Pär Comstedt

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No. 1161 ISSN 0346-6612 ISBN 978-91-7264-521-9 Editor: The Dean of the Faculty of Medicine

Biology of Borrelia garinii Spirochetes

Pär Comstedt

Department of Molecular Biology Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University, Sweden 2008

To Erik Bengtsson

Table of contents

ABSTRACT...... 1

Papers in this thesis...... 2

Papers not included in this thesis...... 3

Abbreviations and terms ...... 4

INTRODUCTION...... 5

The history of Lyme borreliosis ...... 5

Zoonoses...... 6

Vector-borne diseases ...... 7

Birds as reservoirs for zoonoses...... 8

Classification...... 9 The Lyme Borrelia genospecies...... 11 General characteristics ...... 11

Geographical distribution...... 12

Clinical manifestations of Lyme borreliosis...... 13

Diagnosis and treatment ...... 14

Prevention...... 14 High risk areas...... 14 The Lyme borreliosis vaccine ...... 15

Genetic manipulation...... 16

In vitro cultivation ...... 17

Typing...... 17 Phenotypic ...... 17 Genotypic...... 18

The infection cycle ...... 18

The tick vector...... 19 Ixodes ricinus complex ...... 19 Ixodes uriae ...... 21

Transfer of infection from the tick to the host ...... 22

Spirochete protein expression in the tick and the skin ...... 23

Reservoir hosts...... 24 Reservoir hosts in North America...... 24 Reservoir hosts in Europe...... 25 Poor reservoir hosts ...... 26 The complicated biology of B. garinii...... 27

Borrelia and birds ...... 28 Experimental infections of birds...... 28 Borrelia spirochetes and terrestrial birds...... 29 The impact of bird migration ...... 30 B. garinii and seabirds ...... 31 Interaction between the marine and the terrestrial infection cycles ...... 32

The complement system ...... 33 Regulation of the alternative pathway ...... 34 Complement evasion...... 34 Differences among Lyme Borrelia spirochetes ...... 35

AIMS OF THE THESIS...... 37

RESULTS AND DISCUSSION...... 39

Paper I...... 39 Birds as carriers of ticks...... 39 Passerine birds as hosts for B. garinii infected ticks...... 40

Reservoir competence of passerine birds ...... 40 Clinical importance of Lyme Borrelia spirochetes spread by birds ...... 41

Paper II...... 43 Presence of Borrelia spirochetes in the Arctic...... 43 Similarities to other Borrelia isolates...... 43

Paper III...... 45 Prevalence and densities of spirochetes among ticks and seabirds ...... 45 Genetic polymorphism ...... 46 Phylogeny...... 46

Paper IV...... 49 The marine and the terrestrial infection cycle ...... 49 Complement resistance properties...... 49

Paper V...... 51 OspE expression and complement resistance...... 51 Factor H binding proteins of B. garinii ...... 51

CONCLUSIONS...... 53

SAMMANFATTNING ...... 55

Acknowledgements...... 57

References...... 58

ABSTRACT

Lyme borreliosis is a tick-transmitted infectious disease. The causative agents are spiral-shaped bacteria and the most common sign of infection is a skin rash at the site of the tick bite. If not treated with antibiotics, the bacteria can disseminate and cause a variety of different manifestations including arthritis, carditis or neurological problems. The disease is a zoonosis and the bacteria are maintained in nature by different vertebrate reservoir host animals. In Europe, three different Borrelia genospecies cause Lyme borreliosis: B. burgdorferi, B. afzelii and B. garinii. The latter depends in part on birds as its reservoir host. B. garinii bacteria have been found in a marine enzootic infection cycle worldwide and also among terrestrial birds. This thesis suggests that passerine birds and seabirds constitute an important reservoir for B. garinii bacteria also with clinical importance. We have found bacteria very similar to Lyme borreliosis causing isolates in ticks infesting migrating passerine birds. The birds not only transport infected ticks, but are competent reservoir hosts, as measured by their ability to infect naïve ticks. Their role as a reservoir host is dependent on their foraging behavior, where ground-dwelling birds are of greater importance than other species. When comparing B. garinii isolates from Europe, the Arctic and North Pacific, and including isolates from seabirds, passerine birds, Ixodes ricinus ticks and Lyme borreliosis patients, we found that phylogenetic grouping was not necessarily dependent on geographical or biological origin. B. garinii from seabirds were very heterogeneous and found in all different groups. Therefore, the marine and the terrestrial infection cycles are likely to overlap. This was supported by the fact that B. garinii isolated from seabirds can establish a long- term infection in mice. Bacteria from the genospecies B. garinii are overrepresented among neuroborreliosis patients. Interestingly, many clinical B. garinii isolates are sensitive to human serum and have shown weak binding to the complement inhibitor protein factor H. By transforming a serum-sensitive B. garinii isolate with a shuttle vector containing the gene for the factor H binding protein OspE from complement-resistant B. burgdorferi, serum resistance could be increased. In addition, neurovirulent B. garinii strains recently isolated from neuroborreliosis patients were shown to express a factor H binding protein, not found in bacteria that had been kept in culture for a long time. This protein may contribute to the virulence of neuroborreliosis-causing B. garinii strains. When testing B. garinii isolates from Lyme borreliosis patients and seabirds for resistance to human serum, all members of the latter group were sensitive to even low levels of serum. This suggests that seabird isolates are not capable of infecting humans. In agreement with this, B. garinii isolated from seabirds do not appear to bind human factor H.

1 Papers in this thesis

I. Comstedt, P., Bergström, S., Olsen, B., Garpmo, U., Marjavaara, L., Mejlon, H., Barbour, A. G. and Bunikis, J. (2006). Migratory passerine birds as reservoirs of Lyme borreliosis in Europe. Emerging Infectious Diseases 12, 1087-1095.

II. Larsson, C., Comstedt, P., Olsen, B. and Bergström, S. (2007). First record of Lyme disease Borrelia in the Arctic. Vector-Borne and Zoonotic Diseases 7, 453-456.

III. Comstedt, P., Bunikis, J., Eliasson, I., Asokliene, L., Olsen, B., Wallensten, B., Bergström, S. Complex population structure of Lyme borreliosis group spirochete Borrelia garinii in subarctic Eurasia. Manuscript

IV. Comstedt, P., Larsson, C., Meri, T., Meri, S. and Bergström, S. Borrelia garinii isolated from seabirds can infect rodents but is sensitive to normal human serum. Manuscript.

V. Alitalo, A., Meri, T., Comstedt, P., Jeffrey L., Tornberg, J., Strandin, T., Lankinen, H., Bergström, S., Cinco, M., Vuppala, S. R., Akins, D. R. and Meri, S. (2005). Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis. European Journal of Immunology 35, 3043-3053

2 Papers not included in this thesis

Pinne, M., Östberg, Y., Comstedt, P. and Bergström, S. (2004). Molecular analysis of the channel-forming protein P13 and its paralogue family 48 from different Lyme disease Borrelia species. Microbiology 150, 549-559.

Östberg, Y., Berg, S., Comstedt, P., Wieslander, A. and Bergström, S. (2007). Functional analysis of a lipid galactosyltransferase synthesizing the major envelope lipid in the Lyme disease spirochete Borrelia burgdorferi. FEMS Microbiology Letters 272, 22-29.

3 Abbreviations and terms

ACA acrodermatitis chronica atrophicans AP alternative pathway CSF cerebrospinal fluid EM erythema migrans LB Lyme borreliosis MAbs monoclonal antibodies MAC membrane attack complex OspA outer surface protein A OspC outer surface protein C PCR polymerase chain reaction qPCR quantitative polymerase chain reaction RF relapsing fever rOspA recombinant outer surface protein A rRNA ribosomal ribonucleic acid tRNA transfer ribonucleic acid

Lyme Borrelia genospecies: The 12 genospecies transmitted by Ixodes ticks LB-causing genospecies: B. burgdorferi, B. afzelii and B. garinii

4 INTRODUCTION

The history of Lyme borreliosis In 1883 the German physician Buchwald described the first known case of acrodermatitis chronica atrophicans (ACA), a late skin manifestation of Lyme borreliosis (LB) (Buchwald, 1883). Later a case of erythema migrans (EM), the characteristic skin rash at the initial stage of LB, was described by Dr. Afzelius at a dermatologic meeting in Stockholm, Sweden 1909 (Berger, 1993). Over the course of 12 years he encountered six cases, and suggested that the reason for the rash was the bite of a tick or another insect (Afzelius, 1921). Some colleagues were not very enthusiastic about the wild hypothesis: “the disease is of no importance for its own sake and is rather to be looked on as a freak,” Prof. James Victor Strandbergs stated (Strandberg, 1920). He could of course not imagine that 70 years later the same disease would become the most common vector-borne zoonosis in temperate regions and be diagnosed in 130 of 100,000 inhabitants in endemic regions of Europe (WHO, 1995). On the other hand, Prof. Strandberg did admit that EM could be problematic. He describes a female patient with a lesion that had extended “across the breast and could only with difficulty be hidden by the application of powder when the patient wore a décolleté dress“ (Strandberg, 1920). At that time the connection between EM and ACA was also recognized (Herxheimer & Hartman, 1902). In 1948, physicians in Europe started to use penicillin to treat erythema migrans, because spirochete-like structures had been found in skin specimens (Hellerström, 1951).

Later the same century the topic was again of immediate interest, but now in another part of the world. In 1977 Steere and co-workers described a new, previously unrecognized illness among inhabitants of the town Old Lyme, Connecticut. The disease had been recognized since 1975 and the symptoms involved, fatigue, chills, fever, headache, stiff neck, backache, myalgias, nausea, vomiting etc. Some other odd but interesting symptoms were an expanding red

5 annular lesion at the site of a previous tick bite, severe arthritis most often in knee joints and in some cases neurological or cardiac abnormalities (Steere et al., 1980). Since there was a dramatic increase in cases during summer and early fall they suggested that an arthropod vector, probably a tick from the genus Ixodes, was responsible for the infection now known as “Lyme disease” (Steere et al., 1977a; Steere et al., 1977b). A few years later, in 1981, Willy Burgdorfer encountered long, irregularly coiled spirochetes when examining the midguts of Ixodes scapularis ticks. The original goal was to isolate a virulent strain of Rickettsia rickettsii, the causative agent of spotted fever, a disease that had caused eight deaths on Long Island a few years earlier (Burgdorfer, 2006). Burgdorfer then recalled a discussion with Dr. Hellerström back in 1950, who had suggested that the skin manifestation Erythema chronicum migrans was caused by a tick bite (Hellerström, 1951), an idea to which no one had paid attention for the last 30 years. The relationship between the new treponema-like spirochete and Lyme disease was confirmed by infection in rabbits that developed the characteristic red skin rash a few weeks later. In addition, Western blot and indirect immunofluorescence using sera from a patient recovering from Lyme disease identified the spirochete (Burgdorfer et al., 1982). The vectors responsible for the infection were identified as I. scapularis in the Northeast and Midwest and in the West I. pacificus (Steere et al., 1978; Wallis et al., 1978). The Lyme disease spirochete was now named Borrelia burgdorferi. Later, early findings of the pioneer Afzelius were also acknowledged by naming a common etiological agent of Lyme disease in Europe “B. afzelii” (Canica et al., 1993). The disease is now recommended to be designated Lyme borreliosis worldwide (Åsbrink & Hovmark, 1993).

Zoonoses More than half of all pathogens infectious to humans are zoonoses, i.e. diseases of animals that can be transmitted to humans. A recent study presented 816 zoonotic species including viruses, bacteria, fungi, protozoa and helminths (parasitic flatworms or roundworms) (Woolhouse & Gowtage-Sequeria, 2005). The majority of emerging or reemerging pathogens are known to be zoonotic. For most of the zoonotic pathogens, humans are considered to be a “dead end”. This is true also for Lyme Borrelia spirochetes, where humans do not contribute

6 to infect ticks. One of the oldest known zoonotic bacteria is Mycobacterium tuberculosis, the causative agent of tuberculosis. It has probably evolved as an infection in both animals and humans 20,000 years ago and became more specialized for humans 10,000 years ago after domestication of animals. Bone deformations as a result of the disease have been documented in animals and humans since 3700 BC. Also, DNA from M. tuberculosis has been found in a 1000-year-old South American mummy (Källenius & Svensson, 2001).

Vector-borne diseases Some diseases are transmitted to humans by a vector. The vector is normally an arthropod (insects, spiders etc). Malaria is probably the most well-known vector- borne disease in modern times. The disease is caused by Plasmodium parasites and humans are infected by a mosquito bite. It infects 500 million people and harvesting 100 million casualties every year (Snow et al., 2005). Most of the victims are in Africa and the disease is mostly associated with tropical or sub- tropical climates but was once present also in Europe. In Sweden the last case of malaria was diagnosed in 1930 (Wahlgren, 1999). Other well-known vector- borne diseases include sleeping sickness where the tsetse fly acts as vector for Trypanosoma spp. (Vickerman et al., 1988). Some zoonoses are also vector-borne. The vector then acquires the infection when feeding on an infected reservoir animal. Subsequent feeding on humans then transfers the etiological agent and causes the disease. Different vector-borne zoonotic diseases have had an enormous impact on civilizations throughout history. Bubonic plague caused by Yersinia pestis is a disease normally associated with rats and flies. Occasionally, the flies can transmit the infection to humans. This has happened several times in history and the results have been devastating. From 1347 to 1351, a massive pandemic swept through Asia and Europe. When it ended, 100 million people had died worldwide (Prentice & Rahalison, 2007).

In some cases ticks function as vectors for bacteria. This is true for the intracellular family members of Anaplasmataceae, causig human granulocytic anaplasmosis (Dumler et al., 2007). Other intracellular bacteria are Rickettsia spp., causing typhus and spotted fever in humans (Winkler, 1990). Relapsing fever Borrelia species such as B. duttonii, B. crocidurae, B. hispanica and B. persica are

7 present in Africa, Europe and Asia, and are transmitted by Ornithodoros ticks. In North America B. hermsii is the most common RF species, transmitted by O. hermsi (Felsenfeld, 1971). Different viruses are also transmitted by ticks. Tick- borne encephalitis (TBE) virus is transmitted by I. ricinus or I. persulcatus in Europe and Russia, respectively. Infection can lead to acute central nervous system disease, but also death or long term neurological damage (Dumpis et al., 1999).

Birds as reservoirs for zoonoses Birds play an important role in the ecology of many pathogens also infective to humans; their relatively long lifespan and the fact that they can fly aid in dispersing infections worldwide. Also, birds of diverse species tend to congregate at migration stopover sites and thereby allow for horizontal transfer of the pathogen. In some cases the bird itself is a relatively poor host but, spreading pathogens to new distant foci where it can infect a more suitable reservoir host may be of great importance (Olsen et al., 2006). There are three mechanisms by which birds can be involved in spreading pathogens (Hubalek, 2004):

1. Biological carriers. The bird is infected and the pathogen multiplies in the avian body. One of the more well-known diseases for which birds act as biological carriers is West Nile virus (Woolhouse & Gowtage- Sequeria, 2005). Also, influenza A virus has been found in some birds in Europe (De Marco et al., 2003), Vibrio cholera (Ogg et al., 1989). Two bacterial species causing human gastroenteritis, Camplylobacter jejuni and Salmonella typhimurium, have also been isoalated from wild migrating birds (Palmgren et al., 1997). These two bacterial species can also contaminate water through the faeces of birds.

2. Mechanical carriers. The bird carries the pathogen on or in the body, but there is no amplification. Fungal spores from Candida albicans have been shown to be spread by gulls feeding on contaminated food (Buck, 1986). Aspergillus fumigatus, the causative agent of aspergillosis (fungi infection of the lungs) simply sticks to feathers or other avian body parts as a means of transportation (Hubalek, 1994).

3. Transporters. The bird has attached infected ectoparasites. The medically most important ectoparasites on birds are ticks (Ixodes spp. and Argas spp.). TBE virus, has been found in ticks feeding on birds of many different species (Hubalek, 1994). These birds often origin from Russia or Eastern Europe. Also isolates of Ehrlichia phagocytophila identical to clinical strains causing human granulocytic anaplasmosis have been isolated from ticks feeding on wild birds (Bjöersdorff et al., 2001). There are also reports of Rickettsia sibirica (North Asian tick typhus), Coxiella brunettii (Q-fever) and Babesia microti (babesiosis) in ticks parasitizing on birds (Alekseev et al., 2003; Somov & Soldatov, 1964; Syrucek & Raska, 1956).

When it comes to the Lyme Borrelia spirochetes, birds function both as biological carriers and transporters of infected ticks, as described in later chapters.

Classification Borrelia bacteria belong to the Spirochete phylum in the class Spirochaetes and the order Spirochetales. Examples of other human disease-causing spirochetes are Treponema pallidum (causing syphilis), T. denticola (periodontal diseases and tooth loss) and Leptospira interorrgans (leptospirosis) (Paster & Dewhirst, 2000). The genus Borrelia is comprised of many different genospecies but the two groups causing disease in humans are relapsing fever Borrelia genospecies and Lyme Borrelia genospecies. Most of the RF genospecies (B. duttonii, B. hermsii, and B. crocidurae) are transmitted by soft-bodied ticks (Argasidae), but one species (B. recurrentis) is transmitted by the human body louse. The latter is responsible for the epidemic outbreaks of RF (Felsenfeld, 1971). The Lyme Borrelia genospecies (see Table 1) on the other hand are only transmitted by hard-bodied ticks of the genera Ixodes (Sonenshine, 1991).

Table 1. Lyme Borrelia genospecies, tick vectors, geographical distribution and main reservoir animals. Lyme Borrelia Bacterial geographical Main reservoir Principal tick vector genospecies distribution animals I. scapularis, I. pacificus N. America Rodents, birds B. burgdorferi* I. ricinus Europe Rodents, birds I. persulcatus Russia Rodents, birds I. ricinus Europe, W. Russia Rodents B. afzelii* I. persulcatus E. Russia, China, Japan Rodents I. ricinus Europe, W. Russia Rodents, birds Europe, E. Russia, N.E. and N.W. America, S. B. garinii* I. uriae Seabirds Pacific Ocean, Arctic, Sub-Antarctic Islands I. persulcatus Russia, China, Japan Rodents, birds I. spinipalpis, I. pacificus N. America Rodents B. bissettii** I. ricinus Europe Rodents B. lusitaniae** I. ricinus S. Europe, N. Africa Birds, lizards B. spielmanii** I. ricinus Europe Rodents I. ricinus Europe Birds B. valaisiana I. columnae Japan Birds B. andersonii I. dentatus N. America Rabbits B. japonica I. ovatus Japan Rodents B. tanukii I. tanuki Japan, Nepal Rodents B. sinica I. ovatus China, Nepal Rodents B. turdi I. turdus Japan Birds

* Genospecies frequently infecting humans. ** Genospecies occasionally infecting humans. E., East. N., North. S., South. W., West. (Dsouli et al., 2006; Herzberger et al., 2007; Kurtenbach et al., 2002a; Majlathova et al., 2006; Masuzawa et al., 2001; Masuzawa, 2004; Sonenshine, 1993; Strle et al., 1997)

10 The Lyme Borrelia genospecies The Lyme Borrelia group of spirochetes includes 12 known genospecies. B. burgdorferi, B. garinii and B. afzelii are all known to cause disease in humans (Stanek & Strle, 2003). Lately a few clinical cases due to B. bissettii, B. lusitaniae and B. spielmanii infections also have been reported (Collares-Pereira et al., 2004; Fingerle et al., 2007; Maraspin et al., 2002). On the other hand, B. japonica, B. andersonii, B. tanukii, B. turdi, B. valaisiana and B. sinica are also found in ticks and animals but are not reported to infect humans (Wilske, 2005). The tick vector, geographical distribution and reservoir animals differ between the genospecies, and are summarized in Table 1. However, since some genospecies are newly discovered and others only found in isolated areas or in a few examined animals, the Table is not comprehensive. In some cases, spirochetes have only been detected by polymerase chain reaction (PCR) in a few individual animals, and these have not been included. Also, using PCR as a method for identifying reservoir animals is unreliable, since the reservoir capacity has not been evaluated; this is therefore better done with xenodiagnosis or quantitative PCR (qPCR) on feeding larvae.

Figure 1. Electron micrograph of a B. garinii spirochete, isolated from cerebrospinal fluid of an LB patient. Photo: Pär Comstedt and Akemi Takade.

General characteristics The Borrelia spirochetes have an obligate parasitic lifestyle whereby they are only able to survive in a tick or a vertebrate host. They sort with Gram-negative bacteria but lack lipopolysaccharides. They are coiled, up to 30 µm long and less than 1 µm thick (Figure 1). Variation can however occur between genospecies. Motility is mediated by 7 to 11 flagellae located in the periplasmic space. The generation time is determined to be 12 to 24 hours in vitro, which is extremely long compared to other bacteria (Barbour, 1984). B. burgdorferi (strain B31 MI) has a segmented genome with a linear chromosome and 21 plasmids, 9 circular

11 and 12 linear. This is the largest number of extrachromosomal elements known for any bacterium. The total size is only about 1,5M base pairs, and encodes limited biosynthetic genes, suggesting that many nutritional components are taken up from the host. Several of the genes are part of paralogous gene families or encode proteins with no homology in other bacteria. The function of these proteins are therefore largely unknown (Casjens et al., 2000; Fraser et al., 1997).

The plasmids carry genes that are necessary for the bacteria to fulfill the infection cycle and therefore are not dispensable. Some studies therefore suggest referring to them as mini chromosomes (Barbour, 1993; Bergström et al., 1992). The outer membrane is fluid and has a protein content of 46% and a lipid content of 51% (Coleman et al., 1986). One special feature of the outer membrane is the lack of lipopolysaccharides (Takayama et al., 1987). The surface is instead covered with lipoproteins, with genes encoding them constituting 8% of the total open reading frames (Fraser et al., 1997). Borrelia spirochetes are also reported to have fever membrane spanning proteins than other Gram-negative bacteria (Walker et al., 1991).

Geographical distribution In North America clinical cases of LB are due to infections of B. burgdorferi. B. bissettii is also present and this genospecies has been isolated occasionally from human LB patients in Europe (Burkot et al., 2001; Strle et al., 1997). In Europe and Asia the situation is more complex due to three causative agents, namely B. garinii, B. afzelii and B. burgdorferi, although the latter is not very common in Asia (Kurtenbach et al., 2006; Masuzawa, 2004; Saint Girons et al., 1998). In addition, these genospecies are spread by two different vectors: I. ricinus in Europe and I. persulcatus in Asia. The habitats of these two ticks overlap in Eastern Europe (Kurtenbach et al., 2006). The Borrelia spirochete prevalence and genospecies distribution in questing I. ricinus ticks can differ dramatically between regions. A metaanalysis based on 112,579 I. ricinus ticks from 24 European countries suggests that 18.6 % of adults and 10.9% of nymphs are infected with Borrelia spirochetes. When compiling 50 separate studies without distinguishing between nymphs and adults, the mean percentages of the different genospecies were: B. afzelii (38%), B. garinii (33%), B. burgdorferi (18%), B. valaisiana (19%) and B.

12 lusitaniae (7%). Also 5% were untypable and 13% were co-infected (Rauter & Hartung, 2005). It is notable that B. afzelii seems to be more or less absent in the British Isles; the reason for this is not completely clear but different behavior among the questing ticks is suggested (Kurtenbach et al., 2006). This is interesting since B. afzelii is the main genospecies found in rodents on the European continent (Hanincova et al., 2003a). For details about genospecies distribution, vector association and host reservoir etc., see Table 1.

Clinical manifestations of Lyme borreliosis Clinically, Lyme borreliosis is most similar to syphilis, both exhibiting multisystem involvement and mimicry of other diseases (Sleigh & Timbury, 1994; Steere, 1989). The most common sign of an LB infection is a red skin rash called erythema migrans observed in 80-89% of the clinical cases in Europe and North America (Mehnert & Krause, 2005; Steere, 1989). The EM expands from the site of the tick bite with a central clearing and without treatment it can reach 1 m in diameter (Stanek & Strle, 2003). The skin lesion is often accompanied by influenza-like symptoms such as nausea, fatigue, arthralgia, myalgia, fever and headache. This is followed by a disseminated infection within days or weeks affecting the nervous system, joints, skin or heart. Subsequently a persistent infection can evolve within weeks or months (Steere, 2001). European patients can develop a chronic skin manifestation called acrodermatitis chronica atrophicans. Live spirochetes have been isolated from such lesions more than 10 years after the onset of the disease (Åsbrink & Hovmark, 1985). In about 10% of the patients, no symptoms of infection can be seen (Steere, 2001).

When culturing Lyme Borrelia spirochetes from clinical samples, an association between the different genospecies and certain tissues can be seen. In a European survey B. garinii was found in 69% of the cerebrospinal fluid (CSF) samples and B. afzelii in 84% of the ACA biopsies. B. burgdorferi is more often associated with arthritis than other symptoms but the pattern is not as clear as for the two other genospecies (Wilske, 2003). The reason for this connection between genospecies and certain tissues is not known.

13 Diagnosis and treatment Tick bite history and general awareness after staying in LB endemic areas are of course crucial for diagnosis. Persons who develop a skin lesion or other illness within 1 month after removing an attached tick should consult a physician. The EM is still one of the most easily detectable signs of an infection (Wormser et al., 2000).

The heterogeneity of the causative strains is a challenge for clinical diagnosis of LB, especially in Europe. The diagnostic methods have to identify the presence of all three causative agents in a reliable manner. Cultivation of spirochetes in BSK medium is time-consuming and has low sensitivity (Wilske, 2005), for details see section about in vitro cultivation. PCR detection of rrs (16S), p66 or flagellin genes are also used, but does not distinguish between living and dead bacteria. This can however be the method of choice in later stages of the disease (Steere, 2001). Antibody detection of surface localized antigens is a common method but can not be applied at the initial stage of infection (Steere, 2001). When used, it is recommended that positive Enzyme-Linked Immunosorbent Assay (ELISA) results are confirmed by Western blot analysis (Wilske, 2005).

Prevention The skin constitutes an effective physical barrier which is overcome when the tick penetrates the dermis. The most efficient way to decrease risk of infection is therefore to avoid tick bites. Properly tucked in clothes not leaving any exposed skin provides effective protection. Still, removing any attached ticks within 24 hours of attachment reduces the chance of infection dramatically. It is therefore important to examine the body for ticks when staying in endemic areas (Piesman et al., 1987). The risk of attracting ticks can also be reduced by application of mosquito or tick repellents.

High risk areas A lower risk for disease in endemic areas can be achieved by limiting the overall nymphal abundance and/or by lowering nymphal infection prevalence (Mather et al., 1996). In North America, the distribution of deer has been found to correlate

14 well with the distribution of I. scapularis (Fish, 1995). Adult females need a big volume of blood before they lay eggs and therefore prefer to infest larger animals such as deer. This increases the overall prevalence of larvae and subsequently also nymphs, the tick stage most often responsible for spreading the infection to humans (Sonenshine, 1993). The presence and composition of different reservoir animals are crucial for the prevalence of Lyme Borrelia-infected nymphs. This subject is discussed in more detail in other chapters.

One of the most important factors influencing the tick life cycle and therefore also the habitat is the vegetation period. This is defined as the period of the year with a mean temperature above 5°C (Daniel, 1993). The vegetation period can be extended locally by great bodies of water generating warmer autumns. In milder climates where deciduous vegetation dominates, many different potential reservoir animals are also present, including both mammals and birds. Another important factor to consider is the relative humidity, which is positively correlated with questing activity of Ixodes ticks (Loye & Lane, 1988).

The Lyme borreliosis vaccine There is no prophylactic vaccine available against LB. However, a vaccine called LYMErix, produced by SmithKline Beecham, was commercially available in the USA from 1998 but withdrawn from the market in 2002. The company cited poor sales, the need for frequent boosters and that it was not suitable for children as reasons. Also, the costs for this preventive approach compared to treatment with antibiotics could have been a motive (Meltzer et al., 1999). However, the real explanation was rather fear of lawsuits, since in rare cases vaccination was blamed as the cause of developing autoimmune arthritis, although this was never proven (Anonymous, 2006; Steere, 2006). The vaccine was based on a recombinant form of outer surface protein A (rOspA). It was originally believed that protection was conferred in the host by OspA-specific antibodies, since immunized mice challenged with B. burgdorferi spirochetes grown in vitro were protected from infection (Fikrig et al., 1990). However, it is now known that the spirochetes express OspA on their surface when grown in vitro and the antibodies kill the spirochetes in the tick gut, before they migrate to the salivary glands (Fikrig et al., 1992). LYMErix was only effective against B.

15 burgdorferi infections, and therefore availability was restricted to the USA, where this genospecies alone is responsible for all clinical cases. In Europe and Asia, B. burgdorferi is less common. Instead B. garinii and B. afzelii are the major causative agents for LB. A vaccine for the European market therefore needs to provide protectivity against all three genospecies. Now, the LYMErix vaccine is available for dogs only (Ma et al., 1996). Different attempts to identify new, suitable vaccine candidates have been tried through the years, but this has not yet led to any commercially available vaccine. In addition, the rOspA vaccine was used in a field study, where wild white-footed mice were vaccinated. This resulted in a reduction of B. burgdorferi in nymphal I. scapularis ticks the following year (Tsao et al., 2004).

Genetic manipulation Performing genetic manipulation in Borrelia spirochetes is a fairly new area. Low transformation frequencies, the need for a complex growth medium, lack of selectable markers, a long generation time and difficulty growing the spirochetes on solid medium constituted big problems. However, when the B. burgdorferi (B31) genome was published in 1997 progress was made (Fraser et al., 1997). The construction of a KanamycinR cassette under the regulation of a flagellin (flaB) promoter, considerably facilitated gene inactivation (Bono et al., 2000; Tilly et al., 2000). The shuttle vector pBSV2 mediating complementation of genetically inactivated genes was constructed using portions of circular plasmid 9 from B. burgdorferi. Later this vector was followed by pBSV2G, using a gentamycin resistance cassette as selectable marker (Stewart et al., 2001). Another problem is the loss of plasmids during prolonged in vitro cultivation and transformation, leaving many spirochetes non-infective to mice (Schwan et al., 1988). A highly transformable and infectious B. burgdorferi strain was constructed by inactivation of a putative restriction-modification gene (Kawabata et al., 2004). The majority of genetic manipulations are done in B. burgdorferi. Even though the genomes of B. afzelii (PKo) and B. garinii (PBi) now have been sequenced (Glöckner et al., 2004; Glöckner et al., 2006).

16 In vitro cultivation The Borrelia spirochetes have a complex nutritional requirement and growth in vitro is possible using a liquid complex medium, Barbour-Stoenner-Kelly (BSK). The medium is supplemented with 6-10% rabbit serum and in some cases 1.4% (w/v) gelatin. Growth is possible at lower temperatures (20°C) but optimal at 30- 37°C (Barbour, 1984). Borrelia spirochetes are microaerophilic and cultivation is often performed in a CO2 incubator (Barbour, 1984). Also, growth is possible in solid medium which might be useful when obtaining clonal isolates and in genetic manipulations in general (Kurtti et al., 1987). Spreading recombinant bacteria in multiple 96-well plates to isolate clones can also be applied to genetic manipulations. When there is an increased risk for contamination (cultivation of spirochetes from ticks or animal tissues) antibiotics can be included in the BSK. Two commonly used antibiotics are phosphomycin (400 µg/ml) and sulphametoxazole (0.05 µg/ml).

Typing Genotyping of Lyme Borrelia isolates can be performed by phenotypic or genotypic methods. However, the phenotypic typing methods have now been replaced in favor of genotypic methods based on DNA sequence comparison.

Phenotypic The phenotypic methods include monoclonal antibodies (MAbs) reacting against different classes of the OspA protein. Using this method seven OspA serotypes have been identified, where OspA serotype 1 corresponds to B. burgdorferi, serotype 2 to B. afzelii and serotypes 3 through 7 to B. garinii (Wilske et al., 1993). The correlations with different genospecies were confirmed with 16S rRNA sequence analysis. Also, the antigenic differences among the OspA serotypes were verified by partial sequence analysis of the gene from representative isolates. The outer surface protein C (OspC) has also been the basis of classification with MAbs. Thirteen different classes were identified in 6 OspA serotypes, indicating that OspC is more heterogeneous than OspA (Wilske et al., 1995). This might be explained by lateral transfer and recombination of the ospC

17 genes between different spirochetes, made possible by its location on circular plasmid 26 (Livey et al., 1995; Sadziene et al., 1993).

Genotypic Genes used for genotyping are p66 (encoding a integral outer membrane protein with channel-forming activity), flaB (a structural protein of the internal flagella of spirochetes), ospA and ospC (Assous et al., 1994; Pinne et al., 2007). These genes are all under positive selection and therefore large sequence variations are not allowed without loss of function. The flaB gene has for example been shown to be 94-99% identical among different LB-causing genospecies (Noppa et al., 1995). These genes can however still be suitable for genospecies identification but not to obtain evolutionary data (Bunikis et al., 2004). In a comparative study the rrs (16S)–rrlA (23S) intergenic spacer (IGS) located on the chromosome, was identified as the most suitable locus for genospecies identification and evolutionary studies (Bunikis et al., 2004). The rrs 16S rRNA gene and the trnI gene for the tRNA-Ile (located in the spacer) are highly conserved and therefore suitable sites for primer binding, an additional reason making this region appropriate for analysis. The amplicon includes the short trnA gene for tRNA- Ala, the sequenced region however, is not under selective pressure and therefore highly polymorphic. A physical map of the region can be found in Paper III in this thesis.

The infection cycle The Lyme Borrelia group of spirochetes’ infection cycle involves various Ixodes ticks and a wide range of vertebrate hosts. To maintain a large pool of infected reservoir animals, young individuals need to be infected and be able to stay infected for a long period of time. The transmission cycle is dependent on tick larvae becoming infected when feeding on an infected host. When they later feed again as nymphs, they transmit the infection to a new host. It is important to emphasize that infected larval or nymphal ticks still will be infected after having molted to the next developing stage, even though bacterial numbers may have decreased dramatically (Piesman et al., 1990). The role of adults in the infection cycle is limited, since male ticks do not feed and female ticks prefer larger

18 animals such as deer, which are not competent as reservoir hosts (Telford et al., 1988). When humans are infected it is considered to be a “dead end host”, not contributing to the spread of the disease.

Temperature is the main abiotic factor influencing the Ixodes tick life cycle (Sonenshine, 1991). A cold climate makes the life cycle slower, this in turn means that the incubation time for the spirochetes will be extended. On the other hand, too high ambient temperatures (exceeding 27° C) have also been shown to negatively influence the spirochetal numbers in infected Ixodes ticks (Shih et al., 1995). The relative humidity has an indirect impact on the choice of blood host since it determines the height at which the different tick stages quest in the herbage and therefore possibly the size of the animals (Randolph & Rogers, 2007).

The tick vector The Lyme Borrelia group of spirochetes is transmitted by hard-bodied ticks (family Ixodidae) of the genus Ixodes, in total comprised of about 245 species (Sonenshine, 1991). The Ixodes species known to transfer LB infection to humans are I. ricinus, I. persulcatus, I. scapularis and I. pacificus. The ticks have different geographical distributions and are associated with different Borrelia genospecies as presented in Table 1.

Ixodes ricinus complex There are four main tick vectors in the Ixodes ricinus complex responsible for transmitting the spirochetes to humans. I. ricinus (the sheep tick) is the dominant vector in Europe (Figure 2), while in Asia the main vector is I. persulcatus (Sonenshine, 1991). In North America two species are responsible for spreading the disease. I. scapularis previously named I. dammini, but also known as the black- legged tick or the deer tick, is common in the northeastern and northern central parts. This tick is catholic in the choice of host, and has been found to feed on at least 125 species of animals (54 mammalian, 57 avian, and 14 lizard species) (Keirans et al., 1996; Sonenshine, 1993). The other tick species common in the Midwest is I. pacificus or the western black-legged tick (Sonenshine, 1993).

19 The questing (host seeking) tick is attracted by heat and CO2 (Sonenshine, 1993). After having completed the blood meal the engorged tick falls to the ground and the molting process starts. All these tick species feed only once during each life stage (larva, nymph and adult). The blood meal is crucial for the molting process (transition to the next stage) and for laying eggs (adult females only). These ticks typically have a lifespan of two years but this is also dependent on the climate and blood host availability (Sonenshine, 1991). Nymphs are most abundant during late spring and early summer, while questing larvae are most abundant during late summer when the eggs have hatched. Adult ticks are frequently found during autumn (Wilson & Spielman, 1985).

Questing I. ricinus ticks are rather tolerant to sudden drops in temperature; this can also explain their presence in quite harsh environments where they are not expected. After more than 48 hours at -20°C, nymphal ticks are still hungry and looking for a blood host (personal observation).

Figure 2. Mouse infested with I. ricinus nymphs. The ticks prefer to feed where the skin is thin, for example close to the eyes or on the ears. (Photo: Pär Comstedt and Christer Larsson)

20 Ixodes uriae This tick species is also known as the “seabird tick” (Figure 3). It is strictly localized to seabird colonies where it completes the entire life cycle by feeding on seabirds (Arthur, 1963; Sonenshine, 1993). The I. uriae tick was probably first described by Charles Darwin in 1832 when he visited the Isle of Paul in the Atlantic Ocean. He described the species-poor fauna; “a species of Feronia and an acarus, which must have come here as parasites on the birds”. The only birds he could find on the island were a species of gannet and a tern (Darwin, 1839).

I. uriae can be found all around the world, both on the southern and the northern hemisphere (Bergström et al., 1999). It is catholic in the choice of seabird host and has been reported to feed on more than 50 different colonial seabird species (Arthur, 1963; McCoy et al., 2005). It is also present in climates not normally associated with ticks as the Arctic and sub-Antarctic (Paper II in this thesis; Bergström et al., 1999; Olsen et al., 1995a).

When present in seabird colonies, they usually occur in great numbers and some birds are therefore heavily infested. To keep the appropriate humidity the ticks tend to stay together in clusters. Under virtually every stone and in every crack of these colonies you can therefore find enormous numbers of ticks, at all developmental stages (personal observation). The tick can withstand temperatures as low as -30° C, which is necessary for survival in the often harsh environments. In milder climates the tick has a 3-year lifespan and feeds once per year. In colder climates (Arctic and Beringia) however, the tick life cycle can be as long as 7 years (Sonenshine, 1993; Steele et al., 1990).

Figure 3. I. uriae ticks collected on the Commander Islands. 1, engorged larva. 2, unfed nymph. 3, engorged nymph. 4, unfed adult female. 5, engorged adult female. 6, adult male. Bar equals 1.0 cm. Photo: Pär Comstedt

Transfer of infection from the tick to the host Most of the knowledge about the Lyme Borrelia spirochetes and their interaction with Ixodes ticks comes from studies on B. burgdorferi and I. scapularis. Therefore, data discussed here will focus on these two species if not otherwise stated. The Ixodes tick feeding process is a slow process. The I. ricinus larva feeds for 3-5 days, the nymph for 4-7 days and the adult tick for 7-11 days. In addition, blood host species and place of infestation influence the required time (Sonenshine, 1991). When the tick feeds on an infected host, the Borrelia spirochetes become localized in the tick midgut. The spirochetes multiply even after the larva has detached and two weeks post-repletion up to 2,700 spirochetes/tick can be found. After the subsequent molting to the nymphal stage, there is an approximately 10-fold drop in concentration of spirochetes (Piesman et al., 1990). The same pattern continues for the following developmental stages. When

22 the nymph has fed for 72 hours, a concentration of 166,000 spirochetes within the tick has been reported (De Silva & Fikrig, 1995). After detachment, the spirochete concentration declines and 75 days later the spirochete density is approximately 60,000 spirochetes/tick. Again, the subsequent molting process to become an adult tick decreases the bacterial concentration 10-fold (Piesman et al., 1990). This oscillation in spirochete concentration during the tick life cycle has been observed also for I. uriae and B. garinii (Paper III in this thesis). This means that the bacterial concentration is at its lowest when the tick is questing for a new host. To be able to infect a new host, the spirochetes need to penetrate the epithelia of the tick midgut and migrate through the hemolymph to the salivary glands. Plasminogen taken up from the host blood and bound to the spirochete surface mediates extracellular proteolytic activity crucial for traversing the tick gut epithelium (Coleman et al., 1997). In the nymph, the migration process takes 36-48 hours and starts when the tick begins to feed and blood fills up the midgut (De Silva & Fikrig, 1995; Ribeiro et al., 1987). During the first 24 hours of nymphal attachment virtually no transmission of spirochetes to the host takes place. After 48 hours transmission occurs but particularly efficient transmission takes place only after 72 hours (Piesman et al., 1987). Removing a feeding tick within 24 hours after attachment is therefore an efficient way of preventing spread of the spirochetes to a new host. The tick uses a regurgitating feeding behavior, where saliva and possibly gut content is mixed with the host body fluids; this also facilitates the transfer of the spirochetes (Kurtenbach et al., 2002b).

Spirochete protein expression in the tick and the skin For the spirochetes to be transmitted to a host from a tick, they need to penetrate the tick gut epithelium, become systemic, invade the salivary glands and enter the dermis of the host. One of the major proteins expressed by the Lyme Borrelia spirochetes in the unfed tick midgut is OspA, a lipoprotein anchored to the bacterial outer membrane (Schwan et al., 1995). OspA has been shown to mediate adherence to the tick midgut epithelia by binding to the tick receptor for OspA (TROSPA) (Pal et al., 2004). This interaction is essential for the colonization of the tick gut. When the tick starts to feed on a host, the

23 incoming blood changes the environment of the midgut. The spirochetes now cease the expression of OspA and instead up-regulate the expression of another lipoprotein, OspC (Schwan et al., 1995). The reciprocal expression of OspA and OspC correlates with the exit of the spirochetes from the midgut, migration through the hemolymph to the salivary glands and entry into the new host. The function of OspC is not completely clear. However, the protein is essential for the initial infection of the mammalian host but not for migration from the midgut to the salivary glands (Grimm et al., 2004; Stewart et al., 2006). Many studies have shown that at least two environmental cues, temperature and pH, seem to have influence on the shift from OspA to OspC expression. The alkaline milieu (pH ~7.4) and low temperature (~23°C) of the unfed tick midgut signal OspA expression (Carroll et al., 1999). Upon feeding the incoming blood acidifies the midgut (pH ~6.8) and elevates the temperature (~35°C), this triggering up-regulation of OspC (Schwan et al., 1995; Yang et al., 2000).

Reservoir hosts A good reservoir host for Lyme Borrelia spirochetes must be tolerant to infection, be abundantly parasitized by ticks, it must be abundant, and it must permit efficient transfer of the pathogen to the tick (Mather et al., 1989).

It is important to notice that the three principle vectors in Europe, Asia and North America, namely I. ricinus, I. persulcatus and I. scapularis, respectively, are generalist ectoparasites. This means that they feed on many different animals and thereby provide the opportunity for transmission of the spirochetes among various animal species (Sonenshine, 1993). It has been suggested that the host complement system also operates in the tick gut and therefore plays a crucial role in the Lyme Borrelia genospecies present in ticks in a given area. In other words, the composition of the reservoir animal population determines the Lyme Borrelia genospecies in the tick population (Kurtenbach et al., 2002a).

Reservoir hosts in North America In many parts of North America (specially the eastern areas) the white-footed mouse (Peromyscus leucopus) is thought to be one of the most important hosts for

24 larvae and nymphal Ixodes ticks (LoGiudice et al., 2003). The white-footed mouse is also very tolerant to B. burgdorferi infection, since it remains spirochetemic for a long time and shows no inflammatory response (Levin et al., 1996). Other mammals such as chipmunks (Tamlas striatus) are of intermediate importance and meadow voles (Microtus pennsyl-vanlcus) seem to be of less importance. This is due to their lower incidence of tick infestation but also lower rate of B. burgdorferi infection (Mather et al., 1989).

The importance of alternative hosts for B. burgdorferi was shown in a field vaccination study. The wildlife B. burgdorferi reservoir, the white-footed mice, was vaccinated with rOspA. This decreased the nymphal tick infection prevalence in the area and therefore also lowered the risk of infection for humans. If on the other hand, mice were not present and only alternative species were at hand, calculations showed that 13% of the nymphs would be infected. Consequently, the contribution of alternative hosts to the B. burgdorferi maintenance was low but could not be ignored (Tsao et al., 2004). Different bird species are alternative reservoir hosts for B. burgdorferi. Their role will be described in more detail in later chapters.

Reservoir hosts in Europe In Europe, different Lyme Borrelia genospecies are associated with different reservoir hosts. B. afzelii spirochetes are frequently isolated from different rodents including; wood mice (Apodemus sylvaticus), yellow-necked mice (A. flavicollis), black-striped mice (A. agrarius) and bank voles (Clethrionomys glareolus) (Hanincova et al., 2003a; Matuschka et al., 1992). Black-striped mice are also abundant in areas close to human settlements, making them even more important in an LB perspective (Matuschka et al., 1992). B. garinii on the other hand, is mostly associated with birds (Paper I in this thesis; Hanincova et al., 2003b), but can also infect rodents (Hu et al., 2001). B. burgdorferi is not so common, and appears to be less discriminating between birds and rodents. In 1998, Kurtenbach and co-workers, suggested that the complement system had an impact on reservoir competence for different host animals. By testing sera from a wide spectrum of animals, they showed that different Lyme Borrelia genospecies were sensitive or resistant to complement proteins from certain species. B. afzelii

25 was sensitive to bird serum but resistant to different rodent sera, while B. garinii showed the opposite pattern. B. burgdorferi showed intermediate resistance to sera from many different animal species, confirming the pattern seen in nature (Kurtenbach et al., 1998b). Because of this, questing nymphs infected with B. afzelii (infection acquired when feeding on B. afzelii infected rodents as larvae), later feeding on pheasants as nymphs, can be cleared of the infection. If the bird is infected with B. garinii the ticks can acquire that infection instead and thereby change the infecting genospecies (Kurtenbach et al., 2002a). As a consequence of this, only 13% of infected I. ricinus ticks in Europe have mixed infections (Rauter & Hartung, 2005).

There are also some regional differences in Europe regarding Lyme Borrelia genospecies prevalence possibly due to the presence of different host reservoir animals (Rauter & Hartung, 2005). However, some studies suggest the opposite: that species variation at different sites in Europe may evolve randomly and are not dependent on the presence or absence of specific hosts. Moreover, it is proposed that all three LB-causing genospecies in Europe share reservoir hosts. This hypothesis has been proposed since xenodiagnosis infection experiments where naïve ticks were allowed to feed on naturally infected rodents, were infected with the same ratios of all three LB-causing genospecies as the ratios found in questing ticks from the same area. If birds were the main reservoir hosts of B. garinii, ticks at this site would be infected with this genospecies to a greater extent than what was found (Richter et al., 1999).

Poor reservoir hosts Some animals are considered to be poor reservoir hosts of Lyme Borrelia spirochetes. They can therefore have a diluting effect on the spirochete-infected ticks in a given area. LoGiudice and co-workers have identified squirrels as important “dilution hosts” characterized by high tick burdens, low reservoir competence, and high population density (LoGiudice et al., 2003). This group also includes the white-tailed deer in North America and roe deer in Europe (Jaenson & Tälleklint, 1992; Telford et al., 1988). Different deer species are often heavily infested with adult female ticks that can lay hundreds of eggs hatching into larvae and taking part in the Lyme Borrelia infection cycle. In this way the

26 overall tick population in the area is increased. However, in a normal-serum killing assay, deer sera killed more than 90% of the B. burgdorferi spirochetes compared to less than 22% for normal sera from mouse and rabbit. It was shown that the alterative complement activation pathway played a major role in the borrelicidal activity of normal deer serum (Nelson et al., 2000). Shrews have been classified as “rescue hosts” capable of maintaining high infection rate in ticks when mouse density is low (LoGiudice et al., 2003). Some studies suggest that co-feeding is important in Lyme Borrelia ecology. In this way unimportant reservoir hosts also can mediate the spread of spirochetes from a feeding infected tick to another feeding, non-infected tick (Gern & Rais, 1996). In conclusion, the presence of different host species and their relative numbers have a great influence on the Lyme Borrelia infection cycle in nature.

The complicated biology of B. garinii The most complicated reservoir host dependence is that of B. garinii. This extremely heterogeneous genospecies often infects humans and is overrepresented among neuroborreliosis patients in Europe (Wilske et al., 1996). It is found in nature in at least three types of ticks (I. ricinus, I. persulcatus and I. uriae), where at least two feed on humans and cause clinical cases. Moreover, the reservoir animals include passerine birds, seabirds, varying hare and rodents (Paper I in this thesis; Hanincova et al., 2003b; Huegli et al., 2002; Jaenson & Tälleklint, 1996; Moran Cadenas et al., 2007; Tarageoa et al., 2007). It has been suggested that different OspA serotypes are associated with certain reservoir host animals. In this way B. garinii OspA serotypes 3, 5, 6 and 7 are suggested to depend on birds as their reservoir host. B. garinii OspA serotype 4 on the other hand, is dependent upon rodents (Kurtenbach et al., 1998b; Kurtenbach et al., 2002a). The geographical distribution includes Europe, Asia, and many coasts worldwide including North America (Kurtenbach et al., 2006; Olsen et al., 1995a; Smith et al., 2006) (see Table 1).

27 Borrelia and birds Today we know that birds spread and maintain Borrelia spirochetes that are very similar to clinical isolates and their role in the ecology and epidemiology of this disease can not be ignored. However, birds were ruled out as potential hosts for Lyme Borrelia spirochetes for a long time, since they have an elevated body temperature compared to many mammals (Barbour, 1984; Welty & Baptista, 1997). The optimal growth temperature for many LB-causing strains had been determine to be 34-37° C, and birds were therefore not considered to be likely hosts (Barbour, 1984). B. garinii and B. valaisiana are the two Lyme Borrelia genospecies most frequently found in birds, or ticks attached to birds, in Europe and Asia (Paper I in this thesis; Hanincova et al., 2003a; Olsen et al., 1995b). Unlike other genospecies, B. garinii is reported to grow at temperatures of up to 41°C; this is also suggested to be an adaptation to avian hosts (Hubalek et al., 1998).

Anderson and co-workers isolated B. burgdorferi from I. scapularis ticks collected from different bird species for the first time in 1984 (Anderson & Magnarelli, 1984). Two years later they also isolated spirochetes from the livers of passerine birds, finally leading to the implication that birds are involved in the ecology and epidemiology of the Lyme Borrelia group of spirochetes (Anderson et al., 1986). Since then a number of studies on wild birds have been done (Paper I in this thesis; Hanincova et al., 2003b; Marie-Angele et al., 2006; Olsen et al., 1995b).

Experimental infections of birds Some experimental studies suggest that birds are relatively poor reservoir hosts for Lyme Borrelia spirochetes. One-week-old chickens infected with B. burgdorferi were only able to transmit the spirochetes to new larvae during the first three weeks post-infection. The reservoir competence also seemed to decline dramatically with age. When three-week-old chickens were infected, only a fraction of feeding ticks were positive for spirochetes one week post-challenge (Piesman et al., 1996).

28 In other studies the Japanese quail has been used as a model system for studying B. garinii infection in birds. Isogai and co-workers reisolated spirochetes from several organs up to two months post-infection (Isogai et al., 1994). Canary finches also have been tested for host reservoir competence. B. burgdorferi spirochetes could be detected by microscopy in blood up to two weeks post- infection and DNA could be detected up to 3 months in several internal organs after experimental infection in this bird species, suggesting a persistent infection (Olsen et al., 1996).

Borrelia spirochetes and terrestrial birds B. burgdorferi does not seem to discriminate between rodents and birds, but has the capacity to infect both groups of animals (Kurtenbach et al., 2002a). Differences in reservoir competence have been reported for some ground foraging bird species in North America, meaning all are not equally competent in transmitting B. burgdorferi to naïve ticks. For example, American robins have been the focus of some studies and are suggested to be an effective reservoir host. Song sparrows have intermediate and gray cat birds have low reservoir capacity. All of these data are based on field xenodiagnosis experiments (Ginsberg et al., 2005). Some of the data are supported by others performing infections in the laboratory. Reservoir competence for American robins can be as high as for rodents, measured by the capacity to infect feeding larvae. The infection waned after two months, and disappeared after 6 months. The birds however, remained susceptible to reinfection. Ticks infected when feeding on the birds could also in turn infect mice, showing the potential influence of birds on the Lyme Borrelia ecology in North America (Richter et al., 2000). The authors conclude: “American robins are fully but transiently competent as hosts for Lyme disease spirochetes”. The results for gray catbirds are also supported by others. When studying natural populations of this bird none out of 12 birds infected xenodiagnostic ticks, compared to 76% for mice captured in the same area (Mather et al., 1989). However, about 60% of the captured gray catbirds were infested with immature ticks, compared to all of the mice, possibly explaining part of the difference. Regardless, this bird species seems to have poor reservoir capacity for B. burgdorferi.

29 Thrushes are considered to be important reservoir hosts for B. garinii and B. valaisiana in Europe (Paper I in this thesis; Mannelli et al., 2005; Olsen et al., 1995b; Tarageoa et al., 2007). B. garinii can persist in thrushes for several months. Since these birds are also known to be infected with clinically important B. garinii strains, their role can not be overlooked (Paper I in this thesis). One reason for their importance as a reservoir host is due to their ground foraging behavior. When screening passerine birds for infected larvae (i.e. infected birds) we found that ground foraging birds were more often infested than other birds, but both groups infected larvae with the same effectiveness. The main factor influencing the importance of a bird species as a reservoir seems therefore to be the foraging and nesting behavior rather than other biological or genetic factors (Paper I in this thesis). In agreement with this, pheasants in England and Wales have been shown to be important reservoir hosts for Lyme Borrelia spirochetes in a xenodiagnostics set-up (Kurtenbach et al., 1998a).

The impact of bird migration Bird migration can not be ignored as a factor influencing the distribution of Lyme Borrelia spirochetes. Enormous numbers of birds migrate in the autumn and spring all over the planet. A migrating passerine bird can fly as long as 950km/day and sub-adult Ixodes ticks feed for 2 to 4 days (Smith et al., 1996). This means that migrating passerine birds can transfer infected ticks from areas where it is endemic to areas where it is not in a short period of time. Birds can, in other words, facilitate long distance spread of Lyme Borrelia spirochetes and thereby establish new foci at distant locations (Gylfe et al., 2000; Smith et al., 1996). According to our theoretical calculations, as many as 5.6 million ticks infected with B. garinii, are brought to Sweden every spring by migrating passerine birds (Paper I in this thesis). Also, in the autumn when the breeding season is over and the birds migrate south, the number of birds have doubled (Olsen et al., 1995b). In addition, latent Borrelia infections in birds can be reactivated by stress, such as migratory restlessness which is induced when the photoperiod is altered. Naïve ticks can thereby be infected and dispersed elsewhere (Gylfe et al., 2000).

30 B. garinii and seabirds In 1993 Olsen et al. isolated Borrelia spirochetes from I. uriae, the seabird tick. The ticks were collected from razorbills on an isolated island in the Baltic Sea where no mammals were present. Since seabirds were the only blood host available for the ticks, a marine enzootic infection cycle was suggested, where B. garinii circulating among colonial seabirds depend on I. uriae as the only vector (Olsen et al., 1993). The global distribution of this marine infection cycle was then studied, since large seabird colonies are found in many places. I uriae ticks were collected from seabirds at nine different locations worldwide and B. garinii spirochetes with identical flagellin genes were found in ticks from both the southern and the northern hemispheres. This suggests that seabirds can act as long distance carriers of the infection (Olsen et al., 1995a). Some seabirds in the shearwater family (Puffinus spp.) are examples of species known to migrate between North and South Pacific Ocean (Spear & Ainley, 1999). In other words, these birds undertake migrations of several thousand kilometers. Since the ticks only feed for a few days, the spirochetes are probably transported as a latent infection in the birds, rather than by infected ticks. A phylogenetic analysis of I. uriae from both the southern and the northern hemisphere also suggests separate reproduction of the ticks (Gylfe et al., 2001). As mentioned before, there is a possibility that such infections are reactivated when the birds are subjected to stress as in the case of migration (Gylfe et al., 2000).

So far, the only Borrelia genospecies found in seabirds and I. uriae ticks is B. garinii. This genospecies is often isolated from I. ricinus and I. persulcatus ticks in Europe and Asia. The marine infection cycle however, increases the habitat to virtually all oceans, including Arctic and sub-Antarctic waters (Paper II in this thesis; Olsen et al., 1995a). B. garinii spirochetes have been isolated from I. ricinus ticks collected from seabirds on Egg and St. Lazaria Islands, outside Alaska (Olsen et al., 1995a), as well as in Newfoundland (Smith et al., 2006). Since only a few studies have been done in this field, the exact distribution of B. garinii is not known. However, my personal opinion is that wherever there are I. uriae ticks one can also find B. garinii spirochetes.

Figure 4. I. uriae ticks in different developmental stages were found under almost each stone in the tufted puffin colony on the Commander Islands. The tufted puffins nest in burrows in the ground. Photo: Ingvar Eliasson.

Many seabird colonies are very crowded and spread of infection among birds is therefore easily facilitated. The I. uriae ticks thrive in cracks, under stones, etc. and can reach enormous numbers (Figure 4). Different studies suggest that as many as 30% of the nymphs and adults in seabird colonies are positive for B. garinii (Olsen et al., 1995a). A homogeneous B. garinii population could be expected in these colonies since the high numbers of ticks and birds in an often relatively limited area would suggest this. Surprisingly, great heterogeneity among the B. garinii spirochetes has been observed in different studies, suggesting a frequent contact with other geographically distant locations (Olsen et al., 1995a).

Interaction between the marine and the terrestrial infection cycles B. garinii spirochetes are found among rodents and passerine birds as well as among seabirds, suggesting that there has to be some overlap between the marine and the terrestrial infection cycles. The marine infection cycle is restricted to different seabirds and the open sea. I. uriae ticks are very catholic in their choice of seabird host, but the ticks are seldom found outside these colonies. Therefore, a link with the terrestrial infection cycle has to take place within the

32 colony. Rodents searching for food and resting migratory passerine birds are possible candidates mediating contact with I. uriae or I. ricinus ticks. The frequency by which I. uriae ticks feed on rodents and other birds is however not known. Laboratory infections show that B. garinii isolated from seabirds can infect rodents, and be reisolated several weeks later (Paper IV in this thesis). In some locations where the I. uriae and I. ricinus habitats overlap and seabirds as well as migrating passerine birds nest side by side, host cross-over would be possible. This is for example true for some islands in the Baltic Sea (Bunikis et al., 1996). In addition, the same genetic variants of B. garinii have been found to circulate among passerine birds and seabirds (Paper III in this thesis).

Even though I. uriae ticks are reported to feed on humans when opportunities are given, this is not a frequent problem (Arthur, 1963; Clifford, 1979; Gylfe et al., 1999; Mehl & Traavik, 1983; Sonenshine, 1993). So far, all tested seabird isolates have been sensitive to normal human serum. This suggests that the ability to infect humans is probably limited (Paper IV in this thesis).

The complement system As mentioned in previous chapters, the complement system is a central factor in the ecology of Lyme Borrelia spirochetes. It is the first obstacle the spirochetes have to overcome when entering the hostile environment of a new host. This is part of the innate immune system, a non-adaptive defense mechanism, which is activated within minutes by a wide range of pathogens. The complement system contains at least 30 proteins, synthesized in the liver and circulating in the body in their inactive form. The proteins act through a cascade mechanism, resulting in a membrane attack complex (MAC), lysing the pathogen by creating a transmembrane pore. In addition, opsonization of pathogens with complement component C3b (a central protein in the complement system) leads to enhanced phagocytosis by neutrophils and macrophages. Also, inflammation is stimulated with recruitment of other immune cells (Janeway et al., 2005; Kindt et al., 2007).

The complement system can be activated through three different pathways. The activation is complex and involves many complement components and regulators. The classical pathway is antibody dependent and may therefore not be

33 present at the initial phase of an infection. Antibodies binding to the bacterial surface recruit complement proteins and induce formation of MAC leading to lysis of the cell. The lectin pathway is part of the initial defense against pathogens, since antibodies are not involved. Lectin binds to polysaccharides on the bacterial surface and complement proteins are recruited to activate the complement cascade (Kindt et al., 2007). The alternative pathway (AP) is dependent on slow spontaneous hydrolysis of complement protein C3 circulating in the plasma. One of the resulting products is C3b, which binds covalently to the bacterial surface. C3b recruits factor B which is cleaved by another serum protein, factor D. The resulting protein complex, C3bBb, has a C3 convertase activity, meaning it can cleave C3 to produce more C3b. The initial steps are therefore amplified. Also, C3bBbC3b is formed which exhibits C5 convertase activity and cleaves C5 into C5a and C5b; the latter binds to the bacterial surface and recruits additional proteins (C6, C7, C8 and C9) to form MAC and lyse the cell. (Janeway et al., 2005; Kindt et al., 2007).

Regulation of the alternative pathway Factor H (FH) is the most important fluid phase regulator of the AP. This regulatory protein decides whether a surface acts as an activator of the AP or not, by binding to C3b and prevent the association with factor B. C3b is then susceptible for cleavage by factor I. (Janeway et al., 2005). FH have affinity for sialic acid present at high levels on the membranes of mammalian cells. Therefore, C3b bound to mammalian cells is rapidly inactivated. Many bacteria and viruses have only low levels of sialic acid on their surface, leaving them vulnerable to complement attack (Kindt et al., 2007). Factor H-like protein 1 (FHL-1), is an alternative splicing product of FH with inhibitory properties of AP in the same manner as FH (Kraiczy & Wurzner, 2006).

Complement evasion Some pathogens, including group A Streptococcus and Onchocerca volvulus, (causing strep throat and river blindness, respectively) can bind FH to their surfaces and thereby escape activation of the AP (Caswell et al., 2008; Meri et al., 2002). Also Borrelia genospecies have been shown to bind FH via at least two classes of

34 proteins; outer surface protein E (OspE) and complement regulatory-acquiring surface protein 1 (CRASP-1) also called BbA68. Binding of FH to the Borrelia spirochete surface, probably mediates protection from attack by the AP (Hellwage et al., 2001; Kraiczy et al., 2001).

Differences among Lyme Borrelia spirochetes LB-causing genospecies have been shown to vary in their capacity to degrade C3b, which correlates with their level of sensitivity to human sera. Thus, the serum-resistant strains can bind FH and degrade C3b while sensitive strains can not (Alitalo et al., 2001). B. afzelii and B. burgdorferi have been classified as human serum resistant, while many B. garinii isolates have been found to be more sensitive to human serum (Alitalo et al., 2001; Kurtenbach et al., 1998b). Nevertheless, these isolates are clinical specimens, and can thus cause infection in humans. CSF isolates of B. garinii have the ospE gene but do not appear to express the protein in vitro. However, a recent study shows that 83.6% of neuroborreliosis patients have anti-OspE antibodies in their sera, suggesting expression of the protein in vivo (Panelius et al., 2007). Some B. garinii CSF isolates appear to have another novel 27 kDa protein with FH binding properties (Paper V in this thesis). Expression of this protein during infection might contribute to the virulence of the neurotropic strains of B. garinii. The differences observed in serum resistance may in part be explained by the fact that plasmids encoding ospE family genes are lost during prolonged in vitro cultivation. Also, the high degree of heterogeneity found among B. garinii isolates may be a reason. However, when only low passage clinical isolates of B. garinii were tested for complement resistance, some were resistant while others were only semi-resistant (Paper IV in this thesis). Many of these isolates were from human CSF, where the level of complement activity is much lower compared to the rest of the body (Lindsberg et al., 1996). Still the spirochetes must survive the time it takes to migrate from the tick to the central nervous system. We also tested seabird isolates of B. garinii for resistance to normal human serum and found that all were sensitive to human complement. This suggests that marine isolates of B. garinii cannot infect humans (Paper IV in this thesis). In agreement with this, B. garinii isolated from seabirds do not express FH-binding proteins (Paper V in this thesis).

35 36 AIMS OF THE THESIS

The aim of this thesis is to clarify the biology of B. garinii spirochetes. This includes the influence of different birds on their ecological distribution. In addition, this thesis sought to find a link between seabird and terrestrial infection cycles.

Specific aims

• Determine the role of passerine birds and seabirds as reservoir hosts for B. garinii spirochetes.

• Clarify the flux of B. garinii spirochetes on the Eurasian continent.

• Determine the role of birds in maintaining B. garinii spirochetes pathogenic to humans.

• Identify a possible overlap between the marine and the terrestrial infection cycles.

• Compare human complement evasion properties for B. garinii spirochetes isolated from Lyme borreliosis patients and seabirds.

37 38 RESULTS AND DISCUSSION

Paper I.

This study focuses on migrating passerine birds as reservoir hosts for Borrelia spirochetes and transporters of infected ticks.

Birds as carriers of ticks This paper is the first large-scale study showing the importance of passerine birds as reservoirs for Lyme Borrelia spirochetes and as carriers of infected ticks. The latter is of importance since the migrating behavior of the birds aids in dispersal of the infected ticks. During spring and autumn of 2001, 13,260 birds were captured at Ottenby bird observatory. Of these, 13,123 were migrating passerine birds comprising 38 different species. These were all examined for infested ticks. The birds were divided into two groups according to their foraging behavior. The ground foragers constituted 46% of the birds and the non-ground foragers (referred to as “other birds”) constituted 54% of the captured birds. In total, 1120 I. ricinus larvae and nymphs were collected and screened for the presence of Lyme Borrelia spirochetes by qPCR.

Aggregation of infestation risk was indicated by the finding that once a bird is infested with one tick, the likelihood of infestation with two or more ticks was higher than would be expected. Not surprisingly, the risk was greater for the ground-foraging birds than for other birds (5.4% vs. 1.4%). The same pattern was also seen when retrospectively analyzing data from more than 15.000 birds captured in 1991 (Olsen et al., 1995b). In total, 14% of the ticks were positive for Lyme Borrelia spirochetes but almost twice as many nymphs than larvae (19.3% vs. 10.1%) were positive. This is probably explained by accumulation of spirochetes during feeding as larvae.

39 Passerine birds as hosts for B. garinii infected ticks The fact that larvae were infected suggests that birds act as reservoir hosts. To exclude the possibility of transovarial transmission, we studied birds infested with larvae by comparing the proportion of birds with infected larvae among singly infested birds (one attached larvae) and the proportion of birds with one or more infected larvae among multiply infested birds (two or more infested larvae). We found a higher prevalence of infection among ticks from multiply infested birds (21.4%) compared to singly infested birds (5.5%), showing that birds were the source of infection. Also, a correlation between the proportion of birds infested with ticks (larvae and nymphs) and the proportion of birds infested with infected ticks, shows that birds function as the reservoir host of the spirochetes.

Three percent of the ground-foraging birds were infested with infected ticks, compared to 0.6% of the other birds. The percentage of birds with infected larvae among the larvae-infested birds was higher among ground-foraging species (34.0%) than among other bird species (7.8%). This, again, suggests a more central role in the Lyme Borrelia biology for the former group of birds.

Reservoir competence of passerine birds The birds’ reservoir competence, determined as the efficiency of spirochete transmission to larvae (spirochetal numbers and infection prevalence), was then evaluated. When analyzing spirochete load and infection prevalence for larvae collected from the same bird, these parameters were found to positively correlate for 56 larvae from 25 birds. A xenodiagnosis experiment where the larvae were allowed to molt and the nymphs subsequently feed on a new host would fully determine the role of birds as reservoir hosts (Richter et al., 2000). However, in large-scale analyses this is essentially impossible; qPCR-based screening of larvae is therefore the method of choice.

To see if the two groups of birds (ground foraging and other birds) differed in reservoir competence, we studied the larvae from individual birds. There were no differences between the two groups regarding the mean value of spirochetal numbers in the larvae: 135 (95% CI 21-862) and 23 (95% CI 2-318), respectively.

40 Also, there was no difference in infection prevalence between the two groups of larvae: 61% (95% CI 46%-77%) and 77% (95% CI 50%-100%), respectively. This suggests that the two groups of birds are equally competent for infecting larvae and their importance as reservoir hosts is rather decided by foraging behavior.

Clinical importance of Lyme Borrelia spirochetes spread by birds The Borrelia genospecies was determined for 88 of the 160 positive samples. The overall dominance of B. garinii and total lack of B. afzelii among larvae is in agreement with other studies focusing on host association of Lyme Borrelia spirochetes (Kurtenbach et al., 2002a). The IGS of 47 B. garinii from I. ricinus ticks infesting birds and 11 clinical isolates (EM) of the same genospecies were compared. Two groups (1 and 2) were defined with 9 and 2 subgroups (1:1-1:9 and 2:1-2:2), respectively (Table 2). Six subgroups (1:1, 1:3, 1:4, 1:9, 2:1 and 2:2) comprising 29 (62%) of the bird tick isolates were also found in the LB patient biopsies. In addition, larvae (indicated with bold letters) were infected with three variants (1:1, 1:3 and 2:1) which also cause disease in humans. Passerine birds are therefore reservoir hosts of clinically important Lyme Borrelia spirochetes.

Table 2. B. garinii isolates from passerine birds and clinical samples. Sub Group group I. ricinus from birds Clinical samples A31 A99 C851 C861 C871 C891 C901 1 Bio56059 Bio56061 D46 D48 D88 E09 F28 F88 K29 L58 2 D83 F89 3 C24 D49 E07 Bio56016 Bio56081 4 C28 C55 G09 K96 Bio56014 Bio56045 1 5 C78 E06 E08 E13 K60 K92 6 D40 D47 K70 K73 7 C51 K20 F87 L78 8 L05 L07 9 K22 Bio56002 Bio30058 Bio56056 1 B02 D122 D132 K89 2 Bio56101 2 A35 B69 Bio56077

Bold, Larval I. ricinus ticks. 1,2 Larvae are collected from the same bird

41 Migratory passerine birds have dual roles in the maintenance of Lyme Borrelia spirochetes, both as long range carriers of infected ticks and as reservoir hosts infecting naïve larvae. Although migratory birds might have fewer infested ticks than rodents, their contribution to Lyme Borrelia ecology can be of equal importance. This is especially true in Europe where birds constitute the major reservoir host for B. garinii (Hanincova et al., 2003b; Kurtenbach et al., 2002a; Olsen et al., 1995b).

42 Paper II.

Here we present data demonstrating an expanded habitat for marine Borrelia infection cycles to the Arctic.

Presence of Borrelia spirochetes in the Arctic B. garinii spirochetes probably have the widest geographical distribution of all Lyme Borrelia genospecies. Spirochetes have been isolated from seabirds and I. uriae ticks in Europe, Asia, North America, as well as islands in the North Atlantic, North Pacific Ocean and sub-Antarctic waters (Gylfe et al., 1999; Olsen et al., 1993; Olsen et al., 1995a; Smith et al., 2006). In addition, we found B. garinii spirochetes among seabirds on Hornøya (70°37’N31°10’E) in the Barents Sea outside the Varanger peninsula (Figure 5.) The finding of Lyme Borrelia spirochetes above the Arctic Circle suggests an even wider distribution than previously predicted.

Figure 5. The seabird colony is located in the Barents Sea outside the Varanger peninsula (filled circle). The Arctic Circle is indicated with a dashed circle.

Similarities to other Borrelia isolates I. uriae ticks collected from nesting seabirds (Brünnichs guillemots, razorbills and puffins) were analyzed for the presence of spirochetes by qPCR. Four different variants (Var1-Var4) were found and all were identified as B. garinii. When 492 bases of the IGS were compared by ClustalW (available at; www.ebi.ac.uk; full

43 alignment, scores are presented as percent sequence identities), the four variants showed 90-97% identity. Interestingly, 100% identity was found between Var1 and a B. garinii isolate from a puffin colony on Faeroe Island (Gylfe et al., 1999). This suggests a seabird-dependent interaction between the two sites. Similarities were found between the other isolates (Var2-Var4) and B. garinii from migrating passerine birds in southern Sweden (95-99% identity), as well as human EM isolates (95-98% identity). In conclusion, seabird colonies in the Arctic are part of the B. garinii Ecology.

44 Paper III.

This work suggests that the wide distribution and heterogeneity of B. garinii on the Eurasian continent is heavily influenced by seabirds and passerine migrating birds.

Prevalence and densities of spirochetes among ticks and seabirds When DNA extracts from 299 I. uriae ticks collected from the tufted puffin colony on the Commander Islands were screened by qPCR for presence of Borrelia spirochetes, 33.1% were positive. There was no significant difference in prevalence of infection among the different stages of ticks. The bacterial densities were heavily increased after each blood meal, as expected. When the nymphs fed, mean bacterial densities increased from 16 to 893. The same was also true for the adults, where an increase in bacterial numbers from 209 to 2489 could be observed. Since the molting process had a negative influence on bacterial numbers, an oscillating pattern appeared. By partial sequencing of the IGS amplicon, all positive samples were identified as B. garinii.

Figure 6. Tufted puffins on their way to the sea. Commander Islands. Photo: Ingvar Eliasson.

None of 86 blood clots from tufted puffins (Figure 6) were positive for Lyme Borrelia spirochetes. Some studies suggest that birds are poor reservoir hosts for Lyme Borrelia spirochetes, while others show that Borrelia infection in birds can

45 persist for months (Gylfe et al., 2000; Piesman et al., 1996). Also, B. garinii has been isolated from adult puffins (Gylfe et al., 1999). Since, the lifespan of I. uriae ticks in these cold environments can be extended to 7 years (Sonenshine, 1993), the ticks might therefore constitute the actual B. garinii reservoir. Possibly newly hatched birds staying in their burrows for weeks could be infected and subsequently infect new ticks. However, this needs to be confirmed by sampling young birds.

Genetic polymorphism Sixteen different isolates from the Commander Islands were used for phylogenetic comparison with 61 other B. garinii isolates of diverse biological and geographical origins. Twenty different genetic variants were identified, where all but three comprised more than one sample. Five variants were represented by the Commander Island isolates. When looking at seabird associated isolates as a group, they represent 32% of the sample collection, but comprise 50% of the IGS types, suggesting great variation. On the other hand, less variation was observed for B. garinii samples from other biological sources. The most complex genetic variant (no. 13) contained B. garinii isolates from I. ricinus ticks infesting migrating birds in Sweden, questing I. ricinus in Lithuania, as well as both skin and cerebrospinal fluid isolates from patients. Together this suggests that B. garinii associated with seabirds constitute a more diverse group of spirochetes than the terrestrial group.

Two of the genetic variants (no. 16 and no. 17) represented B. garinii from both I. ricinus and I. uriae ticks. Samples from migrating birds were also present, suggesting a possible role as carriers. Some of these isolates were also collected from the same area, but from different species of ticks. The marine and the terrestrial infection cycles therefore appear to overlap and possibly share the same B. garinii strains.

Phylogeny We next evaluated the aligned partial rrs-rrl IGS locus for evidence of intragenic recombination, which could confound the identification of monophyletic

46 groups. We found no such evidence and therefore carried out phylogenetic analysis. The 20 different genetic variants grouped into 6 different clusters, with 2 to 5 variants in each. Interestingly, B. garinii circulating among seabirds could be found in all 6 clusters, but only one was composed entirely thereof. The other 5 clusters also involved samples from migrating birds. Further, isolates from clinical samples were found in 3 of the clusters (3, 4 and 6). Commander Island isolates did not group together, but were found in 3 of the 6 clusters (1, 3 and 5). In other words, some of them were more related to B. garinii found in other contexts (LB patients, passerine birds, etc) in Europe, than to isolates from the same seabird colony. There must therefore be frequent exchange between the seabird colony on the Commander Islands and other B. garinii infection cycles in the northern hemisphere. In conclusion, the 77 B. garinii isolates did not necessarily cluster according to geography, biological hosts or vectors.

Seabirds apparently have a great impact on B. garinii ecology. The marine infection cycle (involving seabirds and I. uriae) and the terrestrial infection cycle (involving other Ixodes ticks and various vertebrates) probably are linked and it is possible that the heterogeneity found among B. garinii isolates worldwide is due to birds being a major reservoir host. Bird migration and the wide geographical spread of seabirds are therefore likely to contribute to the genetic variation among B. garinii spirochetes. This can in the end also have an effect on humans.

47 48 Paper IV.

This study attempts to identify a connection between the marine and the terrestrial B. garinii infection cycles.

The marine and the terrestrial infection cycle It has been suggested that many Lyme Borrelia genospecies are adapted to use either mammalian or avian species as reservoir hosts but not both. The exceptions are B. burgdorferi spirochetes which do not seem to discriminate much between the two groups of animals. Further, this species-specificity has been suggested to also apply for the many sub-groups of B. garinii. In this manner, the B. garinii OspA serotype 4 depends on rodents while serotypes 3, 5, 6 and 7 depend on birds as reservoir hosts. Consequently, rodents are not reservoir competent for bird-associated variants of B. garinii (Kurtenbach et al., 2002a). We found that B. garinii isolated from seabirds (OspA serotype 6) are able to establish infection in mice. The spirochetes could be re-isolated 82 days post- challenge from a number of organs, suggesting a persistent infection. Although, the challenge was done by needle inoculation and not xenodiagnosis, this suggests a potential overlap between the marine and the terrestrial infection cycles. The marine and the terrestrial infection cycles are often separated geographically, where the former is found in colder climates and higher latitudes. At some sites however, seabirds breed side by side with passerine birds and rodents are also present (Bunikis et al., 1996). Here, the exchange of spirochetes between the two infection cycles can occur through I. uriae or I. ricinus ticks.

Complement resistance properties Some B. garinii spirochetes circulating among seabirds show similarities to clinical isolates (Paper II in this thesis). However, their potential to infect humans is not known. We therefore compared the normal human serum (NHS) resistance properties for seabird and clinical isolates of B garinii. Many clinical B. garinii isolates have previously been shown to be sensitive to NHS in vitro (Alitalo et al., 2001). Nevertheless these strains cause neuroborreliosis and skin infections in humans. In our study, three groups with varying degrees of NHS resistance were

49 defined. All clinical isolates were either resistant (not affected by 80% NHS) or semi-resistant (sensitive in a dose-dependent manner) to complement attack. All seabird isolates, on the other hand, were sensitive to even low levels of NHS (affected by 5% NHS). In addition, members of the latter group have shown weak or no binding of the complement inhibitor factor H in vitro (Paper V in this thesis). This possibly explains the observed differences in serum resistance.

The spirochetes in the marine infection cycle have the potential to spread to rodents and thereby also influence the terrestrial infection cycle, but are not expected to cause human infections. However, when infecting rodents this might be one step closer to humans.

50 Paper V.

This study presents complement evasion properties of B. garinii spirochetes.

OspE expression and complement resistance Many B. garinii isolates are more sensitive to normal human serum than are B. burgdorferi and B. afzelii when tested in vitro. One serum-sensitive B. garinii isolate (50/97) showed no expression of OspE. However, when transformed with a shuttle vector containing ospE from a serum-resistant B. burgdorferi, OspE protein was expressed and serum resistance increased. This indicates that expression of OspE enhances serum resistance. When comparing ospE genes from several different B. burgdorferi, B. afzelii and B. garinii isolates, sequences from the latter group were found to differ at sites possibly crucial for binding of the complement inhibitor protein factor H (FH). In agreement with this, recombinant OspE from a serum-sensitive B. garinii strain was found to bind FH at much lower levels than recombinant OspE from a serum-resistant B. burgdorferi isolate.

Factor H binding proteins of B. garinii B. garinii isolates grown for a prolonged time in vitro have ospE genes but do not appear to express the protein. When newly isolated B. garinii strains from the CSF of neuroborreliosis patients were tested for FH binding abilities, binding to an uncharacterized 27 kDa protein could be observed. However, the total FH binding capacity mediated by this protein was lower for all the tested neurovirulent B. garinii strains compared to B. afzelii and B. burgdorferi as determined by comparing immunoblot band intensities.

The 27 kDa protein can play a role in the complement evasion of the spirochetes. In addition, a recent study of neuroborreliosis patient sera suggests that OspE indeed is expressed by B. garinii in vivo (Panelius et al., 2007). Therefore, even if FH-binding proteins are expressed at low levels, protection could be sufficient since there is less complement in the CSF than elsewhere in the body.

51 When proteins of B. garinii isolated from seabirds were tested for FH binding abilities, no FH was bound. Also, no expression of proteins with documented FH binding capacities (OspE or CRASP families) was detected. This suggests that B. garinii circulating in the marine infection cycle are vulnerable to attack by human complement proteins and therefore not likely to cause LB. In vitro experiments also support this theory (Paper IV in this thesis)

Many clinical isolates of B. garinii show only intermediate resistance to human serum in vitro, but are apparently still able to cause infection in humans. Therefore, much is left to investigate regarding mechanisms used by Lyme Borrelia spirochetes to escape complement attack.

52 CONCLUSIONS

• Passerine birds are reservoir hosts for B. garinii spirochetes of medical importance.

• Seabirds influence the heterogeneity and ecology of B. garinii.

• A marine Borrelia infection cycle is present in the Arctic.

• Seabirds and passerine birds are infected with the same genetic variants of B. garinii spirochetes.

• B. garinii from seabirds have the potential to spread to the terrestrial infection cycle by infecting mice.

• Seabird isolates of B. garinii can not bind human factor H and are sensitive to human complement; infection of humans is therefore not likely.

• Neuroborreliosis isolates of B. garinii express factor H binding proteins.

53 54 SAMMANFATTNING

Lyme borrelios (vanligtvis kallad ”Borrelia”) är den vanligaste fästingburna bakteriesjukdomen på norra halvklotet. Det finns tre arter av Borrelia-bakterier som orsakar sjukdom hos människor; B. burgdorferi, B. afzelii och B. garinii. Arterna skiljer sig i utbredning och ger delvis olika symptom när de infekterar människor. I Nordamerika finns bara B. burgdorferi, medan alla tre arter finns i Europa och delar av Asien. Sjukdomen Lyme borrelios är en zoonos, vilket betyder att den förekommer hos djur i naturen men ibland av misstag kan spridas även till människor. När larven (fästingens första stadium) suger blod från ett infekterat djur, hamnar bakterierna i fästingens mage. Där stannar de under tiden fästingen mognar till nästa stadium, nymfen. När nymfen sedan suger blod från ett djur (eller en människa) kommer det att bli infekterat. I Europa sprids bakterierna av fästingen Ixodes ricinus och gnagare samt fåglar är de vanligaste reservoardjuren. Borrelia-bakterier förekommer även bland havsfåglar men då sker spridningen med en särskild fästing I. uriae som i regel inte biter människor.

Denna avhandling handlar nästan uteslutande om B. garinii. Borrelia-arten är speciell på flera sätt; bakterierna är mycket heterogena, dvs. bakterierna skiljer sig mycket åt även om de tillhör samma art, de förekommer i stor utsträckning hos havsfåglar och andra fåglar, och när de infekterar människor ger de ofta upphov till neuroborrelios (infektion av det centrala nervsystemet). Dock har det inte varit helt känt om bakterierna som infekterar fåglar även kan infektera människor.

Samspelet mellan B. garinii och fåglar är centralt i avhandlingen. I den första publikationen visar vi att fåglar kan fungera som reservoardjur för B. garinii- bakterier liknande de som infekterar människor. Olika fågelarter har samma reservoarkapacitet, mätt i förmåga att infektera fästingar. Under vår och höst när

55 fåglar flyttar, sprider de mänger med infekterade fästingar som senare kan smitta människor.

I den andra artikeln visar vi att B. garinii även finns hos havsfåglar i arktiska miljöer. Detta var inte känt tidigare och visar på en förekomst även i områden med kallt klimat. Bakterierna som finns här är lika de vi tidigare hittat bland havsfåglar i Östersjön och Färöarna, samt hos Lyme borrelios-patienter. Det visar att B. garinii-bakterier sprids mellan olika havsfågelkolonier.

Den tredje uppsatsen föreslår att B. garinii sprids med havsfåglar runt hela jorden och att detta även kan påverka människor. En del av de Borrelia-bakterier som finns i en och samma havsfågelkoloni i östra Ryssland visar större likhet med Borrelia-bakterier från europeiska Lyme borrelios-patienter, än med andra Borrelia- bakterier från samma koloni. Havsfåglar och andra fåglar har därför stort inflytande på spridningen av B. garinii-bakterier.

I den fjärde artikeln visar vi att B. garinii-bakterier som finns hos havsfåglar kan infektera möss. Detta visar att den marina (havsfåglar) och den landassocierade (fåglar och gnagare) infektionscykeln kan vara sammankopplade. Dessa bakterier är dock mycket känsliga för människans komplementsystem och kan därför troligen inte infektera människor.

I den sista artikeln studerar vi hur bakterierna kan skydda sig mot människans komplementsystem, en del av immunförsvaret som punkterar bakterier direkt när de kommer in i kroppen. Vi identifierar ett nytt Borrelia-protein som binder faktor H (ett humanprotein som hämmar komplementsystemet) och som därmed kan utgöra ett skydd för bakterien. B. garinii-bakterier från havsfåglar kan dock inte binda faktor H, vilket är en trolig anledning till att de är känsliga för människans komplementsystem.

Sammanfattningsvis kan konstateras att fåglar är viktiga reservoardjur för B. garinii-bakterier i Europa och att de infekterade fästingar som de sprider även kan smitta människor.

56 Acknowledgements

Sven Berka Bergström, one thing I can say for sure, it hasn’t been boring to work with you. Thank you for your no-problem-attitude and good friendship. Christer Korven Larsson, a very nice friend and companion on different trips both private and work-related. Thanks to you and Jenny Benny I have seen remarkable things in Västerbotten (the biggest stone in the world, the most northerly located hazel in Sweden, etc.) and thanks for the painting, Häst- Marie, for all the answers to my horse-related questions, Ignas for your relaxed it’s-OK-attitude and help with my computer, Bettan for pretending not to notice when I steal your candy and for all not always useful words in English, Pajala Lizzo for good laughs and talks at djuris, Schlager-Elin for your easy attitude and taking care of the ticks, Rasta-Lelle for your street slang, Ingela for taking care of the lab, Yngve for teaching me a lot about Borrelia and collaboration, Marija for collaboration, Marie B and Patte for nice company in the bunker, Björn Olsen and Alan Barbour, for collaboration, Jonas Bunikis for neverending enthusiasm and collaboration, Seppo Meri and Taru Meri for hospitality, sauna and collaboration, Ingvar Eliasson and Anders Wallensten, for a very nice time in Kamchatka and the Commander Islands, Urban and Albina for collaboration, Pajala Jögga, Ulrich, Jonas, Edmund, Jenny, Teresa, for sharing the lab, Johnny, for all the QIAgen kits and nice motorcycle talks, Jeanette, Sofia, Sara G, Petra E, Skejtar-Micke, Norköpings-Sara, Monkan, Maria, Lisandro, Marie M, Stefan, Gosia, Jocke, Olena, Åsa, Stina, Anna Å, Claudia, for your polite smile when I speak German, Annika, Barbro, Siv for all sequences, Marita for your patience and making everything run smoothly, Ethel, Mariella for your temper, spicing up the department, Lab service, especially Agneta, and the rest of the department for making it a very nice workplace, Lars, Loffe, Lelle and Christine, Eva L, for the fantastic sandwich layer-cake, Finas konditori, for Thursday-buns, FORMAS for paying my motorbike, BOVAC and KEMPE, The Swedish Research Council, the Comstedt clan for being interested in my work and making me long for Småland.

Anna, älskar dig!

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