Characterization of Borrelia burgdorferi strains isolated from Korea by 16S rDNA sequence analysis and PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons.

Lee, Seung Hyun; Kim, Bumjoon J.; Kim, Jong Hyun; Park, Kyung Hee; Yeo, Seon Ju; Kim, Seo Jeong; Kook, Yoon Hoh

doi:10.1099/00207713-50-2-857

Cited by 10 publications

(11 citation statements)

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“…[18] was selected given their wide use [19,20]. It amplifies the variable spacer region between two conserved structures, the 3' end of the 5S rRNA (rrf) and the 5' end of the 23S rRNA (rrl).…”

Section: Controlmentioning

confidence: 99%

Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.

Rodríguez¹,

Burri²,

Noda³

et al. 2015

Ann Agric Environ Med.

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Rodríguez I, Burri C, Noda AA, Douet V, Gern L. Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. Ann Agric Environ Med. 2015; 22(4): 642-646. doi: 10.5604/12321966.1185767 Abstract Introduction. Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tickborne pathogens (TBP) is mainly by molecular techniques based on polymerase chain reactions (PCR). Objective. To design and evaluate a multiplex PCR for the molecular screening of zoonotic TBP for exploratory studies. Material and methods. Control DNA from reference strains, DNA from experimentally-infected biological specimens, and from Rhipicephalus sanguineus ticks collected from domestic and homeless dogs were used. A multiplex PCR assay to detect the presence of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. was designed and optimized using primers previously reported for B. burgdorferi sensu lato and Anaplasma spp., while for Babesia spp. they were designed in silico. The multiplex PCR was evaluated on the DNA from biological samples. Results. A new set of specific primers for Babesia spp. was designed. Adjustment of the master mix reactive concentrations and amplification conditions for the multiplex PCR allowed the successful amplification of the specific amplicons for each microbial group from the control DNA and experimentally-infected biological specimens. The efficiency of the multiplex PCR amplifying three DNA targets was confirmed. Individual and co-infection of Anaplasma spp. and Babesia spp. were detected in the R. sanguineus ticks from dogs. Conclusions. A multiplex PCR assay for the screening of three TBP is available. By using it, B. burgdorferi sensu lato, Anaplasma spp. and Babesia spp. can be detected accurately in one PCR reaction.

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Section: Controlmentioning

confidence: 99%

Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.

Rodríguez¹,

Burri²,

Noda³

et al. 2015

Ann Agric Environ Med.

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“…The MseI and DraI restriction patterns of the 5S-23S intergenic spacer amplicons of Haenam strains differed from those of other strains of B. burgdorferi sensu lato. Furthermore, in the phylogenetic tree based on 16S ribosomal DNA sequences, Haenam strains also formed a distinctive cluster (8).…”

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confidence: 99%

“…In previous studies, they were characterized as Borrelia afzelii, Borrelia garinii, and unclassified Haenam strains (8,10). In the present study, a comparative sequence analysis of the groEL gene from Korean isolates was performed to determine their relationships with the known species of the genus Borrelia.…”

mentioning

confidence: 99%

“…A previous study proposed that groEL gene analysis is useful for the differentiation of B. burgdorferi sensu lato (9). In the present study, groEL gene analysis was compared with the sequence analyses of the 16S rRNA and the flagellin gene to determine the role of the groEL gene in defining evolutionary relationships among strains of B. burgdorferi sensu lato.Twelve Borrelia strains were recently isolated from Ixodes granulatus, Ixodes nipponensis (tick vectors for Lyme spirochetes in rare cases), and Ixodes persulcatus, and 11 strains were isolated from Apodemus agrarius (8,10). In previous studies, they were characterized as Borrelia afzelii, Borrelia garinii, and unclassified Haenam strains (8,10).…”

mentioning

confidence: 99%

“…Twelve Borrelia strains were recently isolated from Ixodes granulatus, Ixodes nipponensis (tick vectors for Lyme spirochetes in rare cases), and Ixodes persulcatus, and 11 strains were isolated from Apodemus agrarius (8,10). In previous studies, they were characterized as Borrelia afzelii, Borrelia garinii, and unclassified Haenam strains (8,10).…”

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confidence: 99%

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Evaluation of groEL Gene Analysis for Identification of Borrelia burgdorferi Sensu Lato

Park¹,

Lee²,

Jeong³

et al. 2004

J Clin Microbiol

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The nucleotide sequences of the groEL genes, the flagellin genes, and the 16S rRNA genes from 22 reference strains of Borrelia were compared. groEL sequence analysis is useful not only in interspecies differentiation but also in intraspecies differentiation of Borrelia afzelii and Borrelia garinii isolates.Borrelia burgdorferi sensu lato, the causative spirochete of Lyme disease, is transmitted to humans and animals through Ixodes ticks (1, 3). Lyme disease is one of the most prevalent tick-borne infectious diseases in Europe and North America and now occurs all over the world (1, 12). Various DNA-based techniques have recently been developed for the species identification of B. burgdorferi because characterization and identification by conventional methods are time-consuming and expensive (2,5,6,11). A previous study proposed that groEL gene analysis is useful for the differentiation of B. burgdorferi sensu lato (9). In the present study, groEL gene analysis was compared with the sequence analyses of the 16S rRNA and the flagellin gene to determine the role of the groEL gene in defining evolutionary relationships among strains of B. burgdorferi sensu lato.Twelve Borrelia strains were recently isolated from Ixodes granulatus, Ixodes nipponensis (tick vectors for Lyme spirochetes in rare cases), and Ixodes persulcatus, and 11 strains were isolated from Apodemus agrarius (8,10). In previous studies, they were characterized as Borrelia afzelii, Borrelia garinii, and unclassified Haenam strains (8,10). In the present study, a comparative sequence analysis of the groEL gene from Korean isolates was performed to determine their relationships with the known species of the genus Borrelia.Twenty-two reference strains (Table 1) and 23 Korean isolates of the genus Borrelia were used in this study. The strains were cultivated at 32°C in Barbour-Stoenner-Kelly II (BSKII) medium. DNA was extracted by a modified version of a previously described method (4). The groEL genes of 22 reference strains and 23 Korean isolates and the flagellin genes and the 16S rRNA genes of 22 reference strains were amplified as presented in Table 2. The nucleotide sequences of the recombinant DNA were determined using the CEQ L DNA Analysis System and the CEQ 2000 Dye Terminator Cycle Sequencing kit (Beckman Coulter Inc., Fullerton, Calif.) with forward and reverse sequencing primers (M13) and sequencing primers ( Table 2). The multiple-alignment algorithm in the MegAlign software package (Windows version 3.12e; DNASTAR, Madison, Wis.) was used to align the sequences. All positions with alignment gaps were excluded from the pairwise sequence comparison. Phylogenetic trees were constructed by the unweighted pair group method with arithmetic averages using the MEGA program (7). A bootstrap analysis (100 replicates) was performed to evaluate the topology of the phylogenetic tree.In this study, interspecies differences in the groEL genes (positions 552 to 861 in B. burgdorferi B31 T numbering; 310 bp) of B. burgdorferi strains sensu lato were compared ...

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Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe

et al. 2004

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The genetic polymorphism of Borrelia burgdorferi and Borrelia afzelii, two species that cause Lyme borreliosis, was estimated by sequence typing of four loci: the rrs-rrlA intergenic spacer (IGS) and the outer-membrane-protein gene p66 on the chromosome, and the outer-membrane-protein genes ospA and ospC on plasmids. The major sources of DNA for PCR amplification and sequencing were samples of the B. burgdorferi tick vector Ixodes scapularis, collected at a field site in an endemic region of the north-eastern United States, and the B. afzelii vector Ixodes ricinus, collected at a similar site in southern Sweden. The sequences were compared with those of reference strains and skin biopsy isolates, as well as database sequences. For B. burgdorferi, 10-13 alleles for each of the 4 loci, and a total of 9 distinct clonal lineages with linkage of all 4 loci, were found. For B. afzelii, 2 loci, ospC and IGS, were examined, and 11 IGS genotypes, 12 ospC alleles, and a total of 9 linkage groups were identified. The genetic variants of B. burgdorferi and B. afzelii among samples from the field sites accounted for the greater part of the genetic diversity previously reported from larger areas of the north-eastern United States and central and northern Europe. Although ospC alleles of both species had higher nucleotide diversity than other loci, the ospC locus showed evidence of intragenic recombination and was unsuitable for phylogenetic inference. In contrast, there was no detectable recombination at the IGS locus of B. burgdorferi. Moreover, beyond the signature nucleotides that specified 10 IGS genotypes, there were additional nucleotide polymorphisms that defined a total of 24 subtypes. Maximum-likelihood and parsimony cladograms of B. burgdorferi aligned IGS sequences revealed the subtype sequences to be terminal branches of clades, and the existence of at least three monophyletic lineages within B. burgdorferi. It is concluded that B. burgdorferi and B. afzelii have greater genetic diversity than had previously been estimated, and that the IGS locus alone is sufficient for strain typing and phylogenetic studies.3These two authors contributed equally to the study.Abbreviations: IGS, intergenic spacer; LB, Lyme borreliosis; MLST, multilocus sequence typing.The GenBank accession numbers for the sequences reported in this paper are: AY275189-AY275212 for B. burgdorferi rrs-rrlA IGS types 1-9 and nt10, as well as subtypes of each IGS genotype; AY363692-AY363702 for B. afzelii rrs-rrlA IGS types 1-9, and nt10 and nt11; AY275213-AY275225 for B. burgdorferi ospC types 1-9 and nt10-nt13; and AY363710-AY363721 for B. afzelii ospC types 1-6, 7A, 7B, 8, 9, nt10 and nt11.Tables showing pairwise nucleotide and amino acid distances between ospC alleles and OspC proteins of both B. burgdorferi and B. afzelii are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.

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Characterization of Borrelia burgdorferi strains isolated from Korea by 16S rDNA sequence analysis and PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons.

Cited by 10 publications

References 23 publications

Multiplex PCR for molecular screening of <i>Borrelia burgdorferi</i> sensu lato, <i>Anaplasma</i> spp. and <i>Babesia</i> spp.

Multiplex PCR for molecular screening of <i>Borrelia burgdorferi</i> sensu lato, <i>Anaplasma</i> spp. and <i>Babesia</i> spp.

Evaluation of groEL Gene Analysis for Identification of Borrelia burgdorferi Sensu Lato

Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe

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