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Molecular characterization of the lyme disease spirochetes Borrelia burgdorferi sensu lato
Wang, G.
Publication date 1999
Link to publication
Citation for published version (APA): Wang, G. (1999). Molecular characterization of the lyme disease spirochetes Borrelia burgdorferi sensu lato.
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MOLECULAR TYPING OF BORRE LIA BURGDORFERI SENSU LATO: TAXONOMIC, EPIDEMIOLOGICAL AND CLINICAL IMPLICATIONS
Guiqing Wang1, Alje P. van Dam', Ira Schwartz2, and Jacob Dankert'
Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands, and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, U.SA.
Clinical Microbiology Reviews, submitted Chapter 2
SUMMARY
Borrelia burgdorferi sensu lato, the bacterium that causes human Lyme borreliosis (LB), has been cultured from various biological and geographic sources worldwide since its first discovery in 1982. Early studies have demonstrated that isolates of B. burgdorferi sensu lato are genetically and phenotypically divergent. Thus, various molecular approaches were developed and used to determine the phenotypic and genetic heterogeneity within the LB related spirochetes, and their potential association with distinct clinical syndromes. These methods include i) serotyping; ii) multilocus enzyme electrophoresis; iii) DNA-DNA relatedness analysis; iv) rRNA gene restriction analysis (ribotyping); v) pulsed-field gel electrophoresis; vi) plasmid fingerprinting; vii) randomly amplified polymorphic DNA fingerprinting analysis; viii) species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and ix) DNA sequence analysis. On the basis of 5S-23S rRNA intergenic spacer RFLP and DNA-DNA hybridization, ten different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi and B. bissettii sp. nov.. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are known to be responsible for causing human disease. B. burgdorferi sensu stricto is present in both North America and Europe, whereas B. garinii and B. afzelii are widely distributed in Eurasia. B. bissettii sp. nov. may represent the fourth Borrelia species for causing human LB since strains with similarity of genetic and phenotypic characteristics to this species have been cultured from patients in Europe. Several studies suggest that different Borrelia species are associated with distinct clinical manifestations of LB. Infection with B. burgdorferi sensu stricto is more commonly associated with arthritis, B. garinii with neuroborreliosis, and B. afzelii with acrodermatitis chronica atrophicans. In addition, Borrelia species are differentially distributed worldwide, and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomie status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especially correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail.
INTRODUCTION
Lyme borreliosis (LB), or Lyme disease represents a new global public health problem ( 18). It is now the most common vector-borne disease in North America (42, 233) and Eurasia (168, 285). In the United States, more than 100,000 LB cases have been reported from 47 states from 1982 through 1996, with more than 16,000 cases in 1996 (42). It is estimated that annually about 50,000 cases occur in Europe (168). In 1982, the bacterium that causes LB was firstly isolated by Dr. Willy Burgdorfer from the hard tick Ixodes dammini collected in Long Island, New York (35). The isolate was subsequently identified as a new species of the genus Borrelia and was named Borrelia burgdorferi in 1984 (108). Since then, hundreds of B. burgdorferi isolates have been cultured worldwide from various geographic regions and biological sources, including Ixodes ticks, their reservoir hosts and specimens from patients with Molecular Typing of B. burgdorferi Sensu Lato
different clinical syndromes. Molecular analysis has indicated that these B. burgdorferi isolates are genetically and phenotypically divergent. A closely related cluster containing several tick-borne Borrelia species and genomic groups associated with LB has been defined (16, 38, 68, 108, 113, 122, 139, 195, 261). The term 'B. burgdorferi sensu lato' is now collectively used to refer to all Borrelia isolates within this cluster, and to distinguish it from the species 'B. burgdorferi sensu stricto' (strict sense of B. burgdorferi) (222). Like other Borrelia, B. burgdorferi sensu lato is a spiral-shaped, gram-negative bacterium with 7 to 11 periplasmic flagellae. It varies from 10 to 30 (im in length and 0.2 to 5 jam in width (20). The genome of the type strain B. burgdorferi sensu stricto B31 contains a linear chromosome of 910,725 bp with an average G+C of 28.6%, and 21 plasmids (9 circular, 12 linear) with a combined size of approximately 613,000 bp (65). The G+C content of plasmid DNA ranges from 23.1% to 32.3% (65). Human infection due to B. burgdorferi sensu lato may involve multiple organs or tissues, resulting in skin, cardiac, neurological and musculoskeletal disorders. Lyme
Table 1. Major cl^m Staged Clinical features North America" Eurasia I Early local infection Erythema migrans, EM Common (60-90%)(44'79) Common (-77%)(25) Systemic symptoms Common (>50%)(l57)e Uncommon (<3 8%)(241,e II Early disseminated Infection Common (>18%)(,57)e Unusual (6%)(24,) e Multiple EM Common (10-20%) (44'79) Common (16-80%) (25'43) (9U73)/ Neuroborreliosis, NB 3_21%(44.79,f 37-61% (797 1119 mo f Meningoradiculitis 2_17%(44.79)f 4 27°/ - ' > Meningitis 0.5-10% (44'79) 0.5.4% («,i73) Carditis Rare Well documented (3%)(25) Borrelial lymphocytoma III Late Lyme borreliosis Lyme arthritis Common(51-57%)(44'238)g Uncommon (-7%)(25) ACA Rare Well documented (3%)(25) Peripheral neuropathy 30-70% late NB(90)(44)f 40-63% ACA patients (117) CNS involvement Well documented (89) <9%NB(173)f (90) Encephalomyelitis rare (0.1%) 4_60/o(9..173)f Meningencephalitis 9%(h) { 0.5-4% (9U73)f
a. Stages of the clinical features were according to Steere (232); b. EM: erythema migrans; ACA: acrodermatitis chronica atrophicans; NB: neuroborreliosis; CNS: central nervous system.; c. Data were mainly based on an earlier report of CDC on LB surveillance from 1984 to 1986 (44) and a population-based study in children in Southern Connecticut (79), except for that indicated specifically; d. Data were mainly based on a population-based study in Southern Sweden (25), except for that indicated specifically; e. Data were based on 76 American (157) and 231 European culture-confirmed patients with EM (241); f. NB patients were used as denominator to calculate the relative prevalence. European data were based on data from 330 NB cases from Germany (173) and 176 NB cases from Denmark (91); g A recent population-based study showed that only 7% of children with LB developed Lyme arthritis (79). About 10% of patients with Lyme arthritis may develop chronic antibiotic treatment-resistant arthritis (87).
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disease (232) was described as a new clinical entity in 1977 because of a geographic clustering of children with rheumatoid-like arthritis in Lyme, Connecticut (235, 236, 237). Retrospective analysis revealed that many of the clinical manifestations of LB had been separately recorded by European clinicians since the end of 19th century (264). Table 1 lists the spectrum of the major clinical manifestations of human LB in North America and Eurasia. It has been shown that multiple EM and Lyme arthritis are more common in the United States than in Europe, whereas neuroborreliosis has more frequently occurred in European patients, especially in children with LB (25, 43, 44, 79, 173). Borrelial lymophocytoma and acrodematitis chronica atrophicans (ACA) are well documented in European LB patients, but are rarely recognized among LB patients in the United States (188, 231). In general, the clincial manifestations of LB shows many features in common with syphilis. As a result of its protean clinical manifestations, LB was described as the new 'greater imitator' of various human diseases (174). Numerous studies indicate that the B. burgdorferi sensu lato population is genetically highly divergent (14, 16, 29, 106, 130, 151, 191, 206, 262, 266). Different Borrelia species may be associated with distinct clinical manifestations of LB (11, 13, 16, 38, 56, 169, 180, 188, 253). In this paper, various molecular methods used for the identification and typing of B. burgdorferi sensu lato are reviewed. The current taxonomie status of B. burgdorferi sensu lato and the epidemiological and clinical implications of typing of B. burgdorferi sensu lato, especially the correlation between the various clinical presentations of LB and the infecting Borrelia species, are discussed.
PHENOTYPIC METHODS FOR TYPING OF B. BURGDORFERI SENSU LATO
Molecular techniques used for the identification and typing of microorganisms can be categorized as either phenotypic or genetic, on the basis of the macromolecular targets employed for analysis (244). For B. burgdorferi sensu lato, phenotypic typing systems like biotyping, phage typing and antibiotic susceptibility analysis, which are used for various bacterial species, are not feasible. SDS-PAGE protein (181, 251, 270) and fatty acid profile analysis (131, 132) have been used for typing of B. burgdorferi sensu lato, but the conclusions based on both of these phenotypic characteristics are not always reliable (261). Therefore, serotyping represents the most commonly used phenotypic method for B. burgdorferi sensu lato.
Serotyping The protein profiles of B. burgdorferi sensu lato isolates are heterogeneous (21, 270, 273, 274). Currently, two serotyping systems, based on the heterogeneity of the outer surface protein A (OspA) and the outer surface protein C (OspC) of B. burgdorferi sensu lato are well established. OspA serotyping. OspA is one of the major outer membrane lipoproteins of B. burgdorferi and has been used for serological diagnosis as well as for vaccine development (96, 116). The molecular mass of OspA ranges from 31to34kDa
20 Molecular Typing of B. burgdorferi Sensu Lato
Species OspA type OspC type
^ |B31 1 |B pac | ^^^-^*"*"^
""*• |Pstm | |PBre |
__^^+ |pKo | [PLud |
" * |Ple | |PLj7 |
burgdorferi sensu lato
===^Z. ITN llTlsl 1
Fig 1. Relationship between species, OspA and OspC serotype of B. burgdorferi sensu lato. OspA serotype J10 of Japanese isolates corresponds to the Euro-pean B. afzelii strains of serotype 2. Modified from references (148, 272). among the different species of B. burgdorferi sensu lato (261, 274). Based on the differential reactivities of 112 European and 24 North American B. burgdorferi sensu lato isolates with eight OspA-specific monoclonal antibodies (MAbs), seven different OspA serotypes (serotype 1 to 7) were defined by Wilske et al in 1993 (274). Later, an additional OspA serotype 8 was reported (271). Among Japanese B. burgdorferi sensu lato isolates, OspA serotypes Jl to Jl 1 were recognized (148, 281). Analysis of phenotypic characteristics in parallel with genetic makers showed that OspA serotypes correlated well with the current classification of B. burgdorferi sensu lato (274). As shown in Fig.l, OspA serotypes 1, 2 and Jil correspond to B. burgdorferi sensu stricto, B. afzelii and B. japonica, respectively, and serotypes 3 to 8 (types Jl, J3 to J10 in Japan) correspond to B. garinii (272, 281). OspA serotypes are likely to be differentially distributed among tick and human isolates (272), and have been associated with distinct manifestations of LB (262, 271, 274). OspC serotyping. OspC is the predominant seroreactive antigen in the early stage of human infection due to B. burgdorferi sensu lato (2, 56, 74, 179). The molecular mass of OspC among the different species of B. burgdorferi sensu lato varies from 20- to 25 kDa (175, 273). Similar to OspA serotyping, analysis on the reactivity of B. burgdorferi sensu lato with a set of MAbs specific for OspC has led to the definition of 16 different OspC serotypes for European and North American isolates (272, 273). Comparison of OspA and OspC serotypes of B. burgdorferi sensu lato suggests that OspC is much more heterogeneous than OspA, especially for B. burgdorferi sensu
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stricto and B. afzelii strains. Whilst only one OspA serotype for each species is known, 6 corresponding OspC serotypes for B. burgdorferi sensu stricto and 4 OspC serotypes for B. afzelii were identified (272). The relationships among OspA, OspC serotypes and the delineated Borrelia species are summarized in Fig. 1. OspA and OspC serotyping provides an easy and reliable approach for the analysis of phenotypic characteristics among B. burgdorferi sensu lato at the species and subspecies level. Identification of the major B. burgdorferi sensu lato species can be made based on the reactivity of Borrelia isolates with the well-characterized species- specific MAbs (Table 2). However, both serotyping systems may be hampered due to the lack of OspA or OspC, the aberrant expression of these proteins and possibly their variation during growth in vitro as well as in vivo (99, 240, 274).
Table 2. Reactivity of B. burgdorferi sensu lato species with different species-specific monoclonal antibodies (MAbs) Reactivity with MAba Borrelia species Reference(s) H9724 H5332 H3TS D6 117.3 01141b B. burgdorferi + + + (16, 144) sensu stricto B. garinii + + or- - + (16, 144) B. afzelii + + (38, 144) B. japonica + + - - - + (144) * the reactivity is referred as positive (+) and negative (-) Multilocus enzyme electrophoresis (MLEE) MLEE is a protein-based typing method the results of which can be directly correlated with the genotype. MLEE is accepted as a promising method to elucidate the population genetics of bacteria (249). For this method, bacterial lysates are subjected to electrophoresis under non-denaturing conditions and the electrophoretic mobility of metabolic enzymes is determined after specific staining. Each electromorph is equated with an allele at the corresponding enzyme genetic locus. Thus, by associating each isolate with an electrophoretic type (ET), MLEE allows the differentiation of isolates by marking them with significant characteristics. MLEE was used to analyze the population genetics of B. burgdorferi sensu lato in 1992 (29). In that study 50 B. burgdorferi sensu lato isolates were grouped into 35 ETs, constituting three main divisions separated at a genetic distance greater than 0.75. Each of these three divisions corresponded either with B. burgdorferi sensu stricto, B. garinii or B. afzelii (29). Later, this method was used by Balmelli et al (14) to determine the overall genetic polymorphism of 54 B. burgdorferi sensu lato isolates from different regions of the world. A total of 12 genetic loci were characterized and 50 ETs were distinguished. Cluster analysis of a matrix of genetic distances for pairs of ETs revealed 11 divisions separated from each other at a genetic distance greater than 0.65. Ten of these 11 divisions corresponded with B. burgdorferi sensu stricto (Division I), B. garinii (divisions II and III), B. afzelii (division VII), B. japonica (division VIII), B. valaisiana (division V), B. lusitaniae (division IV), B. andersonii (division XI) and B. bisseitii sp. nov. (divisions IX and X). Division VI contained only one isolate (CA2). Previously, this isolate had been placed into B. bissettii sp. nov., the
22 Molecular Typing of B. burgdorferi Sensu Lato formerly genomic group DN127 (191, 195). Additional studies are needed to determine whether this strain construites a distinct species.
GENOTYPIC METHODS FOR TYPING OF B. BURGDORFERI SENSU LATO
Molecular typing based on the genetic characteristics of microorganisms can provide more precise information on the diversity of pathogenic bacteria. Several genotyping methods have been developed for B. burgdorferi sensu lato since its delineation into three different genospecies in 1992 (16, 266).
DNA-DNA Reassociation Analysis DNA-DNA reassociation analysis is acknowledged as a superior method for examining relationships between closely related taxa and represents the best applicable procedure for bacterial taxonomy at the present time (227). As recommended by the Ad Hoc Committee on reconciliation of approaches to bacterial systematics, the phylogenetic definition of a species generally would include strains with approximately 70% or more DNA-DNA relatedness and with a ATm of 5°C or less (263). The DNA reassociation value among strains in the genus Borrelia varies, ranging from 30 to 100% (20, 108). It is reported that the level of DNA homology between Borrelia species causing relapsing fever and those causing LB is about 30 to 44% (20). Among different B. burgdorferi sensu lato species, the level of DNA relatedness is about 48 to 70% (16, 108, 191). For example, the type strain of B. afzelii VS461 has 48, 65, 54, 64, and 58% DNA homology to the type strains of B. burgdorferi sensu stricto B31, B. garinii 20047 (16), B. japonica H014 (113), B. valaisiana VS 116 and B. lusitaniae PotiB2, respectively (191). DNA hybridization is currently used as the reference method for species delineation of B. burgdorferi sensu lato (16, 113, 191). On the basis of DNA-DNA relatedness, several different Borrelia species or genomic groups associated with LB have been identified (see the taxonomy section of this review for details).
Ribotyping Ribotyping has been frequently used for both taxonomie purposes and subgroup characterization of microorganisms belonging to different genera and species (189). Grouping of bacteria by ribotyping is based on the profiles obtained by restriction fragment patterns of chromosomal DNA digested with appropriate restriction enzymes and hybridized with a probe derived from a highly conserved ribosomal RNA. The restriction enzymes EcoRl, EcoRW, Pstl, Hindi Hpal and Hinàlll and probes either from 16S + 23S rRNA of Escherichia coli (16, 261), 16S rRNA (140), 23S rRNA (71) or 5S rRNA (128, 220) of the LB spirochete have been successfully used to identify B. burgdorferi sensu lato strains at the species level. An example of the restriction patterns obtained after £coRV digestion of genomic DNAs from isolates belonging to different Borrelia species, is summarized by Postic (190). It is noteworthy that almost all of the LB related spirochetes present a 3.2 kb EcoRV (16, 71), a 3.2 kb Hpal (128, 220) or a 2.2 kb Hindlll (253) restriction fragment. Therefore, these bands can be used as genetic markers for the identification of B. burgdorferi sensu lato species. In
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addition, species-specific Hindlll fragments were also recognized by ribotyping for B. burgdorferi sensu stricto (1.45 kb), B. garinii (1.2 kb) and B. afzelii (4.0 and 1.45 kb) (253). The species-specific bands can be used to distinguish these three human pathogenic species from each other. Ribotyping is a useful tool for studying the genetic relatedness between different Borrelia isolates, mainly at the species level. A large number of B. burgdorferi sensu lato isolates from different sources has been analyzed by ribotyping (16, 71, 126, 160, 164, 253). In these studies, the restriction patterns of B. burgdorferi sensu stricto and B. afzelii appear as very homologous, whereas B. garinii is more heterogeneous. In a study including 51 Borrelia isolates (21 from Far Eastern Russia, Japan and China, 20 from Europe and 10 from North America), the 18 B. burgdorferi sensu stricto isolates belonged to only one ribotype, whilst the 10 B. afzelii isolates showed 3 ribotypes and the 23 B. garinii isolates exhibited 9 ribotypes (71).
Pulsed-Field Gel Electrophoresis (PFGE) PFGE was first described in 1984 as a tool for examining the chromosomal DNA profiles of eukaryotic organisms (219). Subsequently, PFGE has proven to be a highly effective molecular typing technique for many bacterial species (245). For this method, the bacterial genomic DNA is separated by PFGE after digestion with a restriction enzyme having relatively few recognition sites. Discrimination of the species and among strains is based on the large restriction fragment length polymorphism (LRFP) of the chromosomal DNAs. After digestion of the genome with Mlul, the LRFPs of B. burgdorferi sensu lato are both species- and strain-specific. As described by Belfaiza et al (22) and confirmed by others, all three LB causing B. burgdorferi sensu lato species are characterized by one to three species-specific fragments: a band at 135 kb for B. burgdorferi sensu stricto, two bands at 220 and 80 kb for B. garinii, and three bands at 460, 320 and 90 kb for B. afzelii isolates (36, 37, 72, 151, 185, 186). The PFGE method also allows the discrimination between strains within each Borrelia species. For example, 20 B. burgdorferi sensu stricto isolates were separated into 10 Mlul LRFPs, while 24 B. garinii and 6 B. japonica isolates exhibited 4 and 2 LRFPs, respectively (22, 192, 193). Strikingly, only a single Mlul pattern was observed among the 20 B. afzelii isolates (22, 192). In addition, PFGE can be used to construct physical maps of the genomes and to define different groups of B. burgdorferi sensu lato by combination of the chromosomal LRFPs resulting from digestion of a set of restriction enzymes, e.g. Mlul, Sad, BssYlll, Eagl, Smal, Apal, Cspl, SgraAl (39, 51, 170). PFGE represents a reproducible and highly discriminative typing method for B. burgdorferi sensu lato. Characterization of isolates by PFGE usually correlates well with species designation of B. burgdorferi sensu lato by other methods (22, 72). The findings from studies using this method have both taxonomie and clinical implications (22, 151). Mathiesen et al (151) reported that B. burgdorferi sensu lato isolates from patients in the United States, irrespective of their geographic region, belonged to a single rDNA cluster. In this study results obtained from PFGE and ospA sequence analysis were in accordance.
Plasmid Fingerprinting
24 Molecular Typing of B. burgdorferi Sensu Lato
B. burgdorferi sensu lato have unusual double-stranded linear plasmids, in addition to the typical supercoiled circular plasmids (19, 65, 97, 105). Usually, plasmids are present at a low copy number of approximately one per chromosome equivalent (27, 97). There may be as many as 21 or more different plasmids within one spirochete cell (40, 65). Since both the number and the size of plasmids can vary among strains, plasmid profile analysis may be used for strain and species identification of B. burgdorferi sensu lato. Xu et al (279) studied by plasmid fingerprinting 40 B. burgdorferi sensu lato isolates from diverse biological sources and geographical locations. The number of plasmids in the B. burgdorferi sensu lato isolates ranged from 4 to 10 and the size of these plasmids ranged from 13.3 to 57.7 kb. The overall plasmid profiles might correlate to the species of B. burgdorferi sensu lato, although definite species-specific plasmids could not be identified in the three Borrelia species studied. Nevertheless, the usefulness of plasmid fingerprinting for the classification of B. burgdorferi sensu lato seems to be limited since some plasmids can be lost during in vitro cultivation (17, 217,280).
Randomly amplified polymorphic DNA (RAPD) There is an increasing interest in the molecular typing of bacteria by using RAPD fingerprinting (265), or arbitrarily primed PCR (AP-PCR) (269). Both of these techniques use low-stringency PCR amplification with a single primer with an arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments. In an early AP-PCR study, 29 B. burgdorferi sensu lato isolates were
% Similarity S pecies
B. afze Hi
B. valais ia n a or relia s p. A 1 4 S e . jap onica B. bissettii 2501 5
ß. burgdorferi sensu stricto
g arm H
B. herms H B. ans erin a
Fig. 2. Simplified dendrogram of B. burgdorferi sensu lato species based on RAPD fingerprints. Modified from reference with permission from the publisher (262).
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divided into three genospecific groups (266). Later, AP-PCR was used to investigate the evolution ofS. burgdorferi sensu stricto (64). More recently, this approach was evaluated by typing a large collection of B. burgdorferi sensu lato isolates from various biological and geographical sources (262). In this study, a total of 136 B. burgdorferi sensu lato strains were divided into seven genetic clusters, based on the DNA fingerprints generated with four arbitrary primers (Fig. 2). Six clusters contained 135 isolates corresponding to the well-defined species. One isolate did not belong to any of the known LB-related Borrelia species. Furthermore, pathogenic subgroups of B. garinii from LB patients with disseminated infections were identified by this analysis (262). Since RAPD is a highly discriminatory method and is easy to perform, this method is now a powerful tool to distinguish the different Borrelia species from each other as well as to recognize Borrelia strains within each of the species (262).
PCR and PCR-based RFLP analysis
Species-specific PCR. PCR amplification utilizing species-specific primers, in which the conserved 16S rRNA gene (127) (137)or species-specific plasmid gene loci (155, 156) are targeted, can be used directly for species identification of the LB spirochetes. The former approach has been used to differentiate the three human pathogenic species, B. burgdorferi sensu stricto, B. garinii, and B. afzelii (118, 137), as well as B. valaisiana (127) from each other. However, cross-amplification between Borrelia species may occur when using primers derived from the species-specific plasmid sequences (155).
Ribosomal DNA PCR-RFLP. The rrn operon of most B. burgdorferi sensu lato . contains one single copy of 16S rRNA (m) and tandem repeated 23 S (rrlA and rrlB) and 5S rRNA (rrfA and rrß) (Fig. 3) (73, 77, 170, 220). The rrn operon is located at the center of the linear chromosome and is arranged according to the following order: rrs-rrlA-rrfA-rrlB-rrß. Three different PCR-RFLP approaches have been designed for B. burgdorferi sensu lato, based on the unique rRNA structure of these microorganisms — rrfA-rrlB spacer, rrs-rrlA spacer and rrs.
i). rrfA-rrlB spacer PCR-RFLP. rrfA-rrlB spacer PCR-RFLP analysis is a widely used typing system for B. burgdorferi sensu lato (191). Development and application of this method has led to the description of eight different species or genomic groups within B. burgdorferi sensu lato in 1994 by Postic et al (191). PCR amplification of the highly variable rrfA-rrlB intergenic spacer usually yields 225 to 256 bp amplicon among strains from different species of B. burgdorferi sensu lato (145, 191, 259). No amplification occurred with the relapsing fever Borreliae. Digestion of amplicons with Msel resulted in different restriction fragments with species-differentiating characteristics (Fig. 3) (191). Table 3 summarizes the Msel restriction patterns of B. burgdorferi sensu lato identified to date. The results based on the Msel restriction patterns are generally in accordance with those from DNA-DNA hybridization (191).
ii). rrs-rrlA spacer PCR-RFLP. The rrs-rrlA intergenic spacer is about 3.2 kb in B. burgdorferi sensu stricto and 5 kb in B. garinii and B. afzelii (170, 220). Liveris et
26 Molecular Typing of B. burgdorferi Sensu Lato
al reported that amplification of the partial rrs-rrlA spacer, followed by digestion with Hinfl and Ms el, produced both species and subspecies-specific RFLP patterns (128, 130). This method has been applied to typing of B. burgdorferi directly in human tissue specimens and field-collected ticks thus obviating the need for culture isolation (129, 130). These studies demonstrated that a significant proportion of human EM lesions contained mixtures of different B. burgdorferi genotypes. Furthermore, typing of B. burgdorferi isolates by this method may allow differentiation of isolates with varying degrees of pathogenic potential (130, 278).
Msel I restriction
ABCDEFGI L
Fig 3. Schematic representation of the amplification of the ribosomal intergenic spacer followed by the analysis of Msel restriction polymorphism of PCR products. Lane A to L correspond to the Msel restriction patterns in Table 3. Adapted from references (77, 190).
Table 3. Mse I restriction fragments of 5S-23S rRNA (rrfA-rrlB) intergenic spacer of B. burgdorferi sensu lato Ampi icon RFLP Msel restriction Taxon Strain size (bp) pattern fragments size (bp) Reference Ä burgdorferi sensu stricto B31 254 A 108,51,38,29,28 (191) B. garinii 20047 253 B 108,95,50 (191) NT29 253 C 108, 57, 50, 38 (191) B. afzelii VS461 246 D 108, 68, 50, 20 (191) B. japonica H014 236 E 108,78,50 (191) B. vailaisiana VS116 255 F 175,50,23,7 (261) Am501 249 Q 169,51,23,6 (145) B. lusitaniae PotiB2 257 G 108,81,39,29 (191) PotiB3 255 H 108, 79, 52, 16 (191) B. bissettii DN127 257 I 108,51,38,33,27 (191) CA 226 J 108,51,38,29 (191) 25015 253 K 108,51,34,27, 17, 12,4 (191) B. andersonii 19857 266 L 120,67,51,28 (191) CA2 255 M 91,50,40,28,22, 17,7 (191) B. tanukii Hk501 245 0 174,51,20 (145) B. turdi Ya501 248 P 107,51,38,21, 16,8,7 (145) Borrelia sp. A14S 225 R 108,66,51 (259)
Ribosomal spacer DNA RFLP analysis of B. burgdorferi sensu lato has a number of advantages. It is a rather simple and useful tool for rapid screening and species identification of large collections of B. burgdorferi sensu lato isolates. Furthermore, it can be employed for typing uncultured samples collected from ticks and patients with
27 Chapter 2
Table 4.jB^bitigdorfêr^^ Species and strain Biological Geographic Accession Reference (s) source region no. of rrs B. burgdorferi sensu stricto B31 Ixodes scapulans USA U03309 (220) 297 Human, CSF USA X85204 (246) 20004 Ixodes ricinus France M64310 (137) DK7 Human skin, ACA Denmark X89195 (246) B. gahnii 20047 Ixodes ricinus France D67018 (67) PBi Human, CSF Germany X85199 (246) DK27 Human shin, EM Denmark X85193 (246) JL20 Human, CSF Sweden X95198 (246) ChY13p Ixodes persulatus China AB007450 (125) B. afzelii DK1 Human skin, EM Denmark X85109 (246) Dk2 Human skin, ACA Denmark X85198 (246) R-IP3 Ixodes persulatus Russia L46697 (137) HT61 Ixodes persulatus Japan D67019 (67) 17Y Ixodes persulatus Korea U44939 GenBank B. japonica HO 14 Ixodes ovatus Japan L40597 (67) IKA2 Ixodes ovatus Japan L40598 (67) B. valaisiana VS116 Ixodes ricinus Switzerland X98232 (259) UK Ixodes ricinus England X98233 (259) Am501 Ixodes columnae Japan D67021 (67) B lusitaniae PotiB2 Ixodes ricinus Portugal X98228 (122) BR41 Ixodes ricinus Czech Republic X98231 (122) B. andersonii 19857 Rabbit USA L46688 (67, 139) 21038 Ixodes dentanus USA L46701 (67, 139) B. tcmukii Hk501 Ixodes tanuki Japan D67023 (67, 68) OR2eL Ixodes tanuki Japan D67020 (67, 68) B. turdi Ya501 Ixodes turdus Japan D67022 (67, 68) Ac502 Ixodes turdus Japan D67024 (67, 68) B. bissettii sp. nov. DN127 Ixodes pacificus USA L40596 (67, 191) B. hermsii HS1 Ornithodoros hermsii USA U42292 (76)
LB. This facilitates epidemiological monitoring of the distribution of the different B. burgdorferi sensu lato species in Ixodes ticks and reservoir hosts. Using reverse blot hybridization (RLB) of the rrfA-rrlB spacer which was amplified by PCR and subsequently hybridized with species-specific oligonucleotide probes, Rijpkema et al were able to identify four different Borrelia species in ticks collected in the Netherlands (201, 202). The prevalence of B. burgdorferi sensu lato infection in ticks and various reservoir hosts in several European countries was also investigated successfully by RLB (47, 115, 200).
iii). rrs. Several species of B. burgdorferi sensu lato can be distinguished from each other on the basis of Bfa\ restriction patterns of PCR amplified rrs (122, 197), or by single strand conformational polymorphism (SSCP)-PCR analysis of the rrs (267).
28 Molecular Typing of B. burgdorferi Sensu Lato
--- B. burgorferi sensu stricto B31
?s- B. burgorferi sensu stricto DK7
*-- B, burgorferi sensu stricto 297
* B. burgorferi sensu stricto 20004
--- B. HsseltnDN127
: B- japon,ca HO 1 4
loo.-- B. japon,ca IK A2
97--- B. lus,tan,ae PotiB2
- B. lus,tan,ae BR4]
. B. gar,nu PBi
B. gar,nu CH Y ] 3
--- B. gar,nu 20047
.-- B. garinii DK27
B. gar,ni, SL20
B. tanuku Hk50 1
. la nut u OR2eL
--- B. turd, Ya501
,,'. B. turd, Ac502
valaisïan a A m 50 1
-- B. valais,ana V S 1 ] 6
B. vala,s,ana UK
B. afieli, R-IP3
--- B. afzei,, 17Y
-- B. afzei,, HT6 1
B. aflelu DK ]
B. afzelu DK2
B. andersonn 2 1 038
andersonu 19S57
herm sil H S 1 Fig. 4. Phylogenetic analysis of B. burgdorferi sensu lato based on 1368 nucleotides of the 16S rRNA gene sequences. The phylogenetic tree was constructed using the Neibougor- joining mehthod in the MEGA program as described in reference (261). The sources of each isolates are given in Table 4. Numbers at the branch nodes indicate the results of bootstrap analysis.
DNA Sequence Analysis Whole DNA-DNA reassociation analysis is the most robust approach by which B. burgdorferi sensu lato can be classified based on phylogenetic relationships since the results are ultimately based on the entire genome sequence of the organism. Generally, this method is useful for the study of bacterial genetics, evolution, taxonomy, and epidemiology (227). However, DNA sequence analysis of some highly conserved gene loci can be used as a suitable alternative method. For example, rrs, fla, and ospA have been employed for this purpose with B. burgdorferi sensu lato. rrs. Sequence analysis of bacterial rrs has been widely used for assessing the evolutionary history and species identification of B. burgdorferi sensu lato (68, 122, 138, 195, 261). More than 200 rrs sequences from various B. burgdorferi sensu lato species have been determined (many of them partial sequences), and are now available from the GenBank database (24). The DNA sequences of rrs among strains from different B. burgdorferi sensu lato species are highly homologous ranging from 95.3 to 99.6% (122). A phylogenetic tree, based on a 1368 bp of rrs gene from representative
29 Chapter 2 isolates of each Borrelia species from diverse geographic regions and various biological sources (Table 4), was constructed by Neighbour-joining method and is presented in Fig. 4. Borrelia isolates belonging to distinct species fall into individual clusters in the phylogenetic tree. Thus, sequence analysis of the 16S rRNA gene represents a reliable method for inferring the taxa of B. burgdorferi sensu lato (68, 122, 138, 195, 261).
fla. The fla gene (76), now known as flaB , encodes a 41kDa flagellin protein or FlaB (78) and is located on the linear chromosome (65). By phylogenetic analysis based on fla gene sequences the relapsing fever Borreliae can be separated from the LB-related Borreliae, and different B. burgdorferi sensu lato species can also be distinguished from each other (69, 70).
ospA. The ospA gene, located on a 49 to 57 kb linear plasmid (26, 212), is present in almost all B. burgdorferi sensu lato isolates (260, 279). Sequence analysis of ospA genes showed homogeneity within B. burgdorferi sensu stricto and B. afzelii but revealed major subgroups within the B. garinii species (268, 274). Although lateral gene transfer and recombination of ospA between different Borrelia species may occur, the frequency of such events is rather low (57, 207). Thus, cluster analysis of the ospA gene could provide useful phylogenetic information. Usually, the clustering of Borrelia strains in the phylogenetic tree based on sequence of the ospA and its predicted amino acid sequence is in agreement with the classification based on sequence analysis of conserved chromosomal genes like rrs (151), p93 (57) and fla (69), as well as with data obtained by PFGE (36, 151) and RAPD fingerprinting (64, 262). However, a recent study of 8 B. valaisiana isolates showed that ospA sequence analysis might not be appropriate for species identification of B. burgdorferi sensu lato (260).
ospC. Comparative analysis of the deduced amino acids sequences from B. burgdorferi sensu lato revealed a species-specific motif in the conserved amino terminal of the OspC protein (66, 67, 106, 133). However, it is usually inconclusive for assignment of an isolate to a specific Borrelia species because of the overall high variability of ospC sequences (106, 247) and lateral gene transfer between species (133, 199,258).
Other gene loci. Sequence analysis of other gene loci such as p93 gene (57), hbb (252), p39 gene (205), hsp60 and hsp70 (257) can provide additional information for species identification of B. burgdorferi sensu lato.
Comparison and Selection of Typing Methods Molecular typing systems can be characterized in terms of typeability, reproducibility, discriminatory power, ease of performance, and ease of interpretation (8, 244). Characteristics of the various typing methods used for B. burgdorferi sensu lato are shown in Table 5. The choice between various methods depends upon a number of factors, such as the objectives of the study, the level of discriminatory power required, the kinds of DNA preparations and laboratory conditions available,
30 Molecular Typing of B. burgdorferi Sensu Lato
and the technical skills of laboratory personnel. At this point in time, we recommend the ribosomal spacer PCR-RFLP to be considered as the method for Borrelia species identification during molecular epidemiological studies of B. burgdorferi sensu lato infection in Ixodes ticks, reservoir hosts and patients (114, 129). The discriminatory power of PFGE and RAPD is high and both methods can be used to evaluate the genetic heterogeneity within Borrelia species. RAPD is easier to perform than PFGE and can be used to monitor large numbers of isolates. DNA-DNA reassociation analysis is currently the reference method for species delineation, although it is a time- and labor consuming method. Therefore, this method is mainly used for confirmation of the taxonomie status of those isolates that are not identifiable or typeable by ribosomal spacer RFLP and rrs sequence analysis, as well as for the identification of newly recognized Borrelia species or genomic groups.
Table 5. Comparative characteristics of molecular methods used for typing of B . burgdorferi sensu lato Typing Proportion Reproduci- Discrimi Easy Easy system of strains bility natory interpretation performance References typeable power Phenotypic methods Sero typing Most Good Fair Moderate Moderate (273,274) MLEE All Good Good Difficult Moderate (14,29) Genotyping methods DNA-DNA hybridization All Good Excellent Easy Difficult (16,191) ribo typing All Excellent Fair Easy Moderate (16,253) PFGE All Excellent Excellent Moderate Moderate (22,36) plasmid fingerprinting Most Good Good Difficult Moderate (279) RAPD All Good Excellent Moderate Easy (262) PCR-RFLP Most Excellent Fair Easy Easy (128,191) DNA sequencing All Excellent Excellent Moderate Difficult (57,70, 122,268)
TAXONOMY OF B. BURGDORFERI SENSU LATO
Taxonomy and Evolution of B. burgdorferi sensu lato The spirochetes are one of the few major bacterial groups whose natural phylogenetic relationships are evident at the level of gross phenotypic characteristics. They form a coherent taxon composed of six major groups as indicated by phylogenetic analysis (177, 276). The taxonomy of the order Spirochetae is out of the range of this review and can be found in two references (177, 229). Our interest focusses on the taxonomy of the genus Borrelia, especially those Borrelia species within the B. burgdorferi sensu lato complex. The genus Borrelia represents a tight phylogenetic cluster, which is differentiated from other spirochetal phylogenetic groups by base signature analysis of rrs (177). More than 20 species have been identified within the genus so far (20, 218). These Borrelia species are usually categorized into two major categories, the relapsing fever Borreliae and the LB Borreliae, on the basis of the differences between their ecological and genetic characteristics (20). MLEE analysis has revealed a clonal population structure of B. burgdorferi sensu lato based upon the linkage disequilibrium of allele distributions (29), indicating that
31 Chapter 2 in the absence of recombination all genes in strains belonging to the same species share a common evolutionary history. This conclusion has also been drawn from RAPD fingerprinting (15) and comparison of the sequences of the highly conserved chromosomal genes fla and p93 (57). In addition, clonal populations were evidenced by the conserved chromosomal gene order in different B. burgdorferi sensu lato species (39, 170). It is not clear whether such populations are strictly clonal, since localized horizontal gene transfer of the outer membrane protein encoding genes has been reported in B. burgdorferi sensu lato (133, 141, 142, 199, 207, 258). For example, a close examination of the highly variable ospC gene indicated that lateral gene transfer might have occurred quite frequently, not only between members of the same species, but also between strains from different species (133). As a result of such gene transfer and subsequent recombination, the linkage disequilibrium among different species might be disturbed. This would lead to a different topology of phylogenetic relationship based on ospC sequence analysis as compared to that based on rrs gene analysis. Nevertheless, in general, ospC genes from strains of the same species appear to be more closely related to each other than to ospC genes from different species. Furthermore, species-specific motifs recognized in the conserved amino terminal of the OspC proteins (67, 106, 133), indicate that ospC is still clonally inherited. It is assumed that speciation among B. burgdorferi sensu lato is only a recent phenomenon, since divergence of the rrs gene sequences among representatives of different Borrelia species may not exceed 1% (122). Which species of B. burgdorferi sensu lato is most likely to be the ancestor of the recently evolving spirochetes? Assuming a clonal evolution of B. burgdorferi sensu lato, one may assume that the greater than the genetic diversity of the isolates within a given species, the longer the time period available for evolution. Since the diversity of B. burgdorferi sensu lato in Eurasia is much greater than in North America, it is likely that the ancestor of this complex originated in Eurasia. Furthermore, B. garinii, which shows the greatest genetic heterogeneity by most analytical methods, is most likely the species which is closest to the common ancestor of B. burgdorferi sensu lato, as was recently proposed (64, 199). Both B. burgdorferi sensu stricto and B. afzelii would have diverged from this ancestor.
Species within B. burgdorferi Sensu Lato Complex Different Borrelia species can be distinguished from each other by analyses of their phenotypic features, such as reactivity with species-specific MAbs (Table 2), or by genetic typing as outlined in the previous section. Since the discovery of B. burgdorferi in 1982, ten different Borrelia species or genomic groups within the B. burgdorferi sensu lato complex have been identified. Nine are separated at the species level and were designated as B. burgdorferi sensu stricto, B. garinii (16), B. afzelii (16, 38), B.japonica (113), B. valaisiana (261), B. lusitaniae (122), B. andersonii (139), B. tanukii, and B. turdi (6, 68), respectively. More recently, the tenth genomic group DN127 (191) has been reevaluated and proposed as a new species B. bissettii sp. nov. (195). The major ecological and human pathogenic characteristics as well as their geographic distributions of the LB-related B. burgdorferi sensu lato species are summarized in Table 6.
32 Molecular Typing of B. burgdorferi Sensu Lato
Table 6. Borrelia species associated with LB, their ecological and pathogenic characteristics and their graphic distribution Taxon Vector Animal host Human Disease* Distribution References B. burgdorferi 1. scaputaris mammals, EM, arthritis, USA (16, 108) sensu lato I. pacificus birds carditis, I. ricinus neuroborreliosis Europe (16) J. persulatus! Asia? (126) B. garinii I. ricinus birds. EM, arthritis, Europe (16) 1. persulatus small mammals neuroborreliosis Asia B. afzelii I. ricinus small mammals EM, arthritis, Europe (16,38) 1. persulatus neuroborreliosis, Asia ACA B. japonica I. ovatus small mammals No Japan (113) B. valaisiana I. ricinus birds unclear Europe (261) I. persulatus Japan I. columnae B. lusitaniae I. ricinus unknown No Central Europe (122) B. andersonii 1. dentatus rabbit No USA (139) B. bissettii I. pacificus rodents, birds Unclear USA (191, 195) I. neotomae I. scaputaris I. ricinus EM, Slovenia (242) lymphocytoma B. takunii I. takunus small mammals No Japan (68) B. turdi I. turdus small mammals No Japan (6, 68)
* The clinical syndromes associated with distinct Borrelia species are in bold.
A number of atypical Borrelia isolates, not clearly belonging to one of the described species, have been cultured from North America (39, 151, 185, 195), Europe (187, 262) and China (126, 285). One of these was cultured from a patient with LB in Europe (259, 262). It is reasonable to expect that more Borrelia species will be recognized as more isolates are recovered from divergent biological and geographic sources and are studied by various molecular typing methods.
Borrelia Species Pathogenic to Humans Not all strains from the described Borrelia species or genomic groups are pathogenic to humans. Currently, only B. burgdorferi sensu stricto, B. garinii and B. afzelii have been cultured frequently from patients with LB (36, 130, 151, 157, 241, 253, 274). These three major species appear to be responsible for causing different clinical syndromes of human LB (13, 38, 56, 169, 253). A recent study of nine B. burgdorferi sensu lato isolates recovered from patients with LB in Slovenia revealed that they were most closely related to the North American isolate 25015 which belongs to B. bissettii sp. nov. (187, 242). Thus, B. bissettii sp. nov. isolates may represent the fourth Borrelia species that could cause human LB. Interestingly, B. bissettii sp. nov. is the second reported B. burgdorferi sensu lato species (B. burgdorferi sensu stricto is the other) to be present in both the Old and New Worlds (187, 242). B. valaisiana is widely distributed in European countries as well as in Japan (261). It might be pathogenic for humans, since DNA specific for this species has been detected by PCR from patients with LB (203). Although it is uncertain whether the other LB-related Borrelia species cause human disease, their pathogenic role is far less important than that of the three major Borrelia species. For example, B. japonica was only weakly pathogenic in mice (111). Although /. ovatus ticks, the specific vector for B. japonica, is distributed widely in Japan and frequently bite humans, no LB patients with a confirmed I. ovatus tick bite
33 Chapter 2 have been reported, except for one case suspected on the basis of the serological findings (149).
EPIDEMIOLOGICAL IMPLICATIONS
Geographic Distribution of Different Borrelia Species LB is a globally distributed zoonosis (23, 216). Human cases occur predominantly in the northern hemisphere (158, 216).
North America. Three LB associated Borrelia species, B. burgdorferi sensu stricto (108), B. andersonii (139) and B. bissettii sp. nov. (195), are recognized in North America. B. burgdorferi sensu stricto is widely distributed in the northeast, midwest and western regions of the United States (42). It is the only Borrelia species cultured from patients with LB in North America. B. andersonii and B. bissettii sp. nov. were mainly cultured from ticks and small mammals from New York and California, respectively (4,31, 195).
European Countries. B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, and B. bissettii sp. nov. have been documented in Europe. The geographic distribution of these species on the European continent has been reviewed recently based on a total of 1263 records (738 isolates and 525 molecular DNA detections) from 26 countries (100). B. garinii and B. afzelii are the most frequently cultured species in Europe, while B. burgdorferi sensu stricto is mainly distributed in western European countries, but rarely recognized in eastern areas of Europe. B. valaisiana has been cultured or detected in ticks or avian reservoirs from at least eight European countries, including the United Kingdom (120), Ireland (114, 115), the Netherlands (201, 261), Switzerland (104, 181, 182), Germany (127, 211), Italy (48), Croatia (85, 200) and the Czech Republic (194). This species seems abundantly present in ticks in Ireland, Germany and Croatia (115, 127, 200). Only a few B. lusitaniae strains have been isolated in Portugal (191), the Czech Republic, Moldavia and Ukraine (194). Nine isolates with genotypic and phenotypic similarity to B. bissettii sp. nov. have been cultured in Slovenia (187, 242).
Asia. Six B. burgdorferi sensu lato species have been reported in Far Eastern Russia and Asian countries. B. garinii and B. afzelii are largely found throughout the range of /. persulcatus ticks in China (125, 243, 284), Japan (72, 160), Korea (176) and Far Eastern Russia (143, 194, 213). B. japonica, B. tanukii, and B. turdi are limited to Japan (68, 113, 145). One isolate from an /. columnae tick in Japan was classified as B. valaisiana (261). The presence of B. burgdorferi sensu stricto in Asia is controversial. Although most reports suggest that B. burgdorferi sensu stricto is absent (125, 143, 160, 160, 194, 243), two recent studies indicated the presence of this Borrelia species in China (126) and Taiwan (221).
Other areas. LB cases were reported on the southern hemisphere including South America (12), Africa (154, 214) and Australia (153). However, these cases were based
34 Molecular Typing of B. burgdorferi Sensu Lato on serology only. B. burgdorferi sensu lato has not been isolated from local Ixodes ticks or any other suspected vectors and patients (34, 208). Recently, B. garinii was recovered from a patient with LB in Australia, however, the infection may have been acquired in Europe (102).
Vector and Borrelia species specificity The principal vectors of B. burgdorferi sensu lato are ticks of the I. ricinus complex (34). These include /. scapularis (I. dammini) and /. pacificus in the United States, I. ricinus in Europe, and /. persulcatus in Asian Russia, China and Japan (3, 34, 54, 160, 284). These vectors for B. burgdorferi sensu lato are not strictly species-specific, and the specificity displayed by many of the relapsing fever Borreliae for a single tick species is likely not true for the LB spirochetes. For example, the European sheep tick I. ricinus has been recognized as a vector of all three human pathogenic Borrelia species, B. burgdorferi sensu stricto, B. garinii and B. afzelii (80, 183). In contrast, I. scapularis and /. pacificus in the United States and I. ricinus in Europe, are vectors of B. burgdorferi sensu stricto (34, 202, 232). On the other hand, B. japonica is mainly found in /. ovatus in Japan, and B. andersonii appears to be restricted to /. dentatus in the United States. Furthermore, B. burgdorferi sensu stricto, the most widely distributed Borrelia species in /. scalpularis of North America and /. ricinus of Europe, is rarely reported in the /. persulcatus regions in Far Eastern Asia. These findings suggest that a particular vector species does not harbor and transmit a specific Borrelia species. A recent comparative study of the three main clusters of European /. ricinus indicated significant differences in their susceptibility to B. afzelii (60). This finding may explain, in part, the uneven distributions of Borrelia species in Europe. Both /. ricinus and /. persulatus can be infected with B. garinii and B. afzelii (101, 160, 194, 285). Other Borrelia species may also be transmitted by several vector species of the /. ricinus group (e.g., B. bissettii sp. nov. strains have been isolated from I. neolomae (now I. spinipalpis) (167), /. scapularis, and /. pacificus in the United States (191), as well as from /. ricinus in Europe (242)). Although B. valaisiana and B. lusitaniae are mainly cultured from European /. ricinus ticks, the former species has also been cultured from /. columnae ticks in Japan (67, 122, 261). Additional tick vectors as well as hematophagous insects may play a role in maintaining of B. burgdorferi sensu lato in enzootic cycles in nature, and occasionally in transmitting B. burgdorferi sensu lato to humans (34). These include /. uriae, Dermacentor variabilis, D. parumapertus, Amblyomma americanum, Rhipicephalus ssanguineus, and Haemaphysalis leporislustris in North America (34), /. hexagonus, I. uriae, I. tnanguliceps, I. acuminatus, I. frontalis, D. reticulatus and H. punctata in Europe (80, 225), /. tanuki, and /. turdus in Japan (67), as well as /. granulatus, H. bispinosa, H. concinna, and H. longicornis in southern China (284, 285). The relationship between these possible tick vectors and various B. burgdorferi sensu lato species remains unknown. Mixed infections of multiple B. burgdorferi sensu lato species are documented in ticks (127, 156, 160, 184, 194, 200), reservoir hosts (120, 159, 160, 194) as well as in patients with LB (53, 203). The prevalence of mixed infections in ticks varies from 5 to 40% in different geographic regions (127, 156, 184, 194). Such mixed infections
35 Chapter 2 may result directly from feeding a host infected with multiple Borrelia species or through co-feeding transmission by which various spirochetes may exchange among ticks infesting on a single host (83, 161, 178, 198). Alternatively, ticks may acquire different Borrelia species from different individual hosts individuals during their three stages of life cycle, since the spirochetes can survive through the molts and are present in all subsequent stages of the vectors (3, 34).
Host and Borrelia Species Association B. burgdorferi sensu stricto in the United States, and B. garinii in Eurasia have been isolated from a large diversity of mammalian hosts and birds (3, 81, 162, 171). In contrast, B. afzelii in Eurasia and B. burgdorferi sensu stricto in Europe were mainly isolated from rodents (81). Many species of mammals are hosts of B. japonica in Japan (146, 147). However, birds are assumed as the only hosts for B. valaisiana in Europe, since no B. valaisiana isolate has been cultured or detected from mammals or rodents to date (104, 120, 261). Only a few B. lusitaniae strains were isolated from /. ricinus in southern, central and eastern Europe (122, 194). Data for hosts of this species are currently not available. B. andersonii and B. bissettii sp. nov. in North America seem to involve particular and narrow host spectra: cottontail rabbits (Sylvilagus floridanus) (4) and wood mice (Neotomae fuscipes) (31), respectively.
Transmission Cycles of Different Borrelia Species in Nature Increasing data indicate that two specific enzootic transmission cycles (rodent-tick and bird-tick) are involved in maintaining different B. burgdorferi sensu lato species in nature, as suggested by Nakao et al (160) and Humair et al (103). Reservoir hosts infected by various B. burgdorferi sensu lato species may not transmit them to ticks with equal efficiency (55, 119, 120, 150, 209). Hosts acting as filters seem to be predisposed to mainly transmitting one species to ticks, to the detriment of other genomic groups (120). The mechanisms underlying the apparent differential transmission of B. burgdorferi sensu lato by various mammalian and avian hosts remain to be determined, although a recent study revealed that differences in serum complement sensitivity among Borrelia species could be a key factor in LB ecology (121). Different B. burgdorferi sensu lato species may be maintained through distinct transmission cycles in natural foci (103, 104, 120, 160). In North America, B. burgdorferi sensu stricto is mainly maintained by the rodent (white-foot mice, Peromyscus leucopus)-tick cycle, while the tick-infested resident and migrant birds are potentially important in regional spread and in dissemination of the spirochetes (3, 162, 226, 228). B. andersonii and B. bissettii sp. nov. have their own distinct enzootic cycles involving other tick vectors and vertebrate hosts (4, 31, 195). In Eurasia, the major species B. afzelii is mainly circulated between the reservoirs, particular Apodemus mice and Clethrionomys voles, and Ixodes ticks (81, 103, 160), whereas B. garinii may be maintained predominantly through the bird-tick transmission cycle, especially in Northern Europe (171, 172). B. valaisiana has been detected in various avian reservoirs, but never in rodent hosts (104, 120, 261). Therefore, this species may not survive and persist in small mammals, and most likely circulates between its avian reservoirs and ticks in natural foci (120).
36 Molecular Typing of B. burgdorferi Sensu Lato
IMPLICATIONS FOR CLINICAL DISEASE , DIAGNOSIS AND VACCINE DEVELOPMENT
Given the genetic diversity of B. burgdorferi sensu lato and the protean disease manifestations of LB, the use of molecular typing provides valuable data for possible elucidation of the association of particular Borrelia species with the distinct clinical manifestations of LB (253). Molecular typing may also contribute in establishing correlation between the spirochetal subtypes and the occurrence and severity of particular clinical manifestations of LB (130, 262). Such data are important for the clinical and laboratory diagnosis of LB as well as for vaccine development.
Association of Clinical Disease and Infecting Borrelia Species The difficulty in culturing B. burgdorferi sensu lato from clinical specimens, other than skin, has hampered the assessment of the association between Borrelia species and distinct clinical entities. However, there is increasing evidence supporting such an association. Firstly, numerous Borrelia isolates recovered worldwide from LB patients with different clinical syndromes have been subjected to molecular typing analysis. We reviewed the reports on isolates from LB patients present in the MEDLINE database from 1992 through October 1998. Only those cultured isolates that had been subjected to molecular typing analysis and assigned properly to their taxon, and whose clinical source was indicated, were included in the analysis. The distribution of Borrelia species of 472 B. burgdorferi sensu lato isolates recovered from patients is summarized in Table 7. Different species of B. burgdorferi sensu lato are associated with distinct clinical manifestations of LB: Lyme arthritis is associated with B. burgdorferi sensu stricto infection, neuroborreliosis with B. garinii infection, and ACA with B. afzelii infection (13, 16, 38, 151, 169, 180, 188, 191, 253, 267, 274). In a recent study, Wormser et al demonstrated a significant association between the infecting genotype of B. burgdorferi in an EM lesion and the presence of spirochetemia or multiple EM lesions, suggesting that hematogenous dissemination is related to B. burgdorferi genotype (278). A limitation in this analysis may be the differences in culture recovery for different B. burgdorferi sensu lato species or genotypes. Two reports suggest that the distribution of genotypes in tick or human tissue specimens (as measured by sequencing or PCR-RFLP analysis) is different from that obtained by culture (129, 166). Another confounding factor may be publication bias (86). Nevertheless, molecular analysis clearly points to a correlation between genotype and clinical manifestation. Secondly, a number of European studies showed that the antibody responses in LB patients to the three B. burgdorferi sensu lato pathogens varied with disease manifestation (7, 10, 11, 56, 165). For example, Assous et al studied sera from 67 LB patients and found that 55% of the 20 patients with Lyme arthritis had preferential reactivity with a B. burgdorferi sensu stricto isolate, 47% of the 23 patients with neuroborreliosis had more reactivity with a B. garinii strain, and all 8 patients with ACA had greater reactivity with a B. afzelii strain (10, 11). Thirdly, in vitro experiments in which the tissue tropism of Borrelia species was evaluated by incubation of various cultured Borrelia species with human brain tissue have provided evidence that B. garinii invaded brain tissue more efficiently than B. burgdorferi sensu stricto and B. afzelii (283).
37 Chapter 2
Table 7. Distribution of B. burgdorferi sensu lato species in 472 isolates from patients with different clinical syndromes of LBa B. burgdorferi ss B. garinu B. afzelii i^ther « Specimen No. No. (%) No. (%) No. (%) No (%) Skin, EMC 308 96 (312) 50 (16.2) 156 (50.7) 6 (1.9) Cerebrospinal 68 9 (13.2) 46 (67.7) 10 (14.7) 3 (4.4)
Skin, ACAe 74 3 (3.0) 5 (6.8) 66 (89.2) 0 0 Othersf 22 16 (72.8) 3 (13.6) 2 (9.1) 1 (4.5) Total 472 124 (26.3) 104 (22.0) 234 (49.6) 10 (2.1) a. Only isolates with indication of clinical source and belonged to Borrelia species in the published papers were included for analysis. These isolates were recovered from patients from 18 countries including Germany (n=123). Unites States (92), the Netherlands (70), Slovenia (54), Austria (45), Denmark (21), Japan (12), Sweden (10), Russia (7), Switzerland (6), Czech Republic (6), China (4), France (3), Italy (2), Poland (1), Croatia (1), Lusiaenia (1) and Australia (1). Isolates with mixed infections of two species were excluded. Detailed data are available upon request. b EM: erythema migrans; ACA: acrodermatitis chronica atrophicans. c. Isolates were extracted from references (1, 22, 37, 64, 70, 84. 128. 133, 151, 164, 188, 191. 194. 194, 246. 253, 262, 267, 275, 279). d Isolates were extracted from references (1, 16, 36. 84. 128. 133. 137, 246, 253). e. Isolates were extracted from references (22. 84. 128, 133, 188. 225, 253, 262, 267). f. Others included 17 isolates from human blood, 3 from synovial fluid. 1 from myocardial samples and 1 from skin of patient with lymphcytoma (14, 211, 230). g. included 9 Slovenian isolates genetically similar to a North America isolate 25015 which belongs to B. bissettii (195) and 1 isolate from a patient with EM in the Netherlands (242, 259).
The mechanisms by which infection with different Borrelia species may lead to distinct clinical syndromes of LB is unclear. Different serum susceptibility (30, 254) and tissue tropism among Borrelia species may in part account for such differences (49, 248, 283). Although each of the three human pathogenic B. burgdorferi sensu lato species has been associated with different clinical syndromes of LB, the clinical spectrum caused by these species can largely overlap.
Erythema migrans (EM). EM is an expanding red or bluish-red rash, often with central clearing. It occurs in up to 90% of patients with objective evidence of LB (158). Usually, EM appears at the early stage of B. burgdorferi sensu lato infection. All three species of B. burgdorferi sensu lato pathogenic to humans have been repeatedly cultured from the skin biopsies of patients with EM (Table 7). No significant difference between the occurrence of EM and infection of a particular Borrelia species has been reported. This is to be expected since all infection with B. burgdorferi causing Lbresults from an initial tick bite. However, it was noted that EM patients infected with B. burgdorferi sensu stricto in North America complained more frequently of systemic symptoms than EM patients in Eurasia (157, 241) where EM is predominantly caused by infection due to B. afzelii (211, 253).
Lyme carditis. Lyme carditis is a well-known manifestation in both North American and European patients with LB (46, 234, 255). Early studies showed that the
38 Molecular Typing of B. burgdorferi Sensu Lato frequency of this syndrome was considerably higher in North America, where about 4- 10% of untreated LB patients presented with cardiac abnormalities (44, 232), than in Europe where only 0.5-4% of untreated LB patients presented with similar abnormalities (46, 158, 173). However, the incidence has been lower in two recent population-based studies in the United States and in Europe (25, 79). The only Borrelia isolate recovered from a myocardial specimen of an Austrian patient in Europe was typed as B. burgdorferi sensu stricto (211, 230).
Lyme Arthritis and B. burgdorferi sensu stricto infection. Lyme arthritis is the most common musculoskeletal symptom in North America resulting from B. burgdorferi sensu stricto infection. It occurs at either the early disseminated stage or as a more chronic manifestation. About 60% of untreated patients with EM experienced brief attacks of monoarticular or oligoarticular arthritis in the United States (44, 232). In contrast, only 3 tol5% of patients suffered from arthritis in Europe (25, 45, 173) where B. garinii and B. afzelii are more frequently recovered than B. burgdorferi sensu stricto (100, 211). This unequal distribution of Borrelia species may directly be related to the disparity in the reported incidence of Lyme arthritis between North America and European countries. Although different B. burgdorferi sensu lato species pathogenic to humans can be detected or recovered from the joint or synovial tissue in animal experimental studies (215, 282) and in patients with Lyme arthritis (59, 163, 256), the only three Borrelia isolates recovered from the synovial fluids of patients with Lyme arthritis were typed as B. burgdorferi sensu stricto (14, 211).
Neuroborreliosis and B. garinii infection. Neuroborreliosis is the most frequent manifestation of disseminated infection in Europe (43, 75, 110), and is a common symptom in North American LB patients as well (44, 90). Both the central and peripheral nervous system can be involved (1757, 1121). All three species, B. burgdorferi sensu stricto, B. garinii and B. afzelii, are known to cause Lyme neuroborreliosis. In European patients, B. garinii constituted up to 72% (n=55) of the 76 Borrelia isolates or DNAs detected in human CSF samples, whereas 8% (n=6) and 20% (n=20) of the these specimens were identified as B. burgdorferi sensu stricto and B. afzelii, respectively (100). Of 36 CSF isolates mainly recovered from patients with neuroborreliosis in Southern Germany, B. garinii, B. afzelii and B. burgdorferi sensu stricto accounted for 58%, 28% and 11%, respectively (36). These findings in European patients suggests an association between infection of B. garinii and the development of Lyme neuroborreliosis (13, 123, 180, 253, 271). This is not strictly the case, since obviously, B. burgdorferi sensu stricto is responsible for human neuroborreliosis in the United States. It may be noteworthy that the incidence of Bannwarth syndrome in LB patients is relatively lower in the United States (75, 88) than in European countries. This also sugguests differences in pathogenicity between B. burgdorferi sensu stricto and B. garinii.
Acrodermatitis chronica atrophans (ACA) and B. afzelii infection. ACA is a late cutaneous manifestation of LB characterized by chronic and long-lasting progressive red and bluish-red lesions, usually on the extensor surface of the extremities (231). It was described as early as 1883 (264) and is widely encountered throughout Europe (9,
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45, 231), but there have been few reports from the United States (58, 112, 151). Molecular studies of ACA isolates from patients in several European countries have provided evidence suggesting an exclusive association of ACA and B. afzelii infection (11, 13, 253, 267). However, a Danish isolate DK7 derived from an ACA lesion was classified as B. burgdorferi sensu stricto by OspA serotyping (274) as well as by 16S rRNA and ospC gene sequence analysis (246). In a recent study, 22 B. burgdorferi sensu lato isolates from Slovenian patients with ACA were subjected to PFGE and species-specific PCR analysis (188). Results showed that infection in 17 cases was due to B. afzelii, in 4 to B. garinii and in one to B. burgdorferi sensu stricto. One American isolate from a patient with ACA was also typed as B. burgdorferi sensu stricto (151). These studies indicate that B. afzelii is the predominant, but not the exclusive, etiologic agent of ACA.
Implication for Serological Diagnosis The diagnosis of LB is primarily clinical, but serological tests can provide very useful supportive evidence, especially in patients with disseminated or chronic infection. The serological tests usually used to assess to B. burgdorferi antibodies include enzyme-linked immunosorbent assay (ELISA), immunofluorescent assays (IFA), and western blot (WB). ELISA and IFA are most widely used but have low specificity (98, 250). Cross-reactive antibodies can produce a false-positive test in patients with other illnesses, e.g., other spirochetal infections, nonspirochetal bacterial endocarditis, Epstein-Barr virus infection, rheumatoid arthritis, and systemic lupus erythematosus (135, 136). In the United States, a two step protocol for the evaluation of the B. burgdorferi antibodies in sera was recommended by the CDC (5). The performance of this protocol may improve the specificity of serodiagnosis for LB (50, 107, 124), but the high percentage of seronegativity in 20-50% of patients, probably dependent on the stage of LB, remains a problem and limits the value of serological tests (2, 94, 98, 241). In addition, the antigenic heterogeneity of B. burgdorferi sensu lato may influence the sensitivity and specificity of serological tests for LB, especially on the European continent where three pathogenic species of B. burgdorferi sensu lato and at least eight OspA serotypes are well-documented (271, 274). Differences in the regional distributions of Borrelial species may also affect preferential reactivities of sera from patients with LB (33, 165). Several studies have shown differences in the reactivity patterns in Western blot, depending on species, strain or serotype used as the source for antigen (56, 93, 94, 152, 165). This issue should be readdressed since molecular typing results based on hundreds of isolates have indicated that the genetic diversity of B. burgdorferi sensu lato is much greater than previously thought, even in North America (130, 151, 191, 281). In a recent study, the influence of interspecies variability of B. burgdorferi sensu lato on serodiagnosis of LB was evaluated by using IgM and IgG ELISA with antigens prepared from representative isolates of species B. burgdorferi sensu stricto (isolates B31 and PKa2), B. garinii (PBi) and B. afzelii (PKo). Variations resulting from the use of different strains for antigenic preparations were noted in sera from 222 patients with clinically defined LB of all stages, 133 blood donors and 458 forest workers (93). Such differences were also noted in ELISA or WB by using whole cell antigenic preparation (94), recombinant OspC (152), internal flagellin fragment (95) and P39 protein (205) from different species of B.
40 Molecular Typing of B. burgdorferi Sensu Lato
burgdorferi sensu lato. Therefore, serological tests comprised of a combination of antigens derived from two or more strains (from different species in Europe) might be most informative, as a response to the impact of strain heterogeneity, especially in Eurasia.
Implication for Vaccine Development In the past years, different types of vaccines, including a whole cell vaccine (109), live attenuated vaccine from a flagella-less mutant (210), recombinant OspA (61), OspC summit vaccines (286) and OspA-based DNA vaccines (134, 224) have been developed (see also reference (277)). Recombinant OspA vaccine has been highly successful in protecting laboratory animals against challenge by homologous strains of B. burgdorferi sensu lato (61). More recently, the efficacy of this recombinant OspA vaccine was evaluated in two large clinical trials involving more than 21000 people from areas of the United States where LB is endemic. The efficacy of these OspA vaccines was 76 to 92% in the second year after a booster dose (223, 239). Vaccine failure appears to be due to a low OspA antibody level. A recent study might raise questions over safely of these OspA vaccines since it seems theoretically possible that OspA protein could provoke autoimmunity in susceptible individuals (87). The efficacy of this OspA-based vaccine in Europe may be considerably lower given the substantial variations displayed in OspA proteins of B. burgdorferi sensu lato, as revealed by molecular typing analysis. Immunization with recombinant OspA derived from only one isolate may fail to protect against heterogeneous population of organisms that are present in the natural focus. OspA immunization may protect mice from tick-borne infection with heterogeneous B. burgdorferi sensu lato from different geographic regions by blocking spirochetal transmission from the vector to the host (52, 63). However, it is not clear in humans whether the specific antibody in individuals who are immunized with an OspA vaccine derived from one isolate can also provide coverage against a wide range of strains. Passive and active immunization against OspC can serve in protective immunity. Recently, passive immunization with high dose of anti-OspC immune sera appeared to be therapeutic for chronic LB in mice (286). However, the OspC-mediated protective immunity is probably strain-specific (28). Thus, the general applicability of this approach may be limited by the well- documented heterogeneity of OspC among members of the species B. burgdorferi sensu lato (133, 273). One strategy to circumvent OspA and OspC heterogeneity is the use of vaccines made of a cocktail of various recombinant OspA and/or OspC gene products derived from different strains. In Europe, a multiple subunit vaccine is expected to yield protective immunity against infections of different species of B. burgdorferi sensu lato. Gern et al (82) reported that a polyvalent OspA vaccine prepared from three isolates of different species, efficiently protected mice against infection due to either B. burgdorferi sensu stricto, B. garinii or B. afzelii. Other potential candidates for vaccines include decorin binding protein A (DbpA) (41, 92, 204), P35 (62), and P66 (32, 196). Further information on the distribution of various Borrelia species in their natural reservoir hosts and vectors, and knowledge of the Borrelia antigenic compositions is needed to find representative immunogenic antigens for vaccine development.
41 Chapter 2
CONCLUSIONS
Since different Borrelia strains may intrinsically be associated with distinct pathogenic properties, application of molecular typing methods to classification of B. burgdorferi sensu lato will provide the framework for a systematic approach to characterize differences in infectivity as well as pathogenicity between strains. Among the typing methods available for B. burgdorferi sensu lato, OspA serotyping can be used to analyze the phenotypic characteristics. Both PFGE and RAPD fingerprinting analysis are powerful tools in elucidating the genetic relationship between closely related Borrelia isolates within a species. Ribosomal DNA spacer RFLP analysis is simple and can be applied to uncultured specimens and is, therefore, ideally suited for molecular epidemiological and population genetic studies of B. burgdorferi sensu lato. Phylogenetic analysis based on 16S rRNA gene sequence may represent a potential method for inferring the taxa of B. burgdorferi sensu lato (68, 122). However, DNA DNA reassociation data are generally required for designation of a new species because of the high degree of sequence similarity of the 16S rRNA genes between closely related Borrelia species (122, 261). In general, there is excellent concordance in the typing results obtained by different molecular approaches at the species level, with the exception of DNA sequence analysis of the ospC genes. Ten B. burgdorferi sensu lato species have been identified. The pathogenicity of three species is well documented in humans. Distribution of Borrelia species and clinical syndromes of LB are different in North America and Eurasia, possibly due to the differing pathogenicity of Borrelia species. Current data indicate that B. burgdorferi sensu stricto is associated with articular disorders, B. garinii infection with neuroborreliosis, and B. afzelii infection with ACA. Since the recovery of Borrelia from clinical specimens of patients with LB by culture is low, in particular in those patients with Lyme arthritis and carditis, assessment of the correlation between infecting species and clinical manifestations will require other approaches. Approporiate serological tests capable of differentiating the antibody response of patients infected with different Borrelia species may be helpful in this regard. In addition, sensitive molecular typing techniques which do not require large amounts of patient material or cultivation of the spirochete will play an increasingly important role in elucidation of the pathogenic potential of different B. burgdorferi genotypes. Ultimately, development of suitable animal models for investigation of the tissue tropisms of different B. burgdorferi sensu lato species will provide more direct evidence for the correlation between Borrelia species and clinical symptoms of LB.
ACKNOWLEDGMENTS
The authors thank Drs. D. Postic, DA. Mathiesen, and M Volkenandt for providing further information on the B. burgdorferi sensu lato isolates included in Table 7. Drs. E. Fikrig and G. Wormser for comments on the manuscript.
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