Borrelia: Molecular Biology, Host Interaction and Pathogenesis | Book
Caister Academic Press
D. Scott Samuels and Justin D. Radolf The University of Montana, Missoula, USA and University of Connecticut Health Center, Farmington, USA (respectively)
March 2010 Available now!
GB £159 or US $319 (hardback).
xii + 548 (plus colour plates)
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Pathogenic Treponema: Molecular and Cellular Biology
The genus Borrelia
, in the spirochete phylum, is not closely related to any other bacteria and has a highly unusual genome composed of a linear chromosome and multiple circular and linear plasmids that appear to be in a constant state of rearrangement, recombination, and deletion. The determination of the genome sequence of Borrelia
strains has facilitated tremendous advances in understanding this genus at the molecular and cellular level as well as the pathogenesis of Lyme disease and relapsing fever. In recent years there has been an explosion of new insights into the molecular biology, genetics, physiology, and ecology of Borrelia
and its tick/vertebrate life cycle. This research is of particular importance as the incidence of Lyme borreliosis continues to increase.
Written by renowned scientists who have made seminal contributions to the field, this book is a comprehensive guide to the pathogenic Borrelia
, providing researchers, advanced students, clinicians, and other professionals with an encyclopedic overview of the molecular biology of this important genus and the pathogenesis of diseases caused. Leading authorities have contributed chapters on topics such as Borrelia
genomics, DNA replication, gene regulation, transcriptomics, proteomics, metabolism and physiology, cellular structure, motility and chemotaxis, genetic manipulation, evolutionary genetics, ecology, tick interactions, Lyme disease and relapsing fever pathogenesis, animal models, host response, detection, and vaccines.
The volume is essential for anyone involved in Borrelia
research and is strongly recommended for microbiologists, immunologists, and physicians involved in spirochete research, Lyme borreliosis, or relapsing fever. The book is a recommended reference volume for all microbiology libraries.
"everything you need to know about these Spirochetes ... updated information about the state-of-art for research ... a fantastic source of information for scientists working on vector-borne diseases and interested in epidemiology, evolution, genomics ... I truly enjoyed reading this book and would recommend it." from Olivier A E Sparagano (Newcastle University, UK) writing in Parasites and Vectors (2010) 3: 52
"an encyclopaedic account ... contributed by experts ... a fabulous resource for those in the field and full of surprises and insights for the outsider." from Charles Penn (University of Birmingham) writing in Microbiology Today (2010)
"The two editors have successfully produced a comprehensive and multifaceted book ... the book offers students and other interested users a deep view of the world of Borrelia ... as a reference book it is outstanding by virtue of its enormous information content ... The work is a must for the scientific library" from Reinhard Wallich (Heidelberg) writing in Biospektrum (October 2010) 16: 715
"this is a landmark resource in the field of borreliae. It is truly the first and the sole resource that comprehensively synthesizes the state of knowledge gained from decades of research. This book is a must for any investigator in this field and will no doubt be consulted frequently. It should be a required acquisition for any medical school library, research institution, and microbiology laboratory, including especially those with interests in genetics and immunology. ... the editors and the authors have produced a paragon for writing about a genus." from Paul G. Auwaerter (Johns Hopkins University, Baltimore, USA) writing in Clin. Inf. Dis. (2011) 52: 965.
"an excellent resource for history, disease manifestations, basic biology ... a useful resource for information on general topics, such as symptoms of diseases, history, or basic bacterial physiology ... a helpful read" from Brian Stevenson (University of Kentucky, Lexington, USA) writing in Q. Rev. Biol. (2012) 87: 392-393.
"a fantastic source of information" (Parasites and Vectors); "a fabulous resource" (Microbiol. Today); "outstanding" (Biospektrum); "required acquisition for any medical school library, research institution, and microbiology laboratory" (Clin. Inf. Dis.); "an excellent resource" (Q. Rev. Biol.)
David A. Haake
The Worldwide Saga of Lyme Borreliosis
Jorge L. Benach and Juan Carlos García-Moncó
The clinical-epidemiologic studies of a cluster of arthritis in Lyme, Connecticut, intersected with ongoing studies on tick borne diseases in Long Island, New York to set the stage for the discovery of the spirochete agent of Lyme disease. Studies on the microbiology of spirochetes in other parts of the country were instrumental for the cultivation of the newly discovered organism. The multi-national clinical studies in Europe throughout the XXth century on the dermatologic and neurologic manifestations characterized and enlarged the syndrome that we now know as Lyme borreliosis. For reasons that are discussed in this chapter, controversies have arisen among advocate patient groups and the clinicians and scientists that are at the forefront of research in Lyme borreliosis. Polarized opinions on what constitutes this disease and how to treat it have resulted in unprecedented litigation involving medical societies and advocate patient groups with no obvious end in sight.
Borrelia Genomics: Chromosome, Plasmids, Bacteriophages and Genetic Variation
Sherwood R. Casjens, Christian H. Eggers and Ira Schwartz
All members of the Borrelia
genus that have been examined harbor a linear chromosome that is about 900 kbp in length as well as a plethora of both linear and circular plasmids in the 5-220 kbp size range. Genome sequences have been determined for B. burgdorferi
, B. garinii
, B. afzelii
, B. duttonii
and B. recurrentis
. The chromosomes, which carry the vast majority of the housekeeping genes, appear to be very constant in gene content and organization across the genus. The content of the plasmids, which carry most of the genes that encode the differentially-expressed surface proteins that interact with Borrelia's
arthropod and vertebrate hosts, are much more variable. B. burgdorferi
strain B31, the B. burgdorferi
type strain, has been studied in the most detail and harbors twelve linear and nine circular plasmids that comprise about 612 kbp. The plasmids are unusual, as compared to most bacterial plasmids, in that they contain many paralogous sequences, a large number of pseudogenes and, in some cases, essential genes. In addition, a number of the plasmids have features suggesting that they are prophages. Some correlations between genome content and pathogenicity have been deduced and comparative whole genome analyses promise future progress in this arena.
Replication of the B. burgdorferi Genome and Scrambling of the Linear Replicons Through Reverse Telomere Resolution
The highly unusual segmented genomes of Borrelia
species can contain over 20 autonomously replicating DNA molecules. Many of the molecules, including the chromosome, are linear with covalently closed hairpin ends. Current knowledge of the replication and maintenance of DNA molecules will be reviewed, including the process of telomere resolution, whereby the covalently closed hairpin ends are generated from replicative intermediates. Finally, the proposal that reverse telomere resolution is the driving force shaping the ongoing rearrangements and telomere exchanges in the linear replicons of Borrelia
species will be discussed.
Gene Regulation, Transcriptomics and Proteomics
Jon T. Skare, James A. Carroll, X. Frank Yang, D. Scott Samuels and Darrin R. Akins
Lyme disease is a tick-borne infection that can lead to chronic, debilitating complications if not recognized or treated appropriately. Borrelia burgdorferi
, the agent of Lyme disease, is maintained in nature by a complex enzootic cycle involving ticks and various vertebrate hosts. Since the initial finding over a decade ago that outer surface lipoproteins OspA and OspC are differentially expressed in the tick and mammal, many studies have reported that B. burgdorferi
differentially expresses a vast array of genes and proteins to help it adapt to growth in the unfed tick, feeding tick and mammalian host environments. Several global microarray studies have utilized various cultivation systems to identify genes regulated by environmental cues and/or mammalian host factors. The combined studies have been directed at identifying genes that are differentially expressed during the unique enzootic life cycle of this pathogen. While the microarray studies have identified hundreds of genes that are differentially expressed under various environmental conditions, recent advances in genetic methodologies have begun providing insight into the mechanisms underlying the differential gene expression observed. Although how most genes are transcriptionally regulated in the tick and/or mammal is still unclear, several regulatory proteins, including alternative sigma factors, DNA-binding proteins and transcriptional activators/repressors have been identified that either directly or indirectly control the differential expression of many genes in B. burgdorferi
. Studies on the various transcriptional regulators, alternative sigma factors and DNA-binding proteins identified thus far have provided important insights into the adaptive strategies employed by B. burgdorferi
as it cycles between the tick and the mammal. In recent years, functional proteomic-based assays also have become an important tool for examining the complement of proteins synthesized by Lyme disease spirochetes under experimental conditions that mimic various host environments.
Metabolism and Physiology of Borrelia
Frank Gherardini, Julie Boylan, Kevin Lawrence and Jon Skare
In this chapter, we attempt to define key biochemical pathways and metabolic systems that are the underpinning of the physiology of Borrelia
. Energy extracted from the fermentation of a few simple sugars fuels these biochemical reactions and also energizes a V-type ATPase (V-ATPase). This establishes a membrane potential that drives motility and the transport of most solutes. After transport, metabolites and biochemical intermediates, such as simple sugars, fatty acids, purines and pyrimidines, peptides and metals ions, are chemically utilized and/or modified to provide an intracellular pool of compounds necessary for protein, nucleic acid, membrane and cell wall biosynthesis. The extremely limited de novo
biosynthetic capacity of Borrelia
restricts members of this genus to a host-dependent lifestyle but conserves energy and reflects a reduction of the genome that is an interesting example of adaptive biology.
Structure, Function and Biogenesis of the Borrelia Cell Envelope
Sven Bergström and Wolfram R. Zückert
Over the past two decades, modern molecular tools have been crucial in shaping our current concepts of the Borrelia
cell envelope. Although Borrelia
spirochetes are often, but mistakenly described as Gram-negative bacteria due to their diderm, i.e. double-membrane envelopes, a closer examination reveals significant differences in composition and architecture. Probably most striking is the lack of LPS, the presence of major surface lipoproteins at the host-pathogen interface during transmission, persistence and ensuing pathogenic processes and the additional function of periplasmic flagella in defining cell shape. While surface lipoproteins such as the Osps interact with a variety of ligands in different organ tissues, they are also targets of the immune response and several have emerged as vaccine candidates. Some of the identified periplasmic lipoproteins, i.e. the OppAs, are components of substrate transport complexes. Investigations into integral membrane proteins led to the identification of several Borrelia
porins: P13, whose structure and function is unknown, DipA, which is specific for dicarboxylates and P66 (Oms66), which has a dual role as a pore-forming outer membrane protein with an extremely high single channel conductance and an adhesin for β3-integrin. The recently identified Tol homologs BesA, -B and -C appear to form a Type I 'channel' to export exogenous toxic agents such as antibiotics and to maintain infectivity by an unknown mechanism. Initial studies on envelope biogenesis pathways based on diderm proteobacterial model organisms already revealed significant deviations from the norm. This further bolsters the unique status of Borrelia
among microbial pathogens.
The Chic Motility and Chemotaxis of Borrelia burgdorferi
Stuart F. Goldstein, Chunhao Li, Jun Liu, Michael Miller, Md. Abdul Motaleb, Steven J. Norris, Ruth E. Silversmith and Charles W. Wolgemuth and Nyles W. Charon
The motility of Borrelia burgdorferi
is complex, enigmatic, yet quite elegant. Here we address specific and unique aspects of borrelial motility and chemotaxis. For B. burgdorferi
, in contrast to most spirochete species, the periplasmic flagella have a skeletal function that affects the entire shape of the cell. These organelles allow the spirochete to swim by generating backward-moving waves along the length of the cell and they do so by rotating within the periplasmic space. The well studied paradigm of Escherichia coli
and Salmonella enterica
serovar Typhimurium motility and chemotaxis does not directly apply to these unique bacteria. Recent advances are presented and summarized with respect to cell and periplasmic flagella structure, along with an exciting analysis of the in situ
hook-basal body-motor complex. In addition, advances in the dynamics of motility, how B. burgdorferi
swims, their chemotaxis, gene regulation and the role of motility and chemotaxis in the biology of B. burgdorferi
are summarized. Now that a foundation in this field has been established, the future can only continue to be exciting as we sort out the structure, function, regulation and the role of motility and chemotaxis in the life cycle of B. burgdorferi
Genetic Manipulation of Borrelia burgdorferi
Patricia A. Rosa, Felipe Cabello and D. Scott Samuels
Genetic studies in Borrelia burgdorferi
require special consideration of the bacterium's segmented genome, complex growth requirements and evolutionary distance from other genetically tractable bacteria, among other features. Despite these challenges, molecular genetics has been applied with growing success to study the biology and virulence of this important human pathogen. In this chapter we summarize the tools and techniques that are currently available for the genetic manipulation of B. burgdorferi
, placing them in the context of their utility and shortcomings. Our primary objective is to help researchers discern what is feasible and what is not practical when thinking about potential genetic experiments in B. burgdorferi
. We have summarized published methods and highlighted their critical elements, but we have not provided detailed protocols. Although a number of advances have been made since B. burgdorferi
was first transformed nearly 15 years ago, some fairly standard components of a good genetic system remain elusive. We mention these limitations as well and why they persist, if known. We hope to encourage investigators to explore what might be possible, in addition to recognizing what currently can be achieved, through genetic manipulation of B. burgdorferi
Evolutionary Genetics of Borrelia burgdorferi sensu lato
Daniel Dykhuizen and Dustin Brisson
Evolution can inform our understanding of the biology of Borrelia
. Four forces propel evolutionary change: mutation, genetic drift, migration and natural selection. Although these microevolutionary forces act primarily within populations, the same forces create macroevolutionary divergence of species. The prevailing data suggest that Borrelia burgdorferi
sensu lato was once a wide-ranging species in the Northern Hemisphere that rapidly separated into the species present today. The limited neutral divergence within species suggests that the long-term effective population size is small. The combined observations that Borrelia
was once a very wide-ranging species and that the current effective population sizes of the species are very small suggests that Borrelia
populations can expand rapidly when conditions are favorable and shrink rapidly when conditions are unfavorable. Divergence among and within genospecies has progressed primarily by mutation, as horizontal gene transfers are rare; so rare that most observed recombinants seen in nature are likely to have been selected to maintain genetic diversity. Recent migration, as well as range expansions and contractions, have resulted in overlapping patchy distributions of genospecies across the Northern Hemisphere. Most likely, infected birds are responsible for long distance migration, while infected mammals are important in range expansion and migration between local populations. Natural selection, especially that promoting host specificity, continues to shape the geographic distribution as well as the genetic diversity within and between species.
Ecology of Borreliae and Their Arthropod Vectors
Joseph Piesman and Tom G. Schwan
Borreliae are unique among the pathogenic spirochetes by requiring obligate blood-feeding arthropods for their transmission and maintenance in susceptible vertebrate host populations. With one exception, ticks transmit all borreliae and nearly all species are maintained in enzootic foci with humans being only accidental victims to infection. The true relapsing fever spirochetes are transmitted by many species of soft (Argasidae) ticks in the genus Ornithodoros
and in many geographic areas, specific species of ticks transmit only one species of spirochete. Specific tick-spirochete associations range from near-desert conditions to high elevation coniferous forests. The four principal hard tick (Ixodidae) species that transmit Lyme disease spirochetes (Borrelia burgdorferi
sensu lato) include Ixodes scapularis
in eastern North America, I. pacificus
in western North America, I. persulcatus
in Asia and I. ricinus
in Europe. These ticks are basically forest dwellers, spending most of their time hiding in the leaf litter of the forest floor, where humidity is high and the risk of desiccation is low. B. burgdorferi
sensu stricto, B. garinii
and B. afzelii
are the principal genospecies causing human disease. Of these three genospecies, B. burgdorferi
sensu stricto is unique in that it has a Holarctic distribution, spanning both the Old World (Palearctic Region) and the New World (Nearctic Region) in the northern hemisphere. Specific rodents were originally identified as the principal reservoirs of B. burgdorferi
sensu stricto, including the white footed mouse (Peromyscus leucopus
) in North America and the wood mouse (Apodemus sylvaticus
), yellow-necked mouse (Apodemus flavicollis
) and bank vole (Clethrionomys glareolus
) in Europe. Birds also have been identified as reservoirs of B. burgdorferi
sensu stricto on both sides of the Atlantic. Since the discovery of the Lyme borreliosis spirochete in 1982, a plethora of knowledge concerning the ecology of the spirochete, its tick vectors and vertebrate hosts has been brought forth through an incredible focus of research efforts. The task before the public health research community is not solely producing more ecological knowledge regarding the Lyme borreliosis spirochetes cycle in nature. Clearly, our immediate challenge is to synthesize and harness available knowledge of spirochete-tick-host interactions to develop ecologically-based interventions to combat Lyme borreliosis.
Utpal Pal and Erol Fikrig
causes Lyme disease in a variety of animals and humans. The spirochete is maintained in an enzootic cycle that primarily involves small mammals and Ixodes spp
. ticks. In the Northeastern United States, I. scapularis
is the main vector. As only I. scapularis
and closely related ticks transmit B. burgdorferi
, the spirochete-tick interactions are thought to be highly specific. Several spirochete and tick proteins that directly or indirectly contribute to the natural cycle of B. burgdorferi
infection have been identified. This chapter will focus on past discoveries and future challenges that are relevant to our understanding of B. burgdorferi
-tick interactions and on the development of novel preventive measures that interfere with the B. burgdorferi
Pathobiology of Lyme Disease Borrelia
Steven J. Norris, Jenifer Coburn, John M. Leong, Linden T. Hu and Magnus Höök
Lyme disease Borrelia
are host-dependent, tick-transmitted, invasive, nontoxigenic, persistent pathogens that cause disease in humans and other mammals primarily through the induction of inflammatory reactions. During transmission from the infected tick, the bacteria undergo dramatic changes in gene expression, resulting in adaptation to the mammalian environment. Expression of outer surface protein C (OspC) is essential during these early stages of colonization, although the mechanism by which OspC promotes spirochetal infectivity is unknown. Organisms multiply and spread locally and induce an inflammatory response that in humans results in an erythema migrans, the hallmark lesion of localized infection. The spirochetes enter the bloodstream during the primary infection and colonize multiple tissue sites, leading to the disseminated stage of infection. Motility and chemotaxis mechanisms undoubtedly play a role in this process. Several borrelial proteins have been implicated in adherence to host cell surface proteins and extracellular matrix components and are likely to be involved in the homing of Borrelia
to histologic compartments within each tissue, penetration of blood vessels and adherence to and migration through endothelial cells and tissue strata at distant sites. Activation of plasmin on the bacterial surface and induction of host proteases are thought to facilitate dissemination and/or inflammation. Most tissue damage appears to result from host inflammatory reactions. Although the mechanisms are not entirely understood, induction of cytokine/chemokine expression by bacterial lipoproteins and the resulting recruitment and activation of lymphocytes, macrophages and granulocytes play a major role in both local histopathology and constitutional symptoms. Despite their relatively low densities in tissues, Borrelia
cause neurologic, cardiovascular, arthritic and dermatologic manifestations during the disseminated and persistent stages of infection by mechanisms that remain largely a mystery. Immune evasion mechanisms, including the vls
antigenic variation system, complement-regulator acquiring surface proteins (CRASPs), down-regulation of highly antigenic surface proteins (such as OspC) and invasion of protective niches, permit the survival of the pathogens for months to years following infection despite robust antibody and cellular responses.
Pathogenesis of Relapsing Fever
Alan G. Barbour and Betty P. Guo
Relapsing fever is caused by several species of Borrelia
, all but one of which are transmitted from reservoir animals to humans by soft (argasid) ticks. The exception is B. recurrentis
, which is transmitted from one human to another by the body louse and, under certain conditions, may cause large epidemics. Relapsing fever Borrelia
species have a number of properties that facilitate invasion and dissemination in the mammalian host; these include neurotropism and adhesiveness for erythrocytes and platelets. But their most characteristic pathogenetic feature is multiphasic antigenic variation. Surface-exposed lipoproteins largely determine the antigenic identities of the spirochetes. Switching between genes encoding these highly polymorphic proteins is accomplished by a recombination in which the active gene at a single expression site is replaced by a copy of an archived, silent gene. The order of appearance of serotypes during infection is partially the function of the location of the archived gene and the surrounding sequences.
Animal Models of Borreliosis
Stephen W. Barthold, Diego Cadavid and Mario T. Philipp
Animal models have been extremely useful for understanding many features of Lyme and relapsing fever borrelioses, including pathogenesis, host response, host-vector interactions, in vivo
bacterial gene regulation, vaccinology and chemotherapy. Some degree of overlap is necessary with these subjects that are covered in other chapters, but this chapter attempts to focus on descriptive and practical aspects of various animal models used for borreliosis research.
Janis J. Weis and Linda K. Bockenstedt
The goal of this chapter is to provide an understanding of the dynamics of host responses during infection with Borrelia burgdorferi
. Pertinent literature will be reviewed with emphasis on studies that influenced our understanding of both the earliest responses of the mammalian host to infection by B. burgdorferi
and those that are associated with chronic infection and disease. Discussion of host responses in this chapter has direct implications for the pathogenesis of Lyme disease and will refer to aspects of the Pathobiology chapter by Norris et al. Animal models, which are more thoroughly discussed in the chapter by Barthold et al., have been critical to our understanding of the mechanistic aspects of the host response to B. burgdorferi
. This chapter will focus on the most widely studied animal model of Lyme borreliosis, the mouse model, with comparison made to human disease. The initial response to infection occurs before the spirochete and mammalian host have made direct contact, that is, during tick feeding and for this reason, reference also will be made to the Tick Interactions chapter by Pal and Fikrig. The host response is important to two features of Lyme borreliosis: the control of the spirochete and the development of tissue damage and clinical manifestations. Reference to the chapter by Radolf et al. on Lyme Disease in Humans will direct the reader to assessment of these responses in the context of the patient experience.
Detection of Borrelia burgdorferi
Guiqing Wang, Maria E. Aguero-Rosenfeld, Gary P. Wormser and Ira Schwartz
Numerous methods have been developed for detection of Borrelia burgdorferi
in a variety of specimen sources. These can be broadly divided into direct and indirect approaches. B. burgdorferi
can be visualized by microscopy, cultivated from numerous clinical and environmental specimens and detected by PCR-based modalities. These direct detection techniques are extensively used in experimental and epidemiological studies, but have not been widely employed as diagnostic approaches in the clinical setting. Immunological detection of B. burgdorferi
by ELISA and immunoblot is currently the primary tool for laboratory diagnosis of Lyme disease in humans. Recent advances in serologic testing include the development of assays based on recombinant B. burgdorferi
antigens or derived peptides, rather than sonicated whole cell lysates.
Lyme Disease Vaccines
Richard T. Marconi and Christopher G. Earnhart
The incidence of Lyme disease is increasing and its endemic regions are expanding. Preventive strategies that have focused primarily on increased public awareness and tick avoidance have proven ineffective. Vaccination remains a viable approach to be pursued in the prevention of Lyme disease. This chapter summarizes past, present and future efforts directed at the development of Lyme disease vaccines.
Lyme Disease in Humans
Justin D. Radolf, Juan C. Salazar and Raymond J. Dattwyler
Lyme disease is a rapidly emerging tick-borne, complex, multi-system infectious disorder caused by the spirochetal bacterium Borrelia burgdorferi
. The ailment, which affects adults and children alike, is widespread in the Northern Hemisphere and it continues to expand as humans encroach on the sylvatic habitat of the spirochete's mammalian reservoirs. Since first identified in the 1970s the incidence of Lyme disease has increased more than 30-fold and it is now considered the most prevalent arthropod-transmitted infection in both the United States and Europe. B. burgdorferi
is transmitted by ticks of the Ixodes ricinus
complex, including I. scapularis
, I. ricinus
and I. persulcatus
. In North America, B. burgdorferi
sensu stricto is the only species proven to be pathogenic for humans. In Europe, both B. afzelii
and B. garinii
are most commonly associated with human disease. The spirochete's genomic features, as well as its unique molecular architecture, are considered to have a seminal role not only in how it is transmitted from ticks to humans, but also how it triggers immune responses in afflicted individuals. Inflammatory manifestations associated with the disease result from the host's innate and adaptive immune responses to the bacterium, rather than from toxigenic molecules, which borrelia cannot produce. Indeed, the deposition of spirochetes into human dermal tissue generates a local inflammatory response that becomes manifest as erythema migrans (EM), the hallmark skin lesion of Lyme disease in North America. In Europe, two additional dermatologic disorders, borrelial lymphocytoma and acrodermatitis chronicum atrophicans (ACA) are frequently associated with infection. EM is frequently accompanied by 'flu-like' symptoms, including myalgias, arthralgias and fever, which are generally believed to be cytokine-mediated in response to hematogenous spread of the bacterium. If treated appropriately, the prognosis is excellent; however, if untreated, patients may develop a wide range of inflammatory clinical manifestations, most commonly involving the central nervous system, joints and heart. Within days of treatment, the signs and symptoms associated with the disease typically begin to subside, although in some individuals a complete recovery can take several weeks or even months. A minority of treated patients may go on to develop a poorly defined fibromyalgia-like illness, which is not responsive to prolonged antimicrobial therapy. Below we integrate current knowledge regarding the ecological, epidemiological, microbiological and immunological facets of Lyme disease into a conceptual framework that sheds light on the disorder that healthcare providers encounter.
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(EAN: 9781904455585 Subjects: [bacteriology] [microbiology] [medical microbiology] [molecular microbiology] [genomics])