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Antimicrobial Agents and Chemotherapy, May 2000, p. 1315-1321, Vol. 44, No. 5
Department of Veterinary Microbiology, Royal
Veterinary and Agricultural University, Frederiksberg, DK-1870
Frederiksberg C,1 and Danish Veterinary
Laboratory, DK-1790 Copenhagen V,3 and
Armed Forces Research Institute of Medical Sciences, Bangkok
10400, Thailand2
Received 1 June 1999/Returned for modification 26 July
1999/Accepted 8 February 2000
In this study, 176 clinical and environmental Vibrio
cholerae strains of different O serotypes isolated in Thailand
from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB,
aadA2, blaP1, dfrA1, and
dfrA15 gene cassettes, which encode resistance to
gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; In the past, the prevalence of
antibiotic resistance of Vibrio cholerae was low and routine
susceptibility testing was not recommended (29, 35).
However, reports of V. cholerae strains resistant to
commonly used antibiotics are appearing with increasing frequency and
susceptibility testing is now recommended for monitoring of resistance
among toxigenic V. cholerae O1 and O139, the two serotypes
which cause cholera (4, 20, 23, 29, 54).
The importance of non-O1, non-O139 serotypes of V. cholerae
as causes of diarrhea are increasingly being recognized (13, 14,
18, 33, 41). Although high prevalences of
multiple-antibiotic-resistant non-O1, non-O139 strains have been
reported recently, little is known about the mechanisms of resistance
(14, 33, 41). Studies of non-O1, non-O139 strains from
Thailand showed that strains frequently contained small-size plasmids.
However, the plasmids did not appear to encode antibiotic resistance
(14, 18).
Antibiotic resistance genes may be acquired and transmitted through
several mechanisms, of which the acquisition of genes through mobile
genetic elements and dissemination through horizontal transfer are of
special interest. In addition to plasmids and conjugative transposons,
integrons have also been described as vehicles for the acquisition of
resistance genes (24, 44; for reviews, refer to
references 25, 38, and 46). Among the three classes of integrons that have been identified, class 1 integrons are prevalent among clinical isolates. Class 1 integrons were
originally defined as being composed of two conserved segments, the 5'
conserved segment (5'-CS) and the 3' conserved segment (3'-CS), and an
internal variable region which contains gene cassettes encoding
antibiotic resistance determinants (24, 44). The 5'-CS
contains the intI1 gene, which encodes the type 1 integrase. This integrase is responsible for site-specific insertion and excision
of gene cassettes (11). Also, the 5'-CS contains the attI1 site, which is responsible for recombination. The
3'-CS contains the qacE Some information is available about the distribution and importance of
class 1 integrons in encoding antibiotic resistance in bacterial
enteropathogens, and recent studies indicate that class 1 integrons may
be widespread in multiple-drug-resistant clinical isolates (26,
27, 30), e.g., Salmonella enterica Typhimurium
(40, 52). Dalsgaard et al. (15) characterized V. cholerae O1 isolated in Vietnam from 1979 to 1996 and
found that strains isolated after 1990 were resistant to sulfonamides and streptomycin and harbored class 1 integrons containing an aadA2 gene cassette. A comparison of phenotypic and
genotypic characteristics of the Vietnamese O1 strains isolated after
1990 with a distinct O1 strain recently found in Samutsakorn, Thailand, suggests that they belong to the same clone (15, 17).
However, it is unknown if the Thai V. cholerae O1 strain
also contains class 1 integrons (17). Recently, Falbo et al.
(21) found chromosomally located class 1 integrons
containing the aadA1 gene cassette among V. cholerae O1 strains isolated during a cholera outbreak in Albania
and Italy in 1994.
In this background, the objective of the present study was to determine
the distribution and importance of class 1 integrons in encoding
antibiotic resistance among a large collection of different V. cholerae O serotypes isolated from clinical and environmental sources in Thailand. As the vast majority of strains containing class 1 integrons are resistant to sulfonamides, we selected strains with
different patterns of susceptibility to sulfonamides and tested them
for the presence of class 1 integrons. Our study showed that integrons
were present in V. cholerae O1 and non-O1, non-O139 serotypes of mainly clinical origin, containing gene cassettes encoding
resistance to aminoglycosides, Bacterial strains.
A total of 176 V. cholerae
strains, mainly isolated in Thailand, were tested for the presence and
contents of class 1 integrons. The strain collection was selected from
a large number of V. cholerae strains studied previously for
various phenotypic and genotypic characteristics (5, 14,
16-19). The majority of strains were sulfonamide resistant, but
strains showing intermediate resistance or susceptibility to
sulfonamides were also studied. Further strain information is provided
in Results.
Transfer of resistance and characterization of plasmids.
The
nalidixic acid-resistant mutant Escherichia coli K-12 strain
J53-1 (lac+ pro met Nalr)
(50) was used as the recipient in conjugation experiments with the plasmid-containing strains 1075/25, 1076/25, and 64/26 (49). After mating on nonselective L agar incubated at
37°C for 4 to 6 h of growth, exconjugants were harvested and
appropriate dilutions were spread on plates of Fluorocult E. coli O157:H7 agar (Merck, Virum, Denmark) supplemented with
nalidixic acid at 50 µg/ml and adequate concentrations of selected
drugs. Phenotypic appearance was used to differentiate possible
spontaneous nalidixic acid-resistant donors from the exconjugants.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Distribution and Content of Class 1 Integrons in
Different Vibrio cholerae O-Serotype Strains Isolated
in Thailand
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the
antibiotic resistance expressed by the strains. Serotype O139 strains
did not contain class 1 integrons. However, the appearance and
disappearance of the O139 serotype in the coastal city Samutsakorn in
1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2
resistance gene cassette. A 150-kb self-transmissible plasmid found in
three O1 strains isolated in 1982 contained the aadB gene
cassette. Surprisingly, several strains harbored two integrons
containing different cassettes. Thus, class 1 integrons containing
various resistance gene cassettes are distributed among different
V. cholerae O serotypes of mainly clinical origin in Thailand.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 and sul1 genes, which
encode resistance to quaternary ammonium compounds and to sulfonamides,
respectively. However, class 1 integrons do not always contain the
entire 3'-CS (38, 46).
-lactams, and trimethoprim.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
PCR amplification and sequencing of integrons.
Figure
1 shows the general structure of class 1 integrons. For PCR amplification, we initially selected a specific
class 1 primer set, qacE1 and sul1, directed at the 3'-CS of class 1 integrons (37, 38). However, as a few class 1 integrons do not contain the qacE1 and sul1 genes, these
would not be detected using the selected primers. PCR was carried out
by suspension of one bacterial colony in 100 µl of sterile distilled
H2O, which was then boiled for 15 min at 95°C. After
lysis, the suspension was centrifuged at 13,000 rpm (Biofuge 13;
Heraeus Sepatech) for 2 min and stored at 
20°C. For each PCR, 5 µl of the lysis suspension was used. The PCR amplification was
performed by the method of Aarestrup et al. (1) and
optimized to detect amplicons in the range of 300 to 1,500 bp. The
annealing temperature was set at 58°C. Strains yielding a PCR product
with the class 1 primers were further amplified with the integron
primers in-F and in-B (F is forward, and B is backward), which amplify
the region between the 5'-CS and the 3'-CS, yielding products of
various sizes, depending on the number and length of the inserted gene
cassettes (Fig. 1 and Table 1). The in-B
primer anneals at the 3'-CS. Primers in-F and addA-B were used to
determine if the integron contained a gene cassette encoding resistance
to streptomycin and spectinomycin (Table 1) (31). Primers
intI1-F and blaP1-B were used to determine if the integron contained
the -lactam resistance gene cassette using an annealing temperature
of 52°C (55). All of the PCR primers used are listed in
Table 1. S. enterica serotype Typhimurium strain 9720921 and
Acinetobacter sp. strain R4-96 were included as positive and
negative controls, respectively (40). A 100-bp molecular
mass standard (GIBCO BRL, Gaithersburg, Md.) was used as a size marker
during electrophoresis of PCR products.
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Nucleotide sequence accession numbers. The nucleotide sequences of the aadA2, dfrA1 and orf, dfrA15, aadB, and blaP1 gene cassettes have been assigned GenBank accession no. AF221903, AF221901, AF221900, AF221902, and AF221899, respectively.
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RESULTS |
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Materials and Methods
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Discussion
References
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An approximately 800-bp 3'-CS PCR product was obtained from 44 of
the 176 V. cholerae strains studied by using the primers qacE1-F and sul1-B. However, the majority of epidemiologically unrelated clinical strains which showed resistance to sulfonamides did
not, based on lack of PCR amplification, contain the sul1 gene. Only a single environmental strain amplified this region. The
phenotypic characteristics and resistance gene cassettes found in
strains containing class 1 integrons are further described below (see
also Tables 2 and 3).
V. cholerae non-O1, non-O139 strains isolated at the Children Hospital, Bangkok. Sixty-nine V. cholerae non-O1, non-O139 strains recovered from children as part of a cholera surveillance conducted from August 1993 to July 1995 at the Children Hospital in Bangkok were studied (14). These strains represented 37 different O serotypes, of which none contained the cholera toxin gene. The strains were originally tested for susceptibility to 12 antibiotics by disk diffusion including ampicillin, chloramphenicol, ciprofloxacin, colistin, gentamicin, kanamycin, nalidixic acid, neomycin, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole (14). Of 40 strains that were resistant to colistin, streptomycin, and sulfisoxazole, 28 showed further resistance and were designated multiply resistant (14).
Nine of the V. cholerae non-O1, non-O139 strains, all resistant to sulfonamides, contained class 1 integrons (Table 2). A PCR product of approximately 739 bp was obtained from each of five strains using the in-F and in-B primers (Fig. 2 and Table 2). DNA sequencing showed that the 739-bp amplicon of strain VO-556 contained a trimethoprim gene cassette, as well as 5'-CS and 3'-CS sequences (GenBank accession no. AF221900). The gene cassette showed 100% identity to the dfrA15 (dhfrXV) gene cassette encoding resistance to trimethoprim (GenBank accession no. Z83311) (2). RFLP analysis of the 739-bp amplicon using HindIII suggested that each of the five strains contained the dfrA15 gene cassette (Table 3). Repeated PCR analysis of the five strains with the in-F and blaP1-B primers (Fig. 2) and the blaP1-F and in-B primers yielded 762- and 853-bp products, respectively, corresponding to the conserved regions of the integron, together with the blaP1 (pse1) gene cassette, that encode a-lactamase (Table 1) (55). DNA sequencing
of the products (GenBank accession no. AF221899) confirmed these
findings, showing 100% identity to the blaP1 gene (GenBank accession no. Z18955 and AF071555) (9, 55). Surprisingly, we did not obtain two different-size amplicons when using
the in-F and in-B primers. As shown below, the integron containing the
blaP1 gene yielded an 1,197-bp amplicon when the in-F and
in-B primers were used (Table 2). We cannot explain why only one
amplicon was obtained.
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-lactamase PSE-1
(Fig. 2; Tables 1 and 2) (55). DNA sequencing of the
1,197-bp product from strain VO-3198 confirmed these findings, showing
100% identity to the blaP1 gene and the 5'CS and 3'-CS
sequences (GenBank accession no. Z18955 and AF071555) (9,
55). Digestion with MfeI, which had a single
restriction site in the 1,197-bp fragment, but none in the 1,009-bp
amplicon, demonstrated identical fragments for all three of the
strains, suggesting that these strains contained the same 764-bp
-lactamase-encoding blaP1 cassette (Table 3) (40).
V. cholerae non-O1, non-O139 strains recovered from patients in Thailand. Nineteen V. cholerae non-O1, non-O139 strains were selected from 41 strains recovered from stool samples from patients with diarrhea (18), including 13 strains isolated in 1990 during an epidemic of a cholera-like disease among Khmer refugees in a camp in Aranyaprathet, Thailand (5).
Ten sulfonamide-resistant non-O1, non-O139 strains isolated from the Khmer refugees contained class 1 integrons each yielding PCR amplicons of 1,009 and 1,237 bp using the in-F and in-B primers, whereas strain 292/90 yielded a single product of 1,237 bp (Fig. 2 and Table 2). PCR with the in-F and aadA-B primers confirmed the presence of an aadA gene cassette (Fig. 2; Tables 1 and 2). DNA sequencing showed that the 1,237-bp amplicon of strain 292/90 showed 100% identity to the dfrA1 (dhfrI) gene encoding resistance to trimethoprim (GenBank accession no. X00926) (22) and an open reading frame downstream of the dfrA1 gene (GenBank accession no. AF221901). This open reading frame was previously noted by Sundström and Sköld (48). Digestion with ClaI, which had a single restriction site on the 1,237-bp amplicon, suggested that each strain contained the dfrA1 gene cassette (Table 3). The remaining strains were intermediately resistant or sensitive to sulfonamides (18).V. cholerae O1 strains recovered from patients in Samutsakorn, Thailand. Thirty-one clinical V. cholerae O1 strains were selected from 70 strains isolated from 1982 to 1996 in Samutsakorn, a port city 30 km southwest of Bangkok (17). We showed in a previous study that the disappearance of V. cholerae O139 from Samutsakorn was associated with distinct phenotypic and genotypic changes in O1 strains isolated during and after the O139 epidemic, including the development of resistance to streptomycin and sulfisoxazole (17).
Twenty V. cholerae O1 strains isolated during and after the O139 epidemic, but also within a 10-months period just before the appearance of the O139 serotype, showed resistance to sulfonamides and streptomycin and contained class 1 integrons (Fig. 2; Table 2). PCR and RFLP analysis as described above confirmed the presence of the aadA2 aminoglycoside resistance gene cassette (Table 2). One strain isolated in October 1991 showed resistance to sulfonamides and streptomycin but did not contain class 1 integrons. Strains 1075/25, 1076/25, and 64/26 were isolated by Tabtieng et al. (49) during a cholera outbreak among children in 1982 and found to carry a conjugative 150-kb plasmid conferring multiple-drug resistance. Each strain contained class 1 integrons, and the use of the in-F and in-B primers yielded a PCR product of 744 bp (GenBank accession no. AF221902) (Table 2). DNA sequencing and RFLP analysis with SphI revealed that the 744-bp amplicon of each of the strains contained the aadB gene cassette, as well as 5'-CS and 3'-CS sequences. This gene cassette showed 100% identity to the aadB gene, which encodes resistance to gentamicin, kanamycin, and tobramycin (GenBank accession no. L06418) (Table 3) (10). Conjugation of the strains with E. coli K-12 strain J53-1 yielded one type of exconjugant which showed identical antibiograms and contained the 150-kb plasmid, suggesting that the entire antibiotic resistance pattern was plasmid encoded (Table 2). Further, PCR of the exconjugants yielded the 744-bp amplicon, showing that the aadB gene cassette was located and transferred on the 150-kb plasmid.Clinical V. cholerae O139 strains and environmental non-O1, non-O139 strains isolated from shrimp aquaculture and seafood. Class 1 integrons were not found among 11 representative V. cholerae O139 strains tested which showed resistance to furazolidone, streptomycin, trimethoprim, and sulfonamides and included strains isolated from patients in Bangladesh, India, and Thailand from 1992 to 1993 (19). Thus, the antibiotic resistances shown by the O139 strains were not associated with this type of integron.
Of 34 V. cholerae non-O1, non-O139 strains selected among 93 strains isolated from water, sediment, and shrimp in a shrimp production area in Thailand (16), none contained class 1 integrons. The strains tested included 23, 6, and 5 strains showing resistance (inhibition zone diameters of <19 mm), intermediate resistance (inhibition zone diameters between 20 and 22 mm), and susceptibility (inhibition zone diameters of
23 mm) to sulfonamides,
respectively, when tested in disk diffusion assays by the Bauer-Kirby
method (7, 18).
Twelve V. cholerae non-O1, non-O139 strains were selected
among 23 strains isolated from seafood samples in Samutsakorn, Thailand (18). Ten and two strains were intermediate and fully
resistant to sulfonamides, respectively (18). One
sulfonamide-resistant strain contained two class 1 integrons containing
amplicons of 1,009 or 1,197 bp (Table 2). PCR and RFLP analysis
suggested that the amplicons found in the seafood isolate were
identical to amplicons found in strains VO-3198, VO-4967, and VO-5398
from the Children Hospital (Tables 2 and 3). Thus, each of these strains seems to contain the aadA2 and blaP1 gene
cassettes (Fig. 2; Tables 2 and 3).
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DISCUSSION |
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Materials and Methods
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In the present study, we showed that class 1 integrons containing
antibiotic resistance gene cassettes were found mainly in epidemiologically related V. cholerae O serotypes isolated
from outbreaks of diarrhea in Thailand. However, several
epidemiologically unrelated V. cholerae non-O1, non-O139
strains isolated mainly from children with diarrhea also contained
class 1 integrons. Obtained by PCR and DNA sequencing, the findings of
aadB, aadA2, blaP1, dfrA1,
and dfrA15 resistance gene cassettes encoding resistances to
gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; -lactams; and trimethoprim, respectively, represent the first report
of the distribution of class 1 integrons among different V. cholerae O serotypes. The gene cassettes reported here corroborate previous findings of the content of class 1 integrons in
Enterobactericeae and Pseudomonas spp., in which
antibiotic resistance gene cassettes often encode resistances to
aminoglycosides (32, 39, 42), -lactams (40,
45), and trimethoprim (2, 48).
It should be noted that the majority of resistance genes,
including determinants of resistance to ampicillin, chloramphenicol, neomycin, streptomycin, trimethoprim, and tetracycline, were not contained within class 1 integrons. Thus, although widely
distributed, resistance genes on class 1 integrons seem to encode only
a part of the antibiotic resistance reported in V. cholerae. As our PCR analysis was designed to detect
relatively smaller-size amplicons, larger arrays of gene
cassettes may have been missed. However, it should be noted that all of
the strains yielding an amplicon with the primers qacE1-F and sul1-B
also showed a product when the in-F and in-B primers were used.
Thus, it seems unlikely that larger arrays of gene cassettes were present.
There was a clear difference in the distribution and frequency of class 1 integrons among clinical and environmental V. cholerae non-O1, non-O139 strains, as only a single environmental strain recovered from seafood in Samutsakorn contained a class 1 integron. The non-O1, non-O139 strains from the shrimp production area in Thailand were isolated from ponds and coastal areas (16) and showed less antibiotic resistance than the clinical non-O1, non-O139 strains (unpublished results). It is likely that the higher frequency of class 1 integrons in clinical isolates is because such strains became dominant through the selective pressure caused by the therapeutic use of antibiotics.
Bagchi et al. (5) reported that during the cholera-like epidemic among the Khmers in 1982, children and pregnant women were treated with trimethoprim-sulfamethoxazole. Further, Dalsgaard et al. (18) showed that a high percentage (92%) of the non-O1, non-O139 Khmer outbreak strains showed resistance to trimethoprim-sulfamethoxazole. Thus, it is likely that the strains that acquired the dfrA1 gene cassette became predominant through selective pressure. The finding that non-O1, non-O139 strains showing resistance to several antibiotics also contained resistance gene cassettes located on integrons is of concern. The finding of trimethoprim resistance gene cassettes, including the recently described dfrA15 gene cassette (2), is of special concern, as trimethoprim is often used to treat diarrhea among children and pregnant women.
The appearance of the O139 serotype in Samutsakorn in 1993 and its sudden disappearance in 1994 were associated with the emergence of a sulfonamide-resistant V. cholerae O1 strain carrying a class 1 integron which contained the aadA2 resistance gene cassette. We reported earlier that this distinct O1 strain showed unique genotypes, as demonstrated by ribotyping, PFGE typing, and cholera toxin typing (17). Interestingly, Dalsgaard et al. (15) found that O1 strains isolated in Vietnam after 1990 also contained class 1 integrons which harbored the aadA2 gene cassette and showed a ribotype R1 identical to the ribotype seen among O1 strains isolated in Samutsakorn (17). Thus, it is likely that this distinct O1 strain was transferred between Thailand and Vietnam and became established as the main strain causing cholera. It remains to be determined how this strain acquired the class 1 integron and the aadA2 gene cassette and/or if the strain was introduced from a third country.
Although the class 1 integrons were first described by Stokes and Hall in 1989 (44), our findings of the aadB resistance gene among the three O1 strains isolated in 1982 from the cholera outbreak at the pediatric ward in Samutsakorn show that class 1 integrons have been present in V. cholerae in Thailand for a number of years (49). The demonstration of the class 1 integron's being located on a 150-kb self-transmissible plasmid is, to our knowledge, the first report of a plasmid containing integrons in V. cholerae. Tabtieng et al. (49) reported that the 150-kb plasmid in each of the three strains belonged to incompatibility group C and contained genes coding for type II dihydrofolate reductase. The genes encoding the type II dihydrofolate reductase were not found as a class 1 gene cassette. However, after 1982, this plasmid and other plasmids were not found in O1 strains isolated in Samutsakorn or elsewhere, suggesting that the 150-kb plasmid containing the class 1 integrons was lost (17). Other studies have reported multiple-drug-resistant V. cholerae O1 encoded by conjugative incompatibility group C plasmids (51). However, it remains to be shown if these or other plasmids in V. cholerae contain class 1 integrons.
Interestingly, class 1 integrons were not found among any V. cholerae O139 strains. Thus, the 62-kb self-transmissible transposon-like SXT element described by Waldor et al. (53) among O139 strains, some of which were included in the present study, does not seem to carry class 1 integrons. The V. cholerae O1 El Tor strain which re-emerged in India and Bangladesh after the O139 epidemic were resistant to sulfamethoxazole, trimethoprim, and streptomycin (4, 34). Waldor et al. (53) showed that these O1 strains also contained the SXT element. It remains to be determined if the O1 strains from India and Bangladesh contain class 1 integrons.
Our findings show that class 1 integrons containing several different antibiotic resistance gene cassettes were distributed among different clinical V. cholerae O1 and non-O1, non-O139 serotypes in Thailand. Thus, PCR mapping of integrons and DNA sequencing of their genetic contents may be a useful epidemiological tool with which to study the evolution of multiresistance plasmids and dissemination of antibiotic resistance genes within V. cholerae. Although the resistance genes within the integrons encoded only a minor part of the antibiotic resistance detected, further studies in Thailand and elsewhere are needed to determine the importance of class 1 integrons in the horizontal acquisition and dissemination of antibiotic resistance genes.
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ACKNOWLEDGMENTS |
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We are grateful for the technical assistance provided by Anne-Mette Petersen at the Royal Veterinary and Agricultural University. We also thank Lorrin Pang and Peter Echeverria at the Armed Forces Research Institute of Medical Sciences for critical revision of the manuscript and provision of strains.
Dorthe Sandvang and Anders Dalsgaard were supported by the Danish Agricultural and Veterinary Research Council (grant 9600012) and the Danish Council for Development Research (DANIDA grant 90928), respectively.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Bülowsvej 13, 1870 Frederiksberg C, Denmark. Phone: 45-35-282720. Fax: 45-35-282757. E-mail: ad{at}kvl.dk.
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Discussion
References
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