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Infection and Immunity, June 1999, p. 2891-2900, Vol. 67, No. 6
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of a Murine Model of Melioidosis:
Comparison of Different Strains of Mice
I.
Hoppe,1
B.
Brenneke,1
M.
Rohde,2
A.
Kreft,3
S.
Häußler,1
A.
Reganzerowski,1 and
I.
Steinmetz1,*
Institute of Medical
Microbiology1 and Institute of
Pathology,3 Hannover Medical School, 30625 Hannover, and Division of Microbiology, National Research
Centre for Biotechnology, 38124 Braunschweig,2
Germany
Received 9 March 1998/Returned for modification 11 May
1998/Accepted 17 March 1999
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ABSTRACT |
Melioidosis is an infectious disease caused by the saprophytic
gram-negative rod Burkholderia pseudomallei. The aim of
this study was to establish and characterize a murine model of
melioidosis to provide a basis for further investigations on the
pathogenesis of the disease. After intravenous infection with B. pseudomallei, C57BL/6 mice were found to be significantly more
resistant than BALB/c mice. There was a marked organotropism of
B. pseudomallei for the spleen and liver in both strains of
mice, with the highest bacterial load in the spleen. Electron
microscopic investigations of the spleen clearly demonstrated
intracellular replication within membrane-bound phagosomes. Electron
micrographs of the liver provided evidence that B. pseudomallei-containing phagosomes in hepatocytes fuse with
lysosomes, leading to degradation of bacteria. In both strains of mice,
the course of infection was highly dependent on the infective dose and
the bacterial strain used, ranging from death within a few days to
death after several weeks. In comparison with BALB/c mice, the
bacterial counts in C57BL/6 mice were decreased 12 h after
infection, which is suggestive of an innate immune mechanism against
B. pseudomallei in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice. BALB/c mice
developed a more pronounced lymphopenia, granulocytosis, and
splenomegaly at a lower infective dose compared to C57BL/6 mice.
Analysis of the antibody response against B. pseudomallei 11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to
infection with B. pseudomallei.
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INTRODUCTION |
Melioidosis is an infectious disease
of humans and animals caused by the saprophytic gram-negative rod
Burkholderia pseudomallei. Southeast Asia and northern
Australia are main areas where the disease is endemic, the bacterium
being isolated from soil and surface waters (17, 29). There
is evidence that melioidosis may also be endemic in Africa, the Indian
subcontinent, and Central and South America (5), but
the disease remains most likely underdiagnosed in areas of the
tropics where sophisticated laboratory facilities are not available
(5). The clinical manifestations of melioidosis are
extremely variable, ranging from acute or chronic localized infections
to fulminant septicemias (6). Severe septicemic melioidosis is typically associated with underlying diseases such as diabetes mellitus and chronic renal failure (4,
17), but it may also occur in previously healthy individuals.
Epidemiological studies suggest that mild or inapparent infections
which are manifested by seroconversion only are common (26).
The proportion of infected but healthy individuals who may harbor
viable B. pseudomallei is unknown. Long
periods of latency and frequent relapses after antibiotic treatment are
characteristic of melioidosis (6). In most cases, humans and
animals are thought to acquire the infection by inoculation of
environmental organisms into minor cuts or abrasions after contact with
soil and muddy waters (7).
B. pseudomallei has an extremely broad host
range. Beside humans, infections in rodents, sheep, goats, pigs,
horses, and kangaroos have been reported (9, 11, 18, 32). In
vitro studies have shown that B. pseudomallei can invade epithelial cells and grow
intracellularly within phagocytes (16, 24). It has recently been shown that the type II O-antigenic polysaccharide moiety of
the B. pseudomallei lipopolysaccharide
(22) is essential for resistance against
complement-mediated bacteriolysis and contributes to virulence in
hamsters, guinea pigs, and infant diabetic rats (8a).
Other putative virulence factors of B. pseudomallei include a siderophore (38),
several relatively uncharacterized extracellular enzymes
(1), and a heat-labile exotoxin (15). Recently,
we identified a constitutively expressed exopolysaccharide (21, 31) and a heat-stable cytotoxic glycolipid (14), which
might have implications for the pathogenesis of melioidosis.
For many bacterial infections, the mouse model has proven to be
invaluable for studies of bacterial virulence factors and host-parasite
interactions. The aim of this study was to establish and characterize a
murine model of melioidosis for further investigations on the
pathogenesis of the disease. We examined the susceptibility of four
mouse inbred strains after intravenous infection. The highly
susceptible BALB/c strain and the more resistant C57BL/6 strain were
chosen to determine the kinetics of the bacterial colony counts in
various organs over a period of several weeks and to perform electron
microscopic studies to locate B. pseudomallei during infection. Changes in blood cell
composition and the induction of a humoral immune response during
infection were compared in both mouse strains.
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MATERIALS AND METHODS |
Animals.
Female 8- to 10-week-old C57BL/6, BALB/c, C3H/HeN,
and DBA/2 mice were obtained from Charles River Wiga (Sulzfeld,
Germany). Animals were maintained under specific-pathogen-free
conditions and were provided with food and water ad libitum.
Bacteria.
The clinical
L-arabinose-nonassimilating B. pseudomallei NCTC 7431 was obtained from the National
Collection of Type Cultures. B. pseudomallei E8 is an
L-arabinose-nonassimilating environmental strain isolated
from soil in northeast Thailand (34) and was obtained from
A. Simpson, Faculty of Tropical Medicine, Mahidol University, Bangkok,
Thailand. Bacteria were grown on Columbia agar at 37°C for 24 h.
Colonies were then harvested using tryptic soy broth, centrifuged at
10,000 × g, suspended in tryptic soy broth containing
20% glycerol, and frozen immediately in 0.2-ml aliquots at a
concentration of 109 CFU per ml at 
70°C.
Infection of animals and enumeration of CFU in various
organs.
For each infection experiment, an aliquot of the
B. pseudomallei suspension was freshly
thawed from the stock. Bacterial cells were diluted in buffer A (0.01 M
potassium phosphate buffer made isotonic with saline [pH 7.5]) to
obtain the appropriate concentration. By this method, commonly used in
other infection models, excellent reproducibility of the B. pseudomallei infection doses was obtained. A bacterial
suspension of 0.2 ml was injected into the lateral tail vein. The
actual number of bacteria administered was determined for each
experiment by plating 0.1 ml of serial 1:10 dilutions on Columbia agar
and counting CFU after 48 h. At various time points after
injection, the number of CFU present in blood and various organs was
determined. Organs were aseptically removed and homogenized in 0.4 or 1 ml (depending on organ size) of sterile buffer A containing 1%
(wt/vol) Tergitol TMN 10 (Fluka, Buchs, Switzerland) and 0.5% (wt/vol)
BSA (bovine serum albumin; Merck, Darmstadt, Germany) in a tissue
homogenizer (glass potter) (Braun, Melsungen, Germany). A 0.1-ml sample
of appropriate 1:10 dilutions was plated out, and the number of CFU was
determined as described above. Using this method, the detection limit
was 3 to 10 CFU per organ, depending on the size of the organ. For the
determination of blood CFU, an undiluted 0.1 ml sample was plated out
and the number of CFU was determined as described above.
Determination of the LD50.
Groups of six mice
were infected with appropriate dilutions of bacteria as described above
and observed for death for 6 weeks. The 50% lethal dose
(LD50) was calculated by the method of Reed and Muench
(25).
Blood cell parameters.
For differential blood counts,
animals were bled through the orbital plexus, and smears were made,
fixed in 100% methanol, and stained with Wright's and Giemsa stains
(Sigma). For leukocyte counts, blood was analyzed on a Coulter S Plus
IV (Coulter Electronics, Krefeld, Germany).
ELISA methods.
The various antibody isotypes with
specificity for B. pseudomallei antigens
from mouse sera were detected in a previously described enzyme-linked
immunosorbent assay (ELISA) (31), with some modifications.
Briefly, single U-shaped wells of nonflexible polystyrol microtiter
plates were coated for 2 h with 20 µl of B. pseudomallei cells (2 × 108 cells
per ml) which had been heat treated (80°C for 1 h in buffer A)
and sonicated with the microtip of a Branson Sonifier 250 (10 min;
output setting of 9 and 50% duty cycle). After washing steps with
buffer A and a blocking step with buffer A-BSA (buffer A containing 1%
[wt/vol] BSA) for 30 min, mouse sera and a standard were diluted in
buffer A-BSA and 20 µl was incubated for 2 h. For the
determination of the different isotypes, plates were washed and
incubated with 10 µl of either biotin-labeled rabbit anti-mouse immunoglobulin G1 (IgG1; 1:5,000 in buffer A-BSA; Zymed, San Francisco, Calif.), rabbit anti-mouse IgG2a (1:1,000; Southern Biotechnology Associates, Birmingham, Ala.), rabbit anti-mouse IgG2b (1:750; Zymed),
rabbit anti-mouse IgG3 (1:1,000; Zymed), or goat anti-mouse IgM
(1:1,000; Southern Biotechnology Associates) for 60 min. Microtiter plates were then developed with streptavidin coupled to
-galactosidase. 4-Methylumbelliferyl--D-galactopyranoside was used as
substrate, and the fluorescent product was measured as relative
fluorescence units (RFU). A high-titered mouse serum with detectable
antibodies of all isotypes tested in this ELISA was used as a standard.
For each isotype assay, a standard curve was generated from appropriate dilutions of this serum. The RFU value of the highest antibody concentration falling within the linear portion of the different standard curves was arbitrarily set as 10 ELISA units. The specific isotype concentration of each serum sample was determined from the
standard regression curve constructed for each assay and expressed as
ELISA units.
Electron microscopic studies.
At different time points after
infection with B. pseudomallei, C57BL/6 and
BALB/c mice were ether anesthetized and 100 µl of buffer A containing
3% glutaraldehyde and 5% formaldehyde were injected intravenously
into the lateral tail vein. Animals were killed after injection, and
the spleen and liver were removed. Organs were cut into small cubes and
kept in the same fixation buffer used for injection overnight at 4°C.
Tissue cubes were further fixed with 1% aqueous osmium tetroxide for
1 h at room temperature, washed with phosphate-buffered saline,
and dehydrated with a graded series of acetone. Samples were
infiltrated with Spurr's epoxy resin by the method of Spurr
(30). Ultrathin sections were cut with glass knives and
counterstained with uranyl acetate and lead citrate. Sections were
examined in a Zeiss TEM910 transmission electron microscope at an
acceleration voltage of 80 kV and at calibrated magnifications.
Statistical analyses.
The statistical significance of the
difference of the mean between experimental groups was determined
by the two-tailed Student t test. P
values of <0.05 were considered significant. Data are presented as
means ± standard deviations (SD).
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RESULTS |
Susceptibility of different mice strains to intravenous infection
with B. pseudomallei.
To examine the role
of the host in B. pseudomallei infection,
the well-characterized inbred mouse strains C57BL/6
(H-2b haplotype, Bcg/Ity/Lshs
phenotype), C3H/HeN (H-2k;
Bcg/Ity/Lshr), DBA/2 (H-2d;
Bcg/Ity/Lshr), and BALB/c
(H-2d; Bcg/Ity/Lshs) were infected
by the intravenous route with B. pseudomallei NCTC 7431 (originally a clinical
isolate), and LD50 values were determined after 6 weeks.
C57BL/6 mice showed the most resistant phenotype
(LD50 = 105) and were significantly more
resistent compared to the most susceptible BALB/c mice
(LD50 = 103). The LD50s of
C3H/HeN and DBA/2 mice were 6 × 103 and 3 × 103, respectively. From the LD50
experiments, it is obvious that the Nramp1 gene
(36), which is located at the Ity/Bcg/Lsh locus on mouse chromosome 1, does not confer resistance to B. pseudomallei infection. Both B. pseudomallei-resistant C57BL/6 mice and susceptible BALB/c mice are Itys.
B. pseudomallei infection in various
organs of BALB/c and C57BL/6 mice.
To address the question of
whether the differences in susceptibility of different strains of mice
correspond to bacterial burden and to examine possible tissue
specificity, the kinetic of the bacillary load in various organs of
C57BL/6 and BALB/c mice was determined. Both strains of mice were
intravenously infected with different doses of B. pseudomallei NCTC 7431, and the numbers of bacteria in
spleen, liver, lung, kidneys, and blood were determined at different
time points after infection. Figure 1A
shows bacterial counts in spleens and livers of the resistant and
susceptible strains after infection with 2 × 102 CFU.
Only very few bacteria were detected in the lungs and kidneys of BALB/c
mice (data not shown). No bacteria were detected in the lungs and
kidneys of C57BL/6 mice at any time point. Bacteria were not detectable
in the blood in both strains of mice. In the spleen and liver, but
especially in the spleen, the number of CFU at different time points
was significantly higher in BALB/c mice than in C57BL/6 mice.
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FIG. 1.
(A) Course of bacterial counts in BALB/c ( ) and
C57BL/6 ( ) mice in spleen and liver after intravenous infection with
2 × 102 CFU of B. pseudomallei NCTC 7431. Values are given as the
mean ± SD for six mice. Asterisks indicate significant
(P < 0.05) increase or decrease in CFU of the various
organs (*, day 0.5 versus day 1; **, day 1 versus day 12;
***, day 12 versus day 16). Crosses indicate significant
(P < 0.05) differences in organ CFU between BALB/c and
C57BL/6 mice at single time points. (B) Course of bacterial counts in
BALB/c () and C57BL/6 () mice in spleen and liver after
intravenous infection with 5 × 103 CFU of
B. pseudomallei NCTC 7431. In BALB/c mice,
organ CFU were not documented beyond day 12 p.i. because of
progressive death of animals. Values are given as the mean ± SD
for six mice. Asterisks indicate significant (P < 0.05) increase or decrease in CFU of the various organs (*, day
0.5 versus day 1; **, day 1 versus day 8; ***, day 8 versus
day 10). Crosses indicate significant (P < 0.05)
differences in organ CFU between BALB/c and C57BL/6 mice at single time
points. (C) Bacterial counts in BALB/c () and C57BL/6 () mice in
spleen and liver after intravenous infection with 2 × 102 CFU of mouse-passaged B. pseudomallei NCTC 7431. BALB/c mice started to die
beyond day 4 p.i.; C57BL/6 mice started to die beyond day 6. Values are given as the mean ± SD for four to six mice. Asterisks
indicate significant (P < 0.05) differences from the
respective values obtained with the nonpassaged strain NCTC 7431 at the
same time points as shown in panel A. Crosses indicate significant
(P < 0.05) differences in organ CFU between BALB/c and
C57BL/6 mice.
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A steep increase in the bacterial load of the spleen of BALB/c mice was
detected in the first 24 h postinfection (p.i.). Interestingly, the bacterial burden in the spleens and livers of C57BL/6 mice was
lower than in BALB/c mice during this early phase of infection, indicating an innate immune mechanism against B. pseudomallei contributing to the lower susceptibility
of C57BL/6 mice. The course of the bacterial load in BALB/c mice was
indicative of a biphasic pattern with a second increase of CFU around
day 16, whereas C57BL/6 mice completely cleared the bacteria within the first few days.
After infection with 5 × 10
3 CFU of strain NCTC 7431, there was also a marked organotropism for the spleen and liver in both
strains of mice (Fig.
1B), whereas again no significant bacterial
numbers were detected in the lungs and kidneys of most animals
(data
not shown). It was only in BALB/c mice that very few bacteria
(1 to 6 CFU per 0.1 ml) were detectable in the blood in some animals
during the
first 2 days p.i. At other time points no bacteria
could be detected in
the blood in both strains of mice. Figure
1B clearly shows a biphasic
course of the bacterial load in spleens
and livers in both strains. In
C57BL/6, there was a peak at day
1 and no bacteria were detected
between days 6 and 8. The second
peak was reached at day 10, and in the
following days the bacteria
were finally cleared. In BALB/c mice, the
first peak was also
reached at day 1 and again a significant reduction
was seen between
days 6 and day 8. In contrast to C57BL/6 mice, BALB/c
mice did
not clear the bacteria after the second increase and succumbed
to infection. There was a significant difference between the two
mouse
strains in the bacterial load of the spleen and liver in
the first
12 h p.i. After infection with 5 × 10
3 CFU, 3 of
12 BALB/c mice developed paresis of both hind legs
at days 10 and
12 p.i. Interestingly, these neurological signs
were also reported
for infected sheep and goats (
33).
To address the question of whether the biphasic course of infection is
a constitutive characteristic of strain NCTC 7431,
we isolated strain
NCTC 7431 from the spleen of a BALB/c mouse
during the second peak at
day 10 and infected BALB/c and C57BL/6
mice with the mouse-passaged
isolate. Interestingly, in both strains
of mice we observed a dramatic
increase in virulence as well as
a loss of the biphasic pattern (Fig.
1C). Infection of BALB/c
mice with 2 × 10
2 CFU of
strain NCTC 7431 after single mouse passage led to significantly
higher
bacterial numbers in the spleen and liver after 24 h compared
to
the same dose of the nonpassaged strain (Fig.
1A), and animals
succumbed to infection between days 4 and 5. Infection of C57BL/6
mice
with 2 × 10
2 CFU of passaged strain NCTC 7431 led to
very high bacterial numbers
at day 6, and animals died within the
next 2 days. Although the
course of infection changed dramatically
using the mouse-passaged
strain, the previously observed
difference in bacterial numbers
between C57BL/6 and BALB/c mice with
the nonpassaged strain was
also detectable in the first 24 h p.i.
with the much more virulent
passaged strain. Figure
2 summarizes the survival curves of
BALB/c
and C57BL/6 mice after infection with different infection doses
of the nonpassaged and passaged strain NCTC 7431.
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FIG. 2.
Survival curves of BALB/c (white symbols) and C57BL/6
mice (black symbols) after intravenous infection with nonpassaged and
passaged B. pseudomallei NCTC 7431. ,
5 × 103 CFU of NCTC 7431;
 , 3 × 105 CFU of NCTC 7431;  , 5 × 103 CFU
of NCTC 7431;  , 3 × 105 CFU of NCTC 7431;  ,
2 × 102 CFU of mouse-passaged strain NCTC 7431;  ,
2 × 102 CFU of mouse-passaged strain NCTC 7431.
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Since infections with
B. pseudomallei are
acquired from the environment and mammalian reservoirs are
epidemiologically not
important (
7), we used a
B. pseudomallei soil isolate (E8)
to further elucidate
the course of
B. pseudomallei infection in
our model. There was also a marked organotropism for the spleen
and
liver, with very few bacteria in the lung and kidney (data
not shown).
Figure
3 shows the results for the spleen
and liver
after infection of BALB/c and C57BL/6 mice with 5 × 10
3 CFU. In BALB/c mice, the bacterial load of the spleen
and also
the liver was significantly higher from 24 h p.i. onward
compared
to nonpassaged strain NCTC 7431 (Fig.
1B). A biphasic course
of
bacterial numbers was not observed, and animals succumbed to
infection
between days 12 and 17 p.i. There was no significant
difference
in the early course of infection in C57BL/6 mice compared to
the
course of infection of the nonpassaged strain NCTC 7431 (Fig.
1B).
No bacteria were recovered from organs even after more than
1 month
p.i. The previously observed difference in bacterial load
between
C57BL/6 and BALB/c mice detectable at very early time
points (12 h
p.i.) was also evident with strain E8. No bacteria
were cultured from
blood samples from both strains of mice. Paresis
of both hind legs was
observed in approximately 25% of BALB/c
mice. The high virulence of
nonpassaged strain E8 became also
evident when four of six BALB/c mice
died within 6 weeks after
infection with only 10 CFU of strain E8.
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FIG. 3.
Course of bacterial counts in BALB/c () and C57BL/6
() mice in spleen and liver after intravenous infection with 5 × 103 CFU of B. pseudomallei
E8. In BALB/c mice, organ CFU were not documented beyond day 10 p.i. because of progressive death of animals. Values are given as the
mean ± SD for five to six mice. Asterisks indicate significantly
higher (P < 0.05) CFU values compared to the
respective values for nonpassaged strain NCTC 7431 at the same time
points as in Fig. 1B. Crosses indicate significant (P < 0.05) differences in organ CFU between BALB/c and C57BL/6 mice
at single time points.
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The influence of mouse passage on the virulence of strain E8 was
examined in BALB/c and C57BL/6 mice infected with 2 × 10
2 CFU of E8. In contrast to strain NCTC 7431, there was
no significant
increase in bacterial numbers after mouse passage (data
not shown).
BALB/c mice succumbed to infection in a time range (weeks 2 to
3 p.i.) similar to that for the nonpassaged strain. In C57BL/6
mice, sporadic cases of high bacterial counts (10
5 to
10
7 CFU) were detected in the spleen at day 8, whereas most
animals
showed no detectable bacteria. In one experiment, C57BL/6 mice
were infected with 2 × 10
2 CFU of double-passaged
strain E8 and the observation period was
extended to 3 months. Two of
six animals died at days 40 and 54
p.i., respectively. These
animals showed a massive splenomegaly
with macroscopically visible
massive abscesses and bacterial numbers
in the range of 10
7
to 10
8 CFU per
spleen.
Determination of the spleen weight after infection with 5 × 10
3 CFU of strain NCTC 7431 (Fig.
4A) or strain E8 (Fig.
4B) revealed
the
development of a marked splenomegaly in BALB/c mice. However,
after
infection with 5 × 10
3 CFU of strain E8, the increase
in spleen weight was significantly
higher than after infection with
nonpassaged NCTC 7431. After
infection with strain NCTC 7431, C57BL/6
mice exhibited a steeper
increase in spleen weight in the first 3 days
compared to BALB/c
mice. From day 4 on, the spleen weight of C57BL/6
mice decreased
and remained only slightly elevated. When C57BL/6 mice
were infected
with 10
6 CFU of strain 7431 or with 2 × 10
2 CFU of mouse-passaged strain NCTC 7431, a significant
splenomegaly
developed (data not shown). No changes in spleen weight
were noticed
in C57BL/6 mice after infection with 5 × 10
3 CFU of E8 during the observation period. No significant
changes
in the weight of other organs were observed in the course of
infection
with either
B. pseudomallei
strain in both strains of mice.
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FIG. 4.
Development of spleen weight after intravenous infection
with 5 × 103 CFU of B. pseudomallei NCTC 7431 (A) and B. pseudomallei E8 (B) in BALB/c () and C57BL/6 ()
mice. Values are given as the mean ± SD for five to six mice.
Asterisks indicate significant (P < 0.05) differences
between the two strains of mice.
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Hematoxylin-and-eosin-stained sections of the livers and spleens of
BALB/c mice were investigated by light microscopy at different
time
points (data not shown). The formation of small abscesses
was detected
12 h p.i. with strain E8 (5 × 10
3 CFU) in both
organs. At day 3 p.i. (10
2 CFU), both organs showed
the formation of small granulomas in
addition to the presence of
microabscesses. Both type of lesions
were most prominent at day 10 p.i. (10
2 CFU). In some cases, large splenic abscesses
replaced most of
the spleen. The formation of abscesses and granulomas
in spleen
and liver was also observed in C57BL/6 mice and correlated
with
the bacterial burden determined at the different time
points.
Electron microscopic investigations of lesions of the spleen of
C57BL/6 mice 6 days after infection with 2 × 10
2 CFU
of mouse-passaged strain NCTC 7431 (Fig.
5) consistently
showed the presence
of large numbers of intact bacterial cells,
which in many cases
appeared densely packed within a membrane.
Intracellular replication of
B. pseudomallei within phagosomes
could be demonstrated. No bacteria were detected in
blood-containing
sinuses. Electron microscopic analysis of the liver
(Fig.
6) revealed
invasion of
B. pseudomallei into hepatocytes adjacent
to the sinus.
There was evidence for a phagosome-lysosome fusion
process leading
to degradation of
B. pseudomallei. Similar to what we found in
the spleen,
no bacteria were detected in sinuses of the liver.
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FIG. 5.
Electron microscopic investigation of spleens of C57BL/6
mice at day 6 after infection with 2 × 102 CFU of
mouse-passaged strain B. pseudomallei NCTC
7431. Ultrathin sections through lesions of the spleen are shown.
Within these lesions, we consistently detected the accumulation of
B. pseudomallei (A). (B) Higher
magnification of panel A showing densely packed bacteria. (C) Numerous
intracellularly dividing bacteria surrounded by phagosomal membranes
(arrows). R, erythrocyte; Bars represent 5 µm in panel A, 1 µm in
panel B and 0.5 µm in panel C.
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FIG. 6.
Electron microscopic investigation of livers of C57BL/6
mice at day 6 after infection with 2 × 102 CFU of
mouse-passaged B. pseudomallei NCTC 7431. Ultrathin sections through liver hepatocytes are shown. (A) Hepatocytes
showing several phagosomes with degraded bacteria (arrows). In these
hepatocytes, we found evidence for a phagosome-lysosome fusion process
resulting in the degradation of B. pseudomallei (B and C). (B) Earlier stage. (C) Content
of a lysosome floating in a phagosome, resulting in the degradation of
B. pseudomallei. L; lysosome, N; nucleus,
R; erythrocyte. Bars represent 2 µm in panel A and 0.5 µm in panels
B and C.
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Blood cell parameters during B. pseudomallei infection.
To determine changes in
leukocyte count and composition during infection of C57BL/6 and BALB/c
mice, blood was analyzed at various time intervals p.i. with either
strain NCTC 7431 or strain E8. The results of the differential blood
film after infection of BALB/c mice with 5 × 103 CFU
of NCTC 7431 (Fig. 7A) revealed an
initial rise in granulocytes during the first day and a subsequent
slight decrease followed by a continuous increase of granulocytes until
day 12. There was an initial sharp decline in lymphocytes, followed by
a transient normalization and finally development of a lymphopenia. The
results after infection of BALB/c mice with 5 × 103
CFU of E8 (Fig. 8A) revealed a similar
pattern, although the most significant difference was a prominent
lymphocytosis until 12 h p.i. and an earlier rise in granulocytes.
Interestingly, the sharp decline in lymphocytes at 24 h p.i. was
also followed by a transient normalization and finally by the
development of a lymphopenia as observed after infection with strain
NCTC 7431. The normalization of lymphocytes was not accompanied by a
decrease in bacterial numbers of strain E8.
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FIG. 7.
Changes in blood cell parameters after intravenous
infection with 5 × 103 CFU of B. pseudomallei NCTC 7431 in BALB/c (A) and C57BL/6 (B)
mice.  , granulocytes; , lymphocytes; , monocytes. Values are
given as the mean ± SD for six mice. Asterisks indicate
significant (P < 0.05) differences of the various cell
types compared to normal values.
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FIG. 8.
Changes in blood cell parameters after intravenous
infection with 5 × 103 CFU of B. pseudomallei E8 in BALB/c (A) and C57BL/6 (B) mice.
, granulocytes; , lymphocytes;  , monocytes. Values are given
as the mean ± SD for five to six mice. Asterisks indicate
significant (P < 0.05) differences of the various cell
types compared to normal values.
|
|
No granulocytosis developed after infection with strain
NCTC 7431 in C57BL/6 mice. However, at days 1, 10, and 16, a less
pronounced but significant lyphopenia developed (Fig.
7B). When
C57BL/6
mice were challenged with 10
6 CFU of strain NCTC 7431, there was also a dramatic lymphopenia
in the early phase followed by a
transient normalization and finally
the development of a lymphopenia
together with a granulocytosis
(data not shown). After infection of
C57BL/6 mice with 5 × 10
3 CFU of strain E8 (Fig.
8B),
we observed as in BALB/c mice an
initial rise of lymphocytes but no
subsequent lymphopenia. There
was only a slight lymphocytosis and
granulocytosis at different
time points p.i.
Kinetics of antibody response during infection against
B. pseudomallei.
B.
pseudomallei-specific IgM and IgG antibodies were
determined in an ELISA using B. pseudomallei sonicate as an antigen. Sera of C57BL/6
and BALB/c mice infected with strain E8 (2 × 102 CFU) were analyzed at day 11 p.i. Figure
9 shows that C57BL/6 mice mount a
significantly higher IgG1, IgG2a, and IgG3 antibody response. There was
no significant difference for the IgM response between the two strains
of mice. A striking finding was the fact that almost no specific IgG2a
was detected in BALB/c mice. The IgG2a/IgG1 ratio calculated from the
ELISA units of single animals was significantly higher in C57BL/6 mice
at day 11 p.i. than in BALB/c mice (1.7 for C57BL/6 and 0.03 for
BALB/c mice; P < 0.001), indicating a stronger T
helper type 1 (Th1) subset activity in C57BL/6 mice during infection.
In sera of BALB/c and C57BL/6 mice tested before infection, no
significant differences in isotype pattern were observed.
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|
FIG. 9.
Analysis of IgG and IgM responses against B. pseudomallei antigens of BALB/c (open bars) and
C57BL/6 (solid bars) mice at day 11 after intravenous infection with
2 × 102 CFU of B. pseudomallei E8. Values are given as the mean ± SD for five to six mice. Asterisks indicate significant (*,
P < 0.05; **, P < 0.001) differences between the
two strains of mice.
|
|
| |
DISCUSSION |
At present, there are very few studies describing the pathogenesis
of melioidosis. It was demonstrated some decades ago that various
laboratory animals can be infected with B. pseudomallei, and LD50 values were
determined. The hamster proved to be the most susceptible animal
(18). Guinea pigs were found to be moderately susceptible
with a high degree of variation, whereas rabbits, rats, and monkeys
were found to be relatively resistant (18). Recently,
experimental infection in rats made diabetic by the injection of
streptozocin was reported (37). The determination of
LD50s revealed that animals treated this way were much more susceptible to infection than controls. It was reported some time ago
that mice can be infected with B. pseudomallei (19) and recently
determination of LD50s after intraperitoneal infection of
mice was used to differentiate virulent B. pseudomallei from a nonvirulent
L-arabinose-assimilating biotype (28). It was found in a small study that outbred mice were more susceptible to
intraperitoneal infection after administration of gamma interferon neutralizing antibodies (27). However, only mortality rates were recorded, and neither the kinetic of the infection nor the bacterial load in various organs or any immunological parameters were
determined (27).
The comparison of the susceptibility of different genetically defined
inbred mouse strains to the intravenous route of infection in this
study provided the first step in the characterization of a murine model
of melioidosis. The intravenous route of infection was chosen to mimic
systemic melioidosis, which is the most common clinical presentation in
Thailand (4). The observation that C57BL/6 mice were much
more resistant to infection than BALB/c mice demonstrates that
susceptibility to B. pseudomallei infection is not linked to the Ity locus, which has been shown
to determine the susceptibility of mice to pathogens such as
Leishmania donovani, Salmonella typhimurium,
and Mycobacterium bovis (36), since both C57BL/6
and BALB/c mice are Itys. Resistance to
gram-negative bacterial infections is not an inherent characteristic of
C57BL/6 mice compared to BALB/c mice. For example C57BL/6 mice exhibit
more severe inflammation after Helicobacter felis infection
(19) and are more susceptible to Pseudomonas aeruginosa infection (20) than BALB/c mice.
Interestingly, the susceptibility pattern of BALB/c and C57BL/6 mice
determined in this study has also been observed after intravenous
infection of mice with the gram-negative bacterium Yersinia
enterocolitica (13). However, there are several
features of the B. pseudomallei infection
in BALB/c and C57BL/6 mice which differ clearly from those observed in
the yersiniosis model. The organotropism of B. pseudomallei for spleen and liver, especially marked
in BALB/c mice in this study, is less pronounced in systemic infection
with Y. enterocolitica (2), where BALB/c mice
exhibit high bacterial numbers in the lung in addition to spleen and
liver. Interestingly, the resistant and susceptible phenotype in
B. pseudomallei infection is expressed
12 h p.i., indicating that some innate resistance mechanism(s)
against B. pseudomallei must be present at
this early time point. This is in contrast to the literature on the
murine Yersinia model, where differences in the bacterial
load of various organs between BALB/c and C57BL/6 are found only 4 days
p.i. and are attributed to specific T-cell responses (2,
13).
An interesting observation in this study is the fact that strain NCTC
7431 no longer exhibited a biphasic course of infection after one mouse
passage but had a significantly increased rate of replication and led
to death of animals within a few days. It seems likely that the highly
virulent organisms selected during the first passage were now capable
to resist the primary attack of host defense mechanisms. In contrast,
the environmental strain E8, which did not show a biphasic course of
infection, was not significantly more virulent after mouse passages.
The development of histopathological lesions in the spleen and liver,
most prevalent in BALB/c mice, consisting of abscesses together with
granulomatous lesions, is in good accordance with lesions found in
human melioidosis (3, 23, 35). Future studies will need to
elucidate the cellular basis of the observation that BALB/c mice
develop a pronounced splenomegaly compared to C57BL/6. Splenomegaly is
also observed in acute systemic melioidosis of humans (12).
This study provides in vivo evidence that B. pseudomallei is capable of intracellular replication
within phagosomes. This observation is in good accordance with previous
in vitro studies demonstrating intracellular replication in phagocytes (16, 24). The determination of viable bacteria during the course of infection in all experiments revealed a higher capacity of
the liver to restrict growth of B. pseudomallei compared to the spleen. The finding that
hepatocytes are capable to kill B. pseudomallei in phagosomes by fusion with lysosomes
describes a mechanism for how the liver can restrict growth of
B. pseudomallei. Future studies will have
to elucidate the distribution of B. pseudomallei between hepatocytes and Kupffer cells
during infection and their roles in host defense.
In the mouse, IgG2a isotype switching is promoted by gamma interferon,
a Th1 cell-derived cytokine, whereas IgG1 isotype switching is
induced by interleukin-4 released from Th2 cells (10).
The significantly higher IgG2a/IgG1 ratio in C57BL/6 than in BALB/c mice is suggestive of a Th1-type immune response in the more resistant mice. However, additional studies are needed to characterize the cellular type of immune response in the two mouse strains more precisely. The experimental system described in this study should help
to clarify mechanisms of host resistance in melioidosis and should also
be useful in future studies on the virulence of B. pseudomallei and vaccine development.
| |
ACKNOWLEDGMENT |
We thank D. Bitter-Suermann for continuous encouragement.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Medical
Microbiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Phone: 0511-532-4352. Fax: 0511-532-4366. E-mail:
Steinmetz.Ivo{at}mh.hannover.de.
Editor:
P. J. Sansonetti
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Abstract
Introduction
