Genomic Confirmation of Borrelia garinii, United States.

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

Molecular Microbiological and Immune Characterization of a Cohort of Patients Diagnosed with Early Lyme Disease.

Lyme disease is a tick-borne infection caused by the bacteria Borrelia burgdorferi Current diagnosis of early Lyme disease relies heavily on clinical criteria, including the presence of an erythema migrans rash. The sensitivity of current gold-standard diagnostic tests relies upon antibody formation, which is typically delayed and thus of limited utility in early infection. We conducted a study of blood and skin biopsy specimens from 57 patients with a clinical diagnosis of erythema migrans. Samples collected at the time of diagnosis were analyzed using an ultrasensitive, PCR-based assay employing an isothermal amplification step and multiple primers. In 75.4% of patients, we directly detected one or more B. burgdorferi genotypes in the skin. Two-tier testing showed that 20 (46.5%) of those found to be PCR positive remained serologically negative at both acute and convalescent time points. Multiple genotypes were found in three (8%) of those where a specific genotype could be identified. The 13 participants who lacked PCR and serologic evidence for exposure to B. burgdorferi could be differentiated as a group from PCR-positive participants by their levels of several immune markers as well as by clinical descriptors such as the number of acute symptoms and the pattern of their erythema migrans rash. These results suggest that within a Mid-Atlantic cohort, patient subgroups can be identified using PCR-based direct detection approaches. This may be particularly useful in future research such as vaccine trials and public health surveillance of tick-borne disease patterns.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Primordial origin and diversification of plasmids in Lyme disease agent bacteria.

BACKGROUND: With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species. RESULTS: We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types. CONCLUSIONS: The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi.

BACKGROUND: Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. RESULTS: We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). CONCLUSIONS: Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

Rapidly maturing field of proteomics: a gateway to studying diseases.

The proteomics work reported by Smith et al. represents a giant step forward in characterizing the cerebrospinal fluid (CSF) proteome in mouse models of human diseases. Whereas prior studies were limited to analysis of CSF pools, Smith et al. (Proteomics 2014, 14, 1102-1106) base their conclusions on data derived from individual mice, thereby capturing a fuller range of the biological diversity present. These results underscore how far proteomics has come in the past few years, developing into a modern tool with the capacity to remove bottlenecks in the study of neuropsychiatric diseases. Past efforts with mass spectrometry (MS) have been hampered by limitations in access to CSF samples, and small volumes when available. These barriers have been overcome with newer MS platforms and advances in sample preparation. We are far closer than before to producing the production of clinically useful proteomic data for biomarker discovery and for deriving insights into pathogenesis that can lead to more effective treatments for many diseases.

BorreliaBase: a phylogeny-centered browser of Borrelia genomes.

BACKGROUND: The bacterial genus Borrelia (phylum Spirochaetes) consists of two groups of pathogens represented respectively by B. burgdorferi, the agent of Lyme borreliosis, and B. hermsii, the agent of tick-borne relapsing fever. The number of publicly available Borrelia genomic sequences is growing rapidly with the discovery and sequencing of Borrelia strains worldwide. There is however a lack of dedicated online databases to facilitate comparative analyses of Borrelia genomes. DESCRIPTION: We have developed BorreliaBase, an online database for comparative browsing of Borrelia genomes. The database is currently populated with sequences from 35 genomes of eight Lyme-borreliosis (LB) group Borrelia species and 7 Relapsing-fever (RF) group Borrelia species. Distinct from genome repositories and aggregator databases, BorreliaBase serves manually curated comparative-genomic data including genome-based phylogeny, genome synteny, and sequence alignments of orthologous genes and intergenic spacers. CONCLUSIONS: With a genome phylogeny at its center, BorreliaBase allows online identification of hypervariable lipoprotein genes, potential regulatory elements, and recombination footprints by providing evolution-based expectations of sequence variability at each genomic locus. The phylo-centric design of BorreliaBase (http://borreliabase.org) is a novel model for interactive browsing and comparative analysis of bacterial genomes online.

Prevalence of Borrelia miyamotoi in Ixodes ticks in Europe and the United States.

Borrelia miyamotoi, a relapsing fever-related spirochete transmitted by Ixodes ticks, has been recently shown to be a human pathogen. To characterize the prevalence of this organism in questing Ixodes ticks, we tested 2,754 ticks for a variety of tickborne pathogens by PCR and electrospray-ionization mass spectrometry. Ticks were collected from California, New York, Connecticut, Pennsylvania, and Indiana in the United States and from Germany and the Czech Republic in Europe from 2008 through 2012. In addition, an isolate from Japan was characterized. We found 3 distinct genotypes, 1 for North America, 1 for Europe, and 1 for Japan. We found B. miyamotoi infection in ticks in 16 of the 26 sites surveyed, with infection prevalence as high as 15.4%. These results show the widespread distribution of the pathogen, indicating an exposure risk to humans in areas where Ixodes ticks reside.

How Do I Approach the Evaluation and Treatment of Early Lyme Disease?

AbstractThe incidence and geographic spread of Lyme disease are increasing, and more than 476,000 new cases a year are estimated to occur in the United States. Therefore, many clinicians in North America will need to consider how to approach a patient with a concern for Lyme disease. This Curbside Consult addresses common clinical considerations, including discussion of signs of early Lyme disease, available laboratory tests, when to treat and with which antibiotics.

Natural selection and recombination at host-interacting lipoprotein loci drive genome diversification of Lyme disease and related bacteria.

Lyme disease, caused by spirochetes in the Borrelia burgdorferi sensu lato clade within the Borrelia genus, is transmitted by Ixodes ticks and is currently the most prevalent and rapidly expanding tick-borne disease in Europe and North America. We report complete genome sequences of 47 isolates that encompass all established species in this clade while highlighting the diversity of the widespread human pathogenic species B. burgdorferi. A similar set of plasmids has been maintained throughout Borrelia divergence, indicating that they are a key adaptive feature of this genus. Phylogenetic reconstruction of all sequenced Borrelia genomes revealed the original divergence of Eurasian and North American lineages and subsequent dispersals that introduced B. garinii, B. bavariensis, B. lusitaniae, B. valaisiana, and B. afzelii from East Asia to Europe and B. burgdorferi and B. finlandensis from North America to Europe. Molecular phylogenies of the universally present core replicons (chromosome and cp26 and lp54 plasmids) are highly consistent, revealing a strong clonal structure. Nonetheless, numerous inconsistencies between the genome and gene phylogenies indicate species dispersal, genetic exchanges, and rapid sequence evolution at plasmid-borne loci, including key host-interacting lipoprotein genes. While localized recombination occurs uniformly on the main chromosome at a rate comparable to mutation, lipoprotein-encoding loci are recombination hotspots on the plasmids, suggesting adaptive maintenance of recombinant alleles at loci directly interacting with the host. We conclude that within- and between-species recombination facilitates adaptive sequence evolution of host-interacting lipoprotein loci and contributes to human virulence despite a genome-wide clonal structure of its natural populations. IMPORTANCE: Lyme disease (also called Lyme borreliosis in Europe), a condition caused by spirochete bacteria of the genus Borrelia, transmitted by hard-bodied Ixodes ticks, is currently the most prevalent and rapidly expanding tick-borne disease in the United States and Europe. Borrelia interspecies and intraspecies genome comparisons of Lyme disease-related bacteria are essential to reconstruct their evolutionary origins, track epidemiological spread, identify molecular mechanisms of human pathogenicity, and design molecular and ecological approaches to disease prevention, diagnosis, and treatment. These Lyme disease-associated bacteria harbor complex genomes that encode many genes that do not have homologs in other organisms and are distributed across multiple linear and circular plasmids. The functional significance of most of the plasmid-borne genes and the multipartite genome organization itself remains unknown. Here we sequenced, assembled, and analyzed whole genomes of 47 Borrelia isolates from around the world, including multiple isolates of the human pathogenic species. Our analysis elucidates the evolutionary origins, historical migration, and sources of genomic variability of these clinically important pathogens. We have developed web-based software tools (BorreliaBase.org) to facilitate dissemination and continued comparative analysis of Borrelia genomes to identify determinants of human pathogenicity.

Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation.

BACKGROUND: Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. "bavariensis" (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. "finlandensis" (1). RESULTS: Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. CONCLUSIONS: Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues.

Common Neurologic Features of Lyme Disease That May Present to a Rheumatologist.

Lyme disease, caused by Borrelia burgdorferi (Bb) infection, has a broad spectrum of clinical manifestations and severity. Patients with possible Lyme disease may seek out or be referred to rheumatologists. Today, the most common reason to engage a rheumatologist is due to complaints of arthralgia. After skin, neurologic manifestations of Lyme disease are now among the most common. Therefore, it is important for rheumatologists to be aware of clues that suggest neurologic Lyme disease and prompt help from a neurologist experienced with Lyme disease.

Myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia are indistinguishable by their cerebrospinal fluid proteomes.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and fibromyalgia have overlapping neurologic symptoms particularly disabling fatigue. This has given rise to the question whether they are distinct central nervous system (CNS) entities or is one an extension of the other. MATERIAL AND METHODS: To investigate this, we used unbiased quantitative mass spectrometry-based proteomics to examine the most proximal fluid to the brain, cerebrospinal fluid (CSF). This was to ascertain if the proteome profile of one was the same or different from the other. We examined two separate groups of ME/CFS, one with (n = 15) and one without (n = 15) fibromyalgia. RESULTS: We quantified a total of 2083 proteins using immunoaffinity depletion, tandem mass tag isobaric labelling and offline two-dimensional liquid chromatography coupled to tandem mass spectrometry, including 1789 that were quantified in all the CSF samples. ANOVA analysis did not yield any proteins with an adjusted p value <.05. CONCLUSION: This supports the notion that ME/CFS and fibromyalgia as currently defined are not distinct entities.Key messageME/CFS and fibromyalgia as currently defined are not distinct entities.Unbiased quantitative mass spectrometry-based proteomics can be used to discover cerebrospinal fluid proteins that are biomarkers for a condition such as we are studying.

Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii.

It has been known for decades that human Lyme disease is caused by the three spirochete species Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Recently, Borrelia valaisiana, Borrelia spielmanii, and Borrelia bissettii have been associated with Lyme disease. We report the complete genome sequences of B. valaisiana VS116, B. spielmanii A14S, and B. bissettii DN127.

Analysis of cerebrospinal fluid from chronic fatigue syndrome patients for multiple human ubiquitous viruses and xenotropic murine leukemia-related virus.

Recent reports showed many patients with chronic fatigue syndrome (CFS) harbor a retrovirus, xenotropic murine leukemia-related virus (XMRV), in blood; other studies could not replicate this finding. A useful next step would be to examine cerebrospinal fluid, because in some patients CFS is thought to be a brain disorder. Finding a microbe in the central nervous system would have greater significance than in blood because of the integrity of the blood-brain barrier. We examined cerebrospinal fluid from 43 CFS patients using polymerase chain reaction techniques, but did not find XMRV or multiple other common viruses, suggesting that exploration of other causes or pathogenetic mechanisms is warranted.

Identification of endosymbionts in ticks by broad-range polymerase chain reaction and electrospray ionization mass spectrometry.

Many organisms, such as insects, filarial nematodes, and ticks, contain heritable bacterial endosymbionts that are often closely related to transmissible tickborne pathogens. These intracellular bacteria are sometimes unique to the host species, presumably due to isolation and genetic drift. We used a polymerase chain reaction/electrospray ionization-mass spectrometry assay designed to detect a wide range of vectorborne microorganisms to characterize endosymbiont genetic signatures from Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor andersoni Stiles, Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Ixodes ricinus (L.), and Rhipicephalus sanguineus (Latreille) ticks collected at various sites and of different stages and both sexes. The assay combines the abilities to simultaneously detect pathogens and closely related endosymbionts and to identify tick species via characterization of their respective unique endosymbionts in a single test.

Genomic hybrid capture assay to detect Borrelia burgdorferi: an application to diagnose neuroborreliosis in horses.

Antemortem diagnosis of neuroborreliosis in horses has been hindered by both the low sensitivity of PCR testing for Borrelia burgdorferi in CSF and the low specificity of serum:CSF ELISA ratios used to determine intrathecal antibody production against the bacterium. PCR testing of the CSF of an adult horse with acute neurologic disease for the B. burgdorferi flagellin gene was negative. However, we enriched B. burgdorferi DNA through nucleic acid hybrid capture, followed by next-generation sequencing, and identified B. burgdorferi in the CSF of the horse, confirming a diagnosis of neuroborreliosis.

Whole-genome sequences of thirteen isolates of Borrelia burgdorferi.

Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.