Genetic variants of Borrelia afzelii, a pathogen of the ixodes tick borrelioses

Borrelia afzelii is one of two most important pathogens of the ixodes tick borrelioses (ITB) in Russia and neighboring countries. This pathogen circulates in various ecosystems and has a wide range of reservoir hosts and transmitters. The results of studies of genetic heterogeneity of the spirochaetae B. afzelii are considered. A total of 139 primary isolates were studied. The isolates were isolated from three species of Ixodes ticks at different stages of development and obtained from the laboratory of infection transmitters, Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Russran Academy of Medical Sciences, Moscow. The transmitters and reservoir hosts of borrelias were caught in natural foci of Russia (from Kaliningrad region irk the west to south Sakhalin in the east), Czechia, Lithuania, Estonia, Ukraine, and Moldavia. Analysis of genotype sequence similarity obtained by sequencing of the rrf(SS)-rrl(23S) spacer demonstrated that the B. afzelii genospecies incorporated no less than 10 genetic variants of spirochaetae, most variants being geographically widespread.     

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.     

Allele variants of Borrelia afzelii found by sequencing of the chromosome gene p66

Sequencing of gene p66 fragments of 246-337 p.b. was performed in 45 isolates of Aorrelia afzelii isolated from different transmitting agents and reservoir hosts within the habitation area of the spirochaeta. At least seven allele variants of the pathogenic agent were found indifferent natural foci of the disease. The extent of similarity between the nucleotide sequences of the isolates of the same allele variant was 99.9-100%; the extent of similarity between different allele variants was 98.9-99.7%. It was found that the majority of genovariants of A. afzelii (with respect to 5S-23S) incorporated several different allele variants of gene p66, all allele variants being found in the two genogroups (VS461 and NT28) of the pathogenic agent.     

Genetic characterization of pathogenic Borrelia, group A14S, isolated in Ukraine

Borrelia Ir-5215, isolated from ticks Ixodes ricinus in Ukraine (the Crimean autonomous region), was identified by the method of the polymorphism of the fragment length of the restriction amplicon of rRNA spacer region 5S-23S. Its Msel-restriction profile was relatively similar to that of B. afzelii. The sequencing of spacer region rrf (5S)-rrl (23S) and 16S rRNA gene, as well as the analysis of the similarity of nucleotide sequences, obtained in the course of these study, revealed the differences between Borrelia sp, lr-5215 and six European species of Borrelia burgdorferi sensu lato and a high level of similarity (more than 95.1% for 5S-23S rRNA and 99.4% for 16S rRNA gene) to three known representatives of genome group A14S (Borrelia spp. A14S, I-77 and PC-Rq17). This suggests that isolated Borrelia lr-5215 is a new representative of pathogenic B. burgdorferi sensu lato genome group A14S, which is spread, together with Central Europe, also in southern Ukraine.     

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.     

Characterization by genotypic methods of symbiotic Nostoc strains isolated from five species of Gunnera

The genetic diversity of ten symbiotic Nostoc strains isolated from different Gunnera species was investigated. The strains were analyzed using molecular methods with different taxonomic resolutions, including restriction fragment length polymorphisms (RFLP) of the PCR-amplified 16S ribosomal gene and the 16S-23S internal transcribed spacer (ITS) region combined with computer-assisted analyses. The functional gene hetR, assigned to heterocyst differentiation, was used for denaturing gradient gel electrophoresis. A high genetic diversity was observed among the isolates even in the conserved gene coding for the small ribosomal unit. No correlation was observed between clustering of cyanobacteria and the host species of Gunnera.     

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.     

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.     

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.     

Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.     

Difference in amino acid sequences of B. afzelii P66 surface-exposed loop region

In the present work, we performed a phenotyping analysis of 45 B. afzelii 89-a.a. long amino acid sequences of 7 different allele variants, corresponding to the surface-exposed loop region of P66. 45 investigated isolates showed 5 phenotypically different variants; 2 phenotypically different variants of loop region, in particular, also showed mutations in the putative monoclonal antibody H1337 binding site; the similarity between the amino acid sequences taken from different variants is about 96.66% to 98.88%; in one natural locus up to 3 different phenotypes of P66 could circulate simultaneously.     

Genetic variants of Borrelia garinii, a widely spread Eurasian pathogen of ixodic tick borreliosis

As a result of PCR-RFLP analysis and the degree of similarity between the nucleotide sequences analysis of the rrfA-rrlB intergenic spacer DNA of 227 primary isolates of Borrelia garinii and 71 isolates/ amplicons from GenBank database in different regions of Eurasia revealed significant intraspecific heterogeneity among those of Borrelia. It was shown that genospecies B. garinii had within the two genetic subgroups (20047 and NT29) 16 genetic variants, whose geography was likely to be different.     

Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.     

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.     

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.     

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).     

Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India

Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and the nifH and nodC genes. Eight haplotypes assigned to the Bradyrhizobium genus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and the dnaK, glnII, recA, and nifH genes revealed three groups. One of them (26% of isolates) was assigned to Bradyrhizobium liaoningense. A second group (36% of isolates) was identified as B. yuanmingense but likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all described Bradyrhizobium species but showed the same symbiotic genotype as B. liaoningense and B. japonicum bv. glycinearum.     

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.