Key words: Campylobacter jejuni, Phagocytosis, Inflammatory response. Campylobacter ...... Campylobacter jejuni and Campylobacter fetus subsp. fetus. Infect.
Microbiol.
Inflammatory
and
Phagocytic
Experimental
Campylobacter
Pedro L. Pancorbol, Aurelia M. Gallego2, and Gerardo Alvarez de Cienfuegos*,1 1
Response
Immunol.,
1994
to
jejuni Infection
Manuel
38(2), 89-95,
de Pablo1, Carmen
in Mice
Alvarez1, Elena Ortega1,
Department of Microbiology, Faculty of Experimental Sciences, University of Jaen, 23071, Jaên, Spain, and of Clinic Laboratory, Hospital "Ciudad de Jaen", 23007, Jaen, Spain
2Service
Received
June
21, 1993;
in revised
form,
November
1, 1993.
Accepted
November
10, 1993
Abstract: After intraperitoneal inoculation with Campylobacter jejuni BALB/c, Swiss and DBA mice show a peritoneal inflammatory response of different intensity. Only BALB/c mice have a strong peritoneal response. Simultaneous intraperitoneal inoculation of C. jejuni plus FeCl3 increase both inflammatory response and phagocytic activity in Swiss mice, without production of diarrhea. Some thermostable compounds of C. jejuni have a very strong chemotactic activity against peritoneal cells of mice, whereas a diffusible, thermolabile and glutaraldehyde-resistant factor has an inhibitory effect over murine peritoneal cell phagocytosis. Bactericidal activity of peritoneal cells increased after in vitro re-challenge with C. jejuni. Bacteremia is present in all the mice strains tested, but the clearance is quick in DBA and slow in BALB/c and Swiss mice. These experiments confirm that in mice, peritoneal non-specific mechanisms of defense, such as macrophages, play an important role in order to control C. jejuni infection. Key words: Campylobacter jejuni, Phagocytosis, Inflammatory
response
contribute to killing C. jejuni (22), although isolates from extraintestinal places show greater serum resistance than gastrointestinal isolates (9). Kiehlbauch et al (16) performed in vitro studies showing that C. jejuni is readily internalized by mononuclear phagocytes but it survives better inside monocytes or macrophages than in control
Campylobacter jejuni is a major cause of human diarrheal disease throughout the world. Man is usually infected from unpasteurized milk, raw or partially cooked foods, contaminated water (28), and by contact with animal carriers (8). The clinical presentation of infection is variable, from watery diarrhea to bloody diarrhea. Fever, abdominal pain and vomiting may appear. However, extraintestinal infection including meningitis (15), cholecystitis (13), urinary tract infections (11) and bacteremia (26) have been reported. Invasiveness and production of toxins are two potential virulence factors (5, 18). Although there is not universally accepted small animal model, mice have been used to produce an experimental infection by several authors, both via oral (6, 7, 14, 29) and via intraperitoneal (3, 19, 24). It has been established that mice are a suitable model to study the immune mechanism to prevent the illness after inoculation. C. jejuni isolates were generally susceptible to the bactericidal activity in normal human serum by both antibody and complement. Moreover, classical and alternative pathways may
preparations without phagocytes. Banfi et al (2) in their experimental model with guinea pigs reported high rate of survival of C. jejuni phagocytosed by macrophages. Also a recent study (27) shows high survival of C. jejuni inside human neutrophils. Other authors, on the contrary, described that normal levels of serum complement are not essential for host defense against C. jejuni infection in mice, and that macrophages are an important factor in the defense against Campylobacter infection (3). The purpose of this study is to examine the cellular defense mechanism that prevents intraperitoneal C. jejuni infection in mice, such as peritoneal inflammatory response and local phagocytes activation. Abbreviations:
* Address Cienfuegos, imental
correspondence Department
Sciences,
to
Dr . Gerardo
of Microbiology,
University
of Jaen,
Alvarez
Faculty 23071,
Jaen,
HBSS,
de
of Exper-
ly; LPS,
Spain.
saline;
89
Hanks
FCS, balanced
fetal
calf
salt
solution;
lipopolysaccharide; TSA,
Trypticase-soy
serum; PBS,
agar.
GC,
Gonococcus;
i. p., intraperitonealphosphate-buffered
90
P.L.
PANCORBO
ET AL
C.
Materials
and
Methods
jejuni
experimental
peritoneal
leukocytic
jejuni
Bacterial strain. C jejuni strain 38309-87 was isolated from a Mexican patient and kindly provided by Dr. G.M. Ruiz Palacios (Instituto Nacional de Nutricion, Mexico D.F.). Mice. For experimental infection Swiss, DBA and BALB/c male mice weighing 20-25 g were used. Mice received standard food and water ad libitum . Preparation jejuni
of
was
(TSA)
grown
(Difco)
under
the
inocula.
at
42
C
in
with
System).
The
bacteria
finally
PBS
by
adjusted
units
(CFU)
The
bacterial
checked
per
10%
were
obtained
ml
at
using
108
viable
on
blood. by
and
supplemented
of
serial
dilution
Miles
and
Misra
c mice ml
groups.
were
(107
mice
in
ml
CFU)
i.p.
and
bacterial
The
PBS.
BALB/c
last
bacterial
washed
3 times pellet
The LB/c
for with was
mice
were
Ten
to
into
CFU
the
Gonococcus
was hr
and
natants
under
were
filtered
membrane,
and
inoculated
with
VI:
Control
i.p.
inoculated
in
group. with
2
800 •~
through
ml
0.1
with
the
of
viable
age.
The
g
for
of
20
washed 2
pension
at in
0.22 ƒÊm
BALB/c
that
of
total
cells
following
functional
of
infected
i.p.
mice.
infected 10%
cell
assay
status
One
tube
at
with
C
at
were
dilutions
on and
Measure of the peritoneal leukocytic response to
200 •~
of
400
percent-
calculated
of
starting
was HBSS
and
distilled to
above.
CFU hr.
of After and
C. jejuni
in
10-fold with
aid
a
the
(non-associated
hr
at
The
shaken
4
collected
plating
48
double
number
107 for
viable
for
in
supplemented
tubes by
C.
from
incubated
CO2
conditions.
viable
cells
containing
supplemented
Intraphagocytic
the of
supplemented
was
g,
analyze
peritoneal
of
5%
enumerated
in
to
HBSS
FCS
centrifuge
TSA
vigorously
scribed
more
number
made
in
1 ml
incubating
resuspended
jejuni
ml
under
microaerophilic
C.
as
The
supernatant
of
bacteria)
cytolysis.
or least
phagocytes
FCS
37 the
supernatant
were
3
The
activity of peritoneal
mice
heat-inactivated
was
at washed
at
expressed
was
heat-inactivated
jejuni
blood
sus-
stain.
in
peritoneal
BALB/c
culture
centrifugated
PBS.
with
was
dish
percentage.
The
V:
i.p.
those
Assay of the bactericidal
jejuni
each
were Giemsa
mouse
the
monolayers
incubation
with
and
was
albicans
After
cells.
per
percent-
and
counted
adherent
cells
this
is,
number
as
For
monolayers
were
The
mouse
cell
ml.
non-
subtract-
percentage.
HBSS,
stained
hr,
the
per
adherent
the
adherent
incubation,
super-
and
cells
of
supernatant. and
fixed
cells
per
1 hr,
to
Candida
yeast
2
by
cells
heat-dead
included
cells.
C.
for
of
for
phagocytic
with
were
PBS,
expressed
fresh
5 •~108
CO2
yeasts
PBS.
Millipore mice
with ml
and
the
cells After
counted.
obtained
this
viable
for
was
from
phagocytosis
received
800 •~
culture
min,
starting
and
dishes.
and
adherent
cells
test,
106
CO2
non-adherent
incubated
10%
Viable
2 •~
5%
were
with
exclusion
removed
of
cells
petri
under
cells
number
were
C
C
of
cells
blue
salt
withdrawn
(FCS).
plastic
were
number
of
of
ml
37
was
containing
adherent
assay
at
inoculated The
at
of
with
was
condition
a
DBA ml
of
suspension.
bacteria-free
Swiss,
ml
0.1
were
BALB/c of
ml
ml
(Difco).
immediately 0.1
0.1
Two
with
jejuni
broth
at
of
suspension
microaerophilic
centrifuged
ml
centrifugation
C.
(GC)
incubated
2
trypan
into
cells
ing
min g 3
the ml)
serum
then
balanced
fluid
a
mice
and
Hanks
supplemented
calf
(2
to
Briefly,
peritoneal
HBSS
distributed
adherent
of
the
fetal by
suspension
from
800 •~
fresh
counted
age
0.1
treated
The
inoculated
of
mg/
60
with
IV:
bacterial
twenty
for
in i.p.
hr.
by
glutaraldehyde-treated
24
were
consecutive
adjusted
at
resuspended i.p.
of C
were
12
PBS
(10
resuspended
suspension
glutaraldehyde
g.
100
suspension.
Swiss
with ml
inoculated
bacterial
adjusted
FeCl3
Two to
0.1
II:
centrifugation
was were
of
in
phagocytic
with
inoculated
heated
by
pellet
BALB/
infected
ml
III:
PBS
mice
heat-dead
2.5%
0.1
was
with
times.
with
immediately
suspension
and
suspension.
suspension.
washed
DBA
(i.p.)
bacterial
treated
bacterial and
of
water
of
Swiss,
intraperitoneally
were
ml)
I:
and
C.
according
Peritoneal
heat-inactivated
with
Experimental
BA
suspended
ml
of
experimental
dislocation
3
centrifuged,
measure
(23).
cervical with
(HBSS).
and
to
performed elsewhere
by i.p.
solution
37
(20).
of
killed
inoculated
calculated
retrospectively
for
method
g
methods.
TSA
Counts
the
800 •~
colony-forming-
were
count
horse
washed
turbidimetric
suspensions
by
with
PBS
at
were
number
into
was
described
incubation a
Microbi-
7.2,
technique
The
response
infection
was
blood in
pH
centrifugation
with
horse
harvested
(PBS)
C.
agar
(BBL
were
saline
with
of
condition system
phosphate-buffered times
culture
10%
atmospheric
microaerophilic
ology
pure
Trypticase-soy
supplemented
reduced
CampyPak
3
A
infection.
water phagocyte
serial
10%
horse
C
under
42 pellet and
was the
tube
hypotonic
and
phagocyte-adhered
was
determined
as
de-
C. JEJUNI Assay mice
of
bacteremia.
inoculated
suspension (30,
60,
120
150
The
samples
blood
plemented
with
conditions
for
presence
of
horse
48
hr C.
were
At
0.1
C.
jejuni
of
times
blood
were
retroorbital
sinus.
in in
42
BALB/c
of different
ml
the
blood C
TSA
sup-
microaerophilic
to
determine
the
jejuni. Differences
C. jejuni-treated analyzed
significance
and ml
cultivated
at
analysis.
in
controls
min)
were
viable
Statistical
0.1
CFU.
from
10%
DBA
with 107
and collected
tical
i.p.
containing
aseptically
results
Swiss,
via
INFECTION
and
the
Student's
by
was
between
mice
assigned
at
those
mean untreated
test.
Statis-
P •ƒ0.05.
IN MICE
91
The response of DBA mice to C. jejuni experimental infection presents a slow increase of all the cellular populations tested that was significant with regard to the peritoneal and adherent cell number in day 3 post-infection (Fig. 2). Little or no change was observed in the percentage of adherent cells (Fig. 4). The low rate of phagocytic cells found both in treated and in control DBA mice was remarkable (Fig. 5). None of these mice showed diarrhea after C. jejuni experimental infection. BALB/c mice showed an intense peritoneal inflammatory response with a great and quick increase in the number of peritoneal cells, but very
Results Peritoneal Inflammatory Response to C. jejuni Infection The i.p. experimental infection of C. jejuni in mice led to a different inflammatory response in the mouse strains tested. Swiss mice have an early and significant decrease in the total peritoneal cell number and a later but not significant increase that reaches the maximun in day 6 post-infection. In day 9 all the values return to control values (Fig. 1). No significant differences were found in the percentage of adherent and phagocytic peritoneal cells between infected and control Swiss mice (Figs. 4 and 5). In spite of the poor phagocytic response, none of the infected mice showed gastroenteritis signs, although this mouse race is used as an experimental model in the study of diarrhea caused by this bacterium (19).
Fig.
Peritoneal
inflammatory
after
i.p.
C.
inoculation.
cells
per
mouse
values.
1.
* P•ƒ
jejuni (mean 0.05.
of
five
response Values experiments).
in are
the Day
Swiss
mice
number 0:
Control
Fig. i.p.
Peritoneal jejuni
mouse
after cells values.
inflammatory
response
inoculation.
(mean
*P •ƒ0
Fig. of
2. C.
of
five
Values experiments).
are
the Day
in
DBA
number 0:
mice of
after
cells
Control
per
values.
.05.
3. i.p. per
Peritoneal C.
inflammatory
jejuni
mouse * * P•ƒ0.01.
inoculation. (mean
of
five
response Values experiments).
in are
BA the Day
LB/c
mice
number 0:
Control
of
92
P.L.
Fig.
4.
Percentage
strains
after
mice
treated
ments.
of i.p.
C.
with
Day
0:
transitory,
(Fig.
(Figs.
4
may
be
LB/c
mice
jejuni
an
percentages
of
results
the
show
in
this
delayed
very
days
of
BALB/c jejuni
the
treated
of
diarrheic
ing
quickly
with
high
day
4
Also
FeCl3
3). first
the
in
day
mice in
tion
and
the
have
even (Fig.
the
These
activates
the
i.p.
mice
with
cells
by
fourth
heat-dead
per
leukocyte
cells
7).
live are
day
C
mouse
This
bacteria
(Fig.
8);
after
and
is
inoculation between in
spite
of
hardly live
*P •ƒ
until
increase
predominant
BALB/c
jejuni of
led peritoneal
with
mice
bacteria
to
a
with progressive cells,
inflammatory
the
number Day
0
FeCI
3 of
0:
Control
after
response
cells
i.p.
C.
per
mouse
values.
jejuni
in
Swiss
**
inoculation.
mice
(mean * P •ƒ
of 0.001.
Values five * *
experiP•ƒ0.01.
.05.
reach-
counts
proportion than
Peritoneal
treated
showed
response,
peritoneal
caused
6.
infection.
ments).
(Fig.
of C.
P •ƒ0.05.
and
5).
against
infected
peritoneal
smaller
number
Swiss experi-
2
this,
arises, inocula-
9).
hyde-treated
five
a strong
(Fig.
treatment
via
phagocyte
Inoculation
in
107
that
of
have
infected
they
inflammatory
Adherent
active
being
great
resident
than
(Fig.
*
mean
to
illness.
treated
4•~
are
inflammatory
defense
and
post-inoculation
greater
the
a
values.
mouse Fe:
i.p.-infected
specially
activity
of
the
show
Values
different Swiss+
to
response
and
cells
4).
phagocytic
mice
FeCl3.
Control
in
factors.
are
symptoms
0:
cells
inoculation.
5 post-
response
significant
adherent
mechanisms
None
with
phagocytic jejuni
and
host
effective
C.
adherent
4 and
FeCl3-treated
(Fig.
that
treated Day
of i.p.
2
Fig.
peritoneal
after
mice
day
strong
some
counts
6).
of mice
increase
experi-
in
of
FeC13
cell
(Fig.
non-infected
a
cells
with
high
post-infection
Percentage
infection.
and
with
5.
strains
ments.
is
most
ET AL
Fig.
Swiss
five
values
by
the
treated
intense
of
percentage
5);
mouse Fe:
P•ƒ0.01.
There
caused
showed
mice
response
high
and
different
mean
control
the
intraperitoneal
Swiss show
3).
peritoneal
infection
C.
to
in
infection
BA
* *
back
phagocytic
are
values.
increase
in
Swiss+
Values
Control
coming
significant
cells inoculation.
FeC13.
post-infection
and
adherents
jejuni
PANCORBO
glutaraldeincrease reaching
a
maximum in day 4 (Fig. 7). This increase occurs essentially in adherent cells. There is a significant decrease in phagocytic cell rate maintained between the first and the fifth day (Fig. 9). In BALB/c mice inoculated i.p. with C. jejuni culture supernatant the inflammatory response shows a kinetics similar to that caused by live C. jejuni inoculation (Fig. 7), but with less adherent cell rate ( Fig. 8). Moreover, there is a decrease in the phagocytic cell rate, though more brief, only two days, than the one produced by glutaraldehydetreated bacteria ( Fig. 9).
C. JEJUNI
Fig.
7.
after
Peritoneal
i.p.
Values
inflammatory
inoculation are
of
the
experiments).
number Day
response
different of
0:
BALB/c
preparations
cells
Control
in
per
of
mouse
values.
INFECTION
mice C.
(mean
* * P•ƒ0.01.
jejuni. of *
five
IN MICE
Fig.
9.
i.p.
inoculation
Values **
P•ƒ
93
Percentage
are P•ƒ0
of of
mean
.01.*
of
phagocytic
cells
different five
in
BALB/c
preparations
experiments.
mice of
Day
0:
C.
after
Control
jejuni. values.
P•ƒ0.05.
0.05.
Table 1. Bactericidal activity of BALB/c mice peritoneal cells against C. jejuni
Fig. p. are **
8.
Percentage
inoculation
of
mean P•ƒ0
of
of .01.
different
five *
adherent
cells
in
preparations
experiments.
BALB/c of
Day
0:
mice
C.
jejuni.
Control
after
i.
Values values.
P•ƒ0.05.
Survival of C. jejuni in the Peritoneal Cavity None of the mice inoculated i.p. with 107CFU of C. jejuni showed viable bacteria in the peritoneal cavity 24 hr after inoculation. This fact shows the efficacy of control mechanisms against i.p. C. jejuni infection. Bactericidal Activity of Peritoneal Cells The bactericidal activity of murine peritoneal cells after a second challenge with C. jejuni is given in Table 1. Peritoneal leukocytes after a contact with the bacteria were activated towards it and were capable to kill it on a second challenge. This effect
remains
at least
infection
but
8 days has
after
disappeared
the
first in
and day
in vivo 13
post-
infection. Bacteremia
The i.p. inoculation of C. jejuni make an early bacteremic status appear in BALB/c and Swiss mice, whereas DBA mice show a relative resistance to bacteremia. The disappearance of C. jejuni in the blood occurs slowly in BALB/c mice, whereas Swiss mice give an earlier bacteremic clearance (Table 2). The simultaneous treatment with FeC13 and the i. p. C. jejuni infection decreases the bacteremic Swiss mice number. No differences occur in bacteremia between FeC13-treated and non-treated BALB/c mice.
94
P.L.
PANCORBO
Table 2. Bacteremia after i.p. C. jejuni inoculation (10 CFU) in different strains of mice
Discussion
The use of mice as an experimental model for the study of the C. jejuni infection has been proposed by several authors (6, 19, 21, 24). In the present study we have employed 3 mouse strains; two of them have been previously used in this type of investigations (BALB/c and Swiss) but the third (DBA) has not been described as an experimental model to C. jejuni infection. The results obtained show differences between mouse strains in the peritoneal response to infection. BA LB/c mice exhibit a more intense inflammatory peritoneal response, together with the greatest stimulation of the phagocytic capacity of the peritoneal cells. BALB/c mice may be a suitable model to study the role of macrophages in the control of C. jejuni infection. Moreover, BALB/c mice have the advantage of being genetically well known. None of the mice tested showed either symptoms of illness or diarrhea after i.p. inoculation of C. jejuni. This proves the resistance of adult mice to this infection (6, 19, 29). Although Fe3+ is considered as a stimulating factor of bacterial infective capacity (1, 10), Swiss mice treated with FeC13 and simultaneously inoculated with C. jejuni showed no diarrhea, contrary to the reports of McCardell et al (19) in Swiss mice and Stanfield et al (24) in BA LB/c mice, with infective doses of 105 to 1010 CFU of C. jejuni using the same via of inoculation. The smaller infective dose and the absence of virulence factors in our bacterial strain may be the cause of these differences. In our model treatment with FeCl3 produced an increase in the peritoneal inflammatory response and in the activity of peritoneal phagocytes that may explain the control of the infection in the peritoneal cavity. The high rate of peritoneal adherent cells, macrophages and polymorphonuclears found in BA LB/c mice inoculated with heat-dead C. jejuni shows a chemotactic effect on the phagocytic cells
ET AL
by some thermostable bacterial compound. This compound may be the lipopolysaccharide (LPS) used in the thermostable antigen typing scheme (12). This chemotactic effect was described by Ullmann and Krause (25) both in a suspension of living bacteria and culture supernatants. These authors proposed enterotoxin as the chemotactic factor of C. jejuni, but enterotoxin is thermolabile (17) and our data do not support the possibility that enterotoxin is the chemotactic factor. Moreover, the smaller peritoneal inflammatory response induced by living bacteria shows the existence of compounds in the bacterial surface with capacity to inhibit this response which would otherwise become inactivated by heat treatment, thus allowing for thermostable compounds (LPS) to exert their potent chemotactic effect. The inoculation of glutaraldehyde-treated bacteria gave a more delayed and less intense peritoneal inflammatory response, suggesting the lack of chemotactic factors. Moreover, the early decrease in the rate of phagocytic cells can be due to the existence of a bacterial phagocytosis inhibitory factor just as has been reported by some authors (2). Supernatants of bacterial culture produced also the decrease of the rate of phagocytes but more transitory, perhaps due to dilution of the factor in the medium. According to our results C. jejuni may present a diffusible, thermolabile and glutaraldehyde-resistant factor inhibitory of the phagocytic activity. The resistance of mice against C. jejuni infection reported by several authors (6, 19) may be due to the existence of non-specific mechanisms of defense. The role that polymorphonuclears and macrophages play in defense against C. jejuni infection has been reported in both in vitro (2, 16, 27) and in vivo (3) assays. Our results show the important role of phagocytes in the infection of C. jejuni in mice. Twenty-four hr after the inoculation there are no viable bacteria in the peritoneal cavity. Moreover, the phagocytic activity increased after a second challenge, but the phenomenon of in vitro intracellular bacterial survival reporter by Kiehlbauch et al (16) for non-activated murine macrophages was not detected. The presence of bacteremia in mice after the infection with C. jejuni has been reported both via i.p. (7, 24) and oral (6). Our results in the bacteremic assay were similar to those described by Stanfield et al (24), but in our assay there are no differences between FeC13-treated and non-treated BALB/c mice. The fact that treatment with FeC13
C. JEJUNI
INFECTION
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