Analysis of the bmp gene family in Borrelia burgdorferi sensu lato

BmpA, BmpB, BmpC, and BmpD are homologous Borrelia burgdorferi lipoproteins of unknown functions, encoded by the bmp genes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of the bmp region has been characterized for a variety of B. burgdorferi sensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All four bmp genes were present in the same relative order in all B. burgdorferi sensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of the bmp genes in these species, variations in DNA sequence in the bmpD-bmpC and bmpC-bmpA intergenic regions were significantly more common than in the corresponding 3' bmpD and bmpC coding regions. The genetic structure of the chromosomal region containing the bmp genes thus appears to be well conserved across different species of B. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest that bmp gene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced by B. burgdorferi sensu lato.     

Specific immune response to a synthetic peptide derived from outer surface protein C of Borrelia burgdorferi predicts protective borreliacidal antibodies

In a previous study, we described the development of a new specific serodiagnostic test for Lyme disease involving enzyme-linked immunosorbent assay and a synthetic peptide, OspC-I. The OspC-I peptide is derived from part of the outer surface protein C (OspC) amino acid sequence of Borrelia burgdorferi and is located in the region conserved among B. burgdorferi sensu stricto or sensu lato isolates. In this study, we demonstrate that sera containing antibodies against OspC-I from patients with early Lyme disease had borreliacidal activity against isolates of three genospecies of Lyme disease spirochete, B. burgdoreferi B31, B. garinii HPI and B. afzelii HT61. However, the borreliacidal activity against B. burgdorferi, which has not been isolated in Japan, was weaker than that against the other species. Vaccination of mice with OspC-I induced the production of anti-OspC-I antibodies in serum with borreliacidal activity. The immune mouse serum had significantly higher levels of borreliacidal activity against HP1 and HT61, than against B31. Neutralization of borreliacidal activity with anti-IgM antibodies showed that the borreliacidal activity of anti-OspC-I antibodies in serum was due to IgM. Furthermore. mice vaccinated with OspC-I were protected against challenge with HPI and HT61. but not fully protected against infection with B31. These results suggest that OspC-I is not only a specific antigen for use in serodiagnostic tests for Lyme disease, but is also a potential candidate for a Lyme disease vaccine in Japan.     

Whole-genome sequences of thirteen isolates of Borrelia burgdorferi

Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.     

Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.

The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis.     

Molecular characterization of Borrelia spp. isolates from greater metropolitan Chicago reveals the presence of Borrelia bissettii. Preliminary report

Lyme Disease in the US is concentrated in three endemic areas: the Northeast, the upper mid-West, and the Pacific coast. In the mid-West, the range of Lyme disease has expanded to include large parts of Wisconsin and Minnesota. Despite its proximity to the mid-Western focus, Illinois, so far, has not been considered an endemic area. However, more recent data suggest that this situation may be changing. Also, the extent of borrelial diversity in the mid-West remains largely unexplored. Here, we present preliminary results on the molecular characterization of Borrelia isolates from rodents captured in Cook and Lake Counties, both of which are parts of the greater metropolitan Chicago area in Illinois. We investigated the rodent reservoir present in forested areas of suburban Chicago in order to determine the frequency of infection with the Lyme disease agent(s) by culture isolation of Borrelia spirochetes (Picken et al., unpublished). Rodent isolates of Borrelia were identified to the species level by genetic characterization. In total, 19 isolates were obtained over 3 years from NW Cook Co. and Lake Co. Pulsed-field gel electrophoretic analysis of Mlul digested DNA from these isolates showed macrorestriction patterns similar to that of the Californian isolate, strain DN127 (PF type I), New York isolate strain 25015 (PF type II), or a variant of the latter (PF type III). Sequence data generated from the rrf(5S)-rrl(23S) intergenic spacer region of the ribosomal RNA gene cluster confirmed the identity of all the Chicago isolates studied to date as B. bissettii. These strains are unlike our previous Borrelia isolates from NW Illinois and Wisconsin. In addition, there was a predominant association of B. bissettii infection with pratal rodent species such as Microtus pennsylvanicus and Zapus hudsonius. The relationship of this novel enzootic focus to the established mid-Western endemic focus of Lyme disease remains to be elucidated. The geographic range and reservoir diversity of this organism may have hitherto been underestimated.     

Strain variation, based on the hemagglutinin gene, in Norwegian ISA virus isolates collected from 1987 to 2001: indications of recombination.

Infectious salmon anemia (ISA) is caused by a virus that probably belongs to the Orthomyxoviridae and was first recorded in Norway in 1984. The disease has since spread along the Norwegian coast and has later been found in Canada, Scotland, the Faroe Islands, Chile, and the USA. This study presents sequence variation of the hemagglutinin gene from 37 ISA virus isolates, viz. one isolate from Scotland, one from Canada and 35 from Norway. The hemagglutinin gene contains a highly polymorphic region (HPR), which together with the rest of the gene sequence provides a good tool for studies of epizootics. The gene shows temporal and geographical sequence variation, where certain areas are dominated by distinct groups of isolates. Evidence of transmission of ISA virus isolates within and between regions is given. It is suggested that the hemagglutinin gene from different isolates may recombine. Possible recombination sites are found within the HPR and in the 5'-end flanking region close to the HPR.     

Lyme disease Borrelia spp. in ticks and rodents from northwestern China.

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

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.     

Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene

An internal fragment of the ddl gene, encoding the cytoplasmic enzyme D-alanyl-D-alanine ligase, was sequenced from 566 isolates of Streptococcus pneumoniae and single isolates of Streptococcus mitis and Streptococcus oralis. The 52 alleles found among the S. pneumoniae isolates fell into two groups. Group A alleles were very uniform in sequence and were present in both penicillin-susceptible and penicillin-resistant pneumococci. Group B alleles were much more diverse and were found only in penicillin-resistant isolates. The Streptococcus oralis and Streptococcus mitis alleles were less diverged from group A alleles than some of the group B pneumococcal alleles, suggesting that the latter alleles contain interspecies recombinational replacements. The ddl gene was located 783 bp downstream of the penicillin-binding protein 2b gene (pbp2b). Sequencing of the pbp2b-recR-ddl-murF region of three penicillin-resistant pneumococci that had diverged ddl alleles showed that the whole region from pbp2b to ddl (or beyond) was highly diverged (about 8%) compared with the sequences from three penicillin-susceptible isolates. The high levels of diversity in the group B ddl alleles from penicillin-resistant isolates were ascribed to a hitchhiking effect whereby interspecies recombinational exchanges at pbp2b, selected by penicillin usage, often extend into, or through, the ddl gene. The data allow the average size of the interspecies recombinational replacements to be estimated at about 6 kb.     

环介导恒温扩增对莱姆病病原伯氏疏螺旋体的分型鉴定

使用环介导恒温扩增技术,基于莱姆病病原伯氏疏螺旋体的外膜蛋白A(OspA)基因,针对伯氏疏螺旋体不同的基因型设计特异性引物,对国内主要的莱姆病病原伯氏疏螺旋体的3个基因型进行分型鉴定。研究结果表明,设计的引物具有良好的特异性,可以对狭义伯氏疏螺旋体(Borrelia burgdorferi sensu strict)、嘎氏疏螺旋体(B.afzelii)和伽氏疏螺旋体(B.garinii)进行分型鉴定。伯氏疏螺旋体的分型鉴定可以对不同临床症状莱姆病患者的治疗和莱姆病的控制提供一定的依据。     

Whole-genome sequences of two Borrelia afzelii and two Borrelia garinii Lyme disease agent isolates

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.     

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 characterization of Borrelia persica, the agent of tick borne relapsing fever in Israel and the Palestinian Authority

The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5'end of the 16S rRNA gene to the 5'end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5' fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area.     

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Abstract The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae . The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis , rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.     

Polymorphism of the TRAP gene of Plasmodium falciparum

Natural sequence variation of the thrombospondin related anonymous protein (TRAP) gene of Plasmodium falciparum has been investigated by DNA analysis following the polymerase chain reaction amplification, and this shows the gene to be highly polymorphic. The region containing the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), common to TRAP, the circumsporozoite protein, properdin, and thrombospondin, was invariant. Elsewhere in the molecule, over 50 amino acid substitutions are described including the insertion of an in-frame, small-variable tandemly repeating motif between amino acid residues 352 and 353. Only one silent mutation was observed. Most nucleotide changes that occur in the first two codon positions result in conservative amino acid changes. Restriction fragment length polymorphism (RFLP) analysis was used to examine inheritance of TRAP in a cross between the HB3 and 3D7 clones of P. falciparum. Out of nine progeny examined, four possessed the HB3 gene and five the 3D7 gene. The TRAP gene hybridized to chromosome 13. Previous work has shown that a subtelomeric region of chromosome 13 from the 3D7 parent (marked by the HRP-III gene) was favoured strongly in this cross. The TRAP gene, however, is over 1 Mb away from this subtelomeric region and exhibits no such linkage because of chromosome crossovers. Five geographically separate isolates shared the same TRAP sequence as well as the same variant of the Th2R/Th3R region from the circumsporozoite protein. The correlation between independent markers in these isolates suggests that they have a common provenance.     

Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi

Borrelia burgdorferi causes Lyme disease, a multisystem illness that can persist in humans for many years. We describe recombination between homologous genes encoding the major outer surface proteins (Osps) A and B of B. burgdorferi which both deletes osp gene sequences and creates chimaeric gene fusions. Recombinant osp genes occur in multiple strains and encode unique proteins that lack some characteristic Osp epitopes. Antigenic variation in Osp through recombination may be relevant to the persistence of B. burgdorferi in an infected host, and has important implications for the utility of OspA and OspB as diagnostic or vaccine candidates for Lyme disease. We also describe Osp variation arising from nonsense mutations and sequence divergence, which may also represent significant sources of Osp polymorphism.     

Lyme Disease Borrelia spp. in Ticks and Rodents from Northwestern China

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Phylogenetic analysis of coat protein gene of BYDV-MAV strain from wheat

Barley yellow dwarf disease is globally the most important viral disease of wheat. The full-length nucleotide sequence of coat protein (CP) gene of 12 isolates revealed the presence of three distinct clusters. Pakistani isolate of MAV (MAV-PK) has maximum similarity of 99.23% with MAV isolate of Morocco and PAV-Australia following 99.22 and 99.22% with PAV-France. Similar degree of similarity was found in comparison of amino acid sequence. The finding of this study is that MAV-PK has similarity with both MAV-France and PAV-Australia, which is due to the reason that both MAV and PAV belong to the same group and both share maximum nucleotide homology. Low genetic diversity was found not only between MAV isolates but also between MAV and PAV isolates because phylogenetic analysis was done on the CP gene which is highly conserved region in genome of Barley yellow dwarf viruses (BYDVs). Divergence in MAV-PK was due to this recombination which is now most prevalent in Pakistan. MAV-PK has maximum similarity with MAV-Morocco followed by MAV-Sweden and MAV-Cz, which seems to indicate that Pakistani isolate of MAV evolved as the result of recombination between MAV isolates of the USA and PAV isolates of Australia and France. At the same time, recombination of MAV-CZ and MAV-Sweden also occur. This work can be successfully utilised in epidemiological studies of MAV isolate in Pakistan. Further analysis of variation level in these isolates will help scientists to formulate appropriate management strategies like incorporation of BdV 2 gene in wheat against BYDVs.     

Stable marker on flagellin gene sequences related to arabinose non-assimilating pathogenic Burkholderia pseudomallei

Using PCR-based isolation and sequence analysis of the flagellin gene from two distinct biotypes of Burkholderia pseudomallei, a 15-bp deletion was found within the variable domain of the gene in isolates capable of assimilating arabinose (Ara+). This finding led to the development of a PCR-based method in order to differentiate and identify pathogenic B. pseudomallei for epidemiological study. A pair of specific primers was designed covering the 15-bp deletion region at the variable domain. PCR-amplification products of 176 and 191 bp in size were detected from 41 Ara+ isolates and 39 Ara - isolates of B. pseudomallei, respectively. Moreover, flagellin gene fragments of other bacterial species tested in this study were not amplified using these primers. The results suggest that the flagellin gene sequences of both B. pseudomallei biotypes in this region are stable and distinct. This method can be applied and useful for the epidemiological study of B. pseudomallei.     

Comparative genome hybridization reveals substantial variation among clinical isolates of Borrelia burgdorferi sensu stricto with different pathogenic properties

Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that all B. burgdorferi clinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven.