Genetic diversity of Borrelia burgdorferi in lyme disease patients as determined by culture versus direct PCR with clinical specimens

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.     

Lyme disease Borrelia species in northeastern China resemble those isolated from far eastern Russia and Japan

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.     

Genospecies identification and characterization of Lyme disease spirochetes of genospecies Borrelia burgdorferi sensu lato isolated from rodents in Taiwan

Lyme disease spirochetes of the genospecies Borrelia burgdorferi sensu lato were identified and characterized for the first time in Taiwan. Seven isolates, designated TWKM1 to TWKM7, were purified from the ear tissues of three species of rodents captured from seven localities of Taiwan. The immunological characteristics of these Taiwan isolates were compared with those of other genospecies of Lyme disease spirochetes by analyzing the protein profiles and reactivities with B. burgdorferi-specific monoclonal antibodies (MAbs). The genospecies of these Taiwan isolates were also identified by the similarities in their plasmid profiles and differential reactivities with genospecies-specific PCR primers. Although two distinct protein profiles were observed among the seven Taiwan isolates, the MAb reactivities against the outer surface proteins of B. burgdorferi of all of these isolates were consistent with those of B. burgdorferi sensu lato. The similarities of the plasmid profiles also confirmed the identities of these Taiwan isolates. PCR analysis indicated that all of these Taiwan isolates were genetically related to the genospecies B. burgdorferi sensu stricto. These results demonstrate the first identification of Lyme disease spirochetes in Taiwan and also highlight the increasing demand for defining the reservoirs and vector ticks of B. burgdorferi. A serosurvey for Lyme disease infection in the human population of Taiwan may also be required.     

Temporal pattern of Borrelia burgdorferi p21 expression in ticks and the mammalian host

The temporal synthesis of the P21 protein of Borrelia burgdorferi and the development of the humoral response to this antigen was assessed in infected mice. p21 is a member of the ospE-F gene family and its protein, P21, has been shown to be expressed by B. burgdorferi within infected mice but not by spirochetes cultured in vitro. P21 was not detected on B. burgdorferi in unfed or engorged Ixodes dammini (also known as I. scapularis) ticks, further supporting the postulate that P21 synthesis is specific for the mammalian host. In B. burgdorferi-infected mice, ospE mRNA and OspE antibodies were observed at 7 d, whereas p21 mRNA and P21-specific antibodies were detected at 21-28 d, suggesting that p21 is expressed later than ospE. Moreover, ospA mRNA was not discernible until day 14, indicating that ospA, like p21, is not expressed in the early stages of tick-transmitted murine Lyme borreliosis. Because p21 is expressed during infection in mice, we assessed the human humoral response to P21. 28% (34 of 122) of the patients with either early- or late-stage Lyme disease, and 33% (11 of 33) of the individuals with Lyme arthritis had P21 antibodies, suggesting that a P21 response may serve, at least partially, as a marker of infection. Active immunization with recombinant P21 did not protect C3H mice from tick-borne B. burgdorferi infection, and passive transfer of P21 antiserum to infected mice did not alter the course of disease. These data suggest that the antigenic structure of B. burgdorferi changes during the early stages of murine infection.     

Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.     

Borrelia burgdorferi and interleukin-1 promote the transendothelial migration of monocytes in vitro by different mechanisms

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% +/- 3%, 65% +/- 2%, or 25% +/- 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold +/- 2-fold increase in the migration of human CD4(+) T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4(+) T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.     

Screening and chemoprophylaxis for tuberculosis infection in college populations

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragment length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe.     

Ankle arthrodesis with cross screw fixation. Good results in 36/40 cases followed 3-7 years

From May 1991 to May 1994, Lyme borreliosis was studied prospectively in 301 residents living on Asp?, a highly endemic area for the disease. The study included annual questionnaires and blood samples for serology. Immunoglobulin G (IgG) antibodies to Borrelia burgdorferi sensu lato were detected by enzyme-linked immunosorbent assay in 63/301 (21%) of the residents at the start of the study. Seropositivity rates increased with time, and 3 years later 101/301 (34%) were positive. A total of 34 individuals developed physician-verified manifestations of Lyme borreliosis during the study period. Nine individuals developed an erythema migrans, despite a previously treated Lyme borreliosis or pre-existing high levels of IgG antibodies to B. burgdorferi s.l.     

Identification of quantitative trait loci governing arthritis severity and humoral responses in the murine model of Lyme disease

A spectrum of disease severity has been observed in patients with Lyme disease, with approximately 60% of untreated individuals developing arthritis. The murine model of Lyme disease has provided strong evidence that the genetic composition of the host influences the severity of arthritis following infection with Borrelia burgdorferi: infected C3H mice develop severe arthritis while infected C57BL/6N mice develop mild arthritis. Regions of the mouse genome controlling arthritis severity and humoral responses during B. burgdorferi infection were identified in the F2 intercross generation of C3H/HeNCr and C57BL/6NCr mice. Rear ankle swelling measurements identified quantitative trait loci (QTL) on chromosomes 4 and 5, while histopathological scoring identified QTL on a unique region of chromosome 5 and on chromosome 11. The identification of QTL unique for ankle swelling or histopathological severity suggests that processes under distinct genetic control are responsible for these two manifestations of Lyme arthritis. Additional QTL that control the levels of circulating Igs induced by B. burgdorferi infection were identified on chromosomes 6, 9, 11, 12, and 17. Interestingly, the magnitude of the humoral response was not correlated with the severity of arthritis in infected F2 mice. This work defines several genetic loci that regulate either the severity of arthritis or the magnitude of humoral responses to B. burgdorferi infection in mice, with implications toward understanding the host-pathogen interactions involved in disease development.     

Polymerase chain reaction-based risk assessment for Wilms tumor in sporadic aniridia

To investigate the prevalence of Lyme disease infection in Taiwan, we conducted a zoonotic survey for spirochetal infection in the small mammals. Ear tissues of trapped rodents collected from various localities in Taiwan were incubated into BSK-H culture medium and examined for the evidence of spirochetal infection by dark-field microscopy. Spirochetes cultured from six species of wild and peridomestic rodents and seven isolates, designated TWKM 1-7, were purified by serial dilution and membrane filtration. Infection was detected in 16.6% (53 of 320) of captured rodents and the highest infection rate (36.4%) was observed in the brown country rat (Rattus losea, Swinhoe). Higher infection rates based on the geographic distribution were observed in the eastern localities and on Kimmen Island. Reactivity with Borrelia burgdorferi-specific monoclonal antibodies and Western blot analysis indicated that these Taiwan isolates were closely related to the causative agent of Lyme disease, B. burgdorferi sensu lato. These results provide the first evidence of the existence of Lyme disease spirochetes in the Taiwan area.     

Rheumatic manifestations in Lyme borreliosis: personal experience in patients with oligoarthritis of "unknown" origin

Lyme borreliosis is an infectious illness caused by the spirochete Borrelia burgdorferi and is transmitted by tick vectors. A prospective study was performed from January 1990, to investigate whether Lyme arthritis might have been undetected among patients with (unclassified arthritis) oligoarthritis of "unknown" aetiology. 210 patients were tested for antibodies to Borrelia burgdorferi: 82 patients with oligoarthritis of "unknown" aetiology; 52 patients with Reiter's syndrome; 20 patients with seronegative, B-27 positive oligoarthritis and 56 controls. Serological testing for Borrelia burgdorferi was performed by indirect immunofluorescence assay. The occurrence of positive antibodies (1:80) in 11 (13.4%) patients with arthritis of "unknown" aetiology was significantly different from the combined control group (1.6%) (p < 0.05). Four out of 11 patients remembered a tick bite, two out of 11 patients developed erythema migrans after 3 to 10 days. Six weeks later 2 patients developed oligoarthritis and one patient after a month. In the remaining 8 patients arthritis was the first sign of the disease. Knees were most commonly affected (90%). Radiographic abnormalities (osteoporosis, soft tissue swelling) were noted in 3 patients. The synovial fluid findings were typical for inflammatory arthritides in 6 patients. The diagnosis of Lyme borreliosis was made according to following data: origin from an area endemic for Lyme borreliosis, tick bite, erythema migrans, significant levels of the antibodies to the Borrelia burgdorferi and oligoarthritis. It can be concluded that arthritis may be the main manifestation of Borrelia burgdorferi infection.     

Isolation of Borrelia burgdorferi from skin biopsy specimens of patients with erythema migrans

Procedures for the cultural isolation and identification of Borrelia burgdorferi from skin biopsy specimens are described. B. burgdorferi was isolated from 24 of 34 skin biopsy specimens from patients with erythema migrans. Eight of the culture-positive patients had single, primary lesions and 16 had multiple, secondary lesions. The 17 male and 7 female patients were 2 to 70 years old. Biopsy samples were obtained from erythematous or normal-appearing skin within 1 cm of the peripheral aspect of the lesion. Twenty-three of the isolates were detected within 8 days of incubation in Barbour-Stoenner-Kelly medium with no antimicrobial agents. The identities of the isolates were determined by reactivity with monoclonal antibodies H9724 and H5332. Cultivation of B. burgdorferi from skin lesions suggestive of erythema migrans is a practical and clinically relevant procedure. Clinical isolates and corresponding patient sera and urine will contribute to efforts to improve existing immunoserologic testing methods and develop new assays to diagnose Lyme borreliosis.     

Macrophages exposed to Borrelia burgdorferi induce Lyme arthritis in hamsters

The mechanism(s) by which Lyme arthritis is induced has not been elucidated. In this study, we showed that macrophages have a direct, effector role in the pathogenesis of Lyme arthritis. Severe destructive arthritis was induced in recipients of macrophages obtained from Borrelia burgdorferi-vaccinated and nonvaccinated hamsters exposed to Formalin-inactivated B. burgdorferi in vitro and then challenged with the Lyme spirochete. Swelling of the hind paws was detected within 8 h of infection, increased rapidly, and peaked at 21 h. This initial swelling decreased, and by day 4 only slight swelling was detected. Severe swelling of the hind paws was detected 8 days after infection and increased rapidly, with peak swelling occurring on day 11. Histopathologic examination affirmed that macrophages exposed to Formalin-inactivated spirochetes induced a severe destructive Lyme arthritis. The onset and severity of the severe destructive arthritis were dependent on the number of macrophages transferred. By contrast, macrophages not exposed to Formalin-inactivated B. burgdorferi failed to induce severe destructive arthritis in recipients after challenge with B. burgdorferi. Similarly, severe destructive arthritis was not detected in recipients of macrophages injected with spirochetal growth medium. Our results also showed that transferred macrophages could not protect hamsters from infection with B. burgdorferi, as spirochetes were readily recovered from their tissues when cultured. These findings demonstrate that macrophages exposed to B. burgdorferi are directly involved in the induction of Lyme arthritis.     

Erythema migrans-like rash illness at a camp in North Carolina: a new tick-borne disease?

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, has never been isolated from a patient thought to have acquired Lyme disease in any southeastern state. OBJECTIVE: To investigate 14 cases of an erythema migrans (EM)-like rash illness that occurred during 2 summers at an outdoor camp in central North Carolina in an effort to determine the etiologic, epidemiological, and clinical aspects of this illness. METHODS: Using active surveillance, we identified cases of clinically diagnosed EM in residents and staff of the camp. We collected clinical and demographic information; history of exposure to ticks; acute and convalescent serum antibodies to B. burgdorferi, Rickettsia rickettsii, and Ehrlichia chaffeensis; and cultures for spirochetes from biopsy specimens of skin lesions. Serum samples from a group of residents and staff who did not develop rashes were tested for the same antibodies. We speciated ticks removed from people and collected from vegetation. RESULTS: We identified 14 cases of EM-like rash illness during the 2 summers. Of the 14 case-patients, 10 had associated mild systemic symptoms and 1 had documented fever. All 14 case-patients had removed attached ticks, and 8 remembered having removed a tick from the site where the rash developed a median of 12 days earlier (range, 2-21 days). One tick removed from the site where a rash later developed was identified as Amblyomma americanum, the Lone Star tick; 97% of ticks collected from vegetation and 95% of ticks removed from people were A. americanum. No spirochetes were isolated from skin biopsy specimens. Paired serum samples from 13 case-patients did not show diagnostic antibody responses to B. burgdorferi or other tick-borne pathogens. CONCLUSIONS: This investigation suggests the existence of a new tick-associated rash illness. We suspect that the disease agent is carried by A. americanum ticks. In the southern United States, EM-like rash illness should no longer be considered definitive evidence of early Lyme disease.     

An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes

The reservoir competence of passerine birds for the Lyme borreliosis spirochetes was studied in an enzootic focus in Switzerland. Skin aspirates and skin biopsies were used to isolate Borrelia spirochetes from Turdus species. B. burgdorferi sensu lato was isolated and/or PCR-detected in BSK medium containing skin biopsy or skin aspirate from 5 blackbirds (T. merula) and one song thrush (T. philomelos). Seven isolates were obtained from 3 different blackbirds. Either B. garinii or Borrelia from the genomic group VS116 was found in bird skin samples. Mixed infection occurred in 2 cases. Tick xenodiagnosis was used to determine whether blackbirds transmitted Borrelia to ticks. Five xenodiagnoses were performed on 3 different blackbirds. Borrelia DNA was detected in BSK medium inoculated with xenodiagnostic ticks from all the passerines tested. Isolates cultured from xenodiagnostic ticks were obtained from 2 blackbirds. Isolates belonged to group VS116 (n = 10) and to B. garinii (n = 1). Our study has shown that Turdus sp. are infected by B. garinii and by Borrelia from group VS116 and that blackbirds are implicated as reservoirs for these 2 genomic groups of Borrelia, as they transmit living borreliae to ticks. An association seems to exist between birds and Borrelia VS116, and to a lesser extent, B. garinii, similar to the association existing between small rodents and B. afzelii. Our observations emphasize the fact that different enzootic cycles maintain Lyme borreliosis spirochetes in nature.     

Infusion methods for determination of peripheral resistance: influence of infused medium and back pressure

Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B. burgdorferi from one of 108 tick pools representing 733 ticks and eight-four tissue samples from twenty-six rodents collected in the U.K, and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B. burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I. ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi, although only one nymph contained > or = 1000 spirochaetes. To examine further the problem of isolating U.K. B. burgdorferi, twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein: implications for taxonomy and for use of OspC protein as a diagnostic antigen

The nucleotide sequences of the ospC gene from five Danish human Borrelia burgdorferi isolates representing all three B. burgdorferi genospecies (B. burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461) and from the American type strain B31 were determined and compared with the published ospC sequence from the German B. burgdorferi isolate PKo (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). The ospC gene was present in all isolates, regardless of the presence or absence of its product, OspC. The deduced amino acid sequences of OspC from the seven isolates were aligned and revealed pairwise sequence identities ranging from 60.5 to 100%. Differences were scattered throughout the amino acid sequences. A phylogenetic tree was constructed and revealed three distinct phenotypic groups OspCI to OspCIII corresponding to the three delineated genospecies. Immunoblot analysis revealed that the seven OspC proteins tested have both common and specific epitopes. There is significant epitope diversity, since even polyclonal antisera showed serotype-restricted specificity. Therefore, a serodiagnostic assay for Lyme borreliosis utilizing OspC as a test antigen should include all three OspC phenotypes in order to obtain a species-wide sensitivity.     

The risk of acquiring Lyme disease or babesiosis from a blood transfusion

To determine the risk of acquiring Lyme disease or babesiosis from blood transfusion, serum was collected before and 6 weeks after patients received multiple transfusions during cardiothoracic surgery and antibodies to Borrelia burgdorferi and Babesia microti were measured. Of 155 subjects, 149 received 601 total units of packed red blood cells (PRBC) and 48 received 371 total units of platelets. No patient developed clinical or serologic evidence of Lyme disease; 1 (who received 5 units of PRBC) developed clinical and serologic evidence of babesiosis. The risk of acquiring Lyme disease from a transfused unit of PRBC was 0 (95% confidence interval [CI], 0-0.5%) and from a transfused unit of platelets was 0 (95% CI, 0-0.8%); the same risks for babesiosis were 0.17% (95% CI, 0.004%-0.9%) and 0 (95% CI, 0-0.8%), respectively. The risk of acquiring either Lyme disease or babesiosis from a blood transfusion in Connecticut is very low.     

Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.     

Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.