Download Serum Bactericidal Effects on Common Clinical Bacterial Isolates in

Original Article
Serum Bactericidal Effects on Common Clinical Bacterial
Isolates in Thais
Suwanna Trakulsombon, M.Sc.
Nalinee Aswapokee, M.D.
Somwang Danchaivijitr, M.D.
Abstract
Serum from healthy human is known to have bactericidal effect on some bacteria such
as Enterobacteriaceae. This effect may act, in concert with phagocytes and complement to pre­
vent bacteremia from those species. We studied serum bactericidal effect on clinically important
bacteria. Pooled sera were obtained from Thai healthy donors during June to September 1987.
These subjects were free from diarrheal disease for 6 months before sera were taken. Standard
time killing curves were done using 80%, 40% serum and controls with Mueller-Hinton broth and
heated serum. Bacterial isolates wereS. aureus (4 strains),E. coli (5),K. pneumonioe (4),S. typhi
(8), nontyphi Salmonella (16) and Shigella species (10) with 105 CFU/ml at the beginning. It was
found that sera from normal Thai subjects exerted bactericidal effect upon Salmonella typhi and
Shigella species but not for S. aureus, E. coli and K. pneumoniae. This study suggests that specific
antibodies to Salmonella and Shigella may readily present in healthy Thai subjects, and that this
may reflect frequent exposure. It might be suggested that there is protective antibody in adult
sera and vaccination may be unnecessary in this group.
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Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
12
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Vol. 6 No.1 Jan. - Mar. 1989
J Infect Dis Antimicrob Agents
INTRODUCTION
The ability to combat invasion into human body
by microorganisms involves two mechanisms. The
first is a specific immunity and the second is a so­
called natural or non-specific resistance which does
not occur for every invading microorganisms. 1-4
These two host defense mechanisms virtually present
in healthy hosts. However, they may vary among in­
dividuals depending on several known and unidenti­
The variation of host immunity in
fied factors.
human may occur through known factors such as
age, sex, genetic, pregnancy, environment and nutri­
tion. 3 ,4
One of these nonspecific mechanism occur in
serum of healthy human, that is bactericidal activity
against some bacteria. This immunity may act among
various defenses, in concert with phagocytes and
complement to prevent bacteremia from those
species. 5 This bactericidal activity is mainly against
some gram-negative bacteria 6 - 9 as described by
Buchner in 1889. This effect is abolished when
serum has been incubated for 30 minutes at 56°C.
Gram-positive bacteria generally are less sensitive than
gram-negative bacteria to this killing effect. lO - 13 Sera
from Thai people, however, are not known, to the
best of our knowledge, to have this effect to any of
these bacteria. We, therefore, study serum bacterici­
dal effect of Thai adult due to the interest that such
effect in area where prevalence of bacterial infections
are high may be different from elsewhere. It is also
known that the environment, nutrition, hygiene and
sanitation, behavior of Thais differ from those in de­
veloped nation, and that these factors might render
the serum bactericidal activity to be different from
the published information.
MATERIALS AND METHODS
Pooled nonnal human serum (PNHS). Pooled
serum from 144 healthy donors, aged between 20-45
years, were obtained from blood bank, Siriraj Hospi­
tal, Mahidol University, Bangkok, Thailand, during
June to September 1987. The individuals whose
blood were taken did not suffer from diarrheal
disease for 6 months prior to this study. PNHS was
made into aliquot and was stored in -70°C before
use. The Widal agglutination test of this pooled
serum was negative for both 0 and H antigens of Sal­
monella typhi and also Salmonella para A and para B.
Controls were PNHS heated at 56°C for 30 minutes
and Mueller-Hinton broth.
Bacteria. Bacterial strains were recent clinical
isolates from patients hospitalized at Siriraj Hospital.
Some strains were obtained from National Institute
of Health, Thailand. These were Staphylococcus
13
aures (5 isolates), Escherichia coli (5; 2 from blood
2 from urine, 1 from pus), Klebsiella pneumoniae (4),
Salmonella paratyphi (8), S. typhimurium (4), S.
choleraesuis (4), S. typhi (10), Shigella dysenteriae
(2), S. flexneri (8), S. boydii (3) and S. sonnei (2).
Most isolates were from blood. Only Shigella species
and some S. typhi were from stool and lung. These
bacteria were grown in trypticase soy broth, and then
adjusted to 10 6 CFU/ml with Mueller-Hinton broth as
initial inoculum size.
Serum bactericidal test. The serum bactericidal
activity of PNHS against various bacteria were per­
formed by modifying plate count method of
Reantree and Rantg 14 and Olling and et al. 18 The test
for bactericidal activity of serum used throughout
this study was set up as follows: The 0.1 ml of 10 6
CFU Iml culture broth was added into 80% and 40%
PNHS (0.8 or 0.4 ml of PNHS with 0.1 or 0.2 mlof
Mueller-Hinton broth respectively). These were done
in steriled 13 x 100 mm. glass tubes and incubated
at 37°C for 24 hours, at time interval of 1, 1/2 , 1, 2, 4,
6 and 24 hours. A standard plate counting was per­
formed for each test. The viable counts expressed as
log CFU Iml, with the minimum detection scale of
10 2 CFU/ml.
The percentage of bactericidal activity was cal­
culated as previously described 19 as follows:
% bactericidal activity =
100-100 x number CFU of experimental tubes
number of CFU of control tubes
RESULTS
The activity of serum bactericidal. The activity
of PNHS bactericidal against tested bacteria are
shown by standard time-kill curves presented in
Figures 1-9. The viable bacteria counts are presented
as log CFU Iml at each period of serum exposure.
The percentage of bactericidal activity of S. au reus,
E. coli and K. pneumoniae; S. typhi and S. paratyphi;
non-typhi Salmonella; and Shigella species were cal­
culated and reported in tables 1-5.
Growth rate of bacteria in serum. All tested or­
ganisms had normal growth rates when incubated
with heated serum or Mueller-Hinton broth. After
2-4 hours of incubation the growth rate of all tested
bacteria in heated serum (Figures 1-9) were slightly
lower than that incubated in broth. The growth rates
of bacteria in fresh serum were slower than those in
heated serum. This effect was seen after 4-6 hours of
serum exposure from some strains of E. coli, all tested
strains of K. pneumoniae, non-typhi Salmonella and
Shigella group B. The difference in growth rate is
clearly seen in S. typhi, S. paratyphi and Shigella
species with the exception of Shigella group B.
14
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The serum bactericidal activity among genus
and bacterial strains. There were significant dif­
ferences in bactericidal activity by PNHS among the
genera of gram-negative bacilli. PNHS exert strong
bactericidal activity against some in genus Salmonella
and genus Shigella, while activity against Escherichia
and Klebsiella was less remarkable.
No major difference in bactericidal activity was
demonstrated among clinical isolates of each species,
in which patterns of bactericidal activity was quite
similar.
Serum resistance. Some species of clinical iso­
lated bacteria such as S. au reus, K. pneumoniae, E.
coli, non-typhi Salmonella (S. typhimurium and S.
choleraesuis) and Shigella group B. (Figure 1-3, 6-7
and 9) were relatively resistant to killing by PNHS.
This could be seen from the distribution of percent­
age of serum bactericidal activity against these or­
ganisms in Tables 1, 4 and 5. It might be noted that
sera from normal Thai subjects had minimal bacteri­
- - 80% serum
++++-i 40% seru m
....... control (heated serum)
--- -- control (Mueller-Hinton broth)
O'------.---.-----.--I~
24
6
4
2
Time (hours)
Fig. 1 Bactericidal activity of normal human serum against
S. aureus.
10
,,
10
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--E
~
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c..J
e
/
9
9
/
/
8
/
/
/
r- ------/
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/
/
7
/
7
1E298 MUCI
6
/
/
/
/
6
/
C>
0
5
5
"iij
>
.~
JJ
4
3
2
- - 80% serum
++++-l 40% serum
.... control (heated serum)
----- control (Mueller-Hinton broth)
4
3
2
1
0
2
E
-~
u.
c..J
e
4
f24
0
10
9
9
8
8
7
7
6
'f---,
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10
4
6
24
6
C>
0
6
IE 299 Pus I
5
IE 300 H/C I
5
"iij
>
.~
::::I
(I)
4
4
3
3
2
2
1
1
0
'f-----,
2
4
Time (hours)
6
24
r---.
0
2
4
Time (hours)
Fig. 2 Bactericidal activity of normal human serum against E. coli.
6
24
Vol. 6 No.1 Jan. - Mar. 1989
J Infect Dis Antimicrob Agents
10
10
9
9
15
,~
/
/
/
/
/
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Sal group B 1024
4
H/C
::::l
::::l
en
B
U
U
/
- - BO%serum
+-+++ 40% serum
...... control (heated serum)
-- - - control (Mueller-Hinton broth)
3
2
1
0
'j-­
2
4
6
en
3
2
1
- - BO%serum
+-+++ 40% serum
..... control (heated serum)
- - - - control (Mueller-Hinton broth)
0
2
24
Fig. 3 Bactericidal activity of normal human serum against
K. pneumoniae.
,.
9
--
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e
6
E
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7
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.-
/
9
B
7
6
5
H/C
4
4
- - BO%serum
+-+++ 40% serum
...... control (heated serum)
-- - - control (Mueller-Hinton
broth)
3
1
'/------------.
Yo 1
4
2
6
24
Time (hours)
,
10
I
I
I
B
I
I
I
7
6
I
I
,,
,If
Cl
o
Sal Para A 236
5
4
3
2
1
- - BO%serum
+-+++ 40% serum
..... control (heated serum)
-- - - control (Mueller-Hinton broth)
0
2
4
f-­
6
24
Fig. 7 Bactericidal activity of normal human serum against
S. choleraesuis (C).
I
I
e
2
I
9
LL.
3
Time (hours)
Fig. 4 Bactericidal activity of normal human serum against
S. typhi
U
Fig. 6 Bactericidal activity of normal human serum against S.
typhimurium (B)
10
/
0
E
24
/
Sal group 0 202
2
~
r------.
/
,,~'
::::l
en
"
,~
/
6
Time (hours)
Time (hours)
10
4
H/C
- - BO%serum
+-+++ 40% serum
...... control (heated serum)
-- - - control (Mueller-Hinton
broth)
OL-.~---,-----.-----,
2
4
6
Time (hours)
Fig. 5 Bactericidal activity of normal human serum against
S. paratyphi (A)
cidal or bacteriostatic activity against these organisms
only in the first 4 hours of serum exposure. After
6 hours of incubation, complete killing against these
organisms was not found. These organisms, moreo­
ver, were able to regrow to the stationary phase, and
after 24 hours of incubation, the sera exerted no acti­
vity at all.
Serum sensitive. The results reported in Tables
2, 3 and 5 show that fresh PNHS from Thai donors
are able to reduce the viability of S. typhi, S. paraty­
phi and Shigella species with the exception of Shigella
group B. The rate of bactericidal of all strains of S.
typhi., S. paratyphi and Shigella species group A, C
and D in this study were very rapid, i.e. only 2-4
hours were needed for complete killing.
The serum bactericidal activity among different
sites of infection. A comparison of the sensitivity to
16
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.;
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2532
10
10
9
B
5
4
-
BO%serum
40% serum
..... control (he.ted serum)
t+++
~ jL--~~
4
.
,
10
9
__c_on_t~~o_1 _(M_U_el...,'e_r'--.Hi~:roth)
24
10
,I
,/
B
I
I
I
.
I
I
I
l
I
/
I
I
I
I
..........
---_.~
5
4
1
1
OL-~..-,--~-..-,---I'f--
o L--..---.----,----.--I,f---,
4
4
24
Time (hours)
24
Time (hours)
Fig. 8 Bactericidal activity of normal serum against Shigella spp.
10
10
,
9
//
B
]
,,
,./­
~
u..
c..>
o
,
/
'"
.g
~
.~
4
4
en
IShig B 335 stool I
o L---.-..-,--,--~,...----/J--,
4
,
10
,/
,
,
,,
24
10
I
- - BO%serum
t+++ 40% serum
..... control (heated seruml
control (Mueller·Hinton
broth)
-~,--~---I/f---,
4
Time (hours)
4
24
Time (hours)
Fig. 9 Bactericidal activity of normal human serum against Shigella B.
Vol. 6 No.1 Jan. - Mar. 1989
J Infect Dis An timicrob Agents
17
Table 1 Distribution of Serum Bactericidal Activity Against S. aureus, E. coli and K. pneumoniae.
Organisms
Tests·
Yz
1
Percent Antibactericidal Activityt
2
4
6
24
S. aureus
41 H/C
I
300
300
33.3
300
60
0
33.3
14.3
II
42 H/C
I
43 H/C
II
I
65 H/C
II
I
II
95
0
70
55
92
84
98
66.6
99.95
37.5
99.50
0
99.98
94.28
95.0
95.0
0
0
0
0
0
0
0
0
E coli
298 MUC
I
II
314 MUC
I
II
299 PUS
I
300 H/C
II
I
II
301 MUC
I
II
100
14.3
99.99
99.97
76.66
76.66
0
50
99.1
0
70.0
92.5
100
100
99.6
99.5
80
90
99.11
99.1
93
93
90
90
99.97
99.97
0
0
0
0
0
0
99.0
97.5
94.0
70.0
60.0
60.0
99.5
99.5
99.99
99.99
99.99
99.99
99.99
99.99
97.0
97.0
99.99
99.99
95.0
95.0
99.99
99.99
99.07
99.07
0
0
0
0
0
0
0
0
0
0
0
0
0
0
K. pneumoniae
132 H/C
I
133 H/C
II
I
II
138 H/C
I
145 H/C
II
I
II
*1 is nonnal pooled serum (80%) compared with control broth.
II is nonnal pooled serum (80%) compared with control heat-inactivated serum.
tserum exposured time (hours).
H/C is hemoculture
MUC is midstream urine culture
CFU/ml of test
%Antibactericidal = 100 - 100 x - - - - ' ' - - - - ­
CFU/ml of control
serum of E. coli, isolated from different sites of in­
fection was performed. E. coli strains isolated from
blood or pus were more resistant to the bactericidal
effect of normal human serum than that from urine,
as illustrated strain E 300 HIC, E 299 PUS, E 314
MUC and E 298 MUC in Figure 2. No significant dif­
ference was found in serum sensitivity of S. typhi
which isolated from different sources, such as blood,
stool and sputum (Table 2).
DISCUSSION
The bactericidal action of pooled serum from
144 healthy donors from Blood Bank Unit, Siriraj
Hospital, Mahidol University, aged between 20-45
years, against various isolated bacteria commonly en­
countered in clinical situation in Thais has been
studies. Since the serum from individual donor had a
similar bactericidal action to pooled serum (data from
::"n"" 6""1J~1Jn"" 1 JJ. fl. -JJ.<\ fl. 2532
u
18
Table 2 Distribution of Serum Bactericidal Activity Against S. typhi
Organisms
Tests·
Percent Antibactericidal Activityt
2
4
6
Yz
I
99.5
99.0
99.90
99.90
99.9
99.9
90.0
90.0
99.5
99.5
97.5
97.5
99.0
99.0
99.0
98.0
99.5
99.66
98.5
99.5
99.9
99.5
100
100
100
100
99.82
99.7
99.82
99.55
99.82
99.77
100
100
99.9
99.1
99.88
99.9
99.94
99.95
24
Salmonella gr. D
169 H/C
I
II
202 H/C
I
II
100 H/C
I
II
45 bronchitis
I
II
48 pneumonitis
I
II
190 stool
I
II
211 stool
I
II
54 H/C
I
II
178 H/C
I
II
215 H/C
I
II
100
100
99.95
99.77
100
100
100
100
100
100
~
100
100
100
100
100
100
*1 is normal pooled serum (80%) compared with control broth.
II is normal pooled serum (80%) compared with control heat-inactivated serum.
tSerum exposured time (hours)
Table 3 Distribution of Serum Bactericidal Activity Against S. paratyphi
Organisms
Tests·
I
Percent Antibactericidal Activityt
2
4
6
Salmonella gr. A
232 H/C
I
99.85
99.82
100
100
100
100
100
100
II
233 H/C
I
II
236 H/C
I
II
57 H/C
I
II
58 H/C
I
II
59 H/C
I
II
60H/C
I
II
1 H/C
I
II
100
100
99.7
99.4
100
100
99.97
99.97
*1 is normal pooled serum (80%) compared with control broth.
II is normal pooled serum (80%) compared with control heat-inactivated serum.
tSerum exposured time (hours)
100
100
100
100
99.88
99.88
100
100
24
J Infect Dis Antimicrob Agents
19
Vol. 6 No.1 Jan. - Mar. 1989
the preliminary study) and we studied different first two hours. There was no difference in bacteri­
strains of the bacteria rather than sera from different cidal effect of serum at concentrations of 40 and 80%
against serum susceptible strains and serum resistant
individuals.
The bactericidal action of serum is demonstrable strains. The rapid bactericidal effects of even low
over a wide range of serum concentrations, although concentration of sera occurred with some clinically
the rate of killing is known to generally increase with important bacteria, that is S. typhi, S. paratyphi, and
increasing serum concentration. For the study of Shigella species (with the exception of some strains of
serum bactericidal activity in relation to infection, Shigella group B), is interesting. This may be an in­
the majority of investigators have employed systems direct evidence that Thai people possess specific
containing relatively high concentration of serum to antibody to these bacteria when reaching adult
ensure that killing is not limited by the availability age group, or the bactericidal activity can be attri­
of essential components of bactericidal system: thus, buted to complement because this effects was abo­
concentrations in the 20 to 80% range have been suc­ lished by heated at 56°C.
cessfully utilized in a number of studies. 6 ,20-24 From
After 2-4 hours of incubation the growth rates
these reasons, serum bactericidal activity of PNHS of all tested bacteria in heated serum were slightly
was studied in concentration of 40 and 80%.
lower than that incubated in broth. From these re­
Serum bactericidal activity is one of the most sults, we can conclude that there are some growth in­
important host defense mechanism for prevention of hibition factors in heated serum or there are some
systemic invasion of serum sensitive bacteria. The bactericidal antibodies in PNHS of Thais, in addition
ability of serum to inhibit the growth of bacteria is to the bactericidal effect of the complement.
The PNHS from healthy Thai donors is able to
repeatedly shown to be highest within the first 2
hours of serum exposure. 9 ,14,17,25,26 The results in our kill S. typhi, S. paratyphi and Shigella group A, C
studies confirmed those studies, in which complete and D. Contradictory finding were presented by
killing of sensitive bacteria by sera occurred in the other, 29,30 which revealed that their serum was not
Table 4 Distribution of Serum Bactericidal Activity Against S. typhimurium and S. cholerasuis.
Organisms
Tests*
Y2
1
Percent Antibactericidal Activityt
2
4
6
24
Salmonella gr B
1024 H/C
I
2977 H/C
II
I
99.33
-100
85
II
40
3999 H/C
I
2162 H/C
II
I
2681 H/C
II
I
95
66.66
90.00
-100
50
25
99.99
90.0
99.99
98.33
99.96
99.50
99.92
98.66
99.99
87.5
66.66
-100.00
97.5
50.0
90.0
50
70
90
99.95
40.0
99.99
-60
99.99
-75.0
99.95
80.0
II
Salmonella gr C
4910 (18) H/C
0
-100
I
4907 (6) H/C
II
I
4907 (5) H/C
II
I
4910 (2) H/C
II
I
II
* I is normal pooled serum (80%) compared with control broth.
II is normal pooled serum (80%) compared with control heat-inactivated serum.
tSerum exposured time (hours)
80.0
-33.3
99.99
98.66
99.99
99.99
99.99
99.92
99.99
99.5
99.90
99.9
99.99
99.2
99.99
-60
99.99
99.85
99.99
95.71
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
lJ'<n 6
o~
<i
<i
n7il77 hfl~~1 iJiJlUl::lJ7ri'7lJ iJililw
,
iwmll JJ. fl. - 1/. fl. 2532
Table 5 Distributionof Serum Bactericidal Activity Against Shigella spp.
Organisms
Tests·
Y2
1
Percent Antibactericidal Activityt
2
6
4
24
Shigella gr. A
3034 stool
I
0
0
90.0
90.0
99.95
99.90
100.0
100.0
50.0
50.0
900.0
566.6
50.0
33.33
0
100.0
70.0
70.0
60.0
33.33
50.0
50.0
70.0
0
66.66
66.66
80.0
66.6
93.33
33.33
95.0
300.0
99.94
99.85
99.0
92.5
99.4
50.0
99.40
50.0
98.5
85.0
99.11
20.0
99.99
40.0
99.99
70.0
100
100
99.99
87.5
99.99
50
99.99
42.86
50.0
50.0
99.0
98.0
100
100
80.0
80.0
99.77
99.77
99.33
99.0
100
100
100
100
90.0
90.0
50.0
50.0
99.0
99.0
90.0
90.0
99.99
99.96
99.33
99.20
II
Shigella gr. A
4421 stool
I
II
335 stool
I
II
337 stool
I
II
4453 stool
I
II
3068 stool
I
II
51 stool
I
II
45 stool
I
II
46 stool
I
II
70.0
70.0
0
0
50.0
33.33
0
0
50.0
50.0
0
0
50.0
50.0
0
0
99.!77
99.93
99.93
99.99
10.0
99.91
98.71
99.99
0
0
0
0
0
0
0
0
99.99
75.0
99.99
33.33
99.95
44.44
0
0
0
0
0
0
99.99
99.99
100
100
Shigella gr. C
4240 stool
I
II
21 stool
I
II
4131 stool
I
II
Shigella gr. D
3947 stool
I
II
3945 stool
I
II
able to reduce the viability of these organisms. Roan­
tree 14 found that the Shigella and Klebsiella groups
showed a higher percentage of serum sensitive strains
than do the others, whearas there is a higher per­
centage of serum resistant strains of Salmonella, using
serum from the American healthy donors or patients.
This contradictory finding may come from the dif­
ference in the characteristic of nutrition, environ­
ment, hygienic, sanitation and behavior among the
Thais and the other races. The most important fac­
tors may be the differences in prevalence of these or­
ganisms in the environment and the rate of exposure
100
100
99.99
99.99
to them.
The PNHS of Thais is unable to reduce the viabi­
lity of S. aureus, K. pneumoniae and some strain of
E. coli. The lack of this bactericidal activity is simi­
lar to that reported from the sera from Westerns
healthy donors. 8 - 11 ,14
The serum bactericidal activities among the
groups of Salmonella spp. and Shigella spp. are signi­
ficant different. PNHS was unable to kill Salmonella
group Band C and Shigella group B, whereas it exert­
ed high activity against Salmonella group A and D,
Shigella group A, C and D. The mechanism for these
J Infect Dis Antimicrob Agents
phenomena is not known, but may be due to the dif­
ference in some antigenic determinant or the dif­
ference of some biochemical groups or structure in
some compositions on the cell surface of individuals
species or genus. 29
It was found that sera from Thai subjects exert­
ed bactericidal effect upon Salmonella typhi and
Shigella species but not for S. au reus, E. coli and K.
pneumoniae. This study suggests that specific antibo­
dies to Salmonella and Shigella may readily present in
healthy subjects in Thailand.
It may indicate
previous apparent or subclinical infection with S.
typhi or Shigella infections. It is known that humoral
and cellular immunity to S. typhi in those who live or
do not live in endemic area of typhoid infection are
different. n ,28 Previous infection with other bacteria
that share common antigenicity with Salmonella or
Shigella 27 may be the cause of this finding. From
this study, two hypotheses are generated. First, the
rates of Salmonella and Shigella exposure in normal
healthy adults in Thailand are relatively higher than
that of Western countries, and that, this may be pro­
tective immunity against these organisms. Second,
the protective immunity to Salmonella in the blood
may reflects the unnecessary vaccination policy,
especially in adults. It is also the explanation for
observations which were made for many decades
that, in Thailand, there are rare cases of enteric
fever in the middle age group of the Thai people and
that the differential diagnosis of febrile illness in
adults aged over 40 who suffer from enteric fever will
come out with low probability.
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