rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.     

Local variations in the distribution and prevalence of Borrelia burgdorferi sensu lato genomospecies in Ixodes ricinus ticks.

Unfed nymphal and adult Ixodes ricinus ticks were collected from five locations within the 10,000-ha Killarney National Park, Ireland. The distribution and prevalence of the genomospecies of Borrelia burgdorferi sensu lato in the ticks were investigated by PCR amplification of the intergenic spacer region between the 5S and 23S rRNA genes and by reverse line blotting with genomospecies-specific oligonucleotide probes. The prevalence of ticks infected with B. burgdorferi sensu lato was significantly variable between the five locations, ranging from 11.5 to 28.9%. Four genomospecies were identified as B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, and VS116. Additionally, untypeable B. burgdorferi sensu lato genomospecies were identified in two nymphs. VS116 was the most prevalent of the genomospecies and was identified in 50% of the infected ticks. Prevalences of B. garinii and B. burgdorferi sensu stricto were similar (17 and 18%, respectively); however, significant differences were observed in the prevalence of these genomospecies in mixed infections (58.8 and 23.5%, respectively). Notably, the prevalence of B. afzelii was low, comprising 9.6 and 7.4%, respectively, of single and mixed infections. Significant variability was observed in the distribution and prevalence of B. burgdorferi sensu lato genomospecies between locations in the park, and the diversity and prevalence of B. burgdorferi sensu lato genomospecies was typically associated with woodland. The distributions of B. burgdorferi sensu lato genomospecies were similar in wooded areas and in areas bordering woodland, although the prevalence of B. burgdorferi sensu lato infection was typically reduced. Spatial distributions vegetation composition, and host cenosis of the habitats were identified as factors which may affect the distribution and prevalence of B. burgdorferi sensu lato genomospecies within the park.     

Detection and typing of Borrelia burgdorferi sensu lato genospecies in Ixodes persulcatus ticks in West Siberia, Russia

The prevalence of Borrelia burgdorferi sensu lato (s.l.) genospecies in West Siberia as well as in many other regions of Russia remains insufficiently investigated. In the present study a total of 151 adult female ticks Ixodes persulcatus Schulze, collected at three localities in eastern regions of West Siberia, where Lyme disease is endemic, were examined for the presence of the spirochete B. burgdorferi s.l. by polymerase chain reaction targeting the 23S-5S rRNA intergenic spacer regions. Spirochetal DNA was detected in on average 15.2+/-3.0% of the ticks examined. The infection rate of adult ticks with B. burgdorferi s.l. at various localities ranged from 8.6+/-3.4% to 29.0+/-7.6%, being greatest in the northernmost site studied and decreasing southwards. The restriction patterns obtained after MseI digestion of the 23S-5S rRNA intergenic spacer amplicons assigned 23 DNA samples to the following genomic groups: 19 to B. garinii (12 to group NT29 and seven to group 20047(T)), three to B. afzelii, and one to mixed B. afzelii and B. garinii NT29. We have not detected other genospecies, which were found in ticks in Europe, the Russian Far East and Japan. Thus, the ticks examined were associated only with two genospecies of Borrelia burgdorferi s.l. pathogenic to humans (B. garinii and B. afzelii), and B. garinii was the major genospecies infecting adult I. persulcatus in eastern regions of West Siberia.     

Comparison of the spirochete Borrelia burgdorferi S. L. isolated from the tick Ixodes scapularis in southeastern and northeastern United States

Thirty-five strains of the Lyme disease spirochete Borrelia burgdorferi sensu lato (B. burgdorferi s. l.) were isolated from the blacklegged tick vector Ixodes scapularis in South Carolina, Georgia, Florida, and Rhode Island. They were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. PCR-RFLP analysis indicated that the strains represented at least 3 genospecies (including a possible novel genospecies) and 4 different restriction patterns. Thirty strains belonged to the genospecies B. burgdorferi sensu stricto (B. burgdorferi s. s.), 4 southern strains were identified as B. bissettii, and strain SCCH-5 from South Carolina exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Complete sequences of rrf-rrl intergenic spacers from 14 southeastern and northeastern strains were determined and the phylogenetic relationships of these strains were compared. The 14 strains clustered into 3 separate lineages on the basis of sequence analysis. These results were confirmed by phylogenetic analysis based on 16S rDNA sequence analysis.     

Rapid identification of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis of groEL gene

Thirty-one reference strains and 23 Korean isolates of the genus Borrelia were identified through the PCR-RFLP analysis using the groEL gene. This will be useful for the rapid differentiation of B. burgdorferi sensu lato and complements one of the 5S-23S intergenic spacers.     

Characterization of Borrelia afzelii isolated from Ixodes nipponensis and Apodemus agrarius in Chungju,Korea, by PCR-rFLP analyses of ospC gene and rrf (5S)-rrl (23S) intergenic spacer

Nine Borrelia afzelii strains, which had been isolated from two vectors, Ixodes nipponensis and Apodemus agrarius in Chungju, Korea, were characterized by PCR-RFLP analyses of ospC genes and rrf (5S)-rrl (23S) intergenic spacer. DraI restriction patterns of Chungju strains were identical to those of B. afzelii VS461. But MseI restriction patterns of rrf (5S)-rrl (23S) intergenic spacer genes of KK2, KM4, KK5 differed from those of previously reported B. burgdorferi sensu lato strains. Nine Chungju strains were classified with four distinct ospC RFLP patterns, which differed from the eight ospC RFLP patterns (A-1 to A-8) of previously reported B. afzelii. Moreover, five additional restriction patterns were deduced from published ospC sequences of reference strains. These results suggest that Chungju strains are very heterogeneous.     

First molecular detection of Borrelia afzelii in clinical samples in Korea

Borrelia afzelii nucleic acids were detected in the sera of febrile disease patients by a nested PCR that targeted the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. The B. afzelii-specific DNA fragment was detected in 8 out of 283 sera which were proven to have immunoglobulin G or M antibodies against B. burgdorferi antigens through IFA. The results were further confirmed through restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated for the first time that Lyme borreliosis is prevalent in Korea.     

Lyme Disease Borrelia Species in Northeastern China Resemble Those Isolated from Far Eastern Russia and Japan

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.     

Molecular phylogenetic analysis of ixodid ticks based on the ribosomal DNA spacer, internal transcribed spacer 2, sequences

An internal transcribed spacer (ITS2) sequence between the 5.8S and 28S rRNA genes was used to estimate the phyletic relationships among Ixodes spp. tick vectors of Lyme disease-causing Borrelia spirochetes. Analysis indicates that Borrelia burgdorferi sensu lato species associated with Lyme disease are found mainly in ticks of the Ixodes ricinus species complex. Other closely related tick species are not known to transmit the Borrelia-that cause Lyme disease in humans, but they appear to have a specific association with other closely related Borrelia species. There is a high degree of concordance in the phylogenetics of Borrelia taxa and the phylogenetic relationships among Ixodes ticks.     

A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.     

Identification of a new Borrelia species among small mammals in areas of northern Spain where Lyme disease is endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.     

Evolution of the Borrelia burgdorferi outer surface protein OspC.

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.     

Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Francisella tularensis and their co-infections in host-seeking Ixodes ricinus ticks collected in Serbia

To evaluate the prevalence rate of tick-borne bacterial pathogens, unfed adult Ixodes ricinus ticks were collected from vegetation in 2001, 2003, and 2004 at 18 localities throughout Serbia. A total of 287 ticks were examined by PCR technique for the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Francisella tularensis. The highest prevalence rate was that for B. burgdorferi sensu lato (42.5%), followed by A. phagocytophilum (13.9%) and F. tularensis (3.8%). The presence of five B. burgdorferi sensu lato genospecies, namely, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana was identified by restriction fragment length polymorphism (RFLP) analysis. The most frequent B. burgdorferi sensu lato genospecies was B. lusitaniae, followed by B. burgdorferi sensu stricto. Co-infection by B. burgdorferi sensu stricto and B. lusitaniae was frequently observed. Co-infection by B. burgdorferi sensu lato and A. phagocytophilum and co-infection by B. burgdorferi sensu lato and F. tularensis appeared in 24 ticks. Sequencing of p44/msp2 paralogs of Serbian A. phagocytophilum showed that they were unique and distinct from those of A. phagocytophilum in US and UK. This is the first report of B. garinii, B. lusitaniae, B. valaisiana, as well as A. phagocytophilum and F. tularensis infected ticks in Serbia. These findings indicate a public health threat in Serbia of tick-borne diseases caused by B. burgdorferi sensu lato, A. phagocytophilum and F. tularensis.     

Rapid introduction of Lyme disease spirochete, Borrelia burgdorferi sensu stricto, in Ixodes scapularis (Acari: Ixodidae) established at Turkey Point Provincial Park, Ontario, Canada

Borrelia burgdorferi sensu stricto (s.s.) was isolated from questing adult Ixodes scapularis Say ticks collected from Turkey Point Provincial Park (TPPP), Ontario, Canada during 2005-2006. DNA from ten (67%) of 15 pools of ticks was confirmed positive for B. burgdorferi s.s. using polymerase chain reaction (PCR) by targeting the rrf (5S)-rrl (23S) intergenic spacer region and OspA genes. This significant infection rate indicates an accelerated development of B. burgdorferi s.s. in TPPP, because this pathogen was not detected five years previously during sampling of the three motile life stages of I. scapularis. Our study provides the initial report of the presence of B. burgdorferi s.s. in TPPP, which is now endemic for Lyme disease. Ultimately, people and domestic animals are at risk of contracting Lyme disease when they frequent this park.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

The first isolation of Borrelia burgdorferi sensu stricto in Russia

18 Borrelia isolates obtained from adult ticks of the Ixodes ricinus species, collected from different plants in April 2000 in the Khostinsk region of the Krasnodar Territory in the vicinity of the health resort of Matsesta not far from Sochi, were identified by means of PCR and the analysis of polymorphism of the restriction fragments of ribosomal rrf-rrl spacer amplicon. Among them, in addition to Borrelia species, found in Russia earlier (B. garinii, B. afzelii, B. valaisiana and B. lusitaniae), the classical causative agent of Lyme borreliosis (B. burgdorferi sensu stricto) was detected for the first time. The isolated strain (lr-4721) is now kept in the collection of the Borreliosis Center of the Ministry of Health of Russia (the Laboratory of Infections Vectors at the Gamaleya Research Institute of Epidemiology and Microbiology). The probable role of this infective agent in infectious pathology in Russia is discussed.     

Phenotypic and genotypic analysis of<Emphasis Type="Italic">Borrelia</Emphasis> spp. isolated from<Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks by using electrophoretic chips and real-time polymerase chain reaction

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.