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Infect Immun, August 1998, p. 3752-3757, Vol. 66, No. 8
Molecular Genetics Laboratory, International
Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka-1000,
Bangladesh,1 and
Department of
Microbiology and Molecular Genetics, Harvard Medical School,
Boston, Massachusetts 021152
Received 19 February 1998/Returned for modification 15 April
1998/Accepted 5 May 1998
In toxigenic Vibrio cholerae, the CTX genetic element
which carries the genes for cholera toxin (CT) is the genome of a
lysogenic bacteriophage (CTX Cholera caused by toxigenic
Vibrio cholerae is a major public health problem in
developing countries. Epidemiological surveillance of cholera and
comparative molecular analysis of strains collected during outbreaks
have demonstrated clonal diversity among epidemic strains and a
continual emergence of new clones of toxigenic V. cholerae
(5-7, 21). The mechanisms involved in the emergence of new
toxigenic clones have not been adequately explained, although it is
assumed that a combination of genetic changes and natural selection
caused by unidentified environmental factors as well as the immune
status of the host populations is likely to influence the process.
The profuse secretory diarrhea characteristic of cholera is caused by
an enterotoxin, cholera toxin (CT), produced by toxigenic V. cholerae when it colonizes the small intestine (20).
The genes encoding CT (ctxAB) are part of a larger genetic
element (CTX genetic element) consisting of at least six genes
(ctxAB, zot, ace, cep, and
orfU) comprising the "core" region that is flanked by
two or more copies of a repeated sequence (19, 23). It has
been demonstrated recently (24) that in the V. cholerae O1 strain P27459 (genetically modified by marker exchange
and renamed SM44), the entire CTX element constituted the genome of a
filamentous bacteriophage (CTX V. cholerae strains and cholera stools.
Toxigenic V. cholerae strains analyzed in this study to
investigate the induction of lysogenic CTX
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Induction of the Lysogenic Phage Encoding
Cholera Toxin in Naturally Occurring Strains of Toxigenic
Vibrio cholerae O1 and O139
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
). Clinical and environmental strains of
V. cholerae O1 or O139 and stools that were culture
positive for cholera were analyzed to study the induction and
transmission of CTX. To our knowledge, this is the first report of
the examination of CTX in clinical materials and in naturally
occurring strains. DNA probe analysis revealed that 4.25% (6 of 141)
of the isolated V. cholerae strains spontaneously produced
a detectable level of extracellular CTX particles in the culture
supernatants whereas another 34.04% (48 of 141) produced CTX
particles when induced with mitomycin C. CTX isolated from 10 clinical or environmental strains infected a CT-negative recipient
strain, CVD103, both inside the intestines of infant mice and under
laboratory conditions. All culture-positive stools analyzed were
negative for the presence of CTX both in the DNA probe assay and by
in vivo assay for the infection of the recipient strain in infant mice.
These results suggested that naturally occurring strains of toxigenic
V. cholerae are inducible lysogens of CTX but that
cholera pathogenesis in humans is not associated with the excretion of
CTX particles in stools, indicating that induction of the phage may
not occur efficiently inside the human intestine. However, in view of
the efficient transmission of the phage under conditions conducive to
the expression of toxin-coregulated pili, it appears that propagation of CTX in the natural habitat may involve both environmental and
host factors.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
). The phage could be propagated in
recipient V. cholerae strains in which the CTX genome
either integrated chromosomally at a specific site, forming stable
lysogens, or was maintained extrachromosomally as a replicative form
(RF) of the phage DNA (24). Cultures of V. cholerae harboring the RF of CTX produced high titers of the
phage in their supernatants. This study indicated that the propagation
of CTX may be associated with horizontal gene transfer leading to
the origination of novel toxigenic strains of V. cholerae.
More recently, it has been reported that during passage of CTX
lysogens through the infant-mouse intestine, phage excision and
replication occur in vivo (16). The present study was
undertaken to analyze the induction of lysogenic CTX in clinical and
environmental isolates of toxigenic V. cholerae and to
investigate whether cholera pathogenesis in humans is associated with
the excretion of CTX particles in the stools. Furthermore, this
study investigated the ability of CTX particles derived from
naturally occurring V. cholerae strains to infect a
CT-negative recipient strain of V. cholerae O1 inside the
gastrointestinal tracts of infant mice and under laboratory conditions.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
included a total of 125 clinical isolates and 16 environmental isolates belonging to O1 or O139
serogroups. Clinical isolates and stools that were culture positive for
cholera were obtained from patients who attended the treatment center
of the International Centre for Diarrhoeal Disease Research, Bangladesh
(ICDDR,B) located in Dhaka. The clinical strains consisted of 78 strains from the culture collection of ICDDR,B (Table
1) and 47 strains isolated from freshly
collected culture-positive stools, which were also analyzed in this
study (Table 2). The environmental
strains were obtained from surface waters in Dhaka. Strains were stored
either in lyophilized form or in sealed deep nutrient agar at room
temperature. Before use, the identities of the V. cholerae
cultures were confirmed by biochemical reactions and serological test
(27), and the presence of the CTX element was ascertained by
using DNA probes (6). The relevant characteristics of
V. cholerae strains used as controls or as a recipient of
CTX are listed in Table 3.
TABLE 1.
Analysis of toxigenic V. cholerae O1 and O139
strains isolated between 1969 and 1997 in Bangladesh for the induction
of CTX
TABLE 2.
Analysis of 47 culture-positive stools collected during
June and July 1997 in Bangladesh for the presence of
cell-free CTX a
TABLE 3.
Characteristics of V. cholerae O1 reference
strains used in this study
Probes and hybridization.
The gene probes used in this study
to detect the CTX genome were a 0.5-kb EcoRI fragment of
pCVD27 (14), containing part of the ctxA gene,
and an 840-bp region internal to the zot gene amplified by
PCR from the recombinant plasmid pBB241 as described previously
(8). Strand-specific oligonucleotide probes with the
sequences 5'-TCTATCTCTGTAGCCCCTATTACG and
5'-CTCAGACGGGATTTGTTAGGCACG for probing the plus and minus
strands, respectively, were also used to detect the presence of
single-stranded DNA of CTX. Colony blots or Southern blots were
prepared with nylon filters (Hybond; Amersham International plc.,
Ayelesbury, United Kingdom) and processed by standard methods
(17). The polynucleotide probes were labeled by random
priming (10) with a random-primer DNA-labeling kit (Bethesda
Research Laboratories, Gaithersburg, Md.) and
[
-32P]dCTP (3,000 Ci/mmol; Amersham), and
oligonucleotide probes were labeled by 3' tailing with terminal
deoxynucleotide transferase and [-32P]dCTP. Southern
blots and colony blots were hybridized with the labeled probes and
autoradiographed as described by us previously (5-7).
Induction of CTX lysogens.
Toxigenic V. cholerae strains were grown in Luria broth (LB) at 30°C to an
absorbance at 540 nm of 0.2. The cells were collected by
centrifugation, washed, and resuspended in fresh LB. The suspension was
divided into aliquots, to which mitomycin C (Sigma Chemical Co., St.
Louis, Mo.) was added at 20 ng/ml and incubated overnight at 30°C.
The culture supernatants were analyzed for extracellular phage carrying
the CTX element as described in the following section. Strains grown in
a similar way but without mitomycin C were used as controls.
Screening of stools and bacterial cultures for CTX.
Freshly collected culture-positive stools and V. cholerae
strains either isolated from the same stools (Table 2) or obtained from
the culture collection (Table 1) were used. Aliquots of watery stools
or cultures were centrifuged at 6,000 × g to
precipitate solid particles and suspended bacteria, and the
supernatants were sterilized by filtration through 0.22-µm-pore-size
filters (Millipore Corp., Bedford, Mass.). To confirm that the
filtrates from stools as well as the culture supernatants did not
contain any bacterial cell, aliquots of the filtrates were streaked on
Luria agar (LA) plates and incubated overnight at 37°C. The sterile
filtrates were mixed with one-fourth volumes of a solution containing
20% polyethylene glycol 6000 and 10% NaCl and centrifuged at
12,000 × g to precipitate the phage particles. The
precipitate was dissolved in a solution containing 20 mM Tris-Cl (pH
7.5), 60 mM KCl, 10 mM MgCl, and 10 mM NaCl and digested with
pancreatic DNase I (100 U/ml) and RNase A (50 µg/ml) at 37°C for
1 h to remove possible nucleic acids carried over from lysed
bacterial cells. The solution was extracted with phenol-chloroform to
disrupt possible phage particles, and the total nucleic acids were
precipitated with ethanol. Preparations containing CTX DNA were
identified by Southern blot hybridization (22). Aliquots of
watery stools mixed with serial dilutions of CTX-Km (101
to 104 particles/ml) isolated from a mitomycin C-induced
culture of strain SM44 were used as positive controls for the assay of
phage in stools, for both in vitro and in vivo assays. CTX-Km
derived from SM44 was quantified by incubating serially diluted
filter-sterilized culture supernatants with strain RV508 and then
determining the number of Kmr colonies, as described
previously (24).
present in the cell-free
culture supernatants of toxigenic V. cholerae strains and extracts of culture-positive stools was assayed with strain CVD103 (15) as the recipient in vivo in suckling mice and in vitro under laboratory conditions as follows. Aliquots (10 ml) of filtered sterile supernatant fluids from mitomycin C-induced cultures or control
cultures without mitomycin C or filtered stool extracts were used to
precipitate phage particles. The pellet was suspended in 100 µl of
TES buffer (20 mM Tris-HCl [pH 7.5], 10 mM NaCl, 0.1 mM disodium
EDTA). The recipient strain was grown in LB at 37°C; the cells were
precipitated by centrifugation and washed in fresh LB. Approximately
105 bacterial cells mixed with 10 µl of the phage
preparation in a final volume of 50 µl were gastrointestinally
inoculated into groups of 5-day-old Swiss Albino mice obtained from the
breeding facilities of the Animal Resources Branch, ICDDR,B. For each
phage preparation, at least five mice were inoculated. The animals were sacrificed after 24 h, and their intestines were removed and
homogenized in 10 mM phosphate-buffered saline (pH 7.2). The homogenate
was centrifuged at low speed to precipitate debris, the supernatant was
then centrifuged to precipitate bacterial cells, and the pellet was
resuspended in phosphate-buffered saline. Serial dilutions of an
aliquot of the suspension were plated on taurocholate-tellurite-gelatin agar (18) to select V. cholerae colonies. All the
colonies were screened for the presence of the CTX genome by using
the ctxA or zot probe. The ratio of
probe-positive colonies to total colonies recovered was calculated and
expressed as the percentage of recipient cells carrying the CTX
genome (Table 4). For in vitro assays, mixtures of phage and recipient cells as described above were prepared.
Each mixture was inoculated into 5 ml of LB and incubated for 1 h
at 30°C, and aliquots of the culture were plated on LA plates and
incubated overnight at 30°C. For each strain and phage combination,
five different in vitro assays were performed and at least
104 colonies of the recipient strain recovered from each of
these assays were screened for the CTX genome.
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genome in infected cells, representative probe-positive
colonies of the infected recipient strain CVD103 were grown at 37°C
in LB for several generations (6 to 24 h) and were tested for the
presence of the CTX element by colony blot hybridization. Similarly, a
colony of CVD103 infected with CTX-Km derived from strain SM44 was
grown for 6 to 24 h in aliquots of LB either containing kanamycin
(50 µg/ml) or without kanamycin. Serial dilutions of the cultures
were plated on LA plates containing kanamycin and on a duplicate set of
LA plates devoid of the antibiotic to determine the proportion of cells
retaining the phage genome.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Induction and transmission of CTX.
To investigate whether
lysogenic CTX present in toxigenic V. cholerae strains
could be induced to produce extracellular infectious phage particles,
we cultured the strains in the presence of a DNA-damaging agent,
mitomycin C, and then screened the culture supernatant fluids for the
presence of CTX by using DNA probes. Similarly, freshly collected
culture-positive stools were analyzed for the presence of CTX to
understand whether the phage is excreted in the stools of cholera
patients. Another strategy for detecting the presence of infectious
CTX in culture supernatants as well as in culture-positive stools
was to expose a suitable recipient strain, CVD103, to the phage
preparations and then look for infection of the bacterial cells. None
of the culture-positive stools analyzed was positive for the presence
of a detectable level of CTX either in the DNA probe assay or in the
in vivo assay in infant mice. To determine the detection limits of our
assays, we used reconstituted stools containing a known number of phage
particles. This was done by using a genetically marked phage, CTX-Km
(derived from strain SM44), since CTX-Km could be quantified by
titration with a recipient strain RV508, which constitutively expresses
toxin-coregulated pili (TCP) and hence is readily infected by the phage
(24). In the case of stools mixed with exogenous CTX-Km
and used as positive controls, a minimum of 103 phage
particles in 5 ml of stool could be distinctly detected by Southern
blot hybridization (Fig. 1). When assayed
in infant mice, the presence of 102 particles of CTX-Km
in 5 ml of reconstituted stools could be detected and the corresponding
stool extract produced between one and nine colonies (recovered from
five different mice) of Kmr transductants of CVD103.
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whereas another 48 strains (34.04%) produced extracellular CTX when induced with mitomycin C, as shown by Southern blot hybridization (Fig. 2). These 54 strains
included 12 strains isolated from fresh culture-positive stools which
tested negative for the presence of CTX. Strains that produced
extracellular CTX included 7 (19.44%) of 36 El Tor strains and 5 (45.45%) of 11 O139 strains obtained from freshly collected stools and
22 (43.13%) of 51 El Tor strains and 20 (46.51%) of 43 O139 strains
analyzed from previous collections (Tables 1 and 2). As expected for a
filamentous phage, Southern blot hybridization with strand-specific
oligonucleotide probes revealed that the phage DNA was single stranded
and hybridized with the probe specific for the plus strand but not with
the probe for the corresponding minus strand. The bands corresponding
to the CTX DNA were not clearly visible on ethidium bromide-stained agarose gels (data not shown) but could be detected distinctly by
hybridization of the Southern blots with the specific probes (Fig. 2).
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genome play a crucial role in the morphogenesis of the
phage (24). The reasons for the apparent inability of 87 of
the 141 strains to produce CTX particles on induction with mitomycin
C (Table 1) may be that the integrated form of CTX carried by these
strains is defective due to possible mutations in one or more genes
essential for phage morphogenesis. Strains analyzed in this study
included both V. cholerae O1 and O139 carrying variable
number of copies of the CTX element, and a difference was noted in
terms of induction of CTX in strains belonging to these serogroups.
The phage was more often inducible in O139 strains (46.29%) than in El
Tor strains (33.33%). Generally, O139 strains are known to carry more
than one copy of the CTX element (9, 25), and this may
account for the higher prevalence of inducible lysogens among O139
vibrios. In the present study, CTX was induced by the DNA-damaging
agent mytomycin C, which is known to induce many temperate
bacteriophages. However, six strains including two environmental
strains produced a detectable level of extracellular CTX particles,
even without treatment with mitomycin C (Table 1), indicating that
unidentified environmental factors or possible mutations in the phage
or the host bacteria might have caused induction of the CTX in these
strains. Further studies are required to define possible factors which
may play a role in the induction of lysogenic CTX in toxigenic
V. cholerae in the natural habitat.
We used two different methods to determine the presence of infectious
phage particles in culture supernatants or culture-positive stools.
These included an in vitro laboratory method and the infant-mouse system, both of which have been previously described (24).
However, in previous studies (16, 24), the transmission of
CTX was examined between a recipient strain and a donor strain of
V. cholerae when the strains were grown in mixed culture or
inoculated into infant mice, whereas in the present study cell-free
phage particles were used to infect the recipient strain. In addition
to adequate expression of TCP, which is the receptor for the phage, the
infant-mouse system is known to cause significant enrichment of
toxigenic V. cholerae strains (2). Hence, the
infant-mouse assay was expected to detect the presence of a smaller
number of infectious phage particles in the samples. On the other hand,
the in vitro method was also chosen because, from the ecological
perspective, it is important to understand the ability of CTX to
propagate independently of the mammalian host, in view of the
possibility that propagation of CTX in the natural habitat is
associated with the origination of new toxigenic strains through
lysogenic conversion (24).
The use of CVD103, which is a derivative of the V. cholerae
O1 classical strain 569B with ctxA deleted, provided certain
obvious advantages. Since CVD103 is a classical biotype strain, it was supposed to be more susceptible to CTX than are El Tor strains, as
described previously (24), and hence was expected to
facilitate the detection of CTX. Due to deletion of the
ctxA gene, CVD103 would not hybridize with the
ctxA probe which was used in colony blot hybridization to
detect colonies infected by wild-type CTX. Moreover, since CVD103 is
a vaccine prototype strain, its use provided an opportunity to study
the susceptibility of an attenuated vaccine strain to CTX.
In the present study, strain CVD103 was infected by CTX both under
in vitro laboratory conditions and inside the intestines of infant mice
(Table 4). The infant-mouse assay, which detected the presence of
102 CTX-Km particles in 5 ml of reconstituted stool, was
approximately 50 times more sensitive than the Southern hybridization
assay, which detected 103 phage particles, since only
one-fifth of the stool extract (10 µl of 50 µl of total extract)
was used for each assay in mice. The high sensitivity of the assay in
infant mice for both reconstituted stools and culture supernatants
could also be due to the prolonged incubation of infected cells
carrying the RF of CTX inside the mouse intestine, leading to
production of a larger number of infectious phage particles, in
addition to adequate expression of TCP in vivo. It should be clarified
that this study was not designed to compare the susceptibility of the
recipient strain to CTX in vivo and in vitro. Instead, we have
successfully used the infant-mouse model in an assay to detect the
presence of infectious CTX in culture supernatants and clinical
specimens. In addition, the observed ability of wild-type CTX to
transmit its genome into the vaccine prototype strain CVD103 suggested
that there is a need to modify and redesign possible live vaccine
candidates in view of the possible reacquisition of CT genes by
attenuated strains.
Stability of the CTX genome in the recipient strain.
CTX
is unusual among filamentous phages because it can either replicate as
a plasmid or integrate into the V. cholerae chromosome at a
specific attachment site (24). Analysis of representative probe-positive colonies for the presence of CTX genome revealed the
presence of the RF of the CTX genome in freshly infected colonies.
However, the concentration of the RF DNA was too low to be visible in
ethidium bromide-stained gels, but it could be faintly detected by
Southern blot hybridization (data not shown). The phage genome was
rapidly lost from infected cells when the infected cells were cultured
under laboratory conditions, and only 0.9% of the colonies recovered
after 24 h of culture hybridized with the ctxA probe.
The CTX genome did not integrate into the chromosome of CVD103, as
shown by Southern blot analysis of genomic DNA with the ctxA
probe (data not shown) as well as presumed from the rapid loss of
CTX genome from infected cells. When CVD103 was infected with
CTX-Km derived from strain SM44, the infected cells retained the RF
of the phage when cells were grown in the presence of kanamycin.
However, when the infected cells were cultured in the absence of
kanamycin, the RF of CTX-Km was rapidly lost and 60.3% of viable
cells recovered after 12 h of culture became susceptible to
kanamycin (50 µg/ml). Previously, the CTX genome was shown to
integrate into the chromosome when an El Tor strain carrying a resident
attRS sequence was used as the recipient (24). In
the previous study, the phage was marked with kanamycin resistance and
the transfectants were maintained in the presence of kanamycin. Similarly, in the present study, CVD103 cells infected with the CTX-Km retained the RF DNA of the phage when the cells were grown in
the presence of kanamycin but rapidly lost the RF of CTX-Km in the
absence of kanamycin. This indicated that there was a requirement for
appropriate selection pressure for the retention of the
extrachromosomal CTX element in V. cholerae. The lack of
selection of cells carrying the RF of CTX could account for the
subsequent loss of the phage DNA. However, once the phage genome
integrates into the chromosome of the recipient cell, it is likely to
be more stable. There have been several studies which suggested that
the gastrointestinal environment can cause a selective enrichment of
toxigenic V. cholerae strains compared to nontoxigenic
strains (2, 11, 13). It therefore seems possible that in
addition to adequate expression of TCP, which is the receptor for the
phage, the intestinal environment also contributes to selecting
V. cholerae cells harboring the CTX genome. Since the CTX
element itself carries the necessary genes for its own integration at
resident attRS sites of the host chromosome (26),
the origination of novel toxigenic strains of V. cholerae
and their selective enrichment are likely to occur in the intestinal
environment.
Propagation of CTX.
The existence of CTX was discovered
by using a genetically modified V. cholerae strain, SM44, in
which the CTX element was marked with a kanamycin resistance
(Kmr) determinant (12, 24). The presence of the
Kmr marker facilitated the detection of the induction and
transmission of the phage. The present study has investigated for the
first time the prevalence of inducible lysogens of CTX among
naturally occurring strains of toxigenic V. cholerae and the
potential of the extracellular phage particles to infect a CT-negative
recipient strain of V. cholerae. This study is also the
first to examine clinical materials for the presence of CTX and to
investigate whether cholera pathogenesis is normally associated with
the excretion of the phage in stools. Since the CTX element was not
marked with a phenotypically detectable marker in native strains,
detection of the induction of CTX and its transmission in vitro and
in infant mice were studied with specific DNA probes. Although V. cholerae is known to be primarily a human pathogen, it has been suggested that the bacterium can persist in the aquatic environment in
unexplained ecological associations (3). The natural habitat of toxigenic V. cholerae therefore seems to consist of two
compartments: the gastrointestinal tract of the host and the aquatic
environment outside the host. The host compartment is known to provide
signals necessary for the expression of most ToxR-regulated
virulence-associated factors, including CT and TCP (1, 4).
To investigate whether induction of the CTX prophage is also
mediated by possible host factors in the human gastrointestinal tract,
we screened culture-positive stools for the presence of the phage. That
all the stools were negative for the phage suggested that induction of
lysogenic CTX was possibly not associated with cholera pathogenesis
in humans, unless we assume that the CTX particles were very
unstable in the human intestine and rapidly degraded beyond the
detection limit of our assays. However, the present study showed that
when extracellular CTX isolated from naturally occurring strains and the control strain SM44 were used to infect the recipient strain in
vivo, CTX particles were stable and remained infectious in the
gastrointestinal environment of infant mice. It may be mentioned that
induction of the CTX prophage in infant mice has been reported recently based on observed transduction of a recipient strain by a
CTX lysogen inside the intestines of infant mice (16). The present study, however, did not provide any evidence in favor of
the induction of lysogenic CTX inside the human intestine in
association with cholera pathogenesis.
in its natural habitat is likely to involve
the excision and replication of the lysogenic phage followed by
infection of recipient V. cholerae strains, possibly
mediated by appropriate signals in the host intestine. Since the
present study did not provide any conclusive evidence to suggest that the induction of the phage occurs inside the intestine of the human
host, we speculate that induction of CTX in naturally occurring strains of toxigenic V. cholerae may also occur in the
environmental habitat. Our efforts are at present directed toward
understanding the role of possible environmental factors in the
induction and propagation of the CTX phage.
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ACKNOWLEDGMENTS |
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This research was funded by the U.S. Agency for International Development (USAID) under grant HRN-5986-A-00-6005-00 with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). The ICDDR,B is supported by countries and agencies which share its concern for the health problems in developing countries.
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Genetics Laboratory, Laboratory Sciences Division, ICDDR,B. G.P.O. Box 128, Dhaka-1000, Bangladesh. Phone: 880 2 871760. Fax: 880 2 872529 and 880 2 883116. E-mail: faruque{at}icddrb.org.
Editor: J. T. Barbieri
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Infect Immun, August 1998, p. 3752-3757, Vol. 66, No. 8
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