Species identification of Borrelia burgdorferi sensu lato from tick and human isolates in Styria (Austria) by PCR-RFLP analysis

Seventy-one isolates of Borrelia burgdorferi sensu lato (B.b.s.l.) derived from Ixodes ricinus ticks (50 strains) and patients (21 strains) were characterised by PCR-RLFP analysis. In four cases the human isolates were obtained from the cerebrospinal fluid (CSF) of patients with clinical symptoms of neuroborreliosis and in 17 cases from skin biopsies of patients with dermatological manifestation of Lyme borreliosis. Ixodes ricinus isolates originated from 14 localities in three regions (Mur valley, eastern and western Styria) in Styria. Thirty six strains of B.b.s.l. were isolated from nymphal ticks, nine strains from female and five strains from male ticks. Species identification of human isolates revealed three B. garinii and one B. afzelii isolates in CSF. In the PCR-RFLP analysis of 17 skin specimens a pattern for B. afzelii was found in ten cases, while six could be identified as B. garinii and one as a mixed infection of B. afzelii and B. garinii. Genetic characterisation of tick isolates resulted in 24 strains of B. afzelii (48%), 11 strains of B. garinii (40%) and 5 strains of B. burgdorferi s.st. (10%); one isolate showed a mixed infection of B. afzelii and B. garinii. Our findings indicate that B. afzelii and B. garinii predominate over B. burgdorferi s.str. in Ixodes ricinus ticks from Styria, which is similar to findings in neighbouring countries. This also reflects the occurrence of different pathogenic Borrelia strains in human samples.     

Detection and identification of Borrelia burgdorferi sensu lato genospecies in ticks from three different regions in Slovakia

Lyme borreliosis is one of the most common tick-borne diseases that occur in Slovakia. In this study, Borrelia burgdorferi sensu lato was detected and cultivated from questing ticks collected in three areas of Slovakia. Two methods, restriction fragment length polymorphism and reverse line blot, were used for identification of isolates and determination of the prevalence of B. burgdorferi s.l. in the ticks. The prevalence of B. burgdorferi s.l. in I. ricinus detected by reverse line blot was 31.9%. Four genospecies, namely B. garinii, B. valaisiana, B. afzelii and B. burgdorferi sensu stricto were found. B. garinii was the most prevalent genospecies.     

Infection of small mammals with Borrelia burgdorferi sensu lato in Slovenia as determined by polymerase chain reaction (PCR)

Thirty-four small mammals collected in the vicinity of Ljubljana were tested for the presence of Borrelia burgdorferi sensu lato by polymerase chain reaction (PCR) of urinary bladder tissues, using universal flagellin primers and species specific rRNA primers. Seventeen small mammals (50%) were found to be positive, and 7 small mammals were infected with two species of B. burgdorferi sensu lato simultaneously. The most commonly found species was B. afzelii (n = 14), followed by B. burgdorferi sensu stricto (n = 7) and B. garinii (n = 3), as determined by species-specific primers. We conclude that PCR is a rapid and reliable method to detect infection with B. burgdorferi sensu lato in small mammals.     

Molecular epidemiology of the aetiological agents of Lyme borreliosis

Ten species are up to now recognized among Borrelia burgdorferi sensu lato complex. Among those, only three (Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii) are reported to be pathogenic for humans and each responsible for a predominant clinical form of Lyme borreliosis. Each species is characterized by its vectors (Ixodidae), its host spectrum, its organotropism (for the pathogenic ones) and its geographical repartition. Borrelia are strictly parasitic and essentially clonal bacteria. Our goal was to explore the diversity of this bacterial complex. We selected, by several molecular markers, atypical isolates and compared them to already known species representative strains by RFLP or sequencing. The results show an unexpected diversity at a level which could be a species one leading to the conclusion that the structure of the Borrelia burgdorferi sensu lato complex is a high number of small (by their populations) clones among which emerge some large ones usually corresponding to the pathogenic species. Our data also allow to speculate on when, where and how these species evolved and migrated.     

Characterization of Borrelia burgdorferi sensu lato strains isolated from human material in Slovenia

The aim of the study was to assess genotypic and phenotypic diversity among a large number of clinical isolates of Borrelia burgdorferi sensu lato obtained from patients in Slovenia. Plasmid profiles and species identification were determined by PFGE, protein profiles by SDS-PAGE. Of 706 B. burgdorferi sensu lato human isolates 599 (85%) were found to be B. afzelii, 101 (14%) B. garinii and six (1%) B. burgdorferi sensu stricto. The vast majority of strains (605; 86%) were isolated from skin, 58 (8%) from blood, and 43 (6%) from CSF. When analysed by RFLP, B. afzelii strains were unique, while heterogeneity was found within B. garinii and B. burgdorferi sensu stricto species. An unusual plasmid content was found in 52/706 (7%) isolated strains, more often in B. garinii than in B. afzelii strains. A plasmid dimer was found in B. afzelii and B. garinii strains, whereas multiple copies of the large plasmid were associated nearly exclusively with B. garinii strains. Analysis of protein profiles revealed that OspA and OspB are expressed more often by B. afzelii strains, and OspC by B. garinii strains. Heterogeneity of Borrelia strains may play a significant role in the virulence and pathogenesis of the infection. Differences in antigenic components have an important impact on serological testing and vaccine development.     

Prevalence of Borrelia burgdorferi sensu lato in the tick Ixodes ricinus in the Styrian mountains of Austria

A total of 691 Ixodes ricinus (22 male, 39 female, 501 nymphs and 129 larvae), the tick vector of Lyme borreliosis, were collected by flagging from vegetation in 11 areas at altitudes between 789 m and 1350 m above sea level in mixed woodland with pasture land (cattle) in the province of Styria in Austria. The ticks were individually examined for presence of Borrelia burgdorferi sensu lato by dark-field microscopy and 107 of them by real-time PCR. Attempts to cultivate borreliae were made in BSK-H medium. The overall positivity rate of all collected ticks (excepting larvae) was 10.9%: 9.1% in males, 17.9% in females and 10.4% in nymphs. The 129 larvae examined showed no presence of B. burgdorferi s.l. The mean infection rate of I. ricinus collected at the highest altitude in this study, Gaberl at 1350 m a.s.l.--and at the same time the highest one reported in Europe--was 6.4%: 1/9 males, 2/18 females and 6/114 (5.3%) nymphs were positive. Culture attempts were positive in 12 cases and species identification showed eight isolates were B. afzelii and four B. garinii. Three additional positive results found by PCR (negative by culture) were identified twice as B. afzelii and once as B. garinii. This study shows that the risk of acquiring Lyme borreliosis in habitats at higher altitudes is limited, because of the lower density of I. ricinus and its lesser infection rate than at lower altitudes in central Europe, but nevertheless the risk does exist.     

Prevalence of coinfection with Francisella tularensis in reservoir animals of Borrelia burgdorferi sensu lato

INTRODUCTION AND PURPOSE: Studies on Lyme borreliosis and other tick-borne zoonoses in the Austrian and Slovakian borderland, a region endemic for tularemia, revealed a relatively high prevalence of infection with Borrelia burgdorferi s.l. and Francisella tularensis in small terrestrial mammals, as well as in the ticks, during a one-year survey. The occurrence of coinfection with the agents of Lyme borreliosis and tularemia was assessed in different species of rodents. METHODS: Organs of small mammals, live-trapped mostly in six-week intervals from May 1994 to April 1995, were cultured on appropriate media in order to grow borreliae and F. tularensis. RESULTS: Infection with B. burgdorferi s.l. and also with F. tularensis was found in all the most abundant rodent species. A significant difference was observed in the time period of isolation of these agents. Borrelia was cultured from May to January (PCR detected borrelia up to April), while F. tularensis was isolated from August to December. Coinfection was seen in two species of voles, Clethrionomys glareolus trapped in August and Microtus arvalis in October. The Borrelia strains isolated from these animals were identified as B. garinii. Isolates of F. tularensis belonged to the subspecies holarctica, biovar II. CONCLUSIONS: Results obtained indicate that in endemic regions for tularemia the prevalence of infection with borreliae could be modified in different animal species mainly during epizootic outbreaks of tularemia.     

Seasonal variations in detecting Borrelia burgdorferi sensu lato in rodents from north eastern Austria

Austria is well known as an endemic area of Lyme borreliosis. To assess the annual variation of rodent populations that may host agents of Lyme borreliosis we collected rodents in northeastern Austria. Life traps were set out every six weeks during a year consecutively in one each of the three different zones (Hohenau, Ernstbrunn, Vienna Woods) that cover the main habitat characteristics of small mammals in northeastern Austria. Rodents were collected and identified. Samples of heart, urine bladder and brain were removed under aseptic conditions for cultivation of borrelia. Samples of heart muscle were additionally used for molecular detection of borrelia by Real-Time polymerase chain reaction. PCR was performed with borrelia universal primers and with species-specific primers. 938 mice were caught, most frequently Apodemus flavicollis (44%), followed by Clethrionomys glareolus (35%), Microtus arvalis (9%), A. sylvaticus (7%) and Mus musculus (6%). Significant differences were seen in the total number of catch per area (Hohenau, Ernstbrunn, Vienna Woods equal 10:9:2) and in the distribution of the various rodent species in the respective areas. Borrelia strains were grown from only 65 (7%) animals, and more frequently isolated from bladder wall than from heart muscle, and only once from brain. Heart specimens of 223 animals were positive by borrelia PCR (24%), most frequently of the rodent species A. flavicollis (43%) and C. glareolus (38%). Borrelia afzelii was most frequently identified, followed by B. burgdorferi sensu stricto, B. garinii and by mixed infection of B. afzelii with B. burgdorferi sensu stricto. B. garinii was most frequently detected in heart samples of A. sylvaticus (about 20%). In about 3% of PCR positive samples the identification of one of the three mentioned genospecies of borrelia could not be ascertained with the test panel used. The results confirm the rodent species A. flavicollis, A. sylvaticus, M. arvalis and C. glareolus as reservoir animals for B. afzelii, B. garinii and B. burgdorferi sensu stricto, agents of Lyme borreliosis. Notable is the salient presence of B. garinii in heart specimens of A. sylvaticus.     

Ticks (Ixodidae) from passerine birds in the Carpathian region

Birds have been found to be a reservoir host of borrelia. In order to assess the situation in Slovakia ticks were collected from a total of 3057 mist-netted, ringed and released passerine birds in two locations at 500 m (in 2001) and 1000 m (in 2003) above sea level in the Bukovské Vrchy Hills, part of the Carpathian region in the north-east of Slovakia. A total of 75 birds of 16 species were infested with subadult ticks of Ixodes ricinus species (prevalence of parasitization 5%). Sixty-two larvae from 31 birds of 9 species and 80 nymphs from 52 birds of 15 species were found. The highest intensity of parasitization was observed on blackbirds Turdus merula, song thrushes T. philomelos and dunnocks Prunella modularis. Six Ixodes ricinus adult ticks were found on humans working with birds, and one I. ricinus female tick on their dog. In ticks, the presence of Rickettsia sp., Coxiella burnetii, Borrelia burgdorferi sensu lato and members of the Anaplasmataceae and Piroplasmidae, were investigated by polymerase chain reaction, followed by sequence analysis. Rickettsia sp. was found in 1 nymph from the European robin Erithacus rubecula, in 3 adult ticks (1 male, 2 females) from humans and in the tick from the dog. The closely related Ehrlichia- like species "Schotti variant" was detected in 1 nymph from the song thrush. Borrelia afzelii was identified in 1 male and B. garinii in 1 female tick collected on humans. Ixodes ricinus was found to be the vector of a wide spectrum of tick-borne pathogens in a mountainous area of the Carpathians. Because of the low yield of ticks and pathogens the importance of birds as reservoir hosts is still poorly understood.     

Evaluation of molecular methods for detection of Borrelia burgdorferi senso lato in ticks

Borrelia burgdorferi sensu lato (s. l.), the agent of Lyme disease, is distributed widely worldwide. A large number of polymerase chain reaction (PCR) methods have been developed and used for detection of B. burgdorferi s. l. However, there is a lack of a reference standard because of the genetic diversity of the B. burgdorferi s. l. complex. In this study, 4 PCR methods, based on the OspA, flagellin, rrs, and P66 genes, for detection of B. burgdorferi s. l. were evaluated by detection of genomic DNA from 3 reference genospecies and tick samples. The sensitivity of the PCR methods was analyzed using serially diluted gDNA from B. afzelii (Bo23), B. burgdorferi sensu stricto (B31), and B. garinii (PBi). The performance of the PCRs was evaluated by detection of the gDNA of 543 ticks. The results showed that the PCRs targeting the OspA gene, fla gene, rrs gene, and P66 gene detected 37 (6.8%), 74 (13.6%), 16 (2.9%), and 14 (2.6%) tick samples, respectively. The PCR targeting the fla gene was the most sensitive method for the detection of B. burgdorferi s. l.     

Genospecies and their influence on immunoblot results

In Europe at least three human pathogenic species of Borrelia burgdorferi sensu lato are the causative agents of Lyme borreliosis. All three species have been isolated or detected by PCR from skin, CSF and synovial fluid of patients with skin lesions, neuroborreliosis and Lyme arthritis respectively. Studies using strains representing the three species as antigen for the immunoblot revealed that interpretation criteria depend strictly on the strain used as antigen. More than using certain species as antigen it is important to use strains (f.e. B. afzelii strain PKo) expressing certain immunodominant antigens like OspC and p17 which may not be expressed by other strains in vitro. Using strain PKo as antigen the two band criterium can be used without loss of too much sensitivity compared to using B. burgdorferi sensu stricto strain PKa2 and B. garinii strain PBi. The use of recombinant antigens allows selection of highly specific and combination of homologous antigens from different strains; however not all desirable antigens have been recombinantly expressed. Addition of p17 and p58 as antigens may improve the sensitivity of the hitherto described recombinant antigen immunoblots containing the antigens p83/100, p39, OspC and the p41 internal fragment.     

Evolving models of Lyme disease spirochete gene regulation

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease (Lyme borreliosis), is well-adapted to maintain a natural cycle of alternately infecting vertebrates and blood-sucking ticks. During this cycle, B. burgdorferi interacts with a broad spectrum of vertebrate and arthropod tissues, acquires nutrients in diverse environments and evades killing by vertebrate and tick immune systems. The bacterium also senses when situations occur that necessitate transmission between hosts, such as when an infected tick is taking a blood meal from a potential host. To accurately accomplish the requirements necessary for survival in nature, B. burgdorferi must be keenly aware of its surroundings and respond accordingly. In this review, we trace studies performed to elucidate regulatory mechanisms employed by B. burgdorferi to control gene expression, and the development of models or "paradigms" to explain experimental results. Through comparisons of five borrelial gene families, it is readily apparent that each is controlled through a distinct mechanism. Furthermore, those results indicate that current models of interpreting in vitro data cannot accurately predict all aspects of B. burgdorferi environmental sensing and gene regulation in vivo.     

Relapsing borrelioses fevers: forgotten and new ones

Relapsing fever borrelioses are widely spread in the endemic regions of Eurasia, Africa, and America as before and account for significant morbidity and mortality; however, these infections have been recently underestimated. The pathogens of the fevers are the Borrelia species transmitted by ticks of the Ornithodoros genus; they genetically differ from the pathogens of Lyme borreliosis--Borrelia burgdorferi sensu lato transmitted by Ixodes ticks. The species Borrelia miyamotoi belongs to the genetic species of Borrelia, the causative agents of relapsing fevers. The authors found Borrelia of this species in the Ixodes ticks of Russia and first showed that B. miyamotoi were able to induce multiple cases in man, which had been earlier diagnosed as erythema-free Ixodes tick-borne borreliosis. The review considers the pathogenesis, clinical picture, diagnosis, and treatment of "old" relapsing fever borrelioses versus the available data on the "new" infection caused by B. miyamotoi. This must assist Russian physicians and scientists both to treat "old" and new tick-borne relapsing borrelioses and to schedule studies of the "new" B. myamotoi infection.     

Lyme disease--another transfusion risk?

Lyme disease (or Lyme borreliosis) is caused by a spirochetal bacteria, Borrelia burgdorferi. Increased recognition of the disease and increased exposure to the vector (ticks) capable of spreading B. burgdorferi from animal hosts have resulted in a rise in the number of cases of Lyme borreliosis reported in the United States. There are three stages of the clinical course of Lyme borreliosis; however, not all those infected will have typical manifestations of each stage, such as the arthritis of the third stage. Routine blood cultures will rarely document bacteremia and serologic testing is not yet reliable. Early treatment can prevent later stages of Lyme borreliosis. There is evidence that transmission of B. burgdorferi by blood transfusion is possible, but, to date, there has been no documentation of transfusion-associated Lyme borreliosis. Thus, no new recommendations for screening donors to identify possible carriers of B. burgdorferi are suggested at this time.     

Lyme disease—another transfusion risk?

Lyme disease (or Lyme borreliosis) is caused by a spirochetal bacteria, Borrelia burgdorferi. Increased recognition of the disease and increased exposure to the vector (ticks) capable of spreading B. burgdorferi from animal hosts have resulted in a rise in the number of cases of Lyme borreliosis reported in the United States. There are three stages of the clinical course of Lyme borreliosis; however, not all those infected will have typical manifestations of each stage, such as the arthritis of the third stage. Routine blood cultures will rarely document bacteremia and serologic testing is not yet reliable. Early treatment can prevent later stages of Lyme borreliosis. There is evidence that transmission of B. burgdorferi by blood transfusion is possible, but, to date, there has been no documentation of transfusion- associated Lyme borreliosis. Thus, no new recommendations for screening donors to identify possible carriers of B. burgdorferi are suggested at this time.     

中国莱姆病螺旋体菌株OppA2基因中人B细胞表位多态性分析

目的 研究中国莱姆病螺旋体菌株中OppA2基因及其人B细胞抗原表位区的序列多态性,了解不同地域OppA2的变化特征,为中国莱姆病的早期诊断提供线索和依据。 方法 选取59株莱姆病螺旋体分离株,PCR扩增其OppA2基因并测序后,结合3种基因型参考菌株序列,与免疫表位数据库（IEDB）中查询到的B细胞表位序列进行比对分析。 结果 中国莱姆病螺旋体中OppA2基因序列在Borrelia burgdorferi sensu strict（B.b.s.s）, Borrelia garinii（B.g）和Borrelia afzelii（B.a）3个基因型间存在明显的多态性。 但OppA2基因序列在B.b.s.s和B.a型内保守;在B.g型内存在异义突变。 B细胞表位改变主要发生在B.g基因型的亚簇2、亚簇3以及JC2-2分离株。 B.g基因型的亚簇2、亚簇3以及JC2-2菌株的296aa变异和亚簇3的295aa变异使3个B细胞抗原表位受影响;JC2-2菌株的372aa变异使1个B细胞抗原表位受影响;这些表位区的变异使部分B细胞表位序列（4/7,57.1%）呈现多态性。 结论 中国莱姆病螺旋体OppA2基因及其人B细胞抗原表位区在3个基因型间和B.g型内存在多态性;其中有3个B细胞抗原表位区（191～225aa、276～290aa和381～400aa）在B.b.s.s,B.g和B.a三种基因型内高度保守,可作为中国莱姆病早期诊断的候选抗原。      

Growth of infectious and non-infectious B. burgdorferi at different salt concentrations

Borrelia burgdorferi, the causative agent of Lyme disease, grows in vitro in modified Barbour-Stoenner-Kelly (BSK-H) medium. We have studied the effect of increased osmotic strength of culture media on growth of infectious and non-infectious B. burgdorferi strains B31 and N40. Relatively small increases in the NaCl concentration of the medium significantly inhibited growth in infectious as well as non-infectious strains. Growth of low passage, infectious clone B31-4a was more sensitive to increased NaCl concentrations than high passage, non-infectious clone B31-a. Growth of two infectious N40 strains, one low passage (N40-Lp) and one high passage (N40-P31) was more resistant to increased NaCl concentration than growth of infectious B31-4a. Osmotic strength is an important physical parameter for growth of B. burgdorferi in vitro and could influence its ability to adapt and to establish an infection within ticks and mammals.     

Molecular diversity of the ospC gene in Borrelia. Impact on phylogeny,epidemiology and pathology

Lyme borreliosis is a 2 steps disease: i) Localized erythema migrans ii) occasionally a disseminated disease. Three out of the 10 up to now described Borrelia species are pathogenic for man and each of them exhibits its own organotropism: joints for Borrelia burgdorferi sensu stricto (B.b. ss), nervous system for B. garinii, skin for B. afzelii, ospc gene is subject to lateral transfer leading to a huge diversity among corresponding encoded proteins. This allows the spirochete to develop a repertoire of epitopes to escape the host immune response. We noticed that the European endemic ospc repertoire is only a subset of the American one. This bottleneck situation transduces a "founder's event" suggesting B.b. ss has been imported from North America to Europe at historical times. Another valuable observation is the fact that isolates from disseminated forms (called "invasive") of the disease, all are distributed in only ten out of the 70 ospc genotypes. The conclusion is that in human, some OspC conformations are associated with the invasive potential of a given Borrelia isolate.     

Complement regulator-acquiring surface proteins of Borrelia burgdorferi: a new protein family involved in complement resistance

The innate immune system, particularly the host complement system, plays an important role in the elimination of invading pathogens. Borrelia burgdorferi, like other human pathogens, has developed strategies to prevent complement-mediated bacteriolysis. It is now well established that Borrelia differ in their complement resistance. In general terms, B. afzelii isolates are mainly resistant to complement-mediated bacteriolysis, whereas the majority of B. burgdorferi s.s. isolates display an intermediate complement-resistant phenotype. Most of the B. garinii isolates, in contrast, are efficiently killed by complement and, therefore, are classified as complement-sensitive. Complement resistance of B. afzelii and B. burgdorferi s.s. isolates correlates directly with the acquisition of the fluid-phase human complement regulators FHL-1/reconectin and factor H. To date, five distinct complement regulator-acquiring surface proteins (CRASPs) have been identified in B. afzelii and B. burgdorferi s.s. isolates. The individual CRASPs can be differentiated according to their size and their binding properties to FHL-1/reconectin and factor H. The domains that interact with CRASPs are localized at the C-terminal ends of these complement regulators. Thus, CRASPs represent a family of functional proteins involved in complement resistance of Borrelia. Furthermore, an alterable pattern of gene expression was observed for three CRASPs of B. afzelii: BaCRASP-1, BaCRASP-2, and BaCRASP-5 are up-regulated at 37 degrees C and down-regulated at 20 degrees C. The continued characterization of CRASPs at the molecular level is expected to identify new virulence factors and potential vaccine candidates.     

In vitro activity of rokitamycin, a new macrolide, against Borrelia burgdorferi.

The activity of rokitamycin, a new macrolide with a 16-member ring, was tested against Borrelia burgdorferi in vitro. The antibiotic had a lower MIC at which 50% of the isolates are inhibited than erythromycin, the parent 14-member macrolide, but the same MIC at which 50% of the isolates are inhibited as the other recent 14- and 15-member macrolides, like clarithromycin and azithromycin. The MBC was equal to the MIC at which 50% of the isolates are inhibited, so rokitamycin can be considered bactericidal against B. burgdorferi. The sensitivity of the Borrelia strains tested was not correlated with the particular species Burgdorferi sensu stricto, B. garinii, and B. afzelii or with the number of subcultures of the isolates.