First isolation of Borrelia burgdorferi in southern California.

The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, was isolated from the blood of a dusky-footed wood rat, Neotoma fuscipes Baird, in the San Bernardino Mountains of southern California. Antigenic, protein, and molecular analyses demonstrated that the isolate varied slightly from most isolates of B. burgdorferi from northern California and was clearly distinct from other species of Borrelia that are endemic to the state. This is the first reported isolate of B. burgdorferi from southern California and demonstrates that the Lyme disease spirochete is enzootic in mountains near the major human population center of the state.     

Borrelia burgdorferi Not Confirmed in Human-Biting Amblyomma americanum Ticks from the Southeastern United States

The predominant human-biting tick throughout the southeastern United States is Amblyomma americanum. Its ability to transmit pathogens causing Lyme disease-like illnesses is a subject of ongoing controversy. Results of previous testing by the Department of Defense Human Tick Test Kit Program and other laboratories indicated that it is highly unlikely that A. americanum transmits any pathogen that causes Lyme disease. In contrast, a recent publication by Clark and colleagues (K. L. Clark, B. Leydet, and S. Hartman, Int. J. Med. Sci. 10:915–931, 2013) reported detection of Lyme group Borrelia in A. americanum using a nested-flagellin-gene PCR. We evaluated this assay by using it and other assays to test 1,097 A. americanum ticks collected from humans. Using the Clark assay, in most samples we observed nonspecific amplification and nonrepeatability of results on subsequent testing of samples. Lack of reaction specificity and repeatability is consistent with mispriming, likely due to high primer concentrations and low annealing temperatures in this protocol. In six suspect-positive samples, Borrelia lonestari was identified by sequencing of an independent gene region; this is not a Lyme group spirochete and is not considered zoonotic. B. burgdorferi was weakly amplified from one pool using some assays, but not others, and attempts to sequence the amplicon of this pool failed, as did attempts to amplify and sequence B. burgdorferi from the five individual samples comprising this pool. Therefore, B. burgdorferi was not confirmed in any sample. Our results do not support the hypothesis that A. americanum ticks are a vector for Lyme group Borrelia infections.     

Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Lyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection of Borrelia burgdorferi infection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinant in vivo-expressed B. burgdorferi antigens for objective detection of a host immune response to B. burgdorferi infection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinant B. burgdorferi in vivo-expressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (C_q) value for each sample minus the mean background C_q plus 3 standard deviations (ΔC_q) being 4 to 10, whereas ΔC_q was 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation = 0.895, P < 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using the B. burgdorferi VlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes to B. burgdorferi infection.     

First isolations of Borrelia burgdorferi from rodents collected in northern Europe

Spirochetes were found in 13% of Ixodes ricinus collected from an island, near Stockholm where human borreliosis is endemic. Borrelia burgdorferi was cultivated from the kidney and/or spleen of wild rodents (Clethrionomys glareolus and Apodemus flavicollis) from the same island. Spirochetes were identified as Borrelia burgdorferi by indirect immunofluorescence assays using species and genus specific monoclonal antibodies. In these tests the spirochetes could not be differentiated from strains previously cultured from Swedish patients with Ixodes-borne borreliosis. The results show that small rodents in Europe may harbour borreliae and indicate that C. glareolus and A. flavicollis may be important reservoirs for the spirochetes causing Ixodes-borne borreliosis in humans and domestic animals in Europe.     

Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents.

An ear punch biopsy method for the detection and isolation of Borrelia burgdorferi from rodents was developed. The ear punch biopsy proved to be extremely sensitive, detecting spirochetes in 100% (11 of 11) of laboratory hamsters infected by tick bite and 95.8% (23 of 24) of hamsters infected by intraperitoneal inoculation. When cultured at 4 to 6 weeks postinfection, 92 to 100% of the ear punches taken from individual hamsters yielded viable spirochetes. B. burgdorferi was detected in sequential cultures from animals as early as 4 days postinfection and as late as 20 weeks postinfection. A total of 86% (6 of 7) of field-collected white-footed mice (Peromyscus leucopus) which were positive for B. burgdorferi as determined by xenodiagnosis were also positive by the ear punch method. The ear punch biopsy method allows individual rodents to be sampled for B. burgdorferi serially over a long period and thus should prove useful for both field and laboratory experiments.     

Serologic analyses of Peromyscus leucopus, a rodent reservoir for Borrelia burgdorferi, in northeastern United States.

An enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent-antibody test were used to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme disease, in Peromyscus leucopus (white-footed mouse). Of the 661 mice captured in Connecticut, Rhode Island, and New York during 1980 and 1983 to 1987, 166 (25.1%) had antibodies to B. burgdorferi by ELISA. Comparative analyses of 210 serum specimens, collected in areas where Lyme disease is endemic, revealed a threefold difference in sensitivity between the ELISA (38.1% positive) and the indirect fluorescent-antibody method (12.4%). Although prevalence of seropositive P. leucopus was highest during June, elevated amounts of antibody (1:1,280 to 1:2,560) were detected in mice that harbored spirochetes during all seasons. Being reservoirs for B. burgdorferi, these rodents are suitable for monitoring spirochete infections at foci and should be included in field evaluations of control programs aimed at suppressing Lyme disease.     

Lack of Serum Antibodies against Borrelia burgdorferi in Children with Autism

It has been proposed that Borrelia burgdorferi infection is present in ∼25% of children with autism spectrum disorders. In this study, antibodies against Borrelia burgdorferi were assessed in autistic (n = 104), developmentally delayed (n = 24), and healthy control (n = 55) children. No seropositivity against Borrelia burgdorferi was detected in the children with and without autism. There was no evidence of an association between Lyme disease and autism.     

Comparison of infectivities of six tick-derived isolates of Borrelia burgdorferi for rodents and ticks.

The infectivity and dissemination to the skin of six isolates of Borrelia burgdorferi were evaluated by inoculating them into groups of deer mice (Peromyscus maniculatus), hamsters, and Swiss Webster mice. Rodent infection was assayed by culture of ear punch biopsy specimens taken at 4, 8, and 12 weeks postinoculation (p.i.). Spirochetes were detected in biopsy specimens from individuals of all three host species that had been inoculated with four isolates (CA3, CA4, CA7, and CA8). Ear punch biopsy specimens taken from Swiss Webster mice at 12 weeks p.i. yielded an additional reisolate (CA2), even though these animals did not seroconvert. The remaining isolate (CA9) was not recovered from any host. However, two deer mice and all hamsters and Swiss Webster mice inoculated with CA9 seroconverted. All six isolates were of low infectivity to ticks when inoculated intramuscularly into hosts. Only 4 (1.6%) of 250 Ixodes pacificus larvae acquired and transstadially maintained infection from hosts inoculated intramuscularly. Infectivity of three isolates for ticks also was tested in Swiss Webster mice injected intradermally. The mean prevalences of infection in xenodiagnostic ticks fed on these mice at 4 weeks p.i. were 47.9, 1.2, and 2.2% for isolates CA4, CA7, and CA8, respectively. The mean prevalences of infection for ticks fed on the same mice at 12 weeks p.i. were 36.4, 11.8, and 20.4%, respectively. Such differences in the infectivity and rate of dissemination of individual isolates of B. burgdorferi should be considered during studies of reservoir and vector competence.     

Borrelia burgdorferi sensu lato and Ehrlichia spp. in Ixodes ticks from southern Norway

We report the results of a study of the prevalence of Ehrlichia and Borrelia species in 341 questing Ixodes ricinus ticks from two locations in southern Norway. The prevalences of Borrelia burgdorferi sensu lato and Ehrlichia spp. were, respectively, 16 and 11.5% at site 1 and 17 and 6% at site 2. Prevalence and species composition of Borrelia and Ehrlichia varied with location and date of collection. The dominant Borrelia species at both sites was Borrelia afzelii, followed by Borrelia burgdorferi sensu stricto. Borrelia garinii was found in only a single tick. The dominant member of the Ehrlichia group was a recently described Ehrlichia-like organism related to the monocytic ehrlichiae. Variants of Ehrlichia phagocytophila and the agent of human granulocytic ehrlichiosis were also found. The highest prevalences for B. afzelii, B. burgdorferi sensu stricto, and the Ehrlichia-like organism were observed in May. B. afzelii was most prevalent in females, less prevalent in nymphs, and least prevalent in males, while the prevalence of Ehrlichia was highest in nymphs, lower in females, and least in males. Double infections with B. afzelii and B. burgdorferi sensu stricto and with B. afzelii and the Ehrlichia-like organism were significantly overrepresented. Tick densities were highest in May, when densities of more than 200 ticks/100 m2 were observed, and declined during the summer months to densities as low as 20 ticks/100 m2. We conclude that estimates of the prevalence of tick-borne bacteria are sensitive to the choice of date and site for collection of ticks. This is the first study of tick-borne Borrelia and Ehrlichia in Norway and the lowest reported B. garinii prevalence in Northern Europe. The prevalence of the Ehrlichia-like organism is described for the first time in questing ticks.     

Adherence of Borrelia burgdorferi to the proteoglycan decorin.

Lyme disease is a tick-borne infection that can develop into a chronic, multisystemic disorder. The causative agent, Borrelia burgdorferi, is initially deposited by the tick into the host dermis, where it associates with collagen fibers, replicates, and eventually disseminates to other tissues. We have examined the adherence of the spirochete to different components of the collagen fiber and demonstrated that decorin, a proteoglycan which decorates collagen fibers, can support the attachment of B. burgdorferi. No significant direct attachment to isolated type I or III collagens could be detected. Attachment of the spirochetes to decorin was highly specific, and the process could be inhibited by soluble decorin but not by various unlabeled, unrelated components. B. burgdorferi also bound soluble 125I-labeled decorin in a time- and concentration-dependent manner. Spirochete binding of soluble 125I-labeled decorin required intact proteoglycan and could not be inhibited by either isolated core protein or glycosaminoglycan chain. B. burgdorferi expresses two decorin-binding proteins with apparent molecular masses of 19 and 20 kDa as revealed in a Western blot (immunoblot)-type assay. Our results indicate that decorin may mediate the adherence of B. burgdorferi to collagen fibers in skin and other tissues.     

Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in southwestern China

By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.     

Binding of human plasminogen to Borrelia burgdorferi.

We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in the binding of Glu- or Lys-plasminogen to B. burgdorferi. Binding of plasminogen was similar in low-passage (infectious) and high-passage (noninfectious) isolates of B. burgdorferi. Plasminogen bound to the surface of B. burgdorferi could be converted into plasmin by a human urokinase-type plasminogen activator. 125I-labeled plasminogen ligand blots of borrelial membrane proteins demonstrated two prominent binding proteins at approximately 70 and approximately 30 kDa. By Western blot (immunoblot), the 30-kDa protein was found to be outer surface protein A (Osp A) of B. burgdorferi. 125I-labeled plasminogen binding to both the 70-kDa protein and Osp A was inhibited by approximately 90% with a 1,000-fold excess of unlabeled plasminogen. By scanning densitometry, the 70-kDa band bound > 10 time more 125I-labeled plasminogen than did Osp A. An Osp A-deficient mutant of B. burgdorferi and wild-type B. burgdorferi bound equal amounts of 125I-labeled plasminogen. Ligand blots of membrane proteins from an Osp A-deficient mutant showed association of 125I-labeled plasminogen at only the 70-kDa protein. Two-dimensional gel electrophoresis showed that the 70-kDa protein had a pI of approximately 5.3, clearly separable from Osp A. The association of host plasmin(ogen) with borrelial surface proteins provides a mechanism by which B. burgdorferi can digest extracellular matrix and disseminate.     

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.     

Epidemiological aspects and molecular characterization of Borrelia burgdorferi s.l. from southern Germany with special respect to the new species Borrelia spielmanii sp. nov

In 475 Borrelia-infected Ixodes ricinus (2155 ticks investigated) from southern Germany the most common Borrelia burgdorferi sensu lato species was B. garinii (34.3%) followed by B. afzelii (25.1%), B. burgdorferi sensu stricto (22.0%), and B. valaisiana (12.7%). B. spielmanii sp. nov. was detected in 5.9% of the 475 infected ticks. Hints for a focal distribution were found for B. spielmanii sp. nov. and B. garinii OspA type 4. In 242 patient isolates, dominance (66.9%) of B. afzelii for skin could be confirmed, while frequency of B. garinii in cerebrospinal fluid (CSF) isolates (51.1%) was comparable to the frequency in nymphal ticks (51.6%). Four patient isolates from southern Germany and two from Slovenia, all isolated from erythema migrans, could be assigned to B. spielmanii sp. nov. Within this new species high sequence identities were found for rrs, fla, and ospA while rrf-rrl, ospC, and dbpA were less conserved: three new ospC and two new dbpA sequence types were found. This genetic heterogeneity reveals that B. spielmanii sp. nov. did not evolve just recently.     

Heat Shock Protein 70 of the Agent of Human Granulocytic Ehrlichiosis Binds to Borrelia burgdorferi Antibodies

We describe a patient with human granulocytic ehrlichiosis (HGE), a diagnosis confirmed by PCR and immunoblot analysis. Unexpectedly, immunoglobulin G (IgG) directed towards an 80-kDa ehrlichial antigen (without detectable IgM) was present in the patient’s serum in the first week of illness. Lyme disease immunoblots were reactive for IgG (but not IgM), a result indicative of prior exposure to the Lyme disease spirochete. Amino-terminal sequencing revealed that the 80-kDa ehrlichial antigen was an HSP-70 homolog similar to Borrelia burgdorferi HSP-70. We conclude that antibodies against B. burgdorferi HSP-70 may cross-react with the ehrlichial heat shock protein and that this possibility must be considered when serologic test results for HGE and Lyme disease are interpreted.     

Genetic Heterogeneity of Borrelia burgdorferi Sensu Lato in the Southern United States Based on Restriction Fragment Length Polymorphism and Sequence Analysis

Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.     

Colony formation and morphology in Borrelia burgdorferi.

Two strains of Borrelia burgdorferi, B31 and 297, formed colonies when plated onto Barbour-Stoenner-Kelly medium solidified with agarose (1.3%) and incubated in a candle jar at 34 degrees C. Colonies differing in morphology were observed in both strains after 2 to 3 weeks of incubation. Strain B31 colonies were either compact, round (mean diameter, 0.43 mm), and restricted to the surface of the agarose medium or diffuse (mean diameter, 1.80 mm) and penetrating into the solid medium. Strain 297 colonies (mean diameter, 1.43 mm) either showed a raised center surrounded by a diffuse ring of spirochetes or consisted of numerous small spirochetal aggregates. Both colony types expanded into the agarose medium. Scanning electron and light microscopy confirmed that the colonies were formed by spirochetes. Twisted tangles of intertwined spirochetes were visible on the surface, with numerous spherical bodies among them, especially in the central regions. At the periphery, the borreliae were more loosely packed, and individual coils were discernible.     

Rickettsiae and Borrelia burgdorferi in ixodid ticks.

Nymphs and adults of hard-bodied ticks were collected in Connecticut and tested by direct and indirect immunofluorescence staining methods for rickettsiae and Borrelia burgdorferi. Of the 609 Ixodes dammini ticks examined, 59 (9.7%) harbored rickettsialike microorganisms in hemocytes (blood cells). These bacteria reacted with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of with fluorescein-conjugated antiserum to Ehrlichia canis, the etiologic agent of canine ehrlichiosis. Prevalence of infection ranged from 6.8 to 12.7% for males and females, respectively. Although the specific identities of the hemocytic rickettsialike organisms are unknown, they share antigens with ehrlichiae. Electron microscopy revealed rickettsiae in ovarian tissues of I. dammini that also had infected hemocytes. Rickettsialike organisms were also observed in the hemocytes of 5 (6.9%) of 73 Dermacentor variabilis ticks. In analyses for B. burgdorferi, 146 (23.7%) of 617 I. dammini ticks harbored these spirochetes in midguts. Hemocytic rickettsialike microorganisms coexisted with B. burgdorferi in 36 (6.7%) of the 537 nymphs and adults of I. dammini examined. I. dammini, with its broad host range, has the potential to acquire multiple microorganisms.     

Borrelia burgdorferi shows specificity of binding to glycosphingolipids.

Live but not fixed or heat-killed Borrelia burgdorferi bound to galactocerebroside, lactosylceramide, and ceramide trihexoside. In addition, this organism bound to the disialoganglioside GD1a and the trisialoganglioside GT1b but not to gangliosides GM1, GD1b, GM2, and GM3 and not to asialo GM1. This adhesion pattern confirmed earlier findings of binding to galactocerebroside and places this organism within a prokaryotic group which binds to lactosylceramide. The binding to GD1a and GT1b, both of which carry terminal as well as multiple sialic acids, indicates that B. burgdorferi can show specificity of binding within a group of acidic gangliosides. Adhesion could not be inhibited by several concentrations of sugars and sialic acid, indicating more complex binding requirements than for terminal carbohydrates alone. Low-passage strains adhered to the four substrates in greater numbers than strains in culture for long periods of time. OspB mutants in general bound better or at least equally well to several of the glycosphingolipids, and preincubation of substrates with soluble recombinant and affinity-purified Osp did not inhibitor or weakly inhibited the binding of the organisms. These findings suggest that outer surface lipoproteins A and B are not directly involved in adhesion to glycosphingolipids.