Edited by Ali Karami
Edited by Ali Karami
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Lyme Disease, Edited by Ali Karami
Molecular Biology of Borrelia burgdorferi 1
Zoonotic Peculiarities of Borrelia burgdorferi s.l.:
Vectors Competence and Vertebrate Host Specificity 27
Alexandru Movila, Ion Toderas, Helen V. Dubinina,
Inga Uspenskaia and Andrey N. Alekseev
Advancement in Borrelia burgdorferi Antibody Testing:
Comparative Immunoblot Assay (COMPASS) 55
András Lakos and Erzsébet Igari
The Serology Diagnostic Schemes in Borrelia burgdorferi
Sensu Lato Infections – Significance in Clinical Practice 79
Małgorzata Tokarska-Rodak and Maria Kozioł-Montewka
Discovering Lyme Disease in Ticks and Dogs in Serbia –
Detection and Diagnostic Methods 95
Adaptation to Glucosamine Starvation in Borrelia
burgdorferi is Mediated by recA 113
Ryan G. Rhodes, Janet A. Atoyan and David R. Nelson
Porins in the Genus Borrelia 139
Iván Bárcena-Uribarri, Marcus Thein, Mari Bonde,
Sven Bergström and Roland Benz
I began to work with Borrelia burgdorferi (Bb), the causative agent of Lyme disease or
Lyme borreliosis, during my PhD at the University of Copenhagen in Denmark. In
1991 I was looking for an interesting research proposal to learn more about the
molecular genetics world of infectious disease agents.
My advisor shared information about the complex structure of Borrelia spirochetes
and the diseases they cause. Therefore, I started my experimental work in the Panum
Institute with the culturing of Bb in a very specific culture medium. I extracted the
smallest one mega base genome that is mostly linear compared to the circular genomes
of most other microorganism. Two interesting phenomenon were observed about Bb.
The one was that it contained more than a dozen extrachromosomal DNA elements or
plasmids that were mostly linear. The other interesting finding was that this linear
genetic structure had telomeres at the ends, like eukaryotic genomes.
I studied the genetic structure and molecular aspects of Bb mostly on molecular
detection. I went on to study diagnosis and protection against the diseases by
recombinant vaccines and subsequently published my papers on the subject, while
contemplating writing a book on this agent.
Lyme disease is an emerging infectious disease characterized by skin changes, joint
inflammation, and flu-like symptoms caused by the bacterium Borrelia burgdorferi
transmitted by the bite of a deer tick. Early symptoms may include fever, headache,
fatigue, depression, and a characteristic circular skin rash called erythema migrans.
Symptoms resolve in three to four weeks even without treatment, but secondary or
tertiary disease may develop if the initial infection is not treated. The symptoms may
affect the joints, heart, and central nervous system. In most cases, the infection and its
symptoms are eliminated by antibiotics, especially if the illness is treated early.
Delayed or inadequate treatment can lead to the more serious symptoms, which can be
disabling and difficult to treat.
Although Allen Steere realized in 1978 that Lyme disease was a tick-borne disease, the
cause of the disease remained a mystery until 1981 when B. burgdorferi was identified
by Willy Burgdorfer.
Lyme disease diagnosis and treatment is hampered by the lack of biological markers
and no standardized treatment protocols. The treatment of patients is hampered by the
fact that no combination of antibiotics completely eradicates the infection, which can
then posture as a self-perpetuating autoimmune response in the patient. There are
over 100 strains of Borrelia burgdorferi in the US, and 300 strains worldwide.
An interesting opportunity was presented to me in 2011 by Intech – Open Access
Publisher to write a book about Lyme disease. With the collaboration of scientists
around the world we started to prepare the chapters. It took over a year to finalize the
This book presents an overview of new diagnosis and treatment protocols arising from
current research. The addressed topics include the pathophysiology of Lyme disease,
antigenic variability, co-infection of other tick-borne diseases, the mechanisms that
allow the spirochete to evade the immune system, the lack of response to antibiotic
treatment, differential diagnosis of rheumatological, and neurological conditions.
In chapter one, the molecular biology of the Lyme disease agent is discussed in detail
with regards to genome organization with interesting linear and circular plasmids. In
chapter two, zoonotic peculiarities of Borrelia burgdorferi is discussed. Chapters three
and four cover detection, diagnosis, advancement in Borrelia burgdorferi antibody
testing, and the serology diagnostic schemes in Bb. Chapter five discusses the
discovery of Lyme disease in ticks and dogs. In chapter six you will read about
adaptation to glucosamine starvation in Bb. The final chapter covers porins in the
This book will be of use to medical doctors, clinicians, biologists and physicians
specializing in the treatment of Lyme disease.
I would like to extend my sincere appreciation and thanks to Professor Benz Roland,
Professor Nelson David, Dr Movila Alexandru, Dr Lakos András, Dr Tokarska-Rodak
Małgorzata, Dr Savic Sara, also special thanks to Igor Babic as publishing process
manager and my special gratitude to Intech – Open Access Publisher for their
encouraging support in the publication of this book.
Research Center of Molecular Biology
Baqyiatallh University of Medical Sciences, Tehran,
Molecular Biology of Borrelia burgdorferi
Research Center of Molecular Biology, Baqyiatallah
University of Medical Sciences, Tehran
Borrelia may be unique among prokaryote in having a genome that is mainly linear DNA
physical and genetic map of linear chromosome of B. burgdurferi has been published, it consist
of 946 to 952 kb Linear DNA (Sherwood et al;1993, Davidson et al;1992, Barbour et al; 1982).
This bacteria also contains several circular and specially linear plasmids from 5 to 55 kb.
Recently analysis of entire Agrobacterium tumefaciens C58 genome revealed presence of one
2.1-Mb linear and one 3- Mb circular plasmid (Servent et al; 1993) and it has been shown that
rhodococcus fascians contains 4 Mb linear chromosome (Crespi et al; 1992). Presence of several
linear plasmids seems the segmentation of Borrelias DNA to several linear pieces has led to
the suggestion that the relatively small linear chromosome and the linear plasmids actually
are minichromosoms. In B. hermsii it has been shown that total cellular DNA organized into
several complete gnomes (Kitten et al; 1992) and it suggests that linear plasmids are like
small chromosomes (Ferdows et al; 1989). Plasmid profile of B. burgdorferi from different
geographical area has been revealed significant heterogeneity a feature that can be used for
classification of bacteria within given species (Barbour et al; 1987, 1989). Another related
spirochete B. hermsii like B. burgdorferi has several linear and circular plasmids and the genes
responsible for antigenic variation are located in linear plasmids. In B. burgdorferi a 49 kb
linear plasmid carries the genes for Outer Surface Protein A and B (OspA and OspB)
(Barbour et al; 1987, Baril et al; 1989). It has been shown that passage of B. burgdorferi in BSK
medium changes the plasmid profile and loss of plasmids may change the infectivity of
organism (Schwan et al; 1988, Simpson, et al; 1990). Structure of Linear plasmids of B.
burgdorferi shows similarity to eukaryotic virus such as vaccinia and African swine fever
virus in having covalently closed ends like hairpin loops (Hinnebusch et al; 1991).
1.1 Taxonomy and classification
Borrelia burgdorferi belongs to the phylum Spirochaetes. The members of this phylum are
long, thin, helically coiled bacteria that have flagella (axial filaments) running lengthwise
between the peptidoglycan layer and the outer membrane. Movement of the flagellum
produces a screw-like motion that propels the organism.
The phylum Spirochates contains a single class (Spirochaetes), a single order
(Spirochaetales), and three families: Brachyspiraceae, Leptospiraceae, and Spirochaetaceae.
Fig. 1. Spirochaetaceae
The Spirochaetaceae family includes the genus Treponema and the genus Borrelia . Treponema
pallidum is the causative agent of the sexually-transmitted disease syphilis.
The three members of the Borrelia genus Borrelia burgdorferi sensu stricto, Borrelia garinii , and
Borrelia afzelii are collectively known as Borrelia burgdorferi sensu lato, and are the causative
agents of Lyme disease.
1.2 Structure and morphology
Borrelia cells average 0.2 to 0.5 µm by 4 to 18 µm, and have fewer coils than Leptospira. The
periplasmic flagella originate from either end of the spirochete (where they are anchored to
the cytoplasmic membrane) and wind around the protoplasmic cylinder, imparting both
motility and shape to the organism—in contrast to other bacteria, in which the
peptidoglycan layer determines the shape.
The role of flagella in imparting Borrelia 's helical shape was established by inactivation of
the flaB gene, which encodes the major flagellar filament protein, FlaB. This produced
bacteria that lacked periplasmic flagella, were non-motile and rod-shaped.
Whereas the motility of externally-flagellated bacteria is hindered in viscous substances, that
of spirochetes is enhanced, and about 6% of the chromosomal genome encodes proteins
involved in motility and chemotaxis.
Molecular Biology of Borrelia burgdorferi
1.3 Genome organization of Borrelia burgdorferi
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.
The highly unusual segmented genomes of Borrelia species can contain over 20 utonomously
replicating DNA molecules. Many of the molecules, including the chromosome, are linear
with covalently closed hairpin ends.
2. Molecular biology
2.1 The Borrelia burgdorferi genome
The genome of Borrelia burgdorferi consists of a single linear chromosome and several
plasmids, both linear and circular. To date—as of January 2005—only the genome of Borrelia
burgdorferi sensu stricto B31 strain has been fully sequenced.
Distribution of cellular functions of E. coli and B. burgdorferi genes 
Category B. burgdorferi genes (%)
Intermediary metabolism 4.9%
Biosynthesis of small molecules 3.1%
Macromolecule metabolism 22.2%
Cell Structure 37.0%
Cellular processes 7.4%
Other functions 5.6%
Unknown functions 19.8%
Molecular Biology of Borrelia burgdorferi Download full-text
2.2 Chromosomal genome
B. burgdorferi contains a single linear chromosome of approximately 900 kb, and about 90%
of it is comprised of coding sequences. Most of the genes encoded by the chromosomal
genome are homologous to genes of known function.
2.3 Extra-chromosomal genome
The extra-chromosomal genome of B. burgdorferi B31 consists of 12 linear plasmids and nine
circular plasmids that total 610 kb in size.
2.3.1 Linear plasmids
There are two linear plasmids in B. burgdorferi that are absolutely necessary for persistent
infection of a mammalian host. These plasmids, known as lp25 and lp28-1, are relatively
unstable in culture, and are commonly lost after a few generations of in vitro growth.
Bacteria that have lost either of these two plasmids remain capable of in vitro growth, but
lose their ability to cause persistent infection even in immunocompromised mice. The lp25
plasmid contains a gene, pncA, which encodes a nicotinamidase whose function is most
likely the biosynthesis of NAD; by all appearances its activity is dispensable growth in
vitro , but crucial for growth within a host. Transforming the lp25- spirochetes with pncA
on a shuttle vector replaces the requirement of lp25 in vivo. Likewise, reintroduction of the
entire lp25 plasmid (by transformation) into lp25- spirochetes successfully rescues
2.3.2 Circular plasmids
An unusual feature of B. burgdorferi is a series of related 32-kb circular plasmids, termed
cp32s. These have been found to be prophage genomes, and it is believed that they play a
role in the horizontal transfer of DNA among spirochetes that share a common geographical
and ecological niche. [3, 4]
2.3.3 OuterSurface Proteins (Osps)
The Outer Surface Proteins (Osps) of B. burgdorferi are lipoproteins that play an important
role in interacting with interstitial and cellular components of insect and mammalian hosts.
OspA, the most studied of the Osps, is expressed on spirochetes in unfed nymphs and adult
ticks, as well as in culture. OspA mediates adherence to the cells of the tick midgut, which
presumably allows spirochetes to avoid endocytosis by tick gut cells during digestion of the
blood meal. The ability of Borrelia to regulate expression of OspA indicates that it also plays
a role in detachment from the midgut, which allows the bacteria to enter the mammalian
host when the tick takes a second bloodmeal.
During tick feeding, Borrelia in the midgut upregulate expression of another outer surface
protein, OspC, and begin to move toward the salivary glands. This evident correlation
suggests that OspC might play a role in transmission. Once it has entered the mammalian
host, Borrelia downregulates OspA and exhibits variable OspC upregulation patterns.
Although B. burgdorferi possesses only one copy of the ospC gene, sequences vary
significantly from one strain to the next, which accounts for the observed antigenic variation