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## **Meet the editor**

Dr Ali Karami was born in Tehran, Iran in 1957. He is Associate Professor of Molecular Biology and Genetic Engineering and is a Medical Biotechnology expert at the Research Center of Molecular Biology, Baqiyatallah University of Medical Sciences. He received his PhD in 1996 from Copenhagen University Faculty of Medical Science in Denmark. Since 1991 he has been working on

the molecular biology of Borrelia burgdorferi with gene cloning and protein expression for Lyme disease recombinant vaccine and other infectious disease agents. He has published several books and numerous articles in national and international journals. He is currently head of Microarray Research Group and working on rapid molecular detection technologies, recombinant proteins, DNA vaccines and bioinformatics.

Contents

**Preface VII**

Ali Karami

Sara Savic

Chapter 7 **Porins in the Genus** *Borrelia* **139**

Sven Bergström and Roland Benz

Chapter 1 **Molecular Biology of** *Borrelia burgdorferi* **1**

Chapter 2 **Zoonotic Peculiarities of** *Borrelia burgdorferi* **s.l.:**

Inga Uspenskaia and Andrey N. Alekseev

András Lakos and Erzsébet Igari

Alexandru Movila, Ion Toderas, Helen V. Dubinina,

Chapter 3 **Advancement in** *Borrelia burgdorferi* **Antibody Testing: Comparative Immunoblot Assay (COMPASS) 55**

Chapter 4 **The Serology Diagnostic Schemes in** *Borrelia burgdorferi*

Chapter 5 **Discovering Lyme Disease in Ticks and Dogs in Serbia – Detection and Diagnostic Methods 95**

Ryan G. Rhodes, Janet A. Atoyan and David R. Nelson

Iván Bárcena-Uribarri, Marcus Thein, Mari Bonde,

Chapter 6 **Adaptation to Glucosamine Starvation in** *Borrelia burgdorferi* **is Mediated by** *recA* **113**

**Vectors Competence and Vertebrate Host Specificity 27**

**Sensu Lato Infections – Significance in Clinical Practice 79**  Małgorzata Tokarska-Rodak and Maria Kozioł-Montewka

### Contents

#### **Preface XI**

Preface

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

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

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

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

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

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

molecular genetics world of infectious disease agents.

contemplating writing a book on this agent.

disabling and difficult to treat.

by Willy Burgdorfer.

genetic structure had telomeres at the ends, like eukaryotic genomes.

### Preface

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

#### VIII Preface

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 book.

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 genus Borrelia.

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.

> **Ali Karami** Research Center of Molecular Biology Baqyiatallh University of Medical Sciences, Tehran, Iran

VIII Preface

book.

genus Borrelia.

specializing in the treatment of Lyme disease.

encouraging support in the publication of this book.

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

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

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

**Ali Karami**

Iran

Research Center of Molecular Biology

Baqyiatallh University of Medical Sciences, Tehran,

over 100 strains of Borrelia burgdorferi in the US, and 300 strains worldwide.

**1**

Ali Karami

*Iran*

**Molecular Biology of** *Borrelia burgdorferi*

*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).

*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

The phylum Spirochates contains a single class (Spirochaetes), a single order (Spirochaetales), and three families: Brachyspiraceae, Leptospiraceae, and Spirochaetaceae.

**1. Introduction**

**1.1 Taxonomy and classification**

produces a screw-like motion that propels the organism.

*Research Center of Molecular Biology, Baqyiatallah*

*University of Medical Sciences, Tehran*

## **1**

### **Molecular Biology of** *Borrelia burgdorferi*

#### Ali Karami

*Research Center of Molecular Biology, Baqyiatallah University of Medical Sciences, Tehran Iran*

#### **1. Introduction**

*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. 2.

Fig. 3.

Molecular Biology of *Borrelia burgdorferi* 3

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.

The Spirochaetaceae family includes the genus *Treponema* and the genus *Borrelia* . *Treponema*

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

*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

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

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

*pallidum* is the causative agent of the sexually-transmitted disease syphilis.

bacteria that lacked periplasmic flagella, were non-motile and rod-shaped.

Fig. 1. Spirochaetaceae

agents of Lyme disease.

**1.2 Structure and morphology**

peptidoglycan layer determines the shape.

involved in motility and chemotaxis.

Fig. 3.

Molecular Biology of *Borrelia burgdorferi* 5

*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

The extra-chromosomal genome of *B. burgdorferi* B31 consists of 12 linear plasmids and nine

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

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

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

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

**2.2 Chromosomal genome**

**2.3 Extra-chromosomal genome**

**2.3.1 Linear plasmids**

infectivity. [2]

**2.3.2 Circular plasmids**

and ecological niche. [3, 4]

**2.3.3 OuterSurface Proteins (Osps)**

host when the tick takes a second bloodmeal.

circular plasmids that total 610 kb in size.

genome are homologous to genes of known function.

#### **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.

Table 1.

#### **2.2 Chromosomal genome**

4 Lyme Disease

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

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

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*

Distribution of cellular functions of *E. coli* and *B. burgdorferi* genes [1]

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%

Category *B. burgdorferi* genes (%)

**1.3 Genome organization of** *Borrelia burgdorferi*

future progress in this arena.

**2. Molecular biology**

Table 1.

with covalently closed hairpin ends.

**2.1 The** *Borrelia burgdorferi* **genome**

*burgdorferi* sensu stricto B31 strain has been fully sequenced.

*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 infectivity. [2]

#### **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

Molecular Biology of *Borrelia burgdorferi* 7

\*The telomeric sequences of the nine linear plasmids assembled as part of this study were not determined; estimation of the number of missing terminal nucleotides by restriction analysis suggests that less than 1,200 bp is missing in all cases. Comparisons with previously determined sequences of lp 16.9 and one terminus of lp28-1 indicate that 25, 60 and 1,200 bp

lp17 16,828 bp (23.1% GC) lp25 24,182 bp (23.3% GC) lp28-1 26,926 bp (32.3% GC) lp28-2 29,771 bp (31.5% GC) lp28-3 28,605 bp (25.1% GC) lp28-4 27,329 bp (24.4% GC) lp36 36,834 bp (26.8% GC) lp38 38,853 bp (26.1% GC) lp54 53,590 bp (28.1% GC) Coding sequences (71%) Intergenic sequence (29%) 430 coding sequences

70 (16%) with identified database match 110 (26%) match hypothetical proteins 250 (58%) with no database match

16S 444581–446118 23S 438590–441508 5S 438446–438557 23S 435334–438267 5S 435201–435312

**Stable RNA**

**Transfer RNA**

tmRNA 46973–47335 mpB 750816–751175

are missing, respectively.

rRNA sequences

fesmid sequences

 fla sequences hbb sequences

**Chromosomally-encoded genes**

34 species (8 clusters,14 single genes)

**Ribosomal RNA Chromosome coordinates**

between OspC proteins. The host immune system plays an important role in selecting for certain strains by eliminating the immunodominant ones.

Fig. 4.

#### **3. Genome features in** *Borrelia burgdorferi*

Chromosome 910,725 bp (28.6% G+C)

Coding sequences (93%)

RNAs (0.7%)

Intergenic sequence (6.3%)

853 coding sequences 500 (59%) with identified database match 104 (12%) match hypothetical proteins 249 (29%) with no database match

#### **Plasmids**

cp9 9,386 bp (23.6% GC) cp26 26,497 bp (26.3% GC)

between OspC proteins. The host immune system plays an important role in selecting for

certain strains by eliminating the immunodominant ones.

**3. Genome features in** *Borrelia burgdorferi*

Chromosome 910,725 bp (28.6% G+C)

500 (59%) with identified database match 104 (12%) match hypothetical proteins 249 (29%) with no database match

Coding sequences (93%)

Intergenic sequence (6.3%)

853 coding sequences

cp9 9,386 bp (23.6% GC) cp26 26,497 bp (26.3% GC)

RNAs (0.7%)

**Plasmids**

Fig. 4.

lp17 16,828 bp (23.1% GC) lp25 24,182 bp (23.3% GC) lp28-1 26,926 bp (32.3% GC) lp28-2 29,771 bp (31.5% GC) lp28-3 28,605 bp (25.1% GC) lp28-4 27,329 bp (24.4% GC) lp36 36,834 bp (26.8% GC) lp38 38,853 bp (26.1% GC) lp54 53,590 bp (28.1% GC) Coding sequences (71%) Intergenic sequence (29%) 430 coding sequences 70 (16%) with identified database match 110 (26%) match hypothetical proteins 250 (58%) with no database match

#### **Ribosomal RNA Chromosome coordinates**

16S 444581–446118 23S 438590–441508 5S 438446–438557 23S 435334–438267 5S 435201–435312

#### **Stable RNA**

tmRNA 46973–47335 mpB 750816–751175

#### **Transfer RNA**

34 species (8 clusters,14 single genes)

\*The telomeric sequences of the nine linear plasmids assembled as part of this study were not determined; estimation of the number of missing terminal nucleotides by restriction analysis suggests that less than 1,200 bp is missing in all cases. Comparisons with previously determined sequences of lp 16.9 and one terminus of lp28-1 indicate that 25, 60 and 1,200 bp are missing, respectively.

#### **Chromosomally-encoded genes**

Molecular Biology of *Borrelia burgdorferi* 9

Gb\_ba:Borrg16s M88329 *rrs* (16S rRNA) 11/93 1,537bp

Gb\_ba:Bor23srrna M93664 *rr1* (23S rRNA) 212 6/92 398bp

Gb\_ba:Bb23s5s X85745 *rrl rrs* (23S & 5S rRNA) B31 7/95 2,093bp Gb\_ba:Borrg23s M88330 *rr1* (23S rRNA) 1/93 2,926bp

number Description Strain Date of

number Description Strain Date of

Gb\_ba:Borflass L29234 *fla* (flagellin) 212 7/94 193bp

Gb\_ba:Bbfla2 X15661 *fla* (flagellin) B31 2/94 1,011bp

Gb\_ba:Borflab31a L29200 *fla* (flagellin) B31 7/94 193bp

Gb\_ba:Borflag M34710 *fla* (flagellin) B31 5/95 684bp

Gb\_ba:Bbgehofla X56334 *fla* (flagellin) GeHo 4/93 1,426bp

Gb\_ba:Bbfla X15660 *fla* (flagellin) GeHo 2/94 1,011bp

*fla* flagellumassociated 41kD antigen (flagellin)

*fla* flagellumassociated 41kD antigen (flagellin)

rRNA genes). 6/93 616bp

spacer 212 7/94 253bp

spacer B31 7/94 254bp

spacer CA2 7/94 255bp

entry Size

entry Size

B31 9/93 1,435bp

BO 5/94 1,008bp

Genbank mnemomic Accession

Table 2.

Genbank mnemomic

Table 4.

*orf38* (open reading frame); *orf37* (open reading frame); *orf36* (open reading frame); *ylxH* (putative ATP-binding protein); *flhF* (flagella asociated putative GTP-binding protein protein); *flhA* (flagellar protein required for flagellar formation); *flhB* (flagellar protein required for flagellar formation); *fliR* (flagellar protein required for flagellar formation); *fliQ* (flagellar protein required for flagellar formation); *fliP* (flagellar protein required for flagellar formation); *fliZ* (flagellar protein required for flagellar formation); *fliN* (flagellar switch protein); *fliM* (flagellar switch protein); *orf25* (open reading frame); *motB* (flagellar motor rotation protein B); *motA* (flagellar motor rotation protein A); *flgE* (flagellar hook protein); *ylxG* (flagellar synthesis); *orf20* (open reading frame); *orf19* (open reading frame); *orf18* (open reading frame); *fliI* (flagellar synthesis); *fliH* (flagellar synthesis); *fliG* (flagellar switch protein); *fliF* (flagella basal-body M ring protein); *fliE* (flagella basal-body protein); *flgC* (flagella associated rod protein); *flgB* (flagella associated rod protein); *hslU* heat shock protein); *hslV* (heat shock protein); *smg* (?); *orf7* (open reading frame); *ftsZ* (cell division protein); *ftsA* (cell division protein); *divIB* (cell division protein); *ftsW* (cell division protein); *mraY* (phosphotransferase); *murF* (pentapeptide presynthetase)

Genbank mnemomic

Gb\_ba:Bbu28760 U28760

Gb\_ba:Borplsctop L32861

Table 6.

Table 13.

Accession

#### **3.12** *Borrelia burgdorferi* **fusion sequences**

Table 13.

Molecular Biology of *Borrelia burgdorferi* 23

Fig. 6. Electron microscopy of unfixed, negative stained DK1 strain (skin isolate).

#### **3.13** *Borrelia burgdorferi* **promoter sequences**

Table 14.

Fig. 5. Electron microscopy of unfixed, negative stained DK1 strain (skin isolate). This strain consist of two morphologically distinct borrelia A. small and B. larger *borrelia*. Bar 1 mm. Magnification 10,260 x.

Gb\_ba:Borproma M28680 promoter B31 6/90 194bp Gb\_ba:Borpromb M28681 promoter B31 6/90 203bp Gb\_ba:Borpromc M28682 promoter B31 6/90 78bp

Fig. 5. Electron microscopy of unfixed, negative stained DK1 strain (skin isolate). This strain consist of two morphologically distinct borrelia A. small and B. larger *borrelia*. Bar 1 mm.

number Description Strain Date of

entry Size

**3.13** *Borrelia burgdorferi* **promoter sequences**

Genbank mnemomic Accession

Table 14.

Magnification 10,260 x.

Fig. 6. Electron microscopy of unfixed, negative stained DK1 strain (skin isolate).

Molecular Biology of *Borrelia burgdorferi* 25

Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP (June 1982). "Lyme

Barbour, A.G. (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med

Barbour, A. G. & Garon, C. F., 1987. The Gene encoding major surface protein of *Borrelia burgdorferi* are located on a plasmid. Ann. NY. Acad. Sci., 539: 144-153. Barbour, A. G. , 1988. *Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent. : J.*

Barbour A.G. & Garon. C. F., 1987.Linear plasmids of the bacterium Borrelia burgdorferi

Shigekawa, K., and Dower, W.J. (1988) Electroporation of eukaryotes and prokaryotes: a

Barbour, A. G., 1989.Classification of *Borrelia burgdorferi* on the basis of plasmid profiles.

Barbour. A. G., 1989.The Molecular biology of *Borrelia.* Rev .Infec.Disease. 11 suppl 6 s :

Bergstrom. S. Bundoc. V. G & Barbour. A., 1989. Molecular analysis of linear plasmid -

Crespi, M., Messense, E. A Caplan, M. Von Montagu & Desomer, 1992. Fasciation Induction

Davidson .B. E, Mac Dugall J & Girons. I. S., 1992. Physical map of the linear chromosome

Ferdows .M. S, Barbour .A. G., 1989.Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the lyme disease agent. Proc.Natl. Acad.Sci.USA., 86: 5969-5973. Hansen, K., hovmark, A., Lebech,A .m., Lebech,k., Olsson,I., Halkier,l ;et al. 1992.

Hinnebusch. J & Barbour. A. G., 1991.Linear plasmids of Borrelia burgdorferi have a

Schwan.T. G, Burgdorfer. W. & Garon. C. F ., 1988. Change in Infectivity and Plasmid Profile

Servent, A. A., Charachon, S, Y., Bilak,E, J., Karayan, L. & Ramuz .M., 1993. Presence of one

Sherwood, C & Wai .M.H.,1993. Linear chromosomal and genetic map of Borrelia burgdorferi, the lyme disease Agent. Molecular miccrobiol., 8 (5): 967-980.

general approach to the introduction of macromolecules into cells. BioTechniques

encoded major surface protein, OspA and OspB, of the lyme disease spirochaete

by the phytopatogen Rhodococoous fascians depends upon a linear plasmid

of the bacterium *Borrelia burgdorferi*, a causative agent of lyme disease, and

Roxithromycin in Lyme borreliosis: discrepant Results of in vitro and in vivo animal susceptibility study and clinical trial in patients with erythema migrans.

telomeric structure and sequence similar to those of eukaryotic virus. J. Bacteriol.,

of Lyme Disease Spirochete, Borrelia Borgdorferi, as a Result of In vitro cultivation.

linear and one circular chromosome in the Agrobacterium tumefaciens C58

disease-a tick-borne spirochetosis?". Science 216 (4552): 1317–9.

have covalently closed ends. Science., 237: 409-411 .

*Borrelia burgdorferi*. Mol.microbiol.,3: 479-486.

Acta.Derm.Venerol., 72: 297-300.

Inf. Immun ., 56: 1831-1836.

genome. J. bacteriol., 175: 7869-7874.

173: 7233-7239.

Encoding a cytokinin synthase gene. EMBO. J., 11: 795-804.

localization of rRNA Genes. J.Bacteriol., 174: 3766-3774.

**4. References**

57: 521-525.

6: 742-751.

1470-1474.

*Clin.Microbiol., 26: 475-478.*

Zbl.Bakt.Supl.,18: 1-7.

Fig. 7. Plasmids isolated from different strains of *Borrelia burgdorferi* : The Dk1 strain (B), Dk5 strain (C), DK6 strain (D), DK 2 strain (E), DK7 strain (F) and a super coiled circular molecular weight marker (G). Linear molecular markers (A) ( HindIII fragments of Lambda DNA). Samples were separated in 0.3% gel at 14C for 20 hr then stained with ethidium bromide.

Fig. 8. Elrectron micrograph of 25 kb plasmid extracted from DK1 strain . One supercoiled plasmid. magnification 52000 x.

#### **4. References**

24 Lyme Disease

Fig. 7. Plasmids isolated from different strains of *Borrelia burgdorferi* : The Dk1 strain (B), Dk5 strain (C), DK6 strain (D), DK 2 strain (E), DK7 strain (F) and a super coiled circular molecular weight marker (G). Linear molecular markers (A) ( HindIII fragments of Lambda DNA). Samples were separated in 0.3% gel at 14C for 20 hr then stained with ethidium

Fig. 8. Elrectron micrograph of 25 kb plasmid extracted from DK1 strain . One supercoiled

bromide.

plasmid. magnification 52000 x.

**1. Introduction**

**vectors and reservoirs**

**2**

*1Republic of Moldova 2Russia Federation*

**Zoonotic Peculiarities of** *Borrelia*

**and Vertebrate Host Specificity**

 Inga Uspenskaia1 and Andrey N. Alekseev2 *1Institute of Zoology, Moldova Academy of Science 2Zoological Institute, Russia Academy of Science*

*burgdorferi* **s.l.: Vectors Competence**

Alexandru Movila1, Ion Toderas1, Helen V. Dubinina2,

Tick-borne diseases are of increasing public health concern because of range expansions of both vectors and pathogens (Daniel et al., 2003). Lyme borreliosis is the most common arthropod-borne human disease in temperate regions of the northern hemisphere. The causative agents of Lyme borreliosis (and other tick-borne borrelioses) are spirochaetes belonging to the *Borrelia burgdorferi* sensu lato (s.l.) species complex. It is well known that *B. burgdorferi* are unique among the pathogenic spirochaetes by requiring obligate bloodfeeding arthropods for their transmission and maintenance in vertebrate host populations. All known causative agents of borrelioses circulate between ticks (Arachnida, Acari, Ixodoidea) and wide variety of vertebrates species (mammals, birds and reptiles). Consequently, *Borrelia* populations are shaped by the dynamics and demographic processes of host and vector populations, host and vector immune responses and extrinsic abiotic factors (e.g. combination of temperature, humidity and types of climate and landscape)

The main goals of this chapter are to summarize the results of vector competence analyses and vertebrate hosts' specificity for Lyme disease agents, give a general description of *B.*

From the time of *B. burgdorferi* discovery a large number of *Borrelia* isolates has been obtained from various vertebrate species, including humans. Involvements of other species from the *B. burgdorferi* s.l. complex were recognized recently. *Borrelia* spirochaetes are transmitted to reservoirs (including humans) by all 3 developmental stages of ixodid ticks (Fig 1 A), but the nymphal stage appears to be the most important at least in the North America and West Europe (Anderson et al., 1990; Kurtenbach et al., 1998; Kurtenbach et al., 2006).Considering

**2.** *Borrelia burgdorferi* **s.l. genospecies diversity and association with**

affecting host and vector populations (Margos et al., 2011).

*burgdorferi* (s.l.)—tick—vertebrate hosts relationships in natural foci.

### **Zoonotic Peculiarities of** *Borrelia burgdorferi* **s.l.: Vectors Competence and Vertebrate Host Specificity**

Alexandru Movila1, Ion Toderas1, Helen V. Dubinina2, Inga Uspenskaia1 and Andrey N. Alekseev2 *1Institute of Zoology, Moldova Academy of Science 2Zoological Institute, Russia Academy of Science 1Republic of Moldova 2Russia Federation*

#### **1. Introduction**

26 Lyme Disease

A G Barbour, C J Carter, V Bundoc, and J Hinnebusch. The nucleotide sequence of a linear

Fraser CM, Casjens S, Huang WM, et al. (December 1997). "Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi". Nature 390 (6660): 580–6. Bundoc, V.G., and Barbour, A.G. (1989) Clonal polymorphisms of outer membrane protein

Hinnebusch, J., and Tilly, K. (1993) Linear plasmids and chromosomes in bacteria. Mol

Rosa, P., Samuels, D.S., Hogan, D., Stevenson, B., Casjens, S., and Tilly, K. (1996) Directed

Saint Girons, I., Old, I.G., and Davidson, B.E. (1994) Molecular biology of the Borrelia,

Samuels, D.S., and Garon, C.F. (1993) Coumermycin A1 inhibits growth and induces

Tilly, K., Casjens, S., Stevenson, B., Bono, J., Samuels, D.S., Hogan, D. and Rosa, P. (1997) The

Ali Karami , Peter Hindeersson , Niels Hoiby , Saeid Morovvati and Akbar Khalilpour.

Ali KARAMI, Seyed Mohammad Javad HOSSEYNI, Yaser KIARUDI .Molecular

A Karami, P Hindersson, N Høiby, S Morovvati. OspA Sequence Comparison and

Sorouri R, Ranjbar R, Jonaidi Jafari N, Karami A. Rapid detection of Borrelia burgdorferi strains by nested polymerase chain reaction. Pak J Biol Sci. 2009 Mar 1;12(5):463-6. Galdwin, Mark; Trattler, Bill (2009). Spirochetes: Clinical Microbiology Made Ridiculously

Samuels DS; Radolf, JD. (2010). Borrelia: Molecular Biology, Host Interaction and

Protein. Iranian Journal of Public Health 2006;35(2):16-24.

Pathogenesis. Caister Academic Press. ISBN 978-1-904455-58-5.

Simple. MedMaster, Inc. ISBN 978-0-940780-81-1.

targeted inactivation of the *ospC* gene. Mol. Microbiol. 25:361-373.

insertion of a selectable marker into acircular plasmid of *Borrelia burgdorferi*. J

relaxation of supercoiled plasmids in *Borrelia burgdorferi*, the Lyme disease agent.

*Borrelia burgdorferi* circular plasmid cp26: conservation of plasmid structure and

Linear and Circular Plasmids in Skin and Cerebrospinal Fluid Isolates of Borrelia burgdorferi Agent of Lyme Disease. Pakistan Journal of Biological Sciences 2006

Characterization of Borrelia burgdorferi Linear Plasmids by DNA Hybridization, PCR, Two-Dimensional Gel Electrophoresis, and Electron Microscopy. Turk J Biol

Protection Against Borrelia burgdorferi Infection in Gerbils by Recombinant OspA

other prokaryotes.J Bacteriol. 1996 November; 178(22): 6635–6639.

OspB of *Borrelia burgdorferi*. Infect Immun 57: 2733-2741.

bacteria with linear replicons. Microbiology 140: 1803-1816.

Antimicrob Agents Chemother 37: 46-50.

Microbiol 10: 917-922.

Bacteriol 178: 5946-5953.

(9): 15 ,2787-2793

31 (2007) 73-80.

plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of

Tick-borne diseases are of increasing public health concern because of range expansions of both vectors and pathogens (Daniel et al., 2003). Lyme borreliosis is the most common arthropod-borne human disease in temperate regions of the northern hemisphere. The causative agents of Lyme borreliosis (and other tick-borne borrelioses) are spirochaetes belonging to the *Borrelia burgdorferi* sensu lato (s.l.) species complex. It is well known that *B. burgdorferi* are unique among the pathogenic spirochaetes by requiring obligate bloodfeeding arthropods for their transmission and maintenance in vertebrate host populations. All known causative agents of borrelioses circulate between ticks (Arachnida, Acari, Ixodoidea) and wide variety of vertebrates species (mammals, birds and reptiles). Consequently, *Borrelia* populations are shaped by the dynamics and demographic processes of host and vector populations, host and vector immune responses and extrinsic abiotic factors (e.g. combination of temperature, humidity and types of climate and landscape) affecting host and vector populations (Margos et al., 2011).

The main goals of this chapter are to summarize the results of vector competence analyses and vertebrate hosts' specificity for Lyme disease agents, give a general description of *B. burgdorferi* (s.l.)—tick—vertebrate hosts relationships in natural foci.

#### **2.** *Borrelia burgdorferi* **s.l. genospecies diversity and association with vectors and reservoirs**

From the time of *B. burgdorferi* discovery a large number of *Borrelia* isolates has been obtained from various vertebrate species, including humans. Involvements of other species from the *B. burgdorferi* s.l. complex were recognized recently. *Borrelia* spirochaetes are transmitted to reservoirs (including humans) by all 3 developmental stages of ixodid ticks (Fig 1 A), but the nymphal stage appears to be the most important at least in the North America and West Europe (Anderson et al., 1990; Kurtenbach et al., 1998; Kurtenbach et al., 2006).Considering

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

*B. carolinensis Ixodes minor* Rodents,

*B. tanukii Ixodes tanuki* Unknown

*Haemaphysalis longicornis*

*B. yangtze Ixodes granulatus*,

(Rudenko et al., 2011 with modifications)

**3. Ticks–Borreliae interface 3.1 Vectors ecological groups**

**3.2 Vector competence for** *Borrelia burgdorferi* **s.l.**

3-rd group

Vectors Competence and Vertebrate Host Specificity 29

birds

(possibly dogs and cats)

*B. japonica Ixodes ovatus* Rodents Japan Kawabata et al.

*B. sinica Ixodes ovatus* Rodents China Masuzawa et al.

*B. turdi Ixodes turdus* Birds Japan Fukunaga et al.

Genomospecies 2 *Ixodes pacificus* Unknown United States Postic et al. (2007)

the human sensitivity to *B. burgdorferi* s.l. and results of the newest publications, the complex of 18 *Borrelia* species (Table 1) can be divided into 3 major groups (Rudenko et al., 2011): the first and second groups contains 9 species with pathogenic potential and species that have not yet been reported in or isolated from humans, respectively, and the 3rd still not named group proposed as genomospecies 2 represented by two far-western US isolates.

There are two big ecological groups of tick species. Ticks seek hosts by an interesting behaviour called "questing." Questing ticks (=exophilic, polyxenous) crawl up the stems of grass or perch on the edges of leaves on the ground in a typical posture with the front legs extended, especially in response to a host passing by (Fig. 1 A). In contrast to questing ticks, nidicolous (=endophilic, mainly monoxenous) ticks live in secluded enclosures such as caves, burrows and nests of their hosts or harborages near these nests (Fig. 1 B, C) (Sonenshine, 1991).

The primary vectors of Lyme borreliosis spirochaetes to humans in temperate regions of the northern hemisphere are closely taxonomically related tick species: *I. pacificus* in Western North America, *I. persulcatus* in Eurasia, *I. ricinus* in Europe and *I. scapularis* in eastern North

Table 1. Currently known species from the Borrelia burgdorferi sensu lato complex

distribution

Reference

(2009a)

(1993)

(2001)

(1996b)

Japan Fukunaga et al. (1996)

Rodents China Chu et al. (2008)

United States Rudenko et al.

*Borrelia* species Vector Reservoirs Geographical

*B. bavariensis Ixodes ricinus* Rodents Europe Margos et al.

distribution

United States

United States

Birds United States Rudenko et al.

Europe, North Africa

Europe, United States,

Asia

Rodents Europe,

Rodents, birds, lizards, big mammals

Birds, lizards, rodents

Rodents, lizards

*B. spielmanii Ixodes ricinus* Rodents Europe Richter et al. (2004,

Birds, lizards

rabbit

Kangaroo rat, mule deer

Rodents Asia, Europe Canica et al. (1993)

Reference

(2009)

Postic et al. (1998)

Baranton et al. (1992); Alekseev et

(1992); Gorelova et

al. (2010)

al. (1996); Korenberg et al.

Margos et al. (2010)

Le Fleche et al. (1997); Alekseev et

Alekseev et al. (1998, 2010)

al. (2010)

2006)

Asia, Europe Wang et al. (1997);

(2009b)

United States Marconi et al. (1995)

United States Postic et al. (2007)

(2010)

Asia, Europe Baranton et al.

*Borrelia* species Vector Reservoirs Geographical

1-st group

*B. burgdorferi* sensu stricto

2-nd group

*B. afzelii Ixodes ricinus*, *Ixodes*

*B. bissettii Ixodes ricinus*, *Ixodes*

*B. garinii Ixodes ricinus, Ixodes*

*B. lusitaniae Ixodes ricinus, Ixodes*

*B. valaisiana Ixodes ricinus*, *Ixodes*

*B. americana Ixodes pacificus*, *Ixodes minor*

*B. californiensis Ixodes pacificus*, *Ixodes*

*persulcatus*

*granulatus, Ixodes persulcatus*

*B. andersonii Ixodes dentatus* Cotton tail

*jellisoni*, *Ixodes spinipalpis*

*persulcatus*

*Ixodes minor*

*scapularis*, *Ixodes pacificus*,

*Ixodes ricinus*, *Ixodes scapularis*, *Ixodes pacificus,*

*Ixodes persulcatus*

*persulcatus, Ixodes hexagonus, Ixodes nipponensis, Ixodes pavlovskyi, Ixodes trianguliceps*

*B. kurtenbachii Ixodes scapularis* Rodents Europe,

Table 1. Currently known species from the Borrelia burgdorferi sensu lato complex (Rudenko et al., 2011 with modifications)

the human sensitivity to *B. burgdorferi* s.l. and results of the newest publications, the complex of 18 *Borrelia* species (Table 1) can be divided into 3 major groups (Rudenko et al., 2011): the first and second groups contains 9 species with pathogenic potential and species that have not yet been reported in or isolated from humans, respectively, and the 3rd still not named group proposed as genomospecies 2 represented by two far-western US isolates.

#### **3. Ticks–Borreliae interface**

#### **3.1 Vectors ecological groups**

There are two big ecological groups of tick species. Ticks seek hosts by an interesting behaviour called "questing." Questing ticks (=exophilic, polyxenous) crawl up the stems of grass or perch on the edges of leaves on the ground in a typical posture with the front legs extended, especially in response to a host passing by (Fig. 1 A). In contrast to questing ticks, nidicolous (=endophilic, mainly monoxenous) ticks live in secluded enclosures such as caves, burrows and nests of their hosts or harborages near these nests (Fig. 1 B, C) (Sonenshine, 1991).

#### **3.2 Vector competence for** *Borrelia burgdorferi* **s.l.**

The primary vectors of Lyme borreliosis spirochaetes to humans in temperate regions of the northern hemisphere are closely taxonomically related tick species: *I. pacificus* in Western North America, *I. persulcatus* in Eurasia, *I. ricinus* in Europe and *I. scapularis* in eastern North

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

spirochaetes transmission during feeding (Eisen & Lane, 2002).

species to serve as a competent vector of *B. burgdorferi*.

*afzelii, B. valaisiana, B. lusitaniae,* etc.).

unique reservoir (*B. spielmanii)*.

be explained and require further studies.

on the vegetation, ground or in the litter.

**3.3 Ticks behaviour and** *Borrelia* **transmission**

humans depends on the behaviour of the vectors.

Vectors Competence and Vertebrate Host Specificity 31

spp. These includes the avian-associated nidicolous tick species such as *Ixodes uriae* White (Olsen et al., 1995), *Ixodes lividus* Koch (Movila et al., 2008), *Ixodes arboricola* Schulze & Schlottke (Špitalská et al., 2011), *Ixodes auritulus* Neumann (Morshed et al., 2005), *Ixodes turdus* Nakatsuji (Fukunaga et al., 1996a) and mammals-associated nidicolous ticks – *Ixodes trianguliceps* Birula (Gorelova et al., 1996) and *Ixodes neotomae* Cooley (Schwan et al., 1993). Seven tick species evaluated for vector competence appear unable to transmit *B. burgdorferi* s.l.: *Amblyomma americanum* (Linnaeus), *Dermacentor andersoni* Stiles, *Dermacentor occidentalis* Marx, *Dermacentor variabilis* (Say), *Ixodes cookie* Packard, *Ixodes holocyclus* Neumann and *Ixodes ovatus* Neumann. In most cases these ticks acquired borreliae better feeding on infected hosts but transstadial passage was rare or absent, and there was no evidence of

Taken all data together, there are 3 kinds of *Borrelia* species and vectors competence:

1. Those associated with a vector characterized by both a broad spectrum of hosts *(I. ricinus, I. persulcatus* or *I. scapularis)* and by a huge expansion area. These species have large populations of individuals and a variety of different vertebrate hosts which do not fully characterize the concerned *Borrelia* species. These species are genetically quite diverse and usually pathogenic or potentially pathogenic (*B. burgdorferi* s.s., *B. garinii, B.*

2. A second kind of species associated with either a unique reservoir or a unique specialized vector *(B. andersonii, B. turdi, B. tanukii)*, or an unspecialized vector but still a

3. Incompetence vector species. Mátlová et al. (1996) showed that unlike *I. ricinus*, *Dermacentor reticulatus* (Fabricius) reveals a gradual decline and the loss of Lyme borrelioses spirochaetes shortly after infection. This indicates a lack of this ixodid

Thus, the physiological mechanisms of vector competence in various tick species remain to

Ticks can survive for years in their biotopes; however, they spend only a small part of their life in a parasitic phase. Most of the *Ixodes* spp. lifetime is spent outside of the hosts, either

To find a host, *I. ricinus* climbs onto low vegetation and waits at the tip where they quest for a host for time-limited periods. During these periods of questing, ticks stay mainly immobile at the tip of the vegetation. When ticks are questing, they respond to mechanical and chemical stimuli produced by hosts, including humans. When hosts pass close enough, questing ticks grab their hosts but sometimes crawl in the direction of the possible prey. Such behaviour of *I. ricinus* is important since it implies that hosts, including humans, take some active part in the tick-host encounter. So, the successful transmission of pathogens to

Methods used to estimate tick behaviour vary and are generally directed to ascertain locomotor activity, which implies an orientation to physical parameters of the environment such as relative humidity, temperature, light and behaviour during questing for a host.

Fig. 1. The life circles of exophilic and endophilic ticks. A – exophilic tick life cycle, B – mammals-associated endophilic life cycle; C – bird-associated endophilic life cycle. 1 – Larvae, 2 – Nymph, 3 – Adult, 4 – Female with eggs

America. 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 dehumidification is low. Ticks require three hosts, and their life cycle takes between 1–3 years to complete. The immature stages (larvae and nymphs) of the tick quest on low lying vegetation and tend to infest smaller hosts to obtain their blood meals, especially rodents, insectivores and birds. After feeding, they detach from their host and molt to the next development stage (larvae to nymph or nymph to adult tick) on the ground under leafs and other kind of the litter.  Both sexes tend to quest up on vegetations and generally infest different species of ungulates, carnivores and lagomorphs. After feeding, the female lay one batch of thousands of eggs and then die. Only one blood meal is taken during each of the three life stages.

In many ways, these ticks are ideally suited as vectors of zoonotic pathogens, since they feed on wide variety of animals but also include humans within the range of hosts they willing feed upon (Filippova, 1977; Xu et al., 2003).

To date, vector competence for *B. burgdorferi* s.l. has been experimentally confirmed for 12 tick species: *Ixodes affinis* Neumann, *Ixodes jellisoni* Cooley & Kohls, *Ixodes pacificus* Cooley & Kohls, *Ixodes persulcatus* Schulze, *Ixodes ricinus* (Linnaeus), *Ixodes scapularis* Say, *Ixodes angustus* Neumann, *Ixodes dentatus* Marx, *Ixodes hexagonus* Leach, *Ixodes minor* Neumann, *Ixodes muris* Bishopp & Smith and *Ixodes spinipalpis* Hadwen & Nuttall. Published vector competence studies have included only four *B. burgdorferi* s.l. genospecies (*B. burgdorferi* s.s., *B. afzelii*, *B. bissettii*, *B. garinii*) (Eisen & Lane, 2002).

The majority of the remaining confirmed vectors feed primary on rodents and/or lagomorphs (i.e. *I. dentatus*, *I. jellisoni*, *I. muris* and *I. spinipalpis* in North America and *I. minor* in North and South America), whereas *I. affinis* infests a wide variety of mammals in North and South America; *I. hexagonus* being a nidicolous arthropod is found on various medium sized mammals in Europe and north-western Africa (Jaenson et al., 1994). Their host preferences and *modus vivendi* render these tick species unlikely to act as vectors to humans.

In addition to the experimentally confirmed vectors, the presence of *Borrelia* in ticks and their primary hosts suggest vector competence for *B. burgdorferi* s.l. of several other *Ixodes*

Fig. 1. The life circles of exophilic and endophilic ticks. A – exophilic tick life cycle, B – mammals-associated endophilic life cycle; C – bird-associated endophilic life cycle. 1 –

and then die. Only one blood meal is taken during each of the three life stages.

America. 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 dehumidification is low. Ticks require three hosts, and their life cycle takes between 1–3 years to complete. The immature stages (larvae and nymphs) of the tick quest on low lying vegetation and tend to infest smaller hosts to obtain their blood meals, especially rodents, insectivores and birds. After feeding, they detach from their host and molt to the next development stage (larvae to nymph or nymph to adult tick) on the ground under leafs and other kind of the litter.  Both sexes tend to quest up on vegetations and generally infest different species of ungulates, carnivores and lagomorphs. After feeding, the female lay one batch of thousands of eggs

In many ways, these ticks are ideally suited as vectors of zoonotic pathogens, since they feed on wide variety of animals but also include humans within the range of hosts they willing

To date, vector competence for *B. burgdorferi* s.l. has been experimentally confirmed for 12 tick species: *Ixodes affinis* Neumann, *Ixodes jellisoni* Cooley & Kohls, *Ixodes pacificus* Cooley & Kohls, *Ixodes persulcatus* Schulze, *Ixodes ricinus* (Linnaeus), *Ixodes scapularis* Say, *Ixodes angustus* Neumann, *Ixodes dentatus* Marx, *Ixodes hexagonus* Leach, *Ixodes minor* Neumann, *Ixodes muris* Bishopp & Smith and *Ixodes spinipalpis* Hadwen & Nuttall. Published vector competence studies have included only four *B. burgdorferi* s.l. genospecies (*B. burgdorferi* s.s.,

The majority of the remaining confirmed vectors feed primary on rodents and/or lagomorphs (i.e. *I. dentatus*, *I. jellisoni*, *I. muris* and *I. spinipalpis* in North America and *I. minor* in North and South America), whereas *I. affinis* infests a wide variety of mammals in North and South America; *I. hexagonus* being a nidicolous arthropod is found on various medium sized mammals in Europe and north-western Africa (Jaenson et al., 1994). Their host preferences and

In addition to the experimentally confirmed vectors, the presence of *Borrelia* in ticks and their primary hosts suggest vector competence for *B. burgdorferi* s.l. of several other *Ixodes*

*modus vivendi* render these tick species unlikely to act as vectors to humans.

Larvae, 2 – Nymph, 3 – Adult, 4 – Female with eggs

feed upon (Filippova, 1977; Xu et al., 2003).

*B. afzelii*, *B. bissettii*, *B. garinii*) (Eisen & Lane, 2002).

spp. These includes the avian-associated nidicolous tick species such as *Ixodes uriae* White (Olsen et al., 1995), *Ixodes lividus* Koch (Movila et al., 2008), *Ixodes arboricola* Schulze & Schlottke (Špitalská et al., 2011), *Ixodes auritulus* Neumann (Morshed et al., 2005), *Ixodes turdus* Nakatsuji (Fukunaga et al., 1996a) and mammals-associated nidicolous ticks – *Ixodes trianguliceps* Birula (Gorelova et al., 1996) and *Ixodes neotomae* Cooley (Schwan et al., 1993).

Seven tick species evaluated for vector competence appear unable to transmit *B. burgdorferi* s.l.: *Amblyomma americanum* (Linnaeus), *Dermacentor andersoni* Stiles, *Dermacentor occidentalis* Marx, *Dermacentor variabilis* (Say), *Ixodes cookie* Packard, *Ixodes holocyclus* Neumann and *Ixodes ovatus* Neumann. In most cases these ticks acquired borreliae better feeding on infected hosts but transstadial passage was rare or absent, and there was no evidence of spirochaetes transmission during feeding (Eisen & Lane, 2002).

Taken all data together, there are 3 kinds of *Borrelia* species and vectors competence:

Thus, the physiological mechanisms of vector competence in various tick species remain to be explained and require further studies.

#### **3.3 Ticks behaviour and** *Borrelia* **transmission**

Ticks can survive for years in their biotopes; however, they spend only a small part of their life in a parasitic phase. Most of the *Ixodes* spp. lifetime is spent outside of the hosts, either on the vegetation, ground or in the litter.

To find a host, *I. ricinus* climbs onto low vegetation and waits at the tip where they quest for a host for time-limited periods. During these periods of questing, ticks stay mainly immobile at the tip of the vegetation. When ticks are questing, they respond to mechanical and chemical stimuli produced by hosts, including humans. When hosts pass close enough, questing ticks grab their hosts but sometimes crawl in the direction of the possible prey. Such behaviour of *I. ricinus* is important since it implies that hosts, including humans, take some active part in the tick-host encounter. So, the successful transmission of pathogens to humans depends on the behaviour of the vectors.

Methods used to estimate tick behaviour vary and are generally directed to ascertain locomotor activity, which implies an orientation to physical parameters of the environment such as relative humidity, temperature, light and behaviour during questing for a host.

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

to humans.

Vectors Competence and Vertebrate Host Specificity 33

sensitive in their impedance measurements. The attachment of the anomalous female may be an indicator of a higher aggressiveness exhibited by anomalous, even pathogen-free ticks

Fig. 2. Adult *Ixodes persulcatus* ticks, collected in vicinities of St. Petersburg (North-West Russia). Females: normal (a), anomalous (b); males: normal (c), anomalous (d). Arrows indicate prominent cuticular depressions symptomatic of exoskeleton pathologies in

anomalous ticks (Original photos)

The predominant behaviour of *I. ricinus* is the tendency of the tick to ascend vegetation during the day and descent at night (Lees & Milne, 1951), while Okulova (1978) showed that *I. persulcatus* adults climbed up the aconite and fern stems to a maximum height of 80 cm (according to observations of Filippova (1977) some of *I. persulcatus* specimens in Primorye (Russian Far East) climb to a height of 2 m) when the temperature is 23.3o C. The maximum activity of *I. ricinus* larvae and adults was associated with the lightest part of the day, 1 p.m., when the air temperature was highest; the adults increased activity of females was observed between 1 p.m and 8 p.m. (Dubinina & Makrushina, 1997). Babenko (1985) suggested the crepuscular activity of ticks to be adaptive, coinciding with the activity of hosts they feed on.

Alekseev et al. (1998) reported that different *Borrelia* genospecies prevailed during different periods of the tick activity season. The author showed that *Borrelia*-infected nymphs and adults emerged within a day later than uninfected ticks and only after 11 a.m., when the temperature gradient exceeded 0.5o C. On the basis of these data Prof. Alekseev proposed a hypothesis that the response of *Borrelia*-infected *I. persulcatus* ticks to some external factors (plant and animal odors) was different from that of the uninfected ticks.

Lefcort & Durden (1996) studied the behaviour of *I. scapularis* nymphs and adults, and compared specimens that were infected in the laboratory by *B. burgdorferi* s.s. to uninfected ticks. The authors showed a stimulating effect of Borreliae on the activity of tick nymphs.

However the work of Alekseev et al. (2000) may be the most interesting example of the behaviour of infected and naive *I. persulcatus* and *I. ricinus* ticks. The comparison of *I. persulcatus* and *I. ricinus* demonstrated that the entire locomotor activity of *I. persulcatus* nymphs was 4 times and that of adults approximately 2 times as great as that of *I. ricinus*. The activity of infected preimaginal and adults *I. ricinus* was less than that of uninfected ticks. These report support earliest opinion that the *I. persulcatus* ticks are more effective vectors of pathogens than *I. ricinus* (Alekseev & Dubinina, 1994; Kovalevskii & Korenberg, 1995). Interesting, the European *Borrelia* genospecies depressed the nymphal activity of *I. ricinus* and *I. persulcatus* ticks. The data of Alekseev et al. (2000) and Lefcort & Durden (1996) can explain the role of nymphs as major vector of Lyme disease in North America and adults – in Euro-Asia.

The group of prof. Alekseev A.N. (for review see Alekseev et al., 2010) demonstrated that adults having exoskeleton anomalies (Fig. 2) and *Borrelia* infection moved more actively than ticks with normal morphology and with infection. This anomalies ticks phenomenon appeared to be associated with the consequences of the anthropogenic pressure and with environmental pollution, which caused the development of tick population with changed morphology and metabolism. It is clear that the accumulation of these anomalies ticks in the vector population increased the risk of pathogen transmission.

The ability of anomalous and normal ticks to attack humans was tested by Alekseev & Dubinina (2006). During 25 minutes of the experiments, none of the normal females attached onto the human skin, while anomalous females started as earlier as 1 minute after the release and this process was painless. The attachment sites of the ticks were corresponded to the acupuncture points on the human body with the impedance 172 ± 3.2 kΩ (Alekseev & Dubinina, 2006). The fact that ticks attach to different sites on the body of the same person perhaps reflects the temporal changes in impedance (Alekseev et al., 2010). The ticks with anomalous in exoskeleton contain heavy metals in their bodies (especially Cd) are more

The predominant behaviour of *I. ricinus* is the tendency of the tick to ascend vegetation during the day and descent at night (Lees & Milne, 1951), while Okulova (1978) showed that *I. persulcatus* adults climbed up the aconite and fern stems to a maximum height of 80 cm (according to observations of Filippova (1977) some of *I. persulcatus* specimens in Primorye (Russian Far East) climb to a height of 2 m) when the temperature is 23.3o C. The maximum activity of *I. ricinus* larvae and adults was associated with the lightest part of the day, 1 p.m., when the air temperature was highest; the adults increased activity of females was observed between 1 p.m and 8 p.m. (Dubinina & Makrushina, 1997). Babenko (1985) suggested the crepuscular activity of ticks to be adaptive, coinciding with the activity of hosts they feed on. Alekseev et al. (1998) reported that different *Borrelia* genospecies prevailed during different periods of the tick activity season. The author showed that *Borrelia*-infected nymphs and adults emerged within a day later than uninfected ticks and only after 11 a.m., when the temperature gradient exceeded 0.5o C. On the basis of these data Prof. Alekseev proposed a hypothesis that the response of *Borrelia*-infected *I. persulcatus* ticks to some external factors

Lefcort & Durden (1996) studied the behaviour of *I. scapularis* nymphs and adults, and compared specimens that were infected in the laboratory by *B. burgdorferi* s.s. to uninfected ticks. The authors showed a stimulating effect of Borreliae on the activity of tick nymphs.

However the work of Alekseev et al. (2000) may be the most interesting example of the behaviour of infected and naive *I. persulcatus* and *I. ricinus* ticks. The comparison of *I. persulcatus* and *I. ricinus* demonstrated that the entire locomotor activity of *I. persulcatus* nymphs was 4 times and that of adults approximately 2 times as great as that of *I. ricinus*. The activity of infected preimaginal and adults *I. ricinus* was less than that of uninfected ticks. These report support earliest opinion that the *I. persulcatus* ticks are more effective vectors of pathogens than *I. ricinus* (Alekseev & Dubinina, 1994; Kovalevskii & Korenberg, 1995). Interesting, the European *Borrelia* genospecies depressed the nymphal activity of *I. ricinus* and *I. persulcatus* ticks. The data of Alekseev et al. (2000) and Lefcort & Durden (1996) can explain the role of nymphs as major vector of Lyme disease in North America and

The group of prof. Alekseev A.N. (for review see Alekseev et al., 2010) demonstrated that adults having exoskeleton anomalies (Fig. 2) and *Borrelia* infection moved more actively than ticks with normal morphology and with infection. This anomalies ticks phenomenon appeared to be associated with the consequences of the anthropogenic pressure and with environmental pollution, which caused the development of tick population with changed morphology and metabolism. It is clear that the accumulation of these anomalies ticks in the

The ability of anomalous and normal ticks to attack humans was tested by Alekseev & Dubinina (2006). During 25 minutes of the experiments, none of the normal females attached onto the human skin, while anomalous females started as earlier as 1 minute after the release and this process was painless. The attachment sites of the ticks were corresponded to the acupuncture points on the human body with the impedance 172 ± 3.2 kΩ (Alekseev & Dubinina, 2006). The fact that ticks attach to different sites on the body of the same person perhaps reflects the temporal changes in impedance (Alekseev et al., 2010). The ticks with anomalous in exoskeleton contain heavy metals in their bodies (especially Cd) are more

(plant and animal odors) was different from that of the uninfected ticks.

vector population increased the risk of pathogen transmission.

adults – in Euro-Asia.

sensitive in their impedance measurements. The attachment of the anomalous female may be an indicator of a higher aggressiveness exhibited by anomalous, even pathogen-free ticks to humans.

Fig. 2. Adult *Ixodes persulcatus* ticks, collected in vicinities of St. Petersburg (North-West Russia). Females: normal (a), anomalous (b); males: normal (c), anomalous (d). Arrows indicate prominent cuticular depressions symptomatic of exoskeleton pathologies in anomalous ticks (Original photos)

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

Vectors Competence and Vertebrate Host Specificity 35

Fig. 3. Scutual index (= a/b) of *Ixodes ricinus* nymphs (Meiners et al., 2006)

functioning without borreliae action.

Actions of this protein have two directions: the first one channeled on vertebrate immune system suppression, the second one directed on infection enlighten of both pathogenic for human microorganism hosts. The first one is working independently of the presents of pathogenic for human microorganisms. The second one is working in dependence of infection. For example *I. scapularis* salp15 inhibits CD4+ T-cell activation (Anguita et al., 2002) by binding to the CD4 receptor (Garg et al., 2006). Hovius and his colleagues (Hovius et al., 2008) found that salp15 from the same species of tick is able to bind the C-type lectin DC-SIGN on human dendritic cells. Such binding inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilizations. All these mechanisms are

When unfed *I*. *ricinus* attaches to a vertebrate host, *Borrelia* transmission does not occur at the beginning of the blood uptake but later on, and transmission efficiency increases with the duration of the blood meal (Crippa et al., 2002; Kahl et al., 1998). The delay in transmission observed during the first hours of the blood meal might be due to this phenomenon, the migration of the spirochaetes from midgut to salivary glands. Crippa et al. (2002), comparing transmission dynamic of spirochaetes by *B. burgdorferi* s.s.– and *B. afzelii*-infected ticks, reported that this delay might also be influenced by the *Borrelia*  species infecting the ticks. In fact, earlier transmission occurred when ticks were infected by *B*. *afzelii* rather than by *B*. *burgdorferi* s.s. These authors reported that during the first 48 h of attachment to the host, *B*. *burgdorferi* s.s.-infected ticks did not infect the 18 exposed mice, whereas *B*. *afzelii-*infected ticks transmitted infection to 33% of the mice. This study not only showed that *I*. *ricinus* transmits *B*. *afzelii* earlier than *B*. *burgdorferi* s.s., but also that *I*. *ricinus* is a more efficient vector for *B*. *afzelii* than for *B*. *burgdorferi* s.s. Unfortunately, nothing is known on the transmission delay for other pathogenic *Borrelia*  species infecting *I. ricinus*, such as *B. garinii*, *B. valaisiana* and the recently described species *B. spielmanii.*

Whereas *B. burgdorferi* transmission to the host usually does not occur during the first 40 h of the blood meal in the North American vector ticks *I. scapularis* and *I. pacificus* (Peavey & Lane, 1995; Piesman et al., 1987, 1991), while *I. ricinus* nymphs were shown to be capable of transmitting *B. burgdorferi* s.l. to mongolian gerbils as early as within the first 24 h of feeding (Kahl et al., 1998). There is a method that allows assessment of the duration of tick feeding as a basis for determining the individual risk of *B. burgdorferi* transmission to the person bitten by a tick. Piesman & Spielman (1980) were the first to establish the so-called scutal index as a quick and simple measure of engorgement and feeding time in partially fed *I. scapularis* nymphs. The scutal index is the ratio between length of the tick alloscutum (a) and width of scutum (b) (Fig. 3).Falco et al. (1996) reported that the scutual index of *I. scapularis* nymphs detached from humans was on average equality to 34.7 h post-attachment. Yeh et al. (1995) reported that only 10% and 41% of people had found and removed *I. scapularis* nymphs by 24 h and 36 h of attachment, respectively. The situation is probably similar in Europe, but there is one important difference: whereas North American tick bite victims have a very good chance to avoid *Borrelia* infection after a tick bite lasting approximately 36 h, many *I. ricinus* nymphs might already have transmitted borreliae to their host after this feeding time.

Meiners et al. (2006) reported that a scutal index of 1.1 as a cutoff allows a clear distinction between high-risk versus low-risk group ticks. The large majority of *I. ricinus* nymphs with a scutal index <1.1 may have fed for <24 h, and the resulting risk of *B. burgdorferi* transmission and host infection might be low, even in the case of a *B. burgdorferi*-infected tick. The risk of host infection might be distinctly higher when a *B. burgdorferi*-infected *I. ricinus* nymph has a scutal index 1.1–1.5 (corresponding to ~24 – ~40 h feeding duration). Infection risk might be very high, if the detached tick has a scutal index >1.5 indicating that it might have fed for >36 h. Sood et al. (1997) identified a long feeding duration in *I. scapularis* ticks as a major risk indicator of *B. burgdorferi* host infection. Influence of borreliae on the salivary gland genome results the appearance of proteins, which suppress vertebrate immune system and properties of *Borrelia.*

Among vast family of proteins derived from tick salivary glands as a result of its genome activity the most interesting multifunctional one is presented by so called salp15 (14.7 kDa).

When unfed *I*. *ricinus* attaches to a vertebrate host, *Borrelia* transmission does not occur at the beginning of the blood uptake but later on, and transmission efficiency increases with the duration of the blood meal (Crippa et al., 2002; Kahl et al., 1998). The delay in transmission observed during the first hours of the blood meal might be due to this phenomenon, the migration of the spirochaetes from midgut to salivary glands. Crippa et al. (2002), comparing transmission dynamic of spirochaetes by *B. burgdorferi* s.s.– and *B. afzelii*-infected ticks, reported that this delay might also be influenced by the *Borrelia*  species infecting the ticks. In fact, earlier transmission occurred when ticks were infected by *B*. *afzelii* rather than by *B*. *burgdorferi* s.s. These authors reported that during the first 48 h of attachment to the host, *B*. *burgdorferi* s.s.-infected ticks did not infect the 18 exposed mice, whereas *B*. *afzelii-*infected ticks transmitted infection to 33% of the mice. This study not only showed that *I*. *ricinus* transmits *B*. *afzelii* earlier than *B*. *burgdorferi* s.s., but also that *I*. *ricinus* is a more efficient vector for *B*. *afzelii* than for *B*. *burgdorferi* s.s. Unfortunately, nothing is known on the transmission delay for other pathogenic *Borrelia*  species infecting *I. ricinus*, such as *B. garinii*, *B. valaisiana* and the recently described

Whereas *B. burgdorferi* transmission to the host usually does not occur during the first 40 h of the blood meal in the North American vector ticks *I. scapularis* and *I. pacificus* (Peavey & Lane, 1995; Piesman et al., 1987, 1991), while *I. ricinus* nymphs were shown to be capable of transmitting *B. burgdorferi* s.l. to mongolian gerbils as early as within the first 24 h of feeding (Kahl et al., 1998). There is a method that allows assessment of the duration of tick feeding as a basis for determining the individual risk of *B. burgdorferi* transmission to the person bitten by a tick. Piesman & Spielman (1980) were the first to establish the so-called scutal index as a quick and simple measure of engorgement and feeding time in partially fed *I. scapularis* nymphs. The scutal index is the ratio between length of the tick alloscutum (a) and width of scutum (b) (Fig. 3).Falco et al. (1996) reported that the scutual index of *I. scapularis* nymphs detached from humans was on average equality to 34.7 h post-attachment. Yeh et al. (1995) reported that only 10% and 41% of people had found and removed *I. scapularis* nymphs by 24 h and 36 h of attachment, respectively. The situation is probably similar in Europe, but there is one important difference: whereas North American tick bite victims have a very good chance to avoid *Borrelia* infection after a tick bite lasting approximately 36 h, many *I. ricinus* nymphs might already have

Meiners et al. (2006) reported that a scutal index of 1.1 as a cutoff allows a clear distinction between high-risk versus low-risk group ticks. The large majority of *I. ricinus* nymphs with a scutal index <1.1 may have fed for <24 h, and the resulting risk of *B. burgdorferi* transmission and host infection might be low, even in the case of a *B. burgdorferi*-infected tick. The risk of host infection might be distinctly higher when a *B. burgdorferi*-infected *I. ricinus* nymph has a scutal index 1.1–1.5 (corresponding to ~24 – ~40 h feeding duration). Infection risk might be very high, if the detached tick has a scutal index >1.5 indicating that it might have fed for >36 h. Sood et al. (1997) identified a long feeding duration in *I. scapularis* ticks as a major risk indicator of *B. burgdorferi* host infection. Influence of borreliae on the salivary gland genome results the appearance of proteins, which suppress vertebrate immune system and

Among vast family of proteins derived from tick salivary glands as a result of its genome activity the most interesting multifunctional one is presented by so called salp15 (14.7 kDa).

transmitted borreliae to their host after this feeding time.

species *B. spielmanii.*

properties of *Borrelia.*

Fig. 3. Scutual index (= a/b) of *Ixodes ricinus* nymphs (Meiners et al., 2006)

Actions of this protein have two directions: the first one channeled on vertebrate immune system suppression, the second one directed on infection enlighten of both pathogenic for human microorganism hosts. The first one is working independently of the presents of pathogenic for human microorganisms. The second one is working in dependence of infection. For example *I. scapularis* salp15 inhibits CD4+ T-cell activation (Anguita et al., 2002) by binding to the CD4 receptor (Garg et al., 2006). Hovius and his colleagues (Hovius et al., 2008) found that salp15 from the same species of tick is able to bind the C-type lectin DC-SIGN on human dendritic cells. Such binding inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilizations. All these mechanisms are functioning without borreliae action.

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

Fig. 4. *Borrelia* spp. co-feeding transmission

infections, and are often detected by PCR.

Vectors Competence and Vertebrate Host Specificity 37

*B*. *valaisiana* constitute the majority of mixed infections, followed by mixed infections with *B*. *garinii* and *B*. *afzelii.* Such mixed infections are reported less frequently than single

Ticks also can get *Borrelia* during copulation. Sexual transmission of microbial pathogens is a well-known phenomenon in arthropods. It was first described in soft ticks (Ornithodorinae) for relapsing fever *Borrelia* (Gaber et al., 1982, 1984; Wagner-Jevseenko, 1958). Chunikhin et al. (1983) studied the sexual transmission of the (TBE) virus in *I. persulcatus*. The virus was transmitted from the infected male tick to the female tick and was present in spermatocytes and spermatids of male ticks (Stefutkina, 1989). TBE virus was subsequently detected in the ovaries of 10% of the infected female ticks. Alekseev et al. (1999) have demonstrated that *B. garinii (*but not *B. afzelii)* spirochaetes can be transmitted from male to female in *I. persulcatus*  ticks during copulation. Transmission of *B. garinii* to the female tick was observed more often when the male tick carried high numbers of spirochaetes. This observation is in accordance with the fact that ticks which carry high numbers of spirochaetes in the gut show systemic infection (i.e. outside the gut lumen) more often than ticks which contain low

It is known now that the presence of OspC facilitates *B. burgdorferi* invasion of *I. scapularis* salivary glands. Pal et al. (2004), Rudenko et al. (2005) affirmed that in the beginning of infected (OspA) vertebrate' blood consuming borreliae enhanced salp15 production, which lighten tick infection. Just the opposite action, in which salp15 is also included, consist in prevention of *B. burgdorferi* infected (OspC) tick saliva from killing by anti-OspC antibodies in the tick just after beginning of attachment and feeding (Ramamoorthi et al., 2005). Rosa (2005) naming his article "Lyme disease agent borrows a practical coat" meaning that coat helped spirochaetes "to change into a new suit" and transfer themselves into the vertebrate host.

In the interface of field collected *I. ricinus* infected by different quantity of representatives of 4 species *Borrelia* determined by real time PCR and RLB a very interesting fact was discovered: the better surviving of adult ticks infected by mean doses of pathogens (Herrmann & Gern, 2010). This phenomenon was a brilliant confirmation of prof. Alekseev (1984) theory that among other criteria of interface specificity in the couple "vector pathogen" the existence of optimum pathogen doses (mean ones) do exist not only in the pairs *"Leishmania—sand flies"*, plague agent and fleas (Alekseev, 1984), "Tick-borne encephalitis (TBE) virus —*Ixodes persulcatus*" (Alekseev & Kondrashova, 1985), but in the couple *"Borrelia—Ixodes ticks"* (Herrmann & Gern, 2010).

#### **3.4** *Borrelia* **circulation in tick population**

Ticks may acquire various *Borrelia* species through their successive blood meals on various hosts, and maintain the infection to the subsequent stage via transstadial transmission: larvae—nymph—adult.

Transovarial transmission of infection agents, which is an important factor of maintaining the disease foci, depends not only on the female obtaining the infection agents at the previous developmental stage, but also on the female contacting the infected male. The success of these contacts, and thus the probability of transmission, is based on the details of the sexual behaviour of the vectors. According to available data, which are scarce and often insufficiently representative, the frequency of *Borrelia* transovarial transmission in Lyme disease main vectors (*I. ricinus*, *I. persulcatus* and *I. scapularis*) may vary but never was estimated as frequent phenomenon (Du et al., 1990; Dubinina, 2000; Lane & Burgdorfer, 1987; Nakao & Miyamoto, 1992; Nefedova et al., 2004). On the basis of published data, probably, this transmission pathway apparently plays no significant role in the maintenance of *Borrelia* circulation and the dynamics of parameters of infection in adult ticks of the next generation. Nevertheless, transovarially transmitted spirochaetes may also contribute to mixed infections in ticks (Gern, 2009). However, *Borrelia miyamotoi* (a relapsing fever spirochete) is transmitted transovarially in *Ixodes* ticks and occurs sympatrically with *B. burgdorferi* spirochaetes (Piesman, 2002).

Thus, mixed infection with more than one species in ticks can be observed in some European endemic areas. These multiple infections may result from the tick species feeding on a host infected by more than one *Borrelia* species or from infected ticks feeding simultaneously with uninfected ticks on a host and exchanging the *Borrelia* species through co-feeding transmission from infected to uninfected ticks (Fig. 4). Different combinations of mixed infections with 2 or 3 species have been detected in *I*. *ricinus*. *Borrelia garinii* and

It is known now that the presence of OspC facilitates *B. burgdorferi* invasion of *I. scapularis* salivary glands. Pal et al. (2004), Rudenko et al. (2005) affirmed that in the beginning of infected (OspA) vertebrate' blood consuming borreliae enhanced salp15 production, which lighten tick infection. Just the opposite action, in which salp15 is also included, consist in prevention of *B. burgdorferi* infected (OspC) tick saliva from killing by anti-OspC antibodies in the tick just after beginning of attachment and feeding (Ramamoorthi et al., 2005). Rosa (2005) naming his article "Lyme disease agent borrows a practical coat" meaning that coat helped spirochaetes "to change into a new suit" and transfer

In the interface of field collected *I. ricinus* infected by different quantity of representatives of 4 species *Borrelia* determined by real time PCR and RLB a very interesting fact was discovered: the better surviving of adult ticks infected by mean doses of pathogens (Herrmann & Gern, 2010). This phenomenon was a brilliant confirmation of prof. Alekseev (1984) theory that among other criteria of interface specificity in the couple "vector pathogen" the existence of optimum pathogen doses (mean ones) do exist not only in the pairs *"Leishmania—sand flies"*, plague agent and fleas (Alekseev, 1984), "Tick-borne encephalitis (TBE) virus —*Ixodes persulcatus*" (Alekseev & Kondrashova, 1985), but in the

Ticks may acquire various *Borrelia* species through their successive blood meals on various hosts, and maintain the infection to the subsequent stage via transstadial transmission:

Transovarial transmission of infection agents, which is an important factor of maintaining the disease foci, depends not only on the female obtaining the infection agents at the previous developmental stage, but also on the female contacting the infected male. The success of these contacts, and thus the probability of transmission, is based on the details of the sexual behaviour of the vectors. According to available data, which are scarce and often insufficiently representative, the frequency of *Borrelia* transovarial transmission in Lyme disease main vectors (*I. ricinus*, *I. persulcatus* and *I. scapularis*) may vary but never was estimated as frequent phenomenon (Du et al., 1990; Dubinina, 2000; Lane & Burgdorfer, 1987; Nakao & Miyamoto, 1992; Nefedova et al., 2004). On the basis of published data, probably, this transmission pathway apparently plays no significant role in the maintenance of *Borrelia* circulation and the dynamics of parameters of infection in adult ticks of the next generation. Nevertheless, transovarially transmitted spirochaetes may also contribute to mixed infections in ticks (Gern, 2009). However, *Borrelia miyamotoi* (a relapsing fever spirochete) is transmitted transovarially in *Ixodes* ticks and occurs sympatrically with *B.*

Thus, mixed infection with more than one species in ticks can be observed in some European endemic areas. These multiple infections may result from the tick species feeding on a host infected by more than one *Borrelia* species or from infected ticks feeding simultaneously with uninfected ticks on a host and exchanging the *Borrelia* species through co-feeding transmission from infected to uninfected ticks (Fig. 4). Different combinations of mixed infections with 2 or 3 species have been detected in *I*. *ricinus*. *Borrelia garinii* and

themselves into the vertebrate host.

couple *"Borrelia—Ixodes ticks"* (Herrmann & Gern, 2010).

**3.4** *Borrelia* **circulation in tick population**

*burgdorferi* spirochaetes (Piesman, 2002).

larvae—nymph—adult.

Fig. 4. *Borrelia* spp. co-feeding transmission

*B*. *valaisiana* constitute the majority of mixed infections, followed by mixed infections with *B*. *garinii* and *B*. *afzelii.* Such mixed infections are reported less frequently than single infections, and are often detected by PCR.

Ticks also can get *Borrelia* during copulation. Sexual transmission of microbial pathogens is a well-known phenomenon in arthropods. It was first described in soft ticks (Ornithodorinae) for relapsing fever *Borrelia* (Gaber et al., 1982, 1984; Wagner-Jevseenko, 1958). Chunikhin et al. (1983) studied the sexual transmission of the (TBE) virus in *I. persulcatus*. The virus was transmitted from the infected male tick to the female tick and was present in spermatocytes and spermatids of male ticks (Stefutkina, 1989). TBE virus was subsequently detected in the ovaries of 10% of the infected female ticks. Alekseev et al. (1999) have demonstrated that *B. garinii (*but not *B. afzelii)* spirochaetes can be transmitted from male to female in *I. persulcatus*  ticks during copulation. Transmission of *B. garinii* to the female tick was observed more often when the male tick carried high numbers of spirochaetes. This observation is in accordance with the fact that ticks which carry high numbers of spirochaetes in the gut show systemic infection (i.e. outside the gut lumen) more often than ticks which contain low

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

connectivity between woodlands and pastures.

**4.1 Mammals as reservoirs for** *Borrelia* **spp.**

squirrels are usually very heavily infested with ticks.

Vectors Competence and Vertebrate Host Specificity 39

ratio, a characteristic which would consequently hinder the infection prevalence in pasture ticks. This mechanism could be mediated by factors that we have not taken into consideration in this study, such as the pasture isolation from woodlands and the lack of

Only a few mammal species have been identified as reservoirs for *B*. *burgdorferi* s.l. in Europe. Globally, little information is available on the real significance of most animal hosts as sources for infecting ticks with *B*. *burgdorferi* s.l. At present, several species of mice, voles, rats and shrews are recognized as reservoirs of *B*. *burgdorferi* s.l. in Europe (Gern & Humair, 2002). In particular, it was evidenced that the mice *Sylvaemus flavicollis* (Melchior), *Sylvaemus sylvaticus* (Linnaeus), *Apodemus agrarius* (Pallas) and the vole, *Myodes* (=*Clethrionomys) glareolus* (Schreber), play key roles in the ecology of Lyme borreliosis as reservoirs for *B*. *burgdorferi* s.l. in many European countries. Once infected by an infectious tick bite, some reservoir hosts, like *Sylvaemus* mice, have been shown to persistently remain infectious for ticks. Small rodents are frequently parasitized by larval and nymphal *I*. *ricinus,*  and this also contributes to their importance as reservoirs. Less information has been obtained on the roles of other small mammal species in the maintenance cycles of *Borrelia* in nature. Nevertheless, another species of vole *Microtus agrestis* (Linnaeus) in Sweden, and black rats *Rattus rattus* (Linnaeus) and Norway rats *Rattus norvegicus* (Berkenhout) in urbanized environments in Germany and in Madeira, may serve as sources of infection for *I*. *ricinus* ticks. Similarly, only few data have been collected on *B*. *burgdorferi* s.l. in shrews (*Sorex minutus* Linnaeus and *Sorrex araneus* Linnaeus and *Neomys fodiens* (Pennant) or in ticks attached on them. Observations in endemic areas of Germany and France showed that edible dormice *Glis glis* (Linnaeus) and garden dormice *Eliomys quercinus* (Linnaeus) are reservoir hosts for *Borrelia*. Other rodent species, like grey squirrels (*Sciurus carolinensis*  Gmelin) in the UK and red squirrels *Sciurus vulgaris* (Linnaeus) in Switzerland also contribute to the amplification of *Borrelia* in the tick population. Interestingly, red and grey

In other investigations in Ireland, Germany and Switzerland, it was reported that the European hedgehog (*Erinaceus europaeus* Linnaeus) also perpetuates *B*. *burgdorferi* s.l. (Rauter & Hartung, 2005). An enzootic transmission cycle of *B*. *burgdorferi* s.l. involving hedgehogs and another tick vector, *I*. *hexagonus*, has been observed in an urban environment. This shows that gardens can also represent zones at risk of Lyme borreliosis as further discussed below. Examination of the role of lagomorphs *Lepus europaeus* (Pallas), *Lepus timidus* (Linnaeus) and *Oryctolagus cuniculus* (Linnaeus) in the support of the enzootic cycle of *B*. *burgdorferi* s.l. has also elucidated their roles as reservoirs (Gern & Humair, 2002). Assessment of the reservoir competency of large mammals is clearly a difficult task. It necessitates, if xenodiagnosis is applied, capture of the animals and maintenance in a laboratory structure. The consequence of this is that the role of mediumsized and large mammalian species has been studied less and is not yet clearly understood. Red foxes seem to be implicated in the maintenance of borreliae in nature, as described in Germany. However, these animals do not appear to be very potent reservoirs, since spirochaetes were poorly transmitted to ticks feeding on them. According to various reports, ruminants appear to act primarily as sources of blood for ticks. Controversy long surrounded the exact role of

numbers of spirochaetes (Moskvitina et al., 1995). Thus, infection of the generative apparatus of male *I. persulcatus* by *B. garinii* may depend on the presence of high numbers of spirochaetes in the tick gut. The preferred transmission of *B. garinii* among tick partners offers an explanation for the predominance of *B. garinii* over *B. afzelii* in field-collected *I. persulcatus* ticks (Alekseev et al., 2010; Korenberg et al., 1997).

Moreover, Alekseev et al. (1999) showed that tick females never transmit *Borrelia* to the *I. persulcatus* males during copulation act. In *I. ricinus* ticks males transmit to the females mostly *B. afzelii*, but the ticks that are more heavily infected by *B. afzelii* do not copulate at all (Alekseev et al., 2010). Dubinina (2000) studied the copulation peculiarities of *Borrelia*infected/uninfected *I. ricinus* and *I. persulcatus* ticks in laboratory condition. The author reported that *I. ricinus* tick usually copulates on horizontal surfaces, while *I. persulcatus*, on vertical ones. The copulation period of uninfected *I. persulcatus* ticks is 1.3–1.5 times shorter to compare to *I. ricinus*, while borreliae-infection of one or both sexual partners increases the copulation time of *I. persulcatus*.

The *Borrelia*-positive ticks were 35.9% in forest biotopes, 23% in agrarian biocenoses and 36.7% in urbanocenoses. The author found that quite a number of Lyme borreliosis foci in Moldova have been situated in recreation areas where the contact between ticks and humans can be expected to be high (Movila et al., 2006). Similar data exist for other countries (Daniels et al., 1997; Juntila et al., 1999).

#### **4. Vertebrates species specificity**

The efficient persistence of the borreliae in endemic areas requires the involvement of reservoir hosts. Potential hosts for ticks are numerous, and more than 300 vertebrate species have been identified as hosts for *I. ricinus,* including small mammals, birds, larger mammals and reptiles. Among these hosts, some act as blood meal sources and as reservoir hosts for pathogens, others as blood meal sources only. Important, that natural host does not seem to develop clinical manifestations of Lyme disease and it is difficult to evaluate the impact of *Borrelia* infection on their health. Minor clinical manifestations may escape medical attention (Gern, 2009).

Halos et al. (2010) reported that the *B. burgdorferi* s.l. infection prevalence was higher on pastures that had a high percentage of shrubs on the perimeter. This result is consistent with the fact that *B. burgdorferi* s.l. reservoir hosts, i.e., rodents and birds, are particularly concentrated in the shrubby vegetation around pastures (Boyard et al., 2008; Vourc'h et al., 2008). The *B. burgdorferi* s.l. infection prevalence also increased in pastures surrounded by forests with low perimeter length/surface area ratios. The lowest theoretical ratio corresponds to a circle; conversely, a high ratio indicated an indented shape with more edge compared to the surface area, which, again, should favor the small vertebrate abundance. This effect was thus in contrast to what we observed on woodland sites, where the prevalence tended to be associated with fragmented forest (Movila, 2008; Movila et al., 2006). Infected ticks found in pastures could have become infected by feeding on infected reservoir hosts located in the pasture itself or by feeding on infected woodland hosts that then imported the ticks into the pasture (Boyard et al., 2007). Halos et al. (2010) hypothesized that a decreased flow of tick hosts between woodlands and pastures could occur when the forest surrounding the pasture has a high perimeter length/surface area

numbers of spirochaetes (Moskvitina et al., 1995). Thus, infection of the generative apparatus of male *I. persulcatus* by *B. garinii* may depend on the presence of high numbers of spirochaetes in the tick gut. The preferred transmission of *B. garinii* among tick partners offers an explanation for the predominance of *B. garinii* over *B. afzelii* in field-collected *I.*

Moreover, Alekseev et al. (1999) showed that tick females never transmit *Borrelia* to the *I. persulcatus* males during copulation act. In *I. ricinus* ticks males transmit to the females mostly *B. afzelii*, but the ticks that are more heavily infected by *B. afzelii* do not copulate at all (Alekseev et al., 2010). Dubinina (2000) studied the copulation peculiarities of *Borrelia*infected/uninfected *I. ricinus* and *I. persulcatus* ticks in laboratory condition. The author reported that *I. ricinus* tick usually copulates on horizontal surfaces, while *I. persulcatus*, on vertical ones. The copulation period of uninfected *I. persulcatus* ticks is 1.3–1.5 times shorter to compare to *I. ricinus*, while borreliae-infection of one or both sexual partners increases the

The *Borrelia*-positive ticks were 35.9% in forest biotopes, 23% in agrarian biocenoses and 36.7% in urbanocenoses. The author found that quite a number of Lyme borreliosis foci in Moldova have been situated in recreation areas where the contact between ticks and humans can be expected to be high (Movila et al., 2006). Similar data exist for other

The efficient persistence of the borreliae in endemic areas requires the involvement of reservoir hosts. Potential hosts for ticks are numerous, and more than 300 vertebrate species have been identified as hosts for *I. ricinus,* including small mammals, birds, larger mammals and reptiles. Among these hosts, some act as blood meal sources and as reservoir hosts for pathogens, others as blood meal sources only. Important, that natural host does not seem to develop clinical manifestations of Lyme disease and it is difficult to evaluate the impact of *Borrelia* infection on their health. Minor clinical manifestations may escape medical attention

Halos et al. (2010) reported that the *B. burgdorferi* s.l. infection prevalence was higher on pastures that had a high percentage of shrubs on the perimeter. This result is consistent with the fact that *B. burgdorferi* s.l. reservoir hosts, i.e., rodents and birds, are particularly concentrated in the shrubby vegetation around pastures (Boyard et al., 2008; Vourc'h et al., 2008). The *B. burgdorferi* s.l. infection prevalence also increased in pastures surrounded by forests with low perimeter length/surface area ratios. The lowest theoretical ratio corresponds to a circle; conversely, a high ratio indicated an indented shape with more edge compared to the surface area, which, again, should favor the small vertebrate abundance. This effect was thus in contrast to what we observed on woodland sites, where the prevalence tended to be associated with fragmented forest (Movila, 2008; Movila et al., 2006). Infected ticks found in pastures could have become infected by feeding on infected reservoir hosts located in the pasture itself or by feeding on infected woodland hosts that then imported the ticks into the pasture (Boyard et al., 2007). Halos et al. (2010) hypothesized that a decreased flow of tick hosts between woodlands and pastures could occur when the forest surrounding the pasture has a high perimeter length/surface area

*persulcatus* ticks (Alekseev et al., 2010; Korenberg et al., 1997).

copulation time of *I. persulcatus*.

countries (Daniels et al., 1997; Juntila et al., 1999).

**4. Vertebrates species specificity**

(Gern, 2009).

ratio, a characteristic which would consequently hinder the infection prevalence in pasture ticks. This mechanism could be mediated by factors that we have not taken into consideration in this study, such as the pasture isolation from woodlands and the lack of connectivity between woodlands and pastures.

#### **4.1 Mammals as reservoirs for** *Borrelia* **spp.**

Only a few mammal species have been identified as reservoirs for *B*. *burgdorferi* s.l. in Europe. Globally, little information is available on the real significance of most animal hosts as sources for infecting ticks with *B*. *burgdorferi* s.l. At present, several species of mice, voles, rats and shrews are recognized as reservoirs of *B*. *burgdorferi* s.l. in Europe (Gern & Humair, 2002). In particular, it was evidenced that the mice *Sylvaemus flavicollis* (Melchior), *Sylvaemus sylvaticus* (Linnaeus), *Apodemus agrarius* (Pallas) and the vole, *Myodes* (=*Clethrionomys) glareolus* (Schreber), play key roles in the ecology of Lyme borreliosis as reservoirs for *B*. *burgdorferi* s.l. in many European countries. Once infected by an infectious tick bite, some reservoir hosts, like *Sylvaemus* mice, have been shown to persistently remain infectious for ticks. Small rodents are frequently parasitized by larval and nymphal *I*. *ricinus,*  and this also contributes to their importance as reservoirs. Less information has been obtained on the roles of other small mammal species in the maintenance cycles of *Borrelia* in nature. Nevertheless, another species of vole *Microtus agrestis* (Linnaeus) in Sweden, and black rats *Rattus rattus* (Linnaeus) and Norway rats *Rattus norvegicus* (Berkenhout) in urbanized environments in Germany and in Madeira, may serve as sources of infection for *I*. *ricinus* ticks. Similarly, only few data have been collected on *B*. *burgdorferi* s.l. in shrews (*Sorex minutus* Linnaeus and *Sorrex araneus* Linnaeus and *Neomys fodiens* (Pennant) or in ticks attached on them. Observations in endemic areas of Germany and France showed that edible dormice *Glis glis* (Linnaeus) and garden dormice *Eliomys quercinus* (Linnaeus) are reservoir hosts for *Borrelia*. Other rodent species, like grey squirrels (*Sciurus carolinensis*  Gmelin) in the UK and red squirrels *Sciurus vulgaris* (Linnaeus) in Switzerland also contribute to the amplification of *Borrelia* in the tick population. Interestingly, red and grey squirrels are usually very heavily infested with ticks.

In other investigations in Ireland, Germany and Switzerland, it was reported that the European hedgehog (*Erinaceus europaeus* Linnaeus) also perpetuates *B*. *burgdorferi* s.l. (Rauter & Hartung, 2005). An enzootic transmission cycle of *B*. *burgdorferi* s.l. involving hedgehogs and another tick vector, *I*. *hexagonus*, has been observed in an urban environment. This shows that gardens can also represent zones at risk of Lyme borreliosis as further discussed below. Examination of the role of lagomorphs *Lepus europaeus* (Pallas), *Lepus timidus* (Linnaeus) and *Oryctolagus cuniculus* (Linnaeus) in the support of the enzootic cycle of *B*. *burgdorferi* s.l. has also elucidated their roles as reservoirs (Gern & Humair, 2002).

Assessment of the reservoir competency of large mammals is clearly a difficult task. It necessitates, if xenodiagnosis is applied, capture of the animals and maintenance in a laboratory structure. The consequence of this is that the role of mediumsized and large mammalian species has been studied less and is not yet clearly understood. Red foxes seem to be implicated in the maintenance of borreliae in nature, as described in Germany. However, these animals do not appear to be very potent reservoirs, since spirochaetes were poorly transmitted to ticks feeding on them. According to various reports, ruminants appear to act primarily as sources of blood for ticks. Controversy long surrounded the exact role of

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

seem to be infectious to birds and rodents.

based on genetic distance and ecological differences.

2002, with modifications).

west Europe where it is much less frequent (Poupon et al., 2006).

Vectors Competence and Vertebrate Host Specificity 41

North Africa and south-west Europe, where this *Borrelia* species clearly dominates, to north-

The most commonly parasitized birds are blackbirds and song thrushes, both grounddwelling birds, which most likely come into contact with *I. ricinus* subadults. Overall, nymphs were more commonly found on birds than were larvae. This result contrasts with those of previous studies involving rodents, in which nymphs feeding on rodents were less abundant than larvae (Hanincová et al., 2003; Movila et al., 2008; Špitalská et al., 2006).

According to classical theory of Kurtenbach et al. (2002) (Fig. 5), *B. afzelii*, *B. garinii* (NT29 ribotype, OspA type 4\*), *B. japonica* and *B. bissettii* are resistant to the rodent complement, that makes rodents appropriate reservoir hosts for these genospecies. *B. garinii* (except the abovementioned strains), *B. valaisiana* and *B. turdi* are resistant to the bird complement. *B. burgdorferi* s.s. has an intermediate resistance to both bird and rodent complement and often

\* Margos et al. (2008) proposed a new species *B. bavariensis* as separate from *B. garinii* OspA serotype 4

Fig. 5. Schematic diagram of host specificity of *Borrelia burgdorferi* s.l. (Kurtenbach et al.,

large animals, particularly cervids, in the maintenance cycle of *Borrelia* in endemic areas. Currently, most studies seem to indicate that they do not play a role as reservoirs. In fact, studies undertaken in Sweden and in the UK on roe deer *Capreolus capreolus* (Linnaeus), moose *Alces alces* (Linnaeus), red deer *Cervus elaphus* (Linnaeus) and fallow deer *Dama dama*  (Linnaeus) suggested that these species do not infect feeding ticks with *B*. *burgdorferi* s.l. However, according to some recent developments, the possibility exists that they may act as supports for co-feeding transmission of borreliae between infected and uninfected ticks, and therefore may represent amplifying hosts (for review see Gern, 2009).

To date, it was shown that different *Borrelia* genospecies are associated with different vertebrate host. Our data and data of other authors showed that small rodents of the genus *Apodemus* and of the genus *Myodes* as well as red (*S. vulgaris*) and grey squirrels *Sciurus carolinensis* (Gmelin) were usually infected by *B*. *afzelii* and less frequently by *B*. *burgdorferi* s.s. and that they transmitted these 2 *Borrelia* species to ticks feeding on them (Gern, 2009; Movila, 2008). As far as less common *Borrelia* species are concerned, like *B. lusitaniae*. Thus, Dsouli et al. (2006) demonstrated the reservoir role of the lizard *Psammodromus algirus* (Linnaeus) for *B. lusitaniae* in Tunisia, Richter & Matuschka (2006) the roles of the common wall lizard *Podarcis muralis* (Laurenti) and sand lizard *Lacerta agilis* Linnaeus in Germany and Amore et al. (2007) reported that *P. muralis* was a reservoir for this *Borrelia* species in Italy.

#### **4.2 Birds as reservoirs for** *Borrelia* **spp.**

When attention was first directed at the role of birds in the ecology of Lyme borreliosis, their role was minimized (Gern, 2009). However, at the beginning of the 1990s, the reservoir role of birds was clarified in Europe, and now it is commonly accepted that some bird species are reservoirs for *B. burgdorferi* s.l. In 1998, 2 studies clearly defined the reservoir role of birds, one on a passerine bird, the blackbird *Turdus merula* (Linnaeus), the other one on a gallinaceous bird species, the pheasant *Phasianus colchicus* (Linnaeus) (Gern & Humair, 2002). Both studies examined the reservoir role of these bird species using xenodiagnosis. Tick xenodiagnosis consists of infecting uninfected ticks – usually larvae – during feeding on the animal suspected to be reservoir host. The fact that some of the same species of *Borrelia* exist both in the northern and in the southern hemisphere is further evidence that birds participate in the natural circulation of *Borrelia* spirochaetes (Poupon et al., 2006). These results and others have evidenced the contribution of birds to the circulation of *Borrelia* in endemic areas. A transmission cycle of *B*. *burgdorferi* s.l. was discovered in environmental settings other than the biotopes where *I. ricinus* usually live. In fact, it was demonstrated, on a Swedish island, that *B. burgdorferi* spirochaetes could be maintained in seabird colonies among razorbills *Alca torda* (Linnaeus) by an associated tick species, *I*. *uriae*. The involvement of seabirds and *I*. *uriae* (in the marine environment) in the transport of infected *Borrelia garinii* between the northern and the southern hemispheres was described. (Comstedt et al., 2009; Gern et al., 1998; Kurtenbach et al., 2006). In this context, it is interesting to mention that in a laboratory study, reactivation of latent *Borrelia-*infection could be induced in passerines experimentally submitted to stressful conditions simulating migration. This implies that during their migration, birds can infect ticks all along their migration route. Bird migration also allows the transfer and establishment of particular *Borrelia* species, as described for *B. lusitaniae.* In fact, birds migrating between south-west Europe/North Africa to northwestern Europe have been suggested to be responsible for the transfer of *B. lusitaniae* from

large animals, particularly cervids, in the maintenance cycle of *Borrelia* in endemic areas. Currently, most studies seem to indicate that they do not play a role as reservoirs. In fact, studies undertaken in Sweden and in the UK on roe deer *Capreolus capreolus* (Linnaeus), moose *Alces alces* (Linnaeus), red deer *Cervus elaphus* (Linnaeus) and fallow deer *Dama dama*  (Linnaeus) suggested that these species do not infect feeding ticks with *B*. *burgdorferi* s.l. However, according to some recent developments, the possibility exists that they may act as supports for co-feeding transmission of borreliae between infected and uninfected ticks, and

To date, it was shown that different *Borrelia* genospecies are associated with different vertebrate host. Our data and data of other authors showed that small rodents of the genus *Apodemus* and of the genus *Myodes* as well as red (*S. vulgaris*) and grey squirrels *Sciurus carolinensis* (Gmelin) were usually infected by *B*. *afzelii* and less frequently by *B*. *burgdorferi* s.s. and that they transmitted these 2 *Borrelia* species to ticks feeding on them (Gern, 2009; Movila, 2008). As far as less common *Borrelia* species are concerned, like *B. lusitaniae*. Thus, Dsouli et al. (2006) demonstrated the reservoir role of the lizard *Psammodromus algirus* (Linnaeus) for *B. lusitaniae* in Tunisia, Richter & Matuschka (2006) the roles of the common wall lizard *Podarcis muralis* (Laurenti) and sand lizard *Lacerta agilis* Linnaeus in Germany and Amore et al. (2007)

When attention was first directed at the role of birds in the ecology of Lyme borreliosis, their role was minimized (Gern, 2009). However, at the beginning of the 1990s, the reservoir role of birds was clarified in Europe, and now it is commonly accepted that some bird species are reservoirs for *B. burgdorferi* s.l. In 1998, 2 studies clearly defined the reservoir role of birds, one on a passerine bird, the blackbird *Turdus merula* (Linnaeus), the other one on a gallinaceous bird species, the pheasant *Phasianus colchicus* (Linnaeus) (Gern & Humair, 2002). Both studies examined the reservoir role of these bird species using xenodiagnosis. Tick xenodiagnosis consists of infecting uninfected ticks – usually larvae – during feeding on the animal suspected to be reservoir host. The fact that some of the same species of *Borrelia* exist both in the northern and in the southern hemisphere is further evidence that birds participate in the natural circulation of *Borrelia* spirochaetes (Poupon et al., 2006). These results and others have evidenced the contribution of birds to the circulation of *Borrelia* in endemic areas. A transmission cycle of *B*. *burgdorferi* s.l. was discovered in environmental settings other than the biotopes where *I. ricinus* usually live. In fact, it was demonstrated, on a Swedish island, that *B. burgdorferi* spirochaetes could be maintained in seabird colonies among razorbills *Alca torda* (Linnaeus) by an associated tick species, *I*. *uriae*. The involvement of seabirds and *I*. *uriae* (in the marine environment) in the transport of infected *Borrelia garinii* between the northern and the southern hemispheres was described. (Comstedt et al., 2009; Gern et al., 1998; Kurtenbach et al., 2006). In this context, it is interesting to mention that in a laboratory study, reactivation of latent *Borrelia-*infection could be induced in passerines experimentally submitted to stressful conditions simulating migration. This implies that during their migration, birds can infect ticks all along their migration route. Bird migration also allows the transfer and establishment of particular *Borrelia* species, as described for *B. lusitaniae.* In fact, birds migrating between south-west Europe/North Africa to northwestern Europe have been suggested to be responsible for the transfer of *B. lusitaniae* from

therefore may represent amplifying hosts (for review see Gern, 2009).

reported that *P. muralis* was a reservoir for this *Borrelia* species in Italy.

**4.2 Birds as reservoirs for** *Borrelia* **spp.**

North Africa and south-west Europe, where this *Borrelia* species clearly dominates, to northwest Europe where it is much less frequent (Poupon et al., 2006).

The most commonly parasitized birds are blackbirds and song thrushes, both grounddwelling birds, which most likely come into contact with *I. ricinus* subadults. Overall, nymphs were more commonly found on birds than were larvae. This result contrasts with those of previous studies involving rodents, in which nymphs feeding on rodents were less abundant than larvae (Hanincová et al., 2003; Movila et al., 2008; Špitalská et al., 2006).

According to classical theory of Kurtenbach et al. (2002) (Fig. 5), *B. afzelii*, *B. garinii* (NT29 ribotype, OspA type 4\*), *B. japonica* and *B. bissettii* are resistant to the rodent complement, that makes rodents appropriate reservoir hosts for these genospecies. *B. garinii* (except the abovementioned strains), *B. valaisiana* and *B. turdi* are resistant to the bird complement. *B. burgdorferi* s.s. has an intermediate resistance to both bird and rodent complement and often seem to be infectious to birds and rodents.

\* Margos et al. (2008) proposed a new species *B. bavariensis* as separate from *B. garinii* OspA serotype 4 based on genetic distance and ecological differences.

Fig. 5. Schematic diagram of host specificity of *Borrelia burgdorferi* s.l. (Kurtenbach et al., 2002, with modifications).

Zoonotic Peculiarities of *Borrelia burgdorferi* s.l.:

**5. Conclusion**

zoonoses.

manifestation.

**6. Acknowledgment**

Academy of Science for financial support.

in various geographic areas.

Vectors Competence and Vertebrate Host Specificity 43

2. Infected migratory birds may infect the ticks, which are then dropped off in a new location. In support of this mechanism, Gylfe et al. (2000) found a reactivation of *B. garinii* infection in experimentally infected *Turdus iliacus* Linnaeus during migratory restlessness, indicating an increased risk of transmitting *Borrelia* sp. to the tick vector during migration. If the migratory birds are chronic carriers of tick-borne diseases, then they may continue to transfer them to ticks that parasitize them after arrival. Sampling

of blood or tissue from the birds would provide more information of this issue. 3. The tick vectors may transfer *Borrelia* species between each other through co-feeding while being transported (Randolph et al., 1998; Dubinina & Alekseev, 2003).

The description of the various *Borrelia* species in ticks has opened an entire new field of research in the ecology of Lyme borreliosis. It seems prudent to review the importance of the different types of associations between *Borrelia* species, tick vectors, and vertebrate hosts

Host specialization is an important factor in vector-borne disease, and different pathogens show varying levels and patterns of host specialization, which may impact the spread of pathogens. Lyme borreliosis group of spirochaetes, *B. garinii* and *B. valaisiana* are transmitted by avian species while *B. afzelii* is associated with rodents and certain insectivore species. *B. burgdorferi* s.s. is a generalist species, known to infect both rodent and avian species, as well as other hosts. The diversity in host specialisms in the Lyme borreliosis group of spirochaetes makes this an ideal system to examine the interplay between the ecology of the host and the epidemiology of the bacteria. As ticks do not move over large distances independently, the spread of *Borrelia* spp. spirochaetes is likely to be linked to the migration of their hosts. Species that are maintained by rodents are therefore predicted to show more limited migration than those associated with birds. In addition to being of public health importance, the delineation and monitoring of the geographic ranges of the different Lyme borreliosis species also provides an opportunity to examine in more general terms the role of host ecology in the epidemiology of vectored

Clearly, additional field studies are needed of *Borrelia* host specificities, keeping in mind particularly the newly described *Borrelia* species and the great subtype diversity hidden in most *Borrelia* species, as well as the geographic diversity of local ecosystems. Future studies in Lyme borreliosis ecology seeking to identify the *Borrelia* spirochaetes in both ticks and reservoir hosts in various endemic areas are strongly encouraged. Those studies are extremely important for the determination of the complete spectrum of *Borrelia* species involved in human Lyme disease worldwide with its unknown, rare, and unusual clinical

We indebted to Mr. Igor Babic (InTech Open Access Publisher) and Mr. Alexandr Morozov (Institute of Zoology, Moldova) for rendered assistance in this chapter preparation. We acknowledge the perfect original images of Mrs. Anna Movila. The authors thank Moldova

A review analysis by Humair (2002) indicated that in Eurasia, *B. garinii* is strongly associated with grounddwelling and sea birds. In Europe, also *B. valaisiana* and *B. burgdorferi* s.s are associated with birds (Derdáková & Lenčaková, 2005). At the same time, Comstedt et al. (2006) demonstrated the presence of five *Borrelia* genospecies (*B. garinii*, *B. valaisiana*, *B. afzelii*, *B. burgdorferi* s.s. and *B. miyamotoi*) in migratory birds in northern Europe. Poupon et al. (2006) found that in Switzerland, *B. valaisiana*, *B. garinii*, and *B. lusitaniae* were the most frequently found species in ticks collected from migratory birds, with *B. lusitaniae* found in a surprisingly high abundance. In the western Mediterranean Basin, however, a high prevalence of *B. lusitaniae* has been observed in some localities, suggesting that this genospecies has a narrow spectrum of reservoir hosts, restricting its geographical range (de Michelis et al., 2000). *B. lusitaniae* appears to be infrequent in *I. ricinus* in most areas of Europe (Gern et al., 1999), but it has been described as the dominant *Borrelia* species in *I. ricinus* in south-western Europe (de Michelis et al., 2000) and in North Africa (Younsi et al., 2001). Moreover, *B. lusitaniae* was the most frequently detected species after *B. valaisiana* in Moldavian questing *I. ricinus* ticks collected in the "Codri" forest reserve (Movila, 2008). A question for further investigation is whether the dominant *Borrelia* species in birds has shifted from *B. burgdorferi* s.s. to *B. valaisiana* and *B. lusitaniae* during last decades in that area (Movila et al., 2008).