Observations on the influence of media composition on the efficacy of vaccines derived from some Clostridium chauvoei strains


Mhoma, J.R.L.

Bulletin of Animal Health and Production in Africa 33(2): 117-121

1985


Potency tests in guinea pigs with alum-precipitated vaccines prepared with three different C. chauvoei strains grown in liver-liver broth or liver-meat broth (pepsin-hydrochloric acid digest) gave consistently better results with the strains grown in liver-meat broth. Vaccine from the Awasa strain grown in liver-meat broth protected for at least a year. In field trials in cattle and sheep over three years, with vaccine from the Awasa strain grown in liver-meat broth, there were no vaccine breaks or adverse reactions.

Bull.
Anint.
filth
Prod.
Afr.
(1985)
33,
117-121
Observations
on
the
Influence
of
Media
Composition
on
the
Efficacy
of
Vaccines
Derived
from
some
Clostridium
chauvoei
Strains
J.R.L.
MHOMA
Tanzania
Livestock
Research
Organization,
Animal
Diseases
Research
Institute,
Dar
es
Salaam
;
P.O.
Box
9254,
Dar
es
Salaam,
Tanzania
Summary
The
immunogenicity
of
four
different
strains
of
Clostridium
chauvoei
prepared
in
four
different
media
were
investigated.
It
was-
observed
that
in
each
medium
a
vaccine
of
variable
immunogenicity
was
produced
regardless
of
the
bacterial
strain
used:
an
observation
which
suggested
that
the
antigenic
composition
of
the
vaccine
could
be
influenced
by
the
composition
of
the
medium.
Introduction
Arlong
and
Cornevin
(1882)
showed
that
cattle
injected
intravenously
with
virulent
blackquarter
exudates
remained
unaffected
and
later
were
non-susceptible
to
experimental
challenge.
Since
then
various
workers
have
prepared
blackquarter
vaccines
from
Clostridium
chauvoei
strains
using
different
methods.
One
problem
encountered
in
attempts
to
prepare
blackquarter
vaccines
has
been
obtaining
an
immunogenic
strain.
For
example,
Kerry
(1967)
studied
immunological
differences
between
nine
strains
of
Clostridium
chauvoei
in
sheep
and
guinea-pigs
and
reported
the
need
to
select
strains
which
had
a
wide
protective
spectrum
against
heterologous
challenge.
Reed
and
Reynolds
(1977)
reported
an
isolate
of
Clostridium
chauvoei
strain
which
when
incorporated
in
a
vaccine
gave
protection
against
challenge
with
all
the
official
strains
of
Clostridium
chauvoei
issued
by
several
regulatory
authorities,
as
well
as
with
the
field
strains.
Apart
from
the
problem
of
obtaining
an
immunogenic
strain
several
authors
have
commented
on
the
importance
of
preparing
appropriate
media
for
supporting
the
in
vitro
culture
of
fastidious
bacteria
(Rajagopalan,
1947;
Coackley
and
Weston,
1957;
Nilakantan,
1965;
Ramachandran
and
Raman,
1973).
One
of
the
earliest
medium
for
the
cultivation
of
Clostridium
chauvoei
and
other
anaerobes
in
broth
free
from
tissue
debris
was
described
by
M'cEwen
(1926).
To
prepare
a
new
form
of
pellet
vaccine
Roberts
(1946)
used
a
4%
solution
of
Evans
peptone
in
saline
with
0.5%
glucose.
Rajagopalan
(1947)
produced
a
vaccine
from
a
culture
grown
in
glucose
broth
containing
cysteine
hydrochloride.
Meat
piece
medium
(Piercy,
1951)
proved
satisfactory
for
vaccine
production
but
the
preparation
was
costly
and
laborious.
Verma
et
al.
(1953)
indicated
that
the
cheapest
medium
was
the
papain
digest
broth.
Salts
such
as
ferric,
ammonium
and
potassium
are
factors
influencing
production
of
good
bacterial
vaccines.
With
Clostridium
chauvoei
such
salts
have
been
found
to
stimulate
fermentation
and
agressin
production
(Scott,
1930).
Also
Scott
(1926)
found
the
ferric
salts
increased
the
virulence
of
all
the
blackquarter
strains
he
tested.
Another
factor
which
influences
the
preparation
of
a
good
blackquarter
vaccine
is
the
precipitant
used
and
the
optimum
concentration
of
the
precipitant
to
be
added.
Aluminium
hydroxide
and
alum
have
been
routinely
employed.
The
necessary
amount
of
adjuvant
to
be
incorporated
in
the
vaccine
and
their
reactions
have
been
described
by
Scheuber
(1944),
Piercy
(1951),
Nilakantan
(1965)
and
Anbuman
and
Moses
(1975).
Tnis
paper
records
some
observations
on
the
influence
of
the
composition
of
media
on
the
efficacy
of
vaccines
derived
from
some
Clostridium
chauvoei
strains
using
alum
as
a
precipitant.
118
MHOMA
Materials
and
Methods
Media
Preparation
Liver-liver
broth.
This
was
prepared
essentially
as
described
by
Mason
and
Scheuber
(1936).
However,
the
bovine
liver
broth
was
supplemented
with
1%
peptone,
0.3%
NaCI,
0.2%
NaHPO
4
and
1%
glucose.
The
broth
was
sterilized
at
110°C
for
70
minutes.
The
pH
was
adjusted
to
7.4
using
sodium
hydroxide.
Meat-liver
broth.
This
was
prepared
as
a
pepsin
hydrochloric
acid
digest
instead
of
trypsin
acetic
digest
originally
described
by
Mason
and
Scheuber
(1936).
Minced
meat
and
liver
in
the
ratio
1:1
was
placed
in
a
digestion
container
and
10
litres
of
tap
water
were
added
to
it.
The
mixture
was
stirred
and
thereafter
80m1
of
concentrated
hydrochloric
acid
and
4
g
of
pepsin
were
added.
The
mixture
was
allowed
to
digest
in
a
water
bath
at
50°C
for
24
hours. The
resulting
broth
was
filtered
and
the
pH
adjusted
to
7.7.
Finally
100
g
of
yeast
extract
were
added
and
the
broth
was
sterilized
at
120°C
for
30
minutes.
Robertson's
cooked
meat
medium.
This
was
prepared
as
described
by
Cruickshank
(1975)
and
kept
in
25
ml
screw-capped
MacCartney
bottles
in
a
refrigerator.
Von
Nibbler's
medium.
The
method
of
production
was
a
modification
of
that
originally
described
by
M'cEwen
(1926).
A
suspension
of
minced
oxen
brain
and
1%
glucose
in
the
ratio
1:3
was
used
in
this
study.
The
medium
was
sterilised
by
autoclaving
for
1
hour.
Bacterial
Strains
Four
Clostridium
chauvoei
strains
of
different
origins
were
used
in
this
study.
Strains
Ch.
16
and
Ch.
22
were
obtained
from
Institute
De
Investigacao
Veterinaria
Maputo
Mozambique
through
the
courtesy
of
Dr.
Tereza.
Strain
Tukuyu
was
an
isolate
from
a
zebu
cow
in
the
vicinity
of
Tukuyu
town
in
Southern
Highlands
of
Tanzania.
Strain
Awasa
was
kindly
donated
by
the
Veterinary
Institute
Debrezeit,
Ethiopia.
All
strains
except
strain
Tukuyu
were
lyophilised.
For
cultivation,
each
freeze-dried
strain
was
reconstituted
in
sterile
distilled
water,
inoculated
into
each
medium
and
incubated
under
anaerobic
condition
at
37°C.
The
Tukuyu
strain,
kept
as
infected
guinea-
pig
muscle
tissue,
was
minced
in
normal
saline,
inoculated
into
the
different
media
and
incubated
also
under
anaerobic
condition
at
37°C.
All
cultures
were
observed
for
active
growth
after
this
were
tested
for
lethality
in
guinea-pigs
and
sugar
fermentation
characteristics.
Vaccine
Preparation
The
technique
of
preparing
whole
culture
blackquarter
vaccine
has
been
described
it
detail
by
Scheuber
(1944).
In
this
work
30
ml
of
actively
growing
bacterial
culture
was
seeded
in
a
10
litre
flask
containing
vaccine
production
growth
medium
that
had
been
sterilized
and
cooled
at
37°C.
The
pH
was
adjusted
to
7.7.
Purity
of
the
culture
was
checked
on
blood
agar
plates
incubated
anaerobically
at
37°C.
To
the
10
litre
flask
350
ml
of
glucose
solution
were
added,
incubated
at
37°C
and
observed
for
growth
for
2
-
4
days.
When
growth
ceased
purity
was
again
carried
out
and
pH
readjusted
to
7.7.
The
culture
was
inactivated
by
addition
of
0.5%
formalin
and
incubated
at
37°C
for
at
least
5
days.
Then
alum
(potassium
aluminium
sulphate)
was
added
at
a
concentration
of
1%.
A
heavy
brownish
precipitate
was
formed.
The
vaccine
was
then
kept
at
room
temperature
for
4
days
before
transfering
it
to
a
cold
room
4°C
6°C
until
potency
testing.
Using
Clostridium
chauvoei
strains
Awasa,
Tukuyu
and
Ch.
16
with
meat-liver
and
liver-
liver
broths
as
production
media
batches
3/77,
4/77,
and
5/77
of
vaccines
precipitated
with
alum
were
obtained
and
subjected
to
potency testing.
Several
batches
of
vaccine
using
Awasa
strain
and
liver-meat
broth,
with
a
yield
of
about
110,000
doses
were
prepared
and
used
for
field
trials.
Testing
Vaccine
for
Potency
Each
vaccine
batch
was
tested
for
immunogenicity
and
inocuity
in
healthy
guinea-pigs
weighing
between
250
and
300
g.
Media
Composition
and
Efficacy
of
Vaccines
from
C.
chauvoei
strains
119
Twelve
guinea-pigs
were
used
to
test
each
batch
and
the
tests
were
repeated
three
times.
Of
the
12
guinea-pigs
two
were
controls.
A
dose
of
2
ml
of
the
vaccine
was
used
and
the
same
amount
was
used
as
a
booster
dose
at
21
days
interval.
Ten
days
after
the
second
vaccination,
the
animals
were
challenged
with
a
virulent
18
hour
culture
of
Clostridium
chauvoei
strain
Ch.
22
which
was
highly
virulent.
The
survival
of
guinea-pigs
was
observed
for
a
period
of
10
days.
For
safety
of
the
vaccine,
the
same
guinea-pigs
were
observed
for
side-effects.
The
efficacy
of
the
vaccine
was
determined
by
challenging
the
guinea-pigs
12
months
post
vaccination.
In
order
to
obtain
some
indication
of
the
performance
of
the
vaccine
under
field
conditions
four
villages,
two
ranches
and
two
research
stations
having
a
history
of
annual
recurrence
of
blackquarter
infection
were
selected.
The
villages
had
a
total
of
about
25,000
zebu
cattle
and
570
local
sheep.
The
ranches
had
15,000
cattle
consisting
of
zebu,
cross-breeds
of
Friesian,
Angus
and
Jersey
with
zebu.
The
research
stations
had
about
800
cattle
consisting
of
Jersey,
Ayrshire
and
Mpwapwa
breeds.
For
three
consecutive
years
preliminary
field
vaccinations
were
carried
out
in
selected
villages,
ranches,
research
stations
and
to
some
individual
farmers.
In
each
selected
area,
cattle
(3
months
of
age
and
above)
and
sheep
were
given
one
dose
of
the
vaccine
yearly,
each
dose
consisting
of
5
ml
and
2
ml
respectively
for
cattle
and
sheep.
Morbidity
and
mortality
rates
due
to
blackquarter
infection
were
recorded
throughout
the
vaccination
trial
period.
Cattle
and
sheep
in
the
surrounding
areas
were
not
vaccinated
and
as
such
served
as
controls.
Results
The
growth
and
properties
of
individual
tested
strains
in
each
medium
is
summarized
in
Table
1.
The
meat-liver
broth
and
Robertson's
cooked
meat
medium
supported
the
growth
of
all
four
strains.
Strain
Ch.
16
was
less
virulent
than
strain
Ch.
22.
Strain
Awasa
was
highly
virulent
followed
by
strain
Tukuyu.
With
alum
as
a
precipitant
the
precipitate
from
the
vaccine
made
from
meat-
liver
medium
was
more
than
that
from
the
liver-liver
medium.
The
laboratory
potency
inocuity
tests
of
batches
3/77,
4/77
and
5/77
are
summarized
in
Table
2.
Each
medium
produced
a
vaccine
of
variable
immunogenicity
with
the
same
strain.
Batch
3/77
appeared
superior
to
others
with
about
90%
protection.
Challenge
experiments
of
guinea-pigs
inoculated
with
Awasa
strain
12
months
post
vaccination
resulted
in
no
extra
deaths.
Except
fora
slight
temperature
rise
during
the
first
2
-
3
days
post
inoculation
and
transient
swelling
at
the
inoculation
sites
no
other
adverse
reactions
were
observed
on
the
guinea-pigs
for
a
period
of
10
days.
In
the
field
trials,
no
single
outbreak
and
adverse
reaction
was
observed
in
the
vaccinated
areas
throughout
the
period
of
vaccination
but
six
confirmed
outbreaks
were
reported
in
the
surrounding
unvaccinated
villages
and
ranch.
The
conditions
of
the
vaccinated
animals
remained
normal
with
little
or
no
rise
in
body
temperature.
Slight
swellings
at
the
site
of
inoculation
were
seen
in
about
5%
of
the
vaccinated
animals
but
these
disappeared
in
about
a
week's
time
and
only
few
cases
persisted
for
more
than
a
month.
Neither
abscess
formation
nor
sloughing
of
the
skin
over
the
site
of
inoculation
was
observed
from
any
quarter.
Discussion
Cultivation
of
Clostridium
chauvoei
in
media
prepared
by
different
methods
has
been
observed
to
yield
vaccines
of
varying
immunogenicity.
Nilakantan
(1965)
for
example,
examined
the
efficacy
of
vaccines
prepared
in
liver-meat
infusion,
liver-meat
acid
digest
broth
and
liver-papain
digest
broth
and
reported
that
the
vaccines
prepared
in
the
three
media
had
better
immunizing
power
than
a
vaccine
prepared
from
glucose
broth
with
pieces
of
rabbit
kidney.
Amongst
the
media
used
in
this
study
the
meat-liver
Table
1:
Growth,
Lethality
and
Sugar
Fermentation
Characteristics
of
Four
Different
Clostridium
chauvoei
Strains
Growth
Lethality
Sugar
Fermentation
Strain
Robertson's
Von
Liver-liver
Meat-liver
18
hours'
culture
cooked
meat
medium
Hibbler's
medium
broth
broth
Glucose
Maltose
Sucrose
Lactose
Saticin
0.1ml
SC
lml
SC
Ch.
16
VG
PG
VG
VG
2/I0
8/10
++
++
++
++
Ch.
22
VG VG
PG
VG
10/10
10/10
++ ++
++
++
Tukuyu
VG
PG
VG
VG
9/10
9/10
++
++
++
++
Awasa
VG
PG
VG VG
10/10
10/10
++
++ ++
++
VG
=
Vigorous
growth;
PG
=
Poor
growth;
++
=
Acid
and
gas;
=
no
reaction;
Numerator
=
number
of
guinea-pigs
dead;
Denominator
=
number
of
guinea-pigs
inoculated;
SC
=
subcutaneous.
Table
2:
Immunogenicity
Trial
in
Guinea-pigs
of
Vaccines
Derived
from
Some
Clostridium
chauvoei
Strains
No.
of
guinea-pigs
survived/No.
of
guinea-pigs
inoculated
Batch
No.
1st
test
2nd
test
3rd
test
Protection
((
Awasa
strain
3/77
+
liver-liver
broth
9/10
9/10
8/10
86
)
)
Awasa
strain
)
Awasa
strain
)
3/77
+
/
liver-meat
broth
10/10
9/10
9/10
93
)
Ch.
16
strain
4/77
+
liver-liver
broth
8/10
7/10
8/10
76.6
)
)
Ch.
16
strain
4/77
+
)
Ch'
16
strain
)
)
liver-meat
broth
8/10
8/10
8/10
80
)
Tukuvu
strain
5/77
liver-liver
broth
7/10
7/10
7/10
70
Tukuyu
strain
)
Tukuyu
strain
5/77
+
liver-meat
broth
8/I0
7
/
1
0
8/10
76.6
)
VI
AIOHI
AI
OZ
I
Media
Composition
and
Efficacy
of
Vaccines
from
C.
chauvoei
strains
121
broth
and
Robertson's
cooked
meat
medium
appeared
to
be
most
favourable
and
gave
excellent
bacterial
growth
with
all
the
strains
studied.
Each
of
the
media
however,
yielded
.
vaccines
of
varying
potencies
regardless
of
the
bacterial
strain
used
to
prepare
the
vaccine.
This
observation
confirms
earlier
reports
by
Nilakantan
(1965)
and
Ramachandran
and
Raman
(1975)
that
the
immunogenicity
of
blackquarter
vaccine
is
largely
affected
by
the
composition
of
the
medium
used
to
prepare
it.
The
established
local
optimum
growth
conditions
gave
high
yield
of
the
organisms
resulting
to
high
concentration
of
the
antigen
which
probably
created
influence
on
the
potency
of
the
vaccine.
It
has
been
claimed
that
immunogenicity
of
Clostridium
chauvoei
vaccine
is
also
closely
related
to
the
virulence
of
the
bacterial
strain.
Rodrigues
(1945)
for
example,
reported
that
highly
pathogenic
strains
of
Clostridium
chauvoei
were
necessary
for
obtaining
potent
antigens.
This
report
however,
was
later
refuted
by
Coackley
and
Weston
(1957)
who
investigated
a
relatively
avirulent
strain
of
Clostridium
chauvoei
in
sheep
and
guinea-pigs
and
found
it
to
possess
enough
potency
for
vaccine
preparation.
The
observations
made
in
this
study
seem
to
support
those
reported
by
Coackley
and
Watson
(1975)
that
immunogenicity
of
Clostridium
chauvoei
strains
is
not
a
function
of
their
virulence
per•
se.
Although
the
Awasa
strain
which
was
found
to
be
highly
virulent
gave
excellent
results
in
potency
tests,
the
potencies
of
vaccines
from
Ch.
16
and
Tukuyu
strains
were
similar.
This
was
in
spite
of
the
fact
that
Ch.
16
strain
was
less
virulent
than
Tukuyu
strain.
The
difference
observed
in
potency
of
Clostridium'
chauvoei
vaccines
could
perhaps,
beSt
be
explained
in
terms
of
diversity
in
antigenic
composition
of
the
bacteria
and
our
knowledge
about
the
variability
of
the
antigenic
composition
of
Clostridium
chauvoei
strains
cultivated
.
in
media
of
differing
compositions
is
limited.
More
research
is,
therefore,
needed
in
this
area.
Most
investigators
on
the
immunization
against
Clostridium
chauvoei
infection
have
subsequently
recommended
two
(Claus
and
Macheak,
1972),
and
in
some
cases
two
booster
doses
with
blackquarter
vaccine
in
endemic
areas:
the
protection
however,
has
almost
always
been
short-lived
(Red
and
Reynold,
1977).
The
Awasa
strain
derived
vaccine
reported
in
this
paper
conferred
protection
against
challenge
up
to
at
least
a
year
with
a
single
dose
of
the
vaccine.
This
observation
is
interesting
and
needs
further
investigation.
Currently,
large
scale
field
vaccinations
have
been
initiated
in
different
parts
of
Tanzania
with
the
aim
of
ascertaining
whether
in
fact
this
vaccine
can
confer
protection
LI
Lainst
all
Clostridium
chauvoei
strains
prevalent
in
this
country.
Acknowledgements
The
author
expresses
gratitude
to
P.
Rwehabula
and
N.
Msangi
for
technical
assistance.
This
paper
is
published
with
the
permission
of
the
Director
General,
Tanzania
Livestock
Research
Organization.
References
Anbuman,
S.P.
and
Moses,
J.S.
(1975).
Indian
Vet.
J..
52
462.
A
rlong,
S.
and
Cornevin,
T.
(1982).
Comp!.
Rend.
Acad.
xci,
734.
Claus,
K.D.
and
Macheak,
M.E.
(1972).
Am.
J.
Vet.
Res.,
33,
1045.
Coackley,
W.
and
Weston,
J.W.
(1957).
J.
Comp.
Path.,
67,
157.
Cruickshank,
R.
(1973).
Medical
Microbiology,
P.
122.
Edinburgh,
Churchill
Livingstone.
Kerry,
J.B.
(1967).
Res.
Vet.
Sci.,
8,
89.
Mason,
J.H.
and
Scheuber,
J.R.
(1936).
Onderst.
J.
Vet.
Sc•i.
Anita
hid,
7,
143.
M'cEwen,
A.D.
(1926)../.
Comp.
Path.,
56,
128.
Nilakantan,
P.R.
(1965).
Indian
J.
Vet.
Sci.
Aninr.
Hush..
35,
135.
Piercy,
S.E.
(1952).
B.
Vet.
J.,
107,
63.
Rajagopalan,
V.R.
(1947).
.1.
Path.
Bact.,
59,
37.
Ramachandran,
S.
and
Raman,
K.
(1973).
Indian
Vet.
J.,
50,
739.
Reed,
G.A.
and
Reynolds,
C.
(1977).
Aust.
Vet.
J.,
53,
393.
Roberts,
R.S.
(1946)../.
Comp.
Path.,
56,
128.
Rodrigues,
C.
(1945).
Vet.
Bull,
17,
771.
Scheuber,
.1.R.
(1944).
Onderst.
.1.
Vet.
Sc.
Anim.
Ind.,
19,
17.
Scott,
J.P.
(1926).
./.
hif
his..
38,
511.
Scott,
J.P.
(1930)../.
Am.
Vet.
Med
Ass.,
29,
525.
Verma,
N.S.,
Rau,
K.G.
and
Govil,
J.L.
(1953).
Indian.
Vet.
.1.,
30,
33.
Received
,for
publication
on
61/i
September
1983.