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Avian infectious bronchitis vaccine (live)
EUROPEAN PHARMACOPOEIA 5.0
better in group C than in group D and significantly better in
group B than in group A. The test is not valid unless there
is a drop in egg production in group A compared to the
normal level noted before challenge of at least 35 per cent
where challenge has been made with a Massachusetts-type
strain ; where it is necessary to carry out a challenge with a
strain of another serotype for which there is documented
evidence that the strain will not cause a 35 per cent drop in
egg production, the challenge must produce a drop in egg
production commensurate with the documented evidence
and in any case not less than 15 per cent.
LABELLING
The label states :
— the category and age of the chickens for which the
vaccine is intended,
— the strains and serotypes of virus used in the production
of the vaccine and the serotypes against which the vaccine
is intended to protect,
— whether the strain in the vaccine is embryo-adapted or
cell-culture-adapted.
01/2005:0442
AVIAN INFECTIOUS BRONCHITIS
VACCINE (LIVE)
Vaccinum bronchitidis infectivae aviariae
vivum
1. DEFINITION
Avian infectious bronchitis vaccine (live) is a preparation
of one or more suitable strains of different types of avian
infectious bronchitis virus. This monograph applies to
vaccines intended for administration to chickens for active
immunisation against respiratory disease caused by avian
infectious bronchitis virus.
2. PRODUCTION
2-1. PREPARATION OF THE VACCINE
The vaccine virus is grown in embryonated hens’ eggs or in
cell cultures.
2-2. SUBSTRATE FOR VIRUS PROPAGATION
2-2-1. Embryonated hens’ eggs. If the vaccine virus is grown
in embryonated hens’ eggs, they are obtained from flocks
free from specified pathogens (SPF) (5.2.2).
2-2-2. Cell cultures. If the vaccine virus is grown in cell
cultures, they comply with the requirements for cell cultures
for production of veterinary vaccines (5.2.4).
2-3. SEED LOTS
2-3-1. Extraneous agents. The master seed lot complies with
the tests for extraneous agents in seed lots (2.6.24). In these
tests on the master seed lot, the organisms used are not
more that 5 passages from the master seed lot at the start
of the test.
2-4. CHOICE OF VACCINE VIRUS
The vaccine virus shall be shown to be satisfactory with
respect to safety (5.2.6) and efficacy (5.2.7) for the chickens
for which it is intended.
The following tests for safety (section 2-4-1), increase in
virulence (section 2-4-2) and immunogenicity (section
2-4-3) may be used during the demonstration of safety and
immunogenicity.
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2-4-1. Safety
2-4-1-1. Safety for the respiratory tract and kidneys. Carry
out the test in chickens not older than the youngest age
to be recommended for vaccination. Use vaccine virus
at the least attenuated passage level that will be present
between the master seed lot and a batch of the vaccine. Use
not fewer than 15 chickens from an SPF flock (5.2.2) and
from the same origin. Administer to each chicken by the
oculonasal route a quantity of the vaccine virus equivalent
to not less than 10 times the maximum virus titre likely to
be contained in 1 dose of the vaccine. On each of days 5, 7
and 10 after administration of the virus, kill not fewer than 5
of the chickens, take samples of trachea and kidney. Fix
kidney samples for histological examination. Remove the
tracheas and cut 10 rings from each trachea (3 from the top,
4 from the mid-part and 3 from the bottom) ; examine each
ring under low magnification and score for ciliostasis on a
scale from 0 (100 per cent ciliary activity) to 4 (no activity,
complete ciliostasis) ; calculate the mean ciliostasis score
(the maximum for each trachea being 40) for the 5 chickens
killed on each of days 5, 7 and 10. The test is not valid if
more than 10 per cent of the chickens die from causes not
attributable to the vaccine virus. The vaccine virus complies
with the test if :
— no chicken shows notable clinical signs of avian infectious
bronchitis or dies from causes attributable to the vaccine
virus,
— the average ciliostasis score is not more than 25,
— at most moderate inflammatory lesions are seen during
kidney histological examination.
2-4-1-2. Safety for the reproductive tract. If the
recommendations for use state or imply that the vaccine
may be used in females of less than 3 weeks old that
are subsequently kept to sexual maturity, it shall be
demonstrated that there is no damage to development of
the reproductive tract when the vaccine is given to chickens
of the minimum age to be recommended for vaccination.
The following test may be carried out : use not fewer
than 40 female chickens not older than the minimum age
recommended for vaccination and from an SPF flock (5.2.2) ;
use the vaccine virus at the least attenuated passage level
that will be present in a batch of vaccine ; administer to
each chicken by a recommended route a quantity of virus
equivalent to not less than the maximum titre likely to
be present in 1 dose of vaccine ; at least 10 weeks after
administration of the vaccine virus, kill the chickens and
carry out macroscopic examination of the oviducts. The
vaccine virus complies with the test if abnormalities are
present in not more than 5 per cent of the oviducts.
2-4-2. Increase in virulence. The test for increase in
virulence consists of the administration of the vaccine virus,
at the least attenuated virus passage level that will be present
between the master seed lot and a batch of the vaccine, to a
group of five 2-week-old chickens from an SPF flock (5.2.2),
sequential passages, 5 times where possible, to further
similar groups and testing of the final recovered virus for
increase in virulence. If the properties of the vaccine virus
allow sequential passage to 5 groups via natural spreading,
this method may be used, otherwise passage as described
below is carried out and the maximally passaged virus that
has been recovered is tested for increase in virulence. Care
must be taken to avoid contamination by virus from previous
passages. Administer by eye-drop a quantity of the vaccine
virus that will allow recovery of virus for the passages
described below. 2 to 4 days after administration of the
vaccine virus, prepare a suspension from the mucosa of the
trachea of each chicken and pool these samples. Administer
0.05 ml of the pooled samples by eye-drop to each of 5
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 5.0
Avian infectious bronchitis vaccine (live)
other 2-week-old chickens from an SPF flock (5.2.2). Carry
out this passage operation not fewer than 5 times ; verify
the presence of the virus at each passage. If the virus is
not found at a passage level, carry out a second series of
passages. Carry out the test for safety for the respiratory
tract and kidney (section 2-4-1-1) and, where applicable, the
test for safety for the reproductive tract (section 2-4-1-2)
using the unpassaged vaccine virus and the maximally
passaged virus that has been recovered. Administer the virus
by the route to be recommended for vaccination likely to be
the least safe. The vaccine virus complies with the test if no
indication of increase in virulence of the maximally passaged
virus compared with the unpassaged virus is observed. If
virus is not recovered at any passage level in the first and
second series of passages, the vaccine virus also complies
with the test.
2-4-3. Immunogenicity. Immunogenicity is demonstrated
for each strain of virus to be included in the vaccine. A test
is carried out for each route and method of administration
to be recommended using in each case chickens from an
SPF flock (5.2.2) not older than the youngest age to be
recommended for vaccination. The quantity of the vaccine
virus administered to each chicken is not greater than the
minimum virus titre to be stated on the label and the virus is
at the most attenuated passage level that will be present in
a batch of the vaccine. 1 or both of the tests below may be
used during the demonstration of immunogenicity.
2-4-3-1. Ciliary activity of tracheal explants. Use not fewer
than 25 chickens of the same origin and from an SPF flock
(5.2.2). Vaccinate by a recommended route not fewer than
20 chickens. Maintain not fewer than 5 chickens as controls.
Challenge each chicken after 21 days by eye-drop with a
sufficient quantity of virulent avian infectious bronchitis
virus of the same type as the vaccine virus to be tested.
Kill the chickens 4 to 7 days after challenge and prepare
transverse sections from the upper part (3), the middle
part (4) and the lower part (3) of the trachea of each chicken.
Examine all explants as soon as possible and at the latest
2 h after sampling by low-magnification microscopy for
ciliary activity. For a given tracheal section, ciliary activity
is considered as normal when at least 50 per cent of the
internal ring shows vigorous ciliary movement. A chicken is
considered not affected if not fewer than 9 out of 10 rings
show normal ciliary activity.
The test is not valid if :
— fewer than 80 per cent of the control chickens show
cessation or extreme loss of vigour of ciliary activity,
— and/or during the period between the vaccination and
challenge more than 10 per cent of vaccinated or control
chickens show abnormal clinical signs or die from causes
not attributable to the vaccine.
The vaccine virus complies with the test if not fewer than
80 per cent of the vaccinated chickens show normal ciliary
activity.
2-4-3-2. Virus recovery from tracheal swabs.Use not fewer
than 30 chickens of the same origin and from an SPF flock
(5.2.2). Vaccinate by a recommended route not fewer than
20 chickens. Maintain not fewer than 10 chickens as controls.
Challenge each chicken after 21 days by eye-drop with a
sufficient quantity of virulent avian infectious bronchitis
virus of the same type as the vaccine virus to be tested.
Kill the chickens 4 to 7 days after challenge and prepare
a suspension from swabs of the tracheal mucosa of each
chicken. Inoculate 0.2 ml of the suspension into the allantoic
cavity of each of 5 embryonated hens’ eggs, 9 to 11 days old,
from an SPF flock (5.2.2). Incubate the eggs for 6-8 days
after inoculation. Eggs that after 1 day of incubation do
not contain a live embryo are eliminated and considered as
non-specific deaths. Record the other eggs containing a dead
embryo and after 6-8 days’ incubation examine each egg
containing a live embryo for lesions characteristic of avian
infectious bronchitis. Make successively 3 such passages.
If 1 embryo of a series of eggs dies or shows characteristic
lesions, the inoculum is considered to be a carrier of avian
infectious bronchitis virus. The examination of a series of
eggs is considered to be definitely negative if no inoculum
concerned is a carrier. The test is not valid if :
— the challenge virus is re-isolated from fewer than 80 per
cent of the control chickens,
— and/or during the period between vaccination and
challenge more than 10 per cent of the vaccinated or
control chickens show abnormal clinical signs or die from
causes not attributable to the vaccine,
— and/or more than 1 egg in any group is eliminated
because of non-specific embryo death.
The vaccine virus complies with the test if the challenge
virus is re-isolated from not more than 20 per cent of the
vaccinated chickens.
3. BATCH TESTS
3-1. Identification
3-1-1. Vaccines containing one type of virus. The vaccine,
diluted if necessary and mixed with avian infectious
bronchitis virus antiserum specific for the virus type, no
longer infects embryonated hens’ eggs from an SPF flock
(5.2.2) or susceptible cell cultures (5.2.4) into which it is
inoculated.
3-1-2. Vaccines containing more than one type of virus.
The vaccine, diluted if necessary and mixed with type-specific
antisera against each strain present in the vaccine except
that to be identified, infects embryonated hens’ eggs from
an SPF flock (5.2.2) or susceptible cell cultures (5.2.4) into
which it is inoculated whereas after further admixture with
type-specific antiserum against the strain to be identified it
no longer produces such infection.
3-2. Bacteria and fungi
Vaccines intended for administration by injection comply
with the test for sterility prescribed in the monograph
Vaccines for veterinary use (0062).
Vaccines not intended for administration by injection
either comply with the test for sterility prescribed in the
monograph Vaccines for veterinary use (0062) or with the
following test : carry out a quantitative test for bacterial
and fungal contamination ; carry out identification tests for
microorganisms detected in the vaccine ; the vaccine does
not contain pathogenic microorganisms and contains not
more than 1 non-pathogenic microorganism per dose.
Any liquid supplied with the vaccine complies with the
test for sterility prescribed in the monograph Vaccines for
veterinary use (0062).
3-3. Mycoplasmas. The vaccine complies with the test for
mycoplasmas (2.6.7).
3-4. Extraneous agents. The vaccine complies with the tests
for extraneous agents in batches of finished product (2.6.25).
3-5. Safety. Use not fewer than 10 chickens from an SPF
flock (5.2.2) and of the youngest age recommended for
vaccination. Administer by a recommended route to each
chicken, 10 doses of the vaccine. Observe the chickens at
least daily for 21 days. The test is not valid if more than
20 per cent of the chickens show abnormal clinical signs
or die from causes not attributable to the vaccine. The
vaccine complies with the test if no chicken shows notable
clinical signs of disease or dies from causes attributable to
the vaccine.
General Notices (1) apply to all monographs and other texts
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Avian infectious bursal disease vaccine (inactivated)
EUROPEAN PHARMACOPOEIA 5.0
3-6. Virus titre. Titrate the vaccine virus by inoculation
into embryonated hens’ eggs from an SPF flock (5.2.2) or
into suitable cell cultures (5.2.4). If the vaccine contains
more than 1 strain of virus, titrate each strain after having
neutralised the others with type-specific avian infectious
bronchitis antisera. The vaccine complies with the test if
1 dose contains for each vaccine virus not less than the
minimum titre stated on the label.
3-7. Potency. The vaccine complies with the requirements
of 1 of the tests prescribed under Immunogenicity
(section 2-4-3) when administered according to the
recommended schedule by a recommended route and
method. It is not necessary to carry out the potency test
for each batch of the vaccine if it has been carried out on a
representative batch using a vaccinating dose containing not
more than the minimum virus titre stated on the label.
Eggs are collected for hatching 5 to 7 weeks after vaccination
and the test described below is carried out with 3-week-old
chickens from that egg collection. Eggs are collected again
towards the end of the period of lay and the test is repeated
with chickens from that egg collection which are at least
15 days old.
Twenty-five chickens from vaccinated hens and ten control
chickens of the same breed and age from unvaccinated hens
are challenged with an eye-drop application of a quantity
of a virulent strain of infectious avian bursal disease virus
sufficient to produce severe signs of disease, including
lesions of the bursa of Fabricius, in all unvaccinated
chickens. 3 to 4 days after challenge, the bursa of Fabricius
is removed from each chicken. The bursae are examined
for evidence of infection by histological examination or by
testing for the presence of infectious avian bursal disease
antigen in an agar-gel precipitation test. The vaccine
complies with the test if three or fewer of the chickens from
01/2005:0960 vaccinated hens show evidence of infectious avian bursal
disease infection. The test is not valid unless all the chickens
BURSAL DISEASE from unvaccinated hens are affected.
AVIAN INFECTIOUS
VACCINE (INACTIVATED)
Vaccinum bursitidis infectivae aviariae
inactivatum
DEFINITION
Inactivated infectious avian bursal disease vaccine consists of
an emulsion or a suspension of a suitable strain of infectious
avian bursal disease virus type 1 which has been inactivated
in such a manner that immunogenic activity is retained. The
vaccine is for use in breeding domestic fowl to protect their
progeny from infectious avian bursal disease.
PRODUCTION
The virus is propagated in fertilised eggs from healthy flocks,
in suitable cell cultures (5.2.4) or in chickens from a flock
free from specified pathogens (5.2.2).
An amplification test for residual live infectious avian
bursal disease virus is carried out on each batch of antigen
immediately after inactivation and on the final bulk vaccine
or, if the vaccine contains an adjuvant, on the bulk antigen
or the mixture of bulk antigens immediately before the
addition of any adjuvant, to confirm inactivation ; the
test is carried out in fertilised hen eggs or in suitable cell
cultures or, where chickens have been used for production
of the vaccine, in chickens from a flock free from specified
pathogens (5.2.2) ; the quantity of inactivated virus used
in the test is equivalent to not less than ten doses of the
vaccine. No live virus is detected.
The vaccine may contain one or more suitable adjuvants.
CHOICE OF VACCINE COMPOSITION
The vaccine is shown to be satisfactory with respect to safety
(5.2.6) and immunogenicity (5.2.7). The following test may
be used during demonstration of efficacy.
Immunogenicity. Each of at least twenty chickens from
a flock free from specified pathogens (5.2.2), and of the
recommended age for vaccination (close to the point of lay),
is injected with the minimum recommended dose of vaccine
by one of the recommended routes. 4 to 6 weeks later
serum samples are collected from each bird and the antibody
response is measured in a serum-neutralisation (SN) test.
A suitable standard, calibrated in Ph. Eur. Units against
infectious avian bursal disease serum BRP, is included
in the test. The vaccine complies with the test if the mean
antibody level in the sera from the vaccinated birds is at least
10 000 Ph. Eur. Units per millilitre.
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Where there is more than one recommended route of
administration, the test described under Potency is carried
out in parallel with the above immunogenicity test, using
different groups of birds for each recommended route. The
serological response of the birds inoculated by routes other
than that used in the immunogenicity test is not significantly
less than that of the group vaccinated by that route.
IDENTIFICATION
In chickens with no antibodies to infectious avian bursal
disease virus type 1, the vaccine stimulates the production
of specific antibodies.
TESTS
Safety. Inject twice the vaccinating dose by one of the
recommended routes into each of ten chickens, 14 to
28 days old, from flocks free from specified pathogens
(5.2.2). Observe the birds for 21 days. No abnormal local or
systemic reaction occurs. Note : this test may be omitted if
inactivation test C is carried out.
Inactivation
A. For vaccine prepared with embryo-adapted strains of
virus, inject two-fifths of a dose into the allantoic cavity or
onto the chorio-allantoic membrane of ten 9- to 11-day-old
fertilised hen eggs from a flock free from specified
pathogens (SPF eggs) (5.2.2). Incubate the eggs and
observe for 6 days. Pool separately the allantoic liquid or
membranes from eggs containing live embryos, and that
from eggs containing dead embryos, excluding those that
die from non-specific causes within 24 h of the injection.
Inject into the allantoic cavity or onto the chorio-allantoic
membrane of each of ten 9- to 11-day-old SPF eggs 0.2 ml
of the pooled allantoic liquid or crushed chorio-allantoic
membranes from the live embryos and, into each of ten
similar eggs, 0.2 ml of the pooled liquid or membranes
from the dead embryos and incubate for 6 days. Examine
each embryo for lesions of infectious avian bursal disease.
If more than 20 per cent of the embryos die at either
stage repeat that stage. The vaccine complies with the
test if there is no evidence of lesions of infectious avian
bursal disease and if, in any repeat test, not more than
20 per cent of the embryos die from non-specific causes.
Antibiotics may be used in the test to control extraneous
bacterial infection.
See the information section on general monographs (cover pages)