Specificities and sensitivities of four monoclonal antibodies for typing of Borrelia burgdorferi sensu lato isolates.

Borrelia burgdorferi, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana. In the United States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East Coast were differentiated. In Asia, B. japonica has been identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of rrf (5S)-rrl (23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55 B. burgdorferi ss isolates but showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45 B. afzelii isolates. Monoclonal antibody D6 was 100% specific for B. garinii but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new Borrelia isolates.

A mutagenic PCR identifies isolates of Borrelia garinii responsible for Lyme borreliosis.

Borrelia garinii is one of the three major Borreliae responsible for Lyme borreliosis in Europe. We have characterized a protein of B. garinii (VS102) and a genomic fragment from the gene encoding this protein was cloned. The DNA sequence of the fragment showed high homology with a known gene of B. burgdorferi sensu stricto. The protein encoded by this gene in B. burgdorferi sensu stricto is a phosphocarrier protein (histidine-containing protein). A mutation T to G polymorphism at codon 57 was found to be specific to B. garinii. A PCR-based approach that allows the rapid detection of this mutation made it possible to specifically discriminate B. garinii from other B. burgdorferi genospecies with high sensitivity and specificity.

Age-related changes in insulin-like growth factor I and II in human femoral cortical bone: lack of correlation with bone mass.

The concentration of insulin-like growth factor-I (IGF-I) in human cortical bone declines with age, but the relevance of this decline for cortical bone turnover and bone mass is unknown. In the present study, we simultaneously assessed the concentration of IGF-I and -II in cortical bone matrix and histomorphometric parameters of bone mass and bone turnover in 125 samples from the proximal human femur shaft. Bone width decreased by 27% and porosity increased by 100% in female cortical bone between the fourth and the ninth decade. Similar, but weaker, changes tended to occur in male cortical bone. The concentrations of both IGF species were correlated with the percentage of osteons undergoing bone remodeling. However, despite age-related decreases in both IGF species in men and in IGF-I in women, neither of the IGFs accounted for age-related or age-independent variability in cortical porosity or bone width. In conclusion, these data suggest that the local concentrations of IGF-I and -II are related to cortical bone turnover. In contrast, our study provides no evidence for a major role of bone matrix IGF-I and -II as determinants of cortical bone mass in elderly individuals. Whether other components of the IGF system may be stronger determinants of cortical bone loss remains to be determined.

Concentration of transforming growth factor beta in human bone tissue: relationship to age, menopause, bone turnover, and bone volume.

Transforming growth factor beta (TGF-beta) is thought to play an important role in bone metabolism, but its relationship to human bone turnover and bone mass has not been examined yet. In this study, we measured the concentration of TGF-beta in 811 samples of male and female bone from four representative sites of the human skeleton and in the supernatants of 72 short-term human bone marrow cultures from the iliac crest. The concentrations of TGF-beta1 and TGF-beta2 in the bone matrix were positively correlated with histomorphometric indices of bone resorption and bone formation and with serum levels of osteocalcin and bone-specific alkaline phosphatase. We also observed a positive association between the release of TGF-beta in the bone marrow cultures and serum osteocalcin. Changes in the rate of cancellous or cortical bone remodeling with age or menopause were accompanied by corresponding changes in skeletal TGF-beta. In contrast, there was no significant relationship between the concentration of TGF-beta and bone volume at any skeletal site. In conclusion, our study supports the hypothesis that TGF-beta plays an important role in human bone remodeling, but fails to demonstrate an association between the skeletal concentration of TGF-beta and human bone mass.

Occurrence of different genospecies of Borrelia burgdorferi sensu lato in ixodid ticks of Valais, Switzerland.

A total of 825 adult ticks (727 Ixodes ricinus, 72 Dermacentor marginatus and 26 Haemaphysalis punctata) was collected from vegetation in Valais (Switzerland) in 1987 to 1992. They were examined for the presence of Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis. B. burgdorferi sensu lato was detected by indirect immunofluorescence assay, dark field microscopy and/or culture in 221 out of 727 I. ricinus (30.4%) and none in the other two species. From these 221 infected ticks we obtained 50 isolates. Indirect immunofluorescence assay and culture were used for all ticks but dark field examination has also been performed and compared to the two above mentioned methods for 231 I. ricinus. Indirect immunofluorescence assay and culture were used for all ticks but dark field examination has also been performed and compared to the two above mentioned methods for 231 I. ricinus. Indirect immunofluorescence was found the most efficient method for the detection of Borrelia in ticks with 54 positive out of 231, followed by dark field examination with 35 positive and culture with 12 isolates. We found no site free of Borrelia where I. ricinus is present. The rate of infection varied from 9.7 to 47.5%, as detected by the addition of the three methods. Typing of the 50 isolates revealed also a nonhomogeneous distribution of the Borrelia species. Based on the electrophoretic mobility of the OspA and B and immunostaining with species specific monoclonal antibodies (H3TS for B. burgdorferi sensu stricto, D6 for B. garinii and J8.3 for B. afzelii) 4 groups could be observed. Half of the isolates (n = 26) were typed as B. burgdorferi sensu stricto, 19 as B. garinii, 3 as B. afzelii and 2 as group VS116. This forth group formed of two isolates from one location is genetically distinct from the 3 former species described in Europe so far. The Borreliae of this group are unreactive with any of the three monoclonal antibodies used.

Genotypic and phenotypic diversity among nine Swiss isolates of Borrelia burgdorferi.

Nine strains of Borrelia burgdorferi isolated from ticks in the canton of Valais (Switzerland) were characterized genotypically by determining restriction fragment length polymorphisms (RFLP) and plasmid profiles. The strains were also compared with respect to presence and electrophoretic mobility of the outer surface proteins OspA and OspB, and immunoreactivity of OspA and a 12 kD antigen. By both approaches, three different patterns were observed resulting in identical grouping of the strains. However, RFLP's allowed determination of relationships among strains within a group and have shown that geographic distribution does not correlate with genotype.

[Isolation of Borrelia burgdorferi in the cerebrospinal fluid of 3 children with neurological involvement]. / Isolement de Borrelia burgdorferi du liquide céphalorachidien de trois enfants avec une atteinte neurologique.

Isolation of Borrelia burgdorferi from the CSF is relatively rare. The present report describes the first three isolations in Switzerland. Clinically, our first observation confirmed the frequent association of B. burgdorferi with peripheral facial paresis in children. The other two cases illustrate the variety of symptoms in neuro-borreliosis. In the first case the culture was positive after 6 weeks. The results of serologic tests (indirect immunofluorescence and ELISA) for detection of antibodies against B. burgdorferi were negative or non-significant in this child's serum. On the other hand, specific antibodies (IgG) were detected in the serum by western blot. Culture of the second CSF already showed Borrelia growth after 10 days. Immunofluorescence revealed high antibody titers (1/256) against B. burgdorferi in this patient's serum. IgG showed a weakly positive reaction in western blot. The reliability of this result was confirmed by isolation of Borrelia. In neither of the two CSF could intrathecal synthesis of specific antibodies be demonstrated. In the third case, however, immunofluorescence showed IgG antibody titers of 1/128 in the CSF and 1/512 in serum. Intrathecal synthesis of specific antibodies was demonstrated with an index of 13.4 (norm < 2). Western blot confirmed the specificity of the reactions observed with the serum and CSF IgG. Culture of CSF produced significant growth of Borrelia within 7 days. Protein profile and reactions with poly- and monoclonal antibodies confirmed that the three strains belonged to B. burgdorferi.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphism of outer surface proteins of Borrelia burgdorferi as a tool for classification.

A total of 23 isolates of Borrelia burgdorferi were characterized by SDS-PAGE and immunoblot analysis. One isolate came from the CSF of a Lyme neuro-borreliosis patient in Valais (Switzerland) and 22 were tick isolates (2 from I. dammini of Shelter Island, USA and 20 from I. ricinus of Valais, Switzerland). Based on the electrophoretic mobility of outer surface proteins (OspA and OspB), four groups of B. burgdorferi could be defined. Group I isolates possess an OspA of 31 KD and an OspB of 34 KD. The group II isolate showed an OspA of 32 KD and OspB of 35 KD. Group III isolates have a 33 KD OspA and group IV a 33.5 KD OspA. This classification was confirmed by the reactivity of a monoclonal antibody (D6) to a 12 KD antigen that was recognized in group III only. A Lyme patient's serum showed a 2-band pattern (10 and 13 KD) for group I and a one-band pattern (12 KD) for the other 3 groups. Therefore OspA, OspB and other proteins of low molecular weight (10, 12, and 13 KD) seem to be important keys for the classification of B. burgdorferi isolates. This typing system correlates with genetic analysis.

Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis.

Fifty Borellia burgdorferi strains isolated from humans and ticks in Europe and the United States were analyzed by multilocus enzyme electrophoresis. Eleven genetic loci were characterized on the basis of the electrophoretic mobilities of their products. Ten loci were polymorphic. The average number of alleles per locus was 5.9, with a mean genetic diversity of 0.673 among electrophoretic types (ETs). The strains were grouped into 35 ETs constituting three main divisions (I, II, and III) separated at a genetic distance greater than 0.75. Divisions I, II, and III contained 13, 6, and 16 ETs, respectively. These findings, together with previous data from DNA hybridization and restriction enzyme analysis of rRNA genes, suggest that divisions I, II, and III may represent three distinct genomic species. All three divisions contained human clinical ETs. However, in division I, which includes the ET of the type strain of B. burgdorferi, the human pathogenic ETs constituted a single clone. The ETs of division I were from west-central Europe and the United States, whereas divisions II and III contained ETs from west-central and northern Europe but not from the United States. Finally, our data show that the genetic structure of B. burgdorferi populations is clonal.