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PubMed1985

Variable major proteins of Borrelia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides.

The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family.

Amino Acid Sequence↗
PubMed1994

Variability of a bacterial surface protein and disease expression in a possible mouse model of systemic Lyme borreliosis.

During persistent infection of scid mice with Borrelia turicatae, an agent of relapsing fever and neuroborreliosis, there was variation in the surface proteins the bacteria expressed and in disease manifestations over time. Two serotypes, A and B, were isolated from the mice, cloned by limiting dilution, and further characterized. The only discernible difference between the two variants was in the size of the major surface protein they expressed: serotype A had a variable major protein (Vmp) of 23,000, and serotype B had a Vmp of 20,000. When other scid mice were inoculated with clonal populations of A and B, the infections were similar with respect to onset and degree of spirochetemia, involvement of the eye and heart, and occurrence of a peripheral vestibular disorder. However, there were differences between the serotypes in other respects: (a) serotype B but not A caused reddened and significantly enlarged joints, markedly impaired performance on a walking bar, and severe arthritis by histologic examination; (b) serotype A but not B invaded the central nervous system during early infection; and (c) serotype A penetrated monolayers of human umbilical vein endothelial cells more readily than did serotype B. The combination of arthritis, myocarditis, and neurologic disease resembled human Lyme borreliosis. The findings indicate that differences in disease expression are determined by variable surface proteins of the bacterium and that scid mouse infections with B. turicatae provide a model for the study of the pathogenesis of Lyme borreliosis and other persistent spirochetal diseases.

Animals↗
PubMed2001

Ticks and tickborne bacterial diseases in humans: an emerging infectious threat.

Ticks are currently considered to be second only to mosquitoes as vectors of human infectious diseases in the world. Each tick species has preferred environmental conditions and biotopes that determine the geographic distribution of the ticks and, consequently, the risk areas for tickborne diseases. This is particularly the case when ticks are vectors and reservoirs of the pathogens. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 15 ixodid-borne bacterial pathogens have been described throughout the world, including 8 rickettsiae, 3 ehrlichiae, and 4 species of the Borrelia burgdorferi complex. This article reviews and illustrate various aspects of the biology of ticks and the tickborne bacterial diseases (rickettsioses, ehrlichioses, Lyme disease, relapsing fever borrelioses, tularemia, Q fever), particularly those regarded as emerging diseases. Methods are described for the detection and isolation of bacteria from ticks and advice is given on how tick bites may be prevented and how clinicians should deal with patients who have been bitten by ticks.

Animals↗
PubMed2002

Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks.

Borrelia burgdorferi sensu lato (sl), the agent of Lyme disease, is transmitted to the host during the blood meal of Ixodes ticks. In most unfed ticks, spirochetes are present in the midgut and migrate during blood feeding to the salivary glands, from which they are transmitted to the host via saliva. In the present study, the efficiency of Ixodes ricinus ticks to transmit B. afzelii and B. burgdorferi sensu stricto (ss) and their infectivity for mice were examined in relation to the duration of the blood meal. In addition, we investigated whether these two Borrelia species can penetrate intact skin. Three modes of infection of mice were studied: tick-bite infection, inoculation of tick homogenates, and transcutaneous infection by topical application of tick homogenates on mouse skin. Transmission of B. burgdorferi sl from I. ricinus nymphs to mouse increased with duration of tick attachment. B. afzelii-infected ticks start to transmit infection earlier (< or = 48 h) than B. burgdorferi ss-infected ticks. As previously shown for B. burgdorferi ss in Ixodes scapularis, B. burgdorferi ss and B. afzelii in unfed I. ricinus were noninfectious for mice when tick homogenates were inoculated. However, the inoculation of homogenates of ticks fed for 24 h readily produced infection in mice. Therefore, B. burgdorferi ss and B. afzelii spirochetes are potentially infectious in the tick before natural transmission can occur. None of the mice (n = 33) became infected by transcutaneous transmission when tick homogenates were applied on mouse skin, showing that B. burgdorferi ss and B. afzelii are unable to penetrate intact skin, in contrast to relapsing fever spirochetes. This study also shows that B. afzelii is transmitted by I. ricinus to the host earlier than B. burgdorferi ss and that I. ricinus seems to be a more efficient vector of B. afzelii than B. burgdorferi ss.

Administration, Oral↗
PubMed2005

Blood-meal analysis for the identification of reservoir hosts of tick-borne pathogens in Ireland.

The results of analysis of blood-meal remnants in unfed nymphs, despite relatively low detection levels (49.4%, n = 322), support the conclusion from an earlier study that small rodents are relatively unimportant as reservoir hosts of B. burgdorferi s.l. in this particular area, and suggest that songbirds (Passeriformes) are the most significant hosts in this respect. Tick (Ixodes ricinus) abundance was greater in the present study, but the overall Borrelia burgdorferi s.l.-infection prevalence of nymphal ticks was the same (12.2%), and the relative proportions of the various Borrelia burgdorferi s.l. genospecies were similar. B. garinii and B. valaisiana were the most frequent, B. burgdorferi s.s the least frequent, and B. afzelii of intermediate frequency. An unusually high proportion of nymphs (39%) with multiple infections of different B. burgdorferi genospecies was detected, and Borrelia spp. related to relapsing-fever spirochetes were detected in Ireland for the first time. The results of the present study contribute to the validation of blood-meal analysis as a means of determining the host origin of certain pathogens in unfed questing ticks, and raise some questions concerning the extent of B. burgdorferi s.l. host specificity.

Animals↗
PubMed1993

A serological survey and review of clinical Lyme borreliosis in Spain.

A serosurvey for Lyme disease was conducted by the National Reference Center for Microbiology in Spain. All serum specimens received from patients with erythema migrans (EM), arthritis, and cardiac and neurological disorders were tested for antibodies to Borrelia burgdorferi. Sera from 499 patients were tested. Of these patients, 310 had neurological disease, 101 had carditis, 79 had arthropathies, and 9 had EM; for 35 patients, a diagnosis of Lyme disease was confirmed. Of these 35, 68% had neurological disease, 6% had carditis, and 26% had EM. Twelve probable cases of Lyme disease were noted. The male-to-female ratio of patients with confirmed or probable disease was 1.6; all age groups were represented, but 25% of patients were within the age group of 0-9 years. The number of cases of Lyme disease decreases from the north of Spain (41%) to the south (26%), a distribution that is in agreement with the known range of Ixodes ricinus. Borrelia burgdorferi is a frequent cause of infection in some areas of Spain. It is possible that relapsing fever accounts for some of the serological reactivity to B. burgdorferi in residents of the south of Spain.

Adolescent↗
PubMed2001

Telomere resolution in the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.

Borrelia burgdorferi↗
PubMed1987

Pilot study of the prevalence of head lice infestation in a population of Saudi Arabian children.

The prevalence of infestation with the head louse, Pediculus capitis, was assessed among the child population, from birth to 10 years old inclusive, of the rapidly expanding Saudi Arabian city of Jeddah. Over a period of two months, 300 consecutive children attending the general practitioner for any reason were examined specifically to identify those infested with head lice: 37 cases of active infestation were found, which is an overall prevalence of 12%. An interesting distribution, however, was noted in respect of age, ranging from less than 2% in the first year of life, rising rapidly to around 30% in ages six to eight years, thereafter declining steadily to about 16% by age 10 years. The distribution of infestation among males and females was broadly similar. These results show a high head lice infestation, particularly in the early school years, where presumably interpersonal contacts are most frequent, facilitating contagious spread. As the head lice is known to spread several viral and rickettsial diseases, such as relapsing fever and typhus, greater efforts should be made towards patient education in hygiene, and towards identifying and treating the disease when found.

Child↗
PubMed1996

Serodiagnosis of Lyme disease: accuracy of a two-step approach using a flagella-based ELISA and immunoblotting.

An ELISA containing a purified flagellar antigen from Borrelia burgdorferi (FLA-ELISA) was evaluated. The FLA-ELISA, detecting IgM and IgG together, did not have adequate specificity by itself. Good accuracy was obtained, however, when the FLA-ELISA was the first step in a two-step protocol that used immunoblotting as a conditional second test. Samples that scored positive or equivocal by the FLA-ELISA were evaluated with separate IgM and IgG immunoblots. The sensitivity of the two-step process for patients with erythema migrans or with later manifestations of Lyme disease was 64% and 100%, respectively. The specificity for health blood donors was 100% and was 90% for the aggregate of all persons with illness that may cause serologic cross-reactivity (98% if the samples from relapsing fever patients were excluded). Test precision was 96% overall, 99% for Lyme disease case serum samples, 100% for specimens from blood donors, and 88% for samples from persons with other illness.

Antigens, Bacterial↗
PubMed2005

Ectoparasitism and vector-borne diseases in 930 homeless people from Marseilles.

Homeless people are particularly exposed to ectoparasites, but their exposure to arthropod-borne diseases has not been evaluated systematically. A medical team of 27 persons (7 nurses, 6 infectious disease residents or fellows, 2 dermatologists, and 12 infectious disease specialists) visited the 2 shelters in Marseilles, France, for 4 consecutive years. Homeless volunteers were interviewed, examined, and received care; and blood was sampled for cell counts and detection of bacteremia, antibodies to louse-borne (Rickettsia prowazekii, Bartonella quintana, and Borrelia recurrentis), flea-borne (R. typhi, R. felis), mite-borne (R. akari), and tick-borne (R. conorii) bacterial agents. We selected sex- and age-adjusted controls among healthy blood donors. Over 4 years, 930 homeless people were enrolled. Lice were found in 22% and were associated with hypereosinophilia (odds ratio, 5.7; 95% confidence intervals, 1.46-22.15). Twenty-seven patients (3%) with scabies were treated with ivermectin. Bartonella quintana was isolated from blood culture in 50 patients (5.3%), 36 of whom were treated effectively. The number of bacteremic patient increased from 3.4% to 8.4% (p = 0.02) over the 4 years of the study. We detected a higher seroprevalence to Borrelia recurrentis, R. conorii, and R. prowazekii antibodies in the homeless. Our study shows a high prevalence of louse-borne infections in the homeless and a high degree of exposure to tick-borne diseases and scabies. Despite effective treatment for Bartonella quintana bacteremia and the efforts made to delouse this population, Bartonella quintana remains endemic, and we found hallmarks of epidemic typhus and relapsing fever. The uncontrolled louse infestation of this population should alert the community to the possibility of severe re-emerging louse-borne infections.

Adolescent↗
PubMed1995

Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan.

The ixodid tick Ixodes persulcatus is the most important vector of Lyme disease in Japan. Most spirochete isolates obtained from I. persulcatus ticks have been classified as Borrelia burgdorferi sensu lato because of their genetic, biological, and immunological characteristics. However, we found that a small number of isolates obtained from I. persulcatus contained a smaller 38-kDa endoflagellar protein and single 23S-5S rRNA gene unit. Representative isolate HT31T (T = type strain) had the same 23S rRNA gene physical map as Borrelia turicatae. The DNA base composition of strain HT31T was 28.6 mol% G+C. DNA-DNA hybridization experiments revealed that strain HT31T exhibited moderate levels of DNA relatedness (24 to 51%) with Borrelia hermsii, B. turicatae, Borrelia parkeri, and Borrelia coriaceae. However, the levels of DNA reassociation with the previously described Lyme disease borreliae (B. burgdorferi, Borrelia garinii, and Borrelia afzelii) were only 8 to 13%. None of the previously described species examined exhibited a high level of DNA relatedness with strain HT31T. In addition, the 16S rRNA gene sequence (length, 1,368 nucleotides) of strain HT31T was determined and aligned with the 16S rRNA sequences of other Borrelia species. Distance matrix analyses were performed, and a phylogenetic tree was constructed. The results showed that isolate HT31T is only distantly related to both previously described Lyme disease borreliae and relapsing fever borreliae. Thus, the spirochetes isolated from I. persulcatus and closely related isolates should be classified as members of a new Borrelia species. We propose the name Borrelia miyamotoi sp. nov. for this spirochete; strain HT31 is the type strain.

Animals↗
PubMed1999

Successful in vitro cultivation of Borrelia duttonii and its comparison with Borrelia recurrentis.

Borrelia duttonii, the cause of East African tick-borne relapsing fever, has until now been refractory to growth in laboratory media. This spirochaete has only be propagated in mice or by tissue culture, restricting both yield and purity of cells available for research. The successful isolation of five clinical isolates of B. duttonii from patients in Central Tanzania and their comparison with Borrelia recurrentis is reported. Electron microscopy revealed spirochaetal cells with pointed ends, a mean wavelength of 1.8 microns with an amplitude of 0.8 micron, similar to the findings for B. recurrentis. Cells contained 10 periplasmic flagella inserted at each end of the spirochaete, again comparable with the counts of 8-10 flagella found in B. recurrentis. PFGE revealed a chromosome of approximately 1 Mb, a large plasmid of approximately 200 kb, and a small plasmid of 11 kb in all strains of B. duttonii and in B. recurrentis. B. duttonii possessed a further 7-9 plasmids with sizes ranging from 20 to 90 kb. In two isolates of B. duttonii, the profiles were identical. In contrast, all 18 isolates of B. recurrentis fell into one of five plasmid patterns with 3-4 plasmids ranging from 25 to 61.5 kb in addition to those of 11 and 200 kb described above. Analysis of the SDS-PAGE profiles of B. duttonii strains revealed a high-molecular-mass band of 33.4-34.2 kDa in four strains (variable large protein, VLP) and a low-molecular-mass band of 22.3 kDa in the remaining strain (variable small protein, VSP). This resembles the protein profiles found in B. recurrentis. The G + C ratio of B. duttonii was 27.6 mol%. Nucleotide sequence of the rrs gene (16S rRNA) from four B. duttonii isolates revealed 100% identity among these strains and 99.7% homology with three strains deposited by others in GenBank. The rrs gene of eight representative clinical isolates of B. recurrentis confirmed their close similarity with B. duttonii.

Bacteriological Techniques↗
PubMed1995

Expression of the flagellin gene in Borrelia is controlled by an alternative sigma factor.

The flagellin genes from six Borrelia species were cloned, sequenced and characterized at the molecular level. The flagellin genes of two relapsing fever Borrelia species, B. hermsii and B. crocidurae, three Lyme disease genomic species, B. burgdorferi, B. afzelii and B. garinii, and the avian borreliosis agent, B. anserina, were compared and showed an 85-93% sequence identity to each other. Comparison of the fla genes from the different Lyme borreliosis spirochaetes revealed that they were 94-99% identical. Nucleotide sequencing of the fla gene and primer extension on isolated mRNA from both B. hermsii (as transcribed in Escherichia coli) and B. burgdorferi (as transcribed in the natural host) identified the putative transcriptional start points, the ribosomebinding sites and the promoter regions of these genes. The deduced promoter of the Borrelia flagellin gene resembled neither the sigma 70 promoter of prokaryotes, as seen for the genes for the outer-surface proteins A and B in Lyme disease Borrelia and the genes for the variable major proteins 7 and 21 of B. hermsii, nor the sigma 28 consensus promoter region of motility genes from other bacteria. Instead, the promoter of the fla gene in Borrelia has most similarity to the bacteriophage SP01 sigma gp33-34 promoter sequence of Bacillus subtilis.

Amino Acid Sequence↗
PubMed2003

A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey.

A novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium (family Ixodidae, subfamily Metastriata) using Barbour-Stoenner-Kelly (BSK) II medium. Tick samples were taken during the summer of 2000 from the Istanbul area in northwestern Turkey. Sixty-seven of 153 adults (44%) and 72 of 185 nymphs (39%) were infected with the novel spirochaete, whereas none of the 20 larvae examined were infected. The optimal growth temperature of the spirochaete in BSK II medium was 34-37 degrees C, and it could grow at 39 degrees C. Doubling times at 34 and 37 degrees C were 5.3 and 5.1 h, respectively. Six pure cultures of the spirochaete were obtained and characterized by microscopic observation, sequence analysis of the flagellin gene (flaB), SDS-PAGE and Western blotting. The spirochaete was morphologically similar to those of the genus Borrelia and contained a 41 kDa protein reactive with mAb H9724 specific to the flagellin of a Borrelia species. Polyclonal antibody raised to this spirochaete reacted with several antigen bands, whereas no bands were detected with Borrelia burgdorferi, Borrelia hermsii, Borrelia turicatae and Borrelia parkeri. The flaB sequences of the six isolates showed high similarity, with sequence similarity values ranging from 99.2 to 100%; however, the similarity of the isolates' flaB sequences to those of the Lyme-disease-related Borrelia and relapsing-fever-associated Borrelia species was less than 90%. These findings suggest that the unique spirochaete is a member of the genus Borrelia, and differs from previously described Borrelia species.

Animals↗
PubMed1993

In vitro cultivation of Borrelia duttonii on cultures of SflEp cells.

Borrelia duttonii strain 406K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0 x 10(5) organisms per ml from infected mice, were 23-fold and 150-fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.

Animals↗
PubMed1992

Subtelomeric expression regions of Borrelia hermsii linear plasmids are highly polymorphic.

Borrelia hermsii, a relapsing fever agent, undergoes multiphasic antigenic variation to evade its host's immune response. Serotype specificity is determined by variable membrane lipoproteins, Vmps, which are expressed from genes located near the end of a linear plasmid. Using the polymerase chain reaction and primers representing the promoter of the active vmp and a conserved telomeric sequence, we characterized the subtelomeric expression regions of the 25 known serotypes of strain HS1. The distance from the promoter to the telomere fell into three size classes of approximately 1.0, 1.5, and 2.5 kilobases. In the sequenced serotypes the size differences were accounted for by variable lengths of the vmp genes and intervening sequences between 3' end of the vmp gene and the start of a downstream homology block. The degree of nucleotide identity between different vmp genes, or between the different 3' flanking DNA varied from 39-78%. Thus, there is length and sequence variability not only between vmp genes themselves but also between the 3' flanking regions of vmp genes.

Antigenic Variation↗
PubMed2006

Borrelia burgdorferi lipoproteins are secreted to the outer surface by default.

Borrelia spirochaetes are unique among diderm bacteria in their abundance of surface-displayed lipoproteins, some of which play important roles in the pathogenesis of Lyme disease and relapsing fever. To identify the lipoprotein-sorting signals in Borrelia burgdorferi, we generated chimeras between the outer surface lipoprotein OspA, the periplasmic oligopeptide-binding lipoprotein OppAIV and mRFP1, a monomeric red fluorescent reporter protein. Localization of OspA and OppAIV point mutants showed that Borrelia lipoproteins do not follow the '+2' sorting rule which targets lipoproteins to the cytoplasmic or outer membrane of Gram-negative bacteria via the Lol pathway. Fusions of mRFP1 to short N-terminal lipopeptides of OspA, and surprisingly OppAIV, were targeted to the spirochaetal surface. Mutagenesis of the OspA N-terminus defined less than five N-terminal amino acids as the minimal secretion-facilitating signal. With the exception of negative charges, which can act as partial subsurface retention signals in certain peptide contexts, lipoprotein secretion occurs independent of N-terminal sequence. Together, these data indicate that Borrelia lipoproteins are targeted to the bacterial surface by default, but can be retained in the periplasm by sequence-specific signals.

Amino Acid Sequence↗
PubMed2006

Unexpected twist: harnessing the energy in positive supercoils to control telomere resolution.

Negative DNA supercoiling is an important conformational property of bacterial DNA that plays a significant role in a wide variety of DNA transactions. In contrast, positive DNA supercoiling is a by-product of cellular processes that involve helical unwinding or movement of DNA by a fixed translocase, and has generally been considered a necessary evil requiring removal. We now report the first evidence suggesting a physiological role for positive supercoiling; this occurs in telomere resolution in the related Lyme disease and relapsing fever Borrelia spirochetes. Telomere resolution is the process whereby covalently closed hairpin telomeres are generated from replicative intermediates by the telomere resolvase, ResT. We observe a 20-fold and greater stimulation of the reaction by positive supercoiling, which facilitates formation of a previously unobserved reaction intermediate. Our data suggest the possibility that the free energy of positive supercoiling, a resource with no previously described cellular function, may be harnessed and utilized as a regulator of post-replication events.

Bacterial Proteins↗