Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes

We developed a rapid and reliable method for the identification Borrelia burgdorferi sensu lato species in ticks. We used the DNA sequence polymorphism of the spacer region between 5S and 23S rRNA genes, which has been shown to be able to discriminate between eight genomic groups of B. burgdorferi sensu lato (D. Postic, M. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). Spacer DNA was amplified by PCR and was then hybridized to five membrane-bound oligonucleotides. The oligonucleotides were specific for B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and group VS116. A probe which reacted with all genomic groups of B. burgdorferi sensu lato was also used. Ninety-six ticks collected in the field were destructed by bead beating, and the supernatant was used directly in a PCR. B. burgdorferi sensu lato DNA was detected in 6 of 57 adult ticks (11%) and 9 of 39 nymphs (23%). B. garinii was found in three nymphs and four adults, three nymphs carried B. afzelii, and one adult and one nymph carried group VS116. Double infections with B. afzelii and group VS116 were found in two nymphs and one adult. Thus, our method can simultaneously identify three genomic groups of B. burgdorferi sensu lato in ticks collected in the field. This technique provides new ways to study the association of genomic groups present in ticks and the risk of Lyme borreliosis.     

Effect of reduced bronchial circulation on lung fluid flux after smoke inhalation in sheep

Murine monoclonal antibodies directed against proteins of Borrelia burgdorferi B31 (low passage) were generated by the administration of antigen via the bite of borrelia-infected ticks. This strategy was employed as a mechanism to create antibodies against antigens presented by the natural route of tick transmission versus those presented by inoculation with cultured borreliae. One of the resultant antibodies reacted with a 17-kDa antigen from cultured B. burgdorferi, as seen by immunoblot analysis. This antibody was used to screen a B. burgdorferi genomic DNA lambda vector expression library, and an immunoreactive clone was isolated. DNA sequence analysis of this clone, containing a 2.7-kb insert, revealed several open reading frames. These open reading frames were found to be homologs of genes discovered as a multicopy gene family in the 297 strain of B. burgdorferi by Porcella et al. (S. F. Porcella, T. G. Popova, D. R. Akins, M. Li, J. D. Radolf, and M. V. Norgard, J. Bacteriol. 178:3293-3307, 1996). By selectively subcloning genes found in this insert into an Escherichia coli plasmid expression vector, the observation was made that the rev gene product was the protein reactive with the 17-kDa-specific monoclonal antibody. The rev gene product was found to be expressed in low-passage, but not in high-passage, B. burgdorferi B31. Correspondingly, the rev gene was not present in strain B31 genomic DNA from cultures that had been passaged >50 times. Serum samples from Lyme disease patients demonstrated an antibody response against the Rev protein. The generation of an anti-Rev response in Lyme disease patients, and in mice by tick bite inoculation, provides evidence that the Rev protein is expressed and immunogenic during the course of natural transmission and infection.     

Nursing intervention to increase hope and quality of life in newly diagnosed cancer patients

A DNA library of strain HB19 from Borrelia burgdorferi sensu stricto, an agent of Lyme borreliosis, was constructed in the cosmid pLA2917. Genes involved in initiation of DNA replication and resolution of recombination intermediates (Holliday junctions) were found on a 23-kbp region up to 0.7 kbp of the "left" extremity of the linear chromosome in representative species of B. burgdorferi sensu lato. The potential ruvB gene, located at 22 kbp from the left telomere, was identified by the similarity of its deduced amino acid sequence to RuvB (helicases) of other bacteria. B. burgdorferi ruvB is part of an operon which comprises the homologues of ruvA, queA and pfbB. Expression of the B. burgdorferi ruvB and ruvA genes renders a wild-type Escherichia coli sensitive to UV light and mitomycin, indicative of negative complementation. priA, which encodes the potential recognition factor for the primosome assembly site, was found at 15 kbp from the left telomere. RuvB and PriA sequences have motifs characteristic of helicases: a DExH box and an ATP binding site.     

Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice

A Borrelia burgdorferi N40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, of B. burgdorferi 297 and B31. N40-dbpA DNA hybridized with B. burgdorferi N40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting from B. burgdorferi sensu stricto N40 and B31, and from B. burgdorferi sensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than against B. burgdorferi N40 recombinant P39, OspC, or OspA. Sera from mice infected with B. burgdorferi sensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected with B. burgdorferi N40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with cultured B. burgdorferi N40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout the B. burgdorferi sensu lato family, but they are likely to be heterogeneous.     

Optimization of polymerase chain reaction for the detection of Borrelia burgdorferi in biologic specimens

This study describes the use of a newly constructed set of primers that amplifies an 85-base pair (bp) segment of Borrelia burgdorferi chromosomal DNA. This 85-bp product is not produced when other Borrelia species, Leptospira, or other bacteria are subjected to polymerase chain reaction (PCR). We also describe a rapid method of optimizing the amplification of B. burgdorferi DNA from canine ethylenediaminetetraacetic acid-treated blood and urine samples that circumvents some of the problems encountered due to low number of spirochetes in clinical specimens and that removes inhibiting substances, which improves the PCR diagnosis of canine Lyme borreliosis.     

The Borrelia burgdorferi 37-kilodalton immunoblot band (P37) used in serodiagnosis of early lyme disease is the flaA gene product

The 37-kDa protein (P37) of Borrelia burgdorferi is an antigen that elicits an early immunoglobulin M (IgM) antibody response in Lyme disease patients. The P37 gene was cloned from a B. burgdorferi genomic library by screening with antibody from a Lyme disease patient who had developed a prominent humoral response to the P37 antigen. DNA sequence analysis of this clone revealed the identity of P37 to be FlaA, an outer sheath protein of the periplasmic flagella. Recombinant P37 expression was accomplished in Escherichia coli by using a gene construct with the leader peptide deleted and fused to a 38-kDa E. coli protein. The recombinant antigen was reactive in IgM immunoblots using serum samples from patients clinically diagnosed with early Lyme disease that had been scored positive for B. burgdorferi anti-P37 reactivity. Lyme disease patient samples serologically negative for the B. burgdorferi P37 protein did not react with the recombinant. Recombinant P37 may be a useful component of a set of defined antigens for the serodiagnosis of early Lyme disease. This protein can be utilized as a marker in diagnostic immunoblots, aiding in the standardization of the present generation of IgM serologic tests.     

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.     

Molecular analysis of sequence heterogeneity among genes encoding decorin binding proteins A and B of Borrelia burgdorferi sensu lato

Immunization of mice with Borrelia burgdorferi decorin binding protein A (DbpA), one of two gene products of the dbpBA locus, has been shown recently to confer protection against challenge. Hyperimmune DbpA antiserum killed a large number of B. burgdorferi sensu lato isolates of diverse phylogeny and origin, suggesting conservation of the protective epitope(s). In order to evaluate the heterogeneity of DbpA and DbpB and to facilitate defining the conserved epitope(s) of these antigens, the sequences of the dbpA genes from 29 B. burgdorferi sensu lato isolates and of the dbpB genes from 15 B. burgdorferi sensu lato isolates were determined. The predicted DbpA sequences were fairly heterogeneous among the isolates (58.3 to 100% similarity), but DbpA sequences with the highest similarity tended to group into species previously defined by well-characterized chromosomal markers. In contrast, the predicted DbpB sequences were highly conserved (96.3 to 100% similarity). Substantial diversity in DbpA sequence was seen among isolates previously shown to be killed by antiserum against a single DbpA, suggesting that one or more conserved protective epitopes are composed of noncontiguous amino acids. The observation of individual dbpA alleles with sequence elements characteristic of more than one B. burgdorferi sensu lato species was consistent with a role for genetic recombination in the generation of dbpA diversity.     

Binding of human plasminogen and urokinase-type plasminogen activator to the Lyme disease spirochete, Borrelia burgdorferi

Many bacteria that spread in the skin produce enzymes that digest extracellular matrix components. Borrelia burgdorferi spreads from a skin inoculation site to form the characteristic erythema migrans skin lesion. It was determined that B. burgdorferi does not produce collagenase, elastase, hyaluronidase, or other enzymes that digest extracellular matrix components. However, B. burgdorferi bound human plasmin, plasminogen (Pgn), and urokinase-type plasminogen activator (uPA). When spirochetes were sequentially incubated with Pgn and uPA, bioactive plasmin was generated on the surface of B. burgdorferi. B. burgdorferi did not produce an endogenous Pgn activator. Fluorochrome-conjugated uPA and Pgn colocalized to the terminus of the spirochete. In a mouse model, uPA-treated B. burgdorferi were more infectious than control spirochetes. Binding of host uPA and Pgn to form a bioactive extracellular matrix protease on B. burgdorferi represents a mechanism that could facilitate dissemination and localization of spirochetes to sites of vascular injury.     

Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.     

Intra-arterial fluorescein angiography using ophthalmic artery catheter in an experimental model

BACKGROUND: Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists despite antibiotic therapy. METHODS: Using the polymerase chain reaction (PCR), we attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period. The samples were tested in two separate laboratories with four sets of primers and probes, three of which target plasmid DNA that encodes outer-surface protein A (OspA). RESULTS: B. burgdorferi DNA was detected in 75 of 88 patients with Lyme arthritis (85 percent) and in none of 64 control patients. Each of the three OspA primer-probe sets was sensitive, and the results were moderately concordant in the two laboratories (kappa = 0.54 to 0.73). Of 73 patients with Lyme arthritis that was untreated or treated with only short courses of oral antibiotics, 70 (96 percent) had positive PCR results. In contrast, of 19 patients who received either parenteral antibiotics or long courses of oral antibiotics (> or = 1 month), only 7 (37 percent) had positive tests (P < 0.001). None of these seven patients had received more than two months of oral antibiotic treatment or more than three weeks of intravenous antibiotic treatment. Of 10 patients with chronic arthritis (continuous joint inflammation for one year or more) despite multiple courses of antibiotics, 7 had consistently negative tests in samples obtained three months to two years after treatment. CONCLUSIONS: PCR testing can detect B. burgdorferi DNA in synovial fluid. This test may be able to show whether Lyme arthritis that persists after antibiotic treatment is due to persistence of the spirochete.     

Borrelia burgdorferi erpT expression in the arthropod vector and murine host

The expression of a Borrelia burgdorferi gene, erpT, was investigated throughout the spirochaete life cycle in the arthropod vector and the murine host. Three phage clones from a B. burgdorferi DNA expression library synthesized a 30 kDa antigen that was recognized by antibodies in the sera of B. burgdorferi-infected mice but not mice hyperimmunized with B. burgdorferi lysates. Differential antibody binding suggested that this protein was preferentially expressed in vivo. This antigen was designated ErpT, based upon 99.6% homology with the BBF01 sequence in the B. burgdorferi genome. ErpT was not detected on spirochaetes cultured in BSK II medium by indirect immunofluorescence or in B. burgdorferi lysates by immunoblotting, implying that ErpT is not readily produced in vitro. erpT mRNA was not discernible by Northern blot but was identified by RNA polymerase chain reaction in vitro, indicating that erpT is expressed at low levels by cultured spirochaetes. erpT expression was then investigated in the vector and mice because B. burgdorferi do not normally reside in culture medium. RNA polymerase chain reaction and immunofluorescence studies demonstrated that erpT was expressed by a small minority of B. burgdorferi (11/500, 2.2%) within unfed ticks and then repressed during engorgement. erpT mRNA or ErpT antibodies were first detected in B. burgdorferi-infected mice at 4 weeks, suggesting that erpT was not expressed in the early stages of murine infection. Then, during persistent infection, RNA polymerase chain reaction showed that erpT was expressed by B. burgdorferi within the joints, heart and spleen, but not by spirochaetes in the skin. Immunization of mice with ErpT was antigenic but was not protective. These studies demonstrate that B. burgdorferi erpT is differentially expressed throughout the B. burgdorferi life cycle, in both the vector and the mammalian host, and is primarily expressed in extracutaneous sites during murine infection.     

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.     

Duration of Borrelia burgdorferi infectivity in white-footed mice for the tick vector Ixodes scapularis under laboratory and field conditions in Ontario

The duration of Borrelia burgdorferi infectivity in white-footed mice (Peromyscus leucopus) experimentally inoculated or infested with infected Ixodes scapularis nymphs was evaluated. Infectivity was assessed by infesting these mice with unfed I. scapularis larvae at 7, 21, 35 and 49 days post-inoculation (DPI) or post-infestation (PI). At 7 DPI, B. burgdorferi was transmitted from 18 of 24 syringe-inoculated mice and all three tick-infected mice to I. scapularis larvae which fed upon them. However, at 21, 35 and 49 DPI, significantly fewer mice were infective. Borrelia burgdorferi was isolated from tissues of 14 of 22 syringe-inoculated mice about 56 DPI, and from all three tick-infected mice. However, the level of agreement between xenodiagnosis and bacterial culture was no greater than would be expected by chance alone. We also determined if B. burgdorferi infectivity of mice varied in relation to periods of tick feeding in the field. White-footed mice were trapped during April, July and August 1993 from two habitats on Long Point peninsula (Ontario, Canada), where B. burgdorferi is endemic. Mice from each habitat were infested with laboratory-reared I. scapularis larvae. Ticks from each mouse were subsequently examined by immunofluorescent assay for B. burgdorferi infection and mice were cultured for B. burgdorferi. None of 3577 I. scapularis larvae fed on 62 mice captured within the cottonwood dune habitat were infected with B. burgdorferi, although it was isolated from six of these mice. Within the maple forest habitat, 0/24, 8/21 (38%) and 1/21 (5%) mice transmitted B. burgdorferi to I. scapularis larvae during April, July and August, respectively. Most mice from the maple forest with B. burgdorferi-positive tissues (14/21) were collected during July, although the level of agreement between xenodiagnosis and tissue culture was poor. Because B. burgdorferi infectivity in mice appears to be of short duration, overwintered I. scapularis larvae and nymphs may have to feed upon infected hosts at the same time of year in order for a cycle of B. burgdorferi infection to be maintained on Long Point. Infected I. scapularis nymphs, rather than persistently infected vertebrate hosts, likely serve as the overwintering "reservoir" for B. burgdorferi on Long Point.     

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.     

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.     

Borrelia burgdorferi erp proteins are immunogenic in mammals infected by tick bite, and their synthesis is inducible in cultured bacteria

Borrelia burgdorferi, the causative agent of Lyme disease, can contain multiple genes encoding different members of the Erp lipoprotein family. Some arthropod-borne bacteria increase the synthesis of proteins required for transmission or mammalian infection when cultures are shifted from cool, ambient air temperature to a warmer, blood temperature. We found that all of the erp genes known to be encoded by infectious isolate B31 were differentially expressed in culture after a change in temperature, with greater amounts of message being produced by bacteria shifted from 23 to 35 degrees C than in those maintained at 23 degrees C. Mice infected with B31 by tick bite produced antibodies that recognized each of the Erp proteins within 4 weeks of infection, suggesting that the Erp proteins are produced by the bacteria during the early stages of mammalian infection and may play roles in transmission from ticks to mammals. Several of the B31 Erp proteins were also recognized by antibodies from patients with Lyme disease and may prove to be useful antigens for diagnostic testing or as components of a protective vaccine.     

Intrauterine transmission of Borrelia burgdorferi in dogs

To determine whether intrauterine transmission of Borrelia burgdorferi could exist in dogs, 10 female Beagles were inoculated intradermally with approximately 1,000 B burgdorferi on day 1 of proestrus; inoculation was repeated every 2 weeks during the gestation period. Ten female control Beagles were similarly inoculated with phosphate-buffered saline solution. Prior to the start of the study, all females and 3 males used for breeding were seronegative for B burgdorferi on the basis of results of the indirect fluorescent antibody test and immunoblot (western analysis. Similarly, results of culture of blood for B burgdorferi were negative. All 20 of the females were bred naturally. Blood samples were collected weekly for serologic testing and culture. Blood samples were obtained from live pups on day 1 of life, then weekly until pups were 6 weeks old when they were euthanatized. Tissues were obtained for culture and testing by use of polymerase chain reaction (PCR). Of 10 spirochete-inoculated (SI) females, 8 became infected with B burgdorferi as evidenced by spirochete culture results and/or PCR-detected B burgdorferi DNA in the tissues of females or their pups. Of the 10 SI females, 8 delivered litters (3 to 7 pups) that had at least 1 neonatal or 6-week-old pup with B burgdorferi DNA-positive tissues (by PCR), and spirochetes were cultured from tissues from pups of 2 litters.(ABSTRACT TRUNCATED AT 250 WORDS)