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PubMed1993

Acylated proteins in Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae.

Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae produced several lipoproteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of bacteria grown in [3H]palmitate. Five major acylated proteins were demonstrated by sequential alkaline and acid hydrolysis. High-pressure liquid chromatography of isolated proteins confirmed that covalently bound radioactivity was represented by fatty acids.

Acylation↗

PubMedDate not supplied

[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.

Adolescent↗

PubMed1999

Asymptomatic Borrelia-seropositive individuals display the same incidence of Borrelia-specific interferon-gamma (IFN-gamma)-secreting cells in blood as patients with clinical Borrelia infection.

Borrelia Lyme disease is a complex disorder that sometimes becomes chronic. There are contradictory reports of experimental Borrelia infections regarding which type of T cell cytokine responses, i.e. Th1 or Th2, are needed to eradicate the Borrelia spirochaetes. In human borreliosis a predominance of Borrelia-specific Th1-like responses has been shown. In this study, spontaneous, as well as Borrelia-specific, secretion of IFN-gamma (Th1) and IL-4 (Th2) in Borrelia-seropositive healthy asymptomatic individuals (n = 17) was investigated in peripheral blood by a sensitive ELISPOT assay, and compared with previously reported responses in patients with clinical Borrelia infection (n = 25). The seropositive asymptomatic individuals displayed the same predominance of Borrelia-specific IFN-gamma-secreting cells as the patients with clinical Borrelia infection. Interestingly, the proportion of spontaneously IL-4-secreting cells, reflecting the unstimulated in vivo secretion, was lower in the seropositive asymptomatic individuals compared with patients with chronic Borrelia infections (n = 13, P = 0.02), whereas no such difference was found compared with subacute Borrelia infections (n = 12). These findings indicate that IFN-gamma secretion alone is not sufficient to eliminate Borrelia spirochaetes in humans, although IFN-gamma may still have a beneficial role in borreliosis acting in concert with other mechanisms.

Adult↗

PubMed2003

In vitro susceptibility testing of four antibiotics against Borrelia burgdorferi: a comparison of results for the three genospecies Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto.

MICs and minimal bactericidal concentrations (MBCs) were evaluated for the four antibiotics azithromycin, amoxicillin, ceftriaxone, and doxycycline against the three main genospecies of Borrelia burgdorferi sensu lato. In MBC testing, statistically significant differences between the genospecies could be found in 7 out of 12 comparative evaluations (P < 0.05).

Amoxicillin↗

PubMed1995

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia.

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.

Animals↗

PubMed1995

T cell response to Borrelia garinii, Borrelia afzelii, and Borrelia japonica in various congenic mouse strains.

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross-reactivity between Borrelia antigens was observed, but there was no cross-reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed-type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4+8- and Ia- T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8+ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.

Animals↗

PubMed1998

Rate of infection of Ixodes ricinus ticks with Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii and group VS116 in an endemic focus of Lyme disease in Italy.

A study to evaluate the natural rate of infection of Ixodes ricinus with Borrelia burgdorferi sensu lato was carried out in an endemic focus of Lyme disease in the Trieste area in northern Italy. Two-hundred and twenty-seven ticks collected in ten different stations were tested individually for the presence of the spirochetes using polymerase chain reaction techniques able to identify both Borrelia burgdorferi sensu lato and the four genospecies (Borrelia burgdorferi sensu stricto, Borrelia garinii. Borrelia afzelii and group VS116). Multiple infection of individual ticks was found. The infection rate ranged from 0-70%. Infection of Ixodes ricinus with Borrelia burgdorferi group VS116 was found for the first time in Italy in both a high and a low endemic focus of Lyme disease.

Animals↗

PubMed1996

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis.

This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi, belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii. To assess specificity, patient sera were statistically (X2, P < or = 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30-33, 36-37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.

Antibodies, Bacterial↗

PubMed2002

Strain typing of Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii by using multiple-locus variable-number tandem repeat analysis.

Human Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by Borrelia spirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of Borrelia burgdorferi and subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse Borrelia isolates including B. burgdorferi, B. afzelii, and B. garinii. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (D) values ranging from 0.10 to 0.87. The average D value was 0.53 across all VNTR loci. A clear correlation exists between the repeat copy number and the D value (r = 0.62) or the number of alleles (r = 0.93) observed across diverse strains. Cluster analysis by the unweighted pair-group method with arithmetic means resolved the 30 observed unique Borrelia genotypes into five distinct groups. B. burgdorferi, B. afzelii, and B. garinii clustered into distinct affiliations, consistent with current 16S rRNA phylogeny studies. Genetic similarity and diversity suggest that B. afzelii and B. garinii are close relatives and were perhaps recently derived from B. burgdorferi. MLVA provides both phylogenetic relationships and additional resolution to discriminate among strains of Borrelia species. This new level of strain identification and discrimination will allow more detailed epidemiological and phylogenetic analysis in future studies.

Animals↗

PubMed1999

Serologic cross-reactivity of antibodies against Borrelia theileri, Borrelia burgdorferi, and Borrelia coriaceae in cattle.

OBJECTIVE: To evaluate the immune response induced by Borrelia theileri infection and to determine whether B theileri induces cross-reacting antibodies to other bovine borreliae. ANIMALS: Two 3-month-old calves, 1 of which was splenectomized. PROCEDURE: Calves were exposed to Boophilus microplus infected with B theileri. Rectal temperature, PCV, bacteremia, and clinical signs of infection were monitored. Serum was obtained weekly and used to evaluate the humoral response to homologous antigen and B burgdorferi and B coriaceae, using an indirect fluorescent antibody (IFA) test, and to B burgdorferi, using a commercially available ELISA. The identity of cross-reacting antigens was explored, using monoclonal antibodies to genus- and species-specific antigens in an IFA test. RESULTS: B theileri-infected calves produced antibodies that cross-reacted with B burgdorferi and B coriaceae whole-cell antigens. Borrelia theileri whole-cell antigen was recognized by genus-specific monoclonal antibody H9724 but not by species-specific antibody H5332. False-positive reactions were not observed when serum from B theileri-infected calves was tested by use of the ELISA for B burgdorferi. CONCLUSIONS: B theileri induces humoral responses in infected cattle that can be confused with those of other borrelial infections. Care must be taken to definitively distinguish between the various borreliae that may cause disease in cattle. CLINICAL RELEVANCE: Serologic cross-reactivity must be taken into account when making a serodiagnosis of Lyme borreliosis or epizootic abortion in epidemiologic studies involving cattle.

Animals↗

PubMed1997

Immunization with a polyvalent OspA vaccine protects mice against Ixodes ricinus tick bites infected by Borrelia burgdorferi ss, Borrelia garinii and Borrelia afzelii.

Sequence variability of the outer surface protein (Osp) A among Borrelia burgdorferi sl species suggests that a monovalent OspA vaccine may not protect against the various Borrelia present in Eurasia. Here, we confirmed that a monovalent recombinant OspA (rOspA) vaccine does not protect mice against Ixodes ricinus mediated infection with B. burgdorferi ss, Borrelia garinii and Borrelia afzelii. However, when mice were vaccinated with a cocktail of various rOspA from these three species, they were protected, and all challenge ticks that fed on them were cleared of their spirochetes. These results showed that a multiple OspA antigens vaccine, compatible with human use, was very efficient at protecting mice against B. burgdorferi ss, B. garinii, and B. afzelii.

Animals↗

PubMed1997

Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans.

OBJECTIVE: To evaluate the diagnostic performance of two polymerase chain reaction (PCR) procedures using skin biopsies of 20 erythema migrans (EM) and 24 acrodermatitis chronica atrophicans (ACA) patients. METHODS: One assay amplified a fragment of the outer surface protein (Osp) A gene. The second method amplified the spacer region between the 5S and 23S rRNA genes; hybridization of this fragment allowed identification of Borrelia burgdorferi sensu lato species. RESULTS: Among EM patients, both assays detected Borrelia DNA in 15 samples. Among ACA patients, the ospA PCR detected 15 positives and 10 samples were positive by 5S-23S PCR. In 19 samples one species was detected, 15 skin biopsies contained Borrelia afzelii, and Borrelia garinii was found in two patients. Group VS116 was detected in two EM patients, and therefore this group has pathogenic potential. Mixed infections of B. afzelii and B. garinii, group VS116 or B. burgdorferi sensu stricto were found in three EM and three ACA patients. CONCLUSIONS: Diagnosis of EM and ACA by PCR is useful and knowledge of the presence of species may be used to predict the course of disease or the need for further antibiotics.

Journal Article↗

PubMed2001

[Criteria for evaluation of immunoblots using Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto for diagnosis of Lyme borreliosis].

The immunoblot was prepared from genotypes Borrelia afzelii (KC 90), Borrelia garinii (M 192) and Borrelia burgdorferi sensu stricto (B 31). Sera of 63 patients with different forms of Lyme borreliosis were examined and 40 healthy donors in the endemic area of the disease. In class IgM in the group of patients significantly more frequently antibodies against OspC, p39, p41 B. afzelii, p39, p41, p66, p83 B. garinii and OspC1, OspA, B. burgdorferi sensu stricto were found. In class IgG there were antibodies against p39, p41, p93 B. afzelii, p14, p41, p93 B. garinii and OspA, OspC p93 B. burgdorferi sensu lato. Based on the assembled results by means of discrimination analysis and logistic regression the most suitable combinations of antigens for evaluation of immunoblots in different genotypes were determined. Furthermore evaluation was suggested using a combination of antigens of several genotypes which led to an increased sensitivity and specificity of the immunoblot. Tables were prepared for easier evaluation of newly examined sera samples.

Adolescent↗

PubMed2000

Occurrence of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in the Ixodes ricinus ticks from Eastern Slovakia.

A total of 2816 unfed adults nymphs of Ixodes ricinus ticks were collected from vegetation in Kosice (Eastern Slovakia) from 1994 to 1997. Prevalence of Borrelia burgdorferi s. l. in I. ricinus ticks, detected by dark field microscopy, varied and depended upon the year and the habitat of the collected ticks. The lowest prevalence was observed in 1994 (4.8%). During 1995 it increased to 17.2% and during the next two years decreased to 15.5% and 14.2%. The rate of infection varied from 2.1 to 23.3% within 10 examined habitats of the Kosice area. A different value of relative density of ticks was observed in various habitats. It ranged from 9-212 ticks per collecting hour within one flagged area (600 m2) which is 1.5-35.5 ticks per 100 m2. Eight isolates were obtained from the infected ticks. Electrophoresis and immunoblotting with 6 monoclonal antibodies were used for the identification of Borrelia strains. Three tick isolates were identified as B. burgdorferi s. s. and the other three isolates were found to be B. garinii. One strain reacted as a mixed culture of B. burgdorferi s. s., and B. garinii. The strain originated from the Vihorlat Mountains habitat and was detected by PCR-SSCP as B. burgdorferi s. s. with a small amount of B. afzelii. The obtained results emphasize the epidemiological importance not only of B. garinii and B. afzelii but also of B. burgdorferi s. s. in Central Europe.

Animals↗

PubMed1998

Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127).

Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.

Borrelia↗

PubMed1995

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species.

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1-7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340-350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819-825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1-7) for all strains analyzed so far (n = 29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3-7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogeneous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2000

Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene.

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured from Ixodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2 degrees C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.

Animals↗

PubMed2000

Identification of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in Ixodes ricinus ticks from southern Bohemia using monoclonal antibodies.

The prevalence of Borrelia burgdorferi sensu lato (s.l.) in 761 Ixodes ricinus ticks collected in different localities of southern Bohemia was investigated. The minimum infection rate of nymphs, males and females as determined by dark-field microscopy was 4.8% in 1997. The total B. burgdorferi prevalence determined in ticks in 1998 was 11.04%. A significant local variability of the prevalence of B. burgdorferi was not found. Nine isolates were identified at a genospecies level by immunoblotting with prepared monoclonal antibodies (Mabs). The most prevalent genospecies in ticks was B. burgdorferi sensu stricto (s.s.) (6 strains). Two tick isolates reacted with Mabs specific to both B. garinii and B. afzelii and one isolate was detected as a mixed culture of B. burgdorferi s.s. and B. garinii. Considerable variability in protein composition among the strains obtained was found to exist. Five B. burgdorferi genospecies-specific Mabs were prepared and used for the identification of spirochaete isolates. Mabs 9B8 and 6C5 reacted with OspA and OspB proteins of B. burgdorferi s.s., while 4D7 and 1D11 recognised OspA protein of B. garinii and 9D2 specifically reacted with the OspB protein of B. afzelii. In addition, Mab 5H4 reacted with flagellin of all the above-mentioned genospecies of B. burgdorferi s.l.

Animals↗