ANTHRAX SPORE VACCINE (LIVE) FOR VETERINARY USE ...

EUROPEAN PHARMACOPOEIA 5.0 Aujeszky’s disease vaccine (inactivated) for pigs
01/2005:0441
ANTHRAX SPORE VACCINE (LIVE)
FOR VETERINARY USE
Vaccinum anthracis vivum
ad usum veterinarium
DEFINITION
Anthrax spore live vaccine for veterinary use consists of a
suspension of live spores of an attenuated, non-capsulated
strain of Bacillus anthracis.
PRODUCTION
The strain used is either: not lethal to the guinea-pig or the
mouse; or lethal to the guinea-pig but not to the rabbit;
or lethal to some rabbits. B. anthracis is grown in an
appropriate medium. At the end of growth the spores are
suspended in a stabilising solution and counted. The vaccine
may contain an adjuvant.
IDENTIFICATION
B. anthracis present in the vaccine is identified by means of
morphological and serological tests, culture and biochemical
tests.
TESTS
Safety. Carry out the test on one of the species of animals
for which the vaccine is intended. If the vaccine is intended
for several species, including the goat, carry out the test
on goats. Administer subcutaneously or intradermally to
each of 2 animals, of the minimum age recommended for
vaccination and having no antibodies against B. anthracis,
twice the dose stated on the label for the species used and
observe the animals for 14 days. No abnormal systemic
reaction is produced but a local reaction may occur at the
site of injection. The severity of the local reaction may vary
according to the strain of the spores and the adjuvants used
in the preparation, but necrosis does not occur.
Spore count. The number of live spores determined by plate
countisnotlessthan80percentofthatstatedonthelabel.
Bacterial and fungal contamination. Carryoutthetest
by microscopic examination and by inoculation of suitable
media. The vaccine does not contain contaminating bacteria
or fungi.
POTENCY
For a strain of B. anthracis which is not lethal to the
guinea-pig or the mouse, the test may be carried out in
guinea-pigs. For a strain which is lethal to the guinea-pig
but not to the rabbit, the test may be carried out in rabbits.
For a strain which is lethal to some rabbits, carry out the
test in sheep.
If the test is carried out in guinea-pigs or in rabbits, use
10 healthy animals (group a). Inject subcutaneously or
intradermally into each animal 1/10 of the smallest dose
of the vaccine stated on the label for sheep. Observe the
animals for 21 days. If more than 2 animals die from
non-specific causes, repeat the test. Use as controls 3 animals
of the same species and of the same origin.
If the test is carried out in sheep, use 5 healthy animals
(group b). Inject subcutaneously or intradermally into each
animal 1/10 of the smallest dose of the vaccine stated on
thelabelforsheep.Observetheanimalsfor21days.Useas
controls 3 sheep of the same origin. Inject subcutaneously
into each vaccinated animal of group (a) or group (b) at least
100 MLD and into each control animal 10 MLD of a strain of
B. anthracis pathogenic for the species of animal used in the
test. Observe all the animals for 10 days. All the vaccinated
animals survive and all the controls die from anthrax during
the observation period. If a vaccinated animal dies after the
challenge, repeat the test. If in the second test a vaccinated
animal dies, the vaccine fails the test.
LABELLING
The label states:
— the strain used for preparation of the vaccine,
— the number of viable spores per millilitre.
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AUJESZKY’S DISEASE VACCINE
(INACTIVATED) FOR PIGS
Vaccinum morbi Aujeszkyi ad suem
inactivatum
DEFINITION
Aujeszky’s disease vaccine (inactivated) for pigs consists
of a suspension of an appropriate strain of Aujeszky’s
disease virus inactivated without affecting its immunogenic
properties or a suspension of an inactivated fraction of the
virus having adequate immunogenic properties.
PRODUCTION
The virus strain is grown in suitable cell cultures (5.2.4).
The viral suspension is harvested and inactivated; it may be
treated to fragment the virus and the viral fragments may
be purified and concentrated.
The test for inactivation is carried out using two passages in
thesametypeofcellcultureasthatusedintheproduction
of the vaccine or cells shown to be at least as sensitive. The
quantity of inactivated virus used in the test is equivalent to
not less than twenty-five doses of the vaccine. No live virus
is detected.
Suitable adjuvants and antimicrobial preservatives may be
added. The vaccine may be freeze-dried.
CHOICE OF VACCINE COMPOSITION
The vaccine is shown to be satisfactory with respect to safety
and immunogenicity. The following tests may be used during
demonstration of safety (5.2.6) and efficacy (5.2.7).
Safety
A. A test is carried out in each category of animals for which
the vaccine is intended (sows, fattening pigs). The animals
used do not have antibodies against Aujeszky’s disease
virus or against a fraction of the virus. Two doses of
vaccine are injected by a recommended route into each
of not fewer than ten animals. After 14 days, one dose of
vaccine is injected into each of the animals. The animals
are observed for a further 14 days. No abnormal local or
systemic reaction is produced during the 28 days of the
test.Ifthevaccineisintendedforuseinpregnantsows,
for the test in this category of animal the observation
period is prolonged up to farrowing and any effects on
gestation or the offspring are noted.
B. The animals used in the test for immunogenicity are
also used to evaluate safety. The rectal temperature
of each vaccinated animal is measured at the time of
vaccination and 6 h, 24 h and 48 h later. No animal shows
a temperature rise greater than 1.5 °C and the number of
animals showing a temperature greater than 41 °C does
not exceed 10 per cent of the group. No other systemic
reactions (for example, anorexia) are noted. At slaughter,
GeneralNotices(1)applytoallmonographsandothertexts 715

Aujeszky’s disease vaccine (inactivated) for pigs EUROPEAN PHARMACOPOEIA 5.0
the injection site is examined for local reactions. No
abnormal local reactions attributable to the vaccine are
produced.
C. The animals used for field trials are also used to evaluate
safety. A test is carried out in each category of animals
for which the vaccine is intended (sows, fattening pigs).
Not fewer than three groups each of not fewer than
twenty animals are used with corresponding groups of
not fewer than ten controls. The rectal temperature
of each vaccinated animal is measured at the time of
vaccinationand6h,24hand48hlater.Noanimalshows
a temperature rise greater than 1.5 °C and the number of
animals showing a temperature greater than 41 °C does
not exceed 25 per cent of the group. At slaughter, the
injection site is examined for local reactions. No abnormal
local reactions attributable to the vaccine are produced.
Immunogenicity. Not fewer than ten fattening pigs of the
age recommended for vaccination and which do not have
antibodies against Aujeszky’s disease virus or against a
fractionofthevirusareused.Thebodymassofnoneofthe
pigs differs from the average body mass of the group by more
than 20 per cent. Each pig is vaccinated according to the
recommended schedule and by a recommended route. Five
similar pigs are used as controls. At the end of the fattening
period (80 kg to 90 kg), each pig is weighed and then
challenged by the intranasal route with a suitable quantity of
a virulent strain of Aujeszky’s disease virus (challenge with
at least 106 CCID50 of a virulent strain having undergone
not more than three passages and administered in not less
than 4 ml of diluent has been found to be satisfactory). The
titreofchallengevirusisdeterminedinswabstakenfrom
the nasal cavity of each animal daily from the day before
challenge until virus is no longer detected. Each animal is
weighed 7 days after challenge or at the time of death if this
occurs earlier and the average daily gain is calculated as a
percentage. For each group (vaccinated and controls), the
average of the average daily gains is calculated. The vaccine
complies with the test if:
— all the vaccinated pigs survive and the difference between
theaveragesofthedailygainsforthetwogroupsisnot
less than 1.5,
— the geometrical mean titres and the duration of excretion
of the challenge virus are significantly lower in vaccinates
than in controls.
The test is not valid unless all the control pigs display signs
of Aujeszky’s disease and the average of their daily gains
is less than −0.5.
If the vaccine is intended for use in sows for the passive
protection of piglets, the suitability of the strain for
this purpose may be demonstrated by the following
method. Eight sows which do not have antibodies against
Aujeszky’s disease virus or against a fraction of the virus
are vaccinated according to the recommended schedule and
by a recommended route; four sows are kept as controls.
Thepigletsfromthesowsarechallengedwithasuitable
quantity of a virulent strain of Aujeszky’s disease virus at 6
to 10 days of age. The piglets are observed for 21 days. The
vaccine is satisfactory if not less than 80 per cent protection
against mortality is found in the piglets from the vaccinated
sows compared to those from the control sows. The test is
not valid if the average number of piglets per litter for each
groupislessthansix.
BATCH TESTING
The test described under Potency is not necessarily carried
out for routine testing of batches of vaccine. It is carried out
for a given vaccine, on one or more occasions, as decided by
or with the agreement of the competent authority; where
the test is not carried out a suitable, validated, alternative
test is carried out, the criteria for acceptance being set with
reference to a batch of vaccine that has given satisfactory
results in the test described under Potency.
IDENTIFICATION
In animals having no antibodies against Aujeszky’s disease
virus or against a fraction of the virus, the vaccine stimulates
the production of specific antibodies against Aujeszky’s
diseasevirusorthefractionofthevirususedinthe
production of the vaccine.
TESTS
Safety. Inject two doses of the vaccine by a recommended
route into each of not fewer than two pigs of the minimum
age recommended for vaccination and having no antibodies
against Aujeszky’s disease virus or against a fraction of
the virus. Observe the animals for 14 days and then inject
onedoseofthevaccineintoeachpiglet.Observetheanimals
forafurther14days.Noabnormallocalorsystemicreaction
occurs during the 28 days of the test.
Inactivation. Wherever possible, carry out a suitable test
for residual infectious Aujeszky’s disease virus using two
passages in the same type of cell culture as used in the
production of the vaccine or cells shown to be at least
as sensitive. Otherwise, inject one dose of the vaccine
subcutaneously into each of five healthy non-immunised
rabbits. Observe the animals for 14 days after the injection.
No abnormal reaction (in particular a local rash) occurs. If
the vaccine strain is not pathogenic for the rabbit, carry out
the test in two sheep.
Extraneous viruses. On the pigs used for the safety test
carry out tests for antibodies. The vaccine does not stimulate
the formation of antibodies, other than those against
Aujeszky’s disease virus, against viruses pathogenic for pigs
or against viruses that could interfere with the diagnosis
of infectious diseases of pigs (including the viruses of the
pestivirus group).
Sterility. The vaccine complies with the test for sterility
prescribed in the monograph on Vaccines for veterinary
use (0062).
POTENCY
Use not fewer than five pigs each weighing 15 kg to 35 kg
and which do not have antibodies against Aujeszky’s disease
virus or against a fraction of the virus. The body mass of
none of the pigs differs from the average body mass of the
group by more than 25 per cent. Administer to each pig
by a recommended route one dose of the vaccine. Use five
similarpigsascontrols.Threeweekslaterweigheachpig
and then challenge by the intranasal route with a suitable
quantity of a virulent strain of Aujeszky’s disease virus.
Weigh each animal 7 days after challenge or at the time
of death if this occurs earlier and calculate the average
daily gain as a percentage. For each group (vaccinated and
controls), calculate the average of the average daily gains.
Thevaccinepassesthetestifthevaccinatedpigssurviveand
thedifferencebetweentheaveragesofthedailygainsforthe
two groups is not less than 1.1. The test is not valid unless
allthecontrolpigsdisplaysignsofAujeszky’sdiseaseand
theaverageoftheirdailygainsislessthan−0.5.
LABELLING
The label states:
— whether the vaccine strain is pathogenic for the rabbit,
— whether the vaccine is a whole-virus vaccine or a subunit
vaccine.
716 See the information section on general monographs (cover pages)