Detection and characterization of Borrelia bissettii in rodents from the central California coast

This is the first report of Borrelia burgdorferi sensu lato in rodents from San Luis Obispo county, with most isolates obtained from a previously unreported host, Neotoma lepida Thomas. B. burgdorferi sensu lato was identified in seven rodent species, including the California vole, Microtus californicus Peale; dusky-footed woodrat, Neotoma fuscipes Baird; desert woodrat, Neotoma lepida Thomas; brush mouse, Peromyscus boylii Baird; California mouse, Peromyscus californicus Gambel; deer mouse, Peromyscus maniculatus Wagner; and western harvest mouse, Reithrodontomys megalotis Baird. Ear punch biopsies were cultured in BSK-H medium from 179 rodents trapped at six different study sites. Overall, prevalence of rodent infection was 44/179 (24.6%), with 34 of these isolates from N. lepida. Spirochete isolates were obtained from rodents at all study sites, indicating widespread prevalence of B. burgdorferi sensu lato across rodent species and habitats. Nucleotide sequences for 14 of these isolates have been submitted to GenBank. Isolates from three N. lepida and one P. boylii had identical flagellin gene sequences, and phylogenetic analysis placed these spirochetes in B. burgdorferi sensu lato group DN127, now known as B. bissettii Postic, Marti Ras, Lane, Hendson & Baranton. Additional sequencing of the intergenic spacer regions between the 5S and 23S ribosomal genes was performed on three of these isolates. Phylogenetic analysis separated these isolates into two clusters that grouped with Colorado or California isolates. The role of B. bissettii and related species other than B. burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner as human pathogens in the United States warrants further investigation.     

Expanded Diversity among Californian Borrelia Isolates and Description of Borrelia bissettii sp. nov. (Formerly Borrelia Group DN127)

Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.In Eurasia, seven species of the complex Borrelia burgdorferi sensu lato have been reported. Only three of these species are associated with Lyme borreliosis. It has also been shown that each pathogenic species is associated predominantly with a given clinical presentation; Borrelia burgdorferi sensu stricto is associated with arthritis, B. garinii is associated with neuroborreliosis, and B. afzelii is associated with late cutaneous symptoms (2, 39). Up to now, B. burgdorferi sensu stricto is the only species associated with Lyme borreliosis in North America. However, two other B. burgdorferi sensu lato genospecies coexist in the United States, B. andersonii (22) and the genomic group DN127 (3, 32). B. andersonii seems to be restricted to a limited ecosystem involving cottontail rabbits and Ixodes dentatus ticks. In contrast, the genomic group DN127 appears to be involved in several enzootic transmission cycles (6, 29). A recent study demonstrated substantial genetic heterogeneity among Californian and other American strains (24). We took advantage of the unique structure of ribosomal genes in B. burgdorferi sensu lato to analyze the polymorphism of some strains isolated in California. A single copy of the rrs gene is separated by a large spacer (rrs-rrl; 3,000 to 5,000 bp) from two tandemly duplicated copies of rrl and rrf genes (13, 36). These two copies are separated by a small spacer, rrf-rrl, which is approximately 250 bp long. The genetic heterogeneity of the group DN127 was first evidenced by analysis of the restriction patterns of the rrf-rrl spacer (32). However, the results of DNA-DNA hybridization on a limited number of strains (32) allowed us to place them in a single genomic group. To clarify the genetic relationships between diverse North American strains, 20 atypical strains were compared with 13 B. burgdorferi sensu stricto strains. Identification procedures involved restriction polymorphism and sequencing studies of both the variable rrf-rrl spacer and the conserved rrs gene. Sequences of the rrf-rrl spacer and the rrs gene were used in a phylogenetic analysis. Some Californian strains are closely related to the genomic group DN127, for which we propose the name of B. bissettii sp. nov. Other atypical strains which do not fall into this group are designated merely as Borrelia spp. in this study. The latter strains cannot be assigned to specific genomic groups until more isolates representative of each group are available for further characterization.     

Seroprotective groups among isolates of Borrelia burgdorferi.

We demonstrated that different seroprotective groups exist among isolates of Borrelia burgdorferi and Borrelia garinii. The major group was composed of isolates 297, B31, S-1-10, MMTI, IPT, and ATCC 35211 and 21 isolates obtained from California, Illinois, New York, Texas, and Wisconsin. A second group was composed of European isolates PBi and G25. A third group was composed of a single isolate, C-1-11. These groupings were supported by Western immunoblot findings. In addition, the seroprotective groups were confirmed by passive transfer of immune sera and challenge of recipient hamsters with the homologous isolate or other isolates of B. burgdorferi or B. garinii. These studies demonstrate that a monovalent vaccine will not provide complete protection against infection with all isolates of B. burgdorferi.     

Detection of Borrelia bissettii in cardiac valve tissue of a patient with endocarditis and aortic valve stenosis in the Czech Republic

Molecular analysis of a clinical sample confirmed the presence of Borrelia bissettii DNA in cardiac valve tissue from a patient with endocarditis and aortic valve stenosis. This evidence strongly supports the involvement of B. bissettii in Lyme disease in Europe.     

Multiple genetically distinct groups revealed among clinical isolates identified as atypical Aspergillus fumigatus

To investigate whether genetic variants of A. fumigatus are found among clinical isolates, four isolates that were originally identified as poorly sporulating strains of Aspergillus fumigatus were subjected to molecular analysis. DNA sequence analysis of the alkaline protease genes of these isolates showed that each is genetically distinct and each shows substantial variation (7 to 11%) from the A. fumigatus nucleotide sequence. Subsequent morphological examination suggested that all of the isolates could be classified as Aspergillus viridinutans. To clarify the taxonomic status of these four clinical isolates and of two previously identified as atypical A. fumigatus isolates, partial beta-tubulin and 18S rRNA gene sequences were determined. Each of the six atypical strains had a unique beta-tubulin sequence, whereas the sequences of three standard isolates of A. fumigatus, which were included as controls, were identical to the published A. fumigatus beta-tubulin sequence. The very low level of DNA sequence variation detected in standard isolates of A. fumigatus compared with other isolates from members of Aspergillus section Fumigati suggests that it may be a relatively recently evolved species. The 18S rRNA gene of two of the atypical isolates differed from that of A. fumigatus at a single nucleotide position. Phylogenetic analyses do not support the classification of all of these isolates as A. viridinutans. Thus, some of these isolates represent new species which are potential opportunistic pathogens.     

Characterization of Borrelia lusitaniae isolates collected in Tunisia and Morocco

Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato and is infrequently isolated in Europe. In contrast, this species is by far the most predominant in North Africa and in Portugal. In this study, we analyzed the genetic diversity, at several loci, of a large population of isolates from free-living Ixodes ricinus ticks collected in Tunisia and Morocco. We found a moderate diversity of the whole genome by using pulsed-field gel electrophoresis as well as in the ospA gene sequences, compared to a high level of strain homogeneity in the small noncoding ribosomal spacer. In contrast, a high diversity of this locus has been previously reported for Portuguese isolates. We hypothesize that B. lusitaniae strains isolated in North Africa constitute a clone of Portuguese origin.     

Linear and circular plasmid content in Borrelia burgdorferi clinical isolates

The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.     

New variant groups identified from HGV isolates

Summary.  We have determined the primary sequence of the 5^′ noncoding region (5^′ NCR) and putative helicase regions (NS-3) of hepatitis G virus (HGV) and GB virus C (GBV-C) that were isolated in Japan from suspected cases of nonA-nonB and/or nonA-nonB-nonC viral hepatitis by using RT-PCR, and we compared the newly isolated sequences with three established isolates. The addition of a "G" residue was found at the 5^′ terminus of all 8 Japanese isolates. These isolates were more clearly distinguished from the prototype viruses by comparison with the 5^′ NCR sequence than by comparison with the NS-3 region. Our results suggested that at least three distinct genomic variants of HGV exist. Genotyping of HGV by using RT-PCR based on the sequence of the 5^′ NCR seems highly feasible. Accepted November 18, 1996 Received September 11, 1996     

Culturing selects for specific genotypes of Borrelia burgdorferi in an enzootic cycle in Colorado.

In Colorado, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease, is maintained in an enzootic cycle between Ixodes spinipalpis ticks and Neotoma mexicana rats (27). The frequencies of flagellin (fla), 66-kDa protein (p66), and outer surface protein A (ospA) alleles were examined in 71 B. burgdorferi isolates from samples from Colorado. Approximately two-thirds of these samples were isolates from I. spinipalpis ticks that had been cultured in BSK-H medium prior to DNA extraction. The remaining samples were from total DNA extracted directly from infected I. spinipalpis ticks. A portion of each gene was amplified by PCR and screened for genetic variability by single-strand conformation polymorphism (SSCP) analysis. We identified three alleles in the fla gene, seven in the p66 gene, and seven in the ospA gene. Sequencing verified that the amplified products originated from B. burgdorferi template DNA and indicated 100% sensitivity and specificity of the SSCP analysis. The frequencies of the p66 and ospA alleles were significantly different between cultured and uncultured spirochetes. The number of three-locus genotypes and the genetic diversity of alleles at all loci were consistently lower in cultured spirochetes, suggesting that culturing of B. burgdorferi in BSK-H medium may select for specific genotypes.     

Glycerophosphodiester phosphodiesterase gene (glpQ) of Borrelia lonestari identified as a target for differentiating Borrelia species associated with hard ticks (Acari:Ixodidae)

A glpQ ortholog was identified in DNA from Borrelia lonestari-positive Amblyomma americanum, providing further evidence that B. lonestari is more closely related to the relapsing fever group spirochetes than to borreliae that cause Lyme disease. This finding provides a basis for developing diagnostic assays to differentiate species of borrelia transmitted by hard ticks.     

Antigenic characteristics of Borrelia burgdorferi isolates from ixodid ticks in California.

Twenty (1.4%) of 1,421 adult Ixodes pacificus ticks and 2 (20%) of 10 adult Ixodes neotomae ticks collected in five counties of northern California were found to contain spirochetes by direct immunofluorescence examination of their tissues with a polyvalent conjugate. Borreliae isolated from the tissues of nine of these ticks (I. pacificus, 8; I. neotomae, 1) were identified as Borrelia burgdorferi with specific monoclonal antibodies and characterized further by polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. The isolate from I. neotomae was the first to be characterized from a tick other than I. pacificus in western North America. All strains were relatively homogeneous with respect to the kind of OspA proteins they produced, whereas they were heterogeneous with regard to their OspB proteins and to several low-molecular-weight proteins in the 21,500-to-24,000 region. Significant phenotypic variation was observed among isolates obtained within and between populations of I. pacificus. This investigation nearly doubles the number of isolates of B. burgdorferi that have been characterized from ixodid ticks in the far western United States.     

Isolation and characterization of Borrelia parkeri in Ornithodoros parkeri (Ixodida: Argasidae) collected in Colorado

This study describes the identification of Borrelia parkeri spirochetes in Colorado. Two isolates of B. parkeri (6230 and 6232) were recovered from Ornithodoros parkeri Cooley ticks collected at an inactive prairie dog town in Moffat County. Both isolates were partially characterized by sequencing and subsequent parsimony and neighbor-joining analyses of appropriate regions of the 16S ribosomal RNA, flagellin and P66 genes. Analyses of the 16S gene sequences from the Colorado isolates indicated that they were more closely related to B. parkeri and B. tucatae than to B. hermsii or the other species of Borrelia investigated in this study. Additional analyses of amino acid sequences for flagellin and P66, however, clearly demonstrated that isolates 6230 and 6232 were most closely related to B. parkeri. The possible significance of B. parkeri as an agent of human disease is discussed.     

Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from The Netherlands.

Sixty-three Borrelia burgdorferi isolates recovered from Ixodes ricinus ticks collected in 17 locations in The Netherlands and three Dutch human skin isolates were characterized by rRNA gene restriction fragment length polymorphism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting). All three human isolates belonged to B. burgdorferi group VS461. Of the tick isolates, 29 (46%) were B. burgdorferi sensu stricto, 2 (3%) were group VS461, 19 (30%) were Borrelia garinii, and 13 (21%) were different from any previously described genomic species. On the basis of the criteria described, 12 isolates formed a distinct genomic group, designated M19. rRNA gene restriction patterns of the group M19 isolates resembled but were not identical to the B. garinii patterns. Hybridization of digested DNA with a flagellin probe confirmed the separation of group M19 from the B. garinii isolates. One isolate, M63, was different from all the others. In conclusion, the occurrence of B. burgdorferi sensu stricto, B. garinii, and B. burgdorferi group VS461 in ticks from The Netherlands corresponds with the occurrence of these genomic species among tick isolates from other European countries. However, our findings suggest that B. burgdorferi sensu lato probably contains more than three genomic species.     

Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain

Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic.     

DNA and protein analyses of tick-derived isolates of Borrelia burgdorferi from California

Nine isolates of Borrelia burgdorferi from ixodid ticks collected in northern California were characterized. Restriction endonuclease analysis, pulsed-field gel electrophoresis, and Western blot (immunoblot) analysis were used in this study. Four isolates were very similar to each other. The others shared some similarities but were classified as having unique genotypes. A strain from an Ixodes neotomae tick displayed the greatest genetic and antigenic diversity when compared to the isolates collected from Ixodes pacificus ticks. A computerized library based on DNA banding patterns of the isolates by restriction enzyme analysis is also reported. This library was created by using a scanning laser densitometer.     

Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources.

A total of 17 B. burgdorferi isolates from various sources were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, restriction enzyme analysis, Southern hybridization with probes complementary to unique regions of evolutionarily conserved genes (16S rRNA and fla), and direct sequencing of in vitro polymerase chain reaction-amplified fragments of the 16S rRNA gene. Three groups were distinguished on the basis of phenotypic and genotypic traits, the latter traced to the nucleotide sequence level.     

Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis.

Fifty Borellia burgdorferi strains isolated from humans and ticks in Europe and the United States were analyzed by multilocus enzyme electrophoresis. Eleven genetic loci were characterized on the basis of the electrophoretic mobilities of their products. Ten loci were polymorphic. The average number of alleles per locus was 5.9, with a mean genetic diversity of 0.673 among electrophoretic types (ETs). The strains were grouped into 35 ETs constituting three main divisions (I, II, and III) separated at a genetic distance greater than 0.75. Divisions I, II, and III contained 13, 6, and 16 ETs, respectively. These findings, together with previous data from DNA hybridization and restriction enzyme analysis of rRNA genes, suggest that divisions I, II, and III may represent three distinct genomic species. All three divisions contained human clinical ETs. However, in division I, which includes the ET of the type strain of B. burgdorferi, the human pathogenic ETs constituted a single clone. The ETs of division I were from west-central Europe and the United States, whereas divisions II and III contained ETs from west-central and northern Europe but not from the United States. Finally, our data show that the genetic structure of B. burgdorferi populations is clonal.     

Variations in Barbour-Stoenner-Kelly culture medium modulate infectivity and pathogenicity of Borrelia burgdorferi clinical isolates

The effects of variations in Barbour-Stoenner-Kelly (BSK) medium on the infectivity and pathogenicity of Borrelia burgdorferi clinical isolates were assessed by retrospective and prospective studies using a murine model of Lyme borreliosis. Thirty of 35 (86%) mice infected with any of six virulent B. burgdorferi clinical isolates grown in a BSK-H medium developed clinically apparent arthritis. By contrast, arthritis was observed in only 25 of 60 (42%) mice inoculated with two of these B. burgdorferi strains grown in a different lot of BSK-H medium (P < 0.001). In a prospective study, mice inoculated with a B. burgdorferi clinical isolate grown in a BSK medium prepared in-house produced significantly greater disease than those injected with the same isolate cultured in BSK-H medium (P < 0.05). The attenuated pathogenicity is not due to the loss of plasmids during in vitro cultivation. The data suggest that variations in BSK medium have a significant impact on the infectivity and pathogenicity of B. burgdorferi clinical isolates.     

Conservation and heterogeneity of vlsE among human and tick isolates of Borrelia burgdorferi

The vls (variable major protein [VMP]-like sequence) locus of Borrelia burgdorferi encodes an antigenic variation system that closely resembles the VMP system of relapsing fever borreliae. To determine whether vls sequences are present consistently in low-passage, infectious isolates of B. burgdorferi, 22 blood and erythema migrans biopsy isolates from Lyme disease patients in Westchester County, New York, were examined by Southern blot and PCR analysis. Each of the strains contained a single plasmid varying in size from 21 to 38 kb that hybridized strongly with a vlsE probe based on the B. burgdorferi B31 sequence. In contrast, PCR products were obtained with only 10 of the 22 strains when primers corresponding to the 5' and 3' regions of the B31 vlsE sequence outside the variable cassette region were used. Only 2 of 16 B. burgdorferi-infected tick specimens yielded detectable PCR product. Eight of 10 strains that yielded a PCR product under these conditions were type 1 (a genotype with a high rate of dissemination), according to PCR-restriction fragment length polymorphism analysis of intergenic rDNA sequences, whereas the isolates that did not yield vlsE PCR products were either type 2 or type 3. Comparison of the sequences of cloned PCR products from the patient isolates indicated a high degree of identity to the B31 sequence, with most of the differences restricted to the hypervariable regions known to undergo sequence variation. Taken together, these results both reinforce previous evidence that vls sequences are present consistently in low-passage Lyme disease spirochetes and indicate that both highly conserved and heterogeneous subgroups exist with regard to vlsE sequences.