Characterization of Lyme disease spirochetes isolated from ticks and vertebrates in North Carolina

Borrelia burgdorferi isolates obtained from numerous locations and from different hosts in North Carolina, were compared to previously characterized strains of the Lyme disease spirochete and other Borrelia spp. The spirochete isolates were confirmed to be B. burgdorferi sensu stricto based on immunofluorescence (IFA) using a monoclonal antibody to outer surface protein A (Osp A [H5332]) and polymerase chain reaction (PCR) using a species-specific nested primer for a conserved region of the gene that encodes for flagellin. In addition, the isolates tested positive in Western blots with species-specific monoclonal antibodies for outer surface protein A and OspB (84c), and the genus-specific, monoclonal antibody to flagellin (H9724). Infectivity studies with several of these isolates were conducted using Mus musculus and Oryzomys palustris and the isolates exhibited markedly different levels of infectivity. This study demonstrates that B. burgdorferi sensu stricto is present and naturally transmitted on the Outer Banks and in the Coastal Plain and Piedmont regions of North Carolina.     

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.     

Molecular characterization of Borrelia isolates from ticks and mammals from the southern United States

Fifty Borrelia isolates from ticks and rodents from several geographic regions of the southern United States were analyzed by genomic macrorestriction analysis. Significant genetic diversity was observed among them. These isolates segregated into 4 major clusters and 10 subclusters, which are correlated with the genospecies distribution. Nineteen pulsed-field gel electrophoresis (PFGE) types were recognized among the isolates. The genospecies Borrelia andersonii and Borrelia bissettii consisted of 5 and 2 subclusters, respectively. Two subclusters comprised the Borrelia burgdorferi sensu stricto (s. s.) strains. These results indicated that PFGE is a suitable molecular typing method for B. burgdorferi at both the genospecies and strain levels. Seventeen representative isolates from different PFGE groups were analyzed by restriction fragment length polymorphism (RFLP) and sequence analysis of flaB. Twenty-three AluI, 3 CelII, and 11 DdeI RFLP patterns were found among strains from the B. burgdorferi sensu lato (s. l.) complex and the relapsing fever borreliae complex. Three genospecies in the B. burgdorferi s. l. complex and 1 species in the relapsing fever borreliae complex were recognized. Phylogenetic analysis based on nucleotide sequences of flaB indicated that all the Borrelia strains analyzed here could be divided into 2 parts, i.e., B. burgdorferi s. l. complex and the relapsing fever borreliae complex. The flaB appears to be a useful target gene to screen and identify strains from both B. burgdorferi s. l. and relapsing fever borreliae complexes.     

Comparative analysis of Borrelia isolates from southeastern USA based on randomly amplified polymorphic DNA fingerprint and 16S ribosomal gene sequence analyses

Fifty-three southern USA Borrelia isolates were characterized using randomly amplified polymorphic DNA fingerprinting analysis (RAPD). Twenty-nine types were recognized among 37 B. andersonii strains, seven types among eight B. bissettii strains, and seven types among seven B. burgdorferi sensu stricto strains. Strain TXW-1 formed a separate RAPD type. Nearly complete sequences of the rrs genes from 17 representative southern Borrelia were determined. The similarity values were found to be 96-100% within the B. burgdorferi sensu lato (s.l.) complex, 94-99% among the relapsing fever borreliae, and 93-99% between the two complexes. Phylogenetic analysis indicated that all the Borrelia strains we analyzed could be divided into two parts: the B. burgdorferi s.l. complex and the relapsing fever borreliae complex. TXW-1 segregated with the North American relapsing fever borreliae and formed a separate subbranch.     

Diversity of Borrelia burgdorferi sensu lato in Russian Far East

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.     

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

Abstract A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi , has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii . The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA aas well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.     

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.     

Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ～900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Distribution of twelve linear extrachromosomal DNAs in natural isolates of Lyme disease spirochetes

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete, Borrelia burgdorferi (sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the B. burgdorferi type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the B. burgdorferi B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents Borrelia garinii and Borrelia afzelii; however, some of these plasmids had uniform but substantially different sizes from their B. burgdorferi counterparts.     

Heterogeneity of the gene P83/100 of Borrelia borrelia burgdorferi sensu lato complex

The 35 full-length Borrelia burgdorferi sensu lato complex a83/100 gene nucleotide sequences were determined. High level of homology was observed in the nucleotide sequences corresponding to the strains and isolates of Borrelia fzelii. The analysis of the nucleotide sequences revealed two groups of Borrelia garinii. The most variable p83/100 gene region containing species-typical insertions and deletions was demonstrated to be included into the region where the antigenic determinants of protein were encoded. According to the data obtained in this work, the modification of the P83/100 protein structure and immunological properties could be suggested to exist even within species. The results of this work could be used for receiving recombinant P83/100 proteins useful for diagnostic applications.     

RECENT ORNITHOLOGICAL PUBLICATIONS

Baker , R. Robin. 1982. Migration-paths through time and space. Blondel , J. & Isenmann , P. 1981. Guide des oiseaux de Camargue. Boag , D. 1982. The Kingfisher. Dunning , J. S. 1982. South American land birds–a photographic aid to identification. Frith , H. J. 1982. Pigeons and doves of Australia. Gardarsson , A. (ed.). 1982. Fuglar. Ritlandvernder 8. Geroudet , P. 1982. Limicoles, Gangaes et Pigeons 'Europe. Gould , P. J., Forsell , D. J. & Lensink , D. J. 1982. Pelagic distribution and abundance of seabirds in the Gulf of Alaska and eastern Bering Sea. Grant , P. J. 1982. Gulls: a guide to identification. Holden , P. & Sharrock , J T. R. 1982. The RSPB book of British birds. Ilichev , V. D. & Vilks , YE. K. 1978. [Spatial orientation of birds]. Jouventin , P. 1982. Visual and vocal signals in penguins, their evolution and adaptive characters. Advances in Ethology, Suppl. 24 to Journal of Comparative Ethology. Jouventin , P., Massé , L. & Tréhen , P. (eds.) 1982. Colloque sur les ecosystèmes subantarctiques. Station biologique de Paimpont (Université de Rennes). Litvinenko , N. M. & Neufeldt , I. A. (eds.). 1982. Cranes of East Asia. Lockley , R. M. 1983. Flight of the storm petrel. Ogilvie , M. 1982. The wildfowl of Britain and Europe. Pontin , A. J. 1982. Competition and coexistence of species. Prestt , I. 1982. British birds–lifestyles and habitats. Ratti , J. T., Flake , L. D. & Wentz , W. A. (eds.). 1982. Waterfowl ecology and management: selected readings. Rutschke , E. (ed.). 1983. Die Vogelwelt Brandenburgs. Scott , D. A. & Smart , M. (eds.). 1982. Proceedings of the second technical meeting on Western Palearctic migratory bird management. Sharrock , J. T. R. & Grant , P. J. 1982. Birds new to Britain and Ireland. Soothill , E. & Soothill , R. 1982. Wading birds of the world. Géroudet , P. 1982. Limicoles, Gangaes et Pigeons 'Europe. Tomkies , M. 1982. Golden Eagle years. Vïksne , J. (ed.). 1983. Birds of Latvia–territorial distribution and number. Watling , D. 1982. Birds of Fiji, Tonga and Samoa. Alderton , D. 1982. Looking after cage birds Ali , S. 1979. The book of Indian birds. 11th ed. Allaby , M. 1982. Animal artisans. Bezzel , E. 1982. Mein Hobby: Vögel beobachten. Wie-wann-wo? Dathe , H. Neufeldt , I. A. 1982. Atlas der verbreitung Palaearktischer vogel. Frith , H. J. 1982. Waterfowl in Australia. (Australian Natural Science Library revised edition). Grimes , B. 1982. British wild birds. Johnston -Stewart , N. G. B. & Heigham , J. B. 1982. Bridging the bird gap. A field guide to the 64 species in Malawi not described in Robert's Birds of South Africa. Kenyo , J. R. 1982. The Willoughby Gardner Library. A collection of early printed books on natural history. Ripley , S. D. 1982. A synopsis of the birds of India and Pakistan, together with those of Nepal, Bhutan, Bangladesh and Sri Lanka. 2nd ed. Rosenberg , M. E. 1982. Sound and hearing. Scott , B. 1982. The birdwatcher's calendar. Vevers , G. 1982. The colours of animals. Institute of Biology Studies in Biology No. 146. Welty , J. C. 1982. The life of birds. 3rd edition.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains

N.G. FOMUKONG, J.W. DALE, T.W. OSBORN AND J.M. GRANGE. 1992. Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.     

Molecular evidence for a new bacteriophage of Borrelia burgdorferi

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi.     

广东省南雄盆地白垩系—第三系交界恐龙绝灭问题

广东省南雄盆地中的红层可划分为三个群五个组,大致代表了晚白垩世—始新世的沉积.根据绝对年龄、古地磁测定结果和脊椎动物化石组合性质的综合分析,位于地磁极性带 29R 上部的坪岭组和上湖组之间的分界线被确定为 K/T 界线.对晚白垩世恐龙蛋的研究表明,不同"种"的恐龙蛋是在地磁极性带 29R 的中、下部,也就是说在白垩系—第三系交界之前20～30万年期间绝迹的.而且在这一时期内,所有已发现的蛋壳中,绝大多数蛋壳的厚度和显微结构都显示出明显的病理特征,例如根据随机取样统计,Macroolithus yaotunensis 蛋壳异常结构的出现率,最高可达75%.产生病态恐龙蛋壳的生理机制可以根据发生在现生鸟类的相同病理特征来解释.进一步分析恐龙蛋壳的微量元素和稳定同位素组成,结果显示, Pb, Cu, Mn 等9种元素丰度变化在这一时期达到最大峰值, δ~(18)O 也出现正异常.在这一基础上提出,微量元素的污染和气候突然的变化妨碍了正常蛋壳结构的形成,导致了恐龙的绝灭.这一绝灭过程大约经历了20～30万年.     

Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999

The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gómara et al., J. Clin. Microbiol. 38:4394-4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.     

BUCHBESPRECHUNGEN

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Genetic diversity and the absence of regional differences of Borrelia garinii as demonstrated by ospA and ospB gene sequence analysis

Unfed adult Ixodes persulcatus ticks were collected from four locations of Nagano and Hokkaido in Japan. Infected Borrelia garinii were investigated by PCR-RFLP of the ospA and ospB gene sequences. The primer set amplified an approximately 1.6-kb DNA fragment (0.7-kb in some strains), and BsrI, BstYI, or NlaIII digestion of the product resulted in six distinctively different PCR-RFLP groups and two independent borrelial strains. The representatives in each PCR-RFLP group and individuals from the borrelial strains were sequenced, and their deduced amino acid sequences were aligned. A neighbor-joining phylogenetic analysis showed that the B. garinii OspA or OspB sequences were each divided into three major clusters including isolates from both the Nagano and Hokkaido locations. There was no local difference in OspA/B sequences between Nagano and Hokkaido. The osp gene of Borrelia burgdorferi sensu lato is highly heterogeneous, and this was also confirmed by our sequence analysis. Some strains of the different PCR-RFLP groups had closely related OspA sequences, while the OspB sequences of these strains were quite different. These findings suggested intraspecies gene exchange and recombination events between the two genes in B. garinii.