Microbiol. Immunol. Vol. 23 (6), 543-547, 1979
Comparisons
of Staphylococcus aureus Grown
In Vitro or In Vivo
*Dennis L . WATSONand Julie-Anne PRIDEAUX *Department of TropicalVeterinary Science, JamesCookUniversity of North Queensland, Queensland. 4811,Australia (Receivedfor publication,August3, 1978) It
has
been
established
immune
responses
injected
into
with
S.
sheep.
aureus
larly cytosis
of
morphs
from
viable
shown
cytophilic
carried
the
in
the
capacity
of
to
the
ovine
intramuscuof
only
the
in
mammary
ewes
than
cell
of
poly-
infected IgG2
Recent
efficient
the
25%
cytophilic
polymorphs.
phago-
on
Although
it was
more
phagocytose
S. aureus
antibody
that
was
to
infected
enhancement
(13).
(12),
abscess)
vaccine
protection
observed
ewes
IgG2
phagocytic
killed
are
ewes
capacity
given
the
organisms
from
antistaphylococcal
a skin
staphylococcal
enhanced
infected
cytophilic
(resulting
immune
for
IgG2
collected
ewes
between
aureus
with
conferred
studies
a killed
gland
against
live
staphylococcal
have
vaccine
in
staphylococcal
(14).
Because it was
of
the
decided
erties
of
then
using
tial
evidence
dangers to
killed to
(5),
and
described
of Gladstone
in
and
bacteriological
changes
in
either
in
desirable in
cultural
has
been
vitro
or
In of
the
S.
aureus
various
can
6),
technique
grown
properties
4,
experi-
culturing
were
induce (3,
preliminary
sac
and
is substan-
antigens
note,
aureus
propculture
There of
dialysis S.
and
the
somatic
this
employed vivo,
killing
conditions
(2).
in
vivo,
in
vaccines,
immunological
vaccine.
alterations
a modification (5)
of
the
a non-viable
properties
which
Glencross broth,
as
compositional
biochemical
use
of
staphylococci
mixture
that and
the
some
growing
cell-toxoid
indicate
with
reproduce
by
functional
are
associated
attempt
vaccination
the
exotoxins ments
to
live
important
of
in
standard
the
bacteria
measured. A
acute
agulase
non-capsulated clinical and
colonial
42D mastitis
catalase
morphology
purity The
CM
basis
differences
Staphylococcus
had from
The
from
ewes
a live
challenge
the
be
enhanced
that
were
13).
significant
killed leucocytes
polymorphs
polymorphs all
stimulating
for
to
staphylococci
significantly
of
(11,
shown
membrane
highly
or
injection)
with
adjuvant
was
are live
subcutaneous
compared
with
there
when
Polymorphonuclear
(by
staphylococci
that
provoked
using in
peritoneal 1)
was
positive,
and
on
grown cavity
placed
5%
cow,
bacteria sheep.
a dialysis
of was
exhibited
sheep
were
Staphylococcus used
a-
agar.
aureus, these
isolated
from
experiments.
It
and ƒÀ-haemolysis
Cultures
were
and routinely
a case was
co-
typical checked
techniques. cultured
inside
Approximately sac
in
strong
blood
bacteriological
of in
strain
a dairy
standard
vivo
phage
in
which
100 was
543
then
dialysis ml
of
sterilized
sacs nutrient by
(Visking broth autoclaving
20/32)
in
(Oxoid, while
D.L. WATSON ANDJ-A. PRIDEAUX
544
immersed of
S.
in
a flask
aureus
was
was
anesthetized
the
dialysis
The
sac
closed ad
was
nutrient
was
placed
after
sacs
capsule
48
hr
The After
in
mice, the
vitro
broth
and sacs
wall
this
by
procedure
the did
a
overnight
of was
the
findings
of
result
and
laparotomy.
suture
and
the
feed
and
sac
the
sheep
Laboratories),
allowed recovered
other
in
culture A
a right-side
length
sheep and
not
an
conditions.
through
The
to
of
(Abbott
cavity
anesthetized,
contrast
ml sterile
Nembutal
peritoneal
again
0.5
under
wound water
through
workers
(9)
development
an
who of
a
used fibrous
sac. cultures
the
cultured the
of
abdominal
was
grown
inoculation
sac
procedures.
In
in
around
the
surgical
laparotomy.
collodion
the
cooling,
dialysis
injection
in
to
After
the
intravenous
standard
and
broth. into
anchored
using
adjacent
the
by sac
libitum
of
introduced
sacs
in
bacteria
were
were
a shaking were
prepared
re-immersed water
in
bath
routinely
at
checked
in
dialysis
sacs
the
flask
containing
37 for
C for
48
purity
as
hr.
and
described
above.
sterile
Upon
nutrient
recovery
viability
using
from standard
techniques. Controlled out
four
to
determine
from
comparisons
times.
an
The the
aliquot
number of
2.1
0.12 to
shown
6.5 •~ in
bers
of
Fig.
of to
by
1.00 •~
A It
in
in
can the
in
7.2
and
for
typical be
S.
in
vivo
from
grown
in of
vitro
Hirsch
cultures.
and
cultures in vivo
this
48
cultures of
mean
that
greatly
each
was
used
deposited ( •~ 3)
with
was
with
PBS.
The
was
0.76 •~
109/ml
3.8 •~
109/ml
(range
of
the
sampling
exceeded
carried
(7)
washed
pattern at
were
were
incubation
growth
figure
cultures
h
vivo
bacteria
adjusted
the
the
in
Strauss
15 min),
volume
after
and and
The
(3,000 •~g,
(PBS)
example
seen
aureus
method
in vitro
109/ml),
109/ml).
bacteria
of
centrifugation pH
bacteria
1.
growth
staphylococci
saline,
number
(range
of
broth
phosphate-buffered mean
of
spectrophotometric
organisms time
those
in
is
the
in vitro
numgrown
cultures. Bacteria and amino
acid
These
assays
In
(grown
lyophilized.
vivo grown
both Aliquots
composition were bacteria
in vivo (10
and mg)
in
determined
carried had
out
in
significantly
vitro)
were
were
washed
hydrolysed on
duplicate more
an
with
amino
and
the
results
isoleucine
Fig
.
acid
(54%)
1.
aureus
Growth grown
( •~ 3)
with
distilled
6 M HCl
(1,
10)
autoanalyser are
in
leucine
curves in vitro
of (•œ
the
(T.S.M.).
presented and
water and
Table
(44%)
Staphylococcus or in
vivo
(•›).
1. than
NOTES
Table
1.
Amino
Values
in
vitro
grown
more of
tyrosine
and
organisms
2
the
and
valine
significantly in
2
were
greater
vivo
and
were
killed
days
at
in
C.
from
a sheep
washed was
shown
aliquot trypsin The remove fusion
to
tests or
had
have
and
adjusted (2.5
ml)
(Searle mixture
particulate assays
the
were
was dissolved
at
matter. carried
of
37 The
out
in
C
for
then in 3 hr
supernatant 1.5%
Agarose
(Difco Two The
S.
prepared
4 ml
was A
aureus
1 ml
with
a solution
phosphate centrifuged maintained
(Pharmacia)
of
bled
collected of
5 x
this
108
animal
recovered
from
immunodiffu-
washed with
way
from
( •~ 3)
with
PBS.
containing
g,
frozen.
An
0.25
(0.05
(3,000 •~
made
; the
were
following
buffer
the
bacteria
was
in
was
1
Laboratories) weeks later
were
the
bacteria
organisms/ml
of
then
of
animals
employed in
following
injection
1010
mixed
content
and
examination
of
7.2 •~
the
serum
intravenous
were
of
of
addition,
by
sera
in
made
post-mortem
suspension
the
non-viability
Adjuvant a sheep.
monocultures
concentration
for was
In
These
substantially
but
concentration
locations.
antigens A
final
checked
S. aureus
lungs.
had
prepared a
emulsion
Upon and
and
suspension
shaken
to
different
with
organisms. a
were
then
aureus
organisms,
staphylococci.
intervals.
infected
Biochemicals) was
two
staphylococcal
to of
in
pyaemia
grown
grown
An
previously.
spleen,
soluble
vitro
PBS.
S.
vitro
formalin
were
two-week
been
acute
liver,
with in
at
10 weeks
kidneys,
in vivo PBS
which
organisms
heart, sion
then
grown in
of Freund's Complete into two sites of
injections
and
vivo
bacteria of
under
assays.
than
grown
They with.
in
in vitro
addition
( •~ 3)
similar
later
in
vitro
by
37
washed
received
weeks
of duplicate
addition
(9.4 •~ 1010 organisms) and 1 ml this was injected intramuscularly sheep
means
In
to
for
are the
arginine,
Cultures held
presented
serine,
was
Antisera way.
acid composition of Staphylococcus aureus grown in vitro or in vivo cultural conditions
organisms.
threonine,
545
M, 30
g of
pH
8).
min)
to
Immunodifup
with
Veronal
546
D.L. WATSON ANDJ-A. PRIDEAUX Fig. 2.
Immunodiffusion
assays
of
various sheep sera with staphylococcal cell antigen extracts. Well 1, Trypsin extract from in vitro grown S.- aureus. 2, Trypsin-extract from in vivogrown S. aureus. a,f, Normal (non-immune) sheep serum. b,g, Antisera to in vitro grown S. aureus, 2 weeks postimmunization. c,h, Antisera to in vivo grown S. aureus, 2 weeks postimmunization. d,i, Antisera to in vitro grown S. aureus, 4 weeks postimmunization. e,j, Serum from sheep infected with viable S. aureus 10 weeks previously.
buffer (LKB). Diffusion was allowed to proceed for 72 hr in a humid chamber at 30 C. The precipitin lines which developed when antisera and normal (non-immunized) sheep sera were used in immunodiffusion assays against the trypsin-extract are shown in Fig. 2. When trypsin-extract from in vitrogrown cells was used there was a confluent precipitin line for each of the antisera and normal sheep serum. An additional precipitin line showed reactivity against each of the antisera but not against normal sheep serum. When the extract from in vivogrown staphylococci was used as antigen the same two lines as described above were evident. However, there was an extra precipitin line against antisera to in vivogrown bacteria and against serum from an S. aureus-infected sheep. The results obtained from these studies support earlier evidence for the existence of fundamental differences between S. aureus grown in vitro or in vivo. The significantly faster growth rate in vivosuggests the presence of a dialysable growth factor(s) in sheep peritoneal fluid which is absent from standard bacteriological broth, or alternatively, the elimination of toxic by-products when bacteria are grown in vivo. The pore size of the dialysis tubing used was 26 A, a value which provides an indication of the maximum possible diameter of the molecule(s) in question. Differences in amino acid composition of in vitro and in vivo grown organisms are not only significant per se, but also suggested that antigenic differences may exist between staphylococci grown by these two techniques. The demonstration of an antigen in extracts from in vivogrown S. aureus which could not be detected (within the limits of sensitivity of these immunodiffusion assays) in extracts from in vitrogrown bacteria, could be of considerable immunological significance. Since this antigen was also precipitated by sera from the S. aureus-infected sheep it seems likely that it is also produced by staphylococci growing in pathogenic lesions. Antibody against this component may account for some of the differences in immune responses to live and killed S. aureus vaccines described earlier. Indeed recent studies (8) suggest that IgG antibodies to an antiphagocytic surface antigen of S. aureus (strain 7007) were capable of enhancing in vitro phagocytosis of this strain of staphylococci. Experiments are now in progress to determine whether antibodies against the antigen
547
NOTES
of in vivo grown lococci
by
S. aureus
polymorphonuclear
are
similarly
active
in
promoting
phagocytosis
of staphy-
leucocytes.
This work was financially supported by grants from the Australian Research Grants Committee and the Dairying Research Committee of Australia. We thank Ms. W. Beresford, Department of Biochemistry and Nutrition, University of New England, for carrying out the amino acid analyses. REFERENCES
1)
2) 3)
4)
5) 6) 7) 8) 9) 10) 11) 12) 13) 14)
Beever, D.E., Harrison, D.G., Thomson, D.J., Cammell, S.B., and Osbourn, D.F. 1974. A method for the estimation of dietary and microbial protein in duodenal digesta of ruminants. Br. J. Nutr. 32: 99-111. Beining, P.R., and Kennedy, E.R. 1963. Characteristics of a strain of Staphylococcusaureus grown in vivoand in vitro. J. Bacteriol. 85: 732-741. Brock, J.H., and Reiter, B. 1976. Chemical and biological properties of extracellular slime produced by Staphylococcusaureusgrown in high-carbohydrate, high-salt medium. Infect. Immun. 13: 653-660. Brock, J.H., Turvey, A., and Reiter, B. 1973. virulence of two mastitis strains of Staphylococcus aureusin bovine skin: Enhancement by growth in high-carbohydrate, high-salt medium or in raw milk. Infect. Immun. 7: 865-872. Gladstone, G.P., and Glencross, E.J.G. 1960. Growth and toxin production of staphylococci in cellophane sacs in vivo.Brit. J. Exp. Path. 41: 313-333. Haukenes, G. 1967. Serological typing of Staphylococcusaureus. Acta Path. Microbiol. Scand. 70: 590-600. Hirsch, J.G., and Strauss, B. 1964. Studies on heat labile opsonin in rabbit serum. J. Immunol. 92: 145-154. Karakawa, W.W., and Kane, J.A. 1975. Immunochemistry of an acidic antigen isolated from a Staphylococcusaureus.J. Immunol. 114: 310-315. Lam, G.T., Sweeney, F. J., Witmer, C.M., and Wise, R.I. 1963. Abscess-forming factor(s) produced by Staphylococcus aureus.I. Collodion bag implantation technique. J. Bacteriol. 86: 611-615. Moore, S., Spackman, D.H., and Stein, W.H. 1958. Chromatography of amino acids on sulfonated polystyrene resins. An improved system. Anal. Chem. 30: 1185-1190. Watson, D.L. 1975a. Enhancement of in vitro phagocytosis of Staphylococcusaureus by polymorphonuclear leucocytes. Res. vet. Sci. 19: 288-292. Watson, D.L. 1975b. Cytophilic attachment of ovine IgG2 to autologous polymorphonuclear leucocytes. Aust. J. Exp. Biol. Med. Sci. 53: 527-529. Watson, D.L. 1976. The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leucocytes. Immunology 31: 159-165. Watson, D.L., and Lee, C.G. 1978. Immunity to experimental staphylococcal mastitis - Comparison of live and killed vaccines. Aust. vet. J. 54: 374-378.
Comparisons
of Staphylococcus aureus Grown
In Vitro or In Vivo
*Dennis L . WATSONand Julie-Anne PRIDEAUX *Department of TropicalVeterinary Science, JamesCookUniversity of North Queensland, Queensland. 4811,Australia (Receivedfor publication,August3, 1978) It
has
been
established
immune
responses
injected
into
with
S.
sheep.
aureus
larly cytosis
of
morphs
from
viable
shown
cytophilic
carried
the
in
the
capacity
of
to
the
ovine
intramuscuof
only
the
in
mammary
ewes
than
cell
of
poly-
infected IgG2
Recent
efficient
the
25%
cytophilic
polymorphs.
phago-
on
Although
it was
more
phagocytose
S. aureus
antibody
that
was
to
infected
enhancement
(13).
(12),
abscess)
vaccine
protection
observed
ewes
IgG2
phagocytic
killed
are
ewes
capacity
given
the
organisms
from
antistaphylococcal
a skin
staphylococcal
enhanced
infected
cytophilic
(resulting
immune
for
IgG2
collected
ewes
between
aureus
with
conferred
studies
a killed
gland
against
live
staphylococcal
have
vaccine
in
staphylococcal
(14).
Because it was
of
the
decided
erties
of
then
using
tial
evidence
dangers to
killed to
(5),
and
described
of Gladstone
in
and
bacteriological
changes
in
either
in
desirable in
cultural
has
been
vitro
or
In of
the
S.
aureus
various
can
6),
technique
grown
properties
4,
experi-
culturing
were
induce (3,
preliminary
sac
and
is substan-
antigens
note,
aureus
propculture
There of
dialysis S.
and
the
somatic
this
employed vivo,
killing
conditions
(2).
in
vivo,
in
vaccines,
immunological
vaccine.
alterations
a modification (5)
of
the
a non-viable
properties
which
Glencross broth,
as
compositional
biochemical
use
of
staphylococci
mixture
that and
the
some
growing
cell-toxoid
indicate
with
reproduce
by
functional
are
associated
attempt
vaccination
the
exotoxins ments
to
live
important
of
in
standard
the
bacteria
measured. A
acute
agulase
non-capsulated clinical and
colonial
42D mastitis
catalase
morphology
purity The
CM
basis
differences
Staphylococcus
had from
The
from
ewes
a live
challenge
the
be
enhanced
that
were
13).
significant
killed leucocytes
polymorphs
polymorphs all
stimulating
for
to
staphylococci
significantly
of
(11,
shown
membrane
highly
or
injection)
with
adjuvant
was
are live
subcutaneous
compared
with
there
when
Polymorphonuclear
(by
staphylococci
that
provoked
using in
peritoneal 1)
was
positive,
and
on
grown cavity
placed
5%
cow,
bacteria sheep.
a dialysis
of was
exhibited
sheep
were
Staphylococcus used
a-
agar.
aureus, these
isolated
from
experiments.
It
and ƒÀ-haemolysis
Cultures
were
and routinely
a case was
co-
typical checked
techniques. cultured
inside
Approximately sac
in
strong
blood
bacteriological
of in
strain
a dairy
standard
vivo
phage
in
which
100 was
543
then
dialysis ml
of
sterilized
sacs nutrient by
(Visking broth autoclaving
20/32)
in
(Oxoid, while
D.L. WATSON ANDJ-A. PRIDEAUX
544
immersed of
S.
in
a flask
aureus
was
was
anesthetized
the
dialysis
The
sac
closed ad
was
nutrient
was
placed
after
sacs
capsule
48
hr
The After
in
mice, the
vitro
broth
and sacs
wall
this
by
procedure
the did
a
overnight
of was
the
findings
of
result
and
laparotomy.
suture
and
the
feed
and
sac
the
sheep
Laboratories),
allowed recovered
other
in
culture A
a right-side
length
sheep and
not
an
conditions.
through
The
to
of
(Abbott
cavity
anesthetized,
contrast
ml sterile
Nembutal
peritoneal
again
0.5
under
wound water
through
workers
(9)
development
an
who of
a
used fibrous
sac. cultures
the
cultured the
of
abdominal
was
grown
inoculation
sac
procedures.
In
in
around
the
surgical
laparotomy.
collodion
the
cooling,
dialysis
injection
in
to
After
the
intravenous
standard
and
broth. into
anchored
using
adjacent
the
by sac
libitum
of
introduced
sacs
in
bacteria
were
were
a shaking were
prepared
re-immersed water
in
bath
routinely
at
checked
in
dialysis
sacs
the
flask
containing
37 for
C for
48
purity
as
hr.
and
described
above.
sterile
Upon
nutrient
recovery
viability
using
from standard
techniques. Controlled out
four
to
determine
from
comparisons
times.
an
The the
aliquot
number of
2.1
0.12 to
shown
6.5 •~ in
bers
of
Fig.
of to
by
1.00 •~
A It
in
in
can the
in
7.2
and
for
typical be
S.
in
vivo
from
grown
in of
vitro
Hirsch
cultures.
and
cultures in vivo
this
48
cultures of
mean
that
greatly
each
was
used
deposited ( •~ 3)
with
was
with
PBS.
The
was
0.76 •~
109/ml
3.8 •~
109/ml
(range
of
the
sampling
exceeded
carried
(7)
washed
pattern at
were
were
incubation
growth
figure
cultures
h
vivo
bacteria
adjusted
the
the
in
Strauss
15 min),
volume
after
and and
The
(3,000 •~g,
(PBS)
example
seen
aureus
method
in vitro
109/ml),
109/ml).
bacteria
of
centrifugation pH
bacteria
1.
growth
staphylococci
saline,
number
(range
of
broth
phosphate-buffered mean
of
spectrophotometric
organisms time
those
in
is
the
in vitro
numgrown
cultures. Bacteria and amino
acid
These
assays
In
(grown
lyophilized.
vivo grown
both Aliquots
composition were bacteria
in vivo (10
and mg)
in
determined
carried had
out
in
significantly
vitro)
were
were
washed
hydrolysed on
duplicate more
an
with
amino
and
the
results
isoleucine
Fig
.
acid
(54%)
1.
aureus
Growth grown
( •~ 3)
with
distilled
6 M HCl
(1,
10)
autoanalyser are
in
leucine
curves in vitro
of (•œ
the
(T.S.M.).
presented and
water and
Table
(44%)
Staphylococcus or in
vivo
(•›).
1. than
NOTES
Table
1.
Amino
Values
in
vitro
grown
more of
tyrosine
and
organisms
2
the
and
valine
significantly in
2
were
greater
vivo
and
were
killed
days
at
in
C.
from
a sheep
washed was
shown
aliquot trypsin The remove fusion
to
tests or
had
have
and
adjusted (2.5
ml)
(Searle mixture
particulate assays
the
were
was dissolved
at
matter. carried
of
37 The
out
in
C
for
then in 3 hr
supernatant 1.5%
Agarose
(Difco Two The
S.
prepared
4 ml
was A
aureus
1 ml
with
a solution
phosphate centrifuged maintained
(Pharmacia)
of
bled
collected of
5 x
this
108
animal
recovered
from
immunodiffu-
washed with
way
from
( •~ 3)
with
PBS.
containing
g,
frozen.
An
0.25
(0.05
(3,000 •~
made
; the
were
following
buffer
the
bacteria
was
in
was
1
Laboratories) weeks later
were
the
bacteria
organisms/ml
of
then
of
animals
employed in
following
injection
1010
mixed
content
and
examination
of
7.2 •~
the
serum
intravenous
were
of
of
addition,
by
sera
in
made
post-mortem
suspension
the
non-viability
Adjuvant a sheep.
monocultures
concentration
for was
In
These
substantially
but
concentration
locations.
antigens A
final
checked
S. aureus
lungs.
had
prepared a
emulsion
Upon and
and
suspension
shaken
to
different
with
organisms. a
were
then
aureus
organisms,
staphylococci.
intervals.
infected
Biochemicals) was
two
staphylococcal
to of
in
pyaemia
grown
grown
An
previously.
spleen,
soluble
vitro
PBS.
S.
vitro
formalin
were
two-week
been
acute
liver,
with in
at
10 weeks
kidneys,
in vivo PBS
which
organisms
heart, sion
then
grown in
of Freund's Complete into two sites of
injections
and
vivo
bacteria of
under
assays.
than
grown
They with.
in
in vitro
addition
( •~ 3)
similar
later
in
vitro
by
37
washed
received
weeks
of duplicate
addition
(9.4 •~ 1010 organisms) and 1 ml this was injected intramuscularly sheep
means
In
to
for
are the
arginine,
Cultures held
presented
serine,
was
Antisera way.
acid composition of Staphylococcus aureus grown in vitro or in vivo cultural conditions
organisms.
threonine,
545
M, 30
g of
pH
8).
min)
to
Immunodifup
with
Veronal
546
D.L. WATSON ANDJ-A. PRIDEAUX Fig. 2.
Immunodiffusion
assays
of
various sheep sera with staphylococcal cell antigen extracts. Well 1, Trypsin extract from in vitro grown S.- aureus. 2, Trypsin-extract from in vivogrown S. aureus. a,f, Normal (non-immune) sheep serum. b,g, Antisera to in vitro grown S. aureus, 2 weeks postimmunization. c,h, Antisera to in vivo grown S. aureus, 2 weeks postimmunization. d,i, Antisera to in vitro grown S. aureus, 4 weeks postimmunization. e,j, Serum from sheep infected with viable S. aureus 10 weeks previously.
buffer (LKB). Diffusion was allowed to proceed for 72 hr in a humid chamber at 30 C. The precipitin lines which developed when antisera and normal (non-immunized) sheep sera were used in immunodiffusion assays against the trypsin-extract are shown in Fig. 2. When trypsin-extract from in vitrogrown cells was used there was a confluent precipitin line for each of the antisera and normal sheep serum. An additional precipitin line showed reactivity against each of the antisera but not against normal sheep serum. When the extract from in vivogrown staphylococci was used as antigen the same two lines as described above were evident. However, there was an extra precipitin line against antisera to in vivogrown bacteria and against serum from an S. aureus-infected sheep. The results obtained from these studies support earlier evidence for the existence of fundamental differences between S. aureus grown in vitro or in vivo. The significantly faster growth rate in vivosuggests the presence of a dialysable growth factor(s) in sheep peritoneal fluid which is absent from standard bacteriological broth, or alternatively, the elimination of toxic by-products when bacteria are grown in vivo. The pore size of the dialysis tubing used was 26 A, a value which provides an indication of the maximum possible diameter of the molecule(s) in question. Differences in amino acid composition of in vitro and in vivo grown organisms are not only significant per se, but also suggested that antigenic differences may exist between staphylococci grown by these two techniques. The demonstration of an antigen in extracts from in vivogrown S. aureus which could not be detected (within the limits of sensitivity of these immunodiffusion assays) in extracts from in vitrogrown bacteria, could be of considerable immunological significance. Since this antigen was also precipitated by sera from the S. aureus-infected sheep it seems likely that it is also produced by staphylococci growing in pathogenic lesions. Antibody against this component may account for some of the differences in immune responses to live and killed S. aureus vaccines described earlier. Indeed recent studies (8) suggest that IgG antibodies to an antiphagocytic surface antigen of S. aureus (strain 7007) were capable of enhancing in vitro phagocytosis of this strain of staphylococci. Experiments are now in progress to determine whether antibodies against the antigen
547
NOTES
of in vivo grown lococci
by
S. aureus
polymorphonuclear
are
similarly
active
in
promoting
phagocytosis
of staphy-
leucocytes.
This work was financially supported by grants from the Australian Research Grants Committee and the Dairying Research Committee of Australia. We thank Ms. W. Beresford, Department of Biochemistry and Nutrition, University of New England, for carrying out the amino acid analyses. REFERENCES
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Beever, D.E., Harrison, D.G., Thomson, D.J., Cammell, S.B., and Osbourn, D.F. 1974. A method for the estimation of dietary and microbial protein in duodenal digesta of ruminants. Br. J. Nutr. 32: 99-111. Beining, P.R., and Kennedy, E.R. 1963. Characteristics of a strain of Staphylococcusaureus grown in vivoand in vitro. J. Bacteriol. 85: 732-741. Brock, J.H., and Reiter, B. 1976. Chemical and biological properties of extracellular slime produced by Staphylococcusaureusgrown in high-carbohydrate, high-salt medium. Infect. Immun. 13: 653-660. Brock, J.H., Turvey, A., and Reiter, B. 1973. virulence of two mastitis strains of Staphylococcus aureusin bovine skin: Enhancement by growth in high-carbohydrate, high-salt medium or in raw milk. Infect. Immun. 7: 865-872. Gladstone, G.P., and Glencross, E.J.G. 1960. Growth and toxin production of staphylococci in cellophane sacs in vivo.Brit. J. Exp. Path. 41: 313-333. Haukenes, G. 1967. Serological typing of Staphylococcusaureus. Acta Path. Microbiol. Scand. 70: 590-600. Hirsch, J.G., and Strauss, B. 1964. Studies on heat labile opsonin in rabbit serum. J. Immunol. 92: 145-154. Karakawa, W.W., and Kane, J.A. 1975. Immunochemistry of an acidic antigen isolated from a Staphylococcusaureus.J. Immunol. 114: 310-315. Lam, G.T., Sweeney, F. J., Witmer, C.M., and Wise, R.I. 1963. Abscess-forming factor(s) produced by Staphylococcus aureus.I. Collodion bag implantation technique. J. Bacteriol. 86: 611-615. Moore, S., Spackman, D.H., and Stein, W.H. 1958. Chromatography of amino acids on sulfonated polystyrene resins. An improved system. Anal. Chem. 30: 1185-1190. Watson, D.L. 1975a. Enhancement of in vitro phagocytosis of Staphylococcusaureus by polymorphonuclear leucocytes. Res. vet. Sci. 19: 288-292. Watson, D.L. 1975b. Cytophilic attachment of ovine IgG2 to autologous polymorphonuclear leucocytes. Aust. J. Exp. Biol. Med. Sci. 53: 527-529. Watson, D.L. 1976. The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leucocytes. Immunology 31: 159-165. Watson, D.L., and Lee, C.G. 1978. Immunity to experimental staphylococcal mastitis - Comparison of live and killed vaccines. Aust. vet. J. 54: 374-378.
