.
; These and other advantages will become more apparent to those skilled in the art from the following detailèd descrip-tion oi the invention.
The live culture virus vaccine against carnivore distem-per in accordance with the invention is constituted by an attenuated strain of the carnivore distemper virus which is preadapted to the cell culture of green monkey kidney . . :
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~540~3 tissue and gro~n in said culture.
The antidstemper vaccine production is based on the in-oculum system. The lnocolum is an attenuated carnivore di-stemper virus~ e.g. the strain isolated by Rolcborn (Sweden). Such a vi m s is adapted to the cell culture of green monkey kidney tissue in the course of 8 to 10 pas-sages at a temperature of 34.0 ~ 0.2 C. Violation of said temperature conditions is undesirable for it may af-fect the properties of the attenuated virus.
In the course of adaptation, the incubation period of virus development is reduced and the first morphological alterations in the cells are observed ~ or 5 days after the inoculation together with a rise in the titre of the multi-plied virus and an increase in the general yield of the vaccine virus in the cell culture grown in matrasses.
~ daptation of the attenuated strain to the cell culture of green monkey kidney tissue under the set of conditions specified hereabove not only leads to a considerably higher virus yield but also ensures preservation of the strain attenuation level, its safety for the vaccinated animals, as well as its high immunological activity and epizootolo-- gical efficiency.
The inoculum of the adapted strain is first subjected to all-round fesfing, including control tests for the pre-. ' ~.
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sence of alien contclln;narlt-viruses pathoyenic for carnivorous animals, for mlcrobial a~ld mycoplasmatic s-terllity, for -the virus titre, reactogenicity and immunological activity on susceptible animals (minks or polar foxes), after which it is dispensed into containers and lyophillzed. The inocu]urn thus obtained and referred to as a prlmary inoculurn is stored at a temperature of not higher than Minus 20 C. A secondary inoculum is produced from the primary ;noculum by one or two serial passages. The secondary inoculum is grown in a cell culture of green monkey kidney tissue under the same conditions as those at which the primary inoculum is cultivated. The secondary inoculum is dis-pensed into Elasks and stored in a frozen state at a temperature of not higher than minus 30C without prelyophiliæation. The secondary inoculum is tested for the presence of contaminant~
viruses pathogenic for the carnivorous animals as well as for microbial and mycoplasmatic sterility and titre. After control tests show the absence of contaminant-viruses and an adequate level of microbial and mycoplasmatic sterility, the secondary inoculum is used to prepare a vaccine.
` 20 The carnivore distemper vaccine virus is grown by use " of a freshly trypsinized cell culture of green monkey kidney :
tissue, subcultures, cell cultures or subcultures after storage - in liquid nitrogen, and continuous cell lines of green monkey kidney tissue. It should be emphasized . :, ~, .
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that the carnivore distemper vaccine virus can be grown in the cell cultures of green monkey kidney tissue discar-ded, for this or that reason, in the prodiction of live antipolio vaccine but entirely suitable for the cultivati-on of the vaccine strain oE the carnivore distemper virus a~s well as for the manufacture of a preventive preparation to control carnivore distemper.
The continuous cell lines of green moykey kidney tis-sue constitute the best material for pxodicing the proposed antidistemper vaccine, because they enable the preparation to be produced independently of antipolio vaccine prodic-tion and irrespective of whether or not the kidney tissue donors, i.e.. green monkeys, are available. The continuous cell lines are capable of withstanding inlimited storage in llquid nitrogen; they can be used in any season and in any amount to produce an antidistemper vaccine. The above-list-ed cell cultures are cultivated in suitable media, such as 0.5% lactalbumin hydrolysate in Hank's solution or Eag-le's medium or a mixture of equal quantities of said me-dia plus 5% of bovine serum.
Prior to infection of the cultures~ the culture medi-um is removed from the culture glasses5 the cultures are once rinsed with Earle~s solution~ and 3 to 6 ml of the secondary inoculum is introduced. After 1 hour~s adsorption .:.~: :
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''; ' :.` : ., ~5~3 o~ the virus at a temperature between 33.8 and 34.2 C., 300 ml of a maintenance mediu~ is placed in the culture &1-asses, the maintenance medium be~ng composed of 0.5% la-ctalbumlnn hydrolysate in Earle~s 901ution, pH 7-5~?
with 5% amLnopeptlde. As soon as the culture shows the Elrst manLfestatlon of vlrus multiplication, i.e. blurring of the cell picture and cell Doundaries, the starting porti-on of the maintenance medium is remo~ed to get rid of the interEeron prsventing further multiplication of the virus, and 500 ml of a fresh medium of the same composition is placed in the culture glasses instead. After 30 to 40 per-cent of all cells show degenerative alterations, the virus--containing medium is drained off and frozen, and the cul-ture glasses are filled with the same amount of a fresh portion of the same medium. The next portion of the virus-containing fluid is harvested in 2 or 3 days~ time depending on the dynamics of cytopathological changes in the cells.
The virus-containing medium is repeatedly harvested and . ~ ..
~` replaced by fresh portions thereof until all the cells are involved in cytopathological change. Usually it takes 3 to ;
- 5 harvestsO
Each separate harvest of the virus-containing fluid is kept frozen at a temperature between -20 and -30C.
till the time of control tests for sterility and or the .
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~, . ': ' ' ~[)5f~3 presence oE contaminant-viruses pathogenic for the animals to be vaccinated. Following the control tests, provided the results are nega~ive (the tests for the presence of ~oreign viruses are conducted on newborn ancl adult whi~e mi-ce, guinea plgs and rabbits), the separate batches of the virus containing fluid are defrosted, poured into a single Ve99el and the resultant pooled harvest is freed from the cell detritus, e.g. by centrifugation.
To stabilize the virus in the detritus-free fluid, 4.5 to 5.5 percent sorbite by wight and 1.4 to 1.6 percen~
gelatose by weight are added. The mixture thus produced is thoroughly mixed, after which the preparation is ampuled and froæen at a temperature of minus 60 C.
after the freezing procedure, the vials are placed in-to a sublimator and lyophlized, the dried preparation hav-ing a residual moisture content of 3 to 5 percent.
The resultant preparation is tested for sterility, vir-us titre and safety on guines pigs. Should the sterility and safety tests be negative and the virus titre results positi-ve, the vaccine is regarded as ready-for-use preparation that can be employed for preventive immunization of fur bearers and dogs. If bur bearer immunization is to be effec-tive~ the mean Vaccine dose must contain at least 1,000 plaque-forming units (PFU) of said strain. Usually, commer-'~ , ' . :
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-14_ ~5~;3 cial vaccines contain no less than 3,000 plaque-forming un-its per dose. The lyophlizecl vaccine is effective for 1 year if it is stored at a temperature not higher than 4 C. If the preparation is stored at minus 20 C., the vaccine may retain its potency longer provided Its virus content is at least 1,000 plaque-~orming units, as determined by retitra tion.
The present invention will be further understood from the following examples, Examples 1 to 3 illustrating the proposed method of producing the "Vakchum" preparation and Examples 4 to 8 illustrating its practical application.
- E x a m p 1 e 1 "Vakchum" manufacture by use of a primary cell culture of green monkey kidney tissue.
The cell culture was grown in 65 1.5-liter culture glasses in a nutrient medium composed of equal quantities of Hanks' and ~aglels solutions with 0~5% lactalbumin hydrolysate~ pH = 7.0~ with 5% of normal Ibovine serum at a temperature of 37 C. After monolayer cultures were produr ced~ the growth medium was removed and the cell cultures wash-; ed with Earle's solution, whereupon 5 ml of an ~inoculum comprising attenuated Rokborn's strain was introduced into each culture glass, the inoculum having been earlier , , ~. '; ';
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~g~5~3 passaged 8 times in the cell cultures of green monkey kid-ney tissue at a temperature of 34 C1 After 1 hour's adso-rption of the virus at a temperature of 34 C., 300 ml of a nutrlent medium comprising 0.5% lactalbumin hydrolysate in Earle~s solution~ p~l = 7.5, with 5% aminopeptide was introduced into each culture. The infected cultures were lncubated at a temperature of 34 C. The first manifestati-ons of morphological changes in the cells testifying to virus multiplication were observed on the 5th day after the infection. The nutrient medium was thereupon removed and re-placed by a 500 ml frech portion of the same medium. The first harvesting of the virus and replacement of the nutri-ent medium of the foregoing composition were carried out on the 8th day; the second on the 11th day; the third on the 15th day; and the fourth, and last, on the 19th day. All in all, 115 liters of the virus-containing fluid was har-vested ; after the virus-containing fluid was freed from cell detritus, virus stabilizers (5% sorbite and 1.5%
gelatose) were added thereto.
The virus-containing fluid was dispensed into 100 ml vials, 33 ml per vial. All in all, 2~937 vials were filled , which were then placed in a refrigerator for freezing at a temperature of -65 C. After freezing~ the vials were transferred to a sublimator and the preparation was ,:
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suitability o each tissue culture source.
It is an obJect of the present invention to obviate the foregoing disadvantages.
Accordin~ly, the invention seeks to provide a novel method o produclng an antidistemper vaccine whereby the tlssue cult~lres of a slngle animal could yield large series of a highly standard, sufficiently immunogenic, safe and cheap vaccine.
It is an object of the present invention to provide an antidistemper vaccine which would be free from viruses pathogenic for the animals to be vaccinated.
It is another object of the present invention to pro~
vide a vaccine for carnivore distemper prevention which would be highly standard, sufficiently immunogenic, safe and cheap.
It is a further object of the invention to provide a method of producing an antidistemper vaccine exhibiting the above-described properties.
It is yet another object of the present invention to provide a method of producing said vaccine which would en-sure a high yield of wirus from the tissue cultures of a single animal.
It is still another object of the present invention to -:. ~
~,provide a method of producing said vaccine which would uti- ` `
' ~
: , .
' ' ' :: ~
:: ' ' O~V5~
lize ~idely available Material.
These and other ob~ects are attained in an antidistem~
per vaccine ~hich, in accordance ~it~ the invention, is constitutecl by an attenuated strain of the carnivore dis-temper virus aclapted to the culture of green monkey kiclney cells ancl gown ln saicd culture.
Said vaccine will be referred to hereinafter as "Va-kchuml' (a Russian abbreviation for antidistemper vacc-ine ).
"Vakchum" offers the following advantages: it is ade-quately immunogenic, safe ior the vaccinated animal re-gardless of their age, highly standard and free from con-taminant-viruses pathogenic for carnivorous animals.
The proposed method of producing "Vakchum", in accorda_ nce with the invention, includes the steps of preadapting an attenuated strain of the carnivore distemper virus to the tissue culture of green monkey kidney cells~ growing the this adapted vaccine strain in the cell culture of gr-een monkey kidney tissue in the presence of a suitable nu~
trient medium~ removing from the resultant virus-contain-ing fluid the degenerated cell detritus of said culture~
and lyophlizing the liquid vaccine in the presence of a suitable virus thermostabilizer.
The te m "cell culture ot = nkey-kidney tissue" impli-1~540~;;3 es the primary cell culture of momkey-kidney tissue~ the subculture and the continuous cell line, however, it mi-ght turn out more efficient to produce the proposed vacci~
ne from the continuoua cell lines of green monkey embryonal kidney.
The term "continuous cell lines" ls to be construed as Lmplyin~ the cell cultures of green monkey embryonal kid-ney capable of unlimited growth, which may be cultivated in the course of an unlimited number of passages after being stored in liquid nitrogen.
Thanks to the above-described features of the continuo-us cell lines~ ~'Vakchum~ may be produced in any desired quantity no matter how many~ if any, green monkeys are ;
available.
However~ the fore~oing subculture of the green mon-key kidney cells is also an effective feedstock for the production of the proposed vaccine~ as subcultivation of the primary cells over 2 or 3 passages enables the yield of the cell mass, and consequintly the?yield of the vaccine, ~;
to be increased accordingly.
.:
~` In accordance with the invention, use is made of an attenuated strain of the carnivore destemper virus which is preadapted to the cell culture of green monkey kidney tissue~ The adaptation is carried out by several passages~ ~;
e.g. 8 to 10 passages~ at a temperature of fro~ 33.8 to ~
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:,.. . : .: ..... , . . , :: .. . . : ;:. ~ ,, . : i , -Lo5~L~S3 39.2C until the incllhation period of v;ral ~3rowth ;6 cut back -to 4 or 5 days.
Since ~he a-ttenuated strain of the carnivore distemper virus is subjected to 8 to ]0 passa~es in the yreen monkey kidney tissue cul-ture, said strain becomes fully adapted to the cell culture (as confirmed by the shortening of -the incubation period oE vlrus development and by the rise in its -I-itre), and ~he primary level of strain attenlla-tion can be preserved. It is ]ikewise posslble to ernploy an attenuated strain adap ed less stringently (4 or 5 passages), but in such a case the incuba-tion period of virus development is lengthened by S to 10 days and its titres decrease by a substantial margin. The level of virus attenuation may be affected by varying the temperature of the procedure beyond the above-cited limits.
The virus adapted as shown hereabove is used to pre-pare an inoculum from which the vaccine is procuded.
The production of the vaccine is based on the inocula-tion principle.
The inoculum system provides for the preparation of a large amount of vaccine virus meeting all the requirements to the attenuated strain, i.e. it must withstand storage in liquid nitrogen and furnish material for the production of the vaccine as such a need arises.
-~ The proposed vaccine may be produced both from freshly trypsinized cell culture of green monkey kidney tissue and from one withdrawn from storage in liquid nitrogen.
~' -,.
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:: - . - . . , :: - . , . . ,. . -~s~0~;3 The vacclne virus shall be grown in said cultures un-der rigirous temperature conditions (34.0 + 0.2 C.).
In order to increase the virus yield and fully utilize the cell culture of said lcidney tissue, it is recommended to grow the strain in such a way that the virus-contalning 1uld is dralned off and replaced by a fresh nutrient medi-um several times, the operation of virus-containing fluid replacement bcing effected or the first time when 30 to 40 percent of cells show a cytopathigc effect (CPE); and this operation is repeated s i.e. the virus-containing fluid is drained off and a fresh nuerient medium placed instead, to the point of total cell degradation~ usually 3 to 5 ti-mes.
The virus-containing fluid batches collected from the cell culture are pooled , freed from cell defritus by any known method, e.g. by settling followed by decantation~
or using a separator, or else by low-speed centrifugation.
To stabilize the virus, a suitable stabilizer is added to the product virus-contain$ng fluid, e.g. 4~5 to 5.5 per_ cent sorbite by weight and 1.4 to 1.6 percent gelatose by weight, after which the liquid is lyophilized.
At all the stages of "Vakchum" preparation sterile condi-tions are required.
; The lyophlized preparation is subjected to biological ., , ~
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~ests on laboratory ani~als and in a culture tissue.
The ai~ of these tests consists in determining the level of the vaccine ViFUs in the preparation, sterility including control for the level of mycoplasm, sa~ety and the level of contaminan~-viruses pathoginic for carnivorous animals~
In case the test results are satisfactory~ the prepara~
tion ls regarded as a ready-for-use vaccine.
The invention offers severàl advantages over the prior art.
The proposed vaccine against carnivore distemper conta-ins no viruses pathogenic for said animals as it is prepared in a green monkey kidney tissue culture heterogenous for car-nivorous animals. Should said cultures happen to be contami-nated with zoonotic viruses, the latter are readily identi-fied by common biologlcal control methods, and the affected cultures are discorded, whereas the prior art vaccines are susceptible to contamination with viruses which are patho-genic for carnivorous animals.
The high immunogenicity and low reactogenicity of the ;
proposed vaccine are guaranteed by the adaptation conditions under which the strain retains all its attenuated properties, high immunological effectiveness and low reactogenicity.
The "Vakchum" preparation is highly standardized, which ~ -is achieved by the very high yield of the preparation in one series ~from 100~000 to 2,000,000`vaccination doses)~
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1~:35~
whereas the prior art vaccines are not standard enollgh9 what with the small volume of the series.
The proposed vaccine con~ains a minimum of foreing proteins, ensuring the anlmals against allergic reac~ions; and the lack of contaminant-viruses pathogenic for carnivorous animals ~uarantees vaccine safety.
~ further advantage of the present invention consists in the possibility of utilizing the cell cultures of green monkey kidney tissue which are discarded, for this or that reason, in the ~ ;
production of antipolio vaccines but quite fit for "Vakchum"
production. This feature favourably affects the economics of the process and permits cutting back heavily on the cost of the preparation, a substantial commercial advantage of "Vakchum" over the prior art antidistemper vaccines. ~ ~ `
The method of producing "Vakchum" in accordance with the present invention conduces to a far cheaper vaccine than all the prior art ones.
; These and other advantages will become more apparent to those skilled in the art from the following detailèd descrip-tion oi the invention.
The live culture virus vaccine against carnivore distem-per in accordance with the invention is constituted by an attenuated strain of the carnivore distemper virus which is preadapted to the cell culture of green monkey kidney . . :
. ~ :
_9_ :
: : . : .:: : :
~540~3 tissue and gro~n in said culture.
The antidstemper vaccine production is based on the in-oculum system. The lnocolum is an attenuated carnivore di-stemper virus~ e.g. the strain isolated by Rolcborn (Sweden). Such a vi m s is adapted to the cell culture of green monkey kidney tissue in the course of 8 to 10 pas-sages at a temperature of 34.0 ~ 0.2 C. Violation of said temperature conditions is undesirable for it may af-fect the properties of the attenuated virus.
In the course of adaptation, the incubation period of virus development is reduced and the first morphological alterations in the cells are observed ~ or 5 days after the inoculation together with a rise in the titre of the multi-plied virus and an increase in the general yield of the vaccine virus in the cell culture grown in matrasses.
~ daptation of the attenuated strain to the cell culture of green monkey kidney tissue under the set of conditions specified hereabove not only leads to a considerably higher virus yield but also ensures preservation of the strain attenuation level, its safety for the vaccinated animals, as well as its high immunological activity and epizootolo-- gical efficiency.
The inoculum of the adapted strain is first subjected to all-round fesfing, including control tests for the pre-. ' ~.
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sence of alien contclln;narlt-viruses pathoyenic for carnivorous animals, for mlcrobial a~ld mycoplasmatic s-terllity, for -the virus titre, reactogenicity and immunological activity on susceptible animals (minks or polar foxes), after which it is dispensed into containers and lyophillzed. The inocu]urn thus obtained and referred to as a prlmary inoculurn is stored at a temperature of not higher than Minus 20 C. A secondary inoculum is produced from the primary ;noculum by one or two serial passages. The secondary inoculum is grown in a cell culture of green monkey kidney tissue under the same conditions as those at which the primary inoculum is cultivated. The secondary inoculum is dis-pensed into Elasks and stored in a frozen state at a temperature of not higher than minus 30C without prelyophiliæation. The secondary inoculum is tested for the presence of contaminant~
viruses pathogenic for the carnivorous animals as well as for microbial and mycoplasmatic sterility and titre. After control tests show the absence of contaminant-viruses and an adequate level of microbial and mycoplasmatic sterility, the secondary inoculum is used to prepare a vaccine.
` 20 The carnivore distemper vaccine virus is grown by use " of a freshly trypsinized cell culture of green monkey kidney :
tissue, subcultures, cell cultures or subcultures after storage - in liquid nitrogen, and continuous cell lines of green monkey kidney tissue. It should be emphasized . :, ~, .
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that the carnivore distemper vaccine virus can be grown in the cell cultures of green monkey kidney tissue discar-ded, for this or that reason, in the prodiction of live antipolio vaccine but entirely suitable for the cultivati-on of the vaccine strain oE the carnivore distemper virus a~s well as for the manufacture of a preventive preparation to control carnivore distemper.
The continuous cell lines of green moykey kidney tis-sue constitute the best material for pxodicing the proposed antidistemper vaccine, because they enable the preparation to be produced independently of antipolio vaccine prodic-tion and irrespective of whether or not the kidney tissue donors, i.e.. green monkeys, are available. The continuous cell lines are capable of withstanding inlimited storage in llquid nitrogen; they can be used in any season and in any amount to produce an antidistemper vaccine. The above-list-ed cell cultures are cultivated in suitable media, such as 0.5% lactalbumin hydrolysate in Hank's solution or Eag-le's medium or a mixture of equal quantities of said me-dia plus 5% of bovine serum.
Prior to infection of the cultures~ the culture medi-um is removed from the culture glasses5 the cultures are once rinsed with Earle~s solution~ and 3 to 6 ml of the secondary inoculum is introduced. After 1 hour~s adsorption .:.~: :
. . .
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''; ' :.` : ., ~5~3 o~ the virus at a temperature between 33.8 and 34.2 C., 300 ml of a maintenance mediu~ is placed in the culture &1-asses, the maintenance medium be~ng composed of 0.5% la-ctalbumlnn hydrolysate in Earle~s 901ution, pH 7-5~?
with 5% amLnopeptlde. As soon as the culture shows the Elrst manLfestatlon of vlrus multiplication, i.e. blurring of the cell picture and cell Doundaries, the starting porti-on of the maintenance medium is remo~ed to get rid of the interEeron prsventing further multiplication of the virus, and 500 ml of a fresh medium of the same composition is placed in the culture glasses instead. After 30 to 40 per-cent of all cells show degenerative alterations, the virus--containing medium is drained off and frozen, and the cul-ture glasses are filled with the same amount of a fresh portion of the same medium. The next portion of the virus-containing fluid is harvested in 2 or 3 days~ time depending on the dynamics of cytopathological changes in the cells.
The virus-containing medium is repeatedly harvested and . ~ ..
~` replaced by fresh portions thereof until all the cells are involved in cytopathological change. Usually it takes 3 to ;
- 5 harvestsO
Each separate harvest of the virus-containing fluid is kept frozen at a temperature between -20 and -30C.
till the time of control tests for sterility and or the .
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.
~, . ': ' ' ~[)5f~3 presence oE contaminant-viruses pathogenic for the animals to be vaccinated. Following the control tests, provided the results are nega~ive (the tests for the presence of ~oreign viruses are conducted on newborn ancl adult whi~e mi-ce, guinea plgs and rabbits), the separate batches of the virus containing fluid are defrosted, poured into a single Ve99el and the resultant pooled harvest is freed from the cell detritus, e.g. by centrifugation.
To stabilize the virus in the detritus-free fluid, 4.5 to 5.5 percent sorbite by wight and 1.4 to 1.6 percen~
gelatose by weight are added. The mixture thus produced is thoroughly mixed, after which the preparation is ampuled and froæen at a temperature of minus 60 C.
after the freezing procedure, the vials are placed in-to a sublimator and lyophlized, the dried preparation hav-ing a residual moisture content of 3 to 5 percent.
The resultant preparation is tested for sterility, vir-us titre and safety on guines pigs. Should the sterility and safety tests be negative and the virus titre results positi-ve, the vaccine is regarded as ready-for-use preparation that can be employed for preventive immunization of fur bearers and dogs. If bur bearer immunization is to be effec-tive~ the mean Vaccine dose must contain at least 1,000 plaque-forming units (PFU) of said strain. Usually, commer-'~ , ' . :
. ' '':
-14_ ~5~;3 cial vaccines contain no less than 3,000 plaque-forming un-its per dose. The lyophlizecl vaccine is effective for 1 year if it is stored at a temperature not higher than 4 C. If the preparation is stored at minus 20 C., the vaccine may retain its potency longer provided Its virus content is at least 1,000 plaque-~orming units, as determined by retitra tion.
The present invention will be further understood from the following examples, Examples 1 to 3 illustrating the proposed method of producing the "Vakchum" preparation and Examples 4 to 8 illustrating its practical application.
- E x a m p 1 e 1 "Vakchum" manufacture by use of a primary cell culture of green monkey kidney tissue.
The cell culture was grown in 65 1.5-liter culture glasses in a nutrient medium composed of equal quantities of Hanks' and ~aglels solutions with 0~5% lactalbumin hydrolysate~ pH = 7.0~ with 5% of normal Ibovine serum at a temperature of 37 C. After monolayer cultures were produr ced~ the growth medium was removed and the cell cultures wash-; ed with Earle's solution, whereupon 5 ml of an ~inoculum comprising attenuated Rokborn's strain was introduced into each culture glass, the inoculum having been earlier , , ~. '; ';
-. : i -15~
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~g~5~3 passaged 8 times in the cell cultures of green monkey kid-ney tissue at a temperature of 34 C1 After 1 hour's adso-rption of the virus at a temperature of 34 C., 300 ml of a nutrlent medium comprising 0.5% lactalbumin hydrolysate in Earle~s solution~ p~l = 7.5, with 5% aminopeptide was introduced into each culture. The infected cultures were lncubated at a temperature of 34 C. The first manifestati-ons of morphological changes in the cells testifying to virus multiplication were observed on the 5th day after the infection. The nutrient medium was thereupon removed and re-placed by a 500 ml frech portion of the same medium. The first harvesting of the virus and replacement of the nutri-ent medium of the foregoing composition were carried out on the 8th day; the second on the 11th day; the third on the 15th day; and the fourth, and last, on the 19th day. All in all, 115 liters of the virus-containing fluid was har-vested ; after the virus-containing fluid was freed from cell detritus, virus stabilizers (5% sorbite and 1.5%
gelatose) were added thereto.
The virus-containing fluid was dispensed into 100 ml vials, 33 ml per vial. All in all, 2~937 vials were filled , which were then placed in a refrigerator for freezing at a temperature of -65 C. After freezing~ the vials were transferred to a sublimator and the preparation was ,:
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3;33 dried by lyophliæation. Each vial contained 100 doses of the virus of titre 10 5 ~ 4 plaque~forming units per milliliter. The entire series contained 276, 300 vaccine doses. Control tests gave Eavourable results~ and the va-ccLne was distributed for actual use u~cler a vaccination pro&,ram.
E x a m p 1 e 2 "Vakchum" production in a cell subculture of green monkey kidney tissue priorly stored in liquid nitrogen.
; The methods of cell culture infection and virus culti-vation duplicated the procedures described in detail in the previous example.
97 liters of a virus-containing 1uid was harvested.
After the fluid was pured into 100 ml vials~ 33 ml per vial, and after the vaccine was lyophilized, 2,050 vials were obtained each containing 100 vaccine doses. The enti-re series consisted of 205~ 000 vaccine doses of titre plaque-forming units per milliliter. The vaccine succe-ssfully passed control tests and was distributed for pre- ~
ventive vaccination. ~ `
E x a m p 1 e 3 ~ -"Vackchum" manufacture in a continuous cell line of ;~ -green monkey embryonal kidney tissue.
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~L~5~3 The continuous cell line culture was grown ln 30 1.5-Li-ter flasks~ The continuous cell line was grown in the me-dium of Example 1 except that 10% calf serum was added to the medium.
The culture was infected with the Rokborn strain of the carnlvore distemper virus preadapted to the primary ce~l culture of green monkey kidney tissue and the virus was grown in said culture in a medium with 0,5% lactalbumin hydrolysate in Earle~s solution~ pH 7.5~ with 5 percent aminopeptide by weigh at a temperature of 34 C. according to Example l to yield 16.7 liters of vaccine. All in all ~ 509 flasks were obtained~ each containing 100 vaccine doses of titre lO plaque~forming units per ml. The whole series comprises 50900 vaccination doses. The vaccine was successfully tested and distributed for preven-tive vaccination.
. ` ' '~ .
E x a m p 1 e 4 Testing of "Vakchum" for safety for the mink and polar fox young.
The vaccine was employed in a dose of 1 ml of a prepa- ;
, ration containing 3~000 plaque-forming units in 1 ml.
which was prediluted in distilled water.
24 min~ cubs and 12 polar fox cubs were vaccinated `with "Vakchum", with 16 mink cubs and 3 polar fox cubs left . .
:: `': "' ` -18 , ::
.
, ,. ., ~: ~. ,,, ,.. ' ' ~5~ ;3 unvaccinated as controls. Observations were conducted dally for 14 days. '~roughout ~he entire experimental period all the animals remained clinically healthy.
E x a m p 1 e 5 (a) Checking the "Vakchum" preparation for immunoge-nicity on polar fox cubs in a dose equal to that of Examp-le 4.
3 polar foxes were vaccinated with "Vakchum"~ 50 days later these 3 foxes and 2 controls (unvaccinated aga-inst distemper ) were infected with 1,500 lethal doses of the Gauyaskii strain. All 5 animals were observed dai- ~ ~;
ly. On the 15th day after the infection both controls were killed by distemper; the vaccinated foxes were subsequent-ly observed for another week. Throughout the entire obser- ~`
vation period (21 days after the infection) the vaccinated ~ `
animals remained clinically healthy. ~;
;~, (b) Checking the "Vakchum~' preparation for immunoge-nicity on adult polar foxes using the dose of Example 4.
` 19 polar foxes received shots of "Vakchum~'. 21 days after the inoculation all the vaccinated animals and 6 con-` trols (unvaccinated against distemper) were infected with 1~000 lethal doses of the Gauyaskii strain of the carnivo-re distemper virus. The observation period lasted for 21 days. On the 14th - 16th day the controls~(unvaccinated :,, '.,`
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~(~541~S;3 foxes) were Icilled by distemper, whereas all 19 vaccinated animals were in good ghealth.
E x a m p 1 e 6 Checking the extent of immunization of mink cubs indu-ced by "Vakchum" adminlstered in the dose of Example 4 In the course of summer vaccination, 5,333 Silver--Steelblu mink cubs were inoculated. In the period of vaccination and in the week that followed, all the animals were observed daily. All the vaccinated animals remained heàlthy.
'' ` ' E x a m p 1 e 7 Checking the length of immunity induced by "Valtchum"
administered in the dose of Example 4 (a) 6 polar foxes were vaccinated with the "Vakchum~' preparation. 9 months after the vaccination, these 6 ani-mals and 2 controls (unvaccinated foxes) were infected with 1,000 lethal do~es of the highly pathogenic Gauyaskii stra-in of ehe carnivor distemper virus. 2 control foxes sho-wed symptoms of distemper on the 10th and 11th day, respe-ctively, and died, whereas the 6 foxes vaccinated against distemper showed no signs of the disease.
., (b) 3 polar foxes were vaccinated with "Vakchum" in the dose of Example 4. 27 months later these 3 animals and :, '' `, ;":'"' '' ':
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6 controls (unvaccinate(l agalnst distemper) were infected with 1,000 lethal doses of the Ga~yaskii strain of carni-vore distemper virus. All 6 controls developed distemper and died on the 12th-13th day, whereas the 3 foxes vacci-nated wLth "Valcchum" 27 months prcviously remained unaffe~
cted.
E x 1 m p 1 e 8 Testlng in a distemper ~ocus.
At a fur farm with a high incedence of mink distemper a vaccination program was carried out using the "Vakchum"
preparation administered in the dose of Example 4. First, ~ ;
23 animals were vaccinated, putting an end to distemper in the group. Then another 12, 170 minks were vaccinated~
checking the morbidity entirely. Since then this farm has been free of distemper instances.
` Large~scale field testing of the "Vakchum" prepara-tion.
For 3 years~ over 7,000~000 doses of "Vackhum" were used to vaccinate fur bearers (mink~ polar fox, red fox and sable) at 36 farms located in different climatic zones. The vaccination programm demonstrated "Vakchum~s"
safety, high immunological and epixootological efficiency ., .
:~ :
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and ~lso its economic advantages. ApplicatLon of this preparation eradicated distemper at the farms whlch had earlier been known for a high incidence of this disease.
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E x a m p 1 e 2 "Vakchum" production in a cell subculture of green monkey kidney tissue priorly stored in liquid nitrogen.
; The methods of cell culture infection and virus culti-vation duplicated the procedures described in detail in the previous example.
97 liters of a virus-containing 1uid was harvested.
After the fluid was pured into 100 ml vials~ 33 ml per vial, and after the vaccine was lyophilized, 2,050 vials were obtained each containing 100 vaccine doses. The enti-re series consisted of 205~ 000 vaccine doses of titre plaque-forming units per milliliter. The vaccine succe-ssfully passed control tests and was distributed for pre- ~
ventive vaccination. ~ `
E x a m p 1 e 3 ~ -"Vackchum" manufacture in a continuous cell line of ;~ -green monkey embryonal kidney tissue.
-: :' :` ' :
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: . : . .: . . :
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~L~5~3 The continuous cell line culture was grown ln 30 1.5-Li-ter flasks~ The continuous cell line was grown in the me-dium of Example 1 except that 10% calf serum was added to the medium.
The culture was infected with the Rokborn strain of the carnlvore distemper virus preadapted to the primary ce~l culture of green monkey kidney tissue and the virus was grown in said culture in a medium with 0,5% lactalbumin hydrolysate in Earle~s solution~ pH 7.5~ with 5 percent aminopeptide by weigh at a temperature of 34 C. according to Example l to yield 16.7 liters of vaccine. All in all ~ 509 flasks were obtained~ each containing 100 vaccine doses of titre lO plaque~forming units per ml. The whole series comprises 50900 vaccination doses. The vaccine was successfully tested and distributed for preven-tive vaccination.
. ` ' '~ .
E x a m p 1 e 4 Testing of "Vakchum" for safety for the mink and polar fox young.
The vaccine was employed in a dose of 1 ml of a prepa- ;
, ration containing 3~000 plaque-forming units in 1 ml.
which was prediluted in distilled water.
24 min~ cubs and 12 polar fox cubs were vaccinated `with "Vakchum", with 16 mink cubs and 3 polar fox cubs left . .
:: `': "' ` -18 , ::
.
, ,. ., ~: ~. ,,, ,.. ' ' ~5~ ;3 unvaccinated as controls. Observations were conducted dally for 14 days. '~roughout ~he entire experimental period all the animals remained clinically healthy.
E x a m p 1 e 5 (a) Checking the "Vakchum" preparation for immunoge-nicity on polar fox cubs in a dose equal to that of Examp-le 4.
3 polar foxes were vaccinated with "Vakchum"~ 50 days later these 3 foxes and 2 controls (unvaccinated aga-inst distemper ) were infected with 1,500 lethal doses of the Gauyaskii strain. All 5 animals were observed dai- ~ ~;
ly. On the 15th day after the infection both controls were killed by distemper; the vaccinated foxes were subsequent-ly observed for another week. Throughout the entire obser- ~`
vation period (21 days after the infection) the vaccinated ~ `
animals remained clinically healthy. ~;
;~, (b) Checking the "Vakchum~' preparation for immunoge-nicity on adult polar foxes using the dose of Example 4.
` 19 polar foxes received shots of "Vakchum~'. 21 days after the inoculation all the vaccinated animals and 6 con-` trols (unvaccinated against distemper) were infected with 1~000 lethal doses of the Gauyaskii strain of the carnivo-re distemper virus. The observation period lasted for 21 days. On the 14th - 16th day the controls~(unvaccinated :,, '.,`
.: ' ` -19- ~ -': ~
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.
:~ , , . , , .,, , . , . - . . . . . . . .
.. :- :. . . . , : : . ~.
~(~541~S;3 foxes) were Icilled by distemper, whereas all 19 vaccinated animals were in good ghealth.
E x a m p 1 e 6 Checking the extent of immunization of mink cubs indu-ced by "Vakchum" adminlstered in the dose of Example 4 In the course of summer vaccination, 5,333 Silver--Steelblu mink cubs were inoculated. In the period of vaccination and in the week that followed, all the animals were observed daily. All the vaccinated animals remained heàlthy.
'' ` ' E x a m p 1 e 7 Checking the length of immunity induced by "Valtchum"
administered in the dose of Example 4 (a) 6 polar foxes were vaccinated with the "Vakchum~' preparation. 9 months after the vaccination, these 6 ani-mals and 2 controls (unvaccinated foxes) were infected with 1,000 lethal do~es of the highly pathogenic Gauyaskii stra-in of ehe carnivor distemper virus. 2 control foxes sho-wed symptoms of distemper on the 10th and 11th day, respe-ctively, and died, whereas the 6 foxes vaccinated against distemper showed no signs of the disease.
., (b) 3 polar foxes were vaccinated with "Vakchum" in the dose of Example 4. 27 months later these 3 animals and :, '' `, ;":'"' '' ':
`` :
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6 controls (unvaccinate(l agalnst distemper) were infected with 1,000 lethal doses of the Ga~yaskii strain of carni-vore distemper virus. All 6 controls developed distemper and died on the 12th-13th day, whereas the 3 foxes vacci-nated wLth "Valcchum" 27 months prcviously remained unaffe~
cted.
E x 1 m p 1 e 8 Testlng in a distemper ~ocus.
At a fur farm with a high incedence of mink distemper a vaccination program was carried out using the "Vakchum"
preparation administered in the dose of Example 4. First, ~ ;
23 animals were vaccinated, putting an end to distemper in the group. Then another 12, 170 minks were vaccinated~
checking the morbidity entirely. Since then this farm has been free of distemper instances.
` Large~scale field testing of the "Vakchum" prepara-tion.
For 3 years~ over 7,000~000 doses of "Vackhum" were used to vaccinate fur bearers (mink~ polar fox, red fox and sable) at 36 farms located in different climatic zones. The vaccination programm demonstrated "Vakchum~s"
safety, high immunological and epixootological efficiency ., .
:~ :
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and ~lso its economic advantages. ApplicatLon of this preparation eradicated distemper at the farms whlch had earlier been known for a high incidence of this disease.
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Claims (12)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A live virus culture vaccine effective against carnivore distemper which comprises a carnivore distemper virus strain which has been adapted to a cell culture of green monkey kidney tissue and grown in such a culture.
2. A method of producing a live virus culture vaccine effective against carnivore distemper, which comprises, under sterile conditions, adapting a carnivore distemper virus strain to a cell culture of green monkey kidney tissue, cultivating the adapted strain in a cell culture of green monkey kidney tissue in the presence of a suitable nitrient medium, removing degen-erated cell detritus from the resultant virus-containing liquid, and lyophilizing the resultant liquid vaccine in the presence of a suitable virus thermostabilizer.
3. A method according to claim 2, in which the cell culture is a primary cell culture of green monkey kidney tissue.
4. A method according to claim 2, in which the cell culture is a cell subculture of green monkey kidney tissue.
5. A method according to claim 2, in which the cell culture is a continuous cell line of green monkey kidney tissue.
6. A method according to claim 2, 3 or 4 in which strain is adapted to the cell culture of green monkey kidney tissue by passing it 8 to 10 times thereover at a temperature of 34.0 + 0.2°C.
7. A method according to claim 2, 3 or 4 wherein the adapted strain is prepared as an inoculum.
3. A method according to claim 2, 3 or 4 in which the cell culture of green monkey kidney tissue in which the adapted strain is cultivated has previously been stored in liquid nitrogen.
9. A method according to claim 2, 3 or 4 in which the adapted strain is cultivated at a temperature of 34.0 + 0.2°C.
10. A method according to claim 2 in which the virus containing liquid is removed and replaced with fresh nutrient medium at least three times until the cells of the culture are completely degenerated.
11. A. method according to claim 10 in which the virus-containing liquid is removed for the first time after 30 to 40 percent of the cells of the culture exhibit a cytopathic effect.
12. A method according to claim 2, 3 or 4 in which the virus-containing liquid is lyophilized in the presence of 4.5 to 5.5 percent by weight sorbite and 1.4 to 1.6 percent by weight gelatose.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU7402019238A SU527072A1 (en) | 1974-04-25 | 1974-04-25 | Method of obtaining 'vakczum'plague vaccine |
| SU2086457 | 1974-12-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1054053A true CA1054053A (en) | 1979-05-08 |
Family
ID=26665514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA225,696A Expired CA1054053A (en) | 1974-04-25 | 1975-04-25 | Live virus culture vaccine against carnivore distemper and method of producing same |
Country Status (4)
| Country | Link |
|---|---|
| CA (1) | CA1054053A (en) |
| DE (1) | DE2517550C3 (en) |
| GB (1) | GB1464494A (en) |
| SE (1) | SE7504610L (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3138531A (en) * | 1961-07-12 | 1964-06-23 | Lilly Co Eli | Canine distemper vaccine |
| DE1235505B (en) * | 1965-01-19 | 1967-03-02 | Behringwerke Ag | Process for the production of a vaccine against distemper |
-
1975
- 1975-04-21 GB GB1643975A patent/GB1464494A/en not_active Expired
- 1975-04-21 DE DE2517550A patent/DE2517550C3/en not_active Expired
- 1975-04-21 SE SE7504610A patent/SE7504610L/en unknown
- 1975-04-25 CA CA225,696A patent/CA1054053A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DE2517550B2 (en) | 1981-07-30 |
| DE2517550C3 (en) | 1982-05-06 |
| DE2517550A1 (en) | 1975-10-30 |
| GB1464494A (en) | 1977-02-16 |
| SE7504610L (en) | 1975-10-27 |
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