Cardiac involvement in non-human primates infected with the Lyme disease spirochete Borrelia burgdorferi

To investigate cardiac involvement in the non-human primate (NHP) model of Lyme disease, we inoculated 39 adult Macaca mulatta with Borrelia burgdorferi sensu stricto strains N40 (BbN40) by needle (N=22, 14 immunocompetent (IC), seven permanently immunosuppressed (IS), and four transiently immunosuppressed (TISP)) or by tick-bite (N=4, all TISP) or strain 297 (Bb297) by needle (N=2 IS), or with B. garinii strains Pbi (N=4, 2 TISP and 2 IS), 793 (N=2, TISP) or Pli (N=2, TISP). Five uninfected NHPs were used as controls. Infection and inflammation was studied in the hearts and the aorta removed at necropsy 2-32 months after inoculation by (1) H&E and trichrome-staining; (2) immunohistochemistry and digital image analysis; (3) Western blot densitometry; and (4) TaqMan RT-PCR. All NHPs inoculated with BbN40 became infected and showed carditis at necropsy. The predominant cells were T cells, plasma cells, and macrophages. There was increased IgG and IgM in the heart independent of immunosuppression. The B-cell chemokine BLC was significantly increased in IS-NHPs. There was increased deposition of the complement membrane attack complex (MAC) in TISP and IS-NHPs. The spirochetal load was very high in all BbN40-inoculated IS-NHPs but minimal if any in IC or TISP NHPs. Double-immunostaining revealed that many spirochetes in the heart of BbN40-IS NHPs had MAC on their membranes. We conclude that carditis in NHPs infected with B. burgdorferi is frequent and can persist for years but is mild unless they are immunosupressed.     

Rapid typing of Borrelia burgdorferi sensu lato species in specimens from patients with different manifestations of Lyme borreliosis

To further investigate the pathogenic potential of different Borrelia burgdorferi genospecies, specimens from 27 patients with different manifestations of Lyme borreliosis were analyzed by PCR and reverse line blotting (RLB). In samples from Lyme arthritis patients, B. burgdorferi sensu stricto was predominantly identified, while in patients with neuroborreliosis or acrodermatitis, Borrelia garinii and Borrelia afzelii, respectively, were exclusively detected. The results demonstrate that PCR-RLB is a valuable tool for epidemiological and pathogenetic studies of Lyme borreliosis.     

DNA microarray assessment of putative Borrelia burgdorferi lipoprotein genes

A DNA microarray containing fragments of 137 Borrelia burgdorferi B31 putative lipoprotein genes was used to examine Lyme disease spirochetes. DNA from B. burgdorferi sensu stricto B31, 297, and N40; Borrelia garinii IP90; and Borrelia afzelii P/Gau was fluorescently labeled and hybridized to the microarray, demonstrating the degree to which the individual putative lipoprotein genes were conserved among the genospecies. These data show that a DNA microarray can globally examine the genes encoding B. burgdorferi lipoproteins.     

Localization of Borrelia burgdorferi in the nervous system and other organs in a nonhuman primate model of lyme disease

Lyme borreliosis is caused by infection with the spirochete Borrelia burgdorferi. Nonhuman primates inoculated with the N40 strain of B. burgdorferi develop infection of multiple tissues, including the central (CNS) and peripheral nervous system. In immunocompetent nonhuman primates, spirochetes are present in low numbers in tissues. For this reason, it has been difficult to study their localization and changes in expression of surface proteins. To further investigate this, we inoculated four immunosuppressed adult Macaca mulatta with 1 million spirochetes of the N40 strain of B. burgdorferi, and compared them with three infected immunocompetent animals and two uninfected controls. The brain, spinal cord, peripheral nerves, skeletal muscle, heart, and bladder were obtained at necropsy 4 months later. The spirochetal tissue load was first studied by polymerase chain reaction (PCR)-ELISA of the outer surface protein A (ospA) gene. Immunohistochemistry was used to study the localization and numbers of spirochetes in tissues and the expression of spirochetal proteins and to characterize the inflammatory response. Hematoxylin and eosin and trichrome stains were used to study inflammation and tissue injury. The results showed that the number of spirochetes was significantly higher in immunosuppressed animals. B. burgdorferi in the CNS localized to the leptomeninges, nerve roots, and dorsal root ganglia, but not to the parenchyma. Outside of the CNS, B. burgdorferi localized to endoneurium and to connective tissues of peripheral nerves, skeletal muscle, heart, aorta, and bladder. Although ospA, ospB, ospC, and flagellin were present at the time of inoculation, only flagellin was expressed by spirochetes in tissues 4 months later. Significant inflammation occurred only in the heart, and only immunosuppressed animals had cardiac fiber degeneration and necrosis. Plasma cells were abundant in inflammatory foci of steroid-treated animals. We concluded that B. burgdorferi has a tropism for the meninges in the CNS and for connective tissues elsewhere in the body.     

Differential survival of Lyme borreliosis spirochetes in ticks that feed on birds

The abilities of the most common European genospecies of Borrelia burgdorferi sensu lato to survive blood meals taken by ticks feeding on birds were analyzed. A pattern of differential survival of the spirochetes in feeding ticks was observed. The result is consistent with the concept of selective transmission of Lyme borreliosis spirochetes.     

Detection of three genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different regions of Poland

Ixodes ricinus ticks, collected in 1996-1998 in different Polish woodlands, were examined to assess the frequency of the occurrence of Lyme borreliosis-associated genospecies. A total of 568 samples of individual adults and 162 samples of individual (n =48) and pooled (of 2 to 7) samples of nymphs were analysed by the polymerase chain reaction (PCR) for Borrelia burgdorferi sensu lato. Spirochetes were detected in 130 adult ticks (22.9%) and in a minimum of 32 (5.3%) nymphs. Further identification of 153 B. burgdorferi s.l.-positive samples by nested PCR using three species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto and B. garinii. Both single-species and mixed infections were noted. Single-species infections were observed in the majority of samples (n = 83/153; 54.2%). Within this group B. afzelii was found in 38/153 samples (24.9%), followed by B. burgdorferi sensu stricto (n = 23/153; 15.0%) and B. garinii (n = 22/153; 14.4%). Dual infections with B. burgdorferi s.s. and B. afzelii were detected in 17/121 (14.0%) adults, while both B. burgdorferi s. s./B. garinii and B. afzelii/B. garinii coinfected 11/121 (9.1%) adult ticks. Triple infection with B. burgdorferi s.s., B. afzelii and B. garinii was noted twice (1.6%). In general, B. afzelii was found in 72/153 (47.1%) tick samples and was the predominant species. B. burgdorferi s. s. and B. garinii were detected in a total of 60/153 (39.2%) and 51/153 (33.3%) samples, respectively. Although, 21 (13.7%) samples were infected by B. burgdorferi s.l. genospecies undetectable by the primers used, results of our study confirm that Lyme borreliosis pathogenic genospecies are well established in tick populations throughout Poland.     

Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots).

The performance of Western blots (immunoblots) prepared with eight strains of Borrelia burgdorferi representing B. burgdorferi sensu stricto, B. garinii, and B. afzelii genospecies was tested with a panel of sera with various clinical presentations collected from eight geographic regions. European sera were generally more reactive to blots prepared with B. garinii or B. afzelii strain antigens, in particular B. garinii 20047 and B. afzelii VS461. North American sera were more reactive with B. burgdorferi sensu stricto strains. Our observation of significant differences in the levels of reactivity of some sera on Western blots of certain strains is potentially important for the development and implementation of generic interpretive criteria. Preferential reactivity of sera from patients with nerve and/or palsy symptoms to B. garinii strains and with cutaneous disease to B. afzelii strains was observed. On the basis of our results, we have concluded that strain 20047 is the best strain to use for the development of a generic Lyme borreliosis Western blot for Europe.     

Distribution of Clinically Relevant Borrelia Genospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR

A LightCycler-based PCR protocol was developed which targets the ospA gene for the identification and quantification of the different Borrelia burgdorferi sensu lato species in culture and in ticks, based on the use of a fluorescently labeled probe (HybProbe) and an internally labeled primer. The detection limit of the PCR was 1 to 10 spirochetes. A melting temperature determined from the melting curve of the amplified product immediately after thermal cycling allowed the differentiation of the three different B. burgdorferi sensu lato genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii) that are clinically relevant in Europe in a single PCR run. This method represents a simplified approach to study the association of different Borrelia species in ticks, the risk of Lyme borreliosis, and the putatively species-specific clinical sequelae. To determine the reliability of the real-time PCR protocol, we studied the prevalence of B. burgdorferi sensu lato infection in Ixodes ricinus ticks. A total of 1,055 ticks were collected by flagging vegetation in five different sites in the region of Konstanz (south Germany) and were examined for the distribution of B. burgdorferi species by real-time PCR. The mean infection rate was 35%. Of 548 adult ticks, 40% were positive, and of 507 nymphs, 30% were positive. The predominant genospecies (with 18% mixed infections) in the examined areas was B. afzelii (53%), followed by B. garinii (18%) and B. burgdorferi sensu stricto (11%); 0.8% of the infecting Borrelia could not be identified.     

Isolation and characterization of Borrelia burgdorferi sensu lato strains in an area of Italy where Lyme borreliosis is endemic

Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to "San Martino" Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.     

Identification of three clinically relevant Borrelia burgdorferi sensu lato genospecies by PCR-restriction fragment length polymorphism analysis of 16S-23S ribosomal DNA spacer amplicons

We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.     

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.     

Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients.

Oligonucleotide primers based on Borrelia burgdorferi sensu lato ospA gene sequences have been designed for use in the PCR to type all (SL primers) or each (GI to GIII primers) of the B. burgdorferi sensu lato genospecies involved in Lyme disease. These genospecies-specific primers were then used in the PCR on 24 biological fluids collected from 18 neuroborreliosis patients. Among the samples tested, 20 contained DNA from Borrelia garinii, 11 contained DNA from B. burgdorferi sensu stricto, and 10 contained DNA from Borrelia afzelii. In toto, 10 patients appeared to have been infected by a single genospecies and 8 were infected by more than one Lyme disease-associated genospecies. Serum specimens from six patients were absorbed with heterologous antigens and tested by Western blotting (immunoblotting). In four cases, residual immunodetection revealed specific epitopes of genospecies also detected by PCR; in two of them, the concordant results indicated pluri-infection of the patients. In the other two cases, Western blotting showed specific antibodies for two genospecies of Borrelia, while PCR detected DNA from only one. In summary, the data underscored the relatively high prevalence of pluri-infections in Lyme disease and confirmed the association of B. garinii with neuroborreliosis.     

Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi-infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.     

Characterization of Lyme borreliosis isolates from patients with erythema migrans and neuroborreliosis in southern Sweden

Southern Sweden is an area of Lyme borreliosis (LB) endemicity, with an incidence of 69 cases per 100,000 inhabitants. The most frequent clinical manifestations are erythema migrans (77%) and neuroborreliosis (16%). There was no record of human Borrelia strains being isolated from patients in this region before the prospective study reported here. Borrelia spirochetes were isolated from skin and cerebrospinal fluid (CSF) from LB patients living in the region. A total of 39 strains were characterized by OspA serotype analysis, species-specific PCR, and signature nucleotide analysis of the 16S rRNA gene. Of 33 skin isolates, 31 (93.9%) were Borrelia afzelii strains and 2 (6.1%) were Borrelia garinii strains. Of six CSF isolates, five (83.3%) were B. garinii and one (16.7%) was B. afzelii. Neither Borrelia burgdorferi sensu stricto strains nor multiple infections were observed. The B. afzelii isolates were of OspA serotype 2. Three B. garinii strains were of OspA serotype 5, and the remaining four strains were of OspA serotype 6. All of the B. garinii strains belonged to the same 16S ribosomal DNA ribotype class. Our findings agree with earlier findings from other geographic regions in Europe where B. afzelii and B. garinii have been recovered predominantly from skin and CSF cultures, respectively. To further study the possible presence in Sweden of the genotype B. burgdorferi sensu stricto, which is known to be present in Europe and to occur predominantly in patients with Lyme arthritis, molecular detection of Borrelia-specific DNA in synovial samples from Lyme arthritis patients should be performed.     

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.     

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.     

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.     

Adaptation of diverse lyme disease spirochetes in a natural rodent reservoir host

We compared the relative reservoir competence of European wood mice for two genospecies of Lyme disease spirochetes by analyzing susceptibility, intrinsic incubation period, and degree and duration of infectivity. Borrelia afzelii, specializing in particular reservoir hosts, is better adapted to those hosts than is the more generalist genospecies B. burgdorferi sensu stricto.     

Development of destructive arthritis in vaccinated hamsters challenged with Borrelia burgdorferi.

We present the first direct evidence that adverse effects, particularly severe destructive arthritis, can develop in vaccinated hamsters after challenge with Borrelia burgdorferi sensu lato isolates. Hamsters were vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant. A severe destructive arthritis was readily evoked in vaccinated hamsters challenged with the homologous B. burgdorferi sensu stricto isolate C-1-11 before high levels of protective borreliacidal antibody developed. Once high levels of C-1-11 borreliacidal antibody developed, hamsters were protected from homologous challenge and development of arthritis. Vaccinated hamsters, however, still developed severe destructive arthritis when challenged with other isolates of the three genomic groups of B. burgdorferi sensu lato (B. burgdorferi sensu stricto isolate 297, Borrelia garinii isolate LV4, and Borrelia afzelii isolate BV1) despite high levels of C-1-11 specific borreliacidal antibody. Vaccines that contained whole spirochetes in adjuvant induced destructive arthritis, but this effect was not dependent on the isolate of B. burgdorferi sensu lato or the type of adjuvant. These studies demonstrate that caution is necessary when employing whole spirochetes in adjuvant for vaccination to prevent Lyme borreliosis. Additional studies are needed to identify the antigen(s) responsible for the induction and activation of arthritis and to define the immune mechanisms involved.