An OspA-based genospecies identification of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan

To further identify the genospecies of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan, we analyzed the genomic identities of these Taiwan isolates (TWKM1-7) by genospecies-specific polymerase chain reaction (PCR) assay, restriction fragment length polymorphism (RFLP) analysis, and gene sequencing based on the OspA gene sequences of B. burgdorferi sensu lato. PCR analysis indicates that all of these Taiwan isolates were genetically related to the genospecies of B. burgdorferi sensu stricto by their differential reactivities with genospecies-specific PCR primers. After cleavage by DraI, three different RFLP patterns in relation to three different genospecies of Lyme disease spirochetes were observed, and all of these Taiwan isolates were affiliated with the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis also reveals that the sequence similarity of PCR-amplified OspA gene of these Taiwan isolates is highly homogeneous, with a homogeneity of more than 99.8% within the genospecies of B. burgdorferi sensu stricto. These results confirm that the genomic identities of these Taiwan isolates belong to the genospecies of B. burgdorferi sensu stricto.     

Borrelia bissettii isolates induce pathology in a murine model of disease

The spirochete Borrelia burgdorferi is a tick-borne pathogen that causes Lyme disease. Although B. burgdorferi sensu lato is a diverse group of bacteria, only three genospecies, B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are known to be pathogenic and commonly recognized to cause human disease. To assess the potential of another common genospecies, Borrelia bissettii, to induce disease, a mouse model was employed. Two Colorado isolates of B. bissettii (CO-Bb) induced lesions of the bladder, heart, and femorotibial joint 8 weeks after inoculation into mice. In contrast, two British Columbia (BC-Bb) isolates, could not be cultured or amplified by PCR from target organs, and did not induce lesions. Consistent with pathology and culture results, the antibody response in mice to BC-Bb was minimal compared to CO-Bb, indicating either transient localized infection or rapid immune clearance of BC-Bb. Although sequence analysis of the rrf (5S)-rrl (23S) intergenic spacer region indicated 99% homology between CO-Bb and BC-Bb, polyacrylamide gel electrophoresis (PAGE) analysis indicated five distinct protein differences between these low-passage isolates. These studies support the prospect that B. bissettii may indeed be the causative agent of Lyme borreliosis cases in Eastern Europe, associated with the atypical Borrelia strain 25015, and in other regions. To our knowledge, this is the first evidence that B. bissettii can induce pathology in a vertebrate host.     

Association of specific subtypes of Borrelia burgdorferi with hematogenous dissemination in early Lyme disease.

To investigate whether genetic diversity of Borrelia burgdorferi sensu stricto may affect the occurrence of hematogenous dissemination, 104 untreated adults with erythema migrans from a Lyme disease diagnostic center in Westchester County, New York, were studied. Cultured skin isolates were classified into 3 groups by a polymerase chain reaction amplification and restriction fragment length polymorphism (RFLP) method. A highly significant association between infecting RFLP type in skin and the presence of spirochetemia was found (P<.001). The same association existed for the presence of multiple erythema migrans lesions (P=.045), providing clinical corroboration that hematogenous dissemination is related to the genetic subtype of B. burgdorferi sensu stricto. There were no significant associations between RFLP type and seropositivity or clinical symptoms and signs except for a history of fever and chills (P=.033). These results suggest that specific genetic subtypes of B. burgdorferi sensu stricto influence disease pathogenesis. Infection with different subtypes of B. burgdorferi sensu stricto may help to explain differences in the clinical presentation of patients with Lyme disease.     

Three major Lyme Borrelia genospecies (Borrelia burgdorferi sensu stricto,B. afzelii and B. garinii) identified by PCR in cerebrospinal fluid from patients with neuroborreliosis in Sweden

The Lyme Borrelia genospecies Borrelia afzelii and B. garinii have previously been isolated using a culture method in Swedish patients with Lyme borreliosis (LB). There are reports suggesting that the genospecies distribution in human tissue specimens as determined by molecular methods is different from that obtained by culture. In the present study, we developed a nested PCR for detection of Lyme Borrelia-specific DNA in cerebrospinal fluid from Swedish patients with LB. The genospecies were subsequently identified by sequence analysis in a total of 7 PCR-positive patients. Two sequences were identified as B. burgdorferi sensu stricto (s. s.), 1 as B. afzelii and 4 as B. garinii. These are the first reported cases in which B. burgdorferi s. s. has been shown to be the causative agent of human LB in Sweden. The results of our study confirm that the use of direct molecular analytical methods for Borrelia genospecies identification in clinical specimens can provide epidemiological information additional to that obtained by culture.     

Genetic diversity of Borrelia burgdorferi sensu lato isolates from Northeastern China

Thirty-two strains of Borrelia burgdorferi sensu lato were isolated from Ixodes persulcatus ticks collected from northeastern China from May to June in 2004 and 2005. Restriction fragment length polymorphism (RFLP) analysis and sequence analysis of 5S-23S rRNA intergenic spacer revealed that 29 (90.6%) belonged to Borrelia garinii, demonstrating B, C, and a unique pattern. The remaining three isolates (9.4%) were Borrelia afzelii with pattern D. The phylogenetic analysis based on 5S-23S rRNA intergenic spacer showed that B. garinii and B. afzelii genospecies clustered into two separate lineages. B. garinii strains were classified into three different branches: All the strains with RFLP pattern C were in the same branch, strain VH10 with a unique RFLP pattern clustered with strains VH9 and MDH2 with pattern B, and the rest of the strains with pattern B constitute another branch. These findings demonstrate the genetic diversity of B. burgdorferi sensu lato isolates from northeastern China.     

Prevalence and genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ticks in Belgium

Borrelia burgdorferi sensu lato is a genetically diverse group of spirochetes that includes the agent of Lyme borreliosis in which genospecies tend to be associated with specific clinical features. The aim of the study was to determine the prevalence and genetic diversity of Borrelia burgdorferi sensu lato in 524 ticks collected in woods of a western province of Belgium. Presence of spirochetes in ticks was determined by phase contrast microscopy. The mean infection rate of ticks was 12.0%. Variability was observed in the prevalence of infection among the five sites examined, ranging from 2.8 to 21.6%. Identification to genospecies was determined by PCR and sequencing. The most common genomospecies were Borrelia afzelii (55%) and Borrelia garinii (21%). For the first time in Belgium, we detected Borrelia valaisiana and Borrelia spielmanii, representing 14% and 2%, respectively. Borrelia burgdorferisensu stricto counted only for 2%. Co-infections were present in 8% of ticks. We emphasize the need for clinical studies to assess the prevalence of specific genospecies-related clinical manifestations of Lyme borreliosis in Belgium.     

Prevalence of Anaplasma phagocytophilum and coinfection with Borrelia burgdorferi sensu lato in the hard tick Ixodes ricinus in the city of Hanover (Germany)

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.     

Antigenic conservation of an immunodominant invariable region of the VlsE lipoprotein among European pathogenic genospecies of Borrelia burgdorferi SL

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.     

Disease severity in a murine model of lyme borreliosis is associated with the genotype of the infecting Borrelia burgdorferi sensu stricto strain

The pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing 2 distinct ribosomal DNA spacer restriction fragment-length polymorphism genotypes (RSTs) was assessed in a murine model of Lyme disease. B. burgdorferi was recovered from 71.5% and 26.6% of specimens from mice infected with RST1 and RST3 isolates, respectively (P<.0001). The average ankle diameter and histologic scores for carditis and arthritis were significantly higher after 2 weeks of infection among mice infected with RST1 isolates than among those infected with RST3 isolates (P<.001). These clinical manifestations were associated with larger numbers of spirochetes in target tissues but not with the serum sensitivity of the individual isolates. Thus, the development and severity of disease in genetically identical susceptible hosts is determined mainly by the pathogenic properties of the infecting B. burgdorferi isolate. The RST1 genotype is genetically homogeneous and thus may represent a recently evolved clonal lineage that is highly pathogenic in humans and animals.     

Molecular and serological evidence of Borrelia burgdorferi sensu lato in wild rodents in the Czech Republic

The aim of the present study was to determine the frequency and spatial distribution of the Borrelia species in wild rodents in the Czech Republic. In total, 293 muscle tissue samples and 106 sera from 293 wild rodents captured in North Bohemia and North-East and South Moravia were examined for the presence of Borrelia spp. and antibodies. Muscle samples were investigated with real-time polymerase chain reaction (PCR) with a recA primer set, with DNA quantification and melting curve analysis, and with restriction fragment length polymorphism (RFLP) analysis of the 5S-23S rDNA intergenic spacer. Infection with Borrelia burgdorferi sensu lato was found in 16.4% of the muscle samples. The most abundant genospecies was Borrelia afzelii (11.3%), followed by Borrelia burgdorferi sensu stricto (4.8%) and Borrelia garinii (0.7%). Borrelia infection was more frequently observed in Clethrionomys glareolus than in Apodemus spp. Sera were analyzed with an enzyme-linked immunosorbent assay (ELISA) test, yielding the total seropositivity rates of 24.5% for anti-Borrelia IgM antibodies and 25.5% for IgG antibodies. Total seroprevalence was higher in Apodemus spp. than in C. glareolus. In conclusion, our data indicate that in the Czech Republic small wild rodents can serve as hosts for B. burgdorferi s. s. as well as for B. afzelii.     

Isolation of Borrelia burgdorferi sensu lato in ticks Ixodes ricinus by polymerase chain reaction (PCR)

Attempts were made to identify the causative orgamsm of Lyme disease in Szczecin from tick Ixodes ricinus as a vector. Ticks were collected in 1997 year in forest areas of Szczecin, from localites associated with numerous attendance of people. The method used in this study was the polymerase chain reaction (PCR) on the flagellin structural gene fla of Borrelia burgdorferi sensu stricto. The flagellin PCR primer set reaction was conservative for B. burgdorferi sensu stricto, B. afzelii and B. garinii. The overall prevalence of B. burgdorferi sensu lato, in tick population studied was 8.8%. The female, nymphs and larves of Ixodes ricinus were infected almost just the some--about 10%, when the male 2.5% only.     

Isolation of Borrelia burgdorferi sensu lato from the skin of the European badger (Meles meles) in Switzerland

No data are available on the role of badgers in the ecology of Lyme borreliosis spirochetes in Europe. In a recent study describing validation of a molecular method allowing host DNA identification and Borrelia burgdorferi sensu lato detection in Ixodes ricinus, the simultaneous presence of B. afzelii DNA and of European badger (Meles meles) DNA was detected in I. ricinus ticks in Switzerland. This suggested that badgers might be reservoir hosts for B. afzelii. Here, we present results obtained in a study on badgers conducted in 1996-1997. Thirty-one tissue samples (ear biopsy: n = 25, aspiration fluid: n = 6) from 8 badgers were placed in BSK medium to isolate B. burgdorferi sensu lato and were then examined by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Globally, six Borrelia isolates (6/31, 19.4%) were obtained from 3/8 (37.5%) badgers. These isolates were identified as B. afzelii (n = 3) and B. valaisiana (n = 3).     

An enzootic transmission cycle of Lyme borreliosis spirochetes in the southeastern United States

Lyme borreliosis, or Lyme disease (LD), is a tick-borne zoonotic infection of biomedical significance, caused by Borrelia burgdorferi sensu lato (s.l.) spirochetes and transmitted by Ixodes species ticks. It usually circulates among wildlife vertebrate reservoirs and vector ticks but may infect humans, causing multisystem problems. In far western and northern North America, the host reservoirs, tick vectors, and genospecies of Borrelia are well known but not so in the southern U.S., where there is controversy as to the presence of "true" LD. Here we report the presence of the LD spirochete B. burgdorferi sensu stricto (s.s.) and Borrelia bissettii, three main reservoir hosts, and two enzootic tick vectors in the southeastern U.S. The two enzootic tick vectors, Ixodes affinis and Ixodes minor, rarely bite humans but are more important than the human biting "bridge" vector, Ixodes scapularis, in maintaining the enzootic spirochete cycle in nature. We also report extraordinary longevities and infections in the reservoir rodents Peromyscus gossypinus, Sigmodon hispidus, and Neotoma floridana.     

Invasion of human tissue ex vivo by Borrelia burgdorferi

Borrelia burgdorferi sensu stricto is an etiological agent of Lyme disease. The lack of an adequate ex vivo system for human tissue infection is an obstacle to fully understanding the molecular mechanisms of invasion of tissue by B. burgdorferi and its adaptation within the human host. Here, we report on the development of such a system. We inoculated blocks of human tonsillar tissue with B. burgdorferi spirochetes, cultured them in a low-shear rotating wall vessel (RWV) bioreactor, and analyzed them using light and electron microscopy, nested polymerase chain reaction (PCR), and quantitative real-time PCR. Also, we evaluated the expression of the outer surface proteins (Osps) OspA and OspC by use of quantitative Western blotting. Light and electron microscopic analysis revealed multiple spirochetes localized extracellularly within the tissue, and their identity was confirmed by PCR. Quantification of spirochetes inside the RWV-cultured tonsillar tissue demonstrated that the number of B. burgdorferi exceeded the initial inoculum by an order of magnitude, indicating that spirochetes replicated in the tissue. Electron microscopic analysis showed that some spirochetes were arranged in cystic structures and that invading spirochetes differentially expressed surface proteins; both of these features have been described for infected tissues in vivo. The system we have developed can be used to study B. burgdorferi pathogenesis under controlled conditions ex vivo, in particular to explore the gene activation responsible for the adaptation of B. burgdorferi to human tissue that leads to Lyme disease.     

Lyme borreliosis

Lyme borreliosis is a multi-organ infection caused by spirochetes of the Borrelia burgdorferi sensu lato group with its species B burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, which are transmitted by ticks of the species Ixodes. Laboratory testing of Lyme borreliosis includes culture, antibody detection using ELISA with whole extracts or recombinant chimeric borrelia proteins, immunoblot, and PCR with different levels of sensitivity and specificity for each test. Common skin manifestations of Lyme borreliosis include erythema migrans, lymphocytoma, and acrodermatitis chronica atrophicans. The last two conditions are usually caused by B garinii and B afzelii, respectively, which are seen more frequently in Europe than in America. Late extracutaneous manifestations of Lyme borreliosis are characterised by carditis, neuroborreliosis, and arthritis. We present evidence-based treatment recommendations for Lyme borreliosis and review the prevention of Lyme borreliosis, including the Lyme vaccines.     

Genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in north-west Poland

Borrelia burgdorferi sensu lato (s. l.), the etiological agent of Lyme diesease, is transmitted by the bite of Ixodes ricinus. During May and September 1999, field surveys on Lyme disease spirochetes were conducted in three locations of a region of north-west Poland, known as recreational districts visited by many people. The ticks Ixodes ricinus were collected in natural habitats by dragging a flanel cloth over the vegetation. Sex and developmental stage of each tick were determined. Based on a polymerase chain reaction test with primers that recognize a chromosomal gene of all strains, out of the total 1414 specimens collected, 126 (8.9%) were found to be infected. The species B. burgdorferi s. l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s. s.), Borrelia garinii, and Borerelia afzelii, witch could be distinguished in nested-PCR tests with species-specific primers. B. burgdorferi s. s. was most prevalent (96% of infected ticks), followed by B. garinii (1.3%), and B. afzelii. was not found. Of the infected ticks, over the 99% were infected with a single species, one specimens was infected with two species. For 4 ticks, the infecting species could not be identied. The difference in rates of prevalence was observed among the tree locations (17%--5.3%--3.2%).     

Diversity of Ixodes-borne Borrelia species--clinical,pathogenetic, and diagnostic implications and impact on vaccine development

Among Borrelia spirochetes carried by hard ticks belonging to the various Ixodes species, at least 10 species can be distinguished. Of these, Borrelia burgdorferi sensu stricto is involved in human Lyme borreliosis in North America and Europe, and Borrelia garinii and Borrelia afzelii in human disease in Europe and Asia. The pathogenetic significance of the other species is uncertain. Although some of the Borrelia species are restricted to certain tick species, Ixodes ricinus, the vector of Lyme borreliosis in Europe, can be infested by at least five different species, including all three pathogenic species. There is evidence that different Borrelia species are preferentially found in different hosts: In Europe, B. afzelii is frequently found in small mammals, whereas B. garinii and Borrelia valaisiana are often found in birds. This could very well be related to differential sensitivity of these species to complement-mediated bactericidal activity of different hosts. Borrelial complement regulator acquiring proteins, among them OspE or Erp proteins, bind to host factor H and related proteins, and this binding protects against activation of complement by the spirochetal surface. The binding is different for proteins originating from different species and is also depending on the host origin of factor H. In Europe, B. garinii is mainly found in neuroborreliosis, whereas in skin disease B. afzelii is more frequently found. The reason is unclear. The majority of human sera cross-react between proteins of different Borrelia species, but some sera react only with proteins from one of the species. This holds especially for reactivity with OspC. A vaccine against B. burgdorferi sensu stricto has been licensed, but was recently redrawn from the market because of commercial reasons. A vaccine protecting against all three pathogenic species is not yet available.     

Use of western-immunoblotting to determine serum IgG-antibodies to antigens of different genotypes of Borrelia--Borrelia burgdorferi sensu stricto,Borrelia afzelii,and Borrelia garinii

The reaction of the sera from 86 patients with Lyme borreliosis was evaluated in the immunoblotting using three genotypes of pathogenic Borrelia strains. The Russian isolates of Borrelia afzelii (strain IP3) and B. garinii 20047T (strain IP90) were compared with the USA typical strain B31--B. burgdorferi sensu stricto. The results were assessed by the criteria recommended for the USA and developed for B. burgdorferi sensu stricto. Certain differences were shown in the reactions of serum IgG with major proteins of three Borrelia genotypes. The sera interacted with p37 of an I-90 isolate and with p39 of both Russian isolates significantly more frequently. The rate of positive results of a serum test using the strain B31 was 18.6% (16 patients); 13 patients were additionally identified when the Russian isolates were applied. It is expedient to use the genotypes circulating in Russia as an antigenic material for immunoblotting. Criteria for positive serum test results may be individual for each genotype of Borreliae.     

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.