Note: Descriptions are shown in the official language in which they were submitted.
1~;Z0856
itle of the Invention
L~VI~G ~I~US CUL~URE VACCI;~E AGAINST CAhINE
DIS~PE~ AND l~HOD OF ~RODUCI~G S~E
Fiel~ of the Invention
The present invention relates to veterinary. and more
particularly to a living virus culture vaccine againæt canine
distemper and the method o~ prepari~g same.
Ganine distemper is an acute contaæious disease of
~ur-bearin~ animal~ and dogs. Youn~ minks~ sil~er a~d blue
fox are especially susceptible to the diæease. The high
lethality and the ab6ence of effective therapeutio meanæ
are the cause of ~reat econimic damage to fur-beari~g animal
breeding.
Background of the Invention ~ ~
Eh~ in the prior art are living virus culture vaccines
used for prophylaxis of canine distemper. ~he vaacine~ contain
attenuated strains of di~tem~er viruse~ adapted to oell cultureæ
of dog or monkey~kidneys,~ or chiok embryo6.
he "Rockborn" vaccine comprisin~ an attenuated strain
of the canine distemper virus, obtai~ed from a wild strain,
solated ~rom~the blood of a dog affectsd with~diæte~per~ i8
m~ prepared on a dog kidney culture céll. ~o prepare the vaccine,
said strain is adapted to-the dog kidney cell culture by over
~s~ 50 passa~es~ and~cultiva~ed on~aid cell cult,ure i~ a ~anks'
solutlon oonthinin~O.5 p~er oent o~ lactalbumin hydrolysa~e and
10 per cent~of horse serum; or~20 per cent of cal~ serum, or
o~ Earle's medium contain mg 0.5 per cent o~ lactalbumin h~dro-
sate and 2 per ¢ent of ~or~e or calf serum. ~he temperature
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112V856
of adapta~ion is maintained at 30-37C, preferably at 35-37C.
~he obtained vaccine is then stabilized and lyophilized (see
U~ Pat. ~o. 3,098,011).
Known in the prior art is also a vaccine against canine
distemper prepared by ten passages of the virus on a cell
culture of dog kidneys in the ~arle's medium containing 5
per cent of lactalbumin hydrolysate and 5 per cent of horse
serum at a temperature of 38.5C (US Pat. ~o. 3,354,038).
Said vaccines have hi~h imm~nogenic activity but there
are also some disadvanta~es inherent in them, one of which
is low reproducibillty o~ the virus in the dog ~ldney cell
culture.
Another disadvantage of said vaccines i6 that dog kidne~ ;
cell culture i~5 used as the substrate for atten~.ation of the
iruB and produotlon of vaccinea, but dog kidneys can be
contaminated with some other viruses, and a special vivarium
is~reguired~to keep the dogs~away from uncontrollable infec-
~tion. ~his envolves~extra e~penditures.
Still another disadvantage of thè YaCCineS iS that it
is~difficult to~-determine thelr biological~activity, since
the~viru6 reproduotion is asso~ciated with an indistinctly
manifest cytopatho~enic action that shows by the 20th day
following the infeotion when nonspecifio degenerative changes
in~the cell oan develop and inter~ere with the assessment
of the result.
S~till another~di6advantage inherent in the known vaccines
prepared o~the dog kidney cell culture is the slow ~rowth
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of the cells (a monolayer of cells is only formed b~ the
5th to 7th da~) and this prolongs the process of preparing
the vaccine ~.
Viral vaccines intended to control canine distemper
animals obtained by cultivating the virus strain on a 9-da~
chick embryo cell culture or on chorioallantoic membranes
of chick embryo can present danger because of the high conta-
mination of chick emb~o with some other viruses, and with
,:,
viruses of the leukemia-sarcomatous group in psrticular (see
US Pat Nos 2,912,361 and 2,965,544).
A vaccine prepared on the chick embr~o cell culture
from the strain known in the USSR~as E~-668 has the Lame
disadvantage. ~ ;
he American vaccine AS~ is harmless~and devoid of said
disadvantage because it is prepared in the cell culture of
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chick embryo not affected by leukemia, but special farms are
reguired where~ such leukemi~-free poultry can be raised,whioh,
nvolvcs additional expenditures. Moreovcr, the vaccinal~
strain of the virus does not produce regular cytopathic~ action
in~thc ohick~cmbryo ccll culture which complicates the process
of thc~vaocinè~produotion.
Endw~ in the~prior art is also a vira ~ accine a~ainst
ca~ine dis~empcr prepared by cultivatin~ the virus of canine
; ;distemper on the oell culture of kidne~s of green monkeys
(e~. U8 Patent No~. 4,00~,974). ~his vaccine is sufficie~tl~
immunogenlc,~areactogcnic, highl~ standard, and harmless.
Howcver, mo~kcy~hu~tin~ bccomes now a difficult problem
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because ol the reduction of their populations. The manufac-
ture o~ this vaccine is bs~ed o~ the utilization of the waste
of poliomyelitis vaccine production and is thus closely con-
nected with this process. It is easy to unders,tand tAat the
co~struction of an independent facility,for the manufacture
of the distemper vaccine based on the cell culture of monkey
~idneys will be ver~ expensive.
~ he selection of the cell substrate for the manufacture
of t~e viral vaccine thus seems dif~icult. The imperative
prerequisite is the absence of contamlnation of the cell
substrate with extraneous viruses and also production of the
vaccine virus in sufficient amounts.
S~mmary of the Invention
" ~
It is an object of thls invention to provide a cheap and
;' harmlesa vaccine against cani~e distemper exhibiting high
immuno~enic and anti8eni~ activity.
Another object~o~the invention is;to~provide a vaccine~
against canine~distemper that will be free from other viruses
,pathogenio~t,o~said animals and~man, especially the viruses
of the leuk~emia-sarcomatous ~group. ~,
A further obj~eo~t~of the i~vention is to provide a highl~
;immunogenic strain of the;~irus of canine di~temper that ca~
be~cultivated on safe and readily available substrate.
Still~another ob3eot~of~the invention i9 to provide a
,me~thod for manufacture of the vaccine against canine distemper
having the above~proper ies. ~ ~
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~ et another ob~ect of the invention i5 to develop a
method for the manu~acture of said ~accine based on inex-
pensive and readily a~ailable materials.
Accordin~ to the invention, there is proposed a living
viru~ culture vacci~e against canine distemper comprising
the attenuated strain EPM Or the virus of canine distemper
deposited under the code number of 10-76, obtained from the
wild virus i~olated from the blood of a m.mk affected by
distemper, by repeated passage on the followin~ cell cultu-
res; (1) dog kidney cell culture, (2) continuouA cel~ culture
of kidneys of human embryo Rh, (3) mixed culture consisting
of cells of dog kidneys and cells of Japanese guall embryos, ada-
pted to the cell culture of Japanese quail embr~os and grown -
~on thi~ culture.
According to~the invention, there is also proposed a
method for preparine a liv1ng virus culture vaccine against
canlne distempDr, compriDi~g cultivation, on ~he cell culture
JDpDnDse quDil Dmbryos:in a suitable medium, of the~virus
of~canine distemper, EPM,:~depoDited under~the~number of 10-76,
obtained:from~the:wild virus i~olated from the~blood of a mink ~:~
afIeotqd~:with~distDmpDr~ by~repeated paDsD~ge on the following
cell~oultures:~(1) dog~kidney céll culture, (2) continuous
cell culture~of~human embr~o kid~ey~ Rh, (3) mixed culture con-
sisti_g of dog~kidne~ cells and cells o~ ~apanese quail embryos,
with subseque~t adaptation to the cell culture of embryos of
Japanese quail embryo~s; callection of the obtained virus-
oontaining fluid; lyophilic drying of the ~irus-containin~
fluid in the preDenoe of a suitable stabilizer .
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Said attenuated strain of the viru~, which is used ~or
the manufacture of the vaccine against canine distemper, i8
given the ~ame of EPM strain of virus o~ canine distemper.
~he capital letters EPM stand for embryo, quail ~Xussian
~ere~ ol~a), and Moscow, respectively.
Said strain is a novel vaccine strain. It is deposited
with the All-Union State In~titute for Veterinary Preparation~
of the Ministry of Agriculture of the USSR, where it has been
assigned the number of 10-76.
he EPM strain of virus of canine distemper has been
obtained from the wild virus isolated from the b}ood of a
mink affected with distemper, by~20-~0-fold passage on a
cell culture of dog kidneys at a temperature ~f 37~1C
with subsequent 5-15 passages on the cell~culture of the
human embryo kidne~ Rh at a temporature of 32t1C
amd ~-7 passages on the mixed cul~ture consisting of kidne~ ;-
oells of the dog~and Japane~se guail embr~o oells, at a tem-
perature of 35+1C~w1th subsequent adaptstion by 4 to 10 ^~
passages on~the~c~ell culture~of Japanese quail (Coturni~
coturn~onioa~ embryos. ~ ~ ~
o prepare~the vacc-ine, said strain is cultivated on
the cell culture of Japanese guail embr~os in a suitable
oulture mediam, e.g. medium 199, or Earle's ~edium contain-
ing 10 per cent of oattle blood ~serum. ~he ~irst harvest of
the vlrus-containin~ fluid~is oollected after de~elopment
o~ the cytopathio~a~otion of the viru~ in 20 to 40 per cent
of;the cells.~he c~ollected culture fluid is dried liophili-
oall~ the;presence o~a suitable stabilizer, e.g., in the
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presence of 4.5-5.5 per cent ~orbitol and 1.4-1.6 per cent
of gelatose.
The vaccine strain EPM 10-76 of the virus of carni-
-vores is a highly attenuated strain well propagating in the
cell culture of Japanese quail embryos characterized by dis-
tinct cytopathic action, which markedly simplifies t~e pro-
cess of production of the vaccine. Said strain is stable in
storage.
~- ~he advantages of the proposed vaccine are as follow~:
a) the vaccine is hqrmless, it does not produce toxic
action, and is manufactured in the cell culture o~ Japanese
quail embryos, which is an inexpensive and readily available
materiaI that~can reliably be controlled for the absence of
extraneous viruses; Japanese quails are naturally ihsusceptib-
le to the leukemia-sarcomatous ~roup viruses and the cell
culture of their embr~os is the optimum ~ubstrate for the
manufacture of the viral vaccine;
(b~ the cell~culture of~Japanese~guail ambryos has high
proliferatlve~activltg which~makes lt~possibl~e;to infect the
cell~suspens~lo~without Sormation of the cell monolayer;
(c) the~ vaoc~ine~has~high a~ti~enic activity, it produces
eff~ective and persistent immunity in the vaccinated ani~als~
and the vacoinatlon in the epi~ootic foci of canine diste~-
per rapidly eradicates the infection;
(d)~the~vaccine;c~n be given parenterally and in aerosol
form, whlch simplifles markedly the prophylactic measures and
ma~es the~m cheapèr;~
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(e) the vaccine- is highly active and the commercial
preparation can therefore be filled in vials or ampoules
in quantities ranging from 1 to 500 doses, which is very
convenient for individual or mass-scale use use of the vaccine.
~ hese and other advantages of the inveAtion will be-
come subsegueAtly clear from the detailed descriptioA of
tbe vaccine and of the method of its manufacture and use.
Deta led Description of the Invention
he method of preparing the living virus culture vaccine
is based on the virus inoculation principle. The principle
co~sists in preparing a large amount of~the vaccinal virus that
meets all requirement6 rOr thc attenuated: str~,in, in keeping~
this virus in the frozen state, and using it ~o prepare the
vaccine.
As has already been stated, the attenuated strain EæM
No. 10-76, is used to E,repare the vaccine again t canine
dist~qm~er.
Said straln~has been obtainéd from the;wild virus of
canine~distemper~isolated~from the blood of~a mink with
distemper.~his;wild ~train i8 attenuated by;multiple ~
pa;ssagei on various~cell~cult~re~ in the following sequence;
t1) 20-40~passage,a on the dog kidney cell culture at a
tem~erature~of 37+1 a;
2) 5-15~passages on a ¢ontinuous cell culture of kidneys
of;human embrya Rh~at~a~temperature of 32'1C;~ and
) 2-7~passages on a~;mixed~culture consisting of ~
dog~kidne;~ ¢ells and cells ~or J~psnese ~uail embryos at z
temperature~of~35 1C.
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The attenuated virus i8 adapted to the cell culture of
Japanése quail embryos by 4 to 10 passage~. ~he obtained
strain is cloned in the cell culture of Japane~e guail
embryos by the limit dilution method. All these operations
give the strain of the virus with marked cypopathic activity
which makes it possible to control the process of preparing
the vaccine, to harvest the virus in optimum terms, to sim-
plify signi~icantly the determination of the infectious ac-
tivity of the virus, and moreover to determine speci~icit~
of the virus in the cell culture and to reveal antibodies
in the blood serum of vaccinated animals. ~he obtained virus
strain is characterized b~ the following:
) specificity in the neutralization te6t on the cell
culture of Japanese quail embryos;
(2) harmlessness for laborstory animals, e.g. mice,
guinea pig8, rabbits, chick embryos, and minks and blue
(3)~in~eCtious aotlvity in tho Japane~e quail embr~o
oell~culture; ~ ~ -
(4) antigenic activity;
(5) imm ~ ogenic activity ~n the neutralization test on
the Japanese`guail embryo cell culture);
(6) absenoo of contaminating virusos;
(7) bacterial sterilit~ and tho absence of P~L0 myco-
plasms;
(8) stabillty in storage;
(9) absence~of oontagiosity.
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he seed virus is prcpared from said strain, char~cteri-
zed by the above listed properties, on the Japanese quail embr~o
cell culture. ~he ~eed Vi~U8 should be characterized b~ the same
properties as specified for the strain of the virus. ~he seed
virus is kept în the rrozen state ~t a temperature not above
minus 20C and used in the required quantities.
A trypsini~ed cell culture of Japane~e qusil embryos
is used for the manufacture of the vaccine. The culture is
~- prepared from 9-10 day old quail embryos which are crushed
and trypsinized by the known method. The obtained cell sus-
pension is inoculated with the seed virus and a suitable
nutrient medium is added. Known media that are usually used to
cultivate cells and viruses, e.g. medium 199, contalning 10
; ; per~cent of cattle~serum (pN 7.0-7,5), are used as the culture'
medium. ~he inoculated medium i8 incubated i~ rollers at a
temperature~f 35+1C. As soon as~a monolayer of cells is formed,~
and 20-40 per cent of cella show cytopathic action`,~the first
harvest of the fluid~containing the virus is collected.tIn
order to~increase the harvest of the virus~and to ensure more
oomplete~ utillzatlon of thè~ce'll culture,~ the harvesting of
the~virus-containing~ fluid'from the same~sample of the culture
is repeated~several times, fresh portions of the nutrient medium
being addcd after eaoh harvest. ~his oper~tion is repeated
until the cell aulture i8 completely degenerated.
In order to recover the intracellular virus, the cell
;cul~ture i8 frozen~and~then defroste~. The collected fluid
containing ~the virus lS testcd~for sterilit~ and infectio~s
activity, and oollectcd~in one~vessel. A~stabilizing agent i8
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added, and the fluid is filled into ampoules or vials and
lyophilized. ~he dried preparation contains 3 per cent by
weight of residual moisture.
l'he l~ophilized preparation can be kept for at least
one year. The lyophilized vaccine should be kept at a tem-
perature not above 4C. If stoxed at lower temperatures.,
this term can be prolonged by several months. h saline
solution prepared on the basis of the Henks solution should
be used to dilute the dry preparatio~ for ~accination purpo-
ses.
~ he obtsined vaccinal preparation is tested for sterility,
harmlessness, specificity, the absence of contaminating
viruses, infectious activity, immunogenic and a~ntigenic ac-
tivities, and residual moisture. After these tests the vaccine
can be used for prophylactic immunization of fur-bearin~ ;
animal and dogs, as well a~ ~or therapeutic purposes
in the course of the first days following the contact of
the animal with a distemper affected one. ~he mean dose to
vaccinate an animal should be not less than 10 ~CD50 (median
tissue cytopathic dose). ~ dose containlng to ~00,000 ~CD
does not produce undesirable reaction in the animals.
~ he animals are immuniz0d by intram~scular inj0ctions
of 1.0 ml oP the vaccine.
~ o make sure the vaccination has been effective, the
immunized animals can be given a serological examination in
10-14 days after the vaccination, and the titres of the
antibodies determined in the neutralizatlon reaction on
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a cell culture of Japanese quRil embr~os, using said vaccinal
strain, ~iving a marked cytopathic effect, a8 the anti~en.
~ntibbdies are revealed in All immunized animals in the titre
of 1:16-~2. ~he maximum accumulation of antibodies (in the
titre of 1:256) is attained by the ~Oth day. The antibodies
are revealed in the blood during 18 months following the
va¢cination. In ~ubsequent challenge of the immunized
animals with the virulent strain Snyder Eill in 1, 3, 6, 12
and 18 months after ~he immunization with the proposed vacci-
ne, none of the animals developed the disease. The proposed
vacoine can thus be considered to have 100 per cent immunoge-
nic and antigenic activit~
. The proposed vacclne wa6 used at a farm;at the moment
of the outbreak of~ distemper among silver fox. ?he vaccina-
tlon~stopped the di6ea6e within~a month and~no cases of distem- '~
per were reported at l6t6r periods. A mink farm was located
by 6ide of the~6ilv6r;;foY farm, and due to high contagiosit~ of~
''the~dlstemp6r viru6, single~ca8e6 of pla~ue~among~minks~were
,reported. ~he~vacoination;of;~the minks,~ that~followed immedia- `~
tel~, prevented~the~outbreak~of~the disea6e~6t~the farm.
The~`obtained~data indicate~the potency of the vaccine
prepared frQm~the~6train EæM 10-76 o~ the~virus of canine
'di6tèmper to eradicate an outbreak of distemper in the course
of three or four weeks.
he prop~o6ed vaccine~oan~also be used to immunize animals ;~
by;~giving~it ln~aerosol form.~
i.e. usual commercial
ba~ches-;o~'t~e vaccine,~can,be used,for aerosol. The aerosol
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can be used to immunize a large number of a~imals within a
short.-~period of time.
Tests of ~he proposed vaccine on animals during prophyla-
xis of diste~per showed that.practically 100 per cent o
immunized animal~ produce antibodies in high titres to re-
liably protect the animal~ from distemper. Moreover, the
vaccine has proved effective as a means of eradicating the
focus of the disease.
The proposed vaccine is comparatively oheap because the
cell culture of Japanese quail embryos i8 used aa the substrate.
his culture has the following advantages over the other known
cultures. ~he~main advantage~is that Japaneæe~quails:are ~atu-
rally immune tow~rd viruse6 of avia~ }eukemia and are resi~tant
toward this~lnfection.
An other important advsntage of the Japaneae quai;~ embryo :.
oell ¢u}ture~is its hi~h prollrerative activity and the mono- ~;
lsyer of the~o~ells~i6 formed~by~the first.or~econd day~ follow-
in~ 3eedi~g~the~.~c~11a.~
8t~ another advantago of said oulture~is that~the cell~ :
suspension-can be~infected. ~hia shortens even more the~time ~:
of the~:prooese:~of~manufaoturing the vaccine. An advantage of : ~.:
t~e~invention~ls~al60 that the fluid containing the viru8
can:be har~ested 8everal time from o~e inoculum ~to five
harve~t~ from one culture~sample). ~ ~
Japaneso~:quails are~readily available and cheap, their
raising;at~speci`al~farma:~is ~imple and doea not require much
inve~stment, All~ hi3;expla~- 8 the high commercial value o~
the propo~ea: VaGoine. ~ ~ :
~o make the invention more understandable to those
-skilled in the art, the ~ollowing examples are given by wa~ of
llustration, in which Exampie 1 shows how to obtain the EP~
10-76 virus of cani~e distemper, Example 2 illustrates the
' method of preparin~ the live culture vaccine, and ~xamples 3
through 9 illustrate the method of using the vaccine.
:~~xample 1. Preparation of Attenuated Sbrain EPM of
Virus o~
t ~ e Dis'temper, Deposited under the
Code Number 10-76, Isolated from the Wild
Virule~t;~Virus
Into 10 test tubes, contalm ng~the primary cell culture of~
dog kidnie~s,~added ars 0.~ ml of~defibrinized blood of a mink
with distemper. After a three-hour contact at room temperature, ^
the oell c'ulture is washed thoroughly with a nutrient medium :
199, 1.5 ml~ Or fresh nutrient medium 199 oontaining 10 per ~ ;~
cent of cattl~e blood~ serum~are~added,~and the cell culture is
inoubsted~at~.a~temperature~of 37C. ~he:nutrient~ medlum ls~
renewed:once'or~twioe~a week.~he fluid:~oo~taining the virus
is:~collseted~in the ~ooursé of 62 da~s~following the inocula-
lon.~ ~he~culturo fluid is used to inoculate~a new portion
.. E~ of ~the dog~kidney o~ell~oul*ure~ To that end, 0.3 ml of the.
:fluid containing the virus and 1 ml of the nu~rient medium '
199~oontaining 10 per oent o~cattle blood serum are added
to:~each of~the ten test tubes containin~ new~portions of
the~primsr~ cell culture of dog kidneys. ~he cell culture is
::: cultivated a~a~:tempera~ure of 37C. ':~
he ~luid containing the viru~t which is~collected 35-40
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days followin~ the infection of the previous cultures, is
used as the inoculum for the first ten passa~es. In th0
next 23 passages of the virus, the fluid containing the virus,
which is collected 15-20 days following the infection, and
also the virus isolated from the infected cells after the
frosting-defrosting c~cle, ~re used as the inoculum.
Next, after infection by a similar method, the virus
is given 12 passages in the continuous cell culture of the
human embryo Rh at32C. The fluid containing the virus,
collected the 15-20th da~ following the previous infection,
is used a6 the inoculum for each next infection.
~ he viruæ i9 further attenuated by pas~age on a mixed
culture consisting of 2 million cells of dog kidneys and
2 million cells of Japanese quail embryo. The process is
carried out at a temperature of ~5C. The rirus i8 given
three passages on this culture. Then, the virus is passed
five times on a cell culture of Japanese quail embryo at a
temperature of 35C in rollers at 4 rpm. Beginning with the
fifth pas~age, the virus regularly produced cytopathic destruc-
; tion of cells, which was obvious 5-7 days following the
infection. This virus was cloned by the limit dilution
method ih the cell culture of Japanese quail embryos and
used as the vaccinal strain. ~he obtained attenuated strain
of the viru~ of canine distemper adapted to the cell culture
of Japanese quail embryos, is tested for the following;
(1) specificity in ~eactions of neutrali~ation on the
Japznese quail ~mbryo cell culture (identification test);
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(2) harmlessness (on laboratory animals, e.g. mice,
&uinea pigs, rabbits, minks, blue fox, and also embryos);
(3) infectious activity in the Jspaneæe guail embryo
cell culture;
(4) anti~enic activity;
(5) absence of contaminating viruses;
(6) immunogenic ~ctivity;
(7) bacterial sterility, absence of mycopl~sms (PP10);
(8) stabilit~ in storage;
(9) absence of contagiosity.
Example 2. Preparation of Seed Virus and Vaccine:
(a) Preparation of Seed Virus. ~he seed virus is prepared
from the strain EPM 10-76 obtained as described in ~xample 1.
~o this end, 9-11 day old embryos of Japanese quails obtained
from a special farm are used. Hundred embryos are trypsinized
b~ the known method to obtain 3,300,000,000 cells that are
suspended in 4.120 litres of nutrient medium 199 containin~
also 10 per cent of cattle blood ~erum and 100 mg/ml of mono-
mycin. 150-ml portions of the cell suspension are placed into
two one-litre flasks and kept as control. 10 ml of a culture
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fluid containing the strain ~P~ 10-76 are added to the re-
maining portion of the cell suspension and 150-ml portions
of the suspension are placed into one litre flasks. The ~us-
pension in the flasks is incubated at a temperature of 35C
n rollers. Three days following the inoculation, the nutri-
en~ medium is removed and fresh portions of the medium are
added into all flasks. ~he medium 199 should contain 100
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~/ml of ~onomycin. On the six-th day following the inoculation,
if th~ fluid containing the viru~ ows the cytopathic acti-~it~,
the fluid is withdrawn from the flasks, its samples are taken
for ~he corresponding tests, and fresh portions of medium 199
are added. ~ext harvestin~ of the virus-containing fluid is
continued until 50 per cent of cells are retained. Now 70 ml
of fresh medium 199 are added, the cell culture is frozen in
dry ice, and then defro~te~ to isolate the intracellular viru8.
Samples are taken at each stage o~ preparing the seeding
virus, which should pass the following tests: for specificity
(iden*ity), harmlessness, infectious activity, antigenic acti-
vity, immunogenecity, absence of contaminating viruses, s*eri-
lity, and the absence of contagiosity. The seeding virus meeting
~aid reguirements is kept frozen at a temperature not above minus~
20C.
(b) Preparing the Vaccine. 9-11 da~ old embryos of Japa-
nese quails obtained from a special controlled farm are used
for the purpose. 200 embryos are trypsinized by the known
method to obtain 6,600,000,000 cells that are suspended in
8.250 litre of ~utrient medium 199. 10 per cent of cattle
blood serum and 100 mg/ml of monomycin are added to the medium.
150-ml portions of the cell suspension are placed into five
one-litre fla~s (750 ml all altogether) and kept for control
purposes. 10 ml of culture fluid containing t~e seeding virus
of strain ~PM 10-76, obtai~ed in item (a) are added to the
remaining cell ~uspen~ion, and its 150-ml portion~ are placed
into one-litre flasks. All the flssks are now cult~vated at
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35C in rollers. On the third day after seedin~, the nutrient
medium is r~moved and fresh medium 199, containing ~00 m~/ml
of monomycin, is added instead. On the sixth day following
the inoculation, a8 the foci of c~topathic activity appear in
30 per cent of the cells, the virus-containing fluid is remo-
ved, its samples are taken for control purpo~es, and fresh
medium 199 is added into all flasks. ~he harvesting is repeated
~ntil 50 per cent of the cells are retained and 70 ml of medium
199 are finally added. The cell culture is now frozen and defrost
~d to isolate the intracell ~irus.
All sterile virus harveæts are ~oined in one vessel,
a sta~ilizing agent consisting of 5 per cent of sorbitol and
1.5 per cent of gelatose is added, the vaccine is filled into
vials or ampoules, and lyophilized. The pool makes one batch
of the vaccine. ~he obtained vaccine can be filled into vials
or ampoules of various capacit~, from 1 to 33 ml, and contain
from 1 to 500 doses in one ampoule or vial, dependin~ on the
consumer demand.
Control ~amples of the vaccine are taken at each stage
of the pxocess. ~he vaccine should be tested for the following:
.
identity, harmlessness, infectious activity, antigenic and immu-
nogenic activity, the absence of contam nating viruses, steri-
lity, and the absence of contagiosity.
A batch of the vaccine meeting these reguirements can be
used to immunize animals. In our example, a batch of the vaccine
contains 360,000 doses.
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Example 3. Testing for Harmlessness
The vaccine was u6ed in a dose of 1 ml containin~r 100
TCD50 f the virus diluted with Xenks solutio~. ~he vaccine
was injected intramuscularly. 24 mink cubs a~d 12 blue fox
cubs were immunized. 16 cubs of mink and 8 of blue fox were
kept as controls. ~he vaccinsted animals were observed daily for
28 days. All the animals remained clinically healthy. Pa~hologi-
cal reactions, ~uch as refusal of food, suppression, alimentary
disorders, were not observed. The animals that were kept in
contact with the vaccinated animal~ did not develop the disease.
Example 4. Testing for Antigenic Activity
~ he animals were vaccinated with the preparation in doses
as specified in Example 3, and blood ~amples were taken from
them in 10-14 days following the vaccination. The titres of
antibodies in the neutralization test on the cell culture of
Japanese quail embryos were determined~ The vaccinal strain
hPM 10-76 of the virus of canine distemper was used as the
antigen. The antibodies were determined in all animals in the
titre of 1;16-32. The maximum accumulation of antibodies (in
the titre of 1:256) was attained b~ the 30th day. The anti-
bodies were found in the cour6e,0f 18 months of observation.
~xample 5. Testing for Immunogenic Activity and
Duration of Immunity
(a~ 24 cub~ of mink and 12 of blue ~ox were given intra-
mu~ularly 1 ml of the dilute vaccine containing 100 TCD50 f
the virua. 16 cubs of mink and 8 of blue fox were kept for
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1120856
control purposes. ~he vaccinatcd animals were challenged a~d the
co~trols were then infected with a virulent virus ~Sn~der Hill
for the minks and Gauiasski for blue fox) on the 30th day follow-
ing the immuniza~ion. ~he animals were obser~ed daily. The
controls (not immunized animals) perished from distemper,
whereas the vaccinated animals did not respend to the admini-
stration of the virulent strain. During the entire time of
observation (21 days following the infection) the vaccinated
snimals remained healthy clinically.
(b) In 18 months after the v~ccination, the animals were
challenged again with the virulent viruses Snyder and Gau~lasski.
None of the repeatedly infected ~nimals perished or developed
distemper.
Example 6. Testing ~he Vaccine in Field Condition~
In the course of the summer vaccination for proph~lactic
purposes, 1,400,000 foxes, 45,500 blue foxes, 300,000 minks
a~d 255,000 dogs were vacclnated. All vaccinated animals
did not develop distemper althou~h outbreaks of the disease
were reported in the surrounding regions.
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3xample 7. ~esting the Vaccine in the Focus of
Canine Distemper
~ he vaccine was tested at a farm where multiple oases
of plague of carnivores were revealed amon~ young silver fox
(proved by laboratory anal~si~ he mortality rate was 120
cubs a day. All animals at the farm were then vaccinated. T~e
~ortality rate deoreased two times in two months, and by the
end of the third ~onth the ani~als stopped perishing~
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Single cases of distemper were reported from a mink farm
located near the silver-fox farm. All animals were vaccinated
and the outbreak of plage waæ thus prevented.
~xample 8. Comparison of the Proposed and the
known Preparations
All animals were immunized with the known vaccine
KF-668 (USSR) at a mink farm with multiple cases of canine
distemper among cubs. After the vaccination the mortality
rate did not diminish and was 180 cubs 8 day. 14,612 animals
were immunized repeatedl~ with the proposed vaccine and
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15,580 animals with the known, ASL vaccine (USA~. ~ollowing
the repeated immunization with said vaccines the mortalit~ \
among the cubs decreased markedly, and in two months the
outbrea~ of distemper was eradicated completel~. Thus the pro-
posed vaccine proved to be no less efficaceou than the kno~n -
vaccine AS~. ~
~xample 9. Testing the Vaccine in Aerosol Form
he vaccine was prepared by the process as described
above (for intramuscular in~ections). It was used in aerosol
form to immunize ~OO cubs of mink, aged 45-60 days. One group
of the animals was exposed to the sprayed aProsol so that
each animal was ~iven one dose of the vaccine whereas in the
othe~ group the animals were given ~o inhale three doses. By
one dose we understand here a usual dose for intramuscular
injection. '~he antibodies to the distemper virus were deter-
mined in the blood serum of the immunized animals by the neutral-
ization resotion on the Japanese quail embr~o cell culture,
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1~2~18S6
using the vaccinal strain EæM 10-76 as the antigen. The anti-
bodies ~re determined in the dilution of 1:8 for the min~
vaccinated with one doSe and in the dilution of 1:32 for the
animals vaccinated with three doses.
We did not observe significant difference in the titres
of tne virus-neutralizing antibodieæ in the animals vaccinate~
intramuscularly (as described in ~xample 4) and tho~e vaccina-
ted with the preparation i~ aerosol form.
~ he duration of immunity in the animals vaccinated with
the prepara~ion in aerosol form was tested by challenging
the animals with the virulent virus Snyder Xill. The animals
vaccinated with the proposed vaccine did not develop distemper
after the infection with the virulent strain o~ the virus.
~ xample 9 shows that the proposed vaccine obtainèd from
the strain ~PM 10-76 of the virus of canine distemper intended
for intramuscular injections i~ as efficaceous for vaccination
of the animals with the preparation in aerosol form.
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