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.In Eurasia, seven species of the complex Borrelia burgdorferi sensu lato have been reported. Only three of these species are associated with Lyme borreliosis. It has also been shown that each pathogenic species is associated predominantly with a given clinical presentation; Borrelia burgdorferi sensu stricto is associated with arthritis, B. garinii is associated with neuroborreliosis, and B. afzelii is associated with late cutaneous symptoms (2, 39). Up to now, B. burgdorferi sensu stricto is the only species associated with Lyme borreliosis in North America. However, two other B. burgdorferi sensu lato genospecies coexist in the United States, B. andersonii (22) and the genomic group DN127 (3, 32). B. andersonii seems to be restricted to a limited ecosystem involving cottontail rabbits and Ixodes dentatus ticks. In contrast, the genomic group DN127 appears to be involved in several enzootic transmission cycles (6, 29). A recent study demonstrated substantial genetic heterogeneity among Californian and other American strains (24). We took advantage of the unique structure of ribosomal genes in B. burgdorferi sensu lato to analyze the polymorphism of some strains isolated in California. A single copy of the rrs gene is separated by a large spacer (rrs-rrl; 3,000 to 5,000 bp) from two tandemly duplicated copies of rrl and rrf genes (13, 36). These two copies are separated by a small spacer, rrf-rrl, which is approximately 250 bp long. The genetic heterogeneity of the group DN127 was first evidenced by analysis of the restriction patterns of the rrf-rrl spacer (32). However, the results of DNA-DNA hybridization on a limited number of strains (32) allowed us to place them in a single genomic group. To clarify the genetic relationships between diverse North American strains, 20 atypical strains were compared with 13 B. burgdorferi sensu stricto strains. Identification procedures involved restriction polymorphism and sequencing studies of both the variable rrf-rrl spacer and the conserved rrs gene. Sequences of the rrf-rrl spacer and the rrs gene were used in a phylogenetic analysis. Some Californian strains are closely related to the genomic group DN127, for which we propose the name of B. bissettii sp. nov. Other atypical strains which do not fall into this group are designated merely as Borrelia spp. in this study. The latter strains cannot be assigned to specific genomic groups until more isolates representative of each group are available for further characterization.     

Borrelia carolinensis sp. nov., a New (14th) Member of the Borrelia burgdorferi Sensu Lato Complex from the Southeastern Region of the United States

Approximately 118 Borrelia isolates were cultured from a variety of rodents, birds, and ticks collected in the southern United States. In addition to a highly diverse group of Borrelia bissettii strains and a homogenous group of Borrelia burgdorferi sensu stricto strains, a group of 16 isolates with unusual characteristics was found. The isolates were cultured from ear biopsy samples of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina. A multilocus sequence analysis of the rrf-rrl intergenic spacer, 16S rRNA, fla, ospA, and p66 genes were used to clarify the taxonomic status of the new group of B. burgdorferi sensu lato isolates. Thirteen species of the B. burgdorferi sensu lato complex were used as controls. Unique restriction fragment length polymorphism patterns of the rrf-rrl intergenic spacer region and fla gene were recognized. Unique signature nucleotides were also found in the 16S rRNA gene. A phylogenetic analysis shows that the 16 new isolates cluster together but separately from the other species in the B. burgdorferi sensu lato complex. Our data strongly support the recognition of the 16 isolates as a new B. burgdorferi sensu lato species. We propose to name this genospecies “Borrelia carolinensis” with respect to the place of its currently known geographic location.     

Genetic analysis of the outer surface protein C gene of Lyme disease spirochaetes (Borrelia burgdorferi sensu lato) isolated from rodents in Taiwan

The outer surface protein C gene (ospC) of Lyme disease spirochaetes (Borrelia burgdorferi sensu lato) was analysed for the first time in Taiwan. The genetic identities of these Taiwan isolates (TWKM1-7) were determined by restriction fragment length polymorphism (RFLP) analysis and sequence similarities of the PCR-amplified ospC gene amplicons. After cleavage by nuclease Dral, differential fragment patterns of PCR-amplified ospC DNA in relation to different genospecies of Lyme disease spirochaetes were observed and all of these Taiwan isolates were genetically affiliated to the genospecies of B. burgdorferi sensu stricto. The phylogenetic analysis on the sequence similarity of these Taiwan isolates revealed a highly homogeneous genotype, ranging from 99.3% to 100%, within the genospecies of B. burgdorferi sensu stricto and was distinguished from other genospecies of Borrelia isolates. The sequence similarity analysis also revealed the high sequence variability of the ospC gene among Borrelia strains that belong to the same genospecies but were isolated from different biological and geographical sources. Thus, these results provide the first investigation on the genetic identity of the ospC gene of these Taiwan isolates and show that these Taiwan isolates were closely related genetically to the genospecies of B. burgdorferi sensu stricto.     

Characterization of Candida parapsilosis complex isolates

Candida parapsilosis former groups II and III have recently been established as independent species, named Candida orthopsilosis and Candida metapsilosis, respectively. We investigated the distribution of C. parapsilosis complex species in 122 isolates from blood and other sources in a southern Spain tertiary-care hospital, and we examined the relationship between species, site of isolation and biofilm positivity. We also evaluated the planktonic MICs and sessile MICs (SMICs) of voriconazole, amphotericin B and anidulafungin. One hundred and eleven isolates (91%) were categorized as C. parapsilosis sensu stricto, whereas ten isolates (8.2%) were categorized as C. orthopsilosis and one (0.8%) as C. metapsilosis. Biofilm positivity was observed in 58.5% (65 of 111) of C. parapsilosis sensu stricto isolates vs. 0% (0 of 11) of C. orthopsilosis and C. metapsilosis isolates (p <0.01). There was no difference in biofilm production among C. parapsilosis sensu stricto isolates from blood and other sources. MIC values showed that all isolates were susceptible to voriconazole and amphotericin B, whereas two isolates (1.8%) of C. parapsilosis sensu stricto were non-susceptible to anidulafungin. However, the MIC₉₀ value of voriconazole was higher (0.125 mg/L) for C. orthopsilosis than for C. parapsilosis sensu stricto (0.03 mg/L). In contrast to planktonic cells, the SMICs show that amphotericin B and anidulafungin are moderately effective against the biofilm of C. parapsilosis sensu stricto, whereas voriconazole is ineffective.     

Virulence characteristics of translocating Escherichia coli and the interleukin-8 response to infection

Four efficiently translocating Escherichia coli (TEC) strains isolated from the blood of humans (HMLN-1), pigs (PC-1) and rats (KIC-1 and KIC-2) were tested for their ability to adhere and translocate across human gut epithelial Caco-2 and HT-29 cells, to elicit a proinflammatory response and for the presence of 47 pathogenic E. coli virulence genes. HMLN-1 and PC-1 were more efficient in adhesion and translocation than rat strains, had identical biochemical phenotype (BPT) and serotype (O77:H18) and phylogenetic group (D). KIC-2 adhered more than KIC-1, belonged to different BPT and serotype but the same phylogenetic group as KIC-1. TEC strains elicited significantly higher IL-8 response in both cell lines (P < 0.05) and monocytic THP-1 (P < 0.0001) cells than non-TEC strains. KIC-2 induced the highest IL-8 response which may be associated with its immunostimulatory flagellin. Apart from adhesin genes fimH and bmaE that were carried by all strains, HMLN-1 and PC-1 carried capsule synthesis gene kpsMT III and KIC-2 carried the EAST1 toxin gene. The lack of known virulence genes and the ability of TEC to efficiently adhere and translocate whilst causing proinflammatory response suggests that these strains may carry as yet unidentified genes that enable their translocating ability.     

Lmb, a Protein with Similarities to the LraI Adhesin Family, Mediates Attachment of Streptococcus agalactiae to Human Laminin

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.     

Detection of azole resistance in Aspergillus fumigatus complex isolates using MALDI-TOF mass spectrometry

ObjectivesThe main goal of this study was to accurately detect azole resistance in species of the Aspergillus fumigatus complex by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).MethodsIdentification of isolates (n = 868) was done with MALDI-TOF MS using both commercial and in-house libraries. To determine azole susceptibility, the EUCAST E.Def. 9.3.2 method was applied as the reference standard. Identification of resistant isolates was confirmed by DNA sequence analysis. Protein spectra obtained by MALDI-TOF MS were analysed to differentiate species within the A. fumigatus complex and to detect azole-resistant A. fumigatus sensu stricto isolates.ResultsCorrect discrimination of A. fumigatus sensu stricto from cryptic species was accomplished in 100% of the cases applying principal component analysis (PCA) to protein spectra generated by MALDI-TOF MS. Furthermore, a specific peak (4586 m/z) was found to be present only in cryptic species. The application of partial least squares (PLS) discriminant analysis allowed 98.43% (±0.038) discrimination between susceptible and azole-resistant A. fumigatus sensu stricto isolates. Finally, based on PLS and SVM, A. fumigatus sensu stricto isolates with different cyp51A gene mutations were correctly clustered in 91.5% of the cases.ConclusionsMALDI-TOF MS combined with peak analysis is a novel tool that allows the differentiation of A. fumigatus sensu stricto from other species within the A. fumigatus complex, as well as the detection of azole-resistant A. fumigatus sensu stricto. Although further studies are still needed, the results reported here show the great potential of MALDI-TOF and machine learning for the rapid detection of azole-resistant Aspergillus fumigatus isolates from clinical origins.     

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.     

Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation

The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.     

PCR-restriction fragment length polymorphism analysis of the ospC gene for detection of mixed culture and for epidemiological typing of Borrelia burgdorferi sensu stricto.

Restriction fragment length polymorphism (RFLP) analysis of the outer surface protein C (ospC) gene amplicon was used for rapid screening for genetic variability within Borrelia burgdorferi sensu stricto species and for detection of multiple borreliae in culture. Primers for the ospC gene amplified a fragment of about 600 bp from Borrelia cultures. After cleavage of the amplified products by MboI and DraI, eight different RFLP types were found among 13 B. burgdorferi sensu stricto strains from various sources and geographical areas, and three RFLP types were found among 10 representative isolates from skin biopsy specimens taken from patients residing on the eastern end of Long Island, New York (B. W. Berger, R. C. Johnson, C. Kodner, and L. Coleman, J. Clin. Microbiol. 30:359-361, 1992). These results suggested that the DNA organization of B. burgdorferi sensu stricto is heterogeneous not only globally but also within a localized geographical area and that the ospC-based typing approach could differentiate the B. burgdorferi sensu stricto. From the results obtained using mixed cultures of two different RFLP types of B. burgdorferi sensu stricto, contamination of at least 0.5% of different types of Borrelia cells in culture could be detected. This method could detect a multiple-B. burgdorferi sensu stricto infection in the bladders of mice experimentally infected with two different RFLP type strains. The present study showed that RFLP analysis of ospC-PCR products is a reliable method for epidemiological typing of B. burgdorferi sensu stricto and could be used for rapid detection of mixed Borrelia culture and multiple B. burgdorferi sensu stricto infections in animals, ticks, and patients.     

Binding of bovine fibronectin to mastitis-causing Streptococcus agalactiae induces adherence to solid substrate but not phagocytosis by polymorphonuclear cells

Streptococcus agalactiae is frequently associated with mastitis in cattle, and fibronectin is a host protein which interferes with infection, particularly at mucosal sites. The binding of bovine fibronectin to S. agalactiae was investigated by enzyme-linked immunosorbent assay (ELISA). Binding was time-dependent and dose-dependent. Most of the strains tested showed low ELISA activity, but some strains (six out of 17) had much higher activity. The highest-binding strains of S. agalactiae were able to adhere to bovine fibronectin-coated polystyrene. Adherence was inhibited by arginine or gelatin, but was slightly favored by soluble fibronectin. Fibronectin did not contribute to the opsonization (deposition of complement C3) and phagocytosis of the strain which bound the highest amount of fibronectin, even when polymorphonuclear cells were activated with serum-derived chemotaxin (C5a). These data suggest that fibronectin might promote tissue adherence but not phagocytosis of S. agalactiae in the mastitis process.     

Molecular characterization of Echinococcus granulosus in a hyperendemic European focus,the Republic of Moldova

Cystic echinococcosis is a zoonosis caused by the tapeworm Echinococcus granulosus sensu lato. The lifecycle of the parasite is mainly domestic, requiring dogs as definitive hosts and livestock species as intermediate hosts. Although human cystic echinococcosis is a high public health priority in the Republic of Moldova, the rare animal data available concerns only infection in cattle. A preliminary slaughterhouse survey was conducted to assess prevalence and perform the first molecular characterization of E. granulosus sensu lato in sheep and cattle. For the survey, 40 sheep and 19 cattle were inspected. Very high prevalence in sheep (82.5 %) and in cattle (78.9 %) was found. Molecular analyses identified genotypes G1 and G3 of E. granulosus sensu stricto in all the liver and lung samples. Based on the concatenated sequences of cox1?+?nad3 (701 bp), 23 different haplotypes were obtained. Mixed infections by different haplotypes/genotypes were frequently identified in both sheep and cattle. The relatively high (20.0 %) cyst fertility observed in cattle argues for the potential contribution of cattle to the lifecycle of E. granulosus sensu stricto, unlike previous observations in Europe. The hyperendemic situation of Moldova can be explained by a high majority of animals slaughtered at home usually without veterinary inspection. Further extensive slaughterhouse surveys with molecular identification also involving pigs and goats are needed to obtain a better overview of the epidemiological situation of E. granulosus sensu lato in this hyperendemic focus in the Republic of Moldova.     

Molecular characterization ofBorrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates

One hundred twenty-nine Slovenian isolates ofBorrelia burgdorferi sensu lato derived from patients (69 strains) orIxodes ricinus ticks (60 strains) were characterized. All of the strains were first- or second-passage isolates obtained in 1992 and 1993 from the same endemic region. The techniques used for the molecular analysis of strains included species-specific polymerase chain reaction (PCR) typing, and pulsed-field gel electrophoretic separation of undigested andMlul-digested genomic DNA. Isolates were identified to the species level by large restriction fragment pattern (LRFP) analysis and the results compared with the species-specific PCR result. Fifty-two patient isolates (75%) were typed asBorrelia afzelii (LRFP MLa1), 6 (9%) asBorrelia garinii (LRFPs MLg1–4), and 11 (16%) asBorrelia burgdorferi sensu stricto. The latter included 9 isolates (13%) with a new LRFP that is not typical ofBorrelia burgdorferi sensu stricto and for which the designation MLx is suggested. In contrast, only 32 of 60 (53%) tick isolates were typed asBorrelia afzelii, while 20 strains (33%) were typed asBorrelia garinii and 8 strains (13%) asBorrelia burgdorferi sensu stricto. Three new LRFPs were found among theBorrelia garinii (MLg5 and 6) andBorrelia burgdorferi sensu stricto (MLb15) tick isolates. Large restriction fragment pattern analysis identified new groups ofBorrelia burgdorferi sensu lato and revealed an apparent difference in the isolation frequency of different species from patients and ticks in the same endemic region.     

Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates

Phylogenetic relationships within the genus Pseudomonas were examined by comparing partial (about 1000 nucleotides) rpoB gene sequences. A total of 186 strains belonging to 75 species of Pseudomonas sensu stricto and related species were studied. The phylogenetic resolution of the rpoB tree was approximately three times higher than that of the rrs tree. Ribogroups published earlier correlated well with rpoB sequence clusters. The rpoB sequence database generated by this study was used for identification. A total of 89 isolates (79.5%) were identified to a named species, while 16 isolates (14.3%) corresponded to unnamed species, and 7 isolates (6.2%) had uncertain affiliation. rpoB sequencing is now being used for routine identification of Pseudomonas isolates in our laboratory.     

Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of theBorrelia strains used as antigens

Sera of 52 Lyme borreliosis patients classified according to their clinical features were analysed by Western blot using as antigensBorrelia strains belonging to three recently described genomic species. The antibody response was demonstrated to be homologous within each genospecies. Serum reactivity was studied for each of the type strainsBorrelia burgdorferi sensu stricto (strain B31^T),Borrelia garinii (strain 20047^T) and group VS461. Seven of 15 sera (46.6 %) of patients with menin-goradiculitis showed preferential reactivity withBorrelia garinii (strain 20047^T), all of 8 sera (100 %) of patients with acrodermatitis chronica atrophicans showed preferential reactivity with group VS461 (strain VS461) and 8 of 16 sera (50 %) of patients with arthritis showed preferential reactivity withBorrelia burgdorferi sensu stricto (strain B31^T). The presence of a strong response to OspA and OspB proteins ofBorrelia burgdorferi sensu stricto was found only in this last group of patients. These results suggest that there are clinical implications of the recently described modifications in the taxonomy ofBorrelia burgdorferi.     

First report of cryptococcosis due to Cryptococcus gattii sensu stricto VGI in an Ivorian HIV negative patient

IntroductionCryptococcus gattii species complex is endemic to tropical and subtropical regions and is described as a causative agent of cryptococcosis in immunocompetent individuals.Case presentationWe describe the first case of cryptococcosis in a HIV-negative patient from Ivory Coast infected by Cryptococcus gattii sensu stricto VGI. Isolates were recovered from cerebrospinal fluid (CSF) prior to systemic antifungal treatment up to 42 days after detection of the presence of yeasts in the CSF. Eighteen isolates were recovered, genetic diversity and antifungal susceptibility analyses were performed. All the isolates belonged to the Cryptococcus gattii sensu stricto (B;VGI) and were identified as a new sequence type (ST) 553 by Multilocus Sequence Typing (MLST) analyses. Susceptibility testing showed that all the strains had a wild-type phenotype for fluconazole, amphotericin B and flucytosine. Treatment with fluconazole (1200 mg/day) was initiated with success.ConclusionThis is the first case report of the presence of C. gattii sensu stricto VGI in a HIV-negative ivorian patient and the second report of the presence of species from the C. gattii complex species in this country.     

Enzyme-linked immunosorbent assays with recombinant internal flagellin fragments derived from different species of Borrelia burgdorferi sensu lato for the serodiagnosis of Lyme neuroborreliosis

The serodiagnosis of early Lyme neuroborreliosis is hampered by false negative results and one of the reasons could be the heterogeneity of strains of Borrelia burgdorferi sensu lato. For this study IgG enzyme-linked immunosorbent assays (ELISAs) with recombinant internal flagellin fragments (p41/i; amino acids 129 – 251) derived from strains PKo (B. afzelii), B31 (B. burgdorferi sensu stricto), and PBi (B. garinii) and ELISAs with whole-cell detergent extracts of strains PKo, PKa2 (B. burgdorferi sensu stricto), and PBi were compared. Cut off absorbance values were defined by the 94th and 92th percentiles of 200 sera from blood donors. Sera from patients with clinically defined neuroborreliosis [n = 88; 41 culture proven and 47 intentionally selected by the criteria specific IgG cerebrospinal fluid/serum index ≥2 and a serum IgG immunofluorescence assay value of <1 : 64] were tested. The sensitivities of the three whole-cell detergent-extract ELISAs were similar (46.6 – 49.2%), whereas those of the recombinant ELISAs were highest with p41/i:PBi (57.1%) and lowest with p41/i:PKo (21.5%). A combination of the p41/i:PBi and the p41/i:B31 test was most sensitive (59.8%). The correlation of absorbance values of different assays was very high for the three whole-cell detergent-extract ELISAs, whereas the absorbance values obtained with the three recombinant tests differed considerably. The greatest differences were observed between p41/i:PKo and any of the other two recombinant antigens. The differences in immune reactivity of patients sera (due to strain heterogeneity?) seems to have more influence on the results of an assay based on a single antigen than on a whole-cell-based test.     

Specificity of Infection-Induced Immunity among Borrelia burgdorferi Sensu Lato Species

The specificity of infection-induced immunity in mice infected with cultured or host-adapted Borrelia burgdorferi sensu lato, the agent of Lyme disease, was examined. Sera obtained from mice following infection with high and low doses of cultured B. burgdorferi sensu stricto, transplantation of infected tissue (host-adapted spirochetes), or tick-borne inoculation all showed protective activity in passive immunization assays. Infection and disease were similar in mice infected with cultured spirochetes or by transplantation. Thus, the adaptive form of inoculated spirochetes did not influence the immune response during active infection. Mice infected with B. burgdorferi sensu stricto and then cured of infection with an antibiotic during early or late stages of infection were resistant to challenge with high doses of homologous cultured spirochetes for up to 1 year. In contrast, actively immune mice infected with different Borrelia species (B. burgdorferi sensu lato, B. burgdorferi sensu stricto cN40, Borrelia afzelii PKo, and Borrelia garinii PBi) and then treated with an antibiotic were resistant to challenge with cultured homologous but not heterologous spirochetes. Similar results were achieved for actively immune mice challenged by transplantation and by passive immunization with sera from mice infected with each of the Borrelia species and then challenged with cultured spirochetes. Arthritis and carditis in mice that had immunizing infections with B. afzelii and B. garinii and then challenged by transplantation with B. burgdorferi sensu stricto were equivalent in prevalence and severity to those in nonimmune recipient mice. These results indicate that protective immunity and disease-modulating immunity that develop during active infection are universal among species related to B. burgdorferi sensu lato but are species specific.     

Genetic diversity of the outer surface protein C gene of southern Borrelia isolates and its possible epidemiological,clinical, and pathogenetic implications

The ospC genes of 20 southern Borrelia strains were sequenced. The strains consisted of B. burgdorferi sensu stricto, B. andersonii, B. bissettii, one undescribed genospecies, MI-8, and one probably new Borrelia species, TXW-1. A high degree of similarity exists between B. burgdorferi sensu stricto and B. bissettii and between B. bissettii and B. andersonii. Lateral transfers of the ospC gene probably occurred between B. burgdorferi sensu stricto and B. bissettii and between B. bissettii and B. andersonii. Internal gene recombination appears to occur among them. The highest degree of genetic diversity among them was observed in the two variable domains (V1 and V2), semivariable domain (SV), and the species-specific epitopes (between amino acids 28 and 31). Differences in ospC sequences among southern strains reflect diversity at the strain and genospecies levels. MI-8, which was recognized as an undescribed genospecies in our previous reports, remains distinguishable in our current analysis of ospC genes and is distinct from B. burgdorferi sensu stricto. Interestingly, another undescribed southern isolate, TXW-1, was not amplified under various PCR conditions. Compared to European B. burgdorferi sensu stricto strains, American B. burgdorferi sensu stricto strains show greater genetic heterogeneity. Southern B. burgdorferi sensu stricto, B. andersonii, and B. bissettii isolates were intermixed with each other in the phylogenetic trees. In the derived trees in our work, at least one southeastern strain of B. burgdorferi, MI-2, most closely aligns with a so-called invasive cluster that possesses many proven human-invasive strains. Transmission experiments show that MI-2 and the strains in this group of southern spirochetes are able to infect mice and hamsters and that the typical vector of Lyme disease, Ixodes scapularis, can acquire the spirochetes from infected mammals. Currently, strain MI-2 appears to be the only southern isolate among the 20 we analyzed that clusters with an OspC invasive group and thus might be invasive for humans.     

Heterogeneity of Borrelia burgdorferi sensu lato demonstrated by an ospA-type-specific PCR in synovial fluid from patients with Lyme arthritis

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis, has been divided into three genospecies: B. burgdorferi sensu stricto (OspA-type 1), B. afzelii (OspA-type 2) and B. garinii (OspA-type 3–7). Whereas in Europe B. afzelii (OspA-type 2) is predominant among human skin isolates and B. garinii (OspA-type 3–7) among human CSF isolates, some previous serological studies suggested that Lyme arthritis is also associated with B. burgdorferi sensu stricto in Europe. In the present study we designed ospA type-specific PCRs and identified four different ospA types associated with Lyme arthritis. Our study group consisted of 20 patients with positive serology (ELISA and immunoblotting) and clinical criteria for Lyme arthritis. B. burgdorferi DNA was detected in 13 patients and in none of 10 control patients from synovial fluid. We identified ospA-type 1 (26.6%), ospA-type 2 (33.3%), ospA-type 4 (6.6%) and ospA-type 5 (33.3%). Our conclusion is that in Europe B. burgdorferi sensu lato strains causing Lyme arthritis are considerably heterogeneous and that there is no prevalence of certain genospecies or OspA-types among this strains. Received: 14 May 1998