Complement inhibitor factor H binding to Lyme disease spirochetes is mediated by inducible expression of multiple plasmid-encoded outer surface protein E paralogs

Borrelia burgdorferi spirochetes can circumvent the vertebrate host's immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.     

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

Lysine-dependent multipoint binding of the Borrelia burgdorferi virulence factor outer surface protein E to the C terminus of factor H

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18-20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala(151)-Lys(166)) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.     

Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.     

Demonstration of the involvement of outer surface protein E coiled coil structural domains and higher order structural elements in the binding of infection-induced antibody and the complement-regulatory protein, factor H

Factor H (fH) is an important regulator of the alternative complement cascade. Several human pathogens have been shown to bind fH to their surface, a process that facilitates immune evasion or cell to cell interaction. Among the pathogens that bind fH are some Borrelia species associated with Lyme disease and relapsing fever. The fH-binding proteins of the Lyme spirochetes form two classes (I and II). In Borrelia burgdorferi B31MI, class I includes the outer surface protein E (OspE) paralogs, L39, N38, and P38, whereas the class II group includes A68 and additional proteins that have not yet been identified. To identify the OspE determinants involved in fH and OspE-targeting infection-induced Ab (iAb) binding, deletion, random, and site-directed mutagenesis of L39 were performed. Mutations in several different regions of L39 abolished fH and or iAb binding, indicating that separable domains and residues of OspE are required for ligand binding. Some of the mutants that lost the ability to bind fH, iAb, or both had only a single amino acid change. Site-directed mutagenesis of three putative coiled coil motifs of OspE revealed that these higher order structures are required for fH binding but not for iAb binding. The data presented within demonstrate that the binding of fH and iAb to the OspE protein is mediated by higher order structures and protein conformation. These studies advance our understanding of fH binding as a virulence mechanism and facilitate ongoing efforts to use fH-binding proteins in the development of microbial vaccines.     

Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy

Abstract This survey evaluates the antibody band patterns of sera taken from clinically defined cases of Lyme borreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii , by means of Western blot. The sera were taken from patients resident in a limited area of Friuli Venezia Giulia (FVG) region. The data indicated that, besides a different feature of the band reactivity which correlated to the different stages of Lyme borreliosis, there was a preferential reactivity to the species Borrelia afzelii and Borrelia garinii . An immunodominant band at 51 kDa, corresponding to a protein visible in the electrophoretic profile of strain BL3 ( B. afzelii ), behaved like a marker of an early infection, because it was present exclusively in the sera of patient with ECM. The overall findings would indicate that B. afzelii and B. garinii are the prevalent genospecies in the FVG area, even if strains belonging to B. sensu stricto have been also isolated in this area. Consequently strains representative of these two species must be used as antigens in Western blot.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

Functional characterization of BbCRASP-2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL-1

Borrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H-like protein-1 (FHL-1) allows spirochetes to resist complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP-2. BbCRASP-2 is distinct from the four previously identified factor H/FHL-1-binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP-2, cspZ, is located on the linear plasmid lp28-3. BbCRASP-2 is highly divergent from the factor H/FHL-1-binding protein BbCRASP-1 and from members of the factor H-binding Erp (OspE/F-related) protein family. Peptide mapping analysis revealed that the factor H/FHL-1 binding site is discontinuous and it was found that C-terminal truncations abrogate factor H and FHL-1 binding. The predominant BbCRASP-2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL-1 bound to BbCRASP-2 maintain cofactor activity for factor I-mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP-2 in serum-sensitive B. burgdorferi mutant B313 increased resistance to complement-mediated lysis. The characterization of BbCRASP-2 now provides a complete picture of the three diverse complement regulator-binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.     

Complement regulator-acquiring surface protein 1 imparts resistance to human serum in Borrelia burgdorferi

Factor H and factor H-like protein 1 (FH/FHL-1) are soluble serum proteins that negatively regulate the alternative pathway of complement. It is now well recognized that many pathogenic bacteria, including Borrelia burgdorferi, bind FH/FHL-1 on their cell surface to evade complement-mediated destruction during infection. Recently, it was suggested that B. burgdorferi open reading frame bbA68, known as complement regulator-acquiring surface protein 1 (CRASP-1), encodes the major FH/FHL-1-binding protein of B. burgdorferi. However, because several other proteins have been identified on the surface of B. burgdorferi that also can bind FH/FHL-1, it is presently unclear what role CRASP-1 plays in serum resistance. To examine the contribution of CRASP-1 in serum resistance, we generated a B. burgdorferi mutant that does not express CRASP-1. The B. burgdorferi CRASP-1 mutant, designated B31cF-CRASP-1, was found to be as susceptible to human serum as a wild-type strain of Borrelia garinii 50 known to be sensitive to human serum. To further examine the role of CRASP-1 in serum resistance, we also created a shuttle vector that expresses CRASP-1 from the native B. burgdorferi gene, which was designated pKFSS-1::CRASP-1. When the pKFSS-1::CRASP-1 construct was transformed into the B. burgdorferi B31cF-CRASP-1 mutant, wild-type levels of serum resistance were restored. Additionally, when pKFSS-1::CRASP-1 was transformed into the serum-sensitive B. garinii 50 isolate, human serum resistance was imparted on this strain to a level indistinguishable from wild-type B. burgdorferi. The combined data led us to conclude that CRASP-1 expression is necessary for B. burgdorferi to resist killing by human serum.     

Study on the infection of taiga ticks with Borrelia in the territory of Novosibirsk Scientific Center SB PAS

In our study, Borrelia were revealed in the taiga ticks Ixodes persulcatus collected on vegetation by flagging, as well as in the ticks removed from the people who asked for help in the vaccination center located in the Novosibirsk Scientific Center of the Siberian Branch of Russian Academy of Science (NS SB RAS). By the isolation of Borrelia on BSK-H medum, the occurrence of B. garinii, B. afzelii, and B. miyamotoi was established in the territory of NSC. B. miyamotoi isolates were unstable and lost their ability to growth in later passages. DNA of the same three species of Borrelia was detected by PCR in the samples of ticks, both collected on vegetation by flagging and removed from humans. DNA of B. garinii was recorded most often; DNA of B. afzelii was less frequent; and the least number of positive samples was shown for B. miyamotoi. In the ticks collected on vegetation by flagging, DNA of B. garinii was found in 38.6%, B. afzelii in 9.9%, and B. miyamoboi in 3.9% of samples. In the ticks removed from people, number of positive samples was lesser; so, DNA of B. garinii was detected in 24.2%, B. afzelii in 6.9%, and B. miyamotoi in 5.6% of samples. Mixed infection with two Borrelia species was recorded, and DNA of B. mivamnotoi more often detected simultaneously with DNA of B. garinii.     

Evaluation of a genotyping method based on the ospA gene to detect Borrelia burgdorferi sensu lato in multiple samples of lyme borreliosis patients

In this study we have developed a new Restriction-Fragment-Length-Polymorphism (RFLP) genotyping method for rapid detection and identification of Borrelia genospecies present as unique species or as co-infection in multiple specimens obtained simultaneously from 29 individual patients affected by early or late Lyme borreliosis (LB). The target of the RFLP-genotyping was the heterogeneous plasmid located ospA gene, thus we developed a method able to detect and differentiate between six clinically relevant Borrelia genospecies circulating in Europe, B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, B. bissettii and B. spielmanii. In this study Borrelia DNA could be detected by PCR in at least one specimen of each patient, except in one case of neuroborreliosis (NB); blood samples gave the highest sensitivity in all patient groups. The genotyping indicated that B. afzelii was present in 8 patients with skin involvement, B. garinii in 2 cases of NB and 4 cases with skin involvement, B. burgdorferi sensu stricto was detected in one patient with skin involvement and another with Lyme arthritis. Different Borrelia species in distinct specimens were identified in one patient with EM. The RFLP analysis of 11 patients revealed mixed patterns, which suggested pluri-infection with different Borrelia species.     

Antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in white-footed mice

Serum samples were obtained from white-footed mice (Peromyscus leucopus) in tick-infested areas of Connecticut during the period 2001 through 2003 and analyzed for antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Emphasis was placed on the evaluations of highly specific recombinant VlsE or protein (p) 44 antigens of B. burgdorferi and A. phagocytophilum, respectively, in a newly developed enzyme-linked immunosorbent assay (ELISA) as well as testing sera with whole-cell antigens by conventional ELISA or indirect fluorescent antibody staining methods. Of the 414 mouse sera analyzed, 310 (75%) had antibodies to whole-cell B. burgdorferi, whereas 157 (38%) were positive to the VlsE antigen. The latter nearly equaled the overall antibody prevalence rate (37%) computed when sera were tested separately with the p44 antigen. Mice were exposed to these pathogens and B. microti (antibody prevalence = 25%) in extreme northern Connecticut as well as the southern coastal areas of the state, thus indicating further geographic expansion of these infections. Fifty-three (13%) sera from widely separated sites had antibodies to all three pathogens. With expression and immunological recognition of VlsE and p44 antigens in P. leucopus, separate incorporation of these fusion proteins in an ELISA was very helpful in confirming past or current infections and in identifying specific foci for B. burgdorferi and A. phagocytophilum.     

Isolation of moderately infectious Borrelia burgdorferi sensu stricto from attenuated cultures by using complement-mediated,antibody-dependent lysis selection technique in a mammalian tissue co-culture system

We investigated the association between complement resistance and phenotypes of pathogenicity of Borrelia burgdorferi sensu lato isolates cultivated in a LEW/N rat tibiotarsal joint-derived tissue feeder layer-supported co-culture system. Guinea pig complement and immune serum raised in LHS/Ss hamsters caused complete lysis of B. burgdorferi sensu stricto isolate 297, B. afzelii and B. garinii in Barbour-Stoenner-Kelly's medium; however, tissue co-cultured B. burgdorferi sensu stricto contained complement escape variants. The arthritogenicity and infectivity of these variants were tested in 3-week-old Syrian hamsters and in a vaccinated hamster model in which formalin-killed B. burgdorferi sensu stricto C-1-11 vaccinated animals develop severe arthritis after challenge with live, pathogenic, low-passage 297 isolate. Non-animal-passaged complement escape variants were infectious in both animal models as demonstrated by re-isolation from the infected animals and competitive PCR. IP injection of animal-passaged complement escape variants caused development of severe arthritis in vaccinated animals 5 weeks post-injection; animal passage of complement escape variants was necessary for isolation of arthritogenic spirochetes from high-passaged, non-arthritogenic, attenuated borrelia cultures. Complement escape variants synthesized outer surface protein E as demonstrated by SDS-PAGE and western blotting analyses. The complement-mediated selection technique in tissue co-culture provides a novel approach to the studies of Lyme disease, enables us to isolate pathogenically distinct borrelia populations from attenuated cultures and prepare a moderately infectious, non-pathogenic live vaccine against this illness.     

Lyme disease in transgenic mice expressing the Borrelia burgdorferi flagellin epitope implicated in human neuroborreliosis

Because of an association of human neuroborreliosis with the development of an antibody response against an antigen in neural tissue that cross-reacts with an epitope on the flagellin protein of Borrelia burgdorferi, C3H transgenic mice were created that expressed the flagellin epitope (amino acids 213–224) as a fusion protein with myelin basic protein. The transgenic mice expressed the flagellin epitope selectively in myelinated regions of the nervous system. Both transgenic and non-transgenic mice developed an antibody response to the flagellin epitope during B. burgdorferi infection and both developed arthritis and carditis. However, no lesions were found in the central nervous system of either type of mouse for up to 8 weeks after infection. The data indicate that expression of the flagellin 213–24 epitope in mice does not result in neurologic disease, suggesting that B. burgdorferi flagellin antibodies may not be directly implicated in neuroborreliosis.     

Evidence of Dbps (decorin binding proteins) among European strains of Borrelia burgdorferi sensu lato and in the immune response of LB patient sera

Decorin binding proteins DbpA and DbpB act as Borrelia burgdorferi (B. burgdorferi) adhesins to decorin, and are able to elicit a persistent antibody response in the mouse; accordingly DbpA protein would seem to be promising in immunoprofilaxis of Lyme borreliosis (LB). This study examines the distribution of Dbp epitopes in European strains of B. burgdorferi, of different genospecies and the presence of antibodies to Dbps in human sera from patients suffering from early and late LB, as revealed by immunoblotting. Different levels of expression of Dbp epitopes were found both among and within genospecies; data from human sera indicate that Dbps are expressed during infection though not as strongly as in the mouse infection.     

Dual binding specificity of a Borrelia hermsii-associated complement regulator-acquiring surface protein for factor H and plasminogen discloses a putative virulence factor of relapsing fever spirochetes

Tick-borne relapsing fever in North America is primarily caused by the spirochete Borrelia hermsii. The pathogen employs multiple strategies, including the acquisition of complement regulators and antigenic variation, to escape innate and humoral immunity. In this study we identified in B. hermsii a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Furthermore, ectopic expression of BhCRASP- 1 converted the serum-sensitive Borrelia burgdorferi B313 strain into an intermediate complement-resistant strain. Finally, we report for the first time that BhCRASP-1 binds plasminogen/plasmin in addition to FH via, however, distinct nonoverlapping domains. The fact that surface-bound plasmin retains its proteolytic activity suggest that the dual binding specificity of BhCRASP-1 for FH and plasminogen/plasmin contributes to both the dissemination/invasion of B. hermsii and its resistance to innate immunity.     

Borrelia burgdorferi sensu lato, the agent of lyme borreliosis: life in the wilds

In Europe, Borrelia burgdorferi sensu lato (sl) the agent of Lyme borreliosis circulates in endemic areas between Ixodes ricinus ticks and a large number of vertebrate hosts upon which ticks feed. Currently, at least 12 different Borrelia species belonging to the complex B. burgdorferi sl have been identified among which seven have been detected in I. ricinus: B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. valaisiana, B. spielmanii and B. bissettii. A few dozens of vertebrate hosts have been identified as reservoirs for these Borrelia species. Specific associations were rather early observed between hosts, ticks and borrelia species, like for example between rodents and B. afzelii and B. burgdorferi ss, and between birds and B. garinii and B. valaisiana. The complement present in the blood of the hosts is the active component in the Borrelia host specificity. Recent studies confirmed trends toward specific association between Borrelia species and particular host, but also suggested that loose associations may be more frequent in transmission cycles in nature than previously thought.     

Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.

Borrelia burgdorferi sensu lato organisms, comprising B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are tick-borne pathogens causing Lyme borreliosis in humans. To identify putative virulence determinants, a B. afzelii DNA library was screened for Congo red dye binding, a property associated with virulence in pathogenic bacteria. One clone was found to carry a 663-nucleotide-long open reading frame encoding a Congo red dye-binding protein with a calculated molecular mass of 25,660 Da. The amino acid sequence deduced from its nucleotide sequence was found to include a consensus bacterial lipidation site present at residues 15 to 18 (Leu-Ser-Gly-Cys). The lipoprotein nature was demonstrated by incorporation of radioactive palmitate; hence, this protein has been termed NlpH, for new lipoprotein H. NlpH is located on the surface of B. afzelii, and the nlpH gene is found on a circular plasmid. The nlpH gene is also found in B. burgdorferi sensu stricto and B. garinii. Immediately upstream of nlpH is located a smaller reading frame encoding a polypeptide containing the casein kinase II phosphorylation recognition sequence, (Ser/Thr)-X-Y-(Glu/Asp), repeated 10 times.     

Prevalence of Lyme disease Borrelia spp. in ticks from migratory birds on the Japanese mainland

Borrelia sp. prevalence in ticks on migratory birds was surveyed in central Japan. In autumn, a total of 1,733 birds representing 40 species were examined for ticks. A total of 361 ticks were obtained from 173 birds of 15 species, and these ticks were immature Haemaphysalis flava (94.4%), Haemaphysalis longicornis, Ixodes columnae, Ixodes persulcatus, Ixodes turdus, and an unidentified Ixodes species. Of these, 27 juveniles of H. flava on Turdus pallidus, Turdus cardis, or Emberiza spodocephala, 2 juveniles of I. persulcatus on T. pallidus, and 1 female H. flava molted from a T. pallidus-derived nymph were positive for the presence of Borrelia by Barbour-Stoenner-Kelly culture passages. In spring, a total of 16 ticks obtained from 102 birds of 21 species were negative for the spirochete. Isolates from 15 ticks were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis; all isolates were identified as Borrelia garinii with pattern B/B' based on the previous patterning. According to the intergenic spacer sequences, 2 of 15 isolates, strains Fi14f and Fi24f, were highly similar to B. garinii strains 935T of Korea and ChY13p of Inner Mongolia, China, respectively. These findings indicate that Lyme disease-causing B. garinii may have been introduced to Japan by migratory birds from northeastern China via Korea. Additionally, a case of transstadial transmission of B. garinii from nymph to adult H. flava suggests that the infected H. flava may transmit Borrelia to large animals.