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PubMed2001

Serial determinations of platelet counts in mice by flow cytometry.

Elucidation of the pathophysiological basis of platelet disorders in murine models requires a reliable method for the frequent determinations of platelet counts in individual mice. Here, we present a rapid, reproducible and accurate flow cytometric method for enumeration of platelets that involves fluorescent staining of platelets in whole blood with specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume. Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and that only five microliters of blood were required for platelet counting. Using this method, we followed platelet counts in mice infected with the relapsing fever spirochete Borrelia turicatae for 26 days, and found that this bacterium induces thrombocytopenia, a common manifestation of human relapsing fever. Therefore, this method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.

Animals↗
PubMedDate not supplied

Antigenic variation of surface proteins of Borrelia species.

During relapsing fever, the etiologic spirochetes employ antigenic variation to avoid early immune clearance. In Borrelia hermsii, an agent of tick-borne relapsing fever, a switch in serotype is associated with a change in the predominant protein on the surface of the cells. The variable major proteins differ from one another in primary structure along their lengths. Genes encoding the different variable major proteins can be found in both "silent" and "active" environments. At the active site the gene is expressed; at the silent site the gene appears to be in storage. In variant cells, which express the new serotype-specific surface protein in a population, a gene from a silent site has replaced the previously sitting gene at the active site through transposition.

Animals↗
PubMedDate not supplied

[Clinical and morphological characteristics of migrating erythema in patients with Ixodes tick-borne Lyme disease].

Patients with erythematous Ixodes tick-borne relapsing fever were examined and their skin biopsy specimens were morphologically studied to reveal clinical, immunological and morphological features of erythematous Ixodes tick-borne relapsing fever. Two types of development of erythema migrans were identified. These include 1) a typical type that appears as an area of homogenous hyperemia or that of annular shape and 2) an atypical one that presents as minor vesicles. There were elevated serum immunoglobulins A levels at the height of the disease. Morphologically, at the early stage of the disease, the center of erythema shows disturbances characterized by epidermal dystrophic processes, koilocytosis, subhorny and epidermal vesicles. The dermis displays solid perivascular lymphocytic infiltrates admixed with fibroblasts, fibrocytes, macrophages, plasmocytes, eosinophils, degranulated labrocytes. The interstitium exhibits scanty infiltrates. These changes are less pronounced at the periphery. Electron microscopy shows the structures morphologically similar to those of Borrelia. The late stage (days 15-23) of the disease is marked by insignificant dystrophy and perivascular fibrosis. There were no interstitial infiltrates. By and large, the pattern of clinical and immunological manifestations in patients with erythema migrans correlates with dermal morphological changes.

Adolescent↗
PubMed1997

[Tick-borne recurrent fever. Description of 5 cases].

BACKGROUND: Tick-borne relapsing fever (FRGT) is a worldwide zoonosis. The disease is caused by spirochetes of the genus Borrelia, and is transmitted to man by ticks of the genus Ornithodoros (O. erraticus in Spain). METHODS: 5 FRTG cases attended in our hospital in a 2 years period are described. Diagnosis was established in all cases by demonstration of borreliae in peripheral blood. Three Borrelia strains were isolated from blood of 3 of our patients and they are maintained by passages through mice. RESULTS: High fever, tachycardia and headache were noted in all cases accompanied in 2 of them by hepatosplenomegaly. Three of the patients complained of arthromyalgias or lumbar myalgia. Treatment with doxycicline was curative in all of them. CONCLUSIONS: FRGT incidence in our country is probably underestimated due to the low suspicion of the disease and the specificity of the clinical symptoms. The isolement of the causative Borrelia strain will allow the development of more specific serological tests necessary to establish the prevalence of this disease as well to assess its implication in chronic diseases, as is observed in other borreliosis (i.e. Lyme disease), that might be missed at this moment.

Adult↗
PubMedDate not supplied

Q fever endocarditis: relapse five years after successful valve replacement for a first unrecognized episode.

A 59-yr-old man presented with mitral endocarditis and negative blood cultures. Antibodies to phase 2 and phase 1 antigens of Coxiella burneti were detected and a diagnosis of Q fever endocarditis was made. Five years earlier, this patient had been successfully treated by aortic valve replacement for a first episode of endocarditis with negative blood cultures. Giemsa and Machiavello stains of the native aortic valve were made retrospectively and showed coccobacilli highly suggestive of Coxiella organisms. It is concluded that the first episode was Q fever endocarditis and that the failure to recognize this aetiology at that time, and the absence of adequate medical therapy, is the cause of the present episode.

Aortic Valve↗
PubMed2002

Comparative reactivity of human sera to recombinant VlsE and other Borrelia burgdorferi antigens in class-specific enzyme-linked immunosorbent assays for Lyme borreliosis.

A comparative study of human sera was conducted to determine which purified preparations of 11 recombinant antigens of Borrelia burgdorferi sensu stricto were diagnostically most important in enzyme-linked immunosorbent assays (ELISAs). To assess sensitivity, 20 serum samples obtained 1-6 weeks after onset of illness from 20 persons who had physician-diagnosed erythema migrans (EM) were tested for IgM and IgG antibodies. In tests for IgM antibody, seropositivity of > or = 25% was recorded when ELISAs had separate preparations of protein (p) 37, p41-G, outer-surface protein (Osp) C, OspE, OspF or VlsE antigens. Sera reacted most frequently (80% positive) with VlsE antigen in analyses for IgG antibodies. When results of both class-specific assays were considered for VlsE, OspC or OspF, 90% of the EM cases were serologically confirmed. Results of specificity testing with a further 59 sera from persons who had syphilis, louse-borne relapsing fever, oral infections, rheumatoid arthritis or human granulocytic ehrlichiosis and 28 normal sera indicated no false positive reactions when VlsE antigen was used in tests for IgM antibody. One of the 11 louse-borne relapsing fever sera cross-reacted with VlsE antigen in tests for IgG antibodies. Minor cross-reactivity also occurred when p37, OspC, OspE or OspF antigens were used. Overall, VlsE was the most suitable antigen for laboratory diagnosis of Lyme borreliosis during the early weeks of B. burgdorferi infection because of its high sensitivity and specificity.

Antibodies, Bacterial↗
PubMed2004

Borrelia turcica sp. nov., isolated from the hard tick Hyalomma aegyptium in Turkey.

Previously, a novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium, which infests tortoises (Testudo graeca), by using Barbour-Stoenner-Kelly (BSK) II medium; the tick samples were taken from the Istanbul area in northwestern Turkey [Güner et al. (2003). Microbiology 149, 2539-2544]. Here is presented a detailed characterization of the spirochaete. Electron microscopy revealed that strain IST7T is morphologically similar to other spirochaetes of the genus Borrelia and possesses 15 to 16 flagellae that emerge from both polar regions. PFGE analysis revealed the genome to comprise a linear chromosome of approximately 1 Mb; two large linear plasmids of approximately 145 and 140 kb, and several small plasmids ranging from 50 to 20 kb in size were also found. The 16S rRNA gene sequence of this Borrelia isolate exhibited 99.4 to 99.8 % identity with other strains isolated from H. aegyptium and less than 99 % similarity with those of other Borrelia species. A phylogenetic tree, generated from 16S rRNA gene sequences, demonstrated that the spirochaete isolates from H. aegyptium clustered together and branched off from both Lyme-disease-related and relapsing-fever-associated Borrelia species. A single copy of the rrs gene was detected in the genome of strain IST7T by Southern hybridization. DNA-DNA hybridization results showed that strain IST7T was distinct from Lyme-disease-related Borrelia, Borrelia burgdorferi and the relapsing-fever-associated species Borrelia hermsii. The G+C content of strain IST7T is 30.0 mol%. From these genetic features, a novel Borrelia species, Borrelia turcica sp. nov., is proposed; the type strain is IST7T (= JCM 11958T = DSM 16138T).

Animals↗
PubMed2000

Molecular and immunological analyses of the Borrelia turicatae Bdr protein family.

Here, we describe the molecular and immunological characterization of the bdr gene family of Borrelia turicatae, a relapsing-fever spirochete. Nine bdr alleles belonging to two different subfamilies were sequenced and localized to linear plasmids. Anti-Bdr antiserum was generated and used to analyze Bdr expression in pre- and postinfection isogenic populations. The analyses presented here provide a detailed characterization of the Bdr proteins in a relapsing-fever spirochete species, enhancing our understanding of these proteins at the genus-wide level.

Alleles↗
PubMed2005

[Diseases produced by Borrelia].

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is a multi-organ infection with dermatological, rheumatological, neurological, and cardiac manifestations. The main characteristic is a skin lesion, named erythema migrans. Relapsing fever, caused by numerous species of Borrelia, is characterized by a periodic cycle of acute and afebrile episodes. The serological diagnosis of these infections has limited value in sensitivity, specificity and predictive values. Lyme borreliosis is usually diagnosed by recognition of a characteristic clinical picture with serological confirmation, and the diagnosis of relapsing fever relies on direct observation of spirochetes in peripherical blood. The elected treatment is almost always tetracycline for the young or for adults but not for pregnant women, although betalactamic (such as penicillin or 3rd generation cephalosporin for the central nervous system) or macrolides are indicated in several situations. The prognosis, with adequate treatment, is good. In the majority of Spanish regions, due to the low incidence of these diseases, the prophylactic antimicrobial treatment after a tick bite is not indicated.

Adult↗
PubMed1999

Body lice as tools for diagnosis and surveillance of reemerging diseases.

Body lice are vectors of three bacteria which cause human disease: Rickettsia prowazekii, the agent of epidemic typhus; Bartonella quintana, the agent of trench fever; and Borrelia recurrentis, the agent of relapsing fever. A recrudescence of body lice is being observed as the numbers of individuals living under social conditions which predispose individuals to infestation have increased. Because this phenomenon may lead to the reemergence of infections transmitted by body lice, we aimed to assess the occurrence and prevalence of the three agents described above in more than 600 body lice collected from infested individuals in the African countries of Congo, Zimbabwe, and Burundi, in France, in Russia, and in Peru. The presence of the three bacteria in each louse was determined by specific PCR amplification, and the identities of the organisms detected were confirmed by determination of the nucleotide base sequences of the amplification products. Using this approach, we were able to confirm the presence of R. prowazekii in lice collected from refugees in Burundi, among whom typhus was epidemic, and the presence of B. quintana in lice collected from all locations except the Congo. B. recurrentis was never found. Molecular approaches are convenient tools for the detection and identification of bacterial DNA in body lice and for the epidemiological study of louse-borne bacteria from countries where no medical and biological laboratory facilities are available.

Animals↗
PubMed1988

Segmented arrangement of Borrelia duttonii DNA and location of variant surface antigen genes.

The DNA of an isolate of Borrelia duttonii, an agent of relapsing fever is present as seven major species ranging in size from 10 kb to greater than 150 kb. Additionally, this isolate contains low copy number species, both smaller and larger than these seven major elements. No one of these individual DNA species obviously corresponds to the bacterial chromosome, unlike the situation in Borrelia hermsii, another relapsing fever Borrelia. Thus it appears that B. duttonii has a unique segmented arrangement of its genetic material. Cloned DNA fragments containing coding sequences specific for variant surface antigens of B. duttonii hybridize to a closely migrating, high copy number subset of these genetic elements.

Animals↗
PubMed1992

Relapsing illness due to Rochalimaea henselae in immunocompetent hosts: implication for therapy and new epidemiological associations.

Two previously healthy, immunocompetent men had persistent Rochalimaea henselae bacteremia with clinical relapses after courses of antibiotics to which the isolates were ultimately demonstrated susceptible in vitro. Both had sustained tick bites prior to their illnesses, thus demonstrating an association not previously identified, although suspected. The first patient had relapsing fever, constitutional symptoms, and an episode of aseptic meningitis despite therapy with amoxicillin, then with doxycycline, and then with ceftriaxone. Thereafter, he spontaneously became asymptomatic during a span of 2 months of persistent bacteremia. Finally, after 2 weeks of therapy with ceftriaxone plus gentamicin, followed by 4 weeks of therapy with oral ciprofloxacin, his bacteremia was cured. The second man had relapsing fever and constitutional symptoms after courses of tetracycline, then of chloramphenicol, and then of doxycycline. He became permanently asymptomatic after serial 2-week courses of chloramphenicol and erythromycin. The greater efficacy of lysis-centrifugation blood cultures in the recovery of R. henselae was noted.

Adult↗
PubMed2000

Evolutionary and molecular analyses of the Borrelia bdr super gene family: delineation of distinct sub-families and demonstration of the genus wide conservation of putative functional domains, structural properties and repeat motifs.

B. turicatae, a causative agent of relapsing fever, carries a polymorphic gene family that is homologous to the bdr gene family of the Lyme disease spirochetes (previously referred to as the rep+ or ORF-E gene family). Here we demonstrate that bdr related genes are widely distributed among pathogenic Borrelia species and exist as large, polymorphic, plasmid carried, gene families. Twenty distinct bdr alleles were identified in isolates of the relapsing fever spirochete, B. hermsii, and were localized to linear plasmids. Cloning and sequence analyses demonstrate that the putative Bdr functional domains (i.e. the phosphorylation motifs and the transmembrane C-terminal domain) are conserved across the genus while other regions of these proteins exhibit variability. An assessment of the evolutionary relationships among all known Bdr protein sequences obtained from five pathogenic Borrelia species revealed that there are distinct Bdr sub-families. The recognition of distinct phyletic clusters serves as the basis of a revised and simplified nomenclature for the bdr proteins that can be applied genus wide. At the biological level the delineation of multiple bdr sub-families within isogeneic populations raises the possibility that there may be functional partitioning among alleles. In summary, the distribution and conservation of the Bdr proteins suggests that they are important in the biology/pathogenesis of the Borrelia at the genus wide level.

Amino Acid Sequence↗
PubMed2001

Structural conservation of neurotropism-associated VspA within the variable Borrelia Vsp-OspC lipoprotein family.

Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to avoid the immune response. One of these proteins, VspA of Borrelia turicatae, is also associated with neurotropism in infected mice. Vsp proteins are highly polymorphic in sequence, which may relate to their specific antibody reactivities and host cell interactions. To determine whether sequence variations affect protein structure, we compared B. turicatae VspA with three related proteins: VspB of B. turicatae, Vsp26 of the relapsing fever agent Borrelia hermsii, and OspC of the Lyme disease spirochete Borrelia burgdorferi. Recombinant non-lipidated proteins were purified by affinity or ion exchange chromatography. Circular dichroism spectra revealed similar, highly alpha-helical secondary structures for all four proteins. In vitro assays demonstrated protease-resistant, thermostable Vsp cores starting at a conserved serine at position 34 (Ser(34)). All proteins aggregate as dimers in solution. In situ trypsin treatment and surface protein cross-linking showed that the native lipoproteins also form protease-resistant dimers. These findings indicate that Vsp proteins have a common compact fold and that their established functions are based on localized polymorphisms. Two forms of VspA crystals suitable for structure determination by x-ray diffraction methods have been obtained.

Amino Acid Sequence↗
PubMed1994

Prevalence of antibodies to Borrelia burgdorferi, Borrelia parkeri and Borrelia turicatae in human settlements of the Cordillera Province, Bolivia.

A seroepidemiological study to determine the prevalence of human Lyme borreliosis and tick-borne relapsing fever was carried out in three communities (Camiri, Boyuibe and Gutierrez) of the Cordillera Province, Santa Cruz Department, south-eastern Bolivia. Anti-B. burgdorferi, anti-B. turicatae and anti-B. parkeri antibodies, tested by the indirect immunofluorescent assay (IFA), were detected in 10.8, 16.1 and 8.2% of the serum samples tested, and confirmed by IFA-ABS in 1.3, 1.3 and 1.0%, respectively. This is the first report of the presence of Lyme borreliosis and tick-borne relapsing fever in Bolivia. For Lyme borreliosis these findings represent a further datum to support its existence in South America.

Adolescent↗
PubMed1999

Comparative analysis and immunological characterization of the Borrelia Bdr protein family.

Multiple circular and linear plasmids of Lyme disease and relapsing fever Borrelia spirochetes carry genes for members of the Bdr (Borrelia direct repeat) protein family. To define their common and divergent attributes, we first comprehensively compared the known homologs. Bdr proteins with predicted sizes ranging from 10.7 to 30. 6 kDa formed five homology groups, based on variable numbers of short direct repeats in a central domain and diverse N- and C-terminal domains. In a further characterization, Western blots were probed with rabbit antisera raised against either of two purified recombinant Bdr proteins from Borrelia burgdorferi B31. The results showed that antibodies cross-react and several Bdr paralogs 19.5 to 30.5 kDa in size are expressed by cultured strain B31 in a temperature-independent manner. In situ proteolysis, immunofluorescence, and growth inhibition assays indicated that Bdr proteins are not surface exposed. Distinct patterns of cross-reacting proteins of 17.5 to 33 kDa were also detected in other B. burgdorferi, Borrelia garinii, and Borrelia afzelii strains as well as in relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae. Last, we examined whether these proteins are antibody targets during Lyme disease. Analysis of 47 Lyme disease patient sera by immunoblotting and enzyme-linked immunosorbent assays showed that 24 (51%) and 20 (43%), respectively, had detectable antibodies to one or more of the Bdr proteins. Together, these data indicate that Bdr proteins constitute a family of cross-reactive Borrelia proteins which are expressed in the course of Lyme disease and in vitro.

Amino Acid Sequence↗
PubMed2006

Molecular analyses of the interaction of Borrelia hermsii FhbA with the complement regulatory proteins factor H and factor H-like protein 1.

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.

Amino Acid Sequence↗
PubMed1982

Variable major proteins of Borrellia hermsii.

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

Antigens, Bacterial↗