Differential Transmission of the Genospecies of Borrelia burgdorferi Sensu Lato by Game Birds and Small Rodents in England

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all tick stages. Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana. Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.     

Lyme disease and current aspects of immunization

Lyme disease is a tick-borne multisystem disease that affects primarily the skin, nervous system, heart and joints. At least three species of Borrelia burgdorferi sensu lato, namely Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, can cause the disease. This review will focus mainly on the pathophysiology of Lyme arthritis, the long-term outcome of Lyme disease, and the recently licensed vaccine against Lyme disease.     

Lyme Disease Borrelia Species in Northeastern China Resemble Those Isolated from Far Eastern Russia and Japan

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

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Epitope mapping of the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi using a panel of monoclonal antibodies and lanthanide competition fluoroimmunoassay

A panel of fourteen different monoclonal antibodies was used for detection and analysis of antigenic determinants located on the outer surface protein A (OspA) of the spirochete Borrelia burgdorferi, which is a causative agent of tick-borne borreliosis (Lyme disease). Two main and several minor partially overlapping antigenic determinants have been found on the surface of the OspA protein of Borrelia burgdorferi sensu stricto (strain 297) by lanthanide competition fluoroimmunoassay. One of the main antigenic determinants is located in the N- and the other in the C-half of the OspA molecule. The involvement of the OspA protein in intact Borrelia burgdorferi sensu stricto (four bacterial strains have been analyzed: 297, B31, FR90-594, and CA90-742) is associated with retention of the above-mentioned two major antigenic determinants, but unlike the case of the isolated OspA they are partially overlapping with each other and with other antigenic determinants. The protein of the spirochete Borrelia afzelii (two bacterial strains have been analyzed: Ip-21 and Pko) contains only one antigenic determinant, which is the same as the main determinant of the OspA protein of Borrelia burgdorferi sensu stricto located in the N-half of the OspA molecule.     

Differential Role of Passerine Birds in Distribution of Borrelia Spirochetes, Based on Data from Ticks Collected from Birds during the Postbreeding Migration Period in Central Europe

Borrelia spirochetes in bird-feeding ticks were studied in the Czech Republic. During the postbreeding period (July to September 2005), 1,080 passerine birds infested by 2,240 Ixodes ricinus subadult ticks were examined. Borrelia garinii was detected in 22.2% of the ticks, Borrelia valaisiana was detected in 12.8% of the ticks, Borrelia afzelii was detected in 1.6% of the ticks, and Borrelia burgdorferi sensu stricto was detected in 0.3% of the ticks. After analysis of infections in which the blood meal volume and the stage of the ticks were considered, we concluded that Eurasian blackbirds (Turdus merula), song thrushes (Turdus philomelos), and great tits (Parus major) are capable of transmitting B. garinii; that juvenile blackbirds and song thrushes are prominent reservoirs for B. garinii spirochetes; that some other passerine birds investigated play minor roles in transmitting B. garinii; and that the presence B. afzelii in ticks results from infection in a former stage. Thus, while B. garinii transmission is associated with only a few passerine bird species, these birds have the potential to distribute millions of Lyme disease spirochetes between urban areas.     

Decorin Binding Proteins of Borrelia burgdorferi Promote Arthritis Development and Joint Specific Post-Treatment DNA Persistence in Mice

Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.     

Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.     

Identification of a New Borrelia Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses

Background

Lyme disease caused by Borrelia burgdorferi sensu lato complex is an important endemic zoonosis whose distribution is closely related to the main ixodid tick vectors. In China, isolated cases of Lyme disease infection of humans have been reported in 29 provinces. Ticks, especially ixodid ticks are abundant and a wide arrange of Borrelia natural reservoirs are present. In this study, we developed a reverse line blot (RLB) to identify Borrelia spp. in ticks collected from sheep and cattle in 7 Provinces covering the main extensive livestock regions in China.

Results

Four species-specific RLB oligonucleotide probes were deduced from the spacer region between the 5S-23S rRNA gene, along with an oligonucleotide probe which was common to all. The species specific probes were shown to discriminate between four genomic groups of B. burgdorferi sensu lato i.e. B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana, and to bind only to their respective target sequences, with no cross reaction to non target DNA. Furthermore, the RLB could detect between 0.1 pg and 1 pg of Borrelia DNA. A total of 723 tick samples (Haemaphysalis, Boophilus, Rhipicephalus and Dermacentor) from sheep and cattle were examined with RLB, and a subset of 667 corresponding samples were examined with PCR as a comparison. The overall infection rate detected with RLB was higher than that of the PCR test. The infection rate of B. burgdoreri sensu stricto was 40% in south areas; while the B. garinii infection rate was 40% in north areas. The highest detection rates of B. afzelii and B. valaisiana were 28% and 22%, respectively. Mixed infections were also found in 7% of the ticks analyzed, mainly in the North. The proportion of B. garinii genotype in ticks was overall highest at 34% in the whole investigation area.

Conclusion

In this study, the RLB assay was used to detect B. burgdorferi sensu lato in ticks collected from sheep and cattle in China. The results showed that B. burdorferi senso stricto and B. afzelii were mainly distributed in the South; while B. garinii and B. valaisiana were dominant in the North. Borrelia spirochaetes were detected in Rhipicephalus spp for the first time. It is suggested that the Rhipicephalus spps might play a role in transmitting Borrelia spirochaetes.     

Consensus Sequence on the Genes Encoding the Major Outer Surface Proteins (OspA and OspB) of Borrelia garinii Isolate

Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii. These B. garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB). In this study, we cloned and sequenced the genes encoding OspA and OspB from B. garinii strain FujiP2 (ribotype IV strain) isolated from I. persulcatus in Shizuoka, Japan. A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B. burgdorferi sensu lato. The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively. The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence. The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B. burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.     

Identification of Borrelia burgdorferi Isolated in Korea Using Outer Surface Protein A (OspA) Serotyping System

Two characteristic strains (935T, 934U) of B. burgdorferi isolated from Ixodes persulcatus and a wild rodent (Apodemus agrarius) in Korea were selected and analyzed by an immunoblot method using the monoclonal antibodies directed to different epitopes of outer surface protein A (OspA). The reactive pattern of strain 934U with these monoclonal antibodies was identical to that of strains belonging to B. afzelii and that of strain 935T was different from other isolates. Monoclonal antibody (5TEE3) which is specific to strain 935T did not react with any other Western and Japanese isolates. So, it was suggested that there exist at least two groups of B. burgdorferi in Korea. One could be classified as B. afzelii and the other is a divergent group from three known species of B. burgdorferi sensu stricto, B. garinii and B. afzelii.     

Perpetuation of the Lyme Disease Spirochete Borrelia lusitaniae by Lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Comparison of Antibody Titers against Borrelial Strains Isolated in Japan by the Microcapsule Agglutination Test for Serological Studies of Early Lyme Disease

Results of a passive microcapsule agglutination test using 2 Japanese strains, Borrelia garinii and Borrelia afzelii, were comparable to that of the passive microcapsule agglutination test using a Swiss strain Borrelia burgdorferi IRS in detecting antibodies of an early stage of Lyme disease patients in Japan. MCAT with microcapsule sensitized strain IRS seems likely to become one of the important tools for the early diagnosis of Lyme disease in the world. The highest titer of MCAT was detected in the serum samples of patients which were taken around 2 weeks after erythema chronicum migrans. The test is very simple and can be employed easily in diagnostic laboratories.     

Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system

Pathogens possess the ability to adapt and survive in some host species but not in others–an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.     

Complete Genome Sequence of Borrelia afzelii K78 and Comparative Genome Analysis

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.     

Genome Sequence of Borrelia afzelii Strain HLJ01, Isolated from a Patient in China

We report here the genome sequence of Borrelia afzelii strain HLJ01, isolated from a patient with Lyme disease in China. It is the first report of the whole genome of a B. burgdorferi sensu lato isolate from a human in China.     

Substantial Rise in the Prevalence of Lyme Borreliosis Spirochetes in a Region of Western Germany over a 10-Year Period

More than a decade after a study on the transmission cycle of Borrelia burgdorferi sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult Ixodes ricinus ticks were collected again in three selected areas of the same low mountain range and examined for infection with B. burgdorferi sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for B. burgdorferi sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for B. burgdorferi sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of B. burgdorferi sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of B. burgdorferi sensu lato revealed high relative prevalences of B. valaisiana (identified in 43.1% of infected ticks) and B. garinii (32.3%), whereas B. afzelii (12.3%) and B. burgdorferi sensu stricto (1.5%) were relatively rare. We conclude that B. burgdorferi sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.     

Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: Relevance for Lyme vaccine design

Lyme borreliosis is the most important vector‐borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC‐MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild‐type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT‐PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 ( http://proteomecentral.proteomexchange.org/dataset/PXD000876 ).